Encyclopedia of Dietary Supplements, Second Edition

Encyclopedia of Dietary Supplements Second Edition

Edited by

Paul M. Coates Joseph M. Betz Marc R. Blackman Gordon M. Cragg Mark Levine Joel Moss Jeffrey D. White

Encyclopedia of Dietary Supplements

Editorial Advisory Board

Stephen Barnes Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. John H. Cardellina II ReevesGroup Consultations, Walkersville, Maryland, U.S.A. Norman H. Farnsworth UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Donald B. McCormick Department of Biochemistry, School of Medicine, and Program in Nutrition and Health Sciences, Division of Biological Sciences, Emory University, Atlanta, Georgia, U.S.A.

Robert M. Russell Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, and Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, U.S.A. Noel W. Solomons Center for Studies of Sensory Impairment, Aging, and Metabolism (CeSSIAM), Guatemala City, Guatemala Roy Upton R American Herbal Pharmacopoeia , Scotts Valley, California, U.S.A. Steven H. Zeisel Director, Nutrition Research Institute, and Director, Nutrition and Obesity Research Center, UNC Gillings School of Public Health, University of North Carolina, Chapel Hill, North Carolina, U.S.A.

Encyclopedia of Dietary Supplements Second Edition Edited by

Paul M. Coates Director, Office of Dietary Supplements National Institutes of Health, Bethesda, Maryland, U.S.A.

Joseph M. Betz Office of Dietary Supplements National Institutes of Health, Bethesda, Maryland, U.S.A.

Marc R. Blackman Research Service, Veterans Affairs Medical Center Washington, D.C., U.S.A. Departments of Medicine, George Washington University Johns Hopkins University and University of Maryland Schools of Medicine

Gordon M. Cragg NIH Special Volunteer, Natural Products Branch Developmental Therapeutics Program Division of Cancer Treatment and Diagnosis National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Mark Levine Molecular and Clinical Nutrition Section Digestive Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland, U.S.A.

Joel Moss Translational Medicine Branch National Heart, Lung, and Blood Institute National Institutes of Health, Bethesda, Maryland, U.S.A.

Jeffrey D. White Director, Office of Cancer Complementary and Alternative Medicine Division of Cancer Treatment and Diagnosis National Cancer Institute, National Institutes of Health Bethesda, Maryland, U.S.A.

First published in 2005 by Marcel Dekker, New York, NY. This edition published in 2010 by Informa Healthcare, Telephone House, 69-77 Paul Street, London EC2A 4LQ, UK. Simultaneously published in the USA by Informa Healthcare, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017, USA. c 2010 Informa UK Ltd, except as otherwise indicated.  No claim to original U.S. Government works. Reprinted material is quoted with permission. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, unless with the prior written permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP, UK, or the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA (http://www.copyright.com/ or telephone 978-750-8400). Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. This book contains information from reputable sources and although reasonable efforts have been made to publish accurate information, the publisher makes no warranties (either express or implied) as to the accuracy or fitness for a particular purpose of the information or advice contained herein. The publisher wishes to make it clear that any views or opinions expressed in this book by individual authors or contributors are their personal views and opinions and do not necessarily reflect the views/opinions of the publisher. Any information or guidance contained in this book is intended for use solely by medical professionals strictly as a supplement to the medical professional’s own judgement, knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures, or diagnoses should be independently verified. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as appropriately to advise and treat patients. Save for death or personal injury caused by the publisher’s negligence and to the fullest extent otherwise permitted by law, neither the publisher nor any person engaged or employed by the publisher shall be responsible or liable for any loss, injury or damage caused to any person or property arising in any way from the use of this book. A CIP record for this book is available from the British Library. ISBN-13: 9781439819289 Orders may be sent to: Informa Healthcare, Sheepen Place, Colchester, Essex CO3 3LP, UK Telephone: +44 (0)20 7017 5540 Email: [email protected] Website: http://informahealthcarebooks.com/ For corporate sales please contact: [email protected] For foreign rights please contact: [email protected] For reprint permissions please contact: [email protected] Typeset by Aptara, Inc. Printed and bound in the United Kingdom

Preface

Given the large number of dietary supplement products in commerce, this book covers only a small fraction of them, with selection based primarily on the frequency of their use and the availability of a sufficient science base to discuss their efficacy and safety. It is clear that the level of scientific information available differs markedly among the various entries. For many ingredients, the chemistry and physiology, preclinical and clinical information, and mechanism of action are well known. For others, by contrast, some or many pieces of these data are missing. The preparation of some commercial products is of high quality and follows good agricultural, laboratory, and manufacturing practices. Again, by contrast, the preparations for others have not been reliable, making them subject to high variability in content and possible contamination. As dietary supplement use becomes more widespread, there are growing concerns about safety of some ingredients, including possible harmful interactions between supplements and prescribed drugs. When known, this information is included in the chapters of this book. These issues should form the basis for future research. The field of dietary supplements is a rich one, and the science related to this large class of ingredients is expanding all the time. All the chapters that appeared in the first edition have been revised and updated for this edition. In addition to providing these updated chapters, we have included 12 additional chapters on topics not previously covered, reflecting the emergence of dietary supplements in the marketplace, as well as the science behind them. There is also a new chapter on the challenges of dietary supplement research. Additional changes involve gathering several related chapters under “umbrella” topics: Carotenoids and Polyphenols. Two of the chapters in this edition of the Encyclopedia, on Ephedra and Androstenedione, were commissioned before their status as dietary supplements in the U.S. market was changed. In 2004, the FDA banned ephedra-containing products from the dietary supplement market in the United States. Also in 2004, the FDA issued warning letters to companies then marketing products containing androstenedione; the regulatory status of these products as dietary supplements has therefore changed. Nevertheless, until recently, both ephedra and androstenedione were widely consumed in the United States. We felt, therefore, that discussion of the science of these ingredients was important. The chapters have been updated to reflect the new regulatory status of these ingredients. Where possible and applicable, chapter names for botanical ingredients have been adapted to conform to the standardized common names in the American Herbal

Welcome to the second edition of Encyclopedia of Dietary Supplements, reflecting the combined efforts of more than 100 authors from 13 countries on 97 topics. Response to the first edition, published in 2005 and then supplemented by a series of online chapters, prompted us to revise and expand the Encyclopedia. There has been considerable expansion in research on many dietary supplements and their ingredients. We expect that this Encyclopedia will continue to be a valuable reference for students and researchers in physiology and chemistry, for health care providers, and for consumers who are interested in understanding the kind of science that is—or is not—behind the claims that are made for dietary supplements that are sold throughout the world, where standards of government regulation differ from country to country. In the United States, sales of products in the dietary supplement market approached $25 billion in 2009. Their form and their labeling are regulated by the Food and Drug Administration (FDA) as a result of legislation passed in 1994 called the Dietary Supplement Health and Education Act (DSHEA). The dietary supplement category in the United States includes vitamins, minerals, and other ingredients that are found in foods, as well as ingredients not ordinarily found in foods—such as extracts of herbs and other natural products—that are used by consumers for their potential health-promoting, disease-preventing, or performance-enhancing properties. Many of these are represented in the chapters of this book. The Encyclopedia is not just for consumers in the U.S. market, although we acknowledge that the term “dietary supplements” is an American expression. We are not aware of any other single term that describes all of the substances that we wish to include in this Encyclopedia, although terms such as food supplements, nutritional supplements, or natural health products have been applied as well. Sometimes the claims for benefit of specific products are borne out by well-documented scientific studies. In other cases, they are not, or the science to support their use is still at an early stage. Enthusiasm for their use may be based on popular legend or on longstanding patterns of use in traditional healing systems. In this book, we hope that readers will be able to examine the types of evidence that have been used to support claims of benefit and safety. The goal of this book is to provide readers with comprehensive, yet accessible, information on the current state of science for individual supplement ingredients or extracts. To this end, each entry reviews basic information available about the ingredient including, where applicable, its chemistry and functions, before detailing the preclinical and clinical literature. Articles conclude with references to the relevant literature. v

vi

Preface

Products Association’s Herbs of Commerce, Second Edition (2000). The accepted scientific names (with authority) and additional synonyms may be found in the individual chapters. We express our thanks to the authors of the individual chapters. This is a challenging and somewhat controversial field, but we believe that our authors have provided a balanced and current view of the literature. We also acknowledge with gratitude the hard work and guidance of Informa Healthcare’s editorial staff, particularly the project editor, Timothy DeWerff.

Finally, we wish to emphasize that the inclusion of chapters on particular dietary supplements in this Encyclopedia does not imply that we endorse them. Paul M. Coates Joseph M. Betz Marc R. Blackman Gordon M. Cragg Mark Levine Joel Moss Jeffrey D. White

Contents

L-Carnitine, Acetyl-L-Carnitine, and Propionyl-L-Carnitine 107 Charles J. Rebouche

Preface . . . . v Contributors . . . . x The Challenges of Dietary Supplement Research and Considerations for Future Studies . . . . xvi

β-Carotene

115 Elizabeth J. Johnson and Robert M. Russell

S-Adenosylmethionine 1 Jos´e M. Mato and Shelly C. Lu

Carotenoids Overview 121 Elizabeth J. Johnson and Robert M. Russell

Aloe Vera 7 Santiago Rodriguez, Steven Dentali, and Devon Powell

Cascara Sagrada 124 Kapil K. Soni and Gail B. Mahady

Androstenedione 15 Benjamin Z. Leder

Chaste Tree 129 Gail B. Mahady, Joanna L. Michel, and Kapil K. Soni

L-Arginine

21 Mauro Maccario, Guglielmo Beccuti, Valentina Gasco, Mariangela Seardo, Gianluca Aimaretti, Emanuela Arvat, Fabio Lanfranco, and Ezio Ghigo

Choline 136 Steven H. Zeisel Chondroitin Sulfate 144 Karla L. Miller and Daniel O. Clegg

Astragalus 29 Roy Upton

Chromium 149 Richard A. Anderson and William T. Cefalu

Bilberry 37 Marilyn Barrett

Coenzyme Q10 157 Gustav Dallner and Roland Stocker

Biotin 43 Donald M. Mock

Conjugated Linoleic Acid 166 Kristina B. Martinez, Arion J. Kennedy, and Michael K. McIntosh

Bitter Orange 52 Steffany Haaz, K. Y. Williams, Kevin R. Fontaine, and David B. Allison

Copper 175 Leslie M. Klevay

Black Cohosh 60 Daniel S. Fabricant, Elizabeth C. Krause, and Norman R. Farnsworth

Cordyceps 185 John Holliday, Matt Cleaver, Mojca Tajnik, Joseph M. Cerecedes, and Solomon P. Wasser

Blue-Green Algae (Cyanobacteria) 75 Wayne W. Carmichael and Mary Stukenberg with Joseph M. Betz

Cranberry 193 Marguerite A. Klein

Boron 82 Curtiss Hunt

Creatine 202 G. S. Salomons, C. Jakobs, and M. Wyss

Caffeine 90 Harris R. Lieberman, Christina E. Carvey, and Lauren A. Thompson

Dong Quai 208 Roy Upton

Calcium 101 Robert P. Heaney

Dehydroepiandrosterone 217 Salvatore Alesci, Irini Manoli, and Marc R. Blackman vii

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Contents

Echinacea Species 226 Rudolf Bauer and Karin Woelkart

Hawthorn 411 Egon Koch, Werner R. Busse, Wiltrud Juretzek, and Vitali Chevts

Elderberry 235 Madeleine Mumcuoglu, Daniel Safirman, and Mina Ferne

5-Hydroxytryptophan 423 Pedro Del Corral, Kathryn S. King, and Karel Pacak

Eleuthero 241 Josef A. Brinckmann

Iron 432 Laura E. Murray-Kolb and John Beard

Ephedra 250 Anne L. Thurn with Joseph M. Betz

Isoflavones 439 Mark Messina

Evening Primrose 256 Fereidoon Shahidi and Homan Miraliakbari

Isothiocyanates 450 Elizabeth H. Jeffery and Anna-Sigrid Keck

Feverfew 267 Dennis V. C. Awang

Kava 459 Michael J. Balick, Katherine Herrera, and Steven M. Musser

Flaxseed 274 Lilian U. Thompson and Julie K. Mason Folate 288 Pamela Bagley and Barry Shane French Maritime Pine 298 Peter J. Rohdewald Garcinia 307 Frank Greenway

Lactobacilli and Bifidobacteria 469 Linda C. Duffy, Stephen Sporn, Patricia Hibberd, Carol Pontzer, Gloria Solano-Aguilar, Susan V. Lynch, and Crystal McDade-Ngutter Licorice 479 Decio Armanini, Cristina Fiore, Jens Bielenberg, and Eugenio Ragazzi α-Lipoic Acid/Thioctic Acid

487

Donald B. McCormick Garlic 314 J. A. Milner Ginger 325 Tieraona Low Dog Ginkgo 332 Kristian Strømgaard, Stine B. Vogensen, Joseph Steet, and Koji Nakanishi Ginseng, American 339 Chong-Zhi Wang and Chun-Su Yuan Ginseng, Asian 348 Lee Jia and Fabio Soldati Glucosamine 363 Karla L. Miller and Daniel O. Clegg Glutamine 370 Steven F. Abcouwer Goldenseal 379 Dennis J. McKenna and Gregory A. Plotnikoff Grape Seed Extract 391 Dallas L. Clouatre, Chithan Kandaswami, and Kevin M. Connolly Green Tea Polyphenols 402 Shengmin Sang, Joshua D. Lambert, Chi-Tang Ho, and Chung S. Yang

Lutein 493 John Paul SanGiovanni, Emily Y. Chew, and Elizabeth J. Johnson Lycopene 504 Rachel Kopec, Steven J. Schwartz, and Craig Hadley Maca 518 Ilias Muhammad, Jianping Zhao, and Ikhlas A. Khan Magnesium 527 Robert K. Rude Melatonin 538 Amnon Brzezinski and Richard J. Wurtman Milk Thistle 550 Elena Ladas, David J. Kroll, and Kara M. Kelly Niacin 562 Christelle Bourgeois and Joel Moss Noni 570 Alison D. Pawlus, Bao-Ning Su, Ye Deng, and A. Douglas Kinghorn Omega-3 Fatty Acids 577 William S. Harris

Contents

Omega-6 Fatty Acids 587 William L. Smith and Bill Lands

St. John’s Wort 727 Jerry M. Cott

Pancreatic Enzymes 598 Naresh Sundaresan, Unwanaobong Nseyo, and Joel Moss

Taurine 738 Robin J. Marles, Valerie A. Assinewe, Julia A. Fogg, Milosz Kaczmarek, and Michael C. W. Sek

Pantothenic Acid 604 Lawrence Sweetman

Thiamin 748 Hamid M. Said

Pau d’Arco 612 Memory P. F. Elvin-Lewis and Walter H. Lewis

Turmeric 754 Janet L. Funk

Phosphorus 626 John J. B. Anderson and Sanford C. Garner

Valerian 766 Dennis V. C. Awang

Polyphenols Overview 632 Navindra P. Seeram

Vitamin A 778 A. Catharine Ross

Proanthocyanidins 635 Catherine Kwik-Uribe, Rebecca Robbins, and Gary Beecher

Vitamin B6 792 James E. Leklem

Pygeum 650 Franc¸ois G. Brackman and Alan Edgar with Paul M. Coates Quercetin 656 Jae B. Park Red Clover 665 Elizabeth C. Krause, Nancy L. Booth, Colleen E. Piersen, and Norman R. Farnsworth Reishi 680 Solomon P. Wasser Riboflavin 691 Richard S. Rivlin Saw Palmetto 700 Edward M. Croom and Michael Chan Selenium 711 Roger A. Sunde Shiitake 719 Solomon P. Wasser

Vitamin B12 812 Lindsay H. Allen Vitamin C 821 Sebastian Padayatty, Michael Graham Espey, and Mark Levine Vitamin D 832 Patsy Brannon, Mary Frances Picciano, and Michelle K. McGuire Vitamin E 841 Maret G. Traber Vitamin K 851 J. W. Suttie Yohimbe 861 Joseph M. Betz Zinc 869 Carolyn S. Chung and Janet C. King Index . . . . 877

ix

Contributors

Guglielmo Beccuti Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Steven F. Abcouwer Departments of Surgery, Cellular and Molecular Physiology, and Ophthalmology, Penn State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, U.S.A.

Gary Beecher Consultant, Lothian, Maryland, U.S.A.

Gianluca Aimaretti Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Joseph M. Betz Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, U.S.A.

Salvatore Alesci Discovery Translational Medicine, Pfizer, Collegeville, Pennsylvania, U.S.A.

Jens Bielenberg Department of Medical and Surgical Sciences, University of Padua, Padua, Italy

Lindsay H. Allen United States Department of Agriculture, Agricultural Research Service—Western Human Nutrition Research Center, Davis, California, U.S.A.

Marc R. Blackman Research Service, Veterans Affairs Medical Center, Washington, D.C., U.S.A., and Departments of Medicine, George Washington University, Johns Hopkins University, and University of Maryland Schools of Medicine

David B. Allison Department of Biostatistics/Nutrition Obesity Research Center, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

Nancy L. Booth Spherix Consulting, Inc, Bethesda, Maryland, U.S.A.

John J. B. Anderson Schools of Public Health and Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A.

Christelle Bourgeois Max F. Perutz Laboratories, Institute of Medical Biochemistry, Medical University of Vienna, Vienna, Austria

Richard A. Anderson Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Beltsville, Maryland, U.S.A.

Franc¸ois G. Brackman Fournier Pharma, Garches, France Patsy Brannon Division of Nutritional Sciences, Cornell University, Ithaca, New York, U.S.A.

Decio Armanini Department of Medical and Surgical Sciences, University of Padua, Padua, Italy

Josef A. Brinckmann Traditional Medicinals, Sebastopol, California, U.S.A.

Emanuela Arvat Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Amnon Brzezinski Department of Obstetrics and Gynecology, The Hebrew University–Hadassah Medical School, Jerusalem, Israel

Valerie A. Assinewe Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada

Werner R. Busse Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

Dennis V. C. Awang MediPlant Consulting Services, White Rock, British Columbia, Canada

Wayne W. Carmichael Department of Biological Sciences, Wright State University, Dayton, Ohio, U.S.A.

Pamela Bagley Biomedical Libraries, Dartmouth College, Hanover, New Hampshire, U.S.A.

Christina E. Carvey Military Nutrition Division, U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts, U.S.A.

Michael J. Balick Institute of Economic Botany, The New York Botanical Garden, Bronx, New York, U.S.A. Marilyn Barrett Pharmacognosy Consulting, Mill Valley, California, U.S.A.

William T. Cefalu Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana, U.S.A.

Rudolf Bauer Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-FranzensUniversity Graz, Graz, Austria

Joseph M. Cerecedes Mycoverse Unlimited Inc., Ashland, Oregon, U.S.A.

John Beard Department of Nutritional Sciences, The Pennsylvania State University, University Park, Pennsylvania, U.S.A. (Deceased).

Michael Chan British Columbia Institute of Technology, Burnaby, British Columbia, Canada x

Contributors

xi

Vitali Chevts Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Emily Y. Chew Division of Epidemiology and Clinical Applications, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Mina Ferne The Israeli Association of Medicinal Plants (EILAM), Israel

Carolyn S. Chung Food and Drug Administration, College Park, Maryland, U.S.A. Matt Cleaver Aloha Medicinals Inc., Carson City, Nevada, U.S.A. Daniel O. Clegg George E. Wahlen Department of Veterans Affairs Medical Center and University of Utah School of Medicine, Salt Lake City, Utah, U.S.A.

Cristina Fiore Department of Medical and Surgical Sciences, University of Padua, Padua, Italy Julia A. Fogg Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada Kevin R. Fontaine Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

Dallas L. Clouatre Glykon Technologies Group, L.L.C., Las Vegas, Nevada, U.S.A.

Janet L. Funk Department of Medicine, College of Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona, U.S.A.

Kevin M. Connolly Glykon Technologies Group, L.L.C., Las Vegas, Nevada, U.S.A.

Sanford C. Garner Carolina, U.S.A.

Pedro Del Corral Grand Forks Human Nutrition Research Center, ARS/USDA, Grand Forks, North Dakota, U.S.A.

Valentina Gasco Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Jerry M. Cott

Fulton, Maryland, U.S.A.

Edward M. Croom School of Pharmacy, University of Mississippi, Oxford, Mississippi, U.S.A. Gustav Dallner Department of Biochemistry and Biophysics, Stockholm University, and Rolf Luft Research Centre for Diabetes, Karolinska Institutet, Stockholm, Sweden Ye Deng Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, U.S.A. Steven Dentali American Herbal Products Association, Silver Spring, Maryland, U.S.A. Linda C. Duffy Natural Products Branch, National Center for Complementary and Alternative Medicine, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, U.S.A. Alan Edgar Fournier Pharma, Garches, France Memory P. F. Elvin-Lewis Department of Biology,Washington University, St. Louis, MO, U.S.A Michael Graham Espey Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A Daniel S. Fabricant Natural Products Association, Washington, D.C., and UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Norman R. Farnsworth UIC/NIH Center for Botanical Dietary Supplements Research, Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and

SRA International, Durham, North

Ezio Ghigo Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy Frank Greenway Division of Clinical Trials, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana, U.S.A. Steffany Haaz Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Craig Hadley Mead Johnson Nutritionals Regulatory Science, Evansville, Indiana, U.S.A. William S. Harris Sanford School of Medicine, University of South Dakota and Sanford Research/USD, Sioux Falls, South Dakota, U.S.A. Robert P. Heaney Creighton University, Omaha, Nebraska, U.S.A. Katherine Herrera Institute of Economic Botany, The New York Botanical Garden, Bronx, New York, U.S.A. Patricia Hibberd Center for Global Health Research, Departments of Medicine, Pediatrics, and Public Health, Tufts University School of Medicine, Boston, MA, U.S.A. Chi-Tang Ho Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunwick, New Jersey, U.S.A. John Holliday Aloha Medicinals Inc., Carson City, Nevada, U.S.A. D. Craig Hopp National Institutes of Health, National Center for Complementary and Alternative Medicine, Bethesda, Maryland, U.S.A. Curtiss Hunt Vienna, Austria C. Jakobs VU University Medical Center, Department of Clinical Chemistry, Metabolic Unit, Amsterdam, The Netherlands

xii

Contributors

Elizabeth H. Jeffery Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, U.S.A.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Lee Jia Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Rockville, Maryland, U.S.A.

David J. Kroll Natural Products Laboratory, Research Triangle Institute (RTI International), Research Triangle Park, North Carolina, U.S.A.

Elizabeth J. Johnson Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, U.S.A. Wiltrud Juretzek Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany Milosz Kaczmarek Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada Chithan Kandaswami State University of New York at Buffalo, Buffalo, New York, U.S.A. Anna-Sigrid Keck Department of Food Science and Human Nutrition, University of Illinois at UrbanaChampaign, Urbana, Illinois, and Research Institute, Carle Foundation Hospital, Urbana, Illinois, U.S.A. Kara M. Kelly Division of Pediatric Oncology, Integrative Therapies Program for Children with Cancer, College of Physicians and Surgeons, Columbia University Medical Center, New York, U.S.A. Arion J. Kennedy Department of Nutrition, University of North Carolina at Greensboro, Greensboro, North Carolina, U.S.A. Ikhlas A. Khan National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi, U.S.A. Janet C. King Children’s Hospital Oakland Research Institute, Oakland, California, U.S.A. Kathryn S. King Program in Adult Endocrinology and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. A. Douglas Kinghorn Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, U.S.A. Marguerite A. Klein Office of Dietary Supplements, Office of the Director, National Institutes of Health, Bethesda, Maryland, U.S.A. Leslie M. Klevay University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, U.S.A. Egon Koch Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

Catherine Kwik-Uribe Mars Chocolate NA, Hackettstown, New Jersey, U.S.A. Elena Ladas Division of Pediatric Oncology, Integrative Therapies Program for Children with Cancer, College of Physicians and Surgeons, New York, U.S.A. Joshua D. Lambert Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania, U.S.A. Bill Lands College Park, Maryland, U.S.A. Fabio Lanfranco Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy Benjamin Z. Leder Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, U.S.A. James E. Leklem Oregon State University, Corvallis, Oregon, U.S.A. Mark Levine Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A Walter H. Lewis Department of Biology, Washington University, St. Louis, MO, U.S.A Harris R. Lieberman Military Nutrition Division, U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts, U.S.A. Tieraona Low Dog University of Arizona Health Sciences Center, Tucson, Arizona, U.S.A. Shelly C. Lu Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, Southern California Research Center for ALPD and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Susan V. Lynch UCSF Crohn’s and Colitis Microbiome Research Core, Division of Gastroenterology, Department of Medicine, University of California, San Francisco, CA, U.S.A. Mauro Maccario Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Rachel Kopec The Ohio State University, Columbus, Ohio, U.S.A.

Gail B. Mahady Department of Pharmacy Practice, College of Pharmacy, PAHO/WHO Collaborating Center for Traditional Medicine, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Elizabeth C. Krause UIC/NIH Center for Botanical Dietary Supplements Research, Program for Collaborative Research in the Pharmaceutical Sciences,

Irini Manoli Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Contributors

xiii

Robin J. Marles Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada

Sciences, School of Pharmacy, University of Mississippi, Mississippi, U.S.A.

Kristina B. Martinez Department of Nutrition, University of North Carolina at Greensboro, Greensboro, North Carolina, U.S.A.

Madeleine Mumcuoglu Razei Bar Industries, Jerusalem, Israel

Julie K. Mason Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada ´ Jos´e M. Mato CIC bioGUNE, Centro de Investigacion Biom´edica en Red de Enfermedades Hep´aticas y Digestivas (CIBERehd), Bizkaia, Spain Donald B. McCormick Department of Biochemistry, School of Medicine, Emory University, Atlanta, Georgia, U.S.A. Crystal McDade-Ngutter Division of Nutrition Research Coordination, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, U.S.A. Michelle K. McGuire School of Molecular Biosciences, Washington State University, Pullman, Washington, U.S.A.

Laura E. Murray-Kolb Department of Nutritional Sciences, The Pennsylvania State University, University Park, Pennsylvania, U.S.A. Steven M. Musser Office of Scientific Analysis and Support, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, Maryland, U.S.A. Koji Nakanishi Department of Chemistry, Columbia University, New York, U.S.A. Unwanaobong Nseyo Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Karel Pacak Program in Adult Endocrinology and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

Michael K. McIntosh Department of Nutrition, University of North Carolina at Greensboro, Greensboro, North Carolina, U.S.A.

Sebastian Padayatty Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A

Dennis J. McKenna Center for Spirituality and Healing, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, U.S.A.

Jae B. Park Phytonutrients, Genomics, and Immunology Laboratory, BHNRC, ARS, United States Department of Agriculture, Beltsville, Maryland, U.S.A.

Mark Messina Department of Nutrition, School of Public Health, Loma Linda University, Loma Linda, California, and Nutrition Matters, Inc., Port Townsend, Washington, U.S.A.

Alison D. Pawlus Groupe d’Etude des Substances V´eg´etales a` Activit´e Biologique, Facult´e de Pharmacie, Institut des Sciences de la Vigne et du Vin de Bordeaux, Universit´e Bordeaux 2, Bordeaux, France

Catherine M. Meyers National Institutes of Health, National Center for Complementary and Alternative Medicine, Bethesda, Maryland, U.S.A.

Mary Frances Picciano Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, U.S.A.

Joanna L. Michel Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Karla L. Miller University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. J. A. Milner Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, Maryland, U.S.A.

Colleen E. Piersen UIC/NIH Center for Botanical Dietary Supplements Research, Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Gregory A. Plotnikoff Penny George Institute for Health and Healing, Abbott Northwestern Hospital, Minneapolis, Minnesota, U.S.A.

Homan Miraliakbari Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

Carol Pontzer Natural Products Branch, National Center for Complementary and Alternative Medicine, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, U.S.A.

Donald M. Mock Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, U.S.A.

Devon Powell International Aloe Science Council, Silver Spring, Maryland, U.S.A.

Joel Moss Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Ilias Muhammad National Center for Natural Products Research, Research Institute of Pharmaceutical

Eugenio Ragazzi Department of Pharmacology and Anaesthesiology, University of Padua, Padua, Italy Charles J. Rebouche Carver College of Medicine, University of Iowa, Iowa City, Iowa, U.S.A. Richard S. Rivlin

Rogosin Institute, New York, U.S.A.

xiv

Contributors

Rebecca Robbins Mars Chocolate NA, Hackettstown, New Jersey, U.S.A. Santiago Rodriguez Lorand Laboratories LLC, Houston, Texas, U.S.A. Peter J. Rohdewald Institute of Pharmaceutical ¨ Chemistry, Westf¨alische Wilhelms-Universit¨at Munster, ¨ Munster, Germany A. Catharine Ross Department of Nutritional Sciences, The Pennsylvania State University, University Park, Pennsylvania, U.S.A. Robert K. Rude Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Robert M. Russell Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, and Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, U.S.A. Daniel Safirman Razei Bar Industries, Jerusalem, Israel Hamid M. Said Department of Medicine and Physiology/Biophysics, University of California School of Medicine, Irvine, California, U.S.A., and Department of Medical Research, VA Medical Center, Long Beach, California, U.S.A.

Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, U.S.A. Fabio Soldati Pharmaton SA, Scientific Coordination, Bioggio, Switzerland Kapil K. Soni Department of Pharmacy Practice, College of Pharmacy, PAHO/WHO Collaborating Center for Traditional Medicine, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Stephen Sporn St. John’s Integrative Medicine Clinic, Springfield, MO, U.S.A. Joseph Steet Department of Biology, Columbia University, New York, U.S.A. Roland Stocker Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia Kristian Strømgaard Department of Medicinal Chemistry, The Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark Mary Stukenberg Department of Biological Sciences, Wright State University, Dayton, Ohio, U.S.A.

G. S. Salomons VU University Medical Center, Department of Clinical Chemistry, Metabolic Unit, Amsterdam, The Netherlands

Bao-Ning Su Analytical Research and Development, Bristol-Myers Squibb, New Brunswick, New Jersey, U.S.A.

Shengmin Sang Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, Kannapolis, North Carolina, U.S.A.

Naresh Sundaresan Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

John Paul SanGiovanni Division of Epidemiology and Clinical Applications, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Steven J. Schwartz The Ohio State University, Columbus, Ohio, U.S.A. Mariangela Seardo Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy Navindra P. Seeram Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island, U.S.A. Michael C. W. Sek Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada Fereidoon Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada Barry Shane Nutritional Sciences and Toxicology, University of California, Berkeley, California, U.S.A. William L. Smith University of Michigan Medical School, Ann Arbor, Michigan, U.S.A. Gloria Solano-Aguilar Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition

Roger A. Sunde Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin, U.S.A. J. W. Suttie Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin–Madison, Madison, Wisconsin, U.S.A. Lawrence Sweetman Mass Spectrometry Laboratory, Institute of Metabolic Disease, Baylor Research Institute, Dallas, Texas, U.S.A. Mojca Tajnik Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia Lauren A. Thompson Military Nutrition Division, U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts, U.S.A. Lilian U. Thompson Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Anne L. Thurn Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, U.S.A. Maret G. Traber Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, Corvallis, Oregon, U.S.A. R Roy Upton American Herbal Pharmacopoeia , Scotts Valley, California, U.S.A.

Stine B. Vogensen Department of Medicinal Chemistry, The Faculty of Pharmaceutical Sciences,

Contributors

xv

University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A.

Chong-Zhi Wang Tang Center for Herbal Medicine Research, University of Chicago, Chicago, Illinois, U.S.A.

M. Wyss DSM Nutritional Products Ltd., Research and Development Base Products, Basel, Switzerland

Solomon P. Wasser Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education and Institute of Evolution, University of Haifa, Haifa, Israel, and N. G. Kholodny Institute of Botany National Academy of Sciences of Ukraine, Kiev, Ukraine

Chung S. Yang Department of Chemical Biology, Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

K. Y. Williams Department of Biostatistics/Nutrition Obesity Research Center, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Karin Woelkart Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-FranzensUniversity Graz, Graz, Austria Richard J. Wurtman Cecil H. Green Distinguished Professor, M.I.T. Department of Brain & Cognitive

Chun-Su Yuan Tang Center for Herbal Medicine Research, University of Chicago, Chicago, Illinois, U.S.A. Steven H. Zeisel Department of Nutrition, UNC Nutrition Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Jianping Zhao National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi, U.S.A.

The Challenges of Dietary Supplement Research and Considerations for Future Studies D. Craig Hopp and Catherine M. Meyers

INTRODUCTION

Table 1 The 10 Most Common CAM Therapies Used in U.S. Adults–2007a

The American public and the popular press have considerable interest in the use of dietary supplements (1,2). In view of observed widespread use, there is a need for further information regarding dietary supplement products and their potential clinical applications. This report presents recently compiled data on dietary supplement use in the United States and discusses primary considerations for further research in this area. These considerations focus largely on the need for a standard approach to product characterization and the need to develop an appropriate knowledge base for individual products, prior to embarking on large multicenter trials assessing product efficacy.

Therapy

Prevalence (%)

Dietary supplements Deep breathing Meditation Chiropractic and osteopathic Massage Yoga Diet-based therapies Progressive relaxation Guided imagery Homeopathic treatment a Source:

17.7 12.7 9.4 8.6 8.3 6.1 3.6 2.9 2.2 1.8

Adapted from Ref. 1.

Table 2 The 10 Most Common Natural Products Used in the United States–2007a

BACKGROUND ON DIETARY SUPPLEMENT USE IN THE UNITED STATES

Prevalence (%) Adults Fish oil/␻-3 Glucosamine Echinacea Flaxseed oil/pills Ginseng Combination herb pills Ginkgo biloba Chondroitin Garlic supplements Coenzyme Q10 Children Echinacea Fish oil/␻-3 Combination herb pills Flaxseed oil/pills

Findings from the 2007 National Health Interview Survey (NHIS), conducted by the Centers for Disease Control and Prevention’s National Center for Health Statistics, have provided extensive information on dietary supplement use by the American public (1). The NHIS is an annual in-person survey of Americans regarding their healthand illness-related experiences. The 2007 NHIS included a complementary and alternative medicine (CAM) section and collected information from nearly 24,000 adults, as well as nearly 9500 children under the age of 18 years. The 2007 NHIS data (Table 1) reveal that approximately 38% of adults, nearly 39 million Americans, use some form of CAM therapy and further that nearly 18% of adults use at least one nonvitamin, nonmineral dietary supplement (1). Similarly, approximately 12% of children less than 18 years of age use some form of CAM therapy, with nearly 4% using at least one dietary supplement (1). The most common reason provided for dietary supplement use is for enhancing wellness (40%). Another 35% of respondents indicate that dietary supplements are used for both wellness and for treatment of a specific condition, whereas only 20% relate that dietary supplements are used to treat a specific condition. The most common health conditions related to CAM product use are those associated with chronic pain, largely of musculoskeletal origin (1). The most commonly used dietary supplements reported in the 2007 NHIS are listed in Table 2 (1). The 10

a Source:

37.4 19.9 19.8 15.9 14.1 13.0 11.3 11.2 11.0 8.7 37.2 30.5 17.9 16.7

Adapted from Ref. 1.

most commonly used products in adult respondents are fish oil or ␻-3 fatty acids, including docosahexaenoic acid, glucosamine, echinacea, flaxseed oil or pills, ginseng combination herb pills, ginkgo biloba, chondroitin, garlic supplements, and coenzyme Q10. Most dietary supplement use reported for children in the United States is focused on four products: echinacea (37.2%), fish oil or ␻-3 fatty acids (30.5%), combination herb pills (17.9%), and flaxseed oil or pills (16.7%) (1). Use of CAM therapies, including dietary supplements, is widespread across all demographic groups of the U.S. population (1,2) and is more prevalent in women xvi

The Challenges of Dietary Supplement Research and Considerations for Future Studies

than in men, with regional variability, in that the use is more prevalent in the West than in the Midwest, Northeast, or Southern regions of the United States. Greater use of CAM therapies is observed between the ages of 30 and 69 years and is also associated with higher levels of education, former smokers, and reported regular levels of physical activity. CAM therapy use is also higher in respondents who report more health conditions or doctor visits, although 20% of CAM users did not report underlying health conditions (1,2). In view of this extensive use, there is a need for further study of dietary supplements. Rigorous testing of individual dietary supplements, however, is frequently limited because of lack of critical information on several product attributes. In particular, lack of information on product characterization, purity, active ingredients, pharmacokinetics, potential mechanisms of action, or biomarkers for activity limits early phase testing of products. Lack of dosing information and definition of appropriate clinical outcome measures also limit planning of clinical trials. A more standardized approach to product characterization and development of a richer knowledge base on individual supplements will be essential to advancing investigative efforts in this field.

PRODUCT INTEGRITY ISSUES FOR DIETARY SUPPLEMENT RESEARCH One of the unique challenges inherent to dietary supplement research is that the product complexity is highly variable. This issue poses a serious challenge to establishing a “standard” list of quality control procedures for these products. Although single-component supplements such as resveratrol or melatonin can be accurately characterized and exactly reproduced, plant extracts are much more complex. Furthermore, as has been widely documented, there can be considerable inconsistency in batch-to-batch, bottle-to-bottle, and brand-to-brand content of “off-the-shelf” dietary supplements (3). For botanical products, there is a high level of complexity and natural variability, which prevent investigators from entirely characterizing or exactly reproducing a particular extract. It is estimated that individual plant species are capable of producing thousands of metabolites at varying concentrations. Additional variables for these products include the observation that the same species grown in different places, or even different years in the same place, will not generate the same metabolic profile. It is therefore apparent that a certain amount of product variability, for some supplements, is to be expected. Despite these obstacles, researchers must still strive to conduct a thorough analysis of products used for research purposes. Extensive characterization of research materials is a necessary initial step so that subsequent study results can be appropriately interpreted and reliably reproduced. It is also apparent that comprehensive characterization, especially for botanical products, can require an enormous amount of effort and expense. Products typically pass through several hands from the grower to the processor and the distributor, prior to arrival at the vendor, and possibly others before reaching consumers. It can be very difficult and sometimes impossible to trace a

xvii

given product back to its origins. Furthermore, the identity of every minor component in an extract is almost never known. However, with some important exceptions, this degree of detail in product characterization is neither necessary nor practical. A pragmatic approach is to establish quality control methods that are appropriate for the complexity of the product, the proposed research plan, and product’s intended use. A clinical trial testing a herbal extract will require a substantial dossier of information to document safety, stability, and reproducibility of that product. This dossier will include detailed knowledge about every step in the chain of custody of that material from the time it was grown to the time it was administered to patients. The U.S. Food and Drug Administration (FDA) released a guidance document for botanical drugs in 2004, which is an appropriate resource on quality control procedures to follow for randomized controlled trials (RCTs) of herbal products (4). Investigators intending to conduct clinical studies are strongly encouraged to contact FDA and determine whether an IND (investigational new drug) application is needed for the study of a product in the United States. If an IND is needed for a given study, FDA will provide specific guidance regarding type of information required and level of detail for product characterization. The characterization requirements for complex products used for in vitro studies are perhaps less clear. Similarly, the requirements for early-stage clinical studies on refined products that are botanically derived, but far less complex than the parent extract from which they originated, are less well defined. In these examples, it might be argued that the focus should be more on accurate product characterization. High-pressure liquid chromatography is the analytical technique most commonly employed for generating a product “fingerprint,” but there are other methods that could be appropriate depending on the sample. This fingerprint, regardless of the method used to generate it, establishes the identity of a given product without having to know the identity of every product component. Furthermore, it sets a reference point that can be used to document batch-to-batch reproducibility and assess product stability over time. Whichever analytical technique is chosen, the fingerprint must be unique enough to distinguish it from related products and sensitive enough to detect significant changes over time. As the particular dietary supplement research progresses into animals and ultimately humans, progressively more information will be needed regarding product origin and its manufacturing process. A realistic balance should be sought between the need for further studies of dietary supplements and the need for extensive product characterization prior to beginning research studies. Such a balance will ensure the feasibility of future research efforts on these products. Investigators need to be cognizant of the need for product characterization even in early stages of dietary supplement research. Part of this effort involves independent product analysis, either by the investigator or third party laboratory, to confirm specifications provided by the supplier. This early-stage testing must be conducted regardless of product complexity. Even “pure” compounds from widely known manufacturers have been noted to be mislabeled, in that the content analysis demonstrated that the marketed product was not consistent

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with label specifications. For products that have not been extensively studied, it may not be feasible to have an independent analysis performed as validated methods may not be available. Moreover, developing or implementing new methods for such products can represent an appropriate independent research endeavor. In such cases, the information provided requires close scrutiny to determine whether additional product concerns remain. Finally, another important product consideration for investigating dietary supplements focuses on familiarity with the product supplier. Whenever possible, investigators should begin cultivating relationships with the product supplier at early stages of their research and start acquiring information that will be required for future studies. It is important to determine early in the course of investigations whether the supplier has stringent quality control procedures in place and whether they will provide the requisite product documentation. This is especially true if the ultimate goal is to develop a knowledge base necessary for performing clinical studies. It is therefore prudent to select suppliers or vendors that have provided products for other research studies and have a track record of supplying test materials with the requisite documentation.

CONSIDERATIONS FOR CLINICAL STUDIES OF DIETARY SUPPLEMENTS Clinical studies are an essential tool for assessing safety and efficacy of therapeutic interventions, whether they are conventional drugs, medical devices, or dietary supplements (5). Similar to standards for assessing efficacy of pharmaceuticals, RCTs play a major role in determining whether a compound or product is safe and effective for a specific indication (5). Prior to initiating (phase III) RCTs, however, there is substantial information that should be collected on a given product. In the pharmaceutical industry, extensive preliminary preclinical and clinical studies (i.e., pharmacokinetics, dosing strategies) are typically undertaken prior to performing large multicenter trials, due to regulatory requirements enforced by FDA. There is a similar need for extensive preliminary studies for dietary supplement investigations, particularly when the research question for the study includes treatment of a disease or condition. It is important to develop a knowledge base for individual dietary supplements, which will provide direction for further clinical investigations. The optimum knowledge base for a product includes information on mechanism(s) of action, clinical chemistry, biomarkers for in vivo effect, appropriate clinical outcome measures, and the target patient population for the product. For many dietary supplements, information is lacking on many aspects of this knowledge base, which has hampered progress in conducting definitive clinical studies. As discussed in the previous section, it is essential to have standardized data collection on product characterization, as well as pharmacokinetics, prior to embarking on clinical trials of dietary supplements. In addition, collecting adequate data regarding dosing, potential toxicity, and development of an appropriate placebo for a given product are also requisite early tasks prior to designing

clinical trials. For some dietary supplements, product taste or odor may significantly limit the ability to generate an acceptable placebo for clinical testing. Understanding the putative mechanism of action of a given product is also an important aspect of the knowledge base, as it strengthens the plausibility of the intervention, and, most importantly facilitates identification of biomarkers to document in vivo effect of the dietary supplement. Availability of a biomarker that can be used to document activity of the agents is of great value. A biomarker facilitates a rational approach to dosing, makes it possible to determine which patients are responding to the intervention, and can assist in identification of outcome measures that are maximally sensitive. The absence of this information can limit expansion of clinical studies beyond early phase testing, particularly for products such as dietary supplements that are generally anticipated to have mild to modest clinical effects. In planning informative large RCTs, it is essential to have standardized outcome measures that are maximally sensitive and can reliably be implemented in the context of a clinical trial (5). It is also important to have adequate preliminary data on the target patient population before embarking on a large clinical trial (5). Although the primary standard for establishing safety and efficacy remains the RCT, early-phase investigations can exploit other design strategies. For example, adaptive trial designs or nof-1 designs could be used for expanding the knowledge base on individual products prior to planning subsequent larger studies. Recent trends in clinical trial design have attempted to facilitate methods for improving trial strategies for medical product development. In clinical studies of new potential therapies, investigators and regulatory agencies have considered adaptations in early-phase trials before planning a large-scale confirmatory phase III RCT (6). To facilitate optimizing final trial design, adaptations in interventional studies may include changes in sample size, enrollment criteria (target subject population), product dose, study end points, and statistical methods for analysis of clinical outcome data (6). As previously discussed, the knowledge base for many products is lacking in several critical aspects, including target subject population, dose, and appropriate end points. Although adaptive design methods provide a mechanism for informed changes to study design after study initiation, appropriate analytic methods must be implemented in the planning of studies such that the scientific validity and integrity of the study are maintained (6). As dietary supplements are frequently used for chronic conditions, individualized medication effectiveness tests (n-of-1 trials) have been considered a potential strategy for specific products (7,8). Unlike the RCT design, n-of-1 trials are individualized within-patient, are randomized and placebo-controlled, and include multiple crossover comparisons of product versus placebo, or versus another active treatment (7,8). Also unlike the RCT, the n-of-1 trial provides a mechanism for assessing intervention effects in individual patients who might not otherwise be included in the targeted RCT subject population (7,8). The use of such less commonly employed designs can provide a means for adequate data collection, markedly enhancing a product’s knowledge base, such

The Challenges of Dietary Supplement Research and Considerations for Future Studies

that more definitive clinical trials can be optimally designed and implemented.

CONCLUSION In developing productive research programs for dietary supplements, it is important to build a hierarchy of evidence for individual supplements, including understanding essentials of individual product characterization, basic product clinical chemistry, and subsequent rigorous testing in the setting of clinical studies. Multiple lines of investigation can then be coordinated for enhancing the knowledge base on a product, with the goal of informing practitioners and the public on safety and efficacy of dietary supplement use.

2.

3.

4.

5. 6.

7.

REFERENCES 8. 1. Barnes PM, Bloom B, Nahin RL. Complementary and alternative medicine use among adults and children: United States,

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2007. National Health Statistics Reports 12. Hyattsville, MD: National Center for Health Statistics, 2008:1–23. Barnes PM, Powell-Griner E, McFann K, et al. Complementary and alternative medicine use among adults: United States, 2002. Advance Data from Vital and Health Statistics: No. 343. Hyattsville, MD: National Center for Health Statistics, 2004. Krochmal R, Hardy M, Bowerman S, et al. Phytochemical assays of commercial botanical dietary supplements. Evid Based Complement Altern Med 2004; 1(3):305–313. U.S. Department of Health and Human Services; Food and Drug Administration; Center for Drug Evaluation and Research (CDER). Guidance for Industry: Botanical Drug Products, 2004:1–52. Friedman LM, Furberg DC, DeMets DL. Fundamentals of Clinical Trials. 3rd ed. New York: Springer-Verlag, 1998:1–125. Chow S-C, Chang M. Adaptive Design Methods in Clinical Trials (Chapman & Hall/CRC Biostatistic Series). Boca Raton, FL: Chapman & Hall/CRC, Taylor & Francis Group, 2007:1– 46. Guyatt GH, Keller JL, Jaeschke R, et al. The n-of-1 randomized controlled trial: Clinical usefulness. Our three-year experience. Ann Intern Med 1990; 112(4):293–299. Nikles CJ, Clavarino AM, Del Mar CB. Using n-of-1 trials as a clinical tool to improve prescribing. Br J Gen Pract 2005; 55(512):175–180.

S -Adenosylmethionine Jos´e M. Mato and Shelly C. Lu

ABBREVIATIONS

converted to cysteine through the “transsulfuration” pathway and discovered the “transmethylation” pathway, that is, the exchange of methyl groups between methionine, choline, betaine, and creatine. In 1951, Cantoni demonstrated that a liver homogenate supplemented with ATP and methionine converted nicotinamide to Nmethylnicotinamide. Two years later, he established that methionine and ATP reacted to form a product, that he originally called “Active Methionine,” capable of transferring its methyl group to nicotinamide, or guanidoacetic acid, to form N-methylmethionine, or creatine in the absence of ATP, which, after determination of its structure, he called “AdoMet” (Fig. 1). Subsequently, Cantoni and his colleagues discovered the enzyme that synthesizes SAMe, methionine adenosyltransferase (MAT); (S)-adenosylhomocysteine (SAH), the product of transmethylation reactions; and SAH hydrolase, the enzyme that converts SAH into adenosine and homocysteine (Hcy). At about the same time, Bennett discovered that folate and vitamin B12 could replace choline as a source of methyl groups in rats maintained on diets containing Hcy in place of methionine, a finding that led to the discovery of methionine synthase (MS). In 1961, Tabor demonstrated that the propylamino moiety of SAMe is converted via a series of enzymatic steps to spermidine and spermine. In the biosynthesis of polyamines, 5 -deoxy-5 -methylthioadenosine (MTA) was identified as an end product. Thus, by the beginning of the 1960s, Laster’s group could finally provide an integrated view, similar to that depicted in Figure 2, combining the transmethylation and transsulfuration pathways with polyamine synthesis. Since then, SAMe has been shown to donate (i) its methyl group to a large variety of acceptor molecules including DNA, RNA, phospholipids, and proteins; (ii) its sulfur atom, via a series of reactions, to cysteine and glutathione (GSH), a major cellular antioxidant; (iii) its propylamino group to polyamines, which are required for cell growth; and (iv) its MTA moiety, via a complex set of enzymatic reactions known as the “methionine salvage pathway,” to the resynthesis of this amino acid. All these reactions can affect a wide spectrum of biological processes ranging from metal detoxication and catecholamine metabolism to membrane fluidity, gene expression, cell growth, differentiation, and apoptosis (2), to establish what Cantoni called the “AdoMet Empire.”

CSF, cerebrospinal fluid; GNMT, glycine N-methyltransferase; GSH, glutathione; HCC, hepatocellular carcinoma; Hcy, homocysteine; MAT, methionine adenosyltransferase; MTA, 5 -deoxy-5 -methylthioadenosine; MTHFR, 5,10-methylenetetrahydrofolate reductase; NASH, nonalcoholic steatohepatitis; SAH, (S)-adenosylhomocysteine; SAMe, (S)-adenosylmethionine.

INTRODUCTION Common and Scientific Name

S-Adenosyl-L-methionine, also known as 5 -[(3-Amino-3carboxypropyl) methylsulfonio]-5 -deoxyadenosine; (S)(5 -desoxyadenosin-5-yl) methionine; [C15 H23 N6 O5 S]+ , is abbreviated in the scientific literature as AdoMet, SAM, or SAMe. In the early literature, before the identification of its structure, SAMe was known as “active methionine.”

General Description SAMe was discovered in 1953 and since then has been shown to regulate key cellular functions such as differentiation, growth, and apoptosis. Abnormal SAMe content has been linked to the development of experimental and human liver disease, and this led to the examination of the effect of SAMe supplementation in various animal models of liver disease and in patients with liver disease. Both serum and cerebrospinal fluid (CSF) levels of SAMe have been reported to be low in depressed patients, which has led to the examination of the effect of SAMe treatment in this condition. The effect of SAMe in the treatment of other diseases, such as osteoarthritis, has also been investigated. This chapter reviews (i) the biochemistry and functions of SAMe; (ii) altered SAMe metabolism in liver disease; (iii) SAMe deficiency in depression; and (iv) the effect of SAMe treatment in liver disease, depression, and osteoarthritis.

BIOCHEMISTRY AND FUNCTIONS SAMe Discovery Although SAMe was discovered by Giulio Cantoni in 1953, the story of this molecule begins in 1890 with Whilhelm His when he fed pyridine to dogs and isolated N-methylpyridine from the urine and emphasized the need to demonstrate both the origin of the methyl group as well as the mechanism for its addition to the pyridine (1). Both questions were addressed by Vincent du Vigneaud who, during the late 1930s, demonstrated that the sulfur atom of methionine was

SAMe Synthesis and Metabolism MAT is an enzyme extremely well conserved through evolution with 59% sequence homology between the human and Escherichia coli isoenzymes. In mammals, there are 1

2

Mato and Lu

S-Adenosylmethionine AdoMet, SAM, SAMe O N N N

+

O

N

S

N

CH3

N O

O

Figure 1 Structure of SAMe. (S)-adenosylmethionine (SAMe) has been shown to donate: (i) its methyl group to a large variety of acceptor molecules including DNA, RNA, phospholipids, and proteins; (ii) its sulfur atom, via a series of reactions, to cysteine and glutathione, a major cellular antioxidant; (iii) its propylamino group to polyamines, which are required for cell growth; and (iv) its MTA moiety, via a complex set of enzymatic reactions known as the “methionine salvation pathway,” to the resynthesis of this amino acid.

MTA Putrescine

Spermidine MTA Spermine

MAT Serine Glycine

Met SAMe

THF

5,10-MTHF MS 5-MTHF

X-

N,N-Dimethyl-Gly

MTs

BHMT Betaine

SAH

GNMT X-CH3

Hcy Ser

CBS

Cystathionine α-Ketobutyrate Cys

GSH

Figure 2 Hepatic metabolism of SAMe. Methionine (Met) is converted into homocysteine (Hcy) via (S)-adenosylmethionine (SAMe) and (S)adenosylhomocysteine (SAH). The conversion of Met into SAMe is catalyzed by methionine adenosyltransferase (MAT). After decarboxylation, SAMe can donate the remaining propylamino moiety attached to its sulfonium ion to putrescine to form spermidine and methylthioadenosine (MTA) and to spermidine to form spermine and a second molecule of MTA. SAMe donates its methyl group in a large variety of reactions catalyzed by dozens of methyltransferases (MTs), the most abundant in the liver being glycine-Nmethyltransferase (GNMT). The SAH thus generated is hydrolyzed to form Hcy and adenosine through a reversible reaction catalyzed by SAH hydrolase. Hcy can be remethylated to form methionine by two enzymes: methionine synthase (MS) and betaine homocysteine methyltransferase (BHMT). In the liver, Hcy can also undergo the transsulfuration pathway to form cysteine via a two-step enzymatic process. In the presence of serine, Hcy is converted to cystathionine in a reaction catalyzed by cystathionine ␤-synthase (CBS). Cystathionine is then hydrolyzed by cystathionase to form cysteine, a precursor of the synthesis of glutathione (GSH). In tissues other than the liver, kidney, and pancreas, cystathionine is not significantly converted to GSH due to the lack of expression of one or more enzymes of the transsulfuration pathway. The expression of BHMT is also limited to the liver. All mammalian tissues convert Met into Hcy, via SAMe and SAH, and remethylate Hcy into Met via the MS pathway. Abbreviations: THF, tetrahydrofolate; 5,10-MTHF, methylenetetrahydrofolate; 5-MTHF, methyltetrahydrofolate; Ser, serine; Gly, glycine; X, methyl acceptor molecule; X-CH3 , methylated molecule.

three isoforms of MAT (MATI, MATII, and MATIII) that are encoded by two genes (MAT1A and MAT2A). MATI and MATIII are tetrameric and dimeric forms, respectively, of the same subunit (␣1 ) encoded by MAT1A, whereas the MATII isoform is a tetramer of a different subunit (␣2 ) encoded by MAT2A. A third gene, MAT2β encodes for a ␤ subunit that regulates the activity of MATII (lowering the Km and Ki for methionine and SAMe, respectively) but not of MATI or MATIII (2). Adult differentiated liver expresses MAT1A, whereas extrahepatic tissues and fetal liver express MAT2A. MAT1A expression is silenced in HCC. It is an intriguing question why there are three different MAT isoforms in the liver. The predominant liver form, MATIII, has lower affinity for its substrates, a hysteretic response to methionine (a hysteretic behavior, defined as a slow response to changes in substrate binding, has been described for many important enzymes in metabolic regulation), and higher V max , contrasting with the other two enzymes. On the basis of the differential properties of hepatic MAT isoforms, it has been postulated that MATIII is the truly liver-specific isoform. Under normal conditions, MATI would, as MATII outside the liver, synthesize most of the SAMe required by the hepatic cells. However, after an increase in methionine concentration, that is, after a protein-rich meal, conversion to the high-activity MATIII would occur and methionine excess will be eliminated (Fig. 2). This will lead to accumulation of SAMe and activation of glycine N-methyltransferase (GNMT), the main enzyme involved in hepatic SAMe catabolism. Consequently, the excess of SAMe will be eliminated and converted to homocysteine via SAH. Once formed, the excess of homocysteine will be used for the synthesis of cysteine and ␣-ketobutyrate as a result of its transsulfuration. This pathway involves two enzymes: cystathionine ␤-synthase (CBS), that is activated by SAMe, and cystathionase. Cysteine is then utilized for the synthesis of GSH as well as other sulfur-containing molecules such as taurine, while ␣-ketobutyrate penetrates the mitochondria where it is decarboxylated to carbon dioxide and propionyl CoA. Because SAMe is an inhibitor of 5,10methylene-tetrahydrofolate-reductase (MTHFR), this will prevent the regeneration of methionine after a load of this amino acid. At the mRNA level, SAMe maintains MAT1A and GNMT expression while inhibiting MAT2A expression. This modulation by SAMe of both the flux of methionine into the transsulfuration pathway and the regeneration of methionine maximizes the production of cysteine and ␣-ketobutyrate, and consequently of ATP, after a methionine load minimizing the regeneration of this amino acid (oxidative methionine metabolism).

ALTERED SAMe METABOLISM AND DISEASE Altered SAMe Metabolism in Liver Disease Accumulating evidence supports the importance of maintaining normal SAMe level in mammalian liver, as both chronic deficiency and excess lead to liver injury, steatosis, and development of hepatocellular carcinoma (HCC) (2,3). Majority of the patients with cirrhosis have impaired SAMe biosynthesis because of lower MAT1A mRNA levels and inactivation of MATI/III (4,5). However, patients with GNMT mutations have been identified and they also

S-Adenosylmethionine

have evidence of liver injury (6). In mice, loss of GNMT results in supraphysiological levels of hepatic SAMe and aberrant methylation (7). The molecular mechanisms responsible for injury and HCC formation are different in MAT1A and GNMT knockout mice but these findings illustrate the importance of keeping SAMe level within a certain range within the cell. In contrast to normal nonproliferating (differentiated) hepatocytes, which rely primarily on MATI/III to generate SAMe and maintain methionine homeostasis, embryonic and proliferating adult hepatocytes as well as liver cancer cells instead rely on MATII to synthesize SAMe (2). Liver cancer cells often have very low levels of GNMT and CBS expression and increased expression of MAT2β, which, as mentioned earlier, lowers the Km for methionine and the Ki for SAMe of MATII. Consequently, proliferating hepatocytes and hepatoma cells tend to utilize methionine into protein synthesis regardless of whether methionine is present in high or low amounts and to divert most homocysteine away from the transsulfuration pathway by regenerating methionine and tetrahydrofolate (THF) (aerobic methionine metabolism). MAT2A/MAT2β-expressing hepatoma cells have lower SAMe levels than cells expressing MAT1A, which also favors the regeneration of methionine and THF. From these results, it becomes evident that proliferating hepatocytes and hepatoma cells do not tolerate well high SAMe levels for converting methionine via the transsulfuration pathway to cysteine and ␣-ketobutyrate. The finding that MAT1A, GNMT, MTHFR, and CBS knockout mice spontaneously develop fatty liver (steatosis) and, in the case of MAT1A- and GNMT-deficient animals, HCC also (3) demonstrates the synchronization of methionine metabolism with lipid metabolism and hepatocyte growth. The medical implications of these observations are obvious, since the majority of cirrhotic patients, independent of the etiology of their disease, have impaired metabolism of methionine and reduced hepatic SAMe synthesis and are predisposed to develop HCC (4,5); and individuals with GNMT mutations that lead to abnormal SAMe catabolism develop liver injury (6). Moreover, the observation that genetic polymorphisms that associate with reduced MTHFR activity and increased thymidylate synthase activity, both of which are essential in minimizing uracyl misincorporation into DNA, may protect against the development of HCC in humans (8) further supports that this synchronization may be an adaptive mechanism that is programmed to fit the specific needs of hepatocytes, and that alterations in the appropriate balance between methionine metabolism and proliferation may be at the origin of the association of cancer with fatty liver disease. An explanation for these observations connecting methionine metabolism with the development of fatty liver and HCC has remained elusive because the association of SAMe with lipid metabolism and hepatocyte proliferation is, at first glance, not intuitive. During the past years, a signaling pathway that senses cellular SAMe content and that involves AMP-activated protein kinase (AMPK) has been identified to operate in hepatocytes (9,10). AMPK is a serine/threonine protein kinase that plays a crucial role in the regulation of energy home-

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ostasis and cell proliferation. AMPK is activated by stress conditions leading to an increase in the AMP/ATP ratio, such as during liver regeneration. Once activated, AMPK shuts down anabolic pathways that mediate the synthesis of proteins, fatty acids, lipids, cholesterol, and glycogen and stimulates catabolic pathways such as lipid oxidation and glucose uptake restoring ATP levels and keeping the cellular energy balance. The finding that in the liver AMPK activity is tightly regulated by SAMe (9,10) has provided a first link between methionine metabolism, lipid metabolism, and cell proliferation. Moreover, excess SAMe can induce aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways (7). Finally, there is evidence indicating that SAMe regulates proteolysis, widening its spectrum of action. In hepatocytes, the protein levels of prohibitin 1 (PHB1) (11), the apurinic/apyrimidininc endonuclease (APEX1) (12), and the dual specificity MAPK phosphatase (DUSP1) (13) are stabilized by SAMe through a process that may involve proteasome inactivation. PHB1 is a chaperone-like protein involved in mitochondrial function, APEX1 is a key protein involved in DNA repair and genome stability, and DUSP1 is a member of a family of mitogen-activated protein kinases (MAPKs) phosphatases, which simultaneously dephosphorylates both serine/threonine and tyrosine residues.

SAMe Deficiency in Depression Major depression has been associated with a deficiency in methyl groups (folate, vitamin B12 , and SAMe) (14,15). Thus, depressed patients often have low plasma folate and vitamin B12 and reduced SAMe content in the CSF. Moreover, patients with low plasma folate appear to respond less well to antidepressants. The mechanism by which low SAMe concentrations may contribute to the appearance and evolution of depression is, however, not well known. SAMe-dependent methylation reactions are involved in the synthesis and inactivation of neurotransmitters, such as noradrenaline, adrenaline, dopamine, serotonin, and histamine; and the administration of drugs that stimulate dopamine synthesis, such as L-dihydroxyphenylalanine, cause a marked decrease in SAMe concentration in rat brain and in plasma and CSF in humans. Moreover, various drugs that interfere with monoaminergic neurotransmission, such as imipramine and desipramine, reduce brain SAMe content in mice (14,15). As in the liver, abnormal SAMe levels may contribute to depression through perturbation of multiple metabolic pathways in the brain. Interestingly, alterations in methionine metabolism that lead to a decrease in the brain SAMe/SAH ratio associate with reduced leucine carboxyl methyltransferase-1 (LCMT-1) and phosphoprotein phosphatase 2AB (PP2AB ) subunit expression, and accumulation of unmethylated PP2A (16). PP2A enzymes exist as heterotrimeric complexes consisting of catalytic (PP2AC ), structural (PP2AA ), and regulatory (PP2AB ) subunits (17). Different PP2AB subunits have been described that determine the substrate specificity of the enzyme. PP2AC subunit is methylated by SAMe-dependent LCMT-1 and demethylated by a specific phosphoprotein phosphatase methylesterase (PME1). PP2AC methylation has no effect on PP2A activity but has a crucial role in the recruitment of specific PP2AB subunits

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to the PP2AA,B complex and therefore PP2A substrate specificity. Downregulation of LCMT-1 and PP2AB and accumulation of unmethylated PP2A are associated with enhanced Tau phosphorylation and neuronal cell death (16).

INDICATIONS AND USAGE SAMe Treatment in Animal Models of Liver Disease The importance of the metabolism of methyl groups in general, and SAMe in particular, to normal hepatic physiology, coupled with the convincing body of evidence linking abnormal SAMe content with the developmental of experimental and human liver disease, led to the examination of the effect of SAMe supplementation in various animal models of liver disease. SAMe administration to alcohol-fed rats and baboons reduced GSH depletion and liver damage (2,18). SAMe improved survival in animal models of galactosamine-, acetaminophen- and thioacetamide-induced hepatotoxicity, and in ischemiareperfusion–induced liver injury (18). SAMe treatment also diminished liver fibrosis in rats treated with carbon tetrachloride (18) and reduced neoplastic hepatic nodules in animal models of HCC (19,20). Similar to the liver, SAMe can block mitogen-induced growth and induce apoptosis in human colon cancer cells (21,22).

SAMe Treatment in Human Diseases SAMe has been used in humans for the past 20 years for the treatment of osteoarthritis, depression, and liver disease. In 2002, the Agency for Healthcare Research and Quality (AHRQ) reviewed 102 individual clinical trials of SAMe (23). Of these 102 studies, 47 focused on depression, 14 focused on osteoarthritis, and 41 focused on liver disease. Of the 41 studies in liver disease, 9 were for cholestasis of pregnancy, 12 were for other causes of cholestasis, 7 were for cirrhosis, 8 were for chronic hepatitis, and 4 were for various other chronic liver diseases.

Pharmacokinetics of SAMe Orally administered SAMe has low bioavailability, presumably because of a significant first-pass effect (degradation in the gastrointestinal tract) and rapid hepatic metabolism. Peak plasma concentrations obtained with an enteric-coated tablet formulation are dose related, with peak plasma concentrations of 0.5 to 1 mg/L achieved three to five hours after single doses ranging from 400 to 1000 mg (23). Peak levels decline to baseline within 24 hours. One study showed a significant gender difference in bioavailability, with women showing three- to sixfold greater peak plasma values than men (23). Plasma-protein binding of SAMe is no more than 5%. SAMe crosses the blood–brain barrier, with slow accumulation in the CSF. Unmetabolized SAMe is excreted in urine and feces. Parenterally administered SAMe has much higher bioavailability. However, this form is currently not approved for use in the United States.

serum bilirubin levels associated with cholestasis of pregnancy (23). Compared with placebo, treatment with SAMe was associated with a significant decrease in pruritus and serum bilirubin levels. Similar results were obtained when six studies were included in a meta-analysis of the efficacy of SAMe to relieve pruritus and decrease bilirubin levels associated with cholestasis caused by various liver diseases other than pregnancy. In 2001, the Cochrane Hepato-Biliary Group analyzed eight clinical trials of SAMe treatment of alcoholic liver disease including 330 patients (24). This metaanalysis found SAMe decreased total mortality [odds ratio (OR) 0.53, 95% confidence interval (CI): 0.22 to 1.29] and liver-related mortality (OR 0.63, 95% CI: 0.25 to 1.58). However, because many of the studies were small and the quality of the studies varied greatly, the Cochrane Group concluded, “SAMe should not be used for alcoholic liver disease outside randomized clinical trials” (24). The AHRQ reached a similar conclusion, “For liver conditions other than cholestasis additional smaller trials should be conducted to ascertain which patient populations would benefit more from SAMe, and what interventions (dose and route of administration) are most effective” (23). The Cochrane Hepato-Biliary Group also concluded that only one trial including 123 patients with alcoholic cirrhosis used adequate methodology and reported clearly on mortality and liver transplantation. In this study (25), mortality decreased from 30% in the placebo group to 16% in the SAMe group (P = 0.077). When patients with more advanced cirrhosis (Child score C) were excluded from the analysis (eight patients), the mortality was significantly less in the SAMe group (12%) as compared with the placebo group (25%, P = 0.025). In this study, 1200 mg/day was administered orally. Unfortunately, new controlled prospective double-blind multicenter studies on the benefits of SAMe for liver diseases are lacking.

SAMe Treatment in Depression Out of the 39 studies in depression analyzed by the AHRQ, 28 studies were included in a meta-analysis of the efficacy of SAMe to decrease symptoms of depression (23). Compared with placebo, treatment with SAMe was associated with an improvement of approximately six points in the score of the Hamilton Rating Scale for Depression measured at three weeks (95% CI: 2.2 to 9.0). This degree of improvement was statistically as well as clinically significant. However, compared with the treatment with conventional antidepressant pharmacology, treatment with SAMe was not associated with a statistically significant difference in outcomes. With respect to depression, the AHRQ report concluded, “Good dose-escalation studies have not been performed using the oral formulation of SAMe for depression” (23). The AHRQ report also concluded, that “Additional smaller clinical trials of an exploratory nature should be conducted to investigate uses of SAMe to decrease the latency of effectiveness of conventional antidepressants and to treat of postpartum depression” (23). Unfortunately, these clinical studies are still lacking.

SAMe Treatment in Liver Diseases Out of the 41 studies in liver disease analyzed by AHRQ, 8 studies were included in a meta-analysis of the efficacy of SAMe to relieve pruritus and decrease elevated

SAMe Treatment in Osteoarthritis Out of the 13 studies in osteoarthritis analyzed by the AHRQ, 10 studies were included in a meta-analysis of

S-Adenosylmethionine

the efficacy of SAMe to decrease pain of osteoarthritis (23). Compared with placebo, one large randomized clinical trial showed a decrease in the pain of osteoarthritis with SAMe treatment. Compared with the treatment with nonsteroidal anti-inflammatory medications, treatment with oral SAMe was associated with fewer adverse effects while comparable in reducing pain and improving functional limitation. In 2009, the Cochrane Osteoarthritis Group analyzed 4 clinical trials including 656 patients, all comparing SAMe with placebo (26). The Cochrane Group concluded, “The effects of SAMe on both pain and function may be potentially clinically relevant and, although effects are expected to be small, deserve further clinical evaluation in adequately sized randomized, parallelgroup trials in patients with knee or hip osteoarthritis. Meanwhile, routine use of SAMe should not be advised” (26).

Adverse Effects The risks of SAMe are minimal. SAMe has been used in Europe for more than 20 years and is available under prescription in Italy, Germany, United Kingdom, and Canada, and over the counter as a dietary supplement in the United States, China, Russia, and India. The most common side effects of SAMe are nausea and gastrointestinal disturbance, which occurs in less than 15% of treated subjects. Recently, SAMe administration to mice treated with cisplatin has been found to increase renal dysfunction (27). Whether SAMe increases cisplatin renal toxicity in humans is not known.

Interactions with Herbs, Supplements, and Drugs Theoretically, SAMe might increase the effects and adverse effects of products that increase serotonin levels, which include herbs and supplements such as Hawaiian Baby Woodrose, St. John’s wort, and L-tryptophan, as well as drugs that have serotonergic effects. These drugs include tramadol (Ultram), pentazocine (Talwin), clomipramine (Anafranil), fluoxetine (Prozac), paroxetine (Paxil), sertraline (Zoloft), amitriptyline (Elavil), and many others. It is also recommended that SAMe should be avoided in patients taking monoamine oxidase inhibitors or within two weeks of discontinuing such a medication.

CONCLUSIONS Although evidence linking abnormal SAMe content with the development of experimental and human liver disease is very convincing, the results of clinical trials of SAMe treatment of liver disease are not conclusive. Consequently, SAMe should not be used outside clinical trials for the treatment of liver conditions other than cholestasis. A new clinical study enrolling a larger number of patients should be carried out to confirm that SAMe decreases mortality in alcoholic liver cirrhosis. This is important because if SAMe improves survival, SAMe will become the only available treatment for patients with alcoholic liver cirrhosis. Although depression has been associated with a deficiency in SAMe, it is not yet clear whether this is a consequence or the cause of depression. To clarify this point, more basic research and the development of new exper-

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imental models are needed. Clinical trials indicate that SAMe treatment is associated with an improvement of depression. Dose studies using oral SAMe should be performed to determine the best dose to be used in depression. New studies should also be carried out where the efficacy of SAMe is compared with that of conventional antidepressants. With respect to osteoarthritis, at present there is no evidence associating a deficiency in SAMe with the appearance of the disease. Moreover, the efficacy of SAMe in the treatment of osteoarthritis is also not convincing at present.

ACKNOWLEDGMENTS This work was supported by grants from NIH DK51719 (to S. C. L.) and AT-1576 (to S. C. L. and J. M. M.) and SAF 2008-04800 (to J. M. M.). CIBERehd is funded by the Instituto de Salud Carlos III.

REFERENCES 1. Finkelstein JD. Homocysteine: A history in progress. Nutr Rev 2000; 58(7):193–204. 2. Mato JM, Lu SC. Role of S-adenosylmethionine in liver health and injury. Hepatology 2007; 45:1306–1312. 3. Mato JM, Mart´ınez-Chantar ML, Lu SC. Methionine metabolism and liver disease. Annu Rev Nutr 2008; 28;273– 293. 4. Duce AM, Ortiz P, Cabrero C, et al. S-Adenosyl-L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology 1988; 8(1):65–68. 5. Avila MA, Berasain C, Torres L, et al. Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma. J Hepatol 2000; 33(6):907–914. 6. Mudd SH, Cerone R, Schiaffimo MC, et al. Glycine Nmethyltransferase deficiency: A novel inborn error causing persistent isolated hypermethioninemia. J Inherit Metab Dis 2001; 24;448–464. 7. Mart´ınez-Chantar ML, V´azquez-Chantada M, Ariz U, et al. Loss of the gycine N-methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice. Hepatology 2008; 47(4):1191–1199. 8. Yuan J-M, Lu SC, Van den Berg D, et al. Genetic polymorphisms in the methylenetetrahydrofolate reductase and thymidylate synthase genes and risk of hepatocellular carcinoma. Hepatology 2007; 46(3):749–758. 9. Mart´ınez-Chantar ML, V´azquez-Chantada M, Garnacho M, et al. S-Adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase. Gastroenterology 2006; 131; 223–232. 10. V´azquez-Chantada M, Ariz U, Varela-Rey M, et al. Evidence for LKB1/AMP-activated protein kinase/endothelial nitric oxide synthase cascade regulated by hepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyte proliferation. Hepatology 2009; 49:608–617. 11. Santamar´ıa E, Avila MA, Latasa MU, et al. Functional proteomics of non-alcoholic steatohepatitis: Mitochondrial proteins as targets of S-adenosylmethionine. Proc Nat Acad Sci U S A 2003; 100(6):3065–3070. 12. Tomasi ML, Iglesias-Ara A, Yang H, et al. Sadenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: Implication in hepatocarcinogenesis. Gastroenterology 2009; 136(3):1025–1036.

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13. Tomasi ML, Ramani K, Lopitz-Osada F, et al. SAdenosylmethionine regulates dual-specificity mitogenactivated protein kinase phosphatase expression in mouse and human hepatocytes. Hepatology 2010, in press. 14. Bottiglieri T. S-Adenosyl-L-methionine (SAMe): From the bench to the bedside—Molecular basis of a pleiotrophic molecule. Am J Clin Nutr 2002; 76(5):1151S–1157S. 15. Miller AL. The methylation, neurotransmitter, and antioxidant connections between folate and depression. Altern Med Rev 2008; 13;216–226. 16. Sontag J-M, Nunbhakdi-Craig V, Montgomery L, et al. Folate deficiency induces in vitro and mouse brain region-specific downregulation of leucine carboxyl methyltransferase-1 and protein phosphatase 2A B␣ subunit expression that correlate with enhanced Tau phosphorylation. J Neurosci 2008; 28(45):11477–11487. 17. Vishrup DM, Shenolikar S. From promiscuity to precision: Protein phosphatases get a makeover. Mol Cell 2009; 33(5):537–545. 18. Mato JM, Alvarez L, Ortiz P, et al. S-Adenosylmethionine synthesis: Molecular mechanisms and clinical implications. Pharmacol Ther 1997; 73(3):265–280. 19. Pascale RM, Simile MM, De Miglio MR, et al. Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine. Alcohol 2002; 27(3):193–198. 20. Lu SC, Ramani K, Ou X, et al. S-Adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model. Hepatology 2009; 50(2):462–471.

21. Chen H, Xia M, Lin M, et al. Role of methionine adenosyltransferase 2A and S-adenosylmethionine in mitogeninduced growth of human colon cancer cells. Gastroenterology 2007; 133(5):207–218. 22. Li TW, Zhang Q, Oh P, et al. S-Adenosylmethionine and methylthioadenosine inhibit cellular FLICE inhibitory protein expression and induce apoptosis in colon cancer cells. Mol Pharmacol 2009; 76(1):192–200. 23. Agency for Healthcare Research and Quality. S-AdenosylL-Methionine for Treatments of Depression, Osteoarthritis, and Liver Disease. Rockville, MD: Agency for Healthcare Research and Quality; 2002. Evidence Report/Technology Assessment 64. http://www.ahrq.gov/clinic/tp/sametp.htm. Accessed August 2002. 24. Rambaldi A, Gluud C. S-Adenosyl-L-methionine for alcoholic liver disease. Cochrane Database Syst Rev 2001; 4:CD002235. 25. Mato JM, C´amara J, Fern´andez de Paz J, et al. SAdenosylmethionine in alcoholic liver cirrhosis: a randomized placebo-controlled, double-blind, multicentre trial. J Hepatol 1999; 30(6):1081–1089. ¨ 26. Rutjes AW, Nuesch E, Reichenbach S, et al. SAdenosylmethionine for osteoarthritis of the knee or hip. Cochrane Database Syst Rev 2009; 4:CD007321. 27. Ochoa B, Bobadilla N, Arrellin G, et al. SAdenosylmethionine-L-methionine increases serum BUN and creatinine in cisplatin-treated mice. Arch Med Res 2009; 40(1):54–58.

Aloe Vera Santiago Rodriguez, Steven Dentali, and Devon Powell

flowers in each of the three branches. Aloe vera does not normally reproduce from seeds but from offshoots often called “pups” that grow out from the mother plant. When the green outer rind of the leaves is cut or damaged, a bitter yellow exudate from pericyclic tubules located between the outer rind and the inner leaf is released. This sap is commonly referred to as “aloe latex” (3) and contains several anthraquinone glycosides that have powerful stimulant laxative properties. When the rind is completely removed, a semitransparent, semicrystalline gel-like layer composed of large thin-walled parenchyma cells is revealed. This inner leaf material is often called “aloe gel,” or “inner leaf fillet,” because of its similarity in shape to a fish fillet. When crushed, it produces a very viscous fluid usually containing approximately 98.5% water. The solids are composed mainly of polysaccharides and other carbohydrates, pectin, and organic acids. As mentioned earlier, aloe latex–derived products are used as a laxative agent and the processed leaf or inner leaf is often employed topically for the treatment of burns and injury. More recent applications range from skin-moisturizing agents to the management of cancers in animals to impregnation in articles of clothing and mattresses for its softening and moisturizing properties. Aloe vera juice is also orally ingested to manage digestive ailments and for its immune-modulating activities and is sold worldwide in beverage form as a food-based drink product available in various flavors. Aloe vera is also used widely in Ayurvedic medicine (4).

INTRODUCTION Aloe vera is one of the oldest known medicinal herbs with a history of use that spans thousands of years. Today, aloe vera is cultivated and used in a large variety of commercial preparations. It is an economic driver in the food, dietary supplement, and personal care industries worldwide. The two main commercial materials derived from aloe vera are aloe vera juice and aloe latex. Aloe vera juice is used for various dietary, cosmetic, and medical purposes such as burn treatment, wound healing, and skincare. It is available in several forms including liquid juice, juice powder, and concentrates. Aloe latex was formerly recognized as an over-the-counter (OTC) laxative drug in the United States. It has seen limited use in dietary supplements as a laxative and in the personal care industry as a skin lightener. Confusion among consumers, researchers, and regulatory bodies has arisen from the fact that products from aloe latex are often referred to as simply “aloe” or “aloe juice” (including in pharmacopoeias and other official documents around the world), which is physically, chemically, and biologically distinct from products made from the charcoal filtered whole leaf or inner leaf aloe vera juice. These latex-free juice products represent the vast majority of aloe products on the market. Regardless, the prominence of, interest in, and use of aloe vera products for centuries attests to the plant’s myriad value and benefits.

BACKGROUND Aloe vera (L.) Burm. f. is one of more than 400 known Aloe species in the Asphodelaceae family, though it is sometimes classified in Aloaceae. Because most aloe species are indigenous to Africa, it is most likely that aloe vera also originated from that continent. However, because of its now worldwide cultivation, its origin is difficult to establish. Linnaeus classified aloe vera as the “true aloe” hence the name “vera,” meaning true in Latin. Although it has also been known as Aloe barbadensis, Aloe chinensis, Aloe indica, Aloe vulgaris, and others, A. vera (L.) Burm. f. has precedence (1). Its standardized common name is “aloe vera” though it has also been called Barbados aloe, Curac¸ao aloe, true aloe, West Indian aloe, Ghrita kumari, or simply aloe (2). The plant is cactus-like in appearance with succulent leaves that grow in a spiral form from a basal rosette (Fig. 1). An inflorescence is produced annually (typically December through March) with yellow flowers in a trident configuration from a single central stalk with many

CULTIVATION Aloe vera is cultivated in subtropical regions around the globe for commercial use and is widely grown by indoor and outdoor plant enthusiasts as an ornamental plant because of its hardiness and beauty. The species is resistant to most insect pests and needs very little maintenance or care to flourish, given appropriate temperature conditions (5). Because of its very low inner leaf solid content of 0.5% to 1.5%, aloe vera plants are highly susceptible to freezing, which causes extensive damage, even killing them when the temperature falls below 32◦ F. For this reason, commercial cultivations are typically carried out in warm weather areas (USDA zones 8–11). Aloe vera is the most cultivated species of the various Aloe species because it produces the largest, thickest leaves and therefore yields the greatest amount of juice. It is cultivated extensively in 7

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ing most of the outer, lower, older leaves. Typically one to four leaves are removed at a time per plant per harvest. This way it is possible to obtain three to six harvests in a year depending on how many leaves are collected from each plant at harvest. Cultivation practices for the industrial production of aloe extracts made from the yellow latex sap are radically different from those used to grow aloe vera for juice. In the case of sap production, plants are not irrigated and are grown in arid regions. The leaves turn brown and thin under these conditions but when cut produce the maximum amount of an anthraquinone glycoside– rich latex, the principal constituents of which are the compounds known as “aloins A and B.” The sap exudates are collected and further processed to produce two main products, aloe latex concentrate also known as “aloin paste” and a product commercially known as “aloin spray dried.”

DESCRIPTION The leaf of aloe vera is normally described as consisting of three major parts that are used in commercial products: the outer mesophyll (rind or cuticle), the interior parenchyma (inner leaf, gel or gel fillet, inner gel, inner leaf gel fillet), and the aloe latex (sap, bitter element, yellow sap, yellow latex). Researchers, raw material manufacturers, and finished goods manufacturers have utilized all three plant parts separately or in combination for aloe vera research and in the formulation of consumer products.

Outer Mesophyll (Rind) Figure 1 Aloe vera flowering. Source: Courtesy of Santiago Rodriguez, Houston, Texas.

Mexico, the Dominican Republic and other Caribbean Islands, Central America, Venezuela, the southern border areas of Texas, New Mexico, Arizona, and California, as well as Tanzania, Uganda, South Africa, Australia, Southern China, Thailand, and India. Some small acreage is also present in the Canary Islands as well as Southern Spain and Southern Italy and recently new commercial operations have been proposed in areas such as Greece, Iran, Pakistan, and other countries in the Middle East. Aloe vera prefers very well-drained soils, such as sandy loam, but can grow in almost any type of soil. Although aloe vera is naturally adapted to survive in very dry climates, water must be supplied to the plant yearround in order to keep the leaves succulent enough for a good commercial juice yield. Aloe vera is typically planted at a density of 10,000 plants per acre yielding approximately 40 metric tons of leaves per year. Commercial growers should not make the mistake of planting these large plants too densely. The suggested row spacing is a standard, 42-inch-wide row with approximately 60 cm of spacing between plants. Harvesting usually begins two years after planting. The plants are harvested year-round by carefully remov-

Aloe vera rind or cuticle is the site of photosynthesis and primarily consists of cellulose, monosaccharides, water soluble and insoluble carbohydrates, chlorophyll, amino acids, proteins, and lipids.

Interior Parenchyma (Inner Leaf) Aloe vera inner leaf is the colorless, mucilaginous parenchyma of the aloe vera plant leaf consisting of water, monosaccharides, water-soluble carbohydrates, water-soluble polysaccharides, and water-insoluble fibrous pulp. The compound ␤-(1–4)-acetylated mannan, a polysaccharide also known as “acemannan” or “acetylated polymannose,” is widely considered to be the biologically most important component of the inner leaf. After removal of fibrous pulp from the inner leaf, the resulting juice contains about 0.5% to 1.5% solids. Histological examination of aloe vera inner leaf pulp has shown it to be composed of large cells made up of 16% cell walls, about 1% microparticles, and 83% of a viscous gel on a dried weight basis. The carbohydrate portion of each of these components was distinct, with the cell walls composed of 34% galacturonic acid (an unusually high level), the microparticles composed of galactose-rich polysaccharides, and the liquid gel contained mannan (6). These findings showed that different pulp structures are associated with different polysaccharides and may therefore confer different biological activities.

Aloe Vera

Harvest of the leaf or the whole plant— root not used

Enzymatic treatment to reduce viscosity

Charcoal filtration step to remove phenolic compounds

(not all manufacturers)

(not all manufacturers)

Cleaning and sanitation of the leaves

Reduction of the leaves to a puree consistency

Removal of the insoluble pulp and rind

Reduction of microbial load by pasteurization

9

Reduction of water content to increase the percent juice solids or to produce juice powder

Preservation and packaging

Flowchart 1 Aloe vera whole leaf processing.

Aloe Latex (Aloe Sap, Aloe Bitters) Aloe latex is a yellow-green bitter exudate that contains the anthraquinone glycosides aloins A and B, formerly known as “barbaloin” and “isobarbaloin,” respectively. The aloin content of aloe latex changes with the season and the age of the leaf but usually makes up 10% to 25% of the dried latex by weight. Products made from aloe latex have been used historically as a laxative. The source plant is most commonly Aloe ferox from Africa or Argentina.

COMMERCIAL RAW MATERIAL PROCESSING Aloe Vera Juice Aloe vera juice can be manufactured from raw leaves in two ways-–from the entire leaf or from only the inner leaf material. In both cases, the leaves are first processed to remove the side thorns and tips. For aloe vera juice made from the entire leaf, the leaves are macerated in a grinder into what is commonly called “guacamole” and then further processed by enzymatic treatment (usually with cellulase) to break up cell walls and then charcoal filtered to remove anthraquinones and other phenolic constituents. The resulting aloe vera juice is commonly referred to as “filtered aloe vera juice” or “purified whole leaf aloe vera juice.” See flowchart 1 for more detail.

Leaf harvest

Enzymatic treatment to reduce viscosity (not all manufacturers)

Cleaning and sanitation of the leaves

Removal of most of the rind and reduction of fillet to puree

Aloin-Rich Materials The commercial production of aloin-rich materials starts with the specialized cultivation practices mentioned earlier. In contrast to aloe juice production, the leaves are

Charcoal filtration step to remove phenolic compounds (not all manufacturers)

Removal of the insoluble pulp and rind

Dicing/slicing of fillet typically for use in beverage or food, the pulp is not removed

When creating juice from only the inner leaf material, the inner leaf is separated from the outer rind either manually with a knife or by machine and then washed to rinse away any aloe latex present. The remaining material is crushed and further processed to produce the aloe vera juice. See flowchart 2 for more detail. At this stage, regardless of starting material, the now-processed aloe vera juice is typically called “singlestrength.” The juice from the leaf or inner leaf can also be further processed to produce concentrates and powders and are often spray or freeze dried. Some heat is usually applied in the industrial production of aloe vera juice to deactivate enzymes that would break down the mannans into oligosaccharides and simple sugars. Heating also serves to control the normal microbial load present on the plant. Enzymatic treatment can be used to further break down cell walls, with filtration removing any remaining insoluble fiber. The resulting filtered juice contains all the major groups of components from the original aloe vera inner leaf.

Reduction of microbial load by pasteurization

Reduction of microbial load by pasteurization

Reduction of water content to increase the percent juice solids or to produce juice powder

Preservation and packaging

Preservation and packaging

Flowchart 2 Aloe vera inner leaf processing.

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Rodriguez et al.

OH

OH HO

HO

OH

OH O

O HO

HO HO

HO

OH

O Aloin A

OH

OH

O Aloin B

gathered and the whole plant is cut at the base, producing a transverse cut on all the leaves of the plant at the same time. These leaves are placed in a V-shaped collection device and the aloin-rich sap is allowed to drain. The resulting yellow sap is then concentrated by applying heat until it becomes viscous and forms a solid upon cooling. This aloin paste product contains approximately 25% of the anthraquinone glycosides aloins A and B (Fig. 2). A more sophisticated production method takes the yellow sap, precipitates the resins by adding acid, and concentrates the soluble fraction under vacuum at reduced temperature. The concentrate is then spray dried to produce “spray-dried aloin,” which typically contains about 50% aloins. Aloin-rich aloe extracts have been used worldwide mostly in laxative preparations though it has other uses including as a bitter flavoring agent, especially in the wine industry.

Acemannan Mannan is a generic name for polysaccharides that are polymers of the sugar mannose. In aloe vera juice, the mannose moieties are connected by ␤-(1–4) linkages, which are partially substituted with acetate units and with galactose-rich side chains on the mannose backbone. This ␤-(1,4)-acetylated-polymannose material is also known by other names such as “aloverose” and “acemannan”; the latter is also a name given to a proprietary substance covered by many patents (7) and has been assigned as a generic name by the United States Adopted Names Council (8). It is based on the chemical name as it refers to the acetylated mannan found in all aloe vera inner leaf fillets (Fig. 3). Acemannan is not sold as a pure material; however, many commercial products contain varying amounts of it depending on the processing of the aloe vera leaf as men-

Figure 3

OH

Figure 2

Structures of Aloin A & B.

tioned earlier. The therapeutic properties of aloe vera juice have been largely attributed to its polysaccharide component and acemannan in particular. This high-molecularweight material is perhaps the most studied component of the aloe vera plant aside from the anthraquinone glycosides. Many industrial methods have been developed to stabilize the aloe vera juice and prevent polysaccharide degradation. Drying the juice at temperatures over 60◦ C has been shown to cause deleterious changes in acemannan and also pectin from the cell walls (9).

ANALYSIS OF COMMERCIAL PRODUCTS Analysis of 32 commercial products showed wide variations in polysaccharide content when compared by molecular weight (10), which was attributed to different manufacturing procedures. A second study of nine commercial powders used a method that hydrolyzed the mannan into mannose as a rapid way to measure the total polysaccharide content in the powder. One sample was found to have an abnormally high concentration of free glucose, four showed signs of spoilage, and all but three were found to have low levels of polymannose present (11). Both studies found all samples to have a significantly lower amount of aloins than the raw unwashed inner leaf fillet, with a high of 16 ppm of aloin A found in one sample in one of the studies. Because of the widespread use of aloe vera juice in personal care products, a voluntary industry limit of 50 ppm aloin content for use in cosmetics as a topical agent has been established (12).

PRECLINICAL STUDIES General Many of the biological properties of aloe vera have been attributed to acemannan. This compound has been studied

Partial molecular model of acemannan [␤-(1,4)-acetylated-polymannose]. Source: Courtesy of Santiago Rodriguez, Houston, Texas.

Aloe Vera

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itate fraction demonstrated wound healing activity (17), suggesting that more than one aloe compound could be useful in treating both inflammation and wounds.

Skin Moisturizing Aloe vera applied topically has a moisturizing effect on skin (18) and has been used for this purpose and as a conditioning agent.

Antidiabetic Activity Rodent studies have shown blood glucose regulating activity of an aloe vera alcoholic extract (19) and processed aloe vera inner leaf juice (20), suggesting its utility in treating non–insulin-dependent diabetes.

Antitumor Activity

Figure 4 Maryland.

Aloe vera plant. Source: Courtesy of Devon Powell, Columbia,

for its ability to effect changes in a mouse macrophage cell line and was found to stimulate cytokine production, nitric oxide release, surface molecule expression, and cause changes to cell morphology. This, coupled with the finding that the effect on cytokine production was dose dependent (13), suggests that acemannan may function in part through macrophage activation. The dermatological activities of aloe vera were investigated in a systematic review that extracted data from 40 studies in a predetermined standardized manner (14). Orally administered to mice, it was found to be effective in promoting wound healing and reducing the incidence of tumors and leishmania parasites by greater than 90% in selected tissues. Its antiviral and antimicrobial properties as well as positive effects on inflammation, frostbite, and burns were reported. Topical application was found not to protect against sunburn in this review. Clinical effectiveness for the use of aloe vera for dermatological conditions was not thought to be sufficiently explored. Another review examined purported biological properties of aloe vera leaf juice, namely promotion of wound healing, and antifungal, antidiabetic, antiinflammatory, anticancer, immunomodulatory, and gastroprotective properties and focused on more recently discovered effects and applications such as the ability of aloe vera juice to increase the bioavailability of coadministered compounds and to enhance skin permeability (15).

Wound Healing The wound healing activity of topical and oral aloe vera was studied in rodent models of anti-inflammatory effects and wound healing (16). Both supernatant and precipitate fractions of a 50% ethanol extract of aloe vera decreased inflammation but only the high-molecular-weight precip-

Acemannan has shown significant antitumor activity via immune system activation. In a mouse model, IP injection of acemannan at the time of implantation of sarcoma cells resulted in a 40% survival rate in the treated animals versus 0% of the controls, most likely because of the production of monokines from macrophage peritoneal stimulation. The data suggested that this acemannan-stimulated synthesis “resulted in the initiation of immune attack, necrosis, and regression of implanted sarcomas in mice” (21). A study involving acemannan treatment as an adjunct to surgery and radiation in confirmed fibrosarcoma in dogs and cats showed tumor shrinkage in one-third of the animals after four to seven weeks of treatment administered by intraperitoneal and intralesional injections (22). An earlier study by the same group showed similar results (23).

Studies on Aloin-Rich Materials Aloin-rich extracts derived from aloe latex belong to the stimulant laxatives drug class. Aloins are inactive until deglycosylated by intestinal flora to form aloe-emodin, the putative active compound (24). Their mechanism of action is believed to involve increasing peristalsis and water accumulation in the colon (25). Aloe latex has been subjected to a human clinical trial as a laxative in combination with other ingredients (26). The potential toxicities of aloin and its metabolites are not well established though studies have shown it does not promote colon cancer in a mouse model (27) and induces cell changes that could be a sign of anticancer activity (28). Selective activity against certain cancers has also been demonstrated by aloe-emodin, a metabolite of aloins A and B (29,30).

CLINICAL STUDIES Wound Healing Although one study showed a delay in healing wound complications after cesarean delivery or gynecological surgery following treatment with “aloe vera dermal wound gel” (31), another recorded a 6-day statistically significant reduction (from 18 to 12 days) in the healing time of partial thickness burns. A systematic review of the literature for the use of topical aloe vera in treating burn wounds included four controlled clinical trials involving 371 patients. A meta-analysis based on the time for healing showed almost nine fewer days required for

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Rodriguez et al.

the aloe vera–treated group over the controls (32). No specific conclusions could be drawn because of the difference in preparations and outcome measures. Further studies with well-characterized materials were called for by the authors as cumulative evidence tended to support the use of aloe vera for the treatment of first- and second-degree burns. A bioadhesive patch of an aloe vera preparation was evaluated in an open uncontrolled trial for the management of mouth ulcers in children with apparent good results (33).

Ulcerative Colitis and Irritable Bowel Syndrome (IBS) Ulcerative colitis is caused by a dysfunction of the immune system (34). A 2004 clinical trial involving 44 patients with mild or moderately active ulcerative colitis compared 100 mL twice-daily oral aloe vera juice treatment with placebo for four weeks. The aloe vera–consuming patients showed positive clinical responses more often than placebo. Clinical remission was seen in 30% of the active group, clinical improvement in 37%, and a clinical response in 47% of patients compared with 7%, 7%, and 14% in the placebo group, respectively. Histological scores and the Simple Clinical Colitis Activity Index did not change in the placebo group but decreased significantly for those who consumed aloe vera. No significant differences were seen between the two groups with regard to laboratory values or sigmoidoscopic scores (35). A human clinical study using aloe vera for treatment of irritable bowel in refractory secondary care patients failed to show a benefit, though the authors could not rule out that diarrhea-predominant patients were helped (36).

Antidiabetic Activity Aloe dried sap has demonstrated hypoglycemic activity in a study involving five patients with non–insulindependent diabetes (37).

Antitumor Activity A preliminary clinical trial on the use of orally administered “aloe vera tincture” for untreatable metastatic solid tumor patients with and without melatonin treatment showed a significantly higher percentage of nonprogressing patients in the group that received the aloe treatment (50% vs. 27%, P < 0.05) (38). Another human trial on 240 patients treated with Aloe aborescens (used because of purported immunostimulating activity from this plant owing to its acemannan component) suggested that oral aloe therapy may be a successful adjunct to chemotherapy in patients with metastatic solid tumors. Tumor regression rate and survival time were improved in this study (39). No conclusions can be drawn from this study because details on characterization of the test material were not provided.

SAFETY A four-week subacute oral toxicity study in mice administered a freeze-dried aloe juice product reported no remarkable subacute toxic effects but did note a decrease in male kidney weights. The report also provided a review of several adverse reaction case studies associated

with aloe vera (40). They ranged from skin irritation from topical use to one report of acute hepatitis in a 73-yearold female taking oral aloe vera capsules for constipation. A second case of acute hepatitis involving a 26-year-old man who had been drinking “aloe vera tea” has also been reported (41). The National Toxicology Program of the U.S. government nominated “aloe vera gel” for study in 1998 (42). No long-term carcinogenicity studies of aloe vera gel in animals were identified at that time. NTP subsequently chose to conduct a two-year carcinogenicity study on mice and rats with a “whole leaf extract” (43) that includes a considerable amount of latex aloins. The majority of aloe vera juice products intended for long-term internal use are either charcoal filtered whole leaf preparation or are made from washed inner leaf juice with aloin concentrations typically under 10 parts per million. The NTP report was still in progress at the time of publication of this chapter.

REGULATORY STATUS United States Aloe and aloin are present in the first approved food additives list published by the U.S. Food and Drug Administration in 1959. Although initially approved in the United States in 1975 as an OTC drug to treat chronic constipation, aloe latex is no longer approved for such use in the United States as of May 9, 2002 (44). Standard quality tests for aloe latex have been described in detail in many official pharmacopeias including the United States Pharmacopeia, Japanese Pharmacopoeia, and the European Pharmacopoeia, though, as mentioned in the introduction, these texts typically define aloe latex as simply “aloe” or incorrectly as “aloe juice.” Aloe vera juice products can be labeled and marketed as dietary supplements. Aloe latex may also be used in dietary supplements in the United States with laxative or constipation claims as long as such claims are not for the treatment of chronic constipation.

Australia Aloe vera inner leaf (called “aloe barbadensis”) is eligible for use as an active or excipient ingredient in Australia in “Listed” medicines in the Australian Register of Therapeutic Goods. Acemannan is approved as a component. Components are not approved as substances for use in their own right and can only be used in conjunction with an approved source. Some aloe vera juice and juice concentrate beverages are viewed as “nontraditional foods” and not as “novel foods” and there are some listed medicines described as “aloe vera drinking gel” or as “aloe vera juice.”

Canada Aloe vera inner leaf, when included as a Natural Health Product (NHP) active ingredient, requires premarket authorization and a product license number for OTC human use. Such products must comply with the minimum specifications outlined in the current NHPD Compendium of Monographs (45).

Aloe Vera

European Community Aloe vera inner leaf was listed as “currently not on the priority list” in the inventory of herbal substances for assessment by the European Medicines Agency as of March 2009. There is an EU regulatory limit established for aloin content of 0.1 ppm in orally ingested products based on a flavoring regulation in which the aloin is defined as an added ingredient as opposed to naturally occurring. The International Aloe Science Council (IASC) (a trade association) has taken a position that these regulations are not applicable to aloe vera juice products.

Japan Aloe vera juice is regulated as a food beverage product in Japan and is not to contain more than 0.60 mg/kg of benzoic acid. Various forms of aloe vera and extracts thereof are used as components of functional food products or in Foods for Specified Health Use such as in fortified waters and fermented yogurt drinks.

South Korea Aloe products, known as “edible aloe concentrate” and “edible aloe gel,” are regulated as food products by the Korean Food and Drug Administration. Juice or concentrate from the inner leaf or dried and powdered inner leaf material containing not-less-than 30 mg/g of total aloe polysaccharides is able to carry the health claim of “smoothing the evacuation” on the basis of 20 to 30 mg delivered as aloe polysaccharides. Processed aloe vera leaf or concentrates thereof, after removal of the inedible parts, and containing 2.0 to 50 mg/g of anthraquinones (as anhydrous barbaloin), is permitted to make the same health claim at the specified daily intake. Aloe vera is also one of the four botanical ingredients allowed to make immune system enhancement claims in South Korea.

CONCLUSION Of the 400 known species of aloe, Aloe vera is the most commonly used in commerce and is cultivated in many different areas of the world. The plant yields two raw materials for use in various consumer products including foods, dietary supplements, cosmetics, and drugs, namely aloe vera juice and aloe latex. Aloe vera juice can be made from processing either the entire leaf or only the inner leaf material. Aloe vera juice is often further processed into a powder or concentrate. Preliminary scientific evidence suggests that aloe vera has therapeutic benefits; however, more studies need to be conducted to definitively demonstrate efficacy. Consumers should be aware and informed when buying aloe vera products; although there are many quality products on the market, there are also many products that may bring little or no benefit to the user. The IASC maintains a certification program using validated analytical methods to determine and ensure products displaying the IASC program seal contain aloe vera of a particular quality. It is recommended that consumers verify that products displaying the IASC seal are current participants in the IASC certification program.

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REFERENCES 1. http://www.ars-grin.gov/cgi-bin/npgs/html/taxon .pl?311403. Accessed January 15, 2010. 2. McGuffin M, Kartesz J, Leung A, et al. In: McGuffin M, ed. Herbs of Commerce. 2nd ed. Silver Spring, MD: American Herbal Products Association, 2000; 10. 3. International Aloe Science Council—Labeling guidance and definitions. http://www.iasc.org/09 0309 IASC labeling guidance.pdf. Accessed January 15, 2010. 4. Ghritkumari (Aloe vera), Ayurved good place for all. http:// ayurved.agoodplace4all.com/ghritkumari.php. Accessed January 15, 2010. 5. Gilman F. Fact Sheet FPS-34. University of Florida, Cooperative Extension Service, 1999. Aloe barbadensis. http://hort .ifas.ufl.edu/shrubs/ALOBARA.PDF. Accessed January 15, 2010. 6. Ni Y, Turner D, Yates KM, et al. Isolation and characterization of structural components of Aloe vera L. leaf pulp. Int Immunopharmacol 2004; 4(14):1745–1755. 7. Reynolds T, Dweck AC. Aloe vera leaf gel: A review update. J Ethnopharmacol 1999; 68(1–3):3–37. 8. Carpenter RH, McDaniel HR, McAnalley BH. Uses of aloe products in the treatment of chronic respiratory diseases. July 28, 1998. US Patent 5,786,342. 9. Femenia A, Garc´ıa-Pascual P, Simal S, et al. Effects of heat treatment and dehydration on bioactive polysaccharide acemannan and cell wall polymers from Aloe barbadensis Miller. Carbohydr Polym 2003; 51(4):397–405. 10. Turner CE, Williamson DA, Stroud PA, et al. Evaluation and comparison of commercially available Aloe vera L. products using size exclusion chromatography with refractive index and multi-angle laser light scattering detection. Int Immunopharmacol 2004; 4(14):1727–1737. 11. Bozzi A, Perrin C, Austin S, et al. Quality and authenticity of commercial aloe vera gel powders. Food Chem 2007; 103(1):22–30. 12. Cosmetic Ingredient Review Expert Panel. Final Report on the Safety Assessment of Aloe andongensis extract, Aloe andongensis leaf juice, Aloe arborescens leaf extract, Aloe arborescens leaf juice, Aloe arborescens leaf protoplasts, Aloe barbadensis flower extract, Aloe barbadensis leaf, Aloe barbadensis leaf extract, Aloe barbadensis leaf juice, Aloe barbadensis polysaccharides, Aloe barbadensis leaf water, Aloe ferox leaf extract, Aloe ferox leaf juice, and Aloe ferox leaf juice extract. Int J Toxicol 2007; 26(S2):1–50. 13. Zhang L, Tizard IR. Activation of a mouse macrophage cell line by acemannan: The major carbohydrate fraction from Aloe vera gel. Immunopharmacology 1996; 35(2):119–128. 14. Feily A, Namazi MR. Aloe vera in dermatology: A brief review. G Ital Dermatol Venereol 2009; 144(1):85–91. 15. Hamman JH. Composition and applications of Aloe vera leaf gel. Molecules 2008; 13(8):1599–1616. 16. Davis RH, Leitner MG, Russo JM, et al. Wound healing. Oral and topical activity of Aloe vera. J Am Podiatr Med Assoc 1989; 79(11):559–562. 17. Davis RH, Parker WL, Samson RT, et al. Isolation of a stimulatory system in an Aloe extract. J Am Podiatr Med Assoc 1991; 81(9):473–478. 18. Dal’Belo SE, Gaspar LR, Maia Campos PM. Moisturizing effect of cosmetic formulations containing Aloe vera extract in different concentrations assessed by skin bioengineering techniques. Skin Res Technol. 2006; 12(4):241–246. 19. Rajasekaran S, Sivagnanam K, Ravi K, et al. Hypoglycemic effect of Aloe vera gel on streptozotocin-induced diabetes in experimental rats. J Med Food 2004; 7(1):61–66. 20. Kim K, Kim H, Kwon J, et al. Hypoglycemic and hypolipidemic effects of processed Aloe vera gel in a mouse model

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of non-insulin-dependent diabetes mellitus. Phytomedicine 2009; 16(9):856–863. Peng SY, Norman J, Curtin G, et al. Decreased mortality of Norman murine sarcoma in mice treated with the immunomodulator, acemannan. Mol Biother 1991; 3(2):79–87. King GK, Yates KM, Greenlee PG, et al. The effect of Acemannan immunostimulant in combination with surgery and radiation therapy on spontaneous canine and feline fibrosarcomas. J Am Anim Hosp Assoc 1995; 31(5):439–434. Harris C, Pierce K, King G, et al. Efficacy of acemannan in treatment of canine and feline spontaneous neoplasms. Mol Biother 1991; 3(4):207–213. Ishii Y, Takino Y, Toyo’oka T, et al. Studies of aloe. VI. Cathartic effect of isobarbaloin. Biol Pharm Bull 1998; 21(11):1226– 1227. Ishii Y, Tanizawa H, Takino Y. Studies of aloe. V. Mechanism of cathartic effect. (4). Biol Pharm Bull 1994; 17(5):651–653. Odes HS, Madar Z. A double-blind trial of a celandin, aloe vera and psyllium laxative preparation in adult patients with constipation. Digestion 1991; 49(2):65–71. Siegers CP, Siemers J, Baretton G. Sennosides and aloin do not promote dimethylhydrazine-induced colorectal tumors in mice. Pharmacology 1993; 47(S1):205–208. Buenz EJ. Aloin induces apoptosis in Jurkat cells. Toxicol In Vitro 2008; 22(2):422–429. Kupchan SM, Karim A. Tumor inhibitors. 114. Aloe emodin: Antileukemic principle isolated from Rhamnus frangula L. Lloydia 1976; 39(4):223–224. Pecere T, Gazzola MV, Mucignat C, et al. Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors. Cancer Res 2000; 60(11):2800–2804. Schmidt JM, Greenspoon JS. Aloe vera dermal wound gel is associated with a delay in wound healing. Obstet Gynecol 1991; 78(1):115–117. Maenthaisong R, Chaiyakunapruk N, Niruntraporn S, et al. The efficacy of aloe vera used for burn wound healing: A systematic review. Burns 2007; 33(6):713–718. Andriani E, Bugli T, Aalders M, et al. The effectiveness and acceptance of a medical device for the treatment of aphthous stomatitis. Clinical observation in pediatric age [in Italian]. Minerva Pediatr 2000; 52(1–2):15–20.

34. Kristensen NN, Claesson MH. Future targets for immune therapy in colitis? Endocr Metab Immune Disord Drug Targets 2008; 8(4):295–300. 35. Langmead L, Feakins RM, Goldthorpe S, et al. Randomized, double-blind, placebo-controlled trial of oral aloe vera gel for active ulcerative colitis. Aliment Pharmacol Ther 2004; 19(7):739–747. 36. Davis K, Philpott S, Kumar D, et al. Randomised doubleblind placebo-controlled trial of aloe vera for irritable bowel syndrome. Int J Clin Pract 2006; 60(9):1080– 1086. 37. Ghannam N, Kingston M, Al-Meshaal IA, et al. Antidiabetic activity of Aloes: preliminary clinical and experimental observations. Horm Res 1986; 24(4):288–294. 38. Lissoni P, Giani L, Zerbini S, et al. Biotherapy with the pineal immunomodulating hormone melatonin versus melatonin plus aloe vera in untreatable advanced solid neoplasms. Nat Immun 1998; 16(1):27–33. 39. Lissoni P, Rovelli F, Brivio F, et al. A randomized study of chemotherapy versus biochemotherapy with chemotherapy plus Aloe arborescens in patients with metastatic cancer. In Vivo 2009; 23(1):171–175. 40. Kwack SJ, Kim KB, Lee BM. Estimation of tolerable upper intake level (UL) of active aloe. J Toxicol Environ Health A 2009; 72(21–22):1455–1462. ´ 41. Curciarello J, De Ortuzar S, Borzi S, et al. Severe acute hepatitis associated with intake of Aloe vera tea [in Spanish]. Gastroenterol Hepatol 2008; 31(7):436–438. 42. Boudreau MD, Beland FA. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller), Aloe vera. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2006; 24(1):103–154. 43. http://ntp.niehs.nih.gov/?objectid=BD4B0DDA-123F7908-7BE69A6EAB26471E. Accessed January 15, 2010. 44. Food and Drug Administration, HHS. Status of Certain Additional Over-the-Counter Drug Category II and III Active Ingredients. Federal Register 2002; 67(90):31125– 31127. 45. http://www.hc-sc.gc.ca/dhp-mps/prodnatur/ applications/licen-prod/monograph/mono aloe-eng.php. Accessed January 15, 2010.

Androstenedione Benjamin Z. Leder

its use in the official East German Olympic athlete doping program. The event that most dramatically sparked widespread curiosity in androstenedione, however, was the media report that the St. Louis Cardinals baseball player Mark McGwire had used androstenedione in the 1999 season (during which he broke the record for most home runs in a season). The publicity that surrounded this supplement also prompted an increased interest in related “prohormones,” such as norandrostenedione and androstenediol. This then led to a proliferation of claims concerning the potential benefits of androstenedione use. Manufacturers credited these products not only with promoting muscle growth and improving athletic performance but also with increasing energy, libido, sexual performance, and general quality of life. Additionally, androstenedione was often packaged in combination with other substances as part of an intensive nutritional approach to performance enhancement. An example of such a combination is shown in Figure 1. Clearly, the use of androstenedione and related compounds during that time went well beyond the accumulation of data that could provide a rational basis for their use.

INTRODUCTION Androstenedione (chemical name: 4-androstene-3,17dione) is a steroid hormone produced primarily in the reproductive system and adrenal glands in men and women. It circulates in the bloodstream and is the immediate precursor to the potent anabolic/androgenic hormone testosterone in the steroid synthesis pathway. Despite this wellknown physiological classification, as well as a growing body of evidence demonstrating that orally administered androstenedione is converted to more potent steroid hormones, the United States Food and Drug Administration originally classified the hormone as a “dietary supplement.” As such, it was available to the general public without a prescription and for nearly a decade could be easily purchased in health clubs, nutrition stores, and over the Internet. This over-the-counter availability of androstenedione came to an end when Food and Drug Administration banned its sale in early 2004. The ban was then codified with the passing of the 2004 Anabolic Steroid Control Act. This law reclassified androstenedione as an anabolic steroid and hence a controlled substance.

GENERAL DESCRIPTION The original and seemingly contradictory classification of androstenedione as a dietary supplement was based on the definition set forth in the 1994 Dietary Supplement Health and Education Act (DSHEA). According to the DSHEA, a substance was defined as a dietary supplement if it was a “product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients: a vitamin, mineral, amino acid, herb or other botanical. . . or a concentrate, metabolite, constituent, extract, or combination of any ingredient described above.” Hence, because androstenedione could be synthesized from plant products, it fell under that umbrella. Furthermore, the DSHEA specified that the Department of Justice could not bring action to remove a product unless it was proven to pose “a significant or unreasonable risk of illness or injury” when used as directed. Not surprisingly, after the passing of the DSHEA, the use of dietary supplements increased dramatically. In fact, by 1999, the dietary supplement industry in the United States was generating annual sales of $12 billion (1). Initially, androstenedione use was primarily confined to athletes in strength and endurance-related sports, an interest that seems to have sprung from reports of

PROHORMONE FACTORS 4-Androstenedione: 100 mg 19-Nor-5-Androstenedione: 50 mg 5-Androstenediol: 50 mg DHEA: 50 mg GH/1GF FACTORS L-Arginine Pyroglutamate: 2500 mg L-Ornithine Alpha-Ketoglutarate: 1250 mg Taurine: 750 mg Colostrum: 250 mg LH BOOSTER Tribulus: 250 mg Acetyl-L-Carnitine: 250 mg L-Carnitine: 100 mg DHT BLOCKERS Saw Palmetto: 200 mg Beta Sitosterol: 200 mg Pygeum Africanum: 50 mg ESTROGEN BLOCKERS Kudzu: 100 mg Chrysin: 250 mg

Figure 1

15

A typical combination dietary supplement product.

16

Leder

O

HO Dehydroepiandrosterone 3β-HSD

O

O

HCOOH CYP19 (aromatase) HO Estrone

O

4.9% of male and 2.4% of female adolescents in the United States had used illegal anabolic steroids (4). Because these substances were so readily available, there was concern that androstenedione use in this particularly susceptible population may have greatly exceeded these numbers. In fact, in a survey administered in five health clubs in Boston, Massachusetts, in 2001, 18% of men and 3% of women respondents admitted to using androstenedione or other adrenal hormone dietary supplements at least once. These percentages suggested that as many as 1.5 million U.S. health club members alone may have used these substances (5).

4-Androstenedione 17β-HSD

17β-HSD

OH

OH HCOOH CYP19 (aromatase) HO

O Testosterone

Estradiol-17β

Figure 2 Androstenedione’s relationship to other steroid hormones. Enzyme abbreviations: 3␤-HSD, 3␤-hydroxysteroid dehydrogenase; 17␤-HSD, 17␤-hydroxysteroid dehydrogenase.

BIOCHEMISTRY AND PHYSIOLOGY Androstenedione is a steroid hormone that is produced primarily in the adrenals, testes, and ovaries. It is classified as a “weak androgen” because it binds to the body’s receptor for androgen hormones in a much less potent fashion than classic anabolic/androgenic steroids such as testosterone (2). It is synthesized from the precursor hormone dehydroepiandrosterone (itself a dietary supplement) and is the direct precursor to testosterone. In normal physiological circumstances, androstenedione can also be converted to potent feminizing hormones such as estrone and estradiol (both members of the “estrogen” class of hormones). The relationship between androstenedione, other steroid hormones, and the enzymes involved in the conversion of androstenedione to testosterone and estrogens is shown in Figure 2. Importantly, the enzymes that convert androstenedione to potent hormones such as testosterone and estradiol are active not only in endocrine glands but also in many peripheral body tissues such as muscle, bone, liver, and brain (3). Thus, if orally administered androstenedione has biological activity, it may act either directly or by conversion to these more potent agents.

ANDROSTENEDIONE USE There were no precise data concerning the prevalence of androstenedione use in the general population during the time that it was widely available. Our best estimates were based on industry sales figures and extrapolations from data on classic anabolic/androgenic steroid use in specific populations. For example, in 1997, it was estimated that

PHARMACOKINETICS AND HORMONAL EFFECTS OF ANDROSTENEDIONE IN MEN Because so many of the claims that surrounded androstenedione were based on the premise that oral administration increases serum testosterone levels, it may be surprising to some that prior to 1999, there was only a single published study investigating the ability of orally administered androstenedione to be converted to more potent steroid hormones (6). In this study, two women were given a single dose of androstenedione, and the levels were subsequently measured over the next several hours. Since 1999, however, numerous small studies (mostly in men) have investigated the effects of the supplement (6–16). In general, these studies report that serum androstenedione levels increase dramatically after oral administration and thus confirm that a significant portion of the supplement is absorbed through the gastrointestinal tract after ingestion. However, the answer to the more important question, namely, whether it is then converted to more potent steroid hormones such as testosterone and estradiol, appears to be complex. In general, these studies suggest that the ability of oral androstenedione to increase estrogen and testosterone levels in men is dose dependent and is possibly related to the age of the study population as well. Specifically, the bulk of the research indicates that when androstenedione is administered to men in individual doses between 50 and 200 mg, serum estrogen levels increase dramatically. However, larger individual doses (e.g., 300 mg) are required to increase serum testosterone levels. For example, King and colleagues studied the effects of a single 100-mg oral dose of androstenedione in 10 men between the ages of 19 and 29 and reported that although serum androstenedione and estradiol levels increased significantly, testosterone levels did not change (13). These investigators then specifically measured the portion of circulating testosterone that is not bound to protein and considered the “bioactive” portion (called “free testosterone”) and similarly saw no effect of the supplement. In a separate study, Leder and colleagues gave 0, 100, or 300 mg of androstenedione to normal healthy men between the ages of 20 and 40 for seven days and took frequent blood samples on days 1 and 7 (14). As in the study by King, they also found that men receiving both the 100- and 300-mg doses of androstenedione experienced dramatic increases in serum estradiol that were often well above the normal male range. Another similarity was that 100-mg dose

Androstenedione

140 120 100 80 0-mg dose group 100-mg dose group 300-mg dose group

60 40 20 0 −20 % Change in serum % Change in serum estradiol testosterone

Figure 3 Percentage change in serum testosterone and estradiol in healthy men after a single androstenedione dose (as measured by eight hours of frequent blood sampling). Source: Adapted from Ref. 14.

17

56 (10). In this study, subjects consuming 100 mg of androstenedione three times daily experienced increases in serum estrogens but not in serum testosterone. However, unlike in the study by King and colleagues discussed in the previous text, free testosterone did increase significantly (even though again by only a small amount). Finally, several studies have compared the hormonal effects of androstenedione with those of other “prohormone” dietary supplements. Broeder and colleagues studied the results of a 100-mg twice-daily dose of oral androstenedione, androstenediol (a closely related steroid hormone), or placebo in men between the ages of 35 and 65 (7). They found that both compounds increased estrogen levels but neither affected total serum testosterone levels. Similarly, Wallace and colleagues studied the effects of 50-mg twice-daily doses of androstenedione and DHEA in normal men and reported no increases in serum testosterone levels with either (16).

EFFECTS ON MUSCLE SIZE AND STRENGTH IN MEN did not affect serum testosterone levels. As shown in Figure 3, however, the novel finding of this study was that 300 mg of androstenedione increased serum testosterone levels significantly, even though by only a modest amount (34%). Leder and colleagues further observed that there was a significant degree of variability among men with regard to their serum testosterone response after androstenedione ingestion. As shown in Figure 4, some subjects, even in the 300-mg dose group, experienced relatively little change in testosterone levels, whereas serum testosterone levels doubled in other men. This finding suggests that there may be individual differences in the way androstenedione is metabolized that could impact any one person’s physiological response to taking the supplement. Brown and colleagues investigated the hormonal response in a group of men between the ages of 30 and

Serum testosterone (ng/dL)

1800 1600 1400

The results of the studies discussed earlier suggest that androstenedione use in men would be less likely to promote the muscle building and performance-enhancing effects associated with testosterone use and more likely to induce the undesirable feminizing effects associated with estrogens. Several studies have assessed the ability of androstenedione (with or without exercise) to increase muscle size and strength and have been uniformly disappointing (7,9,13,15,16). For example, Broeder and colleagues, in the study described earlier, also measured changes in body composition and strength in subjects taking 100 mg androstenedione twice daily in combination with a 12-week intensive weight-training program (7). Despite using sensitive methods that can detect small changes in body composition, they found no differences in muscle mass, fat mass, or strength in the subjects receiving androstenedione compared with those receiving a placebo tablet. Importantly, however, in this study as well as all of these studies referenced earlier, the supplement was given in doses that were not sufficient to increase testosterone levels. It thus remains unknown whether doses of androstenedione sufficient to increase testosterone levels enhance muscle mass or athletic performance.

1200

METABOLISM OF ANDROSTENEDIONE IN MEN

1000 800 600 400 200 0 Baseline

Peak

Figure 4 Individual variability in the peak serum testosterone level achieved after a single 300-mg dose of androstenedione in men. Each line represents one study subject. Source: Adapted from Ref. 14.

One of the consistent findings of the various androstenedione studies in men is the inefficiency of conversion of the supplements to testosterone. Leder and colleagues explored this issue further by investigating the pattern of androstenedione metabolism in healthy men (17). Specifically, they measured the concentration of inactive testosterone metabolites (also called “conjugates”) in the urine of subjects ingesting androstenedione and found an increase of over 10-fold compared with their baseline levels. This finding was in direct contrast to the much more modest changes in serum testosterone they had observed. It suggests that although much of the androstenedione

Serum testosterone (ng/dL)

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published reports investigating the long-term physiological effects in women.

140 120

ADVERSE EFFECTS AND TOXICITY

100 80 60 40 20 0

0

120

240 360 480 Time (minutes)

600

720

Figure 5 Serum testosterone levels during 12 hours of frequent blood sampling in postmenopausal women. Circles represent control subjects receiving no supplement, triangles those receiving 50 mg of androstenedione, and squares those receiving 100 mg. Source: Adapted from Ref. 18.

that is absorbed after oral administration is converted to testosterone, it is then immediately further metabolized to inactive compounds in the liver. The investigators confirmed this hypothesis by directly measuring the concentration of one of these inactive metabolites (testosterone glucuronide) in the serum of these subjects. As expected, they found that testosterone glucuronide levels increased by 500% to 1000% (as opposed to the 34% increase in biologically active serum testosterone after a single 300-mg dose of oral androstenedione). Together, these findings demonstrate the effectiveness of the liver in inactivating steroid molecules when taken orally.

PHARMACOKINETICS AND HORMONAL EFFECTS OF ANDROSTENEDIONE IN WOMEN Since the initial report of androstenedione administration in two women in 1962 (6), research into the effects of the supplement has focused largely on the hormonal response to oral administration in young men. Between 2002 and 2003, however, two studies on women were published. The first of these studies examined the effects of a single dose of 0, 50, or 100 mg of androstenedione in postmenopausal women (18). The findings of this study were surprising. In contrast to the effects observed in men, even these low doses increased testosterone levels significantly in women (Fig. 5). Also, unlike the results seen in men, estradiol levels were unaffected by androstenedione administration. In the other study, 100 mg of androstenedione was administered to young, premenopausal, healthy women. Similar to postmenopausal women, these subjects experienced significant increases in serum testosterone levels after androstenedione administration (estradiol was not measured) (19). Importantly, in both of these studies, the peak testosterone levels achieved by the older and younger women taking androstenedione were often significantly above the normal range. Together, these results predict that the physiological effects of the supplement may be different in men and women, as might their potential toxicities. To date, however, there have been no

Ever since the publicity surrounding androstenedione exploded in 1999, many reports in the lay press have focused on the potential dangerous side effects. Nonetheless, with the exception of a single case description of a man who developed two episodes of priapism in the setting of androstenedione ingestion (20), there have been no published reports of androstenedione-associated serious adverse events. This fact should be only partially reassuring, however, because androstenedione’s prior classification as a dietary supplement (as opposed to a drug) allowed manufacturers to avoid responsibility for rigorously monitoring any potential toxicity of their product. It is well known that oral administration of certain testosterone derivatives can cause severe liver diseases, and anabolic steroid use in general is associated with anecdotal reports of myocardial infarction, sudden cardiac death, and psychiatric disturbances (“roid rage”). Nonetheless, despite androstenedione’s close chemical similarity to these substances, it is important to note that it is not a potent anabolic steroid nor does it have a chemical structure similar to those specific compounds that cause liver problems. Thus, the potential of androstenedione to cause these particular serious side effects appears to be limited. Of more pressing concern to clinicians are the possible long-term effects in specific populations. In clinical trials, the supplement was generally well tolerated, though several studies did report that it reduces highdensity lipoprotein (or “good cholesterol”) levels in men. Importantly, however, even the longest of these studies lasted only several months. It thus remains quite possible that androstenedione use, especially at high doses, could cause subtle physiological changes over prolonged periods that could directly lead to adverse health consequences. In men, for example, the dramatic increase in estradiol levels observed with androstenedione administration could, over time, lead to gynecomastia (male breast enlargement), infertility, and other signs of feminization. In women, because the supplement increases testosterone levels above the normal range, it could cause hirsutism (excess body hair growth), menstrual irregularities, or male-like changes in the external genitalia. In children, increases in both testosterone and estrogen levels could cause precocious puberty or premature closure of growth plates in bone, thereby compromising final adult height.

PURITY OF COMMERCIALLY AVAILABLE ANDROSTENEDIONE During its period of over-the-counter availability, androstenedione was available from multiple manufacturers and could be purchased as a tablet, capsule, sublingual tablet, or even a nasal spray. Often, it was combined with other products that claimed to limit its potential side effects (such as chrysin, for example, which is purported to decrease androstenedione’s conversion to estrogens). Because the manufacture of dietary supplements was not

Androstenedione

Table 1 Analysis of Nine Common Brands of Androstenedione Supplements Amount of androstenedione listed (mg)

Amount of androstenedione found (mg)

100 100 100 100 100 100 50 50

93 83 103 90 88 85 35 0 (no steroid compounds identified) 168 (10 mg of testosterone was also present)

250 Source: From Ref. 21.

subject to the same regulations as pharmaceuticals, the purity and labeling of androstenedione-containing products were often inaccurate. Catlin and colleagues, for example, reported that urine samples from men treated with androstenedione contained 19-norandrosterone, a substance not associated with androstenedione metabolism but rather with the use of a specific banned anabolic steroid (21). Further investigation revealed that the androstenedione product used contained a tiny amount of the unlabeled steroid “19-norandrostenedione.” Though the amount of 19-norandrostenedione was not physiologically significant, it was enough to cause a “positive” urine test for illegal anabolic steroid use when tested in the standard fashion. In fact, it is precisely this type of contamination that may have explained increases in positive tests for 19-norandrosterone among competitive athletes in the past decade. Additionally, it is now common for athletes who test positive for norandrosterone or other androgenic metabolites to point to dietary supplement contamination as the potential explanation. Catlin and colleagues also analyzed nine common brands of androstenedione and showed that there was considerable variation and mislabeling among products in terms of both purity and content (Table 1).

REGULATORY STATUS AND DETECTION Androstenedione was available over-the-counter from 1994 (when the DSHEA was passed) until it was reclassified as an anabolic steroid by the Anabolic Steroid Control Act in 2004. It is important to note that this reclassification came without any evidence that androstenedione increased muscle mass or strength, which was the previous legal definition of an anabolic steroid. Virtually all sports organizations, including the National Football League, the National Collegiate Athletic Association, and the International Olympic Committee, have banned androstenedione. Despite these prohibitions, detection of androstenedione has not been standardized. Specifically, the method used most often to detect testosterone use, measurement of the urinary testosterone-to-epitestosterone ratio, has not proven to be reliable in establishing androstenedione use (22). Further study is still needed to define novel testing procedures that are able to detect androstenedione use reliably.

19

CONCLUSIONS Androstenedione is a steroid hormone, which, until 2004, was a popular over-the-counter dietary supplement. Since then, however, it has been classified as an anabolic steroid, and hence a controlled substance. It is purported to increase strength, athletic performance, libido, sexual performance, energy, and general quality of life. Studies indicate that when taken orally by men, small doses are converted to potent estrogens and larger doses to both testosterone and estrogens. Comparatively, there appears to be a much more physiologically important increase in estrogens compared with testosterone in men. In women, the effects are reversed. Studies have thus far failed to confirm any effect on muscle size or strength, though the dosing regimens were modest. Although documentation of adverse side effects among users of androstenedione is scarce, there is considerable concern over potential longterm toxicity, especially in women and adolescents.

REFERENCES 1. Anonymous. Herbal treatments: The promises and pitfalls. Consum Rep 1999; 64:44–48. 2. Orth DN, Kovacs WJ. The adrenal cortex. In: Wilson D, Foster DW, Kronenberg HM, et al., eds. Williams Textbook of Endocrinology. Philadelphia, PA: W.B. Saunders Company, 1998:517–664. 3. Labrie F, Simard J, Luu-The V, et al. Structure, regulation and role of 3 beta-hydroxysteroid dehydrogenase, 17 betahydroxysteroid dehydrogenase and aromatase enzymes in the formation of sex steroids in classical and peripheral intracrine tissues. Baillieres Clin Endocrinol Metab 1994; 8(2):451–474. 4. Yesalis CE, Barsukiewicz CK, Kopstein AN, et al. Trends in anabolic-androgenic steroid use among adolescents. Arch Pediatr Adolesc Med 1997; 151:1197–1206. 5. Kanayama G, Gruber AJ, Pope HG Jr, et al. Over-the-counter drug use in gymnasiums: An underrecognized substance abuse problem? Psychother Psychosom 2001; 70(3):137–140. 6. Mahesh VB, Greenblatt RB. The in vivo conversion of dehydroepiandrosterone and androstenedione to testosterone in the human. Acta Endocrinol 1962; 41:400–406. 7. Broeder CE, Quindry J, Brittingham K, et al. The Andro Project: Physiological and hormonal influences of androstenedione supplementation in men 35 to 65 years old participating in a high-intensity resistance training program. Arch Intern Med 2000; 160(20):3093–3104. 8. Brown GA, Vukovich MD, Martini ER, et al. Effects of androstenedione-herbal supplementation on serum sex hormone concentrations in 30- to 59-year-old men. Int J Vitam Nutr Res 2001; 71(5):293–301. 9. Brown GA, Vukovich MD, Reifenrath TA, et al. Effects of anabolic precursors on serum testosterone concentrations and adaptations to resistance training in young men. Int J Sport Nutr Exerc Metab 2000; 10(3):340–359. 10. Brown GA, Vukovich MD, Martini ER, et al. Endocrine responses to chronic androstenedione intake in 30- to 56-yearold men. J Clin Endocrinol Metab 2000; 85(11):4074–4080. 11. Earnest CP, Olson MA, Broeder CE, et al. In vivo 4androstene-3,17-dione and 4-androstene-3 beta,17 beta-diol supplementation in young men. Eur J Appl Physiol 2000; 81(3):229–232. 12. Ballantyne CS, Phillips SM, MacDonald JR, et al. The acute effects of androstenedione supplementation in healthy young males. Can J Appl Physiol 2000; 25(1):68–78.

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13. King DS, Sharp RL, Vukovich MD, et al. Effect of oral androstenedione on serum testosterone and adaptations to resistance training in young men. J Am Med Assoc 1999; 281(21):2020–2028. 14. Leder BZ, Longcope C, Catlin DH, et al. Oral androstenedione administration and serum testosterone concentrations in young men. J Am Med Assoc 2000; 283(6): 779–782. 15. Rasmussen BB, Volpi E, Gore DC, et al. Androstenedione does not stimulate muscle protein anabolism in young healthy men. J Clin Endocrinol Metab 2000; 85(1): 55–59. 16. Wallace MB, Lim J, Cutler A, et al. Effects of dehydroepiandrosterone vs. androstenedione supplementation in men. Med Sci Sports Exerc 1999; 31(12):1788–1792. 17. Leder BZ, Catlin DH, Longcope C, et al. Metabolism of orally administered androstenedione in young men. J Clin Endocrinol Metab 2001; 86(8):3654–3658.

18. Leder BZ, Leblanc KM, Longcope C, et al. Effects of oral androstenedione administration on serum testosterone and estradiol levels in postmenopausal women. J Clin Endocrinol Metab 2002; 87(12):5449–5454. 19. Kicman AT, Bassindale T, Cowan DA, et al. Effect of androstenedione ingestion on plasma testosterone in young women: A dietary supplement with potential health risks. Clin Chem 2003; 49(1):167–169. 20. Kachhi PN, Henderson SO. Priapism after androstenedione intake for athletic performance enhancement. Ann Emerg Med 2000; 35(4):391–393. 21. Catlin DH, Leder BZ, Ahrens B, et al. Trace contamination of over-the-counter androstenedione and positive urine test results for a nandrolone metabolite. J Am Med Assoc 2000; 284(20):2618–2621. 22. Catlin DH, Leder BZ, Ahrens BD, et al. Effects of androstenedione administration on epitestosterone metabolism in men. Steroids 2002; 67(7):559–564.

L-Arginine Mauro Maccario, Guglielmo Beccuti, Valentina Gasco, Mariangela Seardo, Gianluca Aimaretti, Emanuela Arvat, Fabio Lanfranco, and Ezio Ghigo

INTRODUCTION

symptoms of high intake are rare, but symptoms of massive dosages may include thickening and coarsening of the skin, muscle weakness, diarrhea, and nausea. The proximal renal tubule accounts for much of the endogenous production of L-Arg from L-citrulline. In the tubule, arginine reacts via the Krebs cycle with the toxic ammonia formed from nitrogen metabolism, producing the nontoxic and readily excretable urea (Fig. 1) (5). If this mechanism does not efficiently handle metabolic byproducts and if L-Arg intake is insufficient, ammonia rapidly accumulates, resulting in hyperammonemia. L-Arg undergoes different metabolic fates. NO, L-citrulline, L-ornithine, L-proline, L-glutamate, and polyamine-like putrescine are formed from L-Arg. Moreover, the high-energy compound NO-creatinine phosphate, which is essential for sustained skeletal muscle contraction, is also formed from L-Arg (Fig. 2). L-Arg, its precursors, and its metabolites are deeply involved in the interaction of different metabolic pathways and interorgan signaling. The amino acid influences the internal environment in different ways: disposal of protein metabolic waste; muscle metabolism; vascular regulation; immune system function; healing and repair of tissue; formation of collagen; and building of new bone and tendons. A leading role for arginine has been shown in the endocrine system, vasculature, and immune response.

Arginine was first isolated in 1895 from animal horn. It is considered a nonessential amino acid under physiological conditions; however, it may be classified as semi-essential (or conditioned) in newborns, young children, or other circumstances characterized by accelerated tissue growth (e.g., infection, sepsis, trauma) when its production may be too slow and not sufficient to meet the requirements (1). Arginine is physiologically active in the L-form (L-Arg) and participates in protein synthesis in cells and tissues. It is essential for the synthesis of urea, creatine, creatinine, and pyrimidine bases. It also strongly influences hormonal release and has an important role in vasculature dynamics, participating in the synthesis of nitric oxide (NO).

BIOCHEMISTRY Dietary arginine is particularly abundant in wheat germ and flour, buckwheat, oatmeal, dairy products (cottage cheese, ricotta cheese, nonfat dry milk, skimmed yogurt), chocolate, beef (roasts, steaks), pork, nuts (coconut, pecans, walnuts, almonds, hazel nuts, peanuts), seeds (pumpkin, sesame, sunflower), poultry (chicken, turkey), wild game (pheasant, quail), seafood (halibut, lobster, salmon, shrimp, snails, tuna), chick peas, and soybeans (2). L-Arg, delivered via the gastrointestinal tract, is absorbed in the jejunum and ileum of the small intestine. A specific amino acid transport system facilitates this process and participates also in the transport of the other basic amino acids, L-lysine and L-histidine. About 60% of the absorbed L-Arg is metabolized by the gastrointestinal enterocytes, and only 40% remains intact reaching the systemic circulation. An insufficient arginine intake produces symptoms of muscle weakness, similar to muscular dystrophy (3). Arginine deficiency impairs insulin secretion, glucose production, and liver lipid metabolism (4). Conditional deficiencies of arginine or ornithine are associated with the presence of excessive ammonia in the blood, excessive lysine, rapid growth, pregnancy, trauma, or protein deficiency and malnutrition. Arginine deficiency is also associated with rash, hair loss and hair breakage, poor wound healing, constipation, fatty liver, hepatic cirrhosis, and hepatic coma (4). Depending on nutritional status and developmental stage, normal plasma arginine concentrations in humans and animals range from 95 to 250 ␮mol/L. Toxicity and

GLUTAMINE

CO2 + NH4+

GLUTAMATE

2ATP

NH4+-GROUPS

2ADP + Piz CARBAMOYL PHOSPHATE

ORNITHINE

CITRULLINE NH2 C=O NH2

ARGININE

ASPARTATE ATP AMP + Ppi + H2O

ARGININOSUCCINATE FUMARATE

Figure 1

21

L-Arginine

and Krebs cycle in the renal tubule.

22

Maccario et al.

Nitric Oxide

Admatine Aldehyde

Urea DAO

Agmatine

Agmatinase

NOS

Nitric Oxide

ADC

Ca2+

ADC

Arginine C NH

CE COCH

HN

NOS

Guanidine Group

A-GAT

Glycine

Proline

Guanidinoacetate

Polyamines

Arginase-I

P-5-C reductase

Protein synthesis

Urea

Urea cycle

Glutamine

OAT

Pyrroline-5-carboxylate

Glutamyl-γ-′semialdehyde P-5-C dehydrogenase

GMT

Creatine

Ornithine

Glu synthase

Glutamate

NH3

Figure 2 L-Arginine metabolites. Abbreviations: ADC, arginine decarboxylase; A:GAT, arginine:glycine amidinotransferase; DAO, diamine oxidase; Glu synthase, glutamine synthase; GMT, guanidinoacetate-N-methyltransferase; NOS, nitric oxide synthase; OAT, ornithine aminotransferase; P-5-C dehydrogenase, pyrroline-5-carboxylate dehydrogenase; P-5-C reductase, pyrroline5-carboxylate reductase.

PHYSIOLOGY Endocrine Actions L-Arg functions as a secretagogue of a number of important hormones at the pituitary, pancreas, and adrenal levels. The effects on growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), and insulin secretion will be discussed in detail.

GH Secretion Among the various factors modulating somatotropin function, arginine is well known to play a primary stimulatory influence. Arginine has been shown to increase basal GH levels and to enhance the GH responsiveness to growth hormone releasing hormone (GHRH) both in animals and in humans throughout their life span (6–9); its GH-stimulating activity occurs after both IV and oral administration and is dose dependent; 0.1 and 0.5 g/kg are the minimal and the maximal IV effective doses, respectively. Moreover, a low orally administered arginine dose has been shown to be as effective as a high IV dose in enhancing the GH response to GHRH both in children and in elderly subjects (10,11). Arginine, directly or indirectly via NO, is likely to act by inhibiting hypothalamic somatostatin (SS) release. It has been shown that arginine—but not isosorbidedinitrate and molsidomine, two NO donors—stimulates GH secretion (12,13), suggesting that it does not exert its effects through the generation of NO. Moreover, arginine does not modify either basal or GHRH-induced GH increase from rat anterior pituitary (14). On the contrary, it potentiates the GH response to the maximal GHRH dose in humans. Arginine can elicit a response even when the response has been previously inhibited by a GHRH ad-

ministration, which induces an SS-mediated negative GH autofeedback (7,8,15). Moreover, arginine counteracts the GH-inhibiting effect of neuroactive substances that act by stimulating SS release; it does not modify the GH-releasing activity of stimuli acting via SS reduction (8). Again, in favor of an SS-mediated mechanism is also the evidence that ornithine, the active form of arginine, is unable to modify plasma GHRH levels in humans (16). Moreover, arginine fails to potentiate the increased spontaneous nocturnal GH secretion, which is assumed to reflect circadian SS hyposecretion and GHRH hypersecretion, respectively (8). Arginine does not influence the strong GH-releasing action of ghrelin, the natural ligand of GH secretagogue receptors, which is supposed to act as a functional antagonist of SS at both the pituitary and the hypothalamic levels (17,18). The GH-releasing activity of arginine is sex dependent but not age dependent, being higher in females than in males but similar in children, young, and elderly subjects (8,19–23). Moreover, it has been clearly demonstrated that arginine totally restores the low somatotrope responsiveness to GHRH in aging, when a somatostatinergic hyperactivity is likely to occur (20–23). This evidence clearly indicates that the maximal secretory capacity of somatotropic cells does not vary with age and that the agerelated decrease in GH secretion is due to a hypothalamic impairment (20–23). This also points out the possible clinical usefulness of this substance to rejuvenate the GH/insulin-like growth factor-I (IGF-I) axis in aging. In fact, the reduced function of the GH/IGF-I axis in aging may account for the changes in body composition, structure, and function. In agreement with this assumption, it has been reported by some, but not all, authors that elderly subjects could benefit from treatment with rhGH to restore IGF-I levels within the young range (21,24). As it has been demonstrated that the GH releasable pool in the aged pituitary is basically preserved and that the agerelated decline in GH secretion mostly reflects hypothalamic dysfunction (21,23), the most appropriate, that is, “physiological,” approach to restore somatotroph function in aging would be a treatment with neuroactive substances endowed with GH-releasing action. Among these GH secretagogues, arginine received considerable attention. In fact, the coadministration of arginine (even at low oral doses) with GHRH (up to 15 days) enhanced the GH responsiveness to the neurohormone in normal aged subjects (11). However, the efficacy of long-term treatment with oral arginine to restore the function of the GH/IGF-I axis in aging has never been shown in elderly subjects. Following the evidence that GHRH combined with arginine becomes the most potent and reproducible stimulus to diagnose GH deficiency throughout the life span (25), GHRH + arginine is, at present, one of the two gold standard tests for the diagnosis of GH deficiency (25,26). In fact, the GH response to a GHRH + arginine test is approximately threefold higher than the response to classical tests and does not vary significantly with age (25,26). Because of its good tolerability and its preserved effect in aging, the GHRH + arginine test is currently considered to be the best alternative choice to the insulin-induced tolerance test (ITT) for the diagnosis of GH deficiency throughout the life span (25).

L-Arginine

PRL Secretion Among the endocrine actions of arginine, its PRLreleasing effect has been shown both in animals and in humans after IV but not after oral administration (10,27). The PRL response to arginine is markedly lower than the response to the classical PRL secretagogues, such as dopaminergic antagonists or thyrotropin-releasing hormone (TRH) (6) but higher than that observed after secretion of GH and other modulators of lactotrope function (17). The mechanisms underlying the stimulatory effect of arginine on PRL secretion are largely unknown, but there is evidence that this effect is not mediated by galanin, a neuropeptide with PRL-releasing effect. In fact, galanin has been shown to potentiate PRL response to arginine, suggesting different mechanisms of action for the two substances (28).

ACTH Secretion Although some excitatory amino acids and their agonists have been demonstrated to differently modulate corticotropin-releasing hormone and arginine vasopressin release in vitro and influence both sympathoadrenal and hypothalamo-pituitary-adrenal (HPA) responses to hypoglycemia in animals (29,30), little is known about arginine influences on HPA axis in humans. Many studies have shown that mainly food ingestion influences spontaneous and stimulated ACTH/cortisol secretion in normal subjects and that central ␣1 -adrenergic-mediated mechanisms are probably involved (31). In humans free fatty acids inhibit spontaneous ACTH and cortisol secretion, but no data exist regarding the effect of each nutrient component on HPA function. Previous studies demonstrated that arginine is unable to exert an ACTH-stimulatory effect in humans via generation of NO (12) and our unpublished preliminary data failed to demonstrate a significant effect of arginine (30 g IV) on either ACTH or cortisol secretion in normal subjects.

Insulin Secretion Arginine is one of the most effective known insulin secretagogue and it may be used with glucose potentiation to determine a patient’s capacity to secrete insulin (32). Arginine acts synergistically with glucose, and to a much lesser extent with serum fatty acids, in stimulating insulin release. A synergistic effect of arginine and glucose on insulin secretion has been shown in humans (33,34), and the combined administration of these two stimuli has been studied in an attempt to test ␤-cell secretory capacity in diabetic patients (35). A protein meal leads to a rapid increase in both plasma insulin and glucagon levels (36). Administration of arginine has a similar effect. An arginine transport system is present in the ␤-cell plasma membrane (37). When arginine enters the ␤ cell, it causes ionic changes that depolarize the ␤ cell and trigger Ca2+ uptake and exocytosis of insulin-containing granules. Several mechanisms for arginine-induced ␤-cell stimulation have been proposed. These include the metabolism of L-Arg leading to the formation of ATP (38,39), the generation of NO (40,41), and the direct depolarization of the plasma membrane potential due to the accumulation of the cationic amino acid (42–44).

23

A sustained Ca2+ influx is directly related to insulin secretion following arginine uptake by ␤ cells. The arginine-induced increase in Ca2+ concentration is inhibited by the activation of ATP-sensitive potassium (K-ATP) channels with diazoxide and seems dependent on the nutritional status. These observations suggest that the K-ATP channels, when fully open, act to prevent membrane depolarization caused by arginine. The presence of a nutrient, such as glucose, produces sufficient closure of K-ATP channels to allow arginine-induced membrane depolarization and activation of the voltage-activated Ca2+ channels (37).

Nonendocrine Actions Cardiovascular System Increasing interest has been recently focused on NO. This mediator, which is synthesized from L-Arg (45) by nitric oxide synthases (NOS) (46), is a potent vasodilator (47) and inhibitor of platelet adhesion and aggregation (48). Three isoforms of NOS are described: neuronal NOS (nNOS—NOS-1), inducible NOS (iNOS—NOS-2), and endothelial NOS (eNOS—NOS-3). NOS-1 and NOS-3 are expressed constitutively and they produce NO at low rates (49). NOS-3 is responsible for a consistent vasodilator tone and, although constitutive, can be regulated by endothelial shear stress (50) and substances such as acetylcholine, histamine, serotonin, thrombin, bradykinin, and catecholamines. Calcium is required for NOS-3 activation (51). NO production is mainly dependent on the availability of arginine and NOS is responsible for the biochemical conversion of L-Arg to NO and citrulline in the presence of cofactors such as reduced nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH4 ), flavin mononucleotide, and flavin adenine nucleotide. Reduced NO production, leading to vasoconstriction and increases in adhesion molecule expression, platelet adhesion and aggregation, and smooth muscle cell proliferation has been demonstrated in atherosclerosis, diabetes mellitus, and hypertension (52–54)—conditions known to be associated with an increased mortality because of cardiovascular disease. Taken together, these observations lead to the concept that interventions designed to increase NO production by supplemental L-Arg might have a therapeutic value in the treatment and prevention of the endothelial alterations of these diseases. Besides several actions exerted mainly through NO production, arginine also has a number of NO-independent properties, such as the ability to regulate blood and cellular pH, and the effect on the depolarization of endothelial cell membranes. The daily consumption of arginine is normally about 5 g/day. Arginine supplementation is able to increase NO production, although the Km for L-Arg is 2.9 ␮mol and the intracellular concentration of arginine is 0.8 to 2.0 mmol. To explain this biochemical discrepancy, named “arginine paradox,” there are theories that include low arginine levels in some diseases (e.g., hypertension, diabetes mellitus, and hypercholesterolemia), and/or the presence of enzymatic inhibitors (55), and/or the activity of the enzyme arginase (which converts arginine to ornithine and urea, leading to low levels of arginine). Recently attention has been given to the methylated forms of L-Arg, generated by the proteolysis of

24

Maccario et al.

methylated proteins; they are represented by asymmetric dimethylarginine (ADMA) and two symmetric dimethylated derivatives: symmetric dimethylarginine (SDMA) and monomethylarginine (MMA) (56). Only ADMA and MMA, but not SDMA, exert inhibitory effects on NOS-3 activity (57). For this reason, ADMA is now recognized as a new emerging cardiovascular risk marker and likely as a causative factor for cardiovascular disease (58). L-Arginine therapy in cardiovascular pathologies showed contradictory results. However, it is now clear that individual response to L-Arg may be influenced by SDMA. In fact, no effects of L-arg therapy are demonstrated in patients with low ADMA levels, whereas in patients with high ADMA level, L-Arg normalizes the L-Arg to ADMA ratio, thus normalizing the endothelial function (59). Several studies demonstrated that L-Arg infusion in normal subjects and patients with coronary heart disease (60), hypercholesterolemia (61), and hypertension (62) is able to improve the endothelial function, but the results, although encouraging, are not conclusive because of the short-term effects of IV arginine. However, arginine does not affect endothelial function in patients with diabetes mellitus. On the other hand, oral L-Arg has a longer half-life and longer-term effects than L-Arg given intra-arterially or intravenously (63). Thus, in the setting of long-term health maintenance or symptom management, the oral route would be preferred. Studies in animals documented that oral L-Arg supplementation is able to reduce the progression of atherosclerosis, preserving endothelium function (64) and inhibiting circulating inflammatory cells (65) and platelets (66) in animals with hypercholesterolemia, and to decrease blood pressure and wall thickness in animals with experimental hypertension (67). On the other hand, studies in humans in vivo are not so widely positive as the animal experimental data. Actually, although the majority of the data is in normal subjects, individuals with a history of cigarette smoking and patients with hypercholesterolemia and claudication demonstrate beneficial effects of oral L-Arg administration on platelet adhesion and aggregation, monocyte adhesion, and endothelium-dependent vasodilation (68,69). Other studies do not show any benefit (70,71); therefore, no definitive conclusions can be drawn. Taken together, the studies show a major effect when L-Arg supplementation was given in subjects with hypercholesterolemia, probably because of an increase in NO production via reduction of the ADMA intracellular concentration, which is increased in the presence of LDL hypercholesterolemia. In conclusion, despite several beneficial effects on intermediate end points, particularly in hypercholesterolemic patients, there is no evidence for a clinical benefit in the treatment or prevention of cardiovascular disease. More data, derived from large-scale prospective studies evaluating the effect of long-term treatment with L-Arg, are needed. Future perspectives of pharmacological intervention are represented by the regulation of the enzyme dimethylarginine dimethylaminohydrolase responsible for the ADMA metabolism (57), the arginase (72), and the endothelial cell L-Arg transporter (73).

Immune System Many studies, in animals as well as in humans, have shown that arginine is involved in immune modulation. In

fact, this amino acid is a component of most proteins and the substrate for several nonprotein, nitrogen-containing compounds acting as immune modulators. There is clear evidence that arginine participates in the cell-mediated immune responses of macrophages and T lymphocytes in humans through the production of NO by inducible nitric oxide synthase (iNOS-–NOS-2), which occurs mostly in the macrophage (74,75), and through the modulation of T-lymphocyte function and proliferation (76,77). At intracellular levels, arginine is metabolized by two different enzymatic pathways: the arginase pathway, by which the guanidino nitrogen is converted into urea to produce ornithine, and the NOS pathway, which results in oxidation of the guanidino nitrogen to produce NO and other substances (78,79). It has been shown that macrophage superoxide production, phagocytosis, protein synthesis, and tumoricidal activity are inhibited by high levels of arginine in vitro and that sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are deficient in free arginine (80). In particular, a decrease in arginine availability due to the activity of macrophagederived arginase rather than the arginine/NO pathway may contribute to the activation of macrophages migrating at inflammatory sites (80). Arginine metabolism in the macrophages is activity dependent. At rest, macrophages exhibit minimal utilization of arginine and lower NOS-2 expression or arginase activity, whereas in activated cells, arginine is transported into the cell, and NOS-2 expression and arginase are induced by cytokines and other stimuli (81). The types of stimuli that induce NOS-2 and arginase are quite different. In vitro and in vivo studies demonstrated that NOS-2 is induced by T-helper I cytokines (IL1, TNF, and ␥ -interferon) produced during activation of the cellular immune response, such as severe infections or sepsis (74,75), whereas arginases are induced by T-helper II cytokines (IL-4, IL-10, and IL-13) and other immune regulators aimed at inducing the humoral immune response (82,83). Thus, in disease processes, where inflammatory response predominates, NOS-2 expression and NO production prevail. Under biological circumstances where Thelper II cytokine expression is prevalent, arginase activity and the production of ornithine and related metabolites would predominate. In vitro studies in animals demonstrated depressed lymphocyte proliferation in cultures containing low levels of arginine and maximal proliferation when arginine is added at physiological plasma concentration (77,84), but the molecular details have not been completely defined. It has also been shown that supplemental arginine increased thymic weight in rodents because of increased numbers of total thymic T lymphocytes. On the other hand, in athymic mice, supplemental arginine increased the number of T cells and augmented delayed-type hypersensitivity responses, indicating that it can exert its effects on peripheral lymphocytes and not just on those within the thymus (76). The immunostimulatory effects of arginine in animal studies have suggested that this amino acid could be an effective therapy for many pathophysiological conditions in humans, able to positively influence the immune response under some circumstances by restoring cytokine balance and reducing the incidence of infection.

L-Arginine

In healthy humans, oral arginine supplementation shows many effects on the immune system, including increase in peripheral blood lymphocyte mitogenesis, increase in the T-helper–T-cytotoxic cell ratio and, in macrophages, activity against microorganisms and tumor cells (85). Furthermore, the delayed-type hypersensitivity response as well as the number of circulating natural killer (NK) and lymphokine-activated killer cells are increased (85–87). Therefore, it has been hypothesized that arginine could be of benefit to patients undergoing major surgery after trauma and sepsis and in cardiovascular diseases, HIV infection, and cancer (88). In fact, short-term arginine supplementation has been shown to maintain the immune function during chemotherapy; arginine supplementation (30 g/day for 3 days) reduced chemotherapy-induced suppression of NK cell activity, lymphokine-activated killer cell cytotoxicity, and lymphocyte mitogenic reactivity in patients with locally advanced breast cancer (89). It must be noted that chronic administration of arginine has also been shown to promote cancer growth by stimulating polyamine synthesis in both animal and human studies (89). On the other hand, NO has been shown to inhibit tumor growth. Thus, the real effect on cancer processes depends on the relative activities of NOS and arginase pathways that show variable expression, depending on the stage of carcinogenesis (91). These data clearly indicate the involvement of arginine in immune responses in both animals and humans. Large clinical trials are needed to clarify the clinical application and efficacy of this amino acid in immunity and immunopathology.

SUPPLEMENTAL ARGININE The available form of supplemental L-Arg is represented by the free base, the Cl− salt (L-Arg hydrochloride-–L-ArgHCl) and the aspartate salt of the amino acid (92). L-Arg is stable under sterilization condition and its administration is safe for mammals in an appropriate dose and chemical form (91). Oral L-Arg (up to 9 g of Arg-HCl per day for adults) has no adverse effects on humans but higher doses can lead to gastrointestinal toxicity, theoretically increasing local production of NO and impairing intestinal absorption of other basic amino acids (91). Moreover, the local NO production may be particularly dangerous if intestinal diseases are present (92). Oral L-Arg supplement is commonly used to increase GH release and consequentially physical performance; moreover, it has been hypothesized that L-Arg supplement could lead to improved muscular aerobic metabolism and less lactate accumulation, enhancing NOmediated muscle perfusion. However, in a clinical trial, arginine supplement in endurance-trained athletes did not show any difference from placebo in endurance performance (maximal oxygen consumption, time to exhaustion), endocrine (GH, glucacon, cortisol, and testosterone concentrations), and metabolic parameters (93). In another study, the association “arginine plus exercise” produced a GH response approximately 50% lower than that observed with exercise alone, suggesting that

25

the acute use of oral L-Arg prior to exercise blunts the GH response to subsequent exercise (94). No effects on NO production, lactate and ammonia metabolism, and physical performance in intermittent anaerobic exercise were shown in well-trained male athletes after short-term arginine supplementation (95). It has been hypothesized that NO production is not modified by arginine supplementation in athletes because they may have higher basal concentrations of NO than general population; in fact, basal NO production can be increased by regular exercise training, without any pharmacological intervention (95). There are many interesting clinical perspectives on arginine supplementation therapy, especially in critical care setting (96), treatment and prevention of pressure ulcers (97), hypertension (59), and asthma and chronic obstructive pulmonary disease (98), but further studies are required to clarify which categories of patients may benefit from this treatment (99).

CONCLUSIONS From an endocrinological point of view, the simple classification of arginine as an amino acid involved in peripheral metabolism is no longer acceptable. In fact, besides other nonendocrine actions, it has been clearly demonstrated that arginine plays a major role in the neural control of anterior pituitary function, particularly in the regulation of somatotrophin secretion. One of the most important concepts regarding arginine is the existence of an arginine pathway at the CNS level, where this amino acid represents the precursor of NO, a gaseous neurotransmitter of major importance. On the other hand, NO does not necessarily mediate all the neuroendocrine or the peripheral arginine actions. In the past years, new discoveries have led to a rapid increase in our knowledge of the arginine/NO system, from a neuroendocrine and nonendocrine point of view. Up to now, there is no evidence for the utility of L-Arg supplement for muscle strength or exercise performance in humans. However, several other aspects still remain to be clarified; the potential clinical implications for arginine have also never been appropriately addressed and could provide unexpected results both in the endocrine and in the cardiovascular fields.

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65. Brandes RP, Brandes S, Boger RH, et al. L-Arginine supplementation in hypercholesterolemic rabbits normalizes leukocyte adhesion to non-endothelial matrix. Life Sci 2000; 66:1519–1524. 66. Coreaux D, Tourneau T, Ezekowitz MD, et al. Enhanced monocyte tissue factor response after experimental balloon angiography in hypercholesterolemic rabbits: Inhibition with L-arginine. Circulation 1998; 98:1176–1182. 67. Sun YP, Zu PQ, Browne AEM, et al. L-Arginine decreases blood pressure and left ventricular hypertrophy in rats with experimental aortic coarctation. J Am Coll Cardiol 1998; 31(suppl A):501A. 68. Adams MR, McCredie R, Jessup W, et al. Oral L-arginine improves endothelium-dependent dilation and reduces monocyte adhesion to endothelial cells in young men with coronary artery disease. Atherosclerosis 1997; 129:261–270. 69. Lerman A, Burnett JC, Higano ST, et al. Long term arginine supplementation improves small vessel coronary endothelial function in humans. Circulation 1998; 97:2123– 2128. 70. Blum A, Hathaway L, Mincemoyer R, et al. Effects of oral L-arginine on endothelium-dependent vasodilation and markers of inflammation in healthy postmenopausal women. J Am Coll Cardiol 2000; 35:271–276. 71. Chin-Dusting JPF, Kaye GM, Lefkovits J, et al. Dietary supplementation with L-arginine fails to restore endothelial function in forearm resistance arteries in patients with severe heart failure. J Am Coll Cardiol 1996; 27:1207– 1213. 72. Santhanam L, Christianson DW, Nyhan D, et al. Arginase and vascular aging. J Appl Physiol 2008; 105(5):1632–1642. 73. Chin-Dusting JP, Willems L, Kaye DM. L-Arginine transporters in cardiovascular disease: A novel therapeutic target. Pharmacol Ther 2007; 116(3):428–436. 74. Hibbs JB Jr, Taintor RR, Vavrin Z, et al. Nitric oxide: A cytotoxic activated macrophage effector molecule. Biochem Biophysiol Res Commun 1988; 157:87–94. 75. Nathan CF, Hibbs JB Jr. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol 1991; 3:65–70. 76. Barbul A, Sisto DA, Wasserkrug HL. Arginine stimulates lymphocyte immune response in healthy humans. Surgery 1981; 90:244–251. 77. Ochoa JB, Strange J, Kearney P, et al. Effects of L-arginine on the proliferation of T lymphocyte subpopulations. JPEN J Parenter Enteral Nutr 2001; 25:23–29. 78. Kepka-Lenhart D, Mistry SK, Wu G, et al. Arginase I: A limiting factor for nitric oxide and polyamine synthesis by activated macrophages? Am J Physiol Regul Integr Comp Physiol 2000; 279:R2237–R2242. 79. Taheri F, Ochoa JB, Faghiri Z, et al. Arginine regulates the expression of the T-cell receptor zeta chain (CD3zeta) in jurkat cells. Clin Cancer Res 2001; 7:958s–965s. 80. Albina JE, Caldwell MD, Henry WL Jr, et al. Regulation of macrophage functions by L-arginine. J Exp Med 1989; 169:1021–1029. 81. Kakuda DK, Sweet MJ, MacLeod CL, et al. CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J 1999; 340:549–553. 82. Modolell M, Corraliza IM, Link F, et al. Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. Eur J Immunol 1995; 25:1101–1104. 83. Hesse M, Modolell M, La Flamme AC, et al. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: Granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol 2001; 167:6533–6544.

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84. Kobayashi T, Yamamoto M, Hiroi T, et al. Arginine enhances induction of T helper 1 and T helper 2 cytokine synthesis by Peyer’s patch alpha beta T cells and antigen-specific mucosal immune response. Biosci Biotechnol Biochem 1998; 62:2334– 2340. 85. Barbul A, Fishel RS, Shimazu S, et al. Intravenous hyperalimentation with high arginine levels improves wound healing and immune function. J Surg Res 1985; 38:328– 334. 86. Daly JM, Reynolds J, Thom A, et al. Immune and metabolic effects of arginine in the surgical patient. Ann Surg 1988; 208:512–523. 87. Park KG, Hayes PD, Garlick PJ, et al. Stimulation of lymphocyte natural cytotoxicity by L-arginine. Lancet 1991; 337:645– 646. 88. Appleton J. Arginine: Clinical potential of a semi-essential amino acid. Altern Med Rev 2002; 7:512–522. 89. Brittenden J, Heys SD, Ross J, et al. Natural cytotoxicity in breast cancer patients receiving neoadjuvant chemotherapy: Effects of L-arginine supplementation. Eur J Surg Oncol 1994; 20:467–472. 90. Park KG. The immunological and metabolic effect of L-arginine in human cancer. Proc Nutr Soc 1993; 52:387–401. 91. Wu G, Bazer FW, Davis TA, et al. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 2009; 37(1):153–168.

92. Grimble GK. Adverse gastrointestinal effects of arginine and related amino acids. J Nutr 2007; 137(6 suppl 2):1693S– 1701S. 93. Abel T, Knechtle B, Perret C, et al. Influence of chronic supplementation of arginine aspartate in endurance athletes on performance and substrate metabolism—a randomized, double-blind, placebo-controlled study. Int J Sports Med 2005; 26(5):344–349. 94. Kanaley JA. Growth hormone, arginine and exercise. Curr Opin Clin Nutr Metab Care 2008; 11(1):50–54. 95. Liu TH, Wu CL, Chiang CW, et al. No effect of shortterm arginine supplementation on nitric oxide production, metabolism and performance in intermittent exercise in athletes. J Nutr Biochem 2009; 20(6):462–468. 96. Marik PE, Zaloga GP. Immunonutrition in critically ill patients: A systematic review and analysis of the literature. Intensive Care Med 2008; 34(11):1980–1990. 97. Schols JM, Heyman H, Meijer EP. Nutritional support in the treatment and prevention of pressure ulcers: An overview of studies with an arginine enriched oral nutritional supplement. J Tissue Viability 2009; 18(3):72–79. 98. Maarsingh H, Pera T, Meurs H. Arginase and pulmonary diseases. Naunyn Schmiedebergs Arch Pharmacol 2008; 378(2):171–184. 99. Coman D, Yaplito-Lee J, Boneh A. New indications and controversies in arginine therapy. Clin Nutr 2008; 27(4):489–496.

Astragalus Roy Upton

INTRODUCTION

CHEMISTRY AND PREPARATION OF PRODUCTS

Astragalus root (Astragalus membranaceus and Astragalus mongholicus) (Figs. 1 and 2; flowers are shown in Fig. 2) is one of the most important plant products used in traditional Chinese medicine (TCM) for supporting immune resistance ( ; wei qi) and energy production ( ; bu qi). Astragalus is also one of the most popular ingredients in botanical dietary supplements for its putative effect of supporting healthy immune function. Despite the widespread use of this botanical among TCM practitioners and its extensive use in botanical supplements, there are few clinical trials supporting its use, though those that are available are positive. Numerous preclinical studies provide evidence for a number of pharmacological effects that are consistent with the traditional and modern use of astragalus.

The primary compounds of interest in astragalus are triterpenes, polysaccharides, and flavonoids. The triterpene astragaloside IV is a relatively unique marker for astragalus species used in Chinese medicine. A variety of preparations are utilized in clinical practice and in herbal supplements. A number of preparations, including crude extracts, isolated polysaccharides, and triterpene saponins, have been subject to study and correlated with activity. Clinically, in China and among some practitioners in the United States, decoctions are frequently given. However, due to the time required for cooking and the subsequent smell and taste of Chinese herb preparations in general (though astragalus is very agreeable), many consumers and practitioners prefer crude powder or extract preparations (capsules, tablets), freeze-dried granules, or liquid extracts. Astragalus is also used as a relatively common ingredient in soups, especially during winter months. Polysaccharides (12–36 kD) have been most often correlated with immune activity, while triterpene saponins have been predominantly associated with cardiovascular and hepatoprotective effects. Astragalus polysaccharides are generally composed of a mixture of D-glucose, D-galactose, and L-arabinose or D-glucose alone. The glucose units appear to be primarily ␣-(1,4)linked with periodic ␣-(1,6)-linked branches (1,2). The triterpene glycosides vary by position, number, and type of sugar residues at positions 3, 6, and 25. Several of these “astragalosides” (e.g., astragaloside IV; Fig. 3) are composed of a single xylopyranosyl substituent at the 3position, which may or may not be acetylated. Others possess either disaccharide or trisaccharide substituents (3– 5). Primary flavonoids of astragalus for which activity has been reported include calycosin, formononetin (Fig. 3), and daidzein (Fig. 3) and additionally include isorhamnetin, kaempferol, and quercetin, among others (6).

BACKGROUND Traditional and Modern Uses In Asia, astragalus is commonly used according to both its traditional Chinese medical indications as a general tonifier and specifically for immune enhancement and for modern biomedical indications such as immune, liver, and cardiovascular support. It has been used for the prevention of the common cold and upper respiratory tract infections and is widely prescribed to children for prevention of infectious disease, though formal clinical English language studies regarding this use are lacking. In the West, astragalus is primarily used as an immune modulator. Astragalus potentiates recombinant interleukin-2 (rIL-2) and recombinant interferon-1 and -2 (rIFN-1 and -2) immunotherapy and by lowering the therapeutic thresholds, may reduce the side effects normally associated with these therapies. The data and opinion of those expert with the use of the botanical suggest that astragalus is useful as a complementary treatment during chemotherapy and radiation therapy and in immune deficiency syndromes. There is some modern evidence for its use in hepatitis and the treatment of cardiovascular disease. In TCM and Western clinical herbal medicine, astragalus is most commonly used in combination with other botanicals and is very seldom used as a single agent. There are numerous studies of some of the classic combinations of astragalus (e.g., astragalus and Angelica sinensis). These have not been reviewed, but use of formulas is more consistent with the use of the astragalus than with the use of the herb alone according to traditional Chinese medical principles.

PRECLINICAL STUDIES Pharmacokinetics Pharmacokinetic data available in English language publications on astragalus, its crude extracts, or its constituents are very limited. In the most detailed study to date, the pharmacokinetics of a decoction of astragalus, the preparation most used traditionally were investigated in four models: four complement in silico, a cacao-2 intestinal cell model, an animal, and a human volunteer (n = 1). Intestinal absorption was demonstrated for several flavonoids including calycosin and formononetin, along with their aglycone metabolites in all four 29

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A

B

C

D

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F

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Figure 1

Different forms and quality of astragalus on the American market. Source: Photo courtesy of Roy Upton, Soquel, CA.

models. Triterpene saponins, used as chemical markers of astragalus (e.g., astragaloside I and IV) in the Pharmacopoeia of the People’s Republic of China and the American Herbal Pharmacopoeia, were lacking, likely due to their low concentrations in the preparation. In the human volunteer, nine flavonoids, including calycosin, formononetin, and the isoflavone daidzein, were detected

in urine (7). In animal models (rats and dogs), astragaloside IV, which has demonstrated cardioprotective activity, showed moderate-to-fast elimination. The half-life in male rats was from 67.2 to 98.1 minutes, in female rats 34.0 to 131.6 minutes, and was linear at the intravenous doses given. The highest concentration of astragaloside IV was found in the lungs and liver. Only 50% of the compound was detected in urine and feces. Binding to plasma protein was also linear at the concentration of 250–1000 ng/mL. Slow systemic clearance of astragaloside IV occurred via the liver at approximately 0.004 L/kg/min (8). In another pharmacokinetic study, a twocompartment, first-order pharmacokinetic model was used to describe the pharmacokinetics of intravenousadministered astragaloside IV. Systemic clearance of this triterpene was reported as moderate and distribution into peripheral tissues was limited (9).

Pharmacodynamics

Figure 2 Astragalus flowers. Source: Photo courtesy of Bill Brevoort, American Herbal Pharmacopoeia.

A large percentage of research on astragalus has focused on its immunostimulatory activity and its purported ability to restore the activity of a suppressed immune system. More recently, interest in its potential as a cardioprotective agent has been shown. Reviews of a limited number of clinical trials and preclinical data provide some evidence for its usefulness in the prevention of the common cold and as an adjunct to cancer therapies. There is limited evidence to suggest a benefit to the cardiovascular

Astragalus

OH

O H OH

HO

O O

HO

OH OH

O

OH Astragaloside IV

O OH HO OCH3

O

HO

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Formononetin

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HO

Figure 3

Daidzein

OH

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Some major constituents of Astragalus.

system, with improvement in clinical parameters associated with angina, congestive heart failure, and acute myocardial infarct. There is also some indication from animal studies supporting its use in the treatment of hepatitis and diabetes.

Immunomodulatory Effects There are relatively strong preclinical data of pharmacological mechanisms that provide support for the putative immunomodulatory effects of astragalus.

Cancer In a rat study, animals were pretreated orally for 50 days with a low or high dose of astragalus extract (3.3 or 10 g/kg/day) prior to IP injection of doxorubicin (cumulative dose of 12 mg/kg over a 2-week period). After 5 weeks of the final injection of doxorubicin, a significant inhibition of cardiac diastolic function was observed. This was accompanied by ascites, catexia, decreased heart weight, and increased mortality. Treatment with astragalus at both doses significantly attenuated the negative effects of doxorubicin on cardiac functions and ascites, while the high dose also improved survival. This protective effective was at least partially associated with the ability of astragalus to attenuate changes in cardiac SERCA2a mRNA expression (10). A broad array of immunomodulatory effects has been demonstrated in numerous preclinical studies that

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suggest a substantial value of astragalus in conjunction with conventional cancer therapies. The most relevant of these was a series of investigations conducted by researchers at the MD Anderson Cancer Center that found that astragalus extract restored to normal the immune response of patients’ mononuclear cells that were grafted into rats immunocompromised by cyclophosphamide. These researchers concluded that astragalus and its polysaccharide fraction reversed the immunosuppressive effect of cyclophosphamide (11–15). In other studies, astragalus and its various fractions were shown to stimulate macrophage phagocytosis (16) and hematopoiesis (17). One study reported on the gastroprotective effects of astragalus extract (characterization not reported) in human peritoneal mesothelial cells (HPMCs) subjected to gastric cancer cell lines. Upon incubation with cancer cell lines, apoptosis of the HPMC cells was observed. The astragalus preparation, via regulation of Bcl-2 and Bax, partially inhibited apoptosis. The authors interpreted these findings as a potential that astragalus may slow down the metastasis of the primary cancer and is therefore a potential treatment for gastric cancer (18). The ability of an astragalus fraction to potentiate the effects of rIL-2 has been demonstrated in in vitro assays. Lymphokine-activated killer (LAK) cells were treated with a combination of the astragalus fraction and 100 units/mL of IL-2. The combination therapy produced the same amount of tumor-cell-killing activity as that generated by 1000 units/mL of rIL-2 on its own, thus suggesting that the astragalus fraction elicited a 10-fold potentiation of rIL-2 in this in vitro model (19). These findings were confirmed in a follow-up study by the MD Anderson researchers using LAK cells from cancer and AIDS patients. In this study, the cytotoxicity of a lower dose of 50 ␮g/mL of rIL-2 given with the astragalus fraction was comparable to that of a higher dose of 500 ␮g/mL of rIL-2 alone against the Hs294t melanoma cell line of LAK cells. With the combination, the effector-target cell ratio could be reduced to one-half to obtain a level of cytotoxicity that was equivalent to the use of rIL-2 alone. In addition, the astragalus fraction was shown to increase the responsiveness of peripheral blood lymphocytes that were not affected by rIL-2. In this study, and in another by the same researchers, it was concluded that the fraction potentiated the activity of LAK cells and allowed for the reduction in rIL-2, thus minimizing the toxicity of rIL-2 therapy (20). Other groups of researchers reported almost identical findings (a 10-fold potentiation) and concluded that astragalus is effective in potentiating IL-2-generated LAK cell cytotoxicity in vitro (21,22). Astragalus was also found to enhance the secretion of tumor necrosis factor (TNF) from human peripheral blood mononuclear cells (PBMCs). A polysaccharide fraction (molecular weight 20,000–25,000) increased secretion of TNF-␣ and TNF-␤ after isolation of adherent and nonadherent mononuclear cells from PBMCs (23). Other investigations support the role of astragalus polysaccharides as immunomodulating agents. In an in vitro study, astragalus polysaccharides significantly induced the proliferation of BALB/c mouse splenocytes resulting in subsequent induction of interleukin 1␤ and tumor necrosis factor-␣ and the activation of murine macrophages. The researchers concluded

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that astragalus had an intermediate-to-high affinity for membrane immunoglobulin (Ig) of ␤ lymphocytes (24).

suggests that it elicits significant anti-inflammatory activity and improves ratios and function of T lymphocytes in SLE (37).

Cardiovascular Effects

Viral Infections

In animal studies, astragalus or its compounds were reported to elicit antioxidant (25), mild hypotensive (26), and both positive (27) (50–200 ␮g/mL) and negative (30 ␮g/mL) inotropic activity (28). The inotropic activity was reported to be due to the modulation of Na+ –K+ ATPase in a manner similar to strophanthin K. Antioxidant (29), calcium channel blocking (30), and fibrinolytic activity (31) have been reported in in vitro studies. Astragaloside IV was studied for potential cardioactivity. Various effects have been reported. Intravenous administration of astragaloside IV reduced the area of myocardial infarct and reduced plasma creatine phosphokinase release in dogs subjected to 3-hour ligation and increased coronary blood flow in anesthetized dogs. In isolated rat heart perfusion investigations, astragaloside IV significantly improved (P < 0.01) postischemic heart function and reduced creatine phosphokinase release from the myocardium. In addition, coronary blood flow during baseline perfusion and reperfusion in ischemic rat hearts was increased, while reperfusion damage was decreased. This activity was shown to be at least partially attributable to coronary dilation via an increase in endothelium-derived nitric oxide. Antioxidant activity via an increase in superoxide dismutase (SOD) activity has also been reported for astragalus and is considered to contribute to its cardioprotective effects (32). Astragaloside IV was also shown to significantly attenuate blood–brain barrier permeability in a rat ischemia/reperfusion model (33).

Prophylaxis against flu and modulation of endogenously produced interferon have been reported in several animal studies utilizing astragalus alone (6).

Hepatoprotective Effects Hepatoprotective effects against numerous hepatotoxic agents (e.g., acetaminophen, carbon tetrachloride, and Escherichia coli endotoxin) have been reported in both animal and in vitro studies. In these experiments, improvement in histological changes in hepatic tissue, including fatty infiltration, vacuolar degeneration, and hepatocellular necrosis, was reported. These effects may be associated with saponin fractions (34). In one clinical study of hepatitis B patients, concomitant use of astragalus with lamivudine and ␣-2b interferon showed greater efficacy than with lamivudine alone (35).

Systemic Lupus Erythematosus Astragalus was also studied for its ability to affect natural killer (NK) cell activity, using an enzyme-release assay. The NK cell activity of PBMCs from 28 patients with systemic lupus erythematosus (SLE) was increased after in vitro incubation with an undefined astragalus preparation. Low levels of NK cell activity were correlated with disease activity. PBMCs from patients with SLE had significantly decreased NK cell activity as compared with those from healthy donors. The extent of stimulation by the astragalus preparation was related to the dose and length of the preincubation period (36). Despite its use as an immune-enhancing agent, which would normally be considered contraindicated in autoimmune disorders, investigation of astragalus may be warranted as evidence

Other Effects In a new line of investigation for astragalus, two triterpenes (astragaloside II and isoastragaloside I) were shown to alleviate insulin resistance and glucose intolerance in mice. The two compounds selectively increased adiponectin secretion on primary adipocytes and potentiated the effects of the insulin-sensitizer rosiglitazone. Chronic administration of the compounds (specific details lacking) to both dietary and genetically obese mice resulted in a significant increase in serum adiponectin, resulting in an alleviation of hyperglycemia, glucose intolerance, and insulin resistance. These effects were diminished in mice lacking adiponectin (38). One study showed that a liquid extract of astragalus (2 g/mL/intravenous) retarded the progression of renal fibrosis in a manner similar to the angiotensin-II-receptor antagonist losartan. The study reported that like losartan, astragalus decreased deposition of fibronectin and type-I collagen by significantly reducing the expression of transforming growth factor-␤1 and ␣-smooth muscle actin (P < 0.05) (39). Astragalus was investigated for its potential effect of reducing atopic dermatitis in mice. Using prednisolone (3 mg/kg/day) as a comparator, an astragalus water extract was administered orally at 100 mg/kg. Astragalus significantly reduced the severity of chemically induced inflammation (2,4-dinitrofluorobenzene) to a degree similar to the comparator but, unlike prednisolone, did not inhibit interleukin-4 production (40).

CLINICAL STUDIES There are both English and Chinese language studies on astragalus. As with much of the literature regarding Chinese herbs, there are few clinical data of high methodological quality. In addition, a positive publication bias regarding Chinese literature has been reported (41), while in primary American medical literature, a negative publication bias against dietary supplement studies has been reported (42).

Immunomodulatory Effects Cancer Among modern herbal practitioners, astragalus is recommended as an immune supportive botanical in conjunction with conventional chemo and radiation therapies for cancer. There is a common belief and some clinical and preclinical evidence that astragalus both reduces side effects associated with conventional cancer therapies and can potentiate the effects of certain therapies. The available evidence is not strong enough to recommend astragalus as a standard part of conventional cancer care. However,

Astragalus

its demonstrated safety, lack of negative interaction with conventional therapies, and its putative benefit in building, preserving, and restoring immunocompetency before and after conventional therapies warrant specific study. There is also potential for use of both oral and injectable preparations, the latter of which are not approved in North America but are widely used throughout Asia. In one clinical study, an astragalus drip (20 mL in 250 mL saline solution daily for 84 days) was administered to cancer patients (n = 60). Compared with the control group (no astragalus), those in the astragalus group showed a slower rate of tumor progression, a lower rate of reduction in peripheral leukocytes and platelets, reduction in suppressor CD8s, improved CD4/CD8 ratios, increased IgG and IgM, and better Karnofsky scores (43). In addition to its use alone, both as a primary treatment and as an adjunct to conventional cancer therapies, astragalus is most often combined with other similar acting immune-enhancing plants. A number of randomized prospective clinical studies of cancer patients were conducted using a combination of astragalus and ligustrum (Ligustrum lucidum) (undisclosed quantities) with positive results, such as mortality reduction in breast and lung cancer patients (44). These effects, of course, must be considered to be due to the cumulative effects of the two botanicals and cannot be presumed to occur with astragalus alone but are more consistent with the manner in which astragalus is used in TCM. An early clinical trial reported that 53 cases of chronic leukopenia responded favorably to an astragalus extract (1:1; 2 mL daily intramuscularly for 1–2 weeks). Improvements in symptoms and white blood cell counts were observed, but specific data were lacking (34).

Cardiovascular Effects Various cardioactive properties have been reported for astragalus, and astragalus is widely used in the treatment of both chronic and acute cardiovascular disease in China. In one study, 92 patients with ischemic heart disease were given an unidentified preparation of astragalus. Marked relief from angina pectoris and other improvements as measured by electrocardiogram (ECG) and impedance cardiogram were reported. Improvement in the ECG index was reported as 82.6%. Overall improvement was significant as compared with the control group (P < 0.05) (45). A similar result in cardiac performance was reported by other groups of researchers. In one study, 43 patients were hospitalized within 36 hours of acute myocardial infarct. After administration of an astragalus preparation (undefined profile), the ratio of preejection period/left ventricular ejection time was decreased, the antioxidant activity of SOD of red blood cells was increased, and the lipid peroxidation content of plasma was reduced (46). In another experiment, 20 patients with angina pectoris were given an undefined astragalus preparation. Cardiac output, as measured by Doppler echocardiogram, increased from 5.09 ± 0.21 to 5.95 ± 0.18 L/min 2 weeks after administration of astragalus (P < 0.01). In this study, neither improvement in left ventricular diastolic function nor inhibition of adenosine triphosphate was observed (47). Intravenous administration of astragalus (undefined preparation) was reported to significantly shorten the duration of ventric-

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ular late potentials in cardiac patients (39.8 ± 3.3 ms vs. 44.5 ± 5.9 ms; P < 0.01) (48). In another investigation, astragaloside IV (intravenous; unspecified amount) was given to patients with congestive heart failure for 2 weeks. Improvement in symptoms such as tightness in the chest, difficult breathing, and reduced exercise capacity were reported. Radionuclide ventriculography showed that left ventricular modeling improved and left ventricular end-diastolic and left ventricular end-systolic volume diminished significantly. The authors concluded that astragaloside IV is an effective positive inotropic agent (49), an action supported by others (27).

Hepatoprotective Effects In China, astragalus is widely used in the treatment of chronic hepatitis where reductions in elevated liver enzymes and improvements in symptoms in humans have been reported. This activity is stated to be associated with polysaccharides that increase interferon production (35).

Viral Infections According to one English language review of the Chinese literature, a prophylactic effect against the common cold was reported in an epidemiological study in China involving 1000 subjects. Administration of astragalus, given either orally or as a nasal spray, reportedly decreased the incidence of disease and shortened cold duration. Studies exploring this protective effect found that oral administration of the preparation to subjects for 2 weeks enhanced the induction of interferon by peripheral white blood cells. Levels of IgA and IgG antibodies in nasal secretions were reported to be increased following 2 months of treatment (34). The effect of astragalus on the induction of interferon was studied in a placebo-controlled study involving 28 people. Fourteen volunteers were given an extract equivalent to 8 g of dried root per day and the rest were given placebos. Blood samples were drawn before treatment, then 2 weeks and 2 months after treatment. Interferon production by leukocytes was statistically increased after both time periods (P < 0.01) in the astragalus group but not the control group (50). In another study, astragalus was shown to potentiate the effects of interferon (rIFN-1) in patients with chronic cervicitis (51).

Dosages r Crude root: 9–30 g daily as a decoction (52). r Decoction: 0.5–1 L daily.

SAFETY PROFILE Side Effects None cited in the literature.

Contraindications None cited in the literature.

Precautions There is some evidence to suggest that astragalus and its putative anti-inflammatory effects are beneficial in those with autoimmune conditions such as lupus. However, astragalus should be used cautiously for the treatment of

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autoimmune diseases or in conjunction with immunosuppressive therapies. Because immunostimulating polysaccharides may stimulate histamine release, allergic symptoms may be aggravated by the use of astragalus. This, however, has not been reported in the literature or from clinical use. According to the principles of TCM, astragalus should not be used during acute infectious conditions unless under the care of a qualified TCM practitioner.

Interactions Both positive and negative interactions may occur. Astragalus potentiates the effects of acyclovir (53); IL-2, -20, -21; and rIFN-1 and -2 therapies (50,51). Because of its immunopromoting effects, astragalus may be incompatible with immunosuppressive agents in general.

Pregnancy, Mutagenicity, and Reproductive Toxicity According to one review, astragalus is reported to have no mutagenic effects (54).

Lactation Based on an authoritative review of the available pharmacologic and toxicologic literature, no limitation is to be expected (6,34,54).

Carcinogenicity Studies suggest an anticarcinogenic activity.

Influence on Driving Based on the available pharmacologic and toxicologic literature, no limitation is to be expected (6,34,54).

Overdose and Treatment Specific data are lacking.

Toxicology Based on a review of the available data and the experience of modern practitioners, astragalus can be considered a very safe herb even when taken within its large dosage range. Investigations of specific fractions including flavonoids, polysaccharide, and triterpene similarly show little toxicity (14,34,54).

indications for which astragalus is applied by herbal practitioners. For its use in cancer therapies, there are no definitive guidelines. The modern experience of practitioners together with the limited clinical and preclinical data pointing to an immunomodulatory effect suggests that there may be some value for these indications, including the concomitant use of astragalus to reduce doxorubicininduced immune suppression. However, more specific investigation in this area is needed. Regarding its putative immunomodulating effects, the following mechanisms of action have been proposed: restoration of immune function, increased stem cell generation of blood cells and platelets, lymphocyte proliferation, rise in numbers of antibody-producing spleen cells, potentiation of rIL-2 and rIFN-1 and -2 immunotherapy, enhancement of phagocytic activity by macrophages and leukocytes, and increased cytotoxicity by NK cells. Potential benefits to cardiovascular health, including relief from angina and congestive heart failure and improvement in clinical parameters following acute myocardial infarct, have been reported. Limited animal studies suggest that astragalus enhances coronary blood flow, may potentiate the release of nitric oxide, and potentiates the effects of endogenous antioxidant systems (e.g., SOD). In Asia, astragalus is also used in conjunction with conventional medical treatments for hepatitis. Both animal and in vitro studies offer support for such treatment. As in the use of astragalus in cancer therapies, further clinical trials are required. Though methodologically sound clinical trials for astragalus are generally lacking, natural health practitioners have a generally high regard for its use as a prophylactic against infectious disease and for its ability to build, maintain, and restore immunocompetency when used as a part of conventional cancer therapies. In addition to the very limited number of formal clinical studies that are available in English language sources, the published medical literature on astragalus has to be considered cautiously, as a number of the supporting studies utilize injectable preparations of isolated fractions that are not consistent with the oral use of astragalus supplements. Still, the existing data do support many of the traditional uses for which astragalus has been employed for centuries.

Regulatory Status In the United States, astragalus is regulated as a dietary supplement.

CONCLUSIONS Astragalus is one of the most frequently used herbal medicines throughout Asia and is a very popular botanical used in western herbal supplements. In China, astragalus is used for a myriad of purposes relating to its high regard as a strengthening tonifier, immune modulator, anti-inflammatory, and anti-hepatotoxic. In the West, astragalus figures prominently in immune supportive formulas. Despite its popularity, there are few clinical trials regarding its use. There is some evidence to support the oral administration of astragalus for the prevention of colds and upper respiratory infections, and as an adjunct to conventional cancer therapies. These are very common

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Astragalus

8. Zhang WD, Zhang C, Liu RH, et al. Preclinical pharmacokinetics and tissue distribution of a natural cardioprotective agent astragaloside IV in rats and dogs. Life Sci 2006; 79:808– 815. 9. Zhang WD, Zhang C, Liu RH, et al. Determination of astragaloside IV, a natural product with cardioactivity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry. J Chromatogra B 2005; 822;170– 177. 10. Su D, Li HY, Yan HR, et al. Astragalus improved cardiac function of adriamycin-injured rat hearts by upregulation of SERCA2a expression. Am J Chin Med 2009; 37(3):519–529. 11. Chu DT, Wong WL, Mavligit GM. Immunotherapy with Chinese medicinal herbs I: immune restoration of local xenogeneic graft-versus-host reaction in cancer patients by fractionated Astragalus membranaceus in vitro. J Clin Lab Immunol 1988; 25(3):119–123. 12. Chu DT, Wong WL, Mavligit GM. Immunotherapy with Chinese medicinal herbs II: reversal of cyclophosphamideinduced immune suppression by administration of fractionated Astragalus membranaceus in vivo. J Clin Lab Immunol 1988; 25:125–129. 13. Chu DT, Sun Y, Lin JR. Immune restoration of local xenogeneic graft-versus-host reaction in cancer patients in vitro and reversal of cyclophosphamide-induced immune suppression in the rat in vivo by fractionated Astragalus membranaceus. Chin J Integr Trad West Med 1989; 9(6): 326–354. 14. Chu DT, Lepe-Zuniga J, Wong WL, et al. Fractionated extract of Astragalus membranaceus, a Chinese medicinal herb, potentiates LAK cell cytotoxicity generated by low dose of recombinant interleukin-2. J Clin Lab Immunol 1988; 26(3):183–187. 15. Shimizu N, Tomoda M, Kanari M, et al. An acidic polysaccharide having activity on the reticuloendothelial system from the root of Astragalus mongholicus. Chem Pharm Bull 1991; 39(11):2969–2972. 16. Tomoda M, Shimuzu N, Ohara N, et al. A reticuloendothelial system-activating glycan from the roots of Astragalus membranaceus. Phytochemistry 1992; 31(1):63–66. 17. Rou M, Renfu X. The effect of Radix Astragali on mouse marrow hemopoiesis. J Tradit Chin Med 1983; 3(3):199–204. 18. Na D, Liu FN, Miao ZF, et al. Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by antiapoptosis. World J Gastroenterol 2009; 15(5):570–577. 19. Chu DT, Sun Y, Lin JR, et al. F3, a fractionated extract of Astragalus membranaceus, potentiates lymphokine-activated killer cell cytotoxicity generated by low dose recombinant interleukin-2. Chin J Integr Trad West Med 1990; 10(1):34–36. 20. Chu DT, Lin JR, Wong WL. The in vitro potentiation of LAK cell cytotoxicity in cancer and AIDS patients induced by F3, a fractionated extract of Astragalus membranaceus. Chung Hua Chung Liu Tsa Chih 1994; 16(3):167–171. 21. Wang Y, Qian XJ, Hadley HR, et al. Phytochemicals potentiate interleukin-2 generated lymphokine-activated killer cell cytotoxicity against murine renal cell carcinoma. Mol Biother 1992; 4(3):143–146. 22. Zhou S, Lu Z, Wang Y, et al. Study on the antineoplastic activity of astragalus polysaccharide. Yao Wu Sheng Wu Ji Shu 1995; 2(2):22–25. 23. Zhao KW, Kong HY. Effect of astragalan on secretion of tumor necrosis factors in human peripheral blood mononuclear cells. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1993; 13(5):263–265. 24. Shao BM, Xu W, Dai H, et al. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochem Biophys Res Commun 2004; 320;1103–1111. 25. Lei C, Yue H, Chen Y, et al. Effects of astragalus saponins on ischemic scope, epicardial ECG, myocardial enzymes in

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acute myocardial infarcted dog heart. Baiqiuen Yike Daxue Xuebao 1995; 21(2):111–113. Hikino H, Funayama S, Endo K. Hypotensive principle of astragalus and hedysarum roots. Planta Med 1976; 30:297– 302. Zhong G, Jiang Y, Wei Y, et al. Positive inotropic action of Astragalus membranaceus saponins on isolated working heart. Baiqiuen Yike Daxue Xuebao 1994; 20(5):448–449. Wang Q, Li Y, Qi H, et al. Inotropic action of Astragalus membranaceus Bunge saponins and its possible mechanism. Zhongguo Zhongyao Zazhi 1993; 17(9):557–559. Sun C, Zhong G, Zhan S, et al. Study on antioxidant effect of astragalus polysaccharide. Zhongguo Yaolixue Tongbao 1996; 12(2):161–163. Guo Q, Peng T, Yang Y, et al. Effect of drugs on Ca2+ influx and CVB3-RNA replication in cultured rat heart cells infected with CVB3. Virol Sin 1996; 11(1):40–44. Zhang WJ, Wojta J, Binder BR. Regulation of the fibrinolytic potential of cultured human umbilical vein endothelial cells: astragaloside IV down regulates plasminogen activator inhibitor-1 and up regulates tissue-type plasminogen activator expression. J Vasc Res 1997; 34(4):273– 280. Zhang WD, Chen H, Zhang C, et al. Astragaloside IV from Astragalus membranaceus shows cardioprotection during myocardial ischemia in vivo and in vitro. Planta Med 2006; 72:4–8. Qu YZ, Li M, Zhao YL, et al. Astragaloside IV attenuates cerebral ischemia-reperfusion-induced increase in permeability of the blood brain barrier in rats. Eur J Pharmacol 2009; 606:137–141. Chang HM, But P. Pharmacology and Applications of Chinese Materia Medica. Singapore: World Scientific, 1987. Wu L, Liu H, Xue P, et al. Influence of a triplex superimposed treatment on HBV replication and mutation during treating chronic hepatitis B. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2001; 15(3):236–238. Zhao XZ. Effects of Astragalus membranaceus and Tripterygium hypoglaucum on natural killer cell activity of peripheral blood mononuclear in systemic lupus erythematosus. Zhongguo Zhong Xi Yi Jie He Za Zhi 1992; 12(11):645, 669– 671. Pan HF, Fang XH, Li WX, et al. Radix Astragali: A promising new treatment option for systemic lupus erythematosus. Med Hypothesis 2008; 71(2)311–312. Xu A, Wang HB, Hoo RLC, et al. Selective elevation of adiponectin production by the natural compounds derived from a medicinal herb alleviates insulin resistance and glucose intolerance in obese mice. Endocrinology 2009; 150(2):625–633. Zuo C, Xie XS, Qiu HY, et al. Astragalus mongholicus ameliorates renal fibrosis by modulating HGF and TGF in rats with unilateral ureteral obstruction. J Zhejiang Univ Sci B 2009; 10(5):380–390. Lee SJ, Oh SG, Seo SW, et al. Oral administration of Astragalus membranaceus inhibits the development of DNFBinduced dermatitis in NC/Nga mice. Biol Pharm Bull 2007; 30(8):1468–1471. Vickers A, Goyal N, Harland R, et al. Do certain countries produce only positive results? A systematic review of controlled trials. Controlled Clin Trials 1998; 19:159–166. Kemper KJ, Hood KL. Does pharmaceutical advertising affect journal publication about dietary supplements? BMC Complement Altern Med 2008; 8(11):1–8. Duan P, Wang ZM. Clinical study on effect of astragalus in efficacy enhancing and toxicity reducing of chemotherapy in patients of malignant tumors. Zhongguo Zhong Xi Yi Jie He Za Zhi 2002; 22(7):515–517.

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44. Morazzoni P, Bombardelli P. Astragalus membranaceus (Fisch) Bunge; Scientific Documentation 30. Milan, Italy: Indena SpA, 1994;1–18. 45. Li SQ, Yuan RX, Gao H. Clinical observation on the treatment of ischemic heart disease with Astragalus membranaceus. Kuo Chung Hsi I Chieh Ho Tsa Chih 1995; 15(2):77–80. 46. Chen LX, Liao JZ, Guo WQ. Astragalus membranaceus on left ventricular function and oxygen free radical in acute myocardial infarction patients and mechanism of its cardiotonic action. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995; 15(3):141–143. 47. Lei ZY, Qin H, Liao JZ. Action of Astragalus membranaceus on left ventricular function of angina pectoris. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1994; 14(4):199–202. 48. Shi HM, Dai RH, Wang SY. Primary research on the clinical significance of ventricular late potentials (VLPs), and the impact of mexiletine, lidocaine, and Astragalus membranaceus on VLPs. Chung Hsi I Chieh Ho Tsa Chih 1991; 11(5): 265–267.

49. Luo HM, Dai RH, Li Y. Nuclear cardiology study on effective ingredients of Astragalus membranaceus in treating heart failure. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995; 15(12):707–709. 50. Hou Y, Zhang Z, Su S, et al. Interferon induction and lymphocyte transformation stimulated by Astragalus membranaceus in mouse spleen cell cultures. Zhonghua Weisheng Wuxue Hemian Yixue Zazhi 1981; 1(2):137–139. 51. Qian ZW, Mao SJ, Cai XC, et al. Viral etiology of chronic cervicitis and its therapeutic response to ␣-recombinant interferon. Chin Med J 1990; 103:647–651. 52. Radix Astragali (huangqi). Pharmacopoeia of the People’s Republic of China. Vol 1. Beijing, China: Chemistry and Industry Press, 1997:442. 53. Zuo L, Dong X, Sun X. The curative effects of Astragalus membranaceus Bunge (A-6) in combination with acyclovir on mice infected with HSV-1. Virol Sin 1995; 10(2):177–179. 54. Wagner H, Bauer R, Peigen X, et al. Radix Astragali [Huang Qi]. Chin Drug Monogr Anal 1997; 1(8):18.

Bilberry Marilyn Barrett

used to treat dysentery, diarrhea, gastrointestinal inflammation, hemorrhoids, vaginal discharges, scurvy, urinary complaints, and to dry up breast milk. More recently, it was found that bilberry was used by World War II pilots to improve their night vision (2,3). Bilberry fruit preparations are still used to improve vision as well as for their benefits to the circulatory system: treating fragility and altered permeability of blood vessels that is either primary or secondary to arterial hypertension, arteriosclerosis, or diabetes (3).

INTRODUCTION Bilberry, Vaccinium myrtillus L., is a shrub with edible fruits that is native to Circumboreal regions from Europe to Asia as well as the Rocky Mountains in North America. Bilberries are related to other edible berries including blueberry, cranberry, huckleberry, and lingonberry. Bilberry fruits contain anthocyanins, which are natural pigments, responsible for the dark blue color of the fruits and for many of the health benefits. In vitro studies have shown that bilberry extracts have antioxidant activity, inhibit platelet aggregation, prevent degradation of collagen in the extravascular matrix surrounding blood vessels and joints, and have a relaxing effect on arterial smooth muscle. Bilberry extracts have also demonstrated anticancer and antibacterial actions, in vitro. Pharmacokinetic studies in animals and humans show that a small percentage of the anthocyanins is absorbed into the body and widely distributed. Human clinical studies have been conducted evaluating the potential benefits of bilberry preparations in treating venous insufficiency and visual disorders ranging from night vision to diabetic retinopathy as well as cancer prevention. No serious toxicities have been associated with preparations of the fruits in animal screens and no serious side effects have been identified in humans.

CHEMISTRY AND PREPARATION Bilberry fruits contain anthocyanins that are natural pigments in the chemical class known as flavonoids. Anthocyanins are glycosides or compounds with sugars attached at the 3 position, while anthocyanidins are aglycones (the same basic structure without the sugars attached) (see chapter 74, “Polyphenol Overview”). The majority (64%) of anthocyanins in the fruit are glycosides of cyanidin and delphinidin (Fig. 1). The quantity of anthocyanin in the fruit ranges from 300 to 700 mg per 100 g. Bilberry fruits also contain flavonols, tannins, phenolic acids, organic acids, sugars, vitamins, and volatile compounds (2). The primary commercial source of bilberry fruits is “wild harvest” from regions in Europe and Scandinavia. The fruits are sold fresh, frozen, or dried. Besides the whole fruit, commercial products include cold macerates, decoctions, and dry extracts. The dry extracts are commonly prepared using alcohol, methanol or ethanol (2). Until recently, a single-wavelength spectrophotometric technique (UV) was commonly used to standardize the anthocyanin content of bilberry products. However, this technique did not detect adulteration of bilberry preparations with substances of similar color (4). A highperformance liquid chromatographic technique that can detect and quantitate both anthocyanins and anthocyanidins has recently been developed enabling a better assurance of product identity and quality (5). Most studies on bilberry have been conducted using extracts characterized as containing 36% anthocyanins or 25% anthocyanidins.

BACKGROUND Bilberries are edible fruits from V. myrtillus L. of the family Ericaceae. Bilberry is the standardized common name for the fruit in the United States, but the fruit is also known as European blueberry, huckleberry, and whortleberry (1). Related to bilberry, and in the same genus of Vaccinium, are other edible berries including blueberry, cranberry, huckleberry, and lingonberry. Bilberry is a shrub, 1–6 dm high, found in heaths, meadows, and moist coniferous forests in Circumboreal regions from Europe to Asia, with populations in the American and Canadian Rocky Mountains (2). The blue-black berries are harvested when ripe, usually during the months of July through September. The berries are oblate-globose, 5–9 mm diameter, with 4–5 locules containing many seeds. The seeds are approximately 1 mm long with a yellow/brown-dimpled surface (2). Both the leaves and fruits of bilberry have been used medicinally since the Middle ages. The leaves were used topically for inflammation, infections, and burns, as well as internally as a treatment for diabetes. According to the herbalist Grieve, the fruits were

PRECLINICAL STUDIES In vitro studies have shown that bilberry extracts have antioxidant activity, inhibit platelet aggregation, prevent 37

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Barrett

R R1 +

HO

O

R2 O R3

OH R OH

R1 R2 OH H

R3 arabinose, glucose, or galactose

Delphinidin 3-O-glycoside OH

OH OH

arabinose, glucose, or galactose

Cyanidin 3-O-glycoside Malvidin 3-O-glycoside

OCH3 OH OCH3 arabinose, glucose, or galactose

Peonidin 3-O-glycoside

OCH3 OH H

Petunidin 3-O-glycoside

OH

Figure 1

arabinose, glucose, or galactose

OH OCH3 arabinose, glucose, or galactose

duced by potassium bromate. Oral administration of the same extract ameliorated the increase in blood urea nitrogen levels and the decreases in kidney malondialdehyde, nitric oxide, and xanthine oxidase levels. The bilberry extract also improved the kidney ORAC levels (11). MyrtoSelectTM , an extract containing approximately 40% anthocyanins, was tested for its effects on gene expression (DNA microarray) in a macrophage cell line stimulated with lipopolysaccharide (LPS). The extract, at a concentration of 75 ␮g/mL, appeared to mitigate the effect of LPS, targeting genes involved in inflammation and immune defense. Pretreatment with the bilberry extract affected 45% of the genes downregulated by LPS and 36% of genes upregulated by LPS (12).

Chemical structures of anthocyanins.

Circulation degradation of collagen in the extravascular matrix surrounding blood vessels and joints, and have a relaxing effect on arterial smooth muscle. These actions are vasoprotective, increasing capillary resistance and reducing capillary permeability (3). Bilberry extracts have also demonstrated anticancer and antibacterial actions in vitro. There is no evidence of toxicity in animals at the effective doses.

Antioxidant Activity Bilberry fruits have demonstrated antioxidant activity in in vitro and in animal models. In the oxygen radical absorbance capacity (ORAC) assay, an in vitro test measuring free radical quenching, bilberry fruits had potent activity compared with other fresh fruits and vegetables (44.6 + 2.3 ␮mol Trolox equivalents (TE)/g) (6). In another assay, a bilberry extract (25% anthocyanins) exhibited antioxidant activity in protecting keratinocytes in culture from damage due to UVA and UVB light (7,8). The bilberry extract attenuated UVA-induced reactive oxygen species formation, peroxidation of membrane lipids, and depletion of intracellular glutathione in concentrations of 10–50 mg/L (7). In the same concentration range, the extract inhibited UVB-induced generation of reactive oxygen and nitrogen species, DNA strand breaks, as well as caspase3 and capase-9 activity (mediators that execute apoptotic cell death) (8). In an animal study, a bilberry extract characterized as containing 38% anthocyanins reduced oxidative stress caused in mice by removal of the animal’s whiskers. The extract administrated orally at a dose of 100 mg/kg for 7 days ameliorated the increase in oxygen radicals (thiobarbituric acid reactive substances), protein carbonyl formation, and lipid peroxidation in the brain, heart, kidney, and liver. The extract also suppressed the stress-induced changes in dopamine levels (9). A bilberry extract (42% anthocyanins) reduced oxidative stress to the liver in a restraint-stress mouse model. The extract, administered at a dose of 200 mg/kg for 5 days, ameliorated the increase in plasma levels of alanine aminotransferase, a liver enzyme. The extract also reduced plasma and liver ORAC levels and increased plasma glutathione and vitamin C levels in the liver (10). A similar study was conducted in mice with kidney damage in-

Bilberry extracts have demonstrated beneficial effects on the circulation, including inhibiting platelet aggregation, reducing capillary permeability, facilitating vasodilation, and inhibiting the development of atherosclerosis and angiogenesis. R Myrtocyan (also known as MirtoSelectTM , containing 36% anthocyanins) inhibited platelet aggregation in vitro induced by adenosine diphosphate (ADP), collagen, and sodium arachidonate in rabbit platelet-rich plasma with IC50 values ranging from 0.36 to 0.81 mg/mL. Myrtocyan administered orally to rats (400 mg/kg) prolonged the bleeding time in the animals, without affecting coagulation pathways. The same dose administered to mice reduced the adhesiveness of platelets to glass (3). Myrtocyan administered to healthy human subjects, 480 mg/day (173 mg anthocyanins/day) for 30–60 days, reduced the aggregation response ex vivo to ADP and collagen (13). In a rabbit skin model, oral treatment with 400 mg anthocyanins per kilogram body weight 30 minutes before topical application of chloroform reduced the capillary permeability caused by the irritant by 66%. In rats, administration of bilberry anthocyanins, 200 mg/kg orally, decreased bradykinin-induced capillary permeability by 39%. The same dose reduced carrageenin-induced rat paw edema by 45% (14). In a rat model of experimentally induced hypertension, 500 mg anthocyanins per kilogram body weight given orally for 12 days completely ameliorated the increase in blood–brain barrier permeability and reduced the increase in aortic vascular permeability by 40% (15). In a hamster cheek pouch model, 100 mg bilberry extract per kilogram daily for 4 weeks reduced the circulatory damage due to ischemic reperfusion (16). A rat model suggested that bilberry anthocyanins (50 mg/kg IP) inhibited the enzymatic degradation of collagen, decreasing the permeability of the blood–brain barrier caused by proteases (17). Bilberry preparations are reported to relax arterial tissues in vitro. Preliminary experiments pointed to a mechanism involving prostaglandins. However, a more recent study using porcine coronary arteries demonstrated a mechanism involving nitric oxide (endothelialderived relaxing factor) (18). A bilberry extract was reported to inhibit the development of atherosclerosis in apolipoprotein E–deficient mice. The mice received diets supplemented with 0.02% of a bilberry extract (52% anthocyanins) for 16 weeks. The

Bilberry

extract reduced lipid deposits and the development of lesions. It did not affect plasma antioxidant capacity or plasma lipid levels (19). A bilberry extract (25% anthocyanins) was tested for its effect on angiogenesis both in vitro and in vivo. The extract at concentrations of 0.3–30 ␮g/mL inhibited tube formation and the migration of human umbilical vein endothelial cells induced by vascular endothelial growth factor A. The extract also inhibited the induction of retinopathy in newborn mice, which was induced with oxygen. Intravitreal administration of 300 ng extract per eye significantly inhibited the area of neovascular tufts (20).

Anticancer Anthocyanins have been reported to mediate several physiological functions that ultimately may result in cancer suppression. Anthocyanins suppress the growth of cancer cell lines in vitro, including HL60 human leukemia calls and HCT116 human colon cancer cells. A bilberry extract induced apoptotic cell bodies and nucleosomal DNA fragmentation in HL60 cells (21). A bilberry extract (Mirtocyan) has also been shown to suppress the activity of receptor tyrosine kinases, which are thought to play a crucial role in carcinogenesis and tumor progression. When tested over a number of tyrosine kinases, the activity was consistent but not specific (22).

Antibacterial Phenolic compounds in bilberry have demonstrated in vitro antimicrobial effects against strains of Salmonella and Staphylococcus possibly through interfering with adhesion of the bacteria. Treatment of bilberry preparations with pectinase released phenolics from the cell wall matrix and increased the antibacterial activity (23). In experiments with Neisseria meningitidis, the bacteria that causes meningitis and septicemia, a bilberry juice fraction was reported to inhibit the binding of the bacteria to epithelial cells in culture. Fractions of the juice also bound to the bacterial pili. The authors concluded that anthocyanins were partly responsible for the activity but that there appeared to be other compounds in bilberry that may also interact directly with the pili or act synergistically with the anthocyanins (24).

Safety Studies (Animal Toxicology) Myrtocyan (25% anthocyanins) has been tested for acute and chronic toxicity in animal studies. There were no deaths with an acute dose in rats up to 20 g/kg orally and in mice up to 25 g/kg. Six months treatment with doses of 125–500 mg/kg in rats and 80–320 mg/kg in dogs found no evidence of toxicity. The preparation was tested in guinea pigs for 2 weeks and in rats for 6 weeks with doses up to 43 mg/kg without incident (2,3).

PHARMACOKINETICS Animal studies show that bilberry anthocyanins are absorbed intact, or after methylation. This is unlike other flavonoid glycosides which are hydrolyzed to their aglycones and metabolized to glucuronidated or sulfated

39

derivatives (25). Following administration of 400 mg/kg orally to rats, peak blood levels of anthocyanins were detected within 15 minutes and afterwards declined rapidly. Only 1% of the anthocyanins was eliminated in the urine and 4% in the bile. The absolute bioavailability of bilberry anthocyanins was estimated to be 1.2–5% (26). A study in mice reported that malvidin 3-glucoside and malvidin 3galactoside were the principal anthocyanins in the plasma 60 minutes after oral administration of 100 mg/kg. When the mice were maintained on a diet containing 0.5% bilberry extract, plasma levels of anthocyanins reached 0.26 ␮M. Anthocyanidins were detected in the liver, kidney, and lung. They were not detected in the spleen, thymus, heart, muscle, brain, white fat, or eyes (25). A pharmacokinetic study with six human subjects detected anthocyanins in the plasma 1.5–6 hours following intake of a bilberry–lingonberry puree. The study examined the production of urinary phenolic acids and found the greatest increase in methylated compounds. The amount of urinary phenolic acids was low, and the authors suggested that the fragmentation of anthocyaninins to phenolic acids was not a major metabolic pathway (27). Another pharmacokinetic study with 20 subjects that consumed 100 g/day of berries, including black currant, lingonberries, and bilberries, for 8 weeks reported an increase in serum quercetin (up to 51% higher) compared with control subjects who did not consume berries (28). A study with 25 subjects administered 1.4–5.6 g Mirtocyan (25% anthocyanins) daily for 7 days reported detection of anthocyanins as well as methyl and glucuronide metabolites in the plasma and urine but not in the liver (29).

CLINICAL STUDIES Human clinical studies have been conducted evaluating the potential benefits of bilberry preparations in treating venous insufficiency and visual disorders ranging from night vision to diabetic retinopathy as well as cancer prevention.

Vascular Health Clinical studies have been conducted evaluating the potential benefits of bilberry preparations in treating venous insufficiency. A review of studies conducted between 1970 and 1985 included 568 patients with venous insufficiency of the lower limbs who were treated with bilberry preparations (30). The studies reported an improvement in circulation and in lymph drainage resulting in a reduction in edema. A more recent placebo-controlled study which included 60 participants with varicose veins reported improvement in edema in the legs and ankles, sensation of pressure, cramps, and tingling or “pins and needles” senR sations with a dose of 160 mg Tegens , three times daily for 1 month (31). Tegens (Inverni della Beffa, Italy) contains a bilberry extract named Myrtocyan or MirtoSelect (25% anthocyanins), manufactured by Indena SpA, Italy.

Visual Health A systematic review was conducted on placebo-controlled studies on the effects of bilberry preparations on night

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vision. Literature searches identified 30 clinical studies, and 12 of those met the inclusion criteria of being placebo controlled. Of the 12 studies, 5 were randomized. Healthy subjects with normal or above average eyesight were tested in 11 out of the 12 studies. Many of the studies were acute, using a single dose, and the longest treatment period was 28 days. Full characterization of the products used in the studies was not available, but assuming 25% anthocyanin content, the doses of anthocyanin ranged from 12 to 2880 mg. The techniques used to measure the extent and rate of dark adaptation ranged from visual acuity, contrast sensitivity, and critical flicker fusion to electroretinographic monitoring of response to light flashes. The four most recent randomized controlled studies with rigorous methodology reported negative results. One randomized controlled study and all seven of the nonrandomized studies reported positive effects. The authors concluded that the present studies do not support the use of bilberry by those who are healthy with normal vision to improve their night vision. However, uncontrolled studies report a benefit for those with eye disorders, including retinal degeneration, myopia, simple glaucoma, and pathological fundus. Furthermore, studies with synthetic anthocyanins suggest a positive benefit for those with myopia, central retinal lesions, and night blindness (32). Two studies on diabetic retinopathy, using a dose of 160 mg Tegens twice daily, demonstrated a trend toward improvement in mild cases of the disease. The first study was a 1-month, placebo-controlled study that included 36 subjects. At the end of the month, 10 of 13 patients in the Tegens group with ophthalmoscopically detectable retinal abnormalities (microaneurisms, hemorrhagic foci, exudates) were improved, while all 15 patients with these abnormalities in the placebo group remained unchanged. A similar trend was observed among those patients with fluoroangiographic abnormalities (33). The second study lasted 1 year and included 40 subjects who were given Tegens or placebo in addition to the usual therapy for retinopathy. As a result, in 50% of patients given bilberry, the retinal lesions and associated edema were improved compared with 20% in the control group (34). A mixture of vitamin E and bilberry (FAR-1, Ditta Farmigea SpA, Italy) showed a trend toward prevention of senile cataracts after 4 months of 180 mg bilberry anthocyanins (25% anthocyanidins) and 100 mg DLtocopheryl acetate twice daily. When the placebo group was changed from placebo to the bilberry preparation, and the trial continued for an additional 4 months, there was no statistical difference between the two groups. The rationale for this study was previous indications that antioxidants might prevent the development of senile cataracts (35). A mechanistic study using Myrtocyan examined changes in pupillary reflexes to light following a single high dose of 240 mg anthocyanosides or placebo in 40 healthy volunteers. The study was conducted to explore the use of bilberry in work situations where exposure to high light intensities dampens pupillary reflexes and leads to vision fatigue. The authors of the study suggested that the pigments in bilberry might increase sensitivity to light and improve blood flow in the capillaries of the eye. Improvement in pupillary reflexes was observed

in both groups, with the improvement in the treatment group being only slightly better than that in the placebo group (36).

Cancer Prevention In an open label study, 25 colorectal cancer patients scheduled to undergo surgery were given 1.4, 3.8, or 5.6 g bilberry extract (Mirtocyan) containing 0.2–2.0 g anthocyanins for 7 days before surgery. Availability of anthocyanins was determined by detection in the plasma, colorectal tissue, and urine but not in the liver. Anthocyanins detected in the body were unaltered, or products of metabolic glucuronidation and O-methylation. Proliferation of cells in the tumor tissue was decreased by 7% compared with before the bilberry intervention (29).

Side Effects and Adverse Effects No side effects were reported in the clinical studies mentioned earlier. In a 1987 postmarketing surveillance study with 2295 subjects, only 94 (4.1%) complained of minor side effects, most of which involved the gastrointestinal track. Most of the participants took 160 mg Tegens twice daily for 1–2 months (3).

Observed Drug Interactions and Contraindications No drug interactions or contraindications have been reported in the literature for bilberry.

CONCLUSIONS Bilberry fruit extracts and anthocyanins have been the subject of pharmacological studies and human clinical trials. In vitro and in vivo studies demonstrate good evidence for the antioxidant activity of bilberry extracts along with strong indications of benefit to the cardiovascular system. Animal and human pharmacokinetic studies demonstrate bioavailability of anthocyanins, but absorption appears to be limited. Human clinical studies on the effects of bilberry extracts on eyesight and vascular diseases suffer from poor methodology, including small sample sizes and short-term exposures. While it appears doubtful that bilberry preparations benefit the night vision of healthy subjects, the benefit for those with diabetic retinopathy and other eye disorders merits exploration. Another area that appears promising is that of benefits to the cardiovascular system, specifically vasculitis or venous insufficiency. Bilberry products have been safely consumed, without significant adverse events or side effects.

REGULATORY STATUS Bilberry is a food and preparations are also used medicinally. In the United States, preparations of bilberry are sold as foods and dietary supplements. The U.S. Pharmacopoeia has published a standard monograph for powdered bilberry extract (37). The German Commission E completed a monograph for bilberry fruits in which preparations of the ripe fruit are indicated orally for

Bilberry

nonspecific, acute diarrhea and topically for mild inflammation for the oral and pharyngeal mucosa (38). The European Scientific Cooperative on Phytotherapy (ESCOP) monograph lists the internal use of bilberry fruit preparations (enriched in anthocyanins) for symptomatic treatment of problems related to varicose veins, such as painful and heavy legs. The ESCOP monograph also lists the dried fruit as supportive treatment of acute, nonspecific diarrhea (39). In Canada, bilberry products are approved as natural health products for traditional use orally as an astringent and as a source of antioxidants as well as for use as a gargle to relieve mild inflammation of the mouth and/or throat (40).

14.

15.

16.

17.

18.

REFERENCES 1. McGuffin M, Kartesz J, Leung A, et al. American Herbal Products Association’s Herbs of Commerce. 2nd ed. Silver Spring, MD: American Herbal Products Association, 2000. 2. Upton R, Graff A, L¨anger R, et al. Bilberry fruit, Vaccinium myrtillus L. Standards of analysis, quality control, and therapeutics. In: American Herbal Pharmacopoeia and Therapeutic Compendium. Santa Cruz, CA: American Herbal Pharmacopoeia, 2001. 3. Morazzoni P, Bombardelli E. Vaccinium myrtillus L. Fitoterapia 1996; 67(1):3–29. 4. Penman KG, Halstead CW, Matthias A, et al. Bilberry adulteration using the food dye amaranth. J Agric Food Chem 2006; 54(19):7378–7382. 5. Cassinese C, de Combarieu E, Falzoni M, et al. New liquid chromatography method with ultraviolet detection for analysis of anthocyanins and anthocyanidins in Vaccinium myrtillus fruit dry extracts and commercial preparations. J AOAC Int 2007; 90(4):911–919. 6. Prior R, Gao G, Martin A, et al. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. J Agric Food Chem 1998; 46(7):2686–2693. 7. Svobodova A, Rambouskova J, Walterova D, et al. Bilberry extract reduces UVA-induced oxidative stress in HaCaT keratinocytes: a pilot study. Biofactors 2008; 33(4):249– 266. 8. Svobodova A, Zdarilova A, Vostalova J. Lonicera caerulea and Vaccinium myrtillus fruit polyphenols protect HaCaT keratinocytes against UVB-induced phototoxic stress and DNA damage. J Dermatol Sci 2009; 56(3):196–204. 9. Rahman MM, Ichiyanagi T, Komiyama T, et al. Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status. J Agric Food Chem 2008; 56(16):7545–7550. 10. Bao L, Yao XS, Yau CC, et al. Protective effects of bilberry (Vaccinium myrtillus L.) extract on restraint stress-induced liver damage in mice. J Agric Food Chem 2008; 56(17):7803– 7807. 11. Bao L, Yao XS, Tsi D, et al. Protective effects of bilberry (Vaccinium myrtillus L.) extract on KBrO3-induced kidney damage in mice. J Agric Food Chem 2008; 56(2):420– 425. 12. Chen J, Uto T, Tanigawa S, et al. Expression profiling of genes targeted by bilberry (Vaccinium myrtillus) in macrophages through DNA microarray. Nutr Cancer 2008; 60(suppl 1):43– 50. 13. Pulliero G, Montin S, Bettini V. Ex vivo study of the inhibitory effects of Vaccinium myrtillus anthocyanosides

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on human platelet aggregation. Fitoterapia 1989; 60(1): 69–75. Lietti A, Cristoni A, Picci M. Studies on Vaccinium myrtillus anthocyanosides. I. Vasoprotective and antiinflammatory activity. Arzneimittelforschung 1976; 26(5):829– 832. Detre Z, Jellinek H, Miskulin M, et al. Studies on vascular permeability in hypertension: action of anthocyanosides. Clin Physiol Biochem 1986; 4(2):143–149. Bertuglia S, Malandrino S, Colantuoni A. Effect of Vaccinium myrtillus anthocyanosides on ischaemia reperfusion injury in hamster cheek pouch microcirculation. Pharmacol Res 1995; 31(3–4):183–187. Robert A, Godeau G, Moati F, et al. Action of anthocyanosides of Vaccinium myrtillus on the permeability of the blood brain barrier. J Med 1977; 8(5):321–322. Bell DR, Gochenaur K. Direct vasoactive and vasoprotective properties of anthocyanin-rich extracts. J Appl Physiol 2006; 100(4):1164–1170. Mauray A, Milenkovic D, Besson C, et al. Atheroprotective effects of bilberry extracts in apo E-deficient mice. J Agric Food Chem 2009; 57(23):11106–11111. Matsunaga N, Chikaraishi Y, Shimazawa M, et al. Vaccinium myrtillus (bilberry) extracts reduce angiogenesis in vitro and in vivo. Evid Based Complement Alternat Med 2010; 7(1):47–56. Katsube N, Iwashita K, Tsushida T, et al. Induction of apoptosis in cancer cells by bilberry (Vaccinium myrtillus) and the anthocyanins. J Agric Food Chem 2003; 51(1):68– 75. Teller N, Thiele W, Marczylo TH, et al. Suppression of the kinase activity of receptor tyrosine kinases by anthocyaninrich mixtures extracted from bilberries and grapes. J Agric Food Chem 2009; 57(8):3094–3101. Puupponen-Pimia R, Nohynek L, Ammann S, et al. Enzymeassisted processing increases antimicrobial and antioxidant activity of bilberry. J Agric Food Chem 2008; 56(3):681– 688. Toivanen M, Ryynanen A, Huttunen S, et al. Binding of Neisseria meningitidis pili to berry polyphenolic fractions. J Agric Food Chem 2009; 57(8):3120–3127. Sakakibara H, Ogawa T, Koyanagi A, et al. Distribution and excretion of bilberry anthocyanines in mice. J Agric Food Chem 2009; 57(17):7681–7686. Morazzoni P, Livio S, Scilingo A, et al. Vaccinium myrtillus anthocyanosides pharmacokinetics in rats. Arzneimittelforschung 1991; 41(2):128–131. Nurmi T, Mursu J, Heinonen M, et al. Metabolism of berry anthocyanins to phenolic acids in humans. J Agric Food Chem 2009; 57(6):2274–2281. Erlund I, Marniemi J, Hakala P, et al. Consumption of black currants, lingonberries and bilberries increases serum quercetin concentrations. Eur J Clin Nutr 2003; 57(1): 37–42. Thomasset S, Berry DP, Cai H, et al. Pilot study of oral anthocyanins for colorectal cancer chemoprevention. Cancer Prev Res (Phila Pa) 2009; 2(7):625–633. Berta V, Zucchi C. Fitoterapia 1988; 59(suppl 1):27. Gatta L. Vaccinium myrtillus anthocyanosides in the treatment of venous stasis: controlled clinical study on sixty patients. Fitoterapia 1988; 59(suppl 1):19–26. Canter PH, Ernst E. Anthocyanosides of Vaccinium myrtillus (bilberry) for night vision—a systematic review of placebo-controlled trials. Surv Ophthalmol 2004; 49(1): 38–50. Perossini M, Chiellini S, Guidi G, et al. Diabetic and hypertensive retinopathy therapy with Vaccinium myrtillus anthocyanosides (Tegens) double-blind placebo-controlled

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clinical trial. Ann Ottalmol Clin Ocul 1987; 113(12):1173– 1190. 34. Repossi P, Malagola R, De Cadilhac C. The role of anthocyanosides on vascular permeability in diabetic retinopathy. Ann Ottalmol Clin Ocul 1987; 113(4):357–361. 35. Bravetti G. Preventive medical treatment of senile cataract with vitamin E and Vaccinium myrtillus anthocyanosides: clinical evaluation. Ann Ottalmol Clin Ocul 1989; 115(2):109– 116. 36. Vannini L, Samuelly R, Coffano M, et al. Study of the pupillary reflex after anthocyanoside administration. Boll Ocul 1986; 65(suppl 6):569–577.

37. United States Pharmacopoeial Convention. Powdered Bilberry Extract (USP 32 NF 27). 2008:964. 38. Blumenthal M, Busse W, Hall T, et al. The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines. Austin, TX: American Botanical Council, 1998. 39. European Scientific Cooperative on Phytotherapy (ESCOP). ESCOP Monographs: The Scientific Foundation for Herbal Medicinal Products. 2nd ed. Exeter, UK: European Scientific Cooperative on Phytotherapy, 2003. 40. Health Canada Natural Health Products Directorate (NHPD). Bilberry. In: NHPD Compendium of Monographs. Ottawa, Canada, 2008.

Biotin Donald M. Mock

INTRODUCTION

pancreatic proteases, dietary avidin binds to dietary biotin (and probably any biotin from intestinal microbes) and prevents absorption, carrying the biotin on through the gastrointestinal tract. Biotin is definitely synthesized by intestinal microbes; however, the contribution of microbial biotin to absorbed biotin, if any, remains unknown. Cooking denatures avidin, rendering this protein susceptible to pancreatic proteases and unable to interfere with the absorption of biotin.

Biotin is usually classified as a B-complex vitamin. “Biotin” is by far the most widely used term for this vitamin. However, discovery of biotin by different approaches has also led to names such as Bios IIB, protective factor X, vitamin H, coenzyme R, factor S, factor W, and vitamin BW . This entry reviews the biochemistry of biotin and summarizes the clinical findings of deficiency. Readers are encouraged to use the references for further information.

SCIENTIFIC NAMES AND STRUCTURE BIOCHEMISTRY

The molecular weight of biotin is 244.31 Da. The structure of biotin was elucidated independently by Kogl and du Vigneaud in the early 1940s and is shown in Figure 1 (1). Biotin is a bicyclic compound. The imidazolidone contains an ureido group (–N–CO–N–). The tetrahydrothiophene ring contains sulfur and has a valeric acid side chain attached to the C2 carbon of the sulfur-containing ring. This chain has a cis configuration with respect to the ring that contains the nitrogen atoms. The two rings are fused in the cis configuration, producing a boat-like structure. With three asymmetric carbons, eight stereoisomers exist; only one [designated D-(+)-biotin or, simply, biotin] is found in nature and is active when covalently joined via an amide bond between the carboxyl group of the valeric acid side chain of biotin and the ε-amino group of a lysine residue of an apocarboxylase. Biocytin (ε-N-biotinyl-L-lysine) is the product of digestion of protein-bound dietary biotin and cellular turnover of biotin-containing carboxylases and histones; biocytin is as active as biotin on a molar basis in mammalian growth studies. Goldberg/Sternbach synthesis or a modification thereof is the method by which biotin is synthesized commercially (1). Additional stereospecific methods have been published (2,3).

Biotin acts as an essential cofactor for five mammalian carboxylases. Each has the vitamin covalently bound to a polypeptide. For monomeric carboxylases, this polypeptide is the apocarboxylase. For the dimeric carboxylases, this monomer with a biotinylation site is designated the chain. The covalent attachment of biotin to the apocarboxylase protein is a condensation reaction catalyzed by holocarboxylase synthetase (EC 6.3.4.10). These apocarboxylase regions contain the biotin motif (methionine– lysine–methionine), a specific sequence of amino acids present in each of the individual carboxylases; this sequence tends to be highly conserved within and between species. One interpretation concerning conservation of this amino acid sequence is that these residues allow the biotinylated peptide to swing the carboxyl (or acetyl) group from the site of activation to the receiving substrate. All five of the mammalian carboxylases catalyze the incorporation of bicarbonate as a carboxyl group into a substrate and employ a similar catalytic mechanism. In the carboxylase reaction, the carboxyl moiety is first attached to biotin at the ureido nitrogen opposite the side chain. Then the carboxyl group is transferred to the substrate. The reaction is driven by the hydrolysis of ATP to ADP and inorganic phosphate. Subsequent reactions in the pathways of the five mammalian carboxylases release CO2 from the product of the enzymatic reaction. Thus, these reaction sequences rearrange the substrates into more useful intermediates but do not violate the classic observation that mammalian metabolism does not result in the net fixation of carbon dioxide (4). The five carboxylases are pyruvate carboxylase (EC 6.4.1.1), methylcrotonyl-CoA carboxylase (EC 6.4.1.4), propionyl-CoA carboxylase (EC 6.4.1.3), and two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), denoted I and II, which are also known as ACC and ␤ ACC. Each

HISTORY Biotin was discovered in nutritional experiments that demonstrated a factor present in many foodstuffs that was capable of curing the scaly dermatitis, hair loss, and neurologic signs induced in rats fed dried egg white. Avidin, a glycoprotein found in egg white, binds biotin very specifically and tightly. From an evolutionary standpoint, avidin probably serves as a bacteriostat in egg white. Consistent with this hypothesis is the observation that avidin is resistant to a broad range of bacterial proteases in both free and biotin-bound form. Because avidin is also resistant to 43

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O C HN

NH

HC

CH

N-H

O

CH (CH 2)4 C N (CH2) 4

H 2C S

H

C-H C=O

O amino group

Figure 1 Protein-bound biotin with arrow showing the amide bond to the ␧-amino acid.

carboxylase catalyzes an essential step in intermediary metabolism (Fig. 2). Pyruvate carboxylase mediates in the incorporation of bicarbonate into pyruvate to form oxaloacetate, an intermediate in the Krebs tricarboxylic acid cycle. Thus, pyruvate carboxylase catalyzes an anaplerotic reaction. In gluconeogenic tissues (i.e., liver and kidney), the oxaloacetate can be converted to glucose. Deficiency of this enzyme (denoted by a block in the metabolic pathway) is likely the cause of the lactic acidosis and hypoglycemia observed in biotin-deficient animals and humans. Methylcrotonyl-CoA carboxylase catalyzes an essential step in the degradation of the branch-chained

3-hydroxypropionate methylcitrate odd-chain fatty acid

isoleucine methionine

amino acid leucine. Deficient activity of this enzyme leads to metabolism of 3-methylcrotonyl CoA to 3hydroxyisovaleric acid and 3-methylcrotonylglycine by an alternate pathway. Thus, increased urinary excretion of these abnormal metabolites reflects deficient activity of this carboxylase. Propionyl-CoA carboxylase catalyzes the incorporation of bicarbonate into propionyl CoA to form methylmalonyl CoA, which undergoes isomerization to succinyl CoA and enters the tricarboxylic acid cycle. In a fashion analogous to methylcrotonyl-CoA carboxylase deficiency, inadequacy of this enzyme leads to increased urinary excretion of 3-hydroxypropionic acid and 3-methylcitric acid and enhanced accumulation of odd-chain fatty acids C15:0 and C17:0. The mechanism is likely the substitution of propionyl CoA for acetyl CoA during fatty acid elongation. Although the proportional increase is large (e.g., 2- to 10fold), the absolute composition relative to other fatty acids is quite small (200-fold. These investigators found no masking of detection or degradation of biotin or biotin sulfoxide. Gel electrophoresis and streptavidin Western blot detected several biotinylated proteins in CSF leading to the conclusion that biotin is bound to protein covalently, reversibly, or both; they speculated that biotin bound to protein likely accounts for the increase in detectable biotin after HPLC and that

47

Biotin

protein-bound biotin plays an important role in biotin nutriture of the brain.

Placental Transport Biotin concentrations are 3- to 17-fold greater in plasma from human fetuses compared with their mothers in the second trimester, consistent with active placental transport (37). Specific systems for transport of biotin from the mother to the fetus have been reported recently (10,38– 40). The microvillus membrane of the placenta contains a saturable transport system for biotin that is Na+ dependent and actively accumulates biotin within the placenta, consistent with SMVT (10,38–40).

Transport into Human Milk More than 95% of the biotin is free in the skim fraction of human milk (41). The concentration of biotin varies substantially in some women (42) and exceeds that in serum by one to two orders of magnitude, suggesting that there is a transport system into milk. The biotin metabolite bisnorbiotin (see discussion of metabolism under pharmacology section) accounts for approximately 50%. In early and transitional human milk, the biotin metabolite and biotin sulfoxide accounts for about 10% of the total biotin plus metabolites (43). With postpartum maturation, the biotin concentration increases, but the bisnorbiotin and biotin sulfoxide concentrations still account for 25% and 8% at 5 weeks postpartum. The concentration of biotin in human milk exceeds the plasma concentration by 10- to 100-fold, implying that a transport system exists. Current studies provide no evidence for a soluble biotinbinding protein or any other mechanism that traps biotin in human milk. The location and the nature of the biotin transport system for human milk have yet to be elucidated.

PHARMACOLOGY Studies in which pharmacologic amounts of biotin were administered orally and intravenously to experimental subjects and tracer amounts of radioactive biotin were administered intravenously to animals show that biotin in pure form is 100% bioavailable when administered orally. The preponderance of dietary biotin detectable by bioassays is bound to macromolecules. Likely biotin is bound to carboxylases and perhaps to histones. The bioavailability of biotin from foodstuffs is not known, whereas that from animal feeds varies but can be well below 50%. After intravenous administration, the vitamin disappears rapidly from plasma; the fastest phase of the three-phase disappearance curve has a half-life of less than 10 minutes. An alternate fate to being covalently bound to protein (e.g., carboxylases) or excretion unchanged in urine is catabolism to an inactive metabolite before excretion in urine (4). About half of biotin undergoes metabolism before excretion. Two principal pathways of biotin catabolism have been identified in mammals. In the first pathway, the valeric acid side chain of biotin is degraded by ␤-oxidation. This leads to the formation of bisnorbiotin, tetranorbiotin, and related intermediates

Table 1 Normal Range of Urinary Excretion of Biotin and Major Metabolites (nmol/24 hr; n = 31 Males and Females) Biotin

Bisnorbiotin

Biotin sulfoxide

18–77

11–39

8–19

that are known to result from ␤-oxidation of fatty acids. The cellular site of this ␤-oxidation of biotin is uncertain. Nonenzymatic decarboxylation of the unstable ␤keto-biotin and ␤-keto-bisnorbiotin leads to formation of bisnorbiotin methylketone and tetranorbiotin methylketone, which appear in urine. In the second pathway, the sulfur in the thiophene ring of biotin is oxidized, leading to the formation of biotin L-sulfoxide, biotin D-sulfoxide, and biotin sulfone. Combined oxidation of the ring sulfur and ␤-oxidation of the side chain lead to metabolites such as bisnorbiotin sulfone. In mammals, degradation of the biotin ring to release carbon dioxide and urea is quantitatively minor. On a molar basis, biotin accounts for approximately half of the total avidin-binding substances in human serum and urine (Table 1). Biocytin, bisnorbiotin, bisnorbiotin methylketone, biotin sulfoxide, and biotin sulfone form most of the balance. Biotin metabolism is accelerated in some individuals by anticonvulsant therapy and during pregnancy, thereby increasing the ratio of biotin metabolites to biotin excreted in urine.

OCCURRENCE AND DIAGNOSIS OF BIOTIN DEFICIENCY The fact that normal humans have a requirement for biotin has been clearly documented in two situations: prolonged consumption of raw egg white and parenteral nutrition without biotin supplementation in patients with short-gut syndrome and other causes of malabsorption (1). Deficiency of this member of the vitamin B group also has been clearly demonstrated in biotinidase deficiency (6). The clinical findings and biochemical abnormalities in cases of biotin deficiency include dermatitis around body orifices, conjunctivitis, alopecia, ataxia, and developmental delay (1). The progression of clinical findings in adults, older children, and infants is similar. Typically, the symptoms appear gradually after weeks to several years of egg white feeding or parenteral nutrition. Thinning of hair progresses to loss of all hair, including eyebrows and lashes. A scaly (seborrheic), red (eczematous) skin rash was present in the majority of reports. In several reports, the rash was distributed around the eyes, nose, mouth, and perineal orifices. The appearance of the rash was similar to that of cutaneous candidiasis; Candida albicans could often be cultured from the lesions. These manifestations on skin, in conjunction with an unusual distribution of facial fat, have been dubbed “biotin deficiency facies.” Depression, lethargy, hallucinations, and paresthesias of the extremities were prominent neurologic symptoms in the majority of adults, while infants showed hypotonia, lethargy, and developmental delay. In cases severe enough to produce the classic cutaneous and behavioral manifestations of biotin deficiency, urinary excretion rates and plasma concentrations

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of biotin are frankly decreased. Urinary excretion of the organic acids discussed in biochemistry section and shown in Figure 2 is frankly increased. The increase is typically 5- to 20-fold or more. However, such a severe degree of biotin deficiency has never been documented to occur spontaneously in a normal individual consuming a mixed general diet. Of greater current interest and debate are the health consequences, if any, of marginal biotin deficiency. Concerns about the teratogenic effects have led to studies of biotin status during human gestation (44–48). These studies provide evidence that a marginal degree of deficiency develops in at least one-third of women during normal pregnancy. Although the degree of biotin deficiency is not severe enough to produce overt manifestations, the deficiency is severe enough to produce metabolic derangements. A similar marginal degree of biotin deficiency causes high rates of fetal malformations in some mammals (30,49,50). Moreover, data from a multivitamin supplementation study provide significant, albeit indirect, evidence that the marginal degree of deficiency that occurs spontaneously in normal human gestation is teratogenic (44). Valid indicators of marginal biotin deficiency have been reported. Asymptomatic biotin shortage was induced in normal adults housed in a general clinical research center by egg white feeding. Decreased urinary excretion of biotin, increased urinary excretion of 3-hydroxyisovaleric acid, and decreased activity of propionyl-CoA carboxylase in lymphocytes from peripheral blood are early and sensitive indicators of biotin deficiency (30,31,51). On the basis of a study of only five subjects, 3-hydroxyisovaleric acid excretion in response to a leucine challenge appears to be an even more sensitive indicator of marginal biotin status (31). The plasma concentration of biotin and the urinary excretion of methylglycine, 3-hydroxypropionic acid, and 3-methylcitric acid do not appear to be good indicators of marginal biotin deficiency (52). In a biotin repletion study, the resumption of a mixed general diet produced a trend toward normalization of biotin status within 7 days. This was achieved when the supplement was started immediately at the time of resuming a normal diet. However, supplementation of biotin at 10 times the dietary reference intake (DRI) (300 ␮g/day) for 14 days reduced 3-hydroxyisovaleric acid excretion completely to normal in only about half of pregnant women who were marginally biotin deficient (47) suggesting a substantial depletion of total body biotin, a substantially increased biotin requirement, or both. On the basis of decreased lymphocyte carboxylase activities and plasma biotin levels, Velazquez et al. (53) have reported that biotin deficiency occurs in children with severe protein-energy malnutrition. These investigators have speculated that the effects of biotin inadequacy may be responsible for part of the clinical syndrome of protein-energy malnutrition. Long-term treatment with a variety of anticonvulsants appears to be associated with marginal biotin deficiency severe enough to interfere with amino acid metabolism (54–56). The mechanism may involve both accelerated biotin breakdown (56–58) and impairment of biotin absorption caused by the anticonvulsants (59).

Biotin deficiency has also been reported or inferred in several other circumstances including Leiner disease (60–62), sudden infant death syndrome (63,64), hemodialysis (65–69), gastrointestinal diseases and alcoholism (1), and brittle nails (70). Additional studies are needed to confirm or refute an etiologic link of these conditions to the vitamin’s deficiency. The mechanisms by which biotin deficiency produces specific signs and symptoms remain to be completely delineated. However, several studies have given new insights on this subject. The classic assumption for most water-soluble vitamins is that the clinical findings of deficiency result directly or indirectly from deficient activities of the vitamin-dependent enzymes. On the basis of human studies on deficiency of biotinidase and isolated pyruvate carboxylase, as well as animal experiments regarding biotin deficiency, it is hypothesized that the central nervous system effects of biotin deficiency (hypotonia, seizures, ataxia, and delayed development) are likely mediated through deficiency of brain pyruvate carboxylase and the attendant central nervous system lactic acidosis rather than by disturbances in brain fatty acid composition (71–73). Abnormalities in metabolism of fatty acids are likely important in the pathogenesis of the skin rash and hair loss (74). Exciting new work has provided evidence for a potential role for biotin in gene expression. These findings will likely provide new insights into the pathogenesis of biotin deficiency (75,76). In 1995, Hymes and Wolf discovered that biotinidase can act as a biotinyl transferase; biocytin serves as the source of biotin, and histones are specifically biotinylated (6). Approximately 25% of total cellular biotinidase activity is located in the nucleus. Zempleni and coworkers have demonstrated that the abundance of biotinylated histones varies with the cell cycle, that these histones are increased approximately twofold compared with quiescent lymphocytes, and that these are debiotinylated enzymatically in a process that is at least partially catalyzed by biotinidase (77–79). These observations suggest that biotin plays a role in regulating DNA transcription and regulation. Biotinylation of histones is emerging as an important histone modification. Recent studies from Hassan and Zempleni provide evidence that biotinylation likely interacts with other covalent modification of histones to suppress gene expression and gene transposition (80). Although the relative importance in biotinidase and holocarboxylase synthetase in the biotinylation and debiotinylation of histones has yet to be fully elucidated, Gravel and Narang have produced evidence that holocarboxylase synthetase is present in the nucleus in greater quantities than in the cytosol or the mitochondria and that holocarboxylase synthetase likely acts in the nucleus to catalyze the biotinylation of histones (81). Moreover, fibroblasts from patients with HCLS deficiency are severely deficient in histone biotinylation (82). Zempleni and coworkers have shown that biotinylation of lysine-12 in histone H4 (K12BioH4) causes gene repression and have proposed a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells (83). They have hypothesized that holocarboxylase synthetase senses biotin and that biotin regulates its own cellular uptake by participating in holocarboxylase synthetase–dependent chromatin

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Biotin

remodeling events at an SMVT promoter locus. Specifically, they hypothesize that nuclear translocation of HCS increases in response to biotin supplementation and then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. This group has shown that nuclear translocation of HCS is a biotin-dependent process potentially involving tyrosine kinases, histone deacetylases, and histone methyltransferases. The nuclear translocation of holocarboxylase synthetase correlates with biotin concentrations in cell culture media and is inversely linked to SMVT expression. Moreover, biotin homeostasis by holocarboxylase synthetase–dependent chromatin remodeling at an SMVT promoter locus is disrupted in holocarboxylase synthetase knockdown cells. Transposable elements such as retrotransposons containing long-terminal repeats constitute about half of the human genome, and the transposition events associated with these elements impair genome stability. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Zempleni and coworkers have provided evidence that the covalent binding of biotin to lysine-12 in histone H4 and lysine-9 in histone H2A mediated by holocarboxylase synthetase is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of biotinylation at those sites depended on biotin supply and on holocarboxylase synthetase activity and was inversely linked with the abundance of long terminal repeat transcripts. Knockdown of holocarboxylase synthetase in Drosophila enhanced retrotransposition. Depletion of biotinylation at those sites in biotin-deficient cells correlated with increased production of transposition events and decreased chromosomal stability. Recently, controversy has arisen concerning the role of biotin as an in vivo covalent modifier of histones. Bailey and coworkers have reported that streptavidin binds to histones independently of biotinylation (84). To further investigate this phenomenon, 293T cells were grown in 14 C-biotin; in contrast to the ready detectability of 14 Cbiotin in carboxylases, 14 C-biotin was undetectable in histones (i.e., represented no more than 0.03% of histones) (84). In a subsequent study, Healy and coworkers demonstrated that histone H2A is nonenzymatically biotinylated by biotinyl-5 -AMP and provided evidence that these enzymes promotes biotinylation of histone H2A by releasing biotinyl-5 -AMP, which then biotinylates lysines in histone H2A somewhat nonspecifically (85). Recently, this group has proposed that biotin is not a natural histone modifier at all. On the basis of studies that fail to find in vivo biotin incorporation into histones using 3 H-biotin uptake, Western blot analysis of histones, or mass spectrometry of affinity purified histone fragments, these investigators concluded that biotin is absent in native histones to a sensitivity of 1 part per 100,000 and that the regulatory impact on gene expression must occur through a mechanism other than histone modification (86). These conclusions are likely to generate a lively debate until definitive evidence is provided using mass spectrometric analysis of in vivo histones harvested at various phases of the cell cycle and at specific locations within particular histones.

Table 2

Adequate Intake for Biotin Consumption

Age 0–6 mo 7–12 mo 1–3 yr 4–8 yr 9–13 yr 14–18 yr 19– >70 yr Pregnancy Lactation

Amount (␮g/day) 5 6 8 12 20 25 30 30 35

Note: Values for males and females in all age groups were combined because they do not differ. Source: From Ref. 88.

INDICATIONS AND USAGE In 1998, the United States Food and Nutrition Board of the National Academy of Sciences reviewed the recommendations for biotin intake (87). The committee concluded that the data were inadequate to justify setting an estimated average requirement. However, adequate intake (AI) was formulated (Table 2). The AI for infants was based on an empirical determination of the biotin content of human milk. Using the value for free biotin determined microbiologically (6 ␮g/L) and an average consumption of 0.78 L/day by infants of age 0–6 months, an AI of 5 ␮g/day was calculated. The AI for lactating women has been increased by 5 ␮g/day to allow for the amount of biotin secreted in human milk. Using the AI for 0–6-month-old infants, the reference body weight ratio method was used to extrapolate AIs for other age groups (see Table 2).

TREATMENT OF BIOTIN DEFICIENCY If biotin deficiency is confirmed, biotin supplementation should be undertaken and effectiveness should be documented. Doses between 100 ␮g and 1 mg are likely to be both effective and safe on the basis of studies supplementing biotin deficiency during pregnancy, chronic anticonvulsant therapy, and biotinidase deficiency.

TOXICITY Daily doses of up to 200 mg orally and up to 20 mg intravenously have been given to treat biotin-responsive inborn errors of metabolism and acquired biotin deficiency. Toxicity has not been reported.

REFERENCES 1. Mock DM, Biotin. In: Ziegler EE, Filer LJ Jr, eds. Present Knowledge in Nutrition. Washington, DC: International Life Sciences Institutes–Nutrition Foundation, 1996:220–235. 2. Miljkovic D, Velimirovic S, Csanadi J, et al. Studies directed towards stereospecific synthesis of oxybiotin, biotin, and their analogs. Preparation of some new 2,5, anhydro-xylitol derivatives. J Carbohydr Chem 1989; 8:457–467. 3. Deroose FD, DeClercq PJ. Novel enantioselective syntheses of (+)-biotin. J Org Chem 1995; 60:321–330.

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4. Mock DM. Biotin. In: Shils ME, Olson JA, Shike M, et al., eds. Modern Nutrition in Health and Disease. Baltimore, MD: Williams & Wilkins, 1999:459–466. 5. Lewis B, Rathman S, McMahon R. Dietary biotin intake modulates the pool of free and protein-bound biotin in rat liver. J Nutr 2001; 131:2310–2315. 6. Wolf B. Disorders of biotin metabolism. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill, Inc., 2001:3151–3177. 7. Wolf B, Heard G, McVoy JRS, et al. Biotinidase deficiency: the possible role of biotinidase in the processing of dietary protein-bound biotin. J Inherit Metab Dis 1984; 7(suppl 2):121–122. 8. Said H. Cell and molecular aspects of the human intestinal biotin absorption process. J Nutr 2008; 139(1):158–162. 9. Prasad PD, Ramamoorthy S, Leibach FH, et al. Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells. Placenta 1997; 18:527–533. 10. Prasad PD, Wang H, Kekuda R, et al. Cloning and functional expression of a cDNA encoding a mammalian sodiumdependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate. J Biol Chem 1998; 273: 7501–7506. 11. Said HM. Recent advances in carrier-mediated intestinal absorption of water-soluble vitamins. Annu Rev Physiol 2004; 66:419–446. 12. Mock DM. Biotin. In: Brown M, ed. Present Knowledge in Nutrition. Blacksburg, VA: International Life Sciences Institute–Nutrition Foundation, 1990:189–207. 13. Wolf B, Grier RE, McVoy JRS, et al. Biotinidase deficiency: a novel vitamin recycling defect. J Inherit Metab Dis 1985; 8(suppl 1):53–58. 14. Chauhan J, Dakshinamurti K. Role of human serum biotinidase as biotin-binding protein. Biochem J 1988; 256:265– 270. 15. Mock DM, Lankford G. Studies of the reversible binding of biotin to human plasma. J. Nutr 1990; 120;375–381. 16. Mock DM, Malik MI. Distribution of biotin in human plasma: most of the biotin is not bound to protein. Am J Clin Nutr 1992; 56:427–432. 17. Nagamine T, Takehara K, Fukui T, et al. Clinical evaluation of biotin-binding immunoglobulin in patients with Graves’ disease. Clin Chim Acta 1994; 226(1):47–54. 18. Mardach R, Zempleni J, Wolf B, et al. Biotin dependency due to a defect in biotin transport. J Clin Invest 2002; 109(12):1617–1623. 19. Bowers-Komro DM, McCormick DB. Biotin uptake by isolated rat liver hepatocytes. In: Dakshinamurti K, Bhagavan HN, eds. Biotin. New York: New York Academy of Sciences, 1985:350–358. 20. Said HM, Ma TY, Kamanna VS. Uptake of biotin by human hepatoma cell line, Hep G(2): a carrier-mediated process similar to that of normal liver. J Cell Physiol 1994; 161(3): 483–489. 21. Balamurugan K, Ortiz A, Said HM. Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system. Am J Physiol Gastrointest Liver Physiol 2003; 285(1):G73– G77. 22. Zempleni J, Mock DM. Uptake and metabolism of biotin by human peripheral blood mononuclear cells. Am J Physiol Cell Physiol 1998; 275(2):C382–C388. 23. Daberkow RL, White BR, Cederberg RA, et al. Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells. J Nutr 2003; 133:2703–2706. 24. Ozand PT, Gascon GG, Al Essa M, et al. Biotin-responsive basal ganglia disease: a novel entity. Brain 1999; 121:1267– 1279.

25. Zeng W, Al-Yamani E, Acierno JS, et al. Mutations in SLC19A3 encoding a novel transporter cause biotinresponsive basal ganglia disease. American Society of Human Genetics Meeting Web site. http://faseb.org/ genetics/ashg01/f101.htm. Accessed April 15, 2010. 26. Subramanian VS, Marchant JS, Said HM. Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2. Am J Physiol Cell Physiol 2006; 291(5):C851–859. 27. Bowman BB, McCormick DB, Rosenberg IH. Epithelial transport of water-soluble vitamins. Ann Rev Nutr 1989; 9:187– 199. 28. Baur B, Baumgartner ER. Na(+)-dependent biotin transport into brush-border membrane vesicles from human kidney cortex. Pflugers Arch 1993; 422:499–505. 29. Balamurugan K, Vaziri ND, Said HM. Biotin uptake by human proximal tubular epithelial cells: cellular and molecular aspects. Am J Physiol Renal Physiol 2005; 288(4):F823–F831. 30. Mock NI, Malik MI, Stumbo PJ, et al. Increased urinary excretion of 3-hydroxyisovaleric acid and decreased urinary excretion of biotin are sensitive early indicators of decreased status in experimental biotin deficiency. Am J Clin Nutr 1997; 65:951–958. 31. Mock DM, Henrich CL, Carnell N, et al. Indicators of marginal biotin deficiency and repletion in humans: validation of 3-hydroxyisovaleric acid excretion and a leucine challenge. Am J Clin Nutr 2002; 76:1061–1068. 32. Baumgartner ER, Suormala T, Wick H. Biotinidase deficiency: factors responsible for the increased biotin requirement. J Inherit Metab Dis 1985; 8(suppl 1):59–64. 33. Baumgartner ER, Suormala T, Wick H. Biotinidase deficiency associated with renal loss of biocytin and biotin. J Inherit Metab Dis 1985; 7(suppl 2):123–125. 34. Spector R, Mock DM. Biotin transport through the blood– brain barrier. J Neurochem 1987; 48:400–404. 35. Spector R, Mock DM. Biotin transport and metabolism in the central nervous system. Neurochem Res 1988; 13(3):213–219. 36. Bogusiewicz A, Stratton SL, Ellison DA, et al. Distribution of biotin in cerebrospinal fluid of children: most of the biotin is bound to protein. FASEB J 2008; 22:1104.4. 37. Mantagos S, Malamitsi-Puchner A, Antsaklis A, et al. Biotin plasma levels of the human fetus. Biol Neonate 1998; 74:72– 74. 38. Karl PI, Fisher SE. Biotin transport in microvillous membrane vesicles, cultured trophoblasts and the isolated perfused cotyledon of the human placenta. Am J Physiol 1992; 262:C302–C308. 39. Schenker S, Hu ZQ, Johnson RF, et al. Human placental biotin transport: normal characteristics and effect of ethanol. Alcohol Clin Exp Res 1993; 17(3):566–575. 40. Hu ZQ, Henderson GI, Mock DM, et al. Biotin uptake by basolateral membrane of human placenta: normal characteristics and role of ethanol. Proc Soc Exp Biol Med 1994; 206(4):404–408. 41. Mock DM, Mock NI, Langbehn SE. Biotin in human milk: methods, location, and chemical form. J Nutr 1992; 122:535– 545. 42. Mock DM, Mock NI, Dankle JA. Secretory patterns of biotin in human milk. J Nutr 1992; 122:546–552. 43. Mock DM, Stratton SL, Mock NI. Concentrations of biotin metabolites in human milk. J Pediatr 1997; 131(3):456–458. 44. Zempleni J, Mock D. Marginal biotin deficiency is teratogenic. Proc Soc Exp Biol Med 2000; 223(1):14–21. 45. Mock DM, Stadler DD, Stratton SL, et al. Biotin status assessed longitudinally in pregnant women. J Nutr 1997; 127(5):710–716. 46. Mock DM, Stadler DD. Conflicting indicators of biotin status from a cross-sectional study of normal pregnancy. J Am Coll Nutr 1997; 16:252–257.

Biotin

47. Mock DM, Quirk JG, Mock NI. Marginal biotin deficiency during normal pregnancy. Am J Clin Nutr 2002; 75(2):295– 299. 48. Mock DM. Marginal biotin deficiency is common in normal human pregnancy and is highly teratogenic in the mouse. J Nutr 2009; 139(1):154–157. 49. Mock DM, Mock NI, Stewart CW, et al. Marginal biotin deficiency is teratogenic in ICR mice. J Nutr 2003; 133:2519–2525. 50. Watanabe T, Endo A. Biotin deficiency per se is teratogenic in mice. J Nutr 1991; 121:101–104. 51. Mock DM, Henrich C, Carnell N, et al. Lymphocyte propionyl-CoA carboxylase and accumulation of odd-chain fatty acid in plasma and erythrocytes are useful indicators of marginal biotin deficiency. J Nutr Biochem 2002; 13(8):462– 470. 52. Mock DM, Henrich-Shell CL, Carnell N, et al. 3hydroxypropionic acid and methylcitric acid are not reliable indicators of marginal biotin deficiency. J Nutr 2004; 134:317– 320. 53. Velazquez A, Martin-del-Campo C, Baez A, et al. Biotin deficiency in protein-energy malnutrition. Eur J Clin Nutr 1988; 43:169–173. 54. Krause K-H, Berlit P, Bonjour J-P. Impaired biotin status in anticonvulsant therapy. Ann Neurol 1982; 12:485–486. 55. Krause K-H, Berlit P, Bonjour J-P. Vitamin status in patients on chronic anticonvulsant therapy. Int J Vitam Nutr Res 1982; 52(4):375–385. 56. Mock DM, Dyken ME. Biotin catabolism is accelerated in adults receiving long-term therapy with anticonvulsants. Neurology 1997; 49(5):1444–1447. 57. Wang K-S, Mock NI, Mock DM. Biotin biotransformation to bisnorbiotin is accelerated by several peroxisome proliferators and steroid hormones in rats. J Nutr 1997; 127(11):2212– 2216. 58. Mock DM, Mock NI, Lombard KA, et al. Disturbances in biotin metabolism in children undergoing long-term anticonvulsant therapy. J Pediatr Gastroenterol Nutr 1998; 26(3):245–250. 59. Said HM, Redha R, Nylander W. Biotin transport in the human intestine: inhibition by anticonvulsant drugs. Am J Clin Nutr 1989; 49:127–131. 60. Nisenson A. Seborrheic dermatitis of infants and Leiner’s disease: a biotin deficiency. J Pediatr 1957; 51:537–548. 61. Nisenson A. Seborrheic dermatitis of infants: treatment with biotin injections for the nursing mother. Pediatrics 1969; 44:1014–1015. 62. Erlichman M, Goldstein R, Levi E, et al. Infantile flexural seborrhoeic dermatitis. Neither biotin nor essential fatty acid deficiency. Arch Dis Child 1981; 567:560–562. 63. Johnson AR, Hood RL, Emery JL. Biotin and the sudden infant death syndrome. Nature 1980; 285:159–160. 64. Heard GS, Hood RL, Johnson AR. Hepatic biotin and the sudden infant death syndrome. Med J Aust 1983; 2(7):305– 306. 65. Yatzidis H, Koutsicos D, Alaveras AG, et al. Biotin for neurologic disorders of uremia. N Engl J Med 1981; 305(13): 764. 66. Livaniou E, Evangelatos GP, Ithakissios DS, et al. Serum biotin levels in patients undergoing chronic hemodialysis. Nephron 1987; 46:331–332. 67. DeBari V, Frank O, Baker H, et al. Water soluble vitamins in granulocytes, erythrocytes, and plasma obtained from chronic hemodialysis patients. Am J Clin Nutr 1984; 39:410– 415.

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68. Yatzidis H, Koutisicos D, Agroyannis B, et al. Biotin in the management of uremic neurologic disorders. Nephron 1984; 36:183–186. 69. Braguer D, Gallice P, Yatzidis H, et al. Restoration by biotin in the in vitro microtubule formation inhibited by uremic toxins. Nephron 1991; 57:192–196. 70. Colombo VE, Gerber F, Bronhofer M, et al. Treatment of brittle fingernails and onychoschizia with biotin: scanning electron microscopy. J Am Acad Dermatol 1990; 23:1127–1132. 71. Sander JE, Packman S, Townsend JJ. Brain pyruvate carboxylase and the pathophysiology of biotin-dependent diseases. Neurology 1982; 32:878–880. 72. Suchy SF, Rizzo WB, Wolf B. Effect of biotin deficiency and supplementation on lipid metabolism in rats: saturated fatty acids. Am J Clin Nutr 1986; 44:475–480. 73. Suchy SF, Wolf B. Effect of biotin deficiency and supplementation on lipid metabolism in rats: cholesterol and lipoproteins. Am J Clin Nutr 1986; 43:831–838. 74. Mock DM. Evidence for a pathogenic role of ␻6 polyunsaturated fatty acid in the cutaneous manifestations of biotin deficiency. J Pediatr Gastroenterol Nutr 1990; 10:222– 229. 75. McMahon RJ. Biotin in metabolism and molecular biology. Annu Rev Nutr 2002; 22:221–239. 76. Zempleni J. Biotin. In: Bowman BB, Russell RM, eds. Present Knowledge in Nutrition. Washington, DC: International Life Sciences Institutes–Nutrition Foundation, 2001. 77. Zempleni J, Mock DM. Chemical synthesis of biotinylated histones and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/streptavidinperoxidase. Arch Biochem Biophys 1999; 371(1):83–88. 78. Zempleni J, Mock DM. Chemical synthesis of biotinylated histones and analysis by SDS-PAGE/streptavidin peroxidase. Biomol Eng 2000; 16(5):181. 79. Stanley JS, Griffin JB, Zempleni J. Biotinylation of histones in human cells: effects of cell proliferation. Eur J Biochem 2001; 268:5424–5429. 80. Hassan YI, Zempleni J. Epigenetic regulation of chromatin structure and gene function by biotin. J Nutr 2006; 136(7):1763–1765. 81. Gravel R, Narang M. Molecular genetics of biotin metabolism: old vitamin, new science. J Nutr Biochem 2005; 16(7):428–431. 82. Narang MA, Dumas R, Ayer LM, et al. Reduced histone biotinylation in multiple carboxylase deficiency patients: a nuclear role for holocarboxylase synthetase. Hum Mol Genet 2004; 13(1):15–23. 83. Zempleni J. Chromatin remodeling events at the SMVT locus. J Nutr 2008; 139(1):163–166. 84. Bailey LM, Ivanov RA, Wallace JC, et al. Artifactual detection of biotin on histones by streptavidin. Anal Biochem 2007; 373:71–77. 85. Healy S, Heightman TD, Hohmann L, et al. Nonenzymatic biotinylation of histone H2A. Protein Sci 2008; 18:314– 328. 86. Healy S, Perez-Cadahia B, Jia D, et al. Biotin is not a natural histone modification. Biochem Biophys Acta 2009; 1789:719– 733. 87. National Research Council. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic acid, biotin, and choline. In: Recommended Dietary Allowances, Food and Nutrition Board, Institute of Medicine, ed. Washington, DC: National Academy Press, 1998:374–389.

Bitter Orange Steffany Haaz, K. Y. Williams, Kevin R. Fontaine, and David B. Allison

cardiovascular, neuromuscular, and antiseptic indications in countries such as China, Curacao, Haiti, India, Mexico, Trinidad, Turkey, and the United States (9). The most common current western use, however, is as a dietary supplement for weight loss.

INTRODUCTION Citrus aurantium (C. aurantium) is the Latin name for a plant commonly referred to as bitter orange, sour orange, Neroli, Chongcao, or Seville orange. It is a source of synephrine and several other biogenic amines, as well as other bioactive phytochemicals and has been used in dietary supplements for weight loss. In this entry, we discuss the available evidence pertaining to safety and efficacy of C. aurantium for weight loss, as examined in animal studies, clinical trials, and case reports.

POTENTIAL CONSTITUENTS Some authors (10) state that C. aurantium contains metasynephrine (m-synephrine, m-s), whereas others (11) state that it contains only para-synephrine (p-synephrine, ps). However, research (I.A. Khan, oral communication, 2005) has shown that C. aurantium naturally contains p-synephrine and does not contain m-synephrine. Allison and colleagues reported that at least one over-thecounter (OTC) product purportedly containing SAs from C. aurantium contains both p-synephrine and m-synephrine (12), raising concerns about possible adulteration and mislabeling. There is also an ortho isomer of synephrine (o-synephrine), whose content in C. aurantium is unknown. p-, m-, and o-synephrine can each exist in D or L forms. p-Synephrine, an undisputed component of C. aurantium, is typically referred to simply as synephrine (13). It is an ␣-adrenergic agonist (14) that also has some ␤adrenergic properties (15). p-Synephrine occurs naturally in the human body in small quantities and might act as a neurotransmitter (16). Under the name oxedrine, it has been used since 1927 (17) in eyedrops. p-Synephrine is thought to be the ingredient in C. aurantium primarily responsible for weight loss. However, neither this nor whether C. aurantium actually produces weight loss in humans is firmly established. m-Synephrine, often referred to as phenylephrine, is an isomer of p-synephrine. To the best of our knowledge, m-synephrine is not contained naturally in C. aurantium. m-Synephrine is also an ␣-adrenergic agonist that has some ␤-adrenergic agonist properties. It has been studied more extensively than p-synephrine and is one of the two most widely used OTC decongestants today (Fig. 1) (13). p-Synephrine and m-synephrine have similar structure to ephedrine, as well as other substances that have some effects on reducing food intake and/or body weight such as epinephrine and norepinephrine (Fig. 1), supporting the conjecture that, to the extent that function follows structure, p-synephrine and m-synephrine, may also reduce food intake and or body weight. The ␣-adrenergic sympathomimetic amine, poctopamine, is also present in C. aurantium, though possibly at inappreciable levels (2). Like both forms of synephrine, it is an ␣-adrenergic agonist with some

GENERAL DESCRIPTION Bitter orange is a member of the Rutaceae family, a hybrid between Pummelo, Citrus grandis, and Mandarin, Citrus reticulata. Native to Asia, various parts of the plant are used throughout the world for a variety of indications. Bitter orange and its components are commercially available in herbal weight loss supplements, ostensibly for their adrenergic agonistic properties (1), often in combination with other ingredients hypothesized to promote weight loss. Its constituent p-octopamine and synephrine alkaloids (SAs) are usually cited as the active ingredients in such products (2). With the banning of ephedra in the United States in 2004, bitter orange has been increasingly included in weight loss supplement formulations. Because of similarities in their constituents and possible mechanisms (both sources of natural alkaloids with sympathomimetic activity), concerns have been raised that bitter orange may carry risks similar to those hypothesized to exist for ephedra (3).

HISTORICAL USE C. aurantium’s origin is in China and appears in writing as far back as 300 BC. Its ancient use has also been documented in Japan and Rome (4). It is native to eastern Africa, Arabia, and Syria and is cultivated in various European, North American, and South American regions. The leaf was historically used as a tonic, laxative, or sedative in Mexico and South America and for insomnia, palpitations, or stomachaches by the European Basque people (5,6). The fruit and peel are also used for stomachaches, as well as high blood pressure (BP), spasm, and a variety of gastrointestinal conditions by both the Basque and practitioners of traditional Chinese medicine (7). While the practice arose in Ancient Egypt, neroli oil is still currently used for aromatherapy and bergamot, a subspecies of C. aurantium, is used for flavoring and aroma in Earl Grey teas (8). Modern uses for C. aurantium include digestive, 52

Bitter Orange

53

Toxicity and Mortality B

A

D

C

E

Figure 1 Chemical structures of (A) p-synephrine, (B) m-synephrine, (C) ephedrine, (D) epinephrine, and (E) norepinephrine.

␤-adrenergic properties. It is used to treat hypertension and as a cardiotonic (13) and has also been examined for its potential role in promoting weight loss (18). Because of their similar properties and the overlap of their inclusion in supplements, we will refer to these substances collectively as synephrine alkaloids (SAs). SAs are used clinically as decongestants (1), during surgical procedures as a vasopressor (19), for acute treatment of priapism (20), and in ophthalmological examinations for pupil dilation (21). Products that contain C. aurantium or its derivatives, including OTC weight loss supplements, will be referred to as C. aurantium products (CAPs). Regulatory oversight for dietary supplements is much less rigorous than for pharmaceuticals, and extensive evidence is not required prior to release of a product on the public market. While a phase of requirements for meeting good manufacturing practices is currently underway, this may help to explain why the quality and quantity of the evidence we have available to evaluate the safety and efficacy of C. aurantium is minimal.

POTENTIAL MECHANISMS As sympathomimetic agents with both ␣- and ␤adrenergic receptor agonist properties, SAs might increase energy expenditure and/or decrease food intake (22). In addition, there is some evidence that adrenergic agonists, including SAs, decrease gastric motility (23). Similar to compounds such as cholecystokinin and other gut peptides which both decrease gastric motility and food intake (24), one might conjecture that SAs may also decrease food intake via reducing gut motility. Activation of lipolysis is a known ␤-adrenergic activity (25) that may be fueled by these components of C. aurantium.

ANIMAL STUDIES Weight Loss SAs reduce food intake in rodents (26), and some studies indicate that SAs can reduce rodent body weight (13,26). SAs have also been shown to promote lipolysis in adipocytes through ␤-adrenergic stimulation (27) and to increase lipoprotein lipase activity in the parametrial fat pad of female hamsters (28). However, among monosodium glutamate–treated obese mice, SAs reduced weight gain but had no effect on body fat percent (29).

Data suggest that m-synephrine (not present in bitter orange) may prolong life in rodents. A 2-year study by the National Toxicology Program (13) evaluated the effects of m-synephrine on spontaneous food intake of rats and mice. At 2 years, there were no significant differences in survival among mice or female rats. However, for male rats, there was a significant reduction in mortality rate, although there was increased mortality in the early phase of the study at the highest dose. It should be noted that too few deaths occurred during the 2-year trial to provide the degree of precision and power desired for a rigorous longevity study (30). Nonetheless, similar results have been reported for ephedrine, another sympathomimetic amine (31). Arbo et al. (32) conducted a subchronic toxicity study in mice and the effects of p-synephrine and C. aurantium L. extract on oxidative stress biomarkers that are believed to be indicators of cell membrane injury (malondialdehyde) and (glutathione and the enzyme glutathione peroxidase) indicative of amphetamine-induced toxicity. The study evaluated adult male CF1 mice treated with 400, 2000, or 4000 mg/kg C. aurantium dried extract and p-synephrine 30 or 300 mg/kg over the course of 28 days. Results showed a reduction in glutathione in mice treated with C. aurantium 400 mg/kg and p-synephrine 30 and 300 mg/kg. Inhibition of glutathione peroxidase activity occurred within mice treated with C. aurantium 400 and 2000 mg/kg and p-synephrine 30 and 300 mg/kg; however, no change occurred within malondialdehyde levels. These two findings suggest the possibility of subchronic toxicity. No significant change in weight occurred in any of the groups, suggesting on the positive side a lack of severe toxicity, and on the negative side a lack of efficacy in producing weight loss. With regard to adverse effects, a study (33) of male Sprague-Dawley rats reported what was believed to be evidence of cardiotoxicity when C. aurantium fruit extracts standardized to 4% and 6% SAs were administered. Increased mortality has been observed among CAPs-treated rats (33) as well as a strain of mice selected to be uniquely susceptible to the effects of adrenergic stimulation (34).

CLINICAL TRIALS Weight Loss Few clinical trials have examined the effects of CAPs alone or in combination with other ingredients on body weight and/or body composition (Table 1). It should be kept in mind that these trials are of short duration and the sample sizes are frequently quite small. Nonetheless, these trials suggest that body weight and/or fat loss may be enhanced by CAPs or SAs. The mechanisms involved are unclear but may be partially due to a suppressing effect of appetite and/or a moderate increase in resting energy expenditure. Armstrong et al. (37) evaluated exercise and herbal preparation containing Ma Huang, bitter orange (5 mg SAs), and guarana over 6 weeks in a randomized, controlled trial. Compared with controls, the intervention group obtained significant reductions in fat mass and a nearly significant reduction in body mass index (kg/m2 )

54 Table 1

Haaz et al.

Summary of Clinical Weight Loss Trials

Reference

Treatment

Design

Sample size

Duration

Results

Comments

Colker et al. (10)

975 mg Citrus aurantium, +528 caffeine and 900 mg St. John’s wort; placebo (with pill) and control (no pill)

Blinded parallel groups RCT

Supplement n = 9; placebo, n = 7; control group (no pills), n = 4

6 wk

Supplement group lost more fat (3.1 kg; P < 0.05) than other groups and increased RMR (2–3%)

Citrus aurantium may assist individuals in losing body fat, due to increased energy and reduced energy intake expenditure. No adverse events were reported

Kalman et al. (36)

Ephedrine and synephrine alkaloids (SAs) (5 mg twice daily) based product vs. placebo with exercise and diet

Prospective, randomized, double blind

30 overweight subjects; BMI > 27

8 wk

3.4 kg weight loss in experimental group vs. 2.05 kg in placebo (P < 0.05)

No adverse events; findings indicate apparent short-term safety and efficacy of ephedrine and synephrine-based compound

Armstrong et al. (37)

Exercise program with assignment to drug (Ma Huang, bitter orange, and guarana) or placebo. Bitter orange standardized for 5 mg synephrine

Randomized trial—unclear if study is blinded

Five overweight males/14 females

44 days

Supplement increased fat loss (2.5 kg; P = 0.033) more than placebo (0.5 kg))

Low statistical power, no marked side effects

Greenway et al. (38): Pilot 1

Two capsules containing pantothenic acid, 40 mg; green tea leaf extract, 200 mg; guarana extract, 550 mg; bitter orange, 150 mg; white willow bark extract, 50 mg; ginger root, 10 mg; proprietary charge thermoblend (L-tyrosine, L-carnitine, naringin), 375 mg

Prospective, randomized, double blind

Eight subjects (1:1 ratio) between supplement group and placebo group

8 wk

Supplement group gained more weight (1.04 ± 0.27 kg; P < 0.04) than placebo and increased RMR (but not at 8 wk)

CAP was not efficacious for weight loss

Greenway et al. (38): Pilot 2

m-Synephrine 20 mg

Prospective, randomized, double blind

Twenty subjects (1:1 ratio) between supplement group and placebo group

8 wk

Supplement group lost weight (0.8 ± 3.4 kg; not significant) in 8 wk, and increased RMR in 8 wk. No control group was used (Greenway, written communication, November 1, 2009)

m-Synephrine was not efficacious for weight loss

Abbreviations: BMI, body mass index; CAP, Citrus aurantium product; RCT, randomized, controlled trial; RMR, resting metabolic rate.

and fat percentage. No significant changes were noted in resting energy expenditure, blood chemistries, or dietary intake between the placebo and experimental groups. In a double-blind, placebo-controlled, randomized trial, Colker et al. found that subjects receiving a combination of C. aurantium, caffeine, and St. John’s wort, along with diet and exercise protocols, lost a statistically significant amount of body weight. Analysis comparing changes in this group with those in placebo or control groups on the same diet and exercise regimen did not show significant differences, though loss of fat mass was significantly greater in the experimental group (35). BP, heart rate, electrocardiographic, blood, and urine analyses were not significantly different between the groups.

Another randomized trial (36) of 30 overweight adults investigated the effects of supplementation, along with a cross-training exercise regimen and dietary education program compared with exercise and dietary education alone on body composition. Supplementation included ephedrine, SAs, caffeine, and calicine. Greater weight and fat loss occurred for the supplement group compared with the exercise–diet only group. Overall, studies indicate a weight loss of 2.4–3.4 kg among participants using SAs, while placebo groups lost 0.94–2.05 kg, suggesting the plausibility of some weight loss benefit from SA supplementation, beyond diet and exercise alone. However, these studies do not separate

Bitter Orange

the effects of C. aurantium or SAs from other ingredients, particularly ephedrine and caffeine.

Metabolic Rate and Cardiovascular Effects Several studies have evaluated the effects of acute administration of SAs on cardiovascular indicators. Kalman et al. (39) tested a product containing 335 mg Ma Huang standardized for 20 mg ephedrine alkaloids, 910 mg guarana standardized for 200 mg caffeine, and 85 mg bitter orange standardized for 5 mg SAs per two capsules. Twentyseven overweight adults were randomized to treatment or placebo for 14 days. BP, heart rate, electrocardiogram, and Doppler echocardiograms were evaluated before and after treatment. Ingestion of this commercial weight loss supplement did not produce any detectable cardiovascular side effects. Penzak et al. (10) examined cardiovascular outcomes in 12 normotensive individuals who were administered 8 oz of Seville orange juice (containing 13–14 mg SAs) and water in a crossover fashion, followed by a repeat ingestion 8 hours later. No changes in cardiovascular indices (BP, maximal arterial pressure, and heart rate) were detected. Thomas et al. (40) evaluated the cardiovascular effects of 10 mg oral SAs in healthy volunteers over a 4-hour period on impedance cardiography and forearm plethysmography. Elevation in total peripheral resistance was observed 30–60 minutes after dosing, although other hemodynamic indexes were not affected. Hemodynamic effects were observed in a crossover design, placebo-controlled study (41) with the administration of Xenadrine, a CAP that contains a variety of other potentially bioactive substances, including green tea extract, cocoa extract, yerba mate, ginger root, grape seed extract, and others. However, these increases in heart rate, and systolic and diastolic BP were not observed with administration of Advantra Z, which contains C. aurantium alone, even at an eightfold higher dose. Haller et al. (42) evaluated a dietary supplement [Ripped Fuel Extreme Cut, containing synephrine from C. aurantium (presumably p-synephrine) and caffeine] in 10 healthy adults (three women) aged 20–31 years. Each subject was given one dose of the dietary supplement under three conditions: (i) resting conditions (without placebo); (ii) moderately intense exercise; and (iii) placebo plus moderately intense exercise in a three-arm, randomized, crossover study. Greater postexercise diastolic BP was seen with the dietary supplement plus exercise than with placebo plus exercise. There were no obvious supplement effects on postexercise HR, systolic BP, or body temperature. Bui et al. (43) reported the effect on BP (systolic and diastolic) and heart rate over 6 hours after one dose of a CAP (Nature’s Way Bitter Orange) on 15 young, healthy adults in this prospective, randomized, double-blind, placebo-controlled, crossover study. Systolic and diastolic BP increased significantly within the 1–5 hours time period in comparison with the placebo group with the peak being 7.3 ± 4.6 mm Hg, while the 4–5 hours time period increase was 2.6 ± 3.8 mm Hg after consumption in comparison with the placebo group with the peak being 4.2 ± 4.5 beats/minute, while

55

heart rate was significantly elevated 2–5 hours after ingestion. In one study of obese adults, increases in resting metabolic rate (RMR) were observed with C. aurantium, both alone and with food, beyond the thermic effect of food (TEF) alone (44). (RMR is a measure of the energy required to maintain basic physiological function while the body is at rest.) However, another recent investigation (45) found that the thermic response to CAPs increased in women only, who had lower TEF than men at baseline. After the intervention, TEF did not differ by gender. BP and pulse rate were not affected, but epinephrine secretion increased. In normal weight adults, an increase in RMR was also found when the extract was taken with a meal (46). No adverse changes in pulse rate or BP were reported. Finally, the effects of two dietary supplement formulas on RMR and other metabolic indicators were evaluated (47). When compared with placebo, Formula A (containing ephedra, guarana, green tea, yohimbe, and quercetin) and Formula B (containing C. aurantium, jing jie, fang feng, guarana, green tea, yohimbe, and quercetin) resulted in increased total RMR, decreased respiratory exchange ratio toward fat burning, and increased body core temperature. Heart rate and RMR increased at each 15-minute interval with Formula A only. BP increased with both, but to a greater extent with Formula A.

CASE REPORTS OF ADVERSE EVENTS Nykamp et al. (48) describe a case of acute lateral-wall myocardial infarction co-occurring with consumption of CAPs in a 55-year-old woman with undetected coronary vascular disease. She reported taking a multicomponent dietary weight loss supplement containing 300 mg of bitter orange over the preceding year. A Consumer Reports article (49) describes a 21-yearold woman who took ephedra-free Xenadrine EFX (which contains C. aurantium). After 3 weeks on the supplement, she suffered a seizure. Her neurologist believes the bitter orange in the supplement was the most likely the cause, though the basis for this conclusion is unknown. Nasir et al. (50) described exercise-induced syncope in a healthy 22-year-old woman that occurred 1 hour after a second dose of Xenadrine EFX, a weight loss supplement that contains, among other compounds, ephedrine and synephrine. The electrocardiography revealed prolongation of the QT interval, which resolved in 24 hours. Bouchard et al. (51) report a case of a 38-year-old male patient with ischemic stroke that occurred after taking a CAP for 1 week. The patient reportedly had no relevant medical history or major atherosclerotic risk factors and took no other medications. Gray and Woolf (52) reported a case of CAPs use by an adolescent with anorexia nervosa and raised concerns that the SAs may have masked bradycardia and hypotension while exacerbating her weight loss. Firenzuoli et al. (53) report a case of a 52-year-old woman that had an allergic reaction after taking a CAPs product. Sultan et al. (54) reported a case of a 52-year-old woman with ischemic colitis that occurred 1 week after consumption of a CAP (Natural Max Skinny Fast, containing bitter orange). She reported no known drug

56 Table 2

Haaz et al.

Summary of Effects, Safety, and Efficacy of Citrus aurantium

Physiological effects

Effects on weight

Effects on body composition

Safety

Variable changes in BP in animals; generally stable BP, heart rate, pulse rate, blood and urine measures in humans; inconsistent changes to resting metabolic rate

Weight loss documented in rodents; weakly supported in humans, as studies used multiple supplements or did not find significant difference from controls

Limited support for loss of fat mass in human studies, noting a trend or using multiple supplements; for animals, some increased lipase activity

Inconsistent mortality data in rodents; some evidence of elevated BP. Results not consistent from study to study, but this may be a function of small sample sizes used in most studies. Several case reports of serious adverse events

allergies and took no other medications. Symptoms resolved over 24–48 hours with conservative management after the supplement was discontinued. Health Canada reported that from January 1, 1998, to February 28, 2004, it received 16 reports in which products containing bitter orange or synephrine were suspected of being associated with cardiovascular events, including tachycardia, cardiac arrest, ventricular fibrillation, transient collapse, and blackout. All cases were considered serious (55). Adverse events from CAPs are currently fairly rare in scientific literature. As CAPs are used more widely in place of ephedrine-containing products, any potentially harmful effects may be clarified over time.

It is important to note that the majority of studies evaluating the safety of CAPs are performed with normotensive subjects. However, because hypertension is a common comorbidity associated with overweight/obesity, studies that evaluate the effects of CAPs on BP should also be conducted with obese hypertensive adults. While C. aurantium extracts have been used in a variety of cultures for thousands of years, they have not been traditionally utilized for long periods of time, or specifically for weight loss (1). As such, there is little, if any, basis for making definitive statements about the intermediate or long-term safety/risk of CAPs used for weight loss. Table 2 summarizes the physiological effects, safety, and weight loss efficacy of C. aurantium.

DISCUSSION The Safety of CAPs

DOSE CONSIDERATIONS

Some have hailed the potential therapeutic value of CAPs (1), while others have warned about possible safety concerns (33). The safety concerns pertain primarily to adverse cardiovascular and cerebrovascular effects. Information on the safety of CAPs comes from the three sources described above: animal studies, clinical trials, and case reports. To date, no large epidemiologic (case control or cohort) studies have evaluated the safety of CAPs. Of course, one cannot extrapolate the safety of CAPs from short-term studies used for one indication (e.g., several days for relief of nasal congestion among the general population) to long-term studies use for another indication (e.g., several months or years for weight loss among obese individuals). Although substantial safetyrelated data exist for CAPs (13,56), there is no published human weight loss trial of CAPs with more than 20 participants or for a duration of more than 7 weeks.

Given the dearth of weight loss trials, the optimal dose (if one exists) of C. aurantium or its SA constituents for weight loss is unknown. Table 3 highlights some relevant dosage information. Although generalizing across species and compounds is difficult and can only provide a limited basis for conjecture, the following comparisons with ephedrine can be made. We analyzed data (12) in which ephedrine or SAs was given to mice. Regression of weight and food intake on dose of ephedrine or SAs yielded slopes (in absolute value) that were approximately four to six times greater for ephedrine than for SAs. Based on linear projections, it would take four to six times the dose of SAs (in these mice) to achieve equivalent reduction in intake and body weight as for ephedrine. In human studies of ephedrine, doses of about 50 mg per day begin to be effective (57). Although an extrapolation, this might suggest a useful clinical dose for SAs as high as 240–360 mg

Table 3

Dosage Information on Citrus aurantium or Synephrine Alkaloids (SAs)

Dose 5–14 mg/day

Citrus aurantium extract with SAs has been used (34–36) and no serious adverse events were reported. These doses purportedly showed efficacy, but products tested included substances beyond C. aurantium, notably ephedrine which we know to be effective for weight loss. We believe that these doses of SAs are very unlikely to be effective when used without ephedrine

32 mg/day

The nasal decongestant Endal (60) contains 20 mg of m-s per tablet and two tablets per dose twice per day are recommended

120 mg/day

Via C. aurantium extract, SAs are marketed in over-the-counter (OTC) products for weight loss. In products, such as Nutres Lipo 6 (61), the directions suggest that for “extreme fat loss” a recommended dosage is two capsules three times per day. The SA content per capsule is 20 mg; this provided a maximal recommended dose of 120 mg/day

300 mg/day

According to Clarke’s Analysis of Drugs and Poisons (62), oxedrine (p-synephrine) is used clinically at ∼300 mg/day

1000 mg/day

Minimum adult lethal dose of m-s (63)

Bitter Orange

per day. From a safety point of view, SAs (per equal weight) have lower potential to raise BP than ephedrine; however, nearly all commercial preparations of SAs also contain caffeine, which might compound any cardiovascular effects. In the absence of caffeine, human studies suggest that 15–30 times the dose of SAs are required to elevate BP to the same degree as ephedrine (58,59). This suggests that such high doses might be well tolerated, but clearly more data are needed, particularly regarding potential synergistic effects of CAPs components. SAs appear to be readily absorbed after oral administration (63). About 80% of oral doses are excreted in the urine within 24 hours. After single oral doses, peak plasma concentrations are typically reached in 1–2 hours. Plasma half-life is ∼2–3 hours. Sympathomimetic drugs for weight loss are typically given TID before meals (64) reducing the evening dose if sleep problems arise.

CONTRAINDICATIONS Topical application (as with aromatherapy or antifungal uses) of CAPs may result in photosensitivity for fairskinned individuals (65) (possibly due to photosensitizing furanocoumarins that occur in the rinds of certain citrus species, especially immature fruits). Although rare, this has also occurred after oral ingestion. To reduce this risk, exposure to ultraviolet light can be minimized. Caution is recommended for use in children, as it may conceivably produce toxic effects (66). Some sources advise that CAPs should be avoided by women who are pregnant or breast-feeding (7,67), while others claim that CAPs can be used safely during pregnancy (66). While effects on BP are unclear, those with hypertension, tachyarrhythmia, or narrow-angled glaucoma may consider refraining from use of CAPs until further evidence confirms their safety (67). CAPs could also possibly exacerbate symptoms for those with stomach or intestinal ulcers (68).

DRUG INTERACTIONS Because CAPs may increase stomach acid, they could potentially reduce the efficacy of acid-lowering drugs, such as antacids and ulcer medications (69). Although a speculative precaution, those taking medications containing SAs, including some cold medications and monoamine oxidase inhibitors (MAOIs), should consider the combined dose of these products with the SAs present in CAPs formulations and possible multiplicative effects (68,69). It has been suggested that CAPs could interfere with the activity of drugs that are metabolized by the liver enzyme cytochrome P450-3A, CYP3A (70,71). A recent comment in Experimental Biology and Medicine noted that some research on drug effects have utilized parts of the plant or methods of administration that may not be applicable to oral consumption of currently marketed dietary supplements (72).

FUTURE RESEARCH The safety and efficacy of CAPs and SAs for weight loss are not well established. While existing literature

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demonstrates plausibility for reducing weight, previous trials were not designed to rigorously evaluate safety and efficacy. Doing so will require better-designed randomized clinical trials with large sample sizes, reliable wellestablished outcome measures, and active surveillance of side effects and adverse events. To better understand the effects of CAPs or SAs specifically, studies will need to test these components without combining them with other ingredients postulated to have antiobesity effects. It would also be worthwhile to examine differences between the types of synephrine-containing compounds that are derived from various sources and how this influences the consistency and potency of supplements.

ACKNOWLEDGMENTS The writing of this entry was supported in part by NIH grant nos. P30DK056336, AR49720–01A1, and T32HL072757. The opinions expressed are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or any other organization with which the authors are affiliated. Disclosure: Dr. Allison has received grants, honoraria, consulting fees, and donations from numerous companies, government agencies, and nonprofit organizations with interests in obesity in general and dietary supplements in particular, including organizations litigating cases involving C. aurantium.

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33. Calapai G, Firenzuoli F, Saitta A. Antiobesity and cardiovascular toxic effects of Citrus aurantium extracts in the rat: a preliminary report. Fitoterapia 1999; 70:586– 592. 34. Iaccarino G, Rockman HA, Shotwell KF, et al. Myocardial overexpression of GRK3 in transgenic mice: evidence for in vivo selectivity of GRKs. Am J Physiol 1998; 275:H1298– H1306. 35. Colker CM, Kalman DS, Torina GC, et al. Effects of Citrus aurantium extract, caffeine, and St. John’s wort on body fat loss, lipid levels, and mood states in overweight healthy adults. Curr Ther Res 1999; 60:145–153. 36. Kalman DS, Colker CM, Shi Q, et al. Effects of a weightloss aid in healthy overweight adults: double-blind, placebocontrolled clinical trial. Curr Ther Res 2000; 61:199–205. 37. Armstrong WJ, Johnson P, Duhme S. The effect of commercial thermogenic weight loss supplement in body composition and energy expenditure in obese adults. J Exerc Physiol 2001; 4:28–35. 38. Greenway F, de Jonge-Levitan L, Martin C, et al. Dietary herbal supplements with phenylephrine for weight loss. J Med Food 2006; 9(4):572–578. 39. Kalman D, Incledon T, Gaunaurd I, et al. An acute clinical trial evaluating the cardiovascular effects of an herbal ephedra-caffeine weight loss product in healthy overweight adults. Int J Obes Relat Metab Disord 2002; 26:1363–1366. 40. Thomas SH, Clark KL, Allen R. et al. (A comparison of the cardiovascular effects of phenylpropanolamine and phenylephrine containing proprietary cold remedies. Br J Clin Pharmacol 1991; 32:705–711. 41. Haller CA, Benowitz NL, Jacob P. Hemodynamic effects of ephedra-free weight-loss supplements in humans. Am J Med 2005; 118:998–1003. 42. Haller AA, Duan M, Jacob P III, et al. Human pharmacology of a performance-enhancing dietary supplement under resting and exercise conditions. Br J Clin Pharmacol 2008; 65(6):833–840. 43. Bui LT, Nguyen DT, Ambrose PJ. Blood pressure and heart rate effects following a single dose of bitter orange. Ann Pharmacother 2006; 40:53–57. 44. Pathak B, Gougeon R. Thermic effect of Citrus aurantium in obese subjects. Curr Ther Res 1999; 60:145–151. 45. Gougeon R, Harrigan K, Tremblay JF, et al. Increase in the thermic effect of food in women by adrenergic amines extracted from Citrus aurantium. Obes Res 2005; 13: 1187–1194. 46. Hedrei P, Gougeon R. Thermogenic Effect of Beta Sympathicomimetic Compounds Extracted from Citrus aurantium. Canada: McGill Nutrition and Food Science Center, Royal Victoria Hospital, 1997. 47. Shugarman AE, Askew EW, Stadler DD, et al. Effect of thermogenic dietary supplements on resting metabolic rate in healthy male and female volunteers. Med Sci Sports Exerc 2004; 31:S164. 48. Nykamp DL, Fackih MN, Compton AL. Possible association of acute lateral-wall myocardial infarction and bitter orange supplement. Ann Pharmacother 2004; 38:812–816. 49. Dangerous supplements: still at large. Consum Rep 2004; 69(5):12–17. 50. Nasir JM, Durning SJ, Ferguson M, et al. Exercise-induced syncope associated with QT prolongation and ephedra-free Xenadrine. Mayo Clin Proc 2004; 79:1059–1062. 51. Bouchard NC, Howland MA, Greller HA, et al. Ischemic stroke associated with use of an ephedra-free dietary supplement containing synephrine. Mayo Clin Proc 2005; 80:541– 545. 52. Gray S, Woolf AD. Citrus aurantium used for weight loss by an adolescent with anorexia nervosa. J Adolesc Health 2005; 37:414–415.

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62. Moffat AC, Osselton MD, Widdop B, et al. Clarke’s Analysis of Drugs and Poisons. London, England: Pharmaceutical Press, 2004. 63. Sweetman SC. Phenylephrine. In: Martindale: The Complete Drug Reference. London, England: Pharmaceutical Press, 2004. 64. Bray GA, Greenway FL. Current and potential drugs for treatment of obesity. Endocr Rev 1999; 20:805–875. 65. Herbal Medicine: Expanded Commission E Monographs. Newton, MA: Integrative Medicine Communications, 1999. 66. American Herbal Products Association’s Botanical Safety Handbook. Boca Raton, FL: CRC Press, 1998. 67. Jellin JM. Natural Medicines Comprehensive Database. Stockton, CA: Therapeutic Research Faculty, 2006. 68. Brinker F. Herb Contraindications & Drug Interactions. Sandy, OR: Eclectic Medical Publications, 2001. 69. Jellin JM. Natural Medicines Comprehensive Database. Stockton, CA: Therapeutic Research Faculty, 2002. 70. Guo LQ, Taniguchi M, Chen QY, et al. Inhibitory potential of herbal medicines on human cytochrome P450-mediated oxidation: properties of umbelliferous or citrus crude drugs and their relative prescriptions. Jpn J Pharmacol 2001; 85: 399–408. 71. Gurley BJ, Gardner SF, Hubbard MA, et al. In vivo assessment of botanical supplementation on human cytochrome P450 phenotypes: Citrus aurantium, Echinacea purpurea, milk thistle, and saw palmetto. Clin Pharmacol Ther 2004; 76:428– 440. 72. Dentali SJ. Comment on Citrus aurantium Minireview. Exp Biol Med (Maywood) 2005; 230:102.

Black Cohosh Daniel S. Fabricant, Elizabeth C. Krause, and Norman R. Farnsworth

United States Pharmacopeia (USP) from the first edition in 1820 to 1936 and in the National Formulary from 1936 to 1950. The eclectic physicians used a preparation of black cohosh called macrotys. It was considered one of the bestknown, specific medicines for heavy, tensive, and aching pains as it was noted to have a direct influence on the female reproductive organs. While the mechanism of action has not been completely elucidated, recent literature suggests that alleviation of climacteric symptoms is mediated through neurotransmitter regulation and not through classical estrogen receptor (ER) endocrine pathways (3,4).

INTRODUCTION Black cohosh is a native eastern North American plant that was used as traditional medicine by Native Americans. Extracts of the roots and rhizomes were used for analgesic, sedative, and anti-inflammatory properties. More recently, root and rhizome black cohosh preparations have had a rich clinical history, spanning almost 60 years of study. These studies have primarily focused on relieving climacteric symptoms associated with menopause as a possible alternative to classical hormone or estrogen replacement therapy.

BACKGROUND CHEMISTRY

The common name for black cohosh [Actaea racemosa L. syn., Cimicifuga racemosa (L.) Nutt. (Ranunculaceae, Buttercup Family)] originated with North American Indians. The term cohosh is thought to be an Algonquian word meaning “rough,” with reference to the texture of the thick, knotted roots and underground stems (rhizomes). A New World plant used by Native Americans, it was most abundant in the Ohio River Valley, but it could also be found from Maine to Wisconsin, south along the Allegheny Mountains to Georgia, and west to Missouri. Various common names have been used to refer to black cohosh, including black snakeroot, bugbane, rattleroot, squawroot, and macrotys. It is a member of the Ranunculaceae or Buttercup family, which includes other medicinal plants such as aconite, goldenseal, and pulsatilla. It has been known by the scientific name C. racemosa and recently has been reassigned to A. racemosa. The generic name Cimicifuga derives from the Latin cimex (a kind of bug) and fugare (to put to flight), which is perhaps indicative of the use of some strongly smelling close relatives to repel insects. The specific epithet racemosa refers to the flowering stalk, termed a raceme. The name rattleroot is indicative of the rattling sound made by the dry seeds in their pods. This plant prefers the shade of rich open hardwood forests, but it will tolerate some sunny spots. Black cohosh has been used clinically for relief of climacteric symptoms for more than 60 years, and its popularity in the United States as a botanical dietary supplement has increased due to the recently recognized potential risks associated with classical estrogen replacement therapy or hormone replacement therapy (1,2). The part of the black cohosh plant used in medicinal preparations is the root and rhizome. It was officially recognized in the

More than 60 triterpene glycosides, most with a 9,19 cycloartane skeleton, and unique to Actaea spp., have been reported from the roots and rhizomes of A. racemosa (5,6). The compound 23-epi-26-deoxyactein (formerly 27-deoxyactein) is the constituent usually selected for standardization of commercial products based on its abundance in the roots and rhizomes (7–12). The pharmacokinetics of 23-epi-26-deoxyactein in serum and urine has recently been reported (13). While triterpenes are structurally similar to steroids and possess a broad range of biological activity (14–17), no significant ligand binding affinity was found toward ER-␤ in the evaluation of 23epi-26-deoxyactein, cimiracemoside F and cimicifugoside, and their respective aglycones (18). This, coupled with the lack of demonstrated estrogenic activity in A. racemosa extracts, has called into question the notion that black cohosh acts through direct ER binding by the triterpenes, as has been hypothesized (19–23). In addition to the triterpene saponins, the roots and rhizomes of black cohosh also contain a number of aromatic acids/polyphenols that possess a wide array of biological activities (5,24–26). Caffeic acid, which is found widely across all species of flowering plants, has shown pregnant mare antigonadotropin activity (27–29), rat uterine antispasmodic activity (30), and smooth muscle relaxant/antispasmolytic activity in rats (31) and guinea pig ileum (32). Ferulic acid, also more or less ubiquitous among flowering plants, has demonstrated luteinizing hormone (LH) release inhibition (33), follicle-stimulating hormone (FSH) release stimulation (33), antiestrogenic activity (34), prolactin stimulation in cows (35) and inhibition in rats (33), and uterine relaxant/antispasmolytic activity in rats (36). Fukinolic acid produced an estrogenic effect on MCF-7 cells with reference to estradiol 60

Black Cohosh

(37). A more recent study refuted this effect and demonstrated a lack of estrogenic effect for 10 other phenolic esters, many of which are unique to Actaea spp. (caffeoylglycolic acid; 2-caffeoylpiscidic acid (cimicifugic acid D); 3,4-dihydroxyphenacyl caffeate (petasiphenone); 3,4dihydroxyphenyl-2-oxopropyl isoferulate (cimiciphenol); 3,4-dihydroxyphenacyl isoferulate (cimiciphenone); cimicifugic acids A, B, E, F; and fukiic acid) from black cohosh (38). Studies on the phenolic acid constituents of black cohosh have shown antioxidant activity (24,39) that may correlate with or prove useful in the determination of the mechanism of action of black cohosh. In addition, a number of plant sterols and fatty acids, generally regarded as ubiquitous in the plant kingdom, are contained in the roots and rhizomes for which the biological activities probably do not relate to the mechanism of action of black cohosh (5). In the past 5 years, novel guanidine alkaloids have been isolated from A. racemosa underground parts (40,41). New phytochemical methodology called pH zone refinement gradient centrifugal partitioning chromatography coupled with a sensitive liquid chromatography–mass spectral dereplication method led to the identification of N-(omega)-methylserotonin as a potential active principle with serotonergic properties (41). Alkaloids have also been reported from other Actaea spp. roots and rhizomes (42,43). There has been some debate over the occurrence of the weakly estrogenic compound formononetin in the plant (44–49). Although there has been at least one report of its occurrence in A. racemosa (46), prior studies using plant material collected from different sites in the Eastern United States at different times of the year failed to find formononetin (47,48). More recent studies on both commercial black cohosh products and wild-crafted material, incorporating both high-performance liquid chromatography with mass spectral and photodiode array detection, confirmed the prior findings of no detectable formononetin in black cohosh (8,49).

BOTANICAL DESCRIPTION A. racemosa syn. C. racemosa is an erect, smooth-stemmed perennial 1–2.5 m in height. Large compound leaves are alternately arranged and triternate on short clasping petioles. Basal leaf petioles are grooved in young specimens. This shallow, narrow sulcus in A. racemosa disappears as the petiole enlarges, whereas it remains present throughout the life of the two related eastern North American species, A. cordifolia DC syn. C. rubifolia Kearney and A. podocarpa DC syn. C. americana Michx (50). Terminal leaflets of A. racemosa are acute and glabrous with sharp serrated margins, often trilobate, occasionally bilobed. Fruits are ovoid follicles occurring sessile on the pedicel. The flowering portion, the raceme, is a long wandlike structure with showy white flowers. The flowers possess numerous characteristic stamen and slender filaments with distinctive white anthers (51). The roots and rhizomes are branched and knotted structures with a dark brown exterior and are internally white and mealy or brown and waxy. The upper rhizome surface has several buds and numerous large stem bases terminated frequently by deep, cup-shaped, radiating scars, each of which show a radiate

61

structure or less frequently fibrous strands. Lower and lateral surfaces exhibit numerous root scars and a few short roots. The fracture is horny, the odor slight, and the taste bitter and acrid (52).

EFFECTS ON CLIMACTERIC SYMPTOMS RELATED TO MENOPAUSE With a history of clinical study spanning almost 60 years, mainly in Europe (53), black cohosh is one of the more popular alternatives to hormone replacement therapy. Most of the clinical research over this span has been performed R on the product known as Remifemin , whose formula has changed over the years. However, a number of other commercial formulations are also available. In 2007, black cohosh was the 50th best-selling dietary supplement in the United States with sales of approximately $52 million (USD), according to the Nutrition Business Journal (54). Black cohosh clinical study outcomes have been evaluated using a variety of tools, including self- or physician assessments of symptom scores and physiological parameters. Typical measurements include psychological, neurovegetative, somatic, and physiological markers of menopause or relief from the climacteric symptoms of menopause. As in all clinical trials, study design is vital, so studies that are adequately powered, incorporate proper controls, and are designed to address confounders relevant to climacteric symptoms such as the placebo effect and botanical product quality should be given more weight than studies that are not as well designed (55–59). Placebo effects in menopausal trials are generally large (60) and reflect underlying fluctuations of symptoms. Therefore, any well-designed study must adjust the appropriate variables (i.e., study duration, number of subjects (n), and/or dosage) to account for such an effect. In the evidence-based medicine model, the gold standard in terms of efficacy involves randomized, controlled trials (RCTs). Many RCTs on black cohosh exist. When high-quality studies are combined, more than 3000 subjects have been randomized, with the more recent studies adding layers of design sophistication. For example, double-blind, multicenter, placebo-controlled trials that provide details regarding clinical material specifications are becoming more prevalent (55–60). A recent phase III, double-blind, randomized, placebo-controlled crossover trial of the effectiveness of black cohosh for the management of hot flashes was conducted over two 4-week periods (one capsule, 20 mg bid) (61). The study used a daily hot flash diary and found that subjects receiving the black cohosh material reported a mean 20% decrease in hot flash score (comparing the fourth treatment week to the baseline week) versus a 27% decrease for patients on placebo (P = 0.53), mean hot flash frequency was reduced 17% in the black cohosh group and 26% on placebo (P = 0.36). Thus, the authors concluded that the study did not provide any evidence that black cohosh reduced hot flashes more than the placebo. Critics of the study point to the short duration and low dose as potential confounders of the results. The Herbal Alternatives for Menopause trial or HALT trial compared the efficacy of 160 mg daily black

62

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cohosh against several other interventions (200 mg daily multibotanical with black cohosh and nine other ingredients; 200 mg daily multibotanical plus dietary soy counseling; 0.625 mg daily conjugated equine estrogen with or without 2.5 mg medroxyprogesterone acetate daily; and placebo) in 351 menopausal and postmenopausal women of ages 45–55 years with two or more vasomotor symptoms per day. Results did not suggest efficacy for any of the herbal interventions when compared with placebo at any time point over the 1-year course of the study (62). The Jacobson study (63), spanning only 60 days of treatment, suggests that the short study duration may have limited the findings (60). In addition, all the study participants had a history of breast cancer. The authors reported that the median number of hot flashes decreased 27% in both the placebo and black cohosh groups. No significant differences were observed between groups. Thus, black cohosh, on the basis of this study, was no more effective than placebo in the treatment of hot flashes. The source and formulation of the extract used in this study was not specified. A more recent open-label study that R treated breast cancer survivors with either Tamoxifen or a combination of BNO 1055, a proprietary black cohosh extract, with Tamoxifen suggested a reduction in the number and severity of hot flashes in the combination treatment group (64). In another randomized, double-blind, placebocontrolled study that lasted 12 weeks, black cohosh was compared with standard conjugated estrogen (CE) therapy (0.625 mg/daily). Patients’ physical and psychological symptoms were measured every 4 weeks. The end result of the study was that the patients treated with black cohosh had significantly lower index scores on both the Kupperman menopausal (KM) and the Hamilton menopausal (HAM-A) scales compared with placebo, indicating a decrease in severity and frequency of hot flashes. In addition, this study showed an increase in the number of estrogenized cells in the vaginal epithelium in the black cohosh treatment arm, which could indicate an estrogenic action in this tissue (65). In 2003, a similar study compared effects of two different preparations of BNO 1055 extract and CE therapy on climacteric symptoms and serum markers of bone metabolism (66). The study outcomes were evaluated using patient self-assessment (diary and menopause rating scale), CrossLaps (to measure bone resorption), bone specific alkaline phosphatase (marker of bone formation), and endometrial thickness (measured by ultrasound). Both BNO 1055 extracts were equipotent to CE therapy and significantly greater than placebo at reducing climacteric complaints. In addition, the study showed that both BNO 1055 preparations had beneficial effects on bone metabolism in serum. Specifically, an increase in bone-specific alkaline phosphatase and no reduction in bone resorption were noted indicating an increase in bone turnover formation. No change in endometrial thickness was observed in either BNO 1055 treatment groups, but it was significantly increased with CE therapy. An increase in superficial vaginal cells was observed in the CE and both BNO 1055 treatment groups. The authors of the study hypothesized that the activity of both BNO 1055 preparations was similar to the effects of selective estrogen recepR tor modulating (SERM), that is, Raloxifene therapy on

bone and neurovegetative climacteric symptoms, without any uterotrophic effects (66). A recent high-quality, double-blind, randomized study evaluated the effects of two dosages (low, 39 mg; high, 127 mg) of a Remifemin extract on menopausal symptoms. Effectiveness was measured using the KM index, self-assessment depression scale (SDS), clinical global impression scale (CGI), serum levels of LH and FSH, sex hormone–binding globulin, prolactin, 17-␤-estradiol, and vaginal cytology. Reductions in the KM and SDS indices were significant. Global efficacy (CGI) was scored at good to very good in 80% (low dosage) and 90% (high dosage) of the patients in the treatment groups (67). No effect on serum hormone levels or vaginal cytology was shown, prompting the authors of the study to suggest that black cohosh does not have a direct estrogenic effect on the serum hormone levels or vaginal epithelium (68). Two recent open-label studies using unspecified types of extracts reported reduced KM index scores. One study reported a significant reduction in 1 month (69), while the other, which also used the HAM-A scale, recorded a 90% improvement in climacteric symptoms in menopausal women after 3 months of black cohosh administration (70). Chung and colleagues (71) examined a combination R of black cohosh and St. John’s wort (Gynoplus ) in a multicenter RCT in 89 peri- or postmenopausal women with climacteric symptoms. Subjects were treated for 12 weeks with either the Gynoplus extract or placebo. In addition to climacteric complaints, investigators also examined effects on vaginal atrophy, serum hormone levels (FSH, LH), and lipid profiles [total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, and triglyceride]. Significant improvements in climacteric symptoms and hot flashes, as well as an increase in HDL, were observed in the Gynoplus group by 4 weeks and maintained after 12 weeks, but there was no significant impact on vaginal atrophy. In a 12-month, randomized, four-arm, double-blind clinical trial of standardized black cohosh, red clover, placebo, and 0.625 mg conjugated equine estrogens plus 2.5 mg medroxyprogesterone acetate (conjugated equine estrogens (CEE) and medroxyprogesterone acetate (MPA); n = 89), black cohosh did not significantly reduce the frequency of vasomotor symptoms as compared with placebo. The primary outcome measures were reduction in vasomotor symptoms (hot flashes and night sweats) by black cohosh and red clover compared with placebo; secondary outcomes included safety evaluation, reduction in somatic symptoms, relief of sexual dysfunction, and overall improvement in quality of life. Reductions in number of vasomotor symptoms after a 12-month intervention were as follows: black cohosh (34%), red clover (57%), placebo (63%), and CEE/MPA (94%), with only CEE/MPA differing significantly from placebo. Secondary measures indicated that both botanicals were safe as administered. In general, there were no improvements in other menopausal symptoms (72). A 12-week trial investigating the effects of black cohosh on menopause-related anxiety disorder found no statistically significant anxiolytic effect of black cohosh versus placebo. However, small sample size, choice of black cohosh preparation, and dosage used may have

Black Cohosh

contributed to the negative results according to the study’s authors (73). More details of the human studies discussed here, as well as others, are presented in Table 1.

BIOCHEMISTRY AND FUNCTIONS Despite the extensive clinical research, the mechanism of action of black cohosh on menopausal and other symptoms remains unclear, which is consistent with the varied results from clinical trials. A majority of the older literature suggest a direct estrogenic effect. More recent hypotheses have proposed an effect on the limbic system (hypothalamus) or an effect on the neurotransmitters involved in regulation of this system as being responsible for the activity of black cohosh. Data fall into the following categories.

Estrogen Receptor Competitive Binding The first report of ER-binding activity of black cohosh indicated this as a possible mechanism of action (74). Additional studies were carried out to substantiate this purported endocrine activity (75,76). However, a factor frequently overlooked regarding black cohosh receptor binding studies is the lipophilic nature of the extracts tested. Chemically, lipophilic extracts and fractions that display ER-binding activity are significantly different from the typical hydroalcoholic extracts used to make products for human consumption. A lipophilic extract of the plant showed relatively weak (35 ␮g/mL) ER binding on rat uteri (75). Another study also confirmed the ERbinding activity of an unspecified lipophilic subfraction on ovariectomized (ovx) rat uterine cells, with no binding activity seen with a hydroalcoholic extract (76). Recent reports have contradicted the ER-binding affinity of black cohosh extracts (4,20,22,77,78). A root extract tested in an in vitro competitive cytosolic ER (from livers of ovx rat) binding assay with diethylstilbesterol (50), an inhibitor of estrogen binding, showed a significant inhibition of estradiol binding in the presence of diethylstilbesterol (77). However, no binding was demonstrated for the black cohosh extract. A hydroalcoholic A. racemosa rhizome extract (50% aqueous ethanol) was assayed for ER binding in intact human breast cancer cell lines MCF-7 and T-47-D. Again no binding affinity was shown for the black cohosh extract. However, binding activity was evident for other hydroalcoholic plant extracts, such as red clover (78). In another study, a high concentration (200 ␮g/ mL) methanol extract of black cohosh displayed no binding affinity for recombinant diluted ER-␣ and ER-␤ (20). A study using BNO 1055 showed contrasting results (79). The extract displayed dose-dependent competition with radio-labeled estradiol in both a porcine and human endometrial cytosolic ER ligand-binding assay system. However, the extract did not displace human recombinant ER-␣ and ER-␤. These contradictory findings prompted the authors to suggest that their product contains estrogenic compounds that have binding affinity for a putative ER-␥ . The absence of a direct estrogenic effect was again confirmed in a human study (21). Postmenopausal women took black cohosh extract for 12 weeks followed by a 12week washout. Black cohosh demonstrated no effect on

63

estrogenic markers in serum and no effect on pS2 or cellular morphology in nipple aspirate fluid (21).

Receptor Expression As with the receptor-binding assays, the nature of the extract or fraction is a decisive factor in the expression of ERs. A lipophilic and hydrophilic black cohosh extract was studied for luciferase expression in a MCF-7 ␣- and ␤-ER expressing subclone (80). The lipophilic extract at 35 ␮g/mL activated transcription of the estrogenregulated genes, while the hydrophilic extract showed no activity. A recent study measuring an extract at a low concentration (4.75 ␮g/L) increased ER levels in human MCF7 cells as did estradiol (81). An unspecified black cohosh extract tested in a transient gene expression assay using HeLa cells co-transfected with an estrogen-dependent reporter plasmid in the presence of human ER-␣ or ER-␤ cDNA failed to show transactivation of the gene (82).

Plasma Hormone Levels The effect of black cohosh on serum concentrations of FSH and LH has been studied extensively. Crude alcoholic extracts suppressed plasma LH with no effect on FSH in ovx rats (75,77). Further fractionation of the crude extract resulted in activity of the lipophilic fraction while the hydrophilic fractions were devoid of this activity (74). A later study in rats using lipophilic and hydrophilic extracts at high doses (140 and 216 mg/rat, IP) resulted in LH suppression with a single injection administration of the lipophilic but not the hydrophilic extract (75). Another study reported LH suppression in ovx rats with an unspecified dose of black cohosh extract (83). A recent study compared the effect of BNO 1055 with that of estradiol on LH levels (79). Extract administered subcutaneously at a dosage of 60 mg/day for 7 days was reported to reduce LH levels in the treated animals. However, another study reported no estrogen agonistic effects on FSH, LH, or prolactin levels in ovx rats using the 7,12Dimethylbenz(a)anthracene model following 7 weeks of daily administration of a 40% isopropanolic extract of the plant (Remifemin) (84).

Hormonal Secretion The effect of black cohosh on prolactin secretion in pituitary cell cultures was measured using an unspecified extract (85). Basal and Thyrotropin-releasing hormone (TRH)-stimulated prolactin levels were significantly reduced at doses of 10 and 100 ␮g/mL. This effect was reversed by the addition of haloperidol (D2 -antagonist) to the cell cultures, suggesting dopaminergic regulation of hormone secretion by black cohosh.

Osteopenia Inhibition The black cohosh extract BNO 1055 (60 mg/rat, SC) has been shown to increase the expression of collagen I and osteocalcin in rats in a manner similar to that produced by 8 ␮g of estradiol in ovx rats (79). An additional study using BNO 1055 demonstrated an osteoprotective effect as shown by a reduced loss of bone mineral density in rat tibia after 3 months of administration (81). A study using an unspecified isopropanol extract of black cohosh showed reduced urinary markers of bone loss. The authors

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Fabricant et al.

Table 1

Selected Black Cohosh Clinical Studies

Author (reference no.)

Year

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

Kesselkaul (110)

1957

R Remifemin 60 drops

2 wk

63

Alleviation of climacteric complaints in 95% of patients

Case series

Schotten (111)

1958

Remifemin 20 drops

3–4 wk

22

Alleviation of neurovegetative and psychic complaints associated with menopause and premenopause

Case series

Foldes (53)

1959

Remifemin, 3 tablets/day

Unknown

41

31 patients of the verum group responded to the treatment with a decrease in menopausal complaints

Placebo, controlled, open, crossover, patient self-assessment

Starfinger (112)

1960

Remifemin, 3–20 drops/day

1 yr

105

Decreased climacteric complaints without incidence of side effects or resulting in nonphysiological bleeding

Case series

Brucker (113)

1960

Remifemin, tablets, variable dose

Variable

87 (517)

Alleviation of menopausal complaints

Case series

Heizer (114)

1960

Remifemin, tablets 3–6/day

2–18 mo

66

Alleviation of menopausal (neurovegetative and psychic) complaints in 47% of patients with intact uteri and 35% with hysterectomies

Case series

Gorlich (115)

1962

Remifemin, tablets, variable dose

Variable

41 (258)

Alleviation of climacteric and vascular symptoms in 85% of patients

Case series

Schildge (116)

1964

Remifemin, fluid extract 60 drops/day

Variable

135

Euphoric and mild sedative-calming effects in all pts

Case series

Stolze (117)

1982

Remifemin, fluid extract 80 drops/day

6–8 wk

629

Alleviation of neurovegetative and psychological menopausal symptoms in 80% of patients

Open, physician and patient self-assessment

Daiber (118)

1983

Remifemin, fluid extract 80 drops/day

12 wk

36

Alleviation of climacteric complaints (hot flashes, insomnia, sweating, and restlessness)

Open, KMI, CGI

Vorberg (119)

1984

Remifemin, fluid extract 80 drops/day

12 wk

50

Significant or highly significant alleviation of menopausal (neurovegetative and psychic) complaints; study included subjects contraindicated to hormone therapy

Randomized, open, KMI, CGI, POMS

Warnecke (120)

1985

Remifemin, fluid extract 80 drops/day

12 wk

20

Significant alleviation of symptoms (psychic and neurovegetative) in the black cohosh, conjugated estrogen, and diazepam groups. Vaginal cytology of treatment group was comparable to estrogenic stimulation

Randomized, open, KMI, HAM-A, SDS, CGI, karyopyknosis index, eosinophil index

Stoll (121)

1987

Remifemin, tablets equivalent to 8 mg extract/day

12 wk

26

Significant alleviation of climacteric symptoms (vaginal atrophy, neurovegetative and psychic complaints) in comparison with estrogen and placebo groups

Double-blinded, randomized, placebocontrolled, KMI, HAM-A, VMI (vaginal epithelium)

Petho (122)

1987

Remifemin, tablets, unspecified dose

6 mo

50

KMI decreased significantly from 17.6 to 9.2, correlates with a significant reduction in neurovegetative symptoms. Severity of subjective self-assessments of subjects physical and psychological symptoms decreased

Open, KMI, patient self-assessment

Lehman-Willenbrock and Riedel (123)

1988

Remifemin, tablets equivalent to 8 mg extract/day

6 mo

15

Significant alleviation of climacteric symptoms in black cohosh and drug treatment groups. No significant change in gonadotropin (FSH, LH) levels

Randomized, open, KMI

Duker et al. (75)

1991

Remifemin, tablets equivalent to 40 mg dried herb/day

2 mo

110

LH suppression

In vitro study using blood from menopausal women taking black cohosh

Black Cohosh

Table 1

65

Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.)

Year

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

Baier-Jagodinski (124)

1995

R Cimisan T Tropfen,

4–8 wk

157

89% of patients showed symptom improvement after 4 wk. At final visit, the efficacy was assessed as very good, 40%; good, 41%; sufficient, 12%; inadequate, 7%

Open, uncontrolled

Mielnik (69)

1997

Uncharacterized extract, 4 mg daily

6 mo

34

Alleviation of climacteric (neurovegetative) symptoms in 76% of patients after 1 mo

Open, KMI

Georgiev and Iordanova (70)

1997

Uncharacterized extract, unspecified dose

3 mo

50

Alleviation of climacteric symptoms in 90% of patients. Increase in vaginal cell proliferation (VMI) in 40% of treated women

Open, KMI, HAM-A, VMI

Nesselhut and Liske (125)

1999

Remifemin, tablets, equivalent to 136 mg dried herb/day

3 mo

28

Good to very good alleviation of 10 menopausal symptoms in 80% of study participants

Open, postmarket surveillance

Jacobson, et al. (63)

2001

Remifemin, tablets equivalent to 40 mg dried herb/day

60 days

42a

No change in median number or intensity of hot flashes

Double blinded, randomized, placebo controlled, patient self-assessment, VAS, MSS

Liske et al. (67)

2002

Unique Cimicifuga racemosa preparation, equivalent to 39 or 127.3 mg/day

6 mo

152

No direct systemic estrogenic effect on serum levels of FSH, LH, SHBG, prolactin, and 17-␤ estradiol. No change in vaginal cytology. Higher dose had a more significant reduction in KM index after 6 mo. Significant reduction with both doses in neurovegetative and psychic complaints

Drug equivalence trial, KMI, SDS, CGI

Hernandez Munoz and Pluchino (66)

2003

BNO 1055

12 mo

136

Combination therapy with tamoxifen (20 mg) reduced severity and incidence of hot flashes

Wuttke et al. (64)

2003

Klimadynon/BNO 1055

Open, randomized, patient self-assessment

3 mo

62

Equipotent to 0.6 CE for relief of climacteric complaints and for bone resorption. No effect on endometrial thickness

Randomized, double blinded, placebo controlled, multicenter, MRS

Verhoeven et al. (126)

2005

125 mg soy extract daily (providing 50 mg isoflavones including 24 mg genistein and 21.5 mg daidzein), 1500 mg evening primrose oil extract (providing 150 mg gamma linoleic acid), 100 mg Actaea racemosa L. extract (providing 8 mg deoxyacetein), 200 mg calcium, 1.25 mg vitamin D, and 10 IU vitamin E, placebo group received 2000 mg olive oil daily

12 wk

124

Subjects were experiencing at least five vasomotor symptoms every 24 hr at study entry. At weeks 6 and 12, all scores in both groups had improved compared with baseline, though the overall difference in scores between the groups was not statistically significant

Multicenter, randomized, placebocontrolled, double-blind study, Kupperman index and Greene Climacteric scale

Nappi et al. (127)

2005

Aqueous isopropanolic extract 40 mg/day

3 mo

64

Postmenopausal women were recruited. Both CR and low-dose TTSE2 significantly reduced the number of hot flushes per day (P < 0.001) and vasomotor symptoms (P < 0.001), starting at the first month of treatment. Such a positive effect was maintained throughout the 3 mo of observation, without any significant difference between the two treatments. An identical effect was evident also for both anxiety (P < 0.001) and depression (P < 0.001), which were significantly reduced following 3 mo of both CR and low-dose TTSE2. Total cholesterol was unchanged by CR treatment but significantly (P < 0.033) reduced by 3 mo of low-dose TTSE2. A slight but significant increase of HDL cholesterol

Randomized, controlled, clinical study

variable dose

R

(continued)

66 Table 1

Fabricant et al.

Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.)

Year

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

(P < 0.04) was found only in women treated with CR, while LDL-cholesterol levels were significantly lowered by 3 mo of both CR (P < 0.003) and low-dose TTSE2 (P < 0.002). Triglyceride levels were not affected by both treatments nor was liver function. FSH, LH, and cortisol were not significantly affected after the 3-mo treatment, while PRL (P < 0.005) and 17-␤-E2 (P < 0.001) were increased slightly only by low-dose TTSE2. Endometrial thickness was not affected by either CR or low-dose TTSE2 Frei-Kleiner et al. (128)

2005

6.5 mg dry rhizome extract; 60% ethanol extraction solvent. Dose = 1 cap daily

12 wk

122

Menopausal women were recruited. The primary efficacy analysis showed no superiority of the tested black cohosh extract compared with placebo. However, in the subgroup of patients with a Kupperman index > or = 20 a significant superiority regarding this index could be demonstrated (P < 0.018). A decrease of 47% and 21% was observed in the black cohosh and placebo group, respectively. The weekly weighted scores of hot flashes (P < 0.052) and the Menopause Rating Scale (P < 0.009) showed similar results. Prevalence and intensity of the adverse events did not differ in the two treatment groups

Multicenter, randomized, placebocontrolled, double-blind, parallel group study

Pockaj et al. (61)

2006

20 mg C. racemosa and rhizome extract standardized to contain 1 mg of triterpene glycosides as calculated by 27-deoxyacetin, placebo

Two 4-wk crossover treatment periods

132

Toxicity was minimal and not different by treatment group. Patients receiving black cohosh reported a mean decrease in hot flash score of 20% (comparing the fourth treatment week with the baseline week) compared with a 27% decrease for patients on placebo (P = 0.53). Mean hot flash frequency was reduced 17% on black cohosh and 26% on placebo (P = 0.36). Patient treatment preferences were measured after completion of both treatment periods by ascertaining which treatment period, if any, the patient preferred. Thirty-four percent of patients preferred the black cohosh treatment, 38% preferred the placebo, and 28% did not prefer either treatment

Double-blind, randomized, crossover clinical trial. Primary end point was the average intrapatient hot flash score (a construct of average daily hot flash severity and frequency) difference between the baseline week and the last study week of the first treatment period. Green Climacteric scale

Newton et al. (HALT) (62)

2006

(i) Black cohosh, 160 mg daily; (ii) multibotanical with black cohosh, 200 mg daily, and 9 other ingredients; (iii) multibotanical plus dietary soy counseling; (iv) conjugated equine estrogen, 0.625 mg daily, with or without medroxyprogesterone acetate, 2.5 mg daily; or (v) placebo

1 yr

351

Women aged 45–55 yr with two or more vasomotor symptoms per day were recruited. Vasomotor symptoms per day, symptom intensity, Wiklund Vasomotor Symptom Subscale score did not differ between the herbal interventions and placebo at 3, 6, or 12 mo or for the average over all the follow-up time points (P > 0.05 for all comparisons) with 1 exception: At 12 mo, symptom intensity was significantly worse with the multibotanical plus soy intervention than with placebo (P > 0.016). The difference in vasomotor symptoms per day between placebo and any of the herbal treatments at any time point was less than one symptom per day; for the average over all the follow-up time points, the difference was less than 0.55 symptom per day. The difference for hormone therapy versus placebo was −4.06 vasomotor symptoms per day for the average over all the follow-up time points (95% CI, −5.93 to −2.19 symptoms per day;

Randomized, double-blind, placebo-controlled trial. Wiklund Vasomotor Symptom scale

Black Cohosh

Table 1

67

Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.)

Year

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

P > 0.001). Differences between treatment groups smaller than 1.5 vasomotor symptoms per day cannot be ruled out. Black cohosh containing therapies had no demonstrable effects on lipids, glucose, insulin, or fibrinogen (124) Raus et al. (129)

2006

Dried aqueous/ethanolic (58% vol/vol) extract CR BNO 1055 of the rhizome of Actaea or CR (black cohosh)

1 yr

400

Postmenopausal women with symptoms related to estrogen deficiency were recruited. The lack of endometrial proliferation and improvement of climacteric complaints as well as only a few gynecologic organ-related adverse events are reported for the first time after a treatment period of 1 yr

Prospective, open-label, multinational, multicenter study. Endovaginal ultrasonography

Sammartino et al. (130)

2006

Group A (n = 40) was treated with 1 tablet/day per os containing a combination of isoflavones [soy germ extracts, Glycine max, no OGM-SoyLife: 150 mg, titrated in isoflavones (40%) = 60 mg], lignans [flaxseed extracts, Linum usitatissimum, no OGM-LinumLife: 100 mg, titrated in lignans (20%) = 20 mg] and C. racemosa [50 mg, titrated in triterpene (2.5%) = R 1.25 mg] (Euclim; Alfa Wassermann, Italy); group B (n = 40) was treated with calcium supplements (Metocal, Rottapharm, Monza, Italy)

Three cycles of 28 days

80

Healthy postmenopausal women were recruited. At baseline no significant difference was detected in KI between groups A and B; however, after three cycles of treatment, KI was significantly (P > 0.05) lower in group A compared with baseline and with group B

Double-blind, randomized, placebo-controlled trial, Kupperman index

Gurley et al. (131)

2006

Milk thistle (300 mg, three times daily, standardized to contain 80% silymarin), black cohosh extract (20 mg, twice daily, standardized to 2.5% triterpene glycosides), rifampin (300 mg, twice daily), and clarithromycin (500 mg, twice daily)

14 days

16

Young adults (8 females) (age, mean ± SD = 26 ± 5 yr; weight, 75 ± 13 kg) compared with the effects of rifampin and clarithromycin, the botanical supplements milk thistle and black cohosh produced no significant changes in the disposition of digoxin, a clinically recognized P-gp substrate with a narrow therapeutic index. Accordingly, these two supplements appear to pose no clinically significant risk for P-gp-mediated herb–drug interactions

Randomized controlled, clinical pharmacokinetic trial

Rebbeck et al. (132)

2007

Varied

Case-control design

949 breast cancer cases; 1524 controls

HRS varied significantly by race, with African American women being more likely than European American women to use any herbal preparation (19.2% vs. 14.7%, P = 0.003) as well as specific preparations including black cohosh (5.4% vs. 2.0%, P > 0.003), ginseng (12.5% vs. 7.9%, P < 0.001) and red clover (4.7% vs. 0.6%, P < 0.001). Use of black cohosh had a significant breast cancer protective effect (adjusted odds ratio 0.39, 95% CI: 0.22–0.70). This association was similar among women who reported use of either black cohosh or Remifemin (a herbal preparation derived from black cohosh; adjusted odds ratio 0.47, 95% CI: 0.27–0.82)

Population-based case–control study

(continued)

68 Table 1

Fabricant et al.

Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.)

Year

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

Hirschberg et al. (133)

2007

Remifemin (batch no. 229690), one tablet twice daily. Each tablet contains 0.018–0.026 mL liquid extract of black cohosh rootstock (0.78–1.14:1) corresponding to 20 mg herbal drug [i.e., 2.5 mg dry extract, extraction agent isopropanol 40% (vol/vol)], 40 mg/day

6 mo

74

None of the women showed any increase in mammographic breast density. Furthermore, there was no increase in breast cell proliferation. The mean change ± SD in proportion of Ki-67-positive cells was 0.5% ± 2.4% (median, 0.0; 95% CI = −1.32–0.34) for paired samples. The mean change in endometrial thickness ± SD was 0.0 ± 0.9 mm (median, 0.0). A modest number of adverse events were possibly related to treatment, but none of these were serious. Laboratory findings and vital signs were normal

Prospective, open, uncontrolled drug safety study

Chung et al. (71)

2007

Gynoplus (264 mg tablet with 0.0364 mL Cimicifuga racemosa rhizome, equivalent to 1 mg terpene glycosides; 84 mg dried Hypericum perforatum extract, equivalent to 0.25 mg hypericin, with 80% methanol)

12 wk

89

Kupperman index (KI) for climacteric complaints. Vaginal maturation indices, serum estradiol, FSH, LH, total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglyceride levels. Significant improvements in climacteric symptoms and hot flashes, as well as an increase in HDL (from 58.32 ± 11.64 to 59.74 ± 10.54) were observed in the Gynoplus group by 4 wk and maintained after 12 wk, compared with the placebo group. There was no significant impact on superficial cell proportion

Randomized, double-blind, placebo-controlled trial

Ruhlen et al. (22)

2007

Remifemin R and CimiPure (2.5% triterpenes; 40 mg capsule contains 1 mg 23-epi-26-deoxyactein)

12 wk followed by 12 wk washout

61

Subjects experienced relief of menopausal symptoms, with reversion to baseline after washout. No effect on serum estrogenic markers. No effect on pS2 or cell morphology in nipple aspirate

Open study

Gurley et al. (134)

2008

Milk thistle (300 mg, three times daily, standardized to contain 80% silymarin), black cohosh extract (40 mg, twice daily, standardized to 2.5% triterpene glycosides), rifampin (300 mg, twice daily), and clarithromycin (500 mg, twice daily)

14 days

19

Young adults [9 women; age (mean ± SD) = 28 ± 6 yr; weight = 76.5 ± 16.4 kg]. Milk thistle and black cohosh appear to have no clinically relevant effect on CYP3A activity in vivo. Neither spontaneous reports from study participants nor their responses to questions asked by study nurses regarding supplement/medication usage revealed any serious adverse events

Randomized controlled, clinical pharmacokinetic trial

Amsterdam et al. (73)

2009

12 wk

28 (15 treatment/ 13 placebo)

The primary outcome measure was changed over time in total HAM-A scores. Secondary outcomes included a change in scores on the Beck Anxiety Inventory, Green Climacteric Scale (GCS), and Psychological General Well-Being Index (PGWBI) and the proportion of patients with a change of 50% or higher in baseline HAM-A scores. There was neither a significant group difference in change over time in total HAM-A scores (P = 0.294) nor a group difference in the proportion of subjects with a reduction of 50% or higher in baseline HAM-A scores at study end point (P = 0.79). There was a significantly greater reduction in the total GCS scores during placebo (vs. black cohosh; P = 0.035) but no group difference in change over time in the GCS subscale scores or in the PGWBI (P = 0.140). One subject (3.6%) taking black cohosh discontinued treatment because of adverse events

Randomized, double-blind, placebo-controlled trial

Black Cohosh

Table 1

69

Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.)

Year

Geller et al. (72)

2009

Extract/formulation/dosage

Study length

N

Outcome measure/result

Study design

12 mo

89

Primary outcome measures were reduction in vasomotor symptoms (hot flashes and night sweats) by black cohosh and red clover compared with placebo; secondary outcomes included safety evaluation, reduction of somatic symptoms, relief of sexual dysfunction, and overall improvement in quality of life. Reductions in number of vasomotor symptoms after a 12-mo intervention were as follows: black cohosh (34%), red clover (57%), placebo (63%), and CEE/MPA (94%), with only CEE/MPA differing significantly from placebo. Black cohosh and red clover did not significantly reduce the frequency of vasomotor symptoms as compared with placebo. Secondary measures indicated that both botanicals were safe as administered. In general, there were no improvements in other menopausal symptoms

Randomized, double-blind, placebo-controlled trial

Studies listed by year of publication. a All with breast cancer history. Abbreviations: CGI, Clinician’s Global Impression scale; HAM-A, Hamilton Anxiety scale; KMI, Kupperman Menopausal Index; MSS, unspecified menopausal index using the Likert scale; Open, open-labeled; POMS, Profile of Mood States Scale; SDS, Self-Assessment Depression scale; VAS, Visual Analog Scale; VMI, Vaginal Maturity Index.

of this study suggested the action was similar to that of the SERM Raloxifene (86). A follow-up study using BNO 1055 versus CE therapy showed beneficial effects of the extract on bone metabolism in humans, specifically an increase in bone-specific alkaline phosphatase in serum (64). While no direct correlation between species has been established, it is of note that studies of Asian Cimicifuga species have demonstrated similar activity and may be of importance for further investigation of this biological activity (87,88).

Uterine Weight/Estrous Induction Uterine and ovarian weight increase, cell cornification, and an increased duration of estrous are generally considered evidence of endometrial estrogenic activity. However, it has recently been proposed that uterine weight is a poor marker for endometrial effects (89). Three studies demonstrating that black cohosh extracts increased the uterine weight of ovx rats have been reported (50,77,90) with two of the studies using an undescribed root extract (77,90). One study on immature mice reported similar findings (50). By contrast, two studies on ovx rats (79,91), as well as four studies on immature mice, reported the converse (79,81,83,92). One of these studies found that although there was no increase in uterine or ovarian weight, the duration of estrous was significantly increased by black cohosh (92). A subsequent study by the authors and collaborators demonstrated no attenuation in uterine weight at variable doses (4, 40, and 400 mg/ kg/day) of a 40% isopropanol extract in ovx rats (4).

Cell Proliferation An unspecified black cohosh extract failed to significantly induce growth of MCF-7 cells when compared with untreated control cells (81). A study using isopropanolic and

ethanolic extracts also failed to induce growth of MCF-7 cells (93).

CNS Effects and Neurotransmitter Binding A murine study using an unspecified extract (25–100 mg/kg, orally) measured effects on body temperature and ketamine-induced sleep time using bromocriptine (D2 agonist) as a positive control. Pretreatment with sulpiride (D2 blocker) suggested a receptor-mediated dopaminergic effect (84). An additional mouse study was carried out to characterize neurotransmitter levels in the striatum and hippocampus after pretreatment with the extract for 21 days (94). Serotonin and dopamine metabolic levels in the striatum were substantially lower in comparison with the control group. These studies have led to the hypothesis that dopaminergic, rather than estrogenic, activity is responsible for the reported success of black cohosh in reducing climacteric symptoms (95,96). A study by the authors and collaborators has pointed to the effects of black cohosh being mediated by serotonin (5-HT) receptors (4). Three different extracts (100% methanol, 40% isopropanol, 75% ethanol) were found to bind to the 5-HT7 -receptor subtype at IC50 ≤ 3.12 ␮g/mL. The 40% isopropanol extract inhibited (3 H)-lysergic acid diethylamide binding to the 5-HT7 receptor with greater potency than (3 H)-8-hydroxy-2(di-N-propylamino)tetralin to the rat 5-HT1A . Analysis of ligand-binding data suggests that the methanol extract functioned as a mixed competitive ligand of the 5HT7 receptor. Further testing of the methanol extract in 293T-5-HT7 transfected HEK cells raised cAMP levels; these raised levels were reversed in the presence of the 5-HT antagonist methiothepin, indicating a receptor-mediated process and possible agonist activity local to the receptor (4).

70

Fabricant et al.

Antioxidant A black cohosh methanol extract protected S30 breast cancer cells against menadione-induced DNA damage at variable concentrations and scavenged DPPH free radicals at a concentration of 99 ␮M (38).

USE IN PREGNANT/LACTATING WOMEN Despite an absence of mutagenic effects reported to date, the use of black cohosh during pregnancy is contraindicated according to WHO suggestions (97). Data are inconclusive regarding the effects on lactation.

DOSAGE (97,98) Recommended doses for black cohosh are as follows: 1. Dried rhizome and root: 1 g up to three times daily. 2. Tincture (1:10): 0.4 mL daily (40–60% alcohol vol/vol). 3. Fluid extract (1:1): 20 drops twice daily (60% ethanol vol/vol, equivalent to 40 mg dried herb). 4. Tablet equivalence: two tablets a day (equivalent to 40 mg dried extract). The Commission E monograph also recommends that usage not be extended for more than 6 months due to a lack of long-term safety data. Experimental data are not available to suggest this 6-month limit.

ADVERSE EFFECTS/SAFETY A majority of adverse event reports (AERs) for black cohosh have been associated with Remifemin products, probably due to its widespread use. Thus, the AER data may speak more to the safety of this particular product rather than black cohosh extracts in general. In clinical trials, minor cases of nausea, vomiting, dizziness, and headaches have been reported (61–73). An analysis of the safety data from published clinical trials, case studies, postmarketing surveillance studies, spontaneous report programs, and phase I studies was carried out (99). The data obtained from more than 20 studies, including more than 2000 patients, suggest that adverse event occurrence with black cohosh is rare, and that such events are mild and reversible, the most common being gastrointestinal upset and rashes. The same review investigated black cohosh preparation and AERs and concluded that adverse events are rare, mild, and reversible (99). That said, black cohosh has garnered a great deal of attention with respect to its safety over the past 5 years, with the emergence of a few case reports citing acute hepatitis, convulsions, cardiovascular, and circulatory insult (100–104). It is important to note that in a number of these reports, no effort was made to positively identify the botanical associated with the event as black cohosh. In one case, depositions taken during a legal proceeding revealed that the lack of alcohol consumption and concomitant medications reported in a published case report (101) was inaccurate (105). Underreporting of adverse effects may also be a common problem with botanical supplement (100–104). However, these case reports have gen-

erated much interest within the research community, so much so that two workshops have been convened by the National Institutes of Health (NIH) on the specific issue of the safety of black cohosh preparations: one workshop sponsored by the National Center for Complementary and Alternative Medicine (NCCAM) and the Office of Dietary Supplements (ODS) in November 2004 and a more recent workshop sponsored by the ODS held in June 2007. The report from the 2004 workshop indicated that there is “no plausible mechanism of liver toxicity.” The 2007 workshop offered no conclusions on safety to contradict those of the 2004 meeting regarding hepatotoxicity of black cohosh preparations. The 2007 workshop did recommend that active steps be taken to monitor liver health in human clinical trials of black cohosh (106). It is also noteworthy that in the 2004 workshop, it was agreed that “suspected hepatotoxicity should not be broadcast when toxicity has not been demonstrated.” Despite concerns by some scientists, a warning statement on commercial black cohosh product labels was mandated in Australia by the Therapeutic Goods Administration (TGA), and the European Medicines Agency (EMEA) released a press statement on July 18, 2006, urging patients to stop taking black cohosh if they develop signs suggestive of liver injury. It is noteworthy that it is not clear and has never been fully disclosed as to how these agencies reached their decision and what the scientific data were that led to these warning statements. While the notion of idiosyncratic hepatotoxicity was raised in the June 2007 workshop by toxicologists from the Food and Drug Administration (FDA), it was acknowledged by these toxicologists that without data from a mandatory adverse event reporting system, no real conclusion on causality regarding idiosyncratic hepatotoxicity can be drawn from case reports. In the September–October 2007 edition of USP’s Pharmacopeial Forum (100), the USP proposed the addition of a cautionary statement for USP quality black cohosh products with regard to liver toxicity. The American Botanical Council (ABC) responded that given the long history of safe black cohosh use and the lack of clear scientific evidence for toxicity, there is not enough information for such a warning. The ABC noted that of the 42 case reports of toxicity cited by the USP, only 18 met criteria for assessment based on a standard-rating scale, and of these, 3 met criteria for “possible” toxicity, and 2 for “probable” toxicity. Many case reports were also said to lack adequate documentation regarding the actual identity of the black cohosh used and possible confounding factors (107).

COMPENDIAL/REGULATORY STATUS Black cohosh products are regulated and marketed in the United States as dietary supplements under the provisions of the Dietary Supplement Health and Education Act (DSHEA) of 1994 (U.S.C. § 321). Dried black cohosh rhizome and roots, powdered black cohosh, black cohosh fluid extract, powdered black cohosh extract, and black cohosh tablets now have official standing in dietary supplement monographs in the United States Pharmacopoeia-– National Formulary (108). In the European Union nations,

Black Cohosh

black cohosh products are approved as nonprescription phytomedicines when administered orally in compliance with the German Commission E monographs (109).

CONCLUSIONS With the elevated concern surrounding side effects related to classical hormone/estrogen therapy for menopause, modulation of certain climacteric symptoms of menopause by both dopaminergic and serotonergic drugs is becoming a more viable and frequent treatment option. A review of the clinical trials associated with black cohosh leads to the conclusion that women using hydroalcoholic extracts of the rhizomes and roots of this plant may gain relief from climacteric symptoms (i.e., hot flashes) in comparison with placebo over the short term, whereas longer studies have not shown the same degree of efficacy. Further clouding the review of these clinical trials is the wide variety and different types of extracts administered in published studies. Early in vitro studies reported that black cohosh extracts acted on ERs or had a sort direct effect on ERs. Now it is becoming clear that the beneficial effect of reducing hot flashes is related, at least in part, to serotonergic or dopaminergic mechanisms that regulate hypothalamic control and possibly mediate estrogenic mechanisms. As mentioned earlier, the controversy surrounding a purported direct estrogenic mechanism of action may also be due to variance in the extracts assayed. Overall, given variation in trial length, extract types, and other potential confounders, the efficacy of black cohosh as a treatment for menopausal symptoms is uncertain and further rigorous trials seem warranted.

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Blue-Green Algae (Cyanobacteria) Wayne W. Carmichael and Mary Stukenberg with Joseph M. Betz

INTRODUCTION In Asia, Africa, and parts of Central/South America, naturally occurring green and blue-green algae have been harvested and consumed for their nutritive properties for centuries. In western cultures, for approximately 30 years, certain fresh water blue-green algae (cyanobacteria) have been accepted as a source of food, in particular Spirulina (Arthrospira) platensis and Spirulina maxima. Beginning in the early 1980s, another blue-green species, Aphanizomenon flos-aquae (AFA), was adopted for similar uses. Both are rich in proteins, vitamins, essential amino acids, minerals, and essential fatty acids. Consumers of blue-green algae report a wide variety of putative effects, such as mental clarity, increased energy, blood and colon cleansing, increased focus, particularly in children with attention deficit disorder, improved digestion, increased eye health, healthier joints, and tissues. In the past 10 years, owing largely to the strong anecdotal consumer testimony about them, studies have been conducted to verify not only their nutritional efficacy but also their potential pharmaceutical benefits as well.

Crypthecodinium, Tetraselmis, Skeletonema, Isochrysis, and Chaetoceros. Within the cyanobacteria, Spirulina (Arthrospira) platensis and S. maxima have been commercially produced as a human and animal food supplement and food coloring for approximately 30 years. Spirulina is cultured in constructed outdoor ponds in Africa, California, Hawaii, Thailand, China, Taiwan, and India. World production in 1995 was approximately 2 × 106 kg. The newest cyanobacterium to be used as a food supplement is AFA, the production of which differs significantly from Spirulina, because it is harvested from a natural lake rather than constructed ponds. Since the early 1980s, this alga has been harvested from Upper Klamath Lake, Oregon, and sold as a food and health food supplement. The popularity of both Spirulina and AFA bluegreen algae products over other seaweeds and green algae may be attributable to the convenience of its packaging and consumption, as well as to its highly directed marketing to the health-conscious consumer. In 1998, the market for AFA as a health food supplement was approximately US $100 million with an annual production greater than 1 × 106 kg (dry weight) (1–18).

BACKGROUND

Chemistry and Preparation Edible blue-green algae are nutrient dense food. The features common to all blue-green algae include a high content of bioavailable amino acids and minerals, such as zinc, selenium, and magnesium. The nutrient profile is subject to variation by habitat, harvest procedure, quality control for contaminating species, proper processing to preserve nutrients, and storage conditions, all of which influence the vitamin content and antioxidant properties delivered by the final product. However, the appeal of blue-green algae is their raw, unprocessed nature and their abundance of carotenoids, chlorophyll, phycocyanins, phytosterols, glycolipids, ␥ -linolenic acid, and other bioactive components (19–21). Approximately, 40 cyanobacteria species and genera produce potent toxins. Spirulina products have not been associated with toxicity reports in humans, largely owing to its being grown under cultured conditions (22). Natural samples and cultured strains of AFA have been reported to produce neurotoxins including paralytic shellfish poisons (neosaxitoxin and saxitoxins) and anatoxin-a. Recent work seems to indicate that a different Aphanizomenon species is the toxin producer. A. flos-aquae has been reported to be dominant or codominant in water blooms containing Microcystis and Anabaena and is found in many eutrophic water bodies. Species of Microcystis can produce a family

Worldwide, algae, for thousands of years, have been a food source and treatment for various physical ailments. In coastal regions of the Far East, recorded use of macroalgae (sea weed) as a food source began approximately 6000 BC, with evidence that many species were used for food and medical treatment by around AD 900. The Spanish recorded the use of microalgae as a food source when they reported that the natives of Lake Texcoco collected cyanobacteria from the waters of the lake to make sundried cakes. In present day Africa, local tribes harvest cyanobacteria in the Lake Chad region, primarily Spirulina, and also use it to make hard cakes, called dihe. In some regions of Chad, people consume from 9 to 13 g/meal, constituting 10% to 60% of the meal. However, the longest recorded use of cyanobacteria as food is the consumption of Nostoc flagelliforme in China, where there are records of its use for some 2000 years and where it is still harvested on a large scale. Use of microalgae in the western culture began in the 1970s. Most commercial producers of microalgae are located in the AsiaPacific rim, where approximately 110 commercial producers of microalgae have an annual production capacity from 3 to 500 tons. These cultivated microalgae include Chlorella, Spirulina, Dunaliella, Nannochloropsis, Nitzschia, 75

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of potent liver toxins called microcystins. Cylindrospermin is a hepatotoxic and nephrotoxic compound produced by several freshwater cyanobacteria, including Cylindrospermopsis raciborskii and Anabaena spp (23). Several species of marine and freshwater cyanobacteria (including a number of Nostoc, Anabaena, and Microcystis species) produce the neurotoxic amino acid BMAA (␤-N-methylaminoL-alanine) (24). Although these toxic substances are probably not naturally present in the target species discussed below, the possibility that they might be present as contaminants in commercial products highlights the need for rigorous quality-control measures. Blue-green algae products most often come in a tablet form as algal material directly compressed. The tablets can contain fillers such as sugars or starches called binders, which give shape and stability to the tablets. Algae supplements also come in a capsule form to neutralize the taste and make the product easier to swallow, or can be bought by the pound in powder form or in liquid extract forms. Some companies combine the algae in “green supplements” that contain other health-enhancing ingredients such as alfalfa sprouts. Supplements come in kosher or vegetarian forms, and can be combined with digestive aids. Recommended dosages of blue-green algae products vary widely, but can be as much as 20 g/day. On the average, companies that produce algal products for consumption as nutritional supplements recommend 500 mg to 1 g/day to start, with a build up over time to several grams a day, often without an upper limit on consumption (25,26).

Efficacy Two types of blue-green algae form the major nutritional supplement groups, Spirulina and AFA. As the traits of each vary slightly, they are addressed separately below.

Spirulina The blue-green alga Spirulina was so named for its helically coiled trichomes or rows of cells. Until recently, Spirulina and Arthrospira were thought to belong to separate genera, and the distinction was thought to be especially important as only the strains of Arthrospira had been proven to be safe for human consumption. These two are now referred to as Arthrospira in scientific circles. Although the name Spirulina has been persisted for commercial labeling, the two are synonymous (27). Spirulina is generally produced in large outdoor ponds under controlled conditions. The safety of Spirulina for human food has been established through long use, and through various toxicological studies done under the auspices of the United Nations Industrial Development Organization (28). Spirulina is 60% to 70% protein by weight and contains many vitamins, especially vitamin B12 and ␤-carotene, and minerals, especially iron and ␥ -linolenic acid (Table 1). Recent reports suggest that a number of therapeutic effects and pharmaceutical uses are potential benefits of Spirulina as well (18). Most studies of the effects of Spirulina on enhanced body function have been performed on animals, and therapeutic effects have been demonstrated in some cases. Conclusive human studies are rare, but those that carry substantive results are cited below.

Table 1

Nutritional Profile of a Commercial Spirulina Product

Composition Per 100 g Macronutrientsa Calories Total fat Total carbohydrate Dietary fiber Protein Essential amino acids (mg) Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Nonessential amino acids (mg) Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Proline Serine Tyrosine Vitaminsb Vitamin A (as 100% ␤-carotene) Vitamin K Thiamine HCl (Vitamin B-1) Riboflavin (Vitamin B-2) Niacin (Vitamin, B-3) Vitamin B-6 (Pyridox.HCl) Vitamin B-12 Mineralsb Calcium Iron Phosphorus Iodine Magnesium Zinc Selenium Copper Manganese Chromium Potassium Sodium Phytonutrientsb Phycocyanin Chlorophyll Superoxide dismutase (SOD) ␥-linolenic acid (GLA) Total carotenoids ␤-Carotene Zeaxanthin Other carotenoids

Spirulina powder

382 7.1 g 15.5 g 6.8 g 55 g 900 3170 5030 2960 1290 2510 2770 740 3500 4110 4130 5670 580 9180 2860 2170 2670 2300 ≥200,000 IU 548 ␮g 0.13 mg 2.55 mg 14.3 mg 0.77 mg 93 ␮g 446 mg 56 mg 1010 mg 39.1 ␮g 305 mg 1.27 mg 19.6 ␮g 0.32 mg 3.0 mg 91.7 ␮g 1620 mg 815 mg 10 g 0.9 g 531,000 IU 1180 mg ≥370 mg ≥120 mg ≥95 mg ∼155 mg

This is a natural product and nutrient data may vary from one lot to anR other. One example of a nutrient profile for Earthrise Spirulina Powder, a commercial Spirulina product, is shown in the above table. a Macronutrient data are based on most recent proximate analysis. b The data indicate minimum values observed over a four-year period except for sodium where the maximum observed value is used.

Blue-Green Algae (Cyanobacteria)

Nutritional Rehabilitation A multicenter study of 182 malnourished children, aged 3 months to 3 years, reported that a 5 g/day dose of Spirulina (Arthrospira) platensis had no added benefit over 90 days when compared to traditional renutrition (29). Four groups of undernourished children under the age of 5 (550 total) were provided with Misola (60% millet flour, 20% soy, 10% peanut, 9% sugar, 1% salt), Misola plus 5 g of S. platensis, traditional meals, or traditional meals plus 5 g of Spirulina. All diets contained about the same number of kilocalories/day. The authors concluded that Misola, Spirulina plus Misola, and Spirulina plus traditional diet are all good food supplements for undernourished children, but that Misola plus Spirulina were superior to the other combinations (30).

Cardiovascular In ischemic heart disease patients, Spirulina supplementation was shown to significantly lower blood cholesterol, triglycerides, and LDL and very-low density lipoprotein cholesterol, and raise HDL (the so-called “good”) cholesterol. A 4 g/day supplementation showed a higher effect in reducing total serum cholesterol and LDL levels than did 2 g/day (31). In a small two-month study of the effects of 1 g/day of Spirulina (species not specified) plus medication versus medication alone on lipid parameters in pediatric hyperlipidemic nephritic syndrome patients, Samuels et al. (32) reported that supplementation of medication with Spirulina helped reduce increased lipid levels in these patients. Several studies in healthy populations have shown positive effects on cardiovascular endpoints. Consumption of Spirulina was found to reduce total lipids, free fatty acids, and triglyceride levels in a human study involving diabetic patients. A reduction in LDL/HDL ratio was also observed (33). A nonplacebo-controlled open label trial of 36 healthy adults administered 4.5 g/day of S. maxima for six weeks reported a hypolipidemic effect, especially on triacylglycerols and LDL = cholesterol, systolic, and diastolic blood pressure were also reduced (34). Effects of 8 g/day Spirulina (species not given) versus placebo on health-related endpoints in 78 healthy elderly Koreans were determined in a 16-week double-blinded trial. In the verum group, significant reductions in total plasma cholesterol and interleukin (IL)-6 concentrations were observed, along with increases in interleukin (IL)-2 concentrations and total antioxidant status (35). Ju´arez-Oropeza et al. (36) reported results of investigations of the effects of S. maxima on vascular reactivity in rats and lipid status and blood pressure in healthy humans. The authors suggest that the results of the rat portion of the study indicate that Spirulina induces a tone-related increase in endothelial synthesis/release of nitric oxide and of a vasodilating cyclooxygenase-dependent arachadonic acid metabolite (or a decrease in synthesis/release of an endothelial vasoconstricting eicosanoid). In the nonplacebocontrolled study of the effects of 4.5 g/day Spirulina on vascular and lipid parameters in 36 human volunteers, the authors reported reductions in blood pressure and plasma lipid concentrations (especially triacylglycerols and LDLcholesterol).

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Immune System Function Spirulina was found to have a positive effect on the immune system. In a paper presented at a meeting of the Japanese Society for Immunology, volunteers consuming a Spirulina drink for two weeks experienced enhanced immune system function, which continued for up to six months after the extract administration was discontinued (37). A follow-up study reported that administration of 50 mL of a hot water extract of S. platensis augmented interferon production and natural killer cell (NK) cytotoxicity in more than 50% of 12 healthy human volunteers (38). Results of a study on immunoglobulin-A in human saliva showed a significant correlation between the immunoglobulin-A level in saliva and the amount of Spirulina consumed (39). Much attention has been focused on the potential mitigation of allergies through Spirulina intake. A group of Russian researchers are pursuing a patent on their success with the normalization of immunoglobulin-E in children living in radioactive environments (40). In a more recent study of allergic rhinitis patients, the production of cytokines, critical in regulating immunoglobulin-E– mediated allergy, was measured. In a randomized doubleblind crossover study versus placebo, allergic individuals were fed daily with either placebo or Spirulina at 1000 or 2000 mg for 12 weeks. Although Spirulina seemed to be ineffective at modulating the secretion of Th-1 cytokines (one type of the so-called “killer” cells), the study reported that at 2000 mg/day, Spirulina significantly reduced IL-4 levels by 32% (41). A six-month double-blind placebocontrolled trial of the effects of 2 g/day of S. platensis on allergic rhinitis in 150 otherwise healthy individuals aged 19 to 49 reported that the cyanobacterium treatment significantly improved symptoms and physical findings including nasal discharge, sneezing, nasal congestion, and itching (42). Cancer The sole human cancer intervention study involving Spirulina intake was done in India on a group of tobacco chewers afflicted with oral leukoplakia. In a study involving 44 subjects in the intervention group and 43 in a placebo group, it was found that supplementation with 1 g of Spirulina per day for one year resulted in complete regression of lesions in 45% of the intervention group and in only 7% of the control group. As supplementation with Spirulina did not result in an increase in retinal ␤-carotene, the authors concluded that other components in Spirulina may be responsible for the regression of lesions observed (43). Other Endpoints A series of four N-of-1 double-blind randomized trials were performed on four individuals who complained of idiopathic chronic fatigue. Each patient was his own control and received three pairs of treatments comprising four weeks of S. platensis and four weeks of placebo in doses of 3 g/day. Outcome measures were severity of fatigue measured on a 10-point scale. The score of fatigue was not significantly different between Spirulina and placebo (44). A small study compared the effects of S. platensis plus a normal diet against soy protein plus normal diet in the prevention of skeletal muscle damage in untrained

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student volunteers. Sixteen subjects were divided into two equal groups (7.5 g/day S. platensis or soy protein). They were administered the Bruce incremental treadmill exercise prior to treatment, took the intervention for three weeks, and were then readministered the treadmill exercise. Results suggested that ingestion of Spirulina (but not soy protein) protected against skeletal muscle damage and may have led to postponement of the time to exhaustion during all-out exercise (45). Most of the research on Spirulina’s efficacy for human nutrition and pharmaceutical use has been concerned with the areas of vitamin and mineral enrichment, immune system function, antioxidant effects, and anticancer and antiviral effects. Although the number of studies referenced by Amha Belay for his Spirulina research review article in 1993 contained 41 references, 18 his review in 2002 contained 98, 17 and this chapter has added additional information, few of the human studies in almost any area can be said to be conclusive. Studies are small or very small, and most are open label nonplacebo-controlled studies. A number of the publications do not provide adequate information (many fail to identify the test cyanobacteria to the species level). Interesting results in both the human studies and in vitro and animal studies show that further research is merited.

Adverse Effects As previously noted, Spirulina products have not been associated with toxicity reports in humans, probably because commercial production is via large-scale culture rather than wild harvest (22).

AFA (Aphanizomenon flos-aquae) In western cultures, certain cyanobacteria have been an accepted source of microalgal biomass for food for approximately 30 years, in particular, as discussed earlier, Spirulina (Arthrospira) platensis and S. maxima. Beginning in the early 1980s, another species, AFA, was adopted for similar uses. Members of this genus are free floating (planktonic) and occur either singly or form feathery or spindle-shaped bundles, are cylindrical in shape, much longer than they are wide, and contain abundant gas vesicles. They occur in temperate climates and are most abundant in summer and fall (46). The only known commercial harvesting of AFA is from Upper Klamath Lake, the largest freshwater lake system in Oregon. In 1998, the annual commercial production of AFA was approximately 1 × 106 kg. As this species is not cultured like Spirulina in outdoor ponds or raceways, it requires very different procedures for harvesting and processing. Other procedures, such as those for removal of detritus and mineral materials, and those for monitoring and reducing the amounts of certain contaminant cyanobacteria, which can produce cyanotoxins, have also become important in quality control and marketing (47). The nutrient profile for AFA is very similar to that for Spirulina (Table 2). Consumers of AFA nutritional supplements report a variety of benefits from enhanced energy to boosted immune system function. Cited below are the peer-reviewed human studies extant in the literature confirming certain of these nutritive and pharmaceutical attributes.

Table 2

Nutritional Profile of a Commercial AFA Product

Nutrient General composition Protein Carbohydrate Calories Minerals (ash) Fat calories Cholesterol Total dietary fiber Sugar profile Dextrose (glucose) Fructose Maltose Sucrose Total sugars Minerals and trace metals Calcium Chloride Chromium Copper Iron Magnesium Manganese Molybdenum Phosphorus Potassium Selenium Sodium Zinc Vitamins Vitamin A (␤-carotene) Thiamin (B1) Riboflavin (B2) Pyridoxine (B6) Cobalamin (B12) Ascorbic acid (C) Niacin Folic acid Choline Pantothenic acid Biotin Vitamin D Vitamin E Vitamin K Amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Asparagine Alanine Glutamine Cystine Glycine Proline Serine Tyrosine Aspartic acid Glutamic acid Total amino acids

Units

Amount

% % % % cal/g mg/g %

55.1 29.1 3.7 6.8 0.3 0.3 5.7

mg/g mg/g mg/g mg/g mg/g

19.4 0.5 5.6 0.8 26.2

mg/g mg/g ␮g/g ␮g/g mg/g mg/g ␮g/g ␮g/g mg/g mg/g ␮g/g mg/g ␮g/g

8.5 2.0 1.2 10.5 0.7 1.8 31.2 4.7 4.7 10.6 0.4 2.5 12.1

IU/g ␮g/g ␮g/g ␮g/g ␮g/g mg/g mg/g ␮g/g mg/g ␮g/g ␮g/g IU/g IU/g ␮g/g

1523 19.0 44.9 14.6 3.7 0.4 0.4 0.6 1.3 3.1 0.2 0.4 0.1 47.7

mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g mg/g

29 9 25 43 29 9 21 29 6 29 49 39 78 3 23 20 25 16 46 49 579

Blue-Green Algae (Cyanobacteria)

Table 2

Nutritional Profile of a Commercial AFA Product (Continued)

Nutrient

Units

Amount

Lipid analysis Total lipid (fat) content Total saturated fat Total unsaturated fat Total essential fatty acids Total Omega-3 essential fatty acids ␣-Linolenic acid (ALA) Eicosapentanoic acid (EPA) Total Omega-6 essential fatty acids Linoleic acid (LA) Arachidonic acid (AA)

4.4% 43% 57% 45% 38% 37% 0.4% 8% 8% 0.1%

44 mg/g 19 mg/g 25 mg/g 20 mg/g 17 mg/g 16 mg/g 0.2 mg/g 3 mg/g 3 mg/g 0.04 mg/g

One example of a nutrient profile for Cell Tech Super Blue-Green Algae, a commercial AFA product, as of 4-22-05, is shown in the above table.

Circulation and Immune Function In a study examining the short-term effects of consumption of moderate amounts (1.5 g/day) of AFA on the immune system, it was discovered that AFA resulted in increased blood cell counts when compared to subjects taking a placebo. When the volunteers were grouped into long-term AFA consumers and na¨ıve volunteers, the na¨ıve volunteers exhibited a minor reduction in natural killer cells, and the long-term consumers exhibited a pronounced reduction. It was further determined that AFA does not activate lymphocytes directly, but that it does increase immune surveillance without directly stimulating the immune system. The authors of this study conclude that AFA has a mild but consistent effect on the immune system and could function as a positive nutritional support for preventing viral infections. They also recommend further research into AFA’s potential role in cancer prevention (48). In a later report, an aqueous extract of AFA found to contain a novel ligand for CD62L (L-selectin). Consumption of the extract by 12 healthy subjects in a doubleblind randomized crossover study caused mobilization of human CD34+ CD133+ and CD34+ CD133− stem cells (49). Eye Disease—Blepharospasm and Meige Syndrome A study to determine whether blue-green algae could be helpful in improving the eyelid spasms associated with essential blepharospasm and Meige syndrome was undertaken by a group of physicians. Although a few patients exhibited a positive effect, for most patients, neither the severity nor the frequency of facial spasms was significantly reduced (50). Adverse Effects No cases of human intoxication by AFA were found in the literature. As noted, around 40 cyanobacterial species have been reported to produce potent natural toxins. Certain A. flos-aquae strains were long thought to produce neurotoxins including the paralytic shellfish poisons neosaxitoxin and saxitoxin and anatoxin-a. A reevaluation of the species using gene sequencing data led to the conclusion that a different Aphanizomenon species was the actual toxin producer (51). While it now seems clear that AFA is not a toxin-producing species, the toxigenic Aphanizomenon species seems to be distinguishable from AFA only by the presence or absence of toxins and by genetic sequencing.

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No evidence exists to suggest that AFA is a toxinproducing strain (51), but water blooms of AFA may also contain Microcystis and Anabaena. Species of Microcystis can produce a family of potent liver toxins called microcystins, while Anabaena species can produce anatoxins and BMAA. The microcystins, especially, are of concern, since hepatic damage caused by this toxin is cumulative. This has led the State of Oregon to set a safe level of microcystin in AFA product from Klamath Lake. Currently this level is set at 1 ␮g/g dry weight of product, and was set to correspond to an average daily adult intake for AFA of 2 g. Recently, the cyanobacterial toxin anatoxin-a was reported in 3 of 39 cyanobacterial dietary supplement product samples at concentrations of 2.5 to 33 ␮g/g (52). Exposure guidelines have been summarized by Burch (23). This points to the need for rigorous quality-control measures in production of products. These measures may range from existing practices such as modifying the harvesting equipment to exclude Microcystis, harvesting the water bloom only at times when Microcystis is at a minimum, or developing and using toxin-detecting methods in an integrated testing scheme (47,53,54).

CONCLUSIONS As a natural source of many vitamins and minerals, proteins, and chlorophyll, it is not surprising that blue-green algae have attracted attention among those interested in natural sources of nutrition. Thousands of people consume blue-green algae in its most popular forms (Spirulina and AFA), and as a result, a large body of anecdotal material has existed for many years concerning the positive health benefits of blue-green algae consumption. The volume of the testimony has contributed to a growing interest in recent years in verifying these benefits through scientific research. Beginning with animal and in vitro studies, and moving toward human studies, scientists have only recently begun to investigate some of the positive health effects attested to by long-term consumers of blue-green algae. These include cholesterol reduction, weight loss, enhanced immune system function, regression of cancerrelated lesions, and enhancement of blood circulation, as well as many vitamin and mineral benefits. It must be stated, however, that there is an overall paucity of welldesigned, controlled human trials using blue-green algal products as interventions. As the industry relies on self-regulation, it is important to be aware of the quality-control issues involved in harvesting and packaging blue-green algae for consumption, particularly in the case of AFA, which is harvested from the wild. The World Health Organization has determined through current knowledge of microcystin toxins and what it calls a tolerable daily intake (TDI), an estimate of the intake over a lifetime that does not constitute an appreciable health risk. This TDI is derived through existing knowledge of toxin tolerance in mice combined with principles used in defining the health risks of other chemicals. It carries with it a degree of uncertainty owing to the lack of long-term data for the effect of microcystin on humans (55). To be sure that their risk has been minimized as much as possible, consumers of blue-green algae supplements would be wise to check to see that the product has been

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tested for toxins, and it has been found to be below the WHO/Oregon Department of Health Regulatory Level of 1 ␮g/g of microcystin (56).

ACKNOWLEDGMENTS The authors would like to thank Jerry Anderson (Cell Tech, Inc.) and Diana Kaylor (Wright State University) for supplying a number of references used in this text.

REFERENCES 1. Hoppe A. Marine algae and their products and constituents in pharmacy. In: Hoppe HA, Levring T, Tanaka Y, eds. Marine Algae in Pharmaceutical Science. New York: Walter de Gruyter, 1979:25–119. 2. Richmond A. Handbook of Microalgal Mass Culture. Boca Raton, FL: CRC Press, 1990. 3. Cannell RJP. Algal biotechnology. Appl Biochem Biotech 1990; 26:85–105. 4. Ciferri O. Spirulina, the edible microorganism. Microbiol Rev 1983; 47:551–578. 5. Farrar WV. Tecuitlatl: A glimpse of Aztec food technology. Nature 1966; 5047:341–342. 6. Ciferri O, Tiboni O. The biochemistry and industrial potential of Spirulina. Ann Rev Microbiol 1985; 39:503–526. 7. Abdulqader G, Barsanti L, Tredici MR. Harvests of Arthrospira platensis from Lake Kossorom (Chad) and its household usage among the Kanembu. J Appl Phycol 2000; 12:493–498. 8. Delpeuch F, Joseph A, Cavelier C. Consumption as food and nutritional composition of blue-green algae among populations in the Kanem region of Chad. Ann Nutr Aliment 1975; 29:497–516. 9. Gao K. Chinese studies on the edible blue-green alga, Nostoc flagelliforme: A review. J Appl Phycol 1998; 10:37–49. 10. Becker EW, Venkataraman LV. Production and processing of algae in pilot plant scale experiences of the Indo-German Project. In: Shelef G, Soeder CJ, eds. Algae Biomass, Production and Use. Amsterdam: Elsevier/North Holland Biomedical Press, 1980:35–50. 11. Lee YK. Commercial production of microalgae in the Asia Pacific rim. J Appl Phycol 1997; 9:403–411. 12. Belay A, Kato T, Ota Y. Spirulina (Arthrospira): Potential application as an animal feed supplement. J Appl Phycol 1996; 8:303–311. 13. Toerien DF, Grobbelaar JU. Algal mass cultivation experiments in South Africa. In: Shelef G, Soeder CJ, eds. Algae Biomass, Production and Use. Amsterdam: Elsevier/North Holland Biomedical Press, 1980:73–80. 14. Li D-M, Qi Y-Z. Spirulina industry in China: present status and future prospects. J Appl Phycol 1997; 9:25–28. 15. Soong P. Production and development of Chlorella and Spirulina in Taiwan. In: Shelef G, Soeder CJ, eds. Algae Biomass, Production and Use. Amsterdam: Elsevier/North Holland Biomedical Press, 1980:97–113. 16. Becker EW, Venkataraman LV. Production and utilization of the blue-green alga Spirulina in India. Biomass 1984; 4:105. 17. Belay A. The potential application of Spirulina (Arthrospira) as a nutritional supplement in health management. JANA 2002; 5(2):27–48. 18. Belay A, Yoshimichi O, Kazuyuki M, et al. Current knowledge on potential health benefits of Spirulina. J Appl Phycol 1993; 5:235–241. 19. Jensen GS, Ginsberg MS, Drapeau C. Blue-green algae as an immuno-enhancer and biomodulator. JANA 2001; 3(4):24– 30.

20. Chen T, Wong Y-S, Zheng W. Purification and characterization of selenium-containing phycocyanin from seleniumenriched Spirulina platensis. Phytochemistry 2006; 67:2424– 2430. 21. Bauersachs T, Compaor´e J, Hopmans EC, et al. Distribution of heterocyst glycolipids in cyanobacteria. Phytochemistry 2009; 70:2034–2039. 22. Carmichael WW. The toxins of cyanobacteria. Sci Am 1994; 270(1):78–86. 23. Burch MD. Effective doses, guidelines and regulations. Adv Exp Med Biol 2008; 619;831–853. 24. Cox PA, Banack SA, Murch SJ, et al. Diverse taxa of cyanobacteria produce ␤-N-methylamino-l-alanine, a neurotoxic amino acid. Proc Natl Acad Sci U S A 2005; 102;5074– 5078. 25. Gilroy GJ, Duncan J, Kauffman KW, et al. Assessing potential health risks from microcystin toxins in blue-green algae supplements. Environ Health Perspect 2000; 108 (5):435–439. 26. Drapeau C. Primordial Food: Aphanizomenon flos-aquae. U.S.A. Prescott, AZ: Unity International, 2003. 27. Vonshak A. Spirulina Platensis (Arthrospira). London: Taylor and Francis, 1997:8–11. 28. Chamorrow-Cevalos G. Toxicological research on the alga— Spirulina. UNIDO, UF/MEX/78/048, 1980. 29. Branger B., Cadudal JL, Delobel M, et al. Spiruline as a food supplement in case of infant malnutrition in Burkina-Faso. Archives de p´ediatrie 2003; 10:424–431. 30. Simpore J, Kabore F, Zongo F, et al. Nutrition rehabilitation of undernourished children utilizing Spiruline and Misola. Nutr J 2006; 5:3. http://www.nutritionj.com/ content/5/1/3. Accessed October 12, 2009. 31. Ramamoorthy A, Premakumari S. Effect of supplementation of Spirulina on hypercholesterolemic patients. J Food Sci Technol 1996; 33:124–128. 32. Samuels R, Mani UV, Iyer UM, et al. Hypocholesterolemic effect of Spirulina in patients with hyperlipidemic nephritic syndrome. J Med Food 2002; 5:91–96. 33. Mani S, Iyer U, Subramanian S. Studies on the effect of Spirulina supplementation in control of diabetes mellitus. In: Subramanian G, Kaushik BD, Venkataraman GS, eds. Cyanobacterial Biotechnology. U.S.A. Enfield, NH: Science Publishers, Inc., 1998:301–304. 34. Torres-Duran PV, Ferreira-Hermosillo A, Ju´arez-Oropeza MA. Antihyperlipemic and antihypertensive effects of Spirulina maxima in an open sample of Mexican population: a preliminary report. Lipids Health Dis 2007; 6:33. http://www.lipidworld.com/content/6/1/33. Accessed November 12, 2009. 35. Park HJ, Lee YJ, Ryu HK, et al. A randomized double-blind, placebo-controlled study to establish the effects of Spirulina in elderly Koreans. Ann Nutr Metab 2008; 52:322–328. 36. Ju´arez-Oropeza MA, Mascher D, Torres-Dur´an PV, et al. Effects of Spirulina on vascular reactivity. J Med Food 2009; 12;15–20. 37. Saeki Y, Matsumoto M, Hayashi A, et al. The effect of Spirulina hot water extract to the basic immune activation. Summary of paper presented at: The 30th Annual Meeting of the Japanese Society for Immunology, Sendai, Japan; November 14–16, 2000. 38. Hirahashi T, Matsumoto M, Hazeki K, et al. Activation of the human innate immune system by Spirulina: Augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis. Int Immunopharmacol 2002; 2:423–434. 39. Ishii K, Katoh T, Okuwaki Y, et al. Influence of dietary Spirulina platensis on IgA level in human saliva. J Kagawa Nutr Univ 1999; 30:27–33. 40. Evets LB, Belookaya T, Lyalikov S, et al. Means to normalize the levels of immunoglobulin E. Russian Federation

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Committee of patents and trade. Patent Number (19) RU (11) 20005486 C1 (51) 5 A 61K35/80. January 15, 1994. 1 page translation. Mao TK, Van de Water J, Gershwin ME. Effects of a Spirulinabased dietary supplement cytokine production from allergic rhinitis patients. J Med Food 2005; 8(3):27–30. Cingi C, Conk-Dalay M, Cakli H, et al. The effects of spirulina on allergic rhinitis. Eur Arch Otorhinolaryngol 2008; 265;1219–1223. Mathew B, Sankaranarayanan R, Nair P, et al. Evaluation of chemoprevention of oral cancer with Spirulina fusiformis. Nutr Cancer 1995; 24:197–202. Baicus C, Baicus A. Spirulina did not ameliorate idiopathic chronic fatigue in four N-of-1 randomized controlled trials. Phytother Res 2007; 21;570–573. Lu H-K, Hsieh C-C, Hsu J-J, et al. Preventive effects of Spirulina platensis on skeletal muscle damage under exerciseinduced oxidative stress. Eur J Appl Physiol 2006; 98:220– 226. Boone DR, Castenholz, RW. The archaea and the deeply branching and phototropic bacteria. In: Castenholz RW, Garrity GM, eds. Bergey’s Manual of Systematic Bacteriology. New York: Springer-Verlag, 2001:569. Carmichael WW, Drapeau C, Anderson D. Harvesting of Aphanizomenon flos-aquae Ralfs ex Born. & Flah.var. flosaquae (cyanobacteria) from Klamath Lake for human dietary use. J Appl Phycol 2000; 12:585–595. Jensen GS, Ginsberg DI, Huerta P, et al. Consumption of Aphanizomenon flos-aquae has rapid effects on the circulation and function of immune cells in humans: a novel approach to nutritional mobilization of the immune system. JANA 2000; 2 (3):50–58.

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49. Jensen GS, Hart AN, Zaske LAM, et al. Mobilization of CD34+ CD133+ and CD34+ CD133− stem cells in vivo by consumption of an extract from Aphanizomenon flos-aquaerelated to modulation of CXCR4 expression by an L-selectin ligand? Cardiovasc Revasc Med 2007; 8:189–202. 50. Vitale S, Miller NR, Mejico LJ, et al. A randomized, placebocontrolled, crossover clinical trial of super blue green algae in patients with essential blepharospasm or Meige syndrome. Am J Ophthalmol 2004; 138 (1):18–32. 51. Li R, Carmichael WW, Yongding L, et al. Taxonomic reevaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences. Hydrobiologia 2000; 438:99–105. 52. Rell´an S, Osswald J, Saker M, et al. First detection of anatoxina in human and animal dietary supplements containing cyanobacteria. Food Chem Toxicol 2009; 47:2189–2195. 53. Chorus I, Bartram J. Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management. London and New York: E & FN Spon, for the World Health Organization, 1999. 54. Scott PM, Niedzwiadek B, Rawn DF, et al. Liquid chromatographic determination of the cyanobacterial toxin beta-nmethylamino-L-alanine in algae food supplements, freshwater fish, and bottled water. J Food Prot 2009; 72:1769–1773. 55. Dietrich D, Hoeger S. Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): A reasonable or misguided approach? Toxicol Pharmacol 2005; 203:273–289. 56. Gilroy DJ, Kauffman KW, Hall RA, et al. Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements. Environ Health Perspect 2000; 108 (5):435–439.

Boron Curtiss Hunt

INTRODUCTION

(common name: tincal) forms of disodium tetraborate (borax, Na2 B4 O7 ), colemanite (2CaO·3B2 O3 ·5H2 O), ulexite (Na2 O·2CaO·5B2 O3 ·16H2 O), boric acid (H3 BO3 ), and monohydrate and tetrahydrate forms of sodium perborate (NaBO3 ) (11). Inorganic boron, within the concentration range expected for human blood (2–61 ␮M B; 22–659 ng B/g wet blood) (12), is essentially present only as the monomeric species orthoboric acid (common name: boric acid) B(OH)3 and borate, that is, B(OH)4 − (13). Boric acid is an exclusively monobasic acid and is not a proton donor, but rather accepts a hydroxyl ion (a Lewis acid) and leaves an excess of protons to form the tetrahedral anion B(OH)4 − (14):

The element boron is essential for all higher plants in phylogenetic kingdom Viridiplantae (1) and at least some organisms in the phylogenetic kingdoms Eubacteria (2), Stramenopila (3), and Animalia (4,5). Specific species in the kingdom Fungi have a demonstrated physiological response to boron, an important finding because Fungi species are thought to share a common ancestor with animals exclusive of plants (6). Physiologic concentrations of the element are needed to support metabolic processes in several species in Animalia. For example, embryological development in fish and frogs does not proceed normally in the absence of boron. There is evidence that higher vertebrates, that is, chicks, rats, and pigs require physiological amounts of boron to assist normal biologic processes including immune function, bone development, and insulin regulation. In humans, boron is under apparent homeostatic control and is beneficial for immune function and calcium and steroid metabolism.

B (OH)3 + 2H2 O ⇔ H3 O+ + B (OH)− 4

pK a = 9.25(25◦ C)

Within the normal pH range of the gut and kidney, B(OH)3 would prevail as the dominant species (pH 1: ∼100% B(OH)3 ; pH 9.3: 50%; pH 11: ∼0%) (15).

Biochemical Forms Many biomolecules contain one or more hydroxy groups and those with suitable molecular structures can react with boron oxo compounds to form boroesters, an important class of biologically relevant boron species. Several types of boron esters exist. Boric acid reacts with suitable dihydroxy compounds to form corresponding boric acid monoesters (“partial” esterification) (Fig. 1) that retain the trigonal-planar configuration and no charge. In turn, a boric acid monoester can form a complex with a ligand containing a suitable hydroxyl to create a borate monoester (“partial” esterification; monocyclic) (Fig. 2), but with a tetrahedral configuration and a negative charge. A compound of similar configuration and charge is also formed when borate forms a complex with a suitable dihydroxy compound. The two types of boromonoesters can react with another suitable dihydroxy compound to give a corresponding spirocyclic borodiester (“complete” esterification) that is a chelate

COMMON CHEMICAL FORMS Boron is the fifth element in the periodic table with a molecular weight of 10.81 and is the only nonmetal in Group III. Organoboron compounds are those organic compounds that contain B–O bonds, and they also include B–N compounds, because B–N is isoelectronic with C–C (7). Organoboron compounds are apparently important in biological systems and are the result of interaction with OH or amine groups. Organoboron complexes occur in plants and are produced in vitro with biomolecules isolated from animal tissues (8).

BORON SPECIATION Environmental Forms Boron does not naturally occur free nor bind directly to any element other than oxygen except for trivial exceptions, for example, NaBF4 (ferrucite) and (K,Cs)BF4 (avogadrite) (7). Its average concentration in the oceans is 4.6 mg/L and is the 10th most abundant element in oceanic salts (9). Weathering of clay-rich sedimentary rock is the major source of total boron mobilized into the aquatic environment (10). Undissociated boric acid (orthoboric acid) is the predominant species of boron in most natural freshwater systems (10) where most concentrations are below 0.4 mg/L and not lowered by typical treatments for drinking water. The most common commercial compounds of boron are anhydrous, pentahydrate, and decahydrate

Figure 1 Boric acid may complex with a suitable dihydroxy ligand to form a boric acid monoester (“partial” esterification) that retains a trigonal-planar configuration and no charge.

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83

Figure 2 Borate may complex with a suitable dihydroxy ligand to form a borate monoester (“partial” esterification; monocyclic) with a tetrahedral configuration and a negative charge.

complex with a tetrahedral configuration and negative charge (16) (Fig. 3). Boric acid and boric acid–like structures, instead of borate, are most likely the reactive species with biological ligands, because it is probably easier for a diol to substitute for a relatively loosely bound water molecule associated with boric acid or a boric acid-like structure than it is for the diol to substitute for a charged hydroxyl ion in borate or a borate-like structure (16).

Procaryotes Boron is an integral component of several biomolecules in which it is thermodynamically stabilized in a covalent bond (17–20) or by forming a boroester (21). Its presence in these molecules is essential; in its absence, they no longer perform their normal physiologic functions. Of great interest is a boron-containing biomolecule produced by a bacterium that is not an antibiotic but rather a cell-to-cell communication signal (20). Communication between bacteria is accomplished through the exchange of extracellular signaling molecules called autoinducers (AIs). This process, termed “quorum sensing,” allows bacterial populations to coordinate gene expression for community cooperative processes such as antibiotic production and virulence factor expression. AI-2 is produced by a large number of bacterial species and contains one boron atom per molecule. Not surprisingly, it is derived from the ribose moiety of biomolecule, (S)-adenosylmethionine (SAM). The gliding bioluminescent marine bacterium, Vibrio harveyi (phylum Proteobacteria), produces and also binds AI-2. In V. harveyi, the primary receptor and sensor for AI-2 is the protein LuxP, which consists of two similar domains connected by a three-stranded hinge. The AI-2 ligand binds in the deep cleft between the two domains to form a furanosyl borate diester complex (Fig. 4) (20). Boron is a structural component of certain antibiotics produced by certain myxobacteria, a distinct and unusual group of bacteria. For example, tartrolon B (Fig. 5) is characterized by a single boron atom in the center of the

Figure 3 Boric acid monoesters or borate monoesters can combine with a suitable dihydroxy compound to form a corresponding spirocyclic borodiester [“complete” (add the close parenthesis) esterification] that is a chelate complex with a tetrahedral configuration and negative charge.

Figure 4 The autoinducer, AI-2, with its integral boron atom is stabilized by a hydrogen network in the binding site of the receptor. The O–O or O–N distances for potential hydrogen bonds are shown in angstroms. Source: From Ref. 20.

molecule (18). Another related antibiotic, boromycin, was discovered to be potent against human immunonodeficiency virus (HIV) (22). It strongly inhibits the replication of the clinically isolated HIV-1 strain and apparently, by unknown mechanisms, blocks release of infectious HIV particles from cells chronically infected with HIV-1.

Animal and Human Tissues Only meager information is available on the speciation of boron in animal or human tissues. However, animal and human biocompounds with vicinal cis-diol moieties bind

Figure 5 Tartrolon B, an example of certain antibiotics produced by certain myxobacteria that require the presence of a single atom of boron for functionality.

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Figure 6 Experimental data indicate that biochemical species with vicinal cis-diols bind strongly to boron: (S)-adenosylmethionine (SAM) ≡ diadenosine hexophosphate (Ap6 A) ≡ Ap5 A > Ap4 A > Ap3 A ≡ NAD+ > Ap2 A > NADH ≡ 5 ATP > 5 ADP > 5 AMP > adenosine (ADS). Species without these moieties do not bind boron well: 3 AMP ≡ 2 AMP ≡ cAMP ≡ adenine (ADN).

boron; those without these moieties typically do not. Of these animal or human biocompounds examined, SAM has the highest known affinity for boron (8). It is the predominant methyl donor in biological methylations and is therefore a versatile cofactor in various physiologic processes. NAD+ , an essential cofactor for five sub-subclasses of oxidoreductase enzymes, also has a strong affinity for boron (23). The di-adenosine-phosphates (Apn A) are structurally similar to NAD+ . Boron binding by Ap4 A, Ap5 A, and Ap6 A is greatly enhanced compared with NAD+ but is still less than that of SAM (8). The Apn A molecules are present in all cells with active protein synthesis and reportedly regulate cell proliferation, stress response, and DNA repair (24). At physiologic pH, the adenine moieties of Apn A are driven together by hydrophobic forces and stack interfacially (25). Stacking of the terminal adenine moieties brings their adjacent ribose moieties into close proximity, a phenomenon that apparently potentiates cooperative boron binding between the opposing riboses (Fig. 6).

Plant-Based Foods All higher plants require boron and contain organoboron complexes. There may have been considerable evolutionary pressure exerted to select for carbohydrate energy sources that do not interact with boron. Sugars often form intramolecular hemiacetals: those with five-membered rings are called furanoses and those with six-membered rings are called pyranoses. In cases where either five- or six-membered rings are possible, the six-membered ring usually predominates for unknown reasons (26). In general, compounds in a configuration in which there are cis-diols on a furanoid ring (e.g., ribose, apiose, and ery-

thritan) form stronger complexes with boron than do those configured to have cis-diols predominately on a pyranoid ring (e.g., the pyranoid form of ␣-D-glucose). D-Glucose reacts with boric acid (27) but the near absence (70 Women, 51–70 Women, >70 Pregnancy Lactation Source: From Ref. 65.

Proposed adequate daily intake (␮g) 0.2 5.5 11 15 25 35 21 24 35 35 25 25 30 30 20 20 30 45

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Anderson and Cefalu

60-min GLUCOSE (mmol/L)

10

8

b WEEK 1 WEEK 4 PLACEBO +CHROMIUM

a

a

a

6

4

2 CONTROLS

HYPERGLY

Figure 2 Effects of dietary Cr on people with good glucose tolerance and those with marginally impaired glucose tolerance consuming 20 ␮g or less of Cr daily. Subjects with good glucose tolerance, controls, are subjects with blood glucose levels less than 5.5 mmol/L (100 mg/dL), 90 minutes after consuming an oral glucose load of 1 g/kg body weight. Subjects defined as marginally hyperglycemic have 90-minute glucose levels between 5.5 and 11.1 mmol/L following an oral glucose load of 1 g/kg. Bars with different superscripts are significantly different at P < 0.05. Subjects with good glucose tolerance are able to maintain normal glucose levels at these low intakes, but not subjects with varying degrees of glucose intolerance. Source: From Ref. 66.

tolerance [90-minute glucose between 5.5 and 11.1 mmol/L (100–200 mg/dL) following an oral glucose load of 1 g/kg body weight]. The average person older than 25 years has blood glucose in this range. Consumption of these same diets by people with good glucose tolerance (90-minute glucose less than 5.5 mmol/L) did not lead to changes in glucose and insulin variables. This is consistent with previous studies demonstrating that the requirement for Cr is related to the degree of glucose intolerance and demonstrates that an intake of 20 ␮g/day of Cr is not adequate for people with marginally impaired glucose tolerance and certainly not for those with impaired glucose tolerance or diabetes.

ADVERSE EFFECTS Safety of Chromium Trivalent Cr, the form of Cr found in foods and nutrient supplements, is considered as one of the least toxic nutrients. The reference dose established by the US Environmental Protection Agency for Cr is 350 times the upper limit of the ESADDI as established in 1980 and affirmed in 1989, 3500 times the new adequate intake for women and 2333 times for men. The ratio of the reference dose to the required levels for most mineral and trace minerals is less than 10. The reference dose is defined as an estimate (with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population, including sensitive subgroups, that is likely to be without an appreciable risk of deleterious effects during a lifetime. With these large safety factors, it is highly unlikely that there would be any reproducible signs of Cr toxicity at daily supplementation ranges of 1000 ␮g or less. There has been no evidence of toxicity in any of the nutritional studies involving Cr supplementation, but there have been

individual case studies reporting detrimental effects that have not been confirmed (67). Since the absorption of Cr is very low, it is likely that there would be indigestion and vomiting before enough Cr was absorbed to cause toxicity. However, Cr does bind to many biological substances including DNA and, at high enough levels, could (like almost all nutrients) lead to signs of toxicity in sensitive individuals. The toxic effects of Cr under nonphysiological conditions have been reviewed (39). The National Toxicology Program (NTP) (68) has completed independent in vitro and in vivo genotoxicity assays and evaluation of Cr picolinate, the most popular form of Cr in nutrient supplements, demonstrating that Cr picolinate did not produce chromosome damage in in vivo mouse assays and had no effect in two bacterial mutation assays (69). The absence of negative effects of Cr picolinate as reviewed by the NTP has led to the addition of Cr picolinate to generally recognized as safe list (68,69). Owing to the low toxicity of trivalent Cr, there is no upper limit established for Cr in the new National Academy of Sciences Guidelines (65).

CONCLUSIONS The effects of Cr on glucose and insulin metabolism are well documented. Normal dietary intake of Cr appears to be suboptimal because several studies have reported beneficial effects of Cr on people with elevated blood glucose or type 2 diabetes eating conventional diets. Stresses that alter blood glucose often lead to increased mobilization of Cr that is subsequently lost from the body via the urine. The mechanism of action of Cr is largely through improvements in insulin sensitivity. Chromium makes insulin more effective and in the presence of Cr in a useable form, lower levels of insulin are required. There is no established upper limit for the supplemental Cr as it has very low toxicity and there have been no documented negative side effects in any of the more than 35 clinical studies. Number of subjects per study ranged from less than 10 to more than 800.

REFERENCES 1. Jeejeebhoy KN, Chu RC, Marliss EB, et al. Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation, in a patient receiving longterm total parenteral nutrition. Am J Clin Nutr 1977; 30(4): 531–538. 2. Brown RO, Forloines-Lynn S, Cross RE, et al. Chromium deficiency after long-term total parenteral nutrition. Dig Dis Sci 1986; 31(6):661–664. 3. Freund H, Atamian S, Fischer JE. Chromium deficiency during total parenteral nutrition. JAMA 1979; 241(5):496–498. 4. Anderson RA. Chromium and parenteral nutrition. Nutrition 1995; 11(suppl 1):83–86. 5. Verhage AH, Cheong WK, Jeejeebhoy KN. Neurologic symptoms due to possible chromium deficiency in long-term parenteral nutrition that closely mimic metronidazoleinduced syndromes. JPEN J Parenter Enteral Nutr 1996; 20(2):123–127. 6. Via M, Scurlock C, Raikhelkar J, et al. Chromium infusion reverses extreme insulin resistance in a cardiothoracic ICU patient. Nutr Clin Pract 2008; 23(3):325–328.

Chromium

7. Anderson RA. Chromium, glucose intolerance and diabetes. J Am Coll Nutr 1998; 17(6):548–555. 8. Anderson RA. Chromium and insulin sensitivity. Nutr Res Rev 2003; 16:267–275. 9. Mertz W, Schwarz K. Relationship of glucose tolerance to impaired intravenous glucose tolerance of rats on stock diets. Am J Physiol 1959; 196:614–618. 10. Anderson RA. Chromium: Physiology, dietary sources and requirements. In: Sadler MJ, Strain JJ, Caballero B, eds. Encyclopedia of Human Nutrition. London: Academic Press, 1998: 388–394. 11. Anderson RA, Kozlovsky AS. Chromium intake, absorption and excretion of subjects consuming self-selected diets. Am J Clin Nutr 1985; 41(6):1177–1183. 12. Anderson RA, Bryden NA, Polansky MM, et al. Dietary chromium effects on tissue chromium concentrations and chromium absorption in rats. J Trace Elem Exp Med 1996; 9:11–25. 13. Anderson RA, Polansky MM, Bryden NA. Stability and absorption of chromium and absorption of chromium histidinate complexes by humans. Biol Trace Elem Res 2004; 101(3):211–218. 14. Seaborn CD, Stoecker BJ. Effect of antacid or ascorbic acid on tissue accumulation and urinary excretion of 51 chromium. Nutr Res 1992; 12:1229–1234. 15. Seaborn CD, Stoecker BJ. Effects of starch, sucrose, fructose and glucose on chromium absorption and tissue concentrations in obese and lean mice. J Nutr 1989; 119(10):1444– 1451. 16. Cefalu WT, Hu FB. Role of chromium in human health and in diabetes. Diabetes Care 2004; 27(11):2741–2751. 17. Cefalu WT, Bell-Farrow AD, Stigner J, et al. Effect of chromium picolinate on insulin sensitivity in vivo. J Trace Elem Exp Med 1999; 12:71–84. 18. Althuis MD, Jordan NE, Ludington EA, et al. Glucose and insulin responses to dietary chromium supplements: A metaanalysis. Am J Clin Nutr 2002; 76(1):148–155. 19. Anderson RA, Cheng N, Bryden NA, et al. Elevated intakes of supplemental chromium improve glucose and insulin variables in individuals with type 2 diabetes. Diabetes 1997; 46(11):1786–1791. 20. Ghosh D, Bhattacharya B, Mukherjee B, et al. Role of chromium supplementation in Indians with type 2 diabetes mellitus. J Nutr Biochem 2002; 13(11):690–697. 21. Jovanovic L, Gutierrez M, Peterson CM. Chromium supplementation for women with gestational diabetes mellitus. J Trace Elem Exp Med 1999; 12:91–98. 22. Ravina A, Slezak L, Mirsky N, et al. Control of steroidinduced diabetes with supplemental chromium. J Trace Elem Exp Med 1999; 12:375–378. 23. Ravina A, Slezak L, Mirsky N, et al. Reversal of corticosteroid-induced diabetes mellitus with supplemental chromium. Diabet Med 1999; 16(2):164–167. 24. Albarracin C, Fuqua B, Geohas J, et al. Combination of chromium and biotin improves coronary risk factors in hypercholesterolemic type 2 diabetes mellitus: A placebocontrolled, double-blind randomized clinical trial. J Cardiometab Syndr 2007; 2(2):91–97. 25. Singer GM, Geohas J. The effect of chromium picolinate and biotin supplementation on glycemic control in poorly controlled patients with type 2 diabetes mellitus: A placebocontrolled, double-blinded, randomized trial. Diabetes Technol Ther 2006; 8(6):636–643. 26. Bartlett HE, Eperjesi F. Nutritional supplementation for type 2 diabetes: A systematic review. Ophthalmic Physiol Opt 2008; 28(6):503–523. 27. Cheng N, Xixing Z, Shi H, et al. Follow-up survey of people in China with type 2 diabetes mellitus consuming supplemental chromium. J Trace Elem Exp Med 1999; 12:55–60.

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28. Wang ZQ, Qin J, Martin J, et al. Phenotype of subjects with type 2 diabetes mellitus may determine clinical response to chromium supplementation. Metabolism 2007; 56(12):1652– 1655. 29. Cefalu WT, Rood J, Pinsonat P, et al. Characterization of the metabolic and physiologic response from chromium supplementation in subjects with type 2 diabetes. Metabolism 2010; 59(5):755–762. 30. Altomare E, Vendemiale G, Chicco D, et al. Increased lipid peroxidation in type 2 poorly controlled diabetic patients. Diabetes Metab 1992; 18(4):264–271. 31. Armstrong AM, Chestnutt JE, Gormley MJ, et al. The effect of dietary treatment on lipid peroxidation and antioxidant status in newly diagnosed noninsulin dependent diabetes. Free Radic Biol Med 1996; 21(5):719–726. 32. Preuss HG, Grojec PL, Lieberman S, et al. Effects of different chromium compounds on blood pressure and lipid peroxidation in spontaneously hypertensive rats. Clin Nephrol 1997; 47:325–330. 33. Anderson RA, Roussel AM, Zouari N, et al. Potential antioxidant effects of zinc and chromium supplementation in people with type 2 diabetes mellitus. J Am Coll Nutr 2001; 20(3):212–218. 34. Cheng HH, Lai MH, Hou WC, et al. Antioxidant effects of chromium supplementation with type 2 diabetes mellitus and euglycemic subjects. J Agric Food Chem 2004; 52(5):1385–1389. 35. Lai MH. Antioxidant effects and insulin resistance improvement of chromium combined with vitamin C and e supplementation for type 2 diabetes mellitus. J Clin Biochem Nutr 2008; 43(3):191–198. 36. Anderson RA. Effects of chromium on body composition and weight loss. Nutr Rev 1998; 56(9):266–270. 37. Pittler MH, Stevinson C, Ernst E. Chromium picolinate for reducing body weight: Meta-analysis of randomized trials. Int J Obes Relat Metab Disord 2003; 27(4):522–529. 38. Lindemann MD, Wood CM, Harper AF, et al. Dietary chromium picolinate additions improve gain: Feed and carcass characteristics in growing-finishing pigs and increase litter size in reproducing sows. J Anim Sci 1995; 73(2):457– 465. 39. Vincent JB. The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent and muscle development agent. Sports Med 2003; 33(3):213–230. 40. Frank A, Danielsson R, Jones B. Experimental copper and chromium deficiency and additional molybdenum supplementation in goats. II. Concentrations of trace and minor elements in liver, kidneys and ribs: Haematology and clinical chemistry. Sci Total Environ 2000; 249(1–3):143–170. 41. Frank A, Anke M, Danielsson R. Experimental copper and chromium deficiency and additional molybdenum supplementation in goats. I. Feed consumption and weight development. Sci Total Environ 2000; 249(1–3):133–142. 42. Anton SD, Morrison CD, Cefalu WT, et al. Effects of chromium picolinate on food intake and satiety. Diabetes Technol Ther 2008; 10(5):405–412. 43. Martin J, Wang ZQ, Zhang XH, et al. Chromium picolinate supplementation attenuates body weight gain and increases insulin sensitivity in subjects with type 2 diabetes. Diabetes Care 2006; 29(8):1826–1832. 44. Davidson JR, Abraham K, Connor KM, et al. Effectiveness of chromium in atypical depression: A placebo-controlled trial. Biol Psychiatry 2003; 53(3):261–264. 45. Paykel ES, Mueller PS, de la Vergne PM. Amitriptyline, weight gain and carbohydrate craving: A side effect. Br J Psychiatry 1973; 123(576):501–507. 46. Attenburrow MJ, Odontiadis J, Murray BJ, et al. Chromium treatment decreases the sensitivity of 5-HT2 A receptors. Psychopharmacology (Berl) 2002; 159(4):432–436.

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47. Docherty JP, Sack DA, Roffman M, et al. A double-blind, placebo-controlled, exploratory trial of chromium picolinate in atypical depression: Effect on carbohydrate craving. J Psychiatr Pract 2005; 11(5):302–314. 48. Piotrowska A, Mlyniec K, Siwek A, et al. Antidepressantlike effect of chromium chloride in the mouse forced swim test: Involvement of glutamatergic and serotonergic receptors. Pharmacol Rep 2008; 60(6):991–995. 49. Vincent JB. The biochemistry of chromium. J Nutr 2000; 130(4):715–718. 50. Clodfelder BJ, Emamaullee J, Hepburn DD, et al. The trail of chromium(III) in vivo from the blood to the urine: The roles of transferrin and chromodulin. J Biol Inorg Chem 2001; 6(5–6):608–617. 51. Davis CM, Vincent JB. Chromium oligopeptide activates insulin receptor kinase activity. Biochemistry 1997; 36:4382– 4385. 52. Davis CM, Sumrall KH, Vincent JB. A biologically active form of chromium may activate a membrane phosphotyrosine phosphatase (PTP). Biochemistry 1996; 35(39):12963– 12969. 53. Moore JW, Maher MA, Banz WJ, et al. Chromium picolinate modulates rat vascular smooth muscle cell intracellular calcium metabolism. J Nutr 1998; 128(2):180–184. 54. Miranda ER, Dey CS. Effect of chromium and zinc on insulin signaling in skeletal muscle cells. Biol Trace Elem Res 2004; 101(1):19–36. 55. Wang H, Kruszewski A, Brautigan DL. Cellular chromium enhances activation of insulin receptor kinase. Biochemistry 2005; 44(22):8167–8175. 56. Horvath EM, Tackett L, Elmendorf JS. A novel membranebased anti-diabetic action of atorvastatin. Biochem Biophys Res Commun 2008; 372(4):639–643. 57. Horvath EM, Tackett L, McCarthy AM, et al. Antidiabetogenic effects of chromium mitigate hyperinsulinemiainduced cellular insulin resistance via correction of plasma membrane cholesterol imbalance. Mol Endocrinol 2008; 22(4):937–950. 58. Pattar GR, Tackett L, Liu P, et al. Chromium picolinate positively influences the glucose transporter system via affecting cholesterol homeostasis in adipocytes cultured under hyperglycemic diabetic conditions. Mutat Res 2006; 610(1–2):93– 100.

59. Chen G, Liu P, Pattar GR, et al. Chromium activates glucose transporter 4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesteroldependent mechanism. Mol Endocrinol 2006; 20(4):857– 870. 60. Kozlovsky AS, Moser PB, Reiser S, et al. Effects of diets high in simple sugars on urinary chromium losses. Metabolism 1986; 35(6):515–518. 61. Anderson RA. Stress effects on chromium nutrition of humans and farm animals. In: Lyons TP, Jacques KA, eds. Proceedings of Alltech’s Tenth Symposium, Biotechnology in the Feed Industry. Nottingham, England: University Press, 1994:267–274. 62. Anderson RA, Bryden NA, Polansky MM. Dietary chromium intake—freely chosen diets, institutional diets and individual foods. Biol Trace Elem Res 1992; 32:117–121. 63. National Research Council. Recommended Dietary Allowance. 10th ed. Washington, DC: National Academy Press, 1989. 64. Davies S, McLaren HJ, Hunnisett A, et al. Age-related decreases in chromium levels in 51,665 hair, sweat, and serum samples from 40,872 patients—implications for the prevention of cardiovascular disease and type II diabetes mellitus. Metabolism 1997; 46(5):469–473. 65. Anonymous. Dietary Reference Intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc. Washington, DC: National Academy Press, 2001:197– 223. 66. Anderson RA, Polansky MM, Bryden NA, et al. Supplemental-chromium effects on glucose, insulin, glucagon, and urinary chromium losses in subjects consuming controlled low-chromium diets. Am J Clin Nutr 1991; 54(5):909–916. 67. Anderson RA, Bryden NA, Polansky MM. Lack of toxicity of chromium chloride and chromium picolinate in rats. J Am Coll Nutr 1997; 16(3):273–279. 68. National Toxicology Program (NTP). Chromium picolinate. http://ntp.niehs.nih.gov (Search: chromium picolinate). 2003. Accessed on April 19, 2010. 69. Heimbach JT, Anderson RA. Chromium: Recent studies regarding nutritional roles and safety. Nutr Today 2005; 40(4):189–195.

Coenzyme Q10 Gustav Dallner and Roland Stocker

INTRODUCTION

ter procedure makes it possible to analyze—under certain conditions—the ratio of oxidized/reduced coenzyme Q amount, reflecting the in vivo situation.

Coenzyme Q is a lipid with broad distribution in nature, present in plants, bacteria, fungi, and all animal tissues. Coenzyme Q refers to a general structure composed of a nucleus, that is, 2,3-dimethoxy-5-methylbenzoquinone, and, substituted at position 6 of this quinone, a side chain consisting of isoprene units (5 carbons), all in trans configuration and with one double bond. In human tissues, the major part of coenzyme Q is coenzyme Q10 , which has 10 isoprene units; only 2% to 7% is present as coenzyme Q9 .

BIOCHEMISTRY AND FUNCTIONS Biosynthesis The biosynthesis of coenzyme Q in animal and human tissues is unique though the initial section, designated the mevalonate pathway, is identical for the production of coenzyme Q, cholesterol, dolichol, and isoprenylated proteins (2). After the branch point, however, the terminal portions of the biosynthetic pathways for each of the products are specific (Fig. 2). The mevalonate pathway consists of eight enzymatic reactions, which lead to the production of farnesyl pyrophosphate, the common initial substrate for all terminal products mentioned earlier. The pathway starts with two enzymatic steps using three molecules of acetyl-CoA, resulting in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The next reaction is a reduction to mevalonate by HMG-CoA reductase. This reaction is considered to be the main regulatory step in the pathway and also in cholesterol synthesis. Statins, drugs very commonly used in the treatment of hypercholesterolemia, are competitive inhibitors of HMG-CoA reductase. Mevalonate is phosphorylated in two steps to mevalonate pyrophosphate, which is then decarboxylated to isopentenyl pyrophosphate. Isopentenyl pyrophosphate is not only an intermediate but also the main building block for the synthesis of dolichol and the side chain of coenzyme Q. It is isomerized to dimethylallyl pyrophosphate, the substrate for farnesyl synthase. This enzyme mediates a two-step reaction, giving rise initially to the enzyme-bound, two-isoprenoid intermediate geranyl pyrophosphate, followed by a new condensation with isopentenyl pyrophosphate to the three-isoprenoid farnesyl pyrophosphate. All branch-point enzymes utilize farnesyl pyrophosphate as substrate and they initiate the terminal part of the synthesis. These enzymes are considered for overall rate limiting and are consequently of utmost importance in the regulation of the biosynthesis of the lipid in question. In cholesterol synthesis, squalene synthase mediates the head-to-head condensation of two molecules of farnesyl pyrophosphate. cis-Prenyltransferase catalyzes the 1 -4 condensation of cis-isopentenyl pyrophosphate to all-trans farnesyl pyrophosphate, which, after additional modifications, generates dolichols with chain length between 16 and 23 isoprene units. trans-Prenyltransferase mediates a series of addition reactions of isopentenyl

NAME AND GENERAL DESCRIPTION Coenzyme Q10 (C59 H90 O4 ) has a molecular weight of 863.3, melting point of 49◦ C, and redox potential of around +100 mV. The lipid is soluble in most organic solvents but not in water. The term coenzyme Q refers to both oxidized and reduced forms. The oxidized form of coenzyme Q, ubiquinone (CoQ), has an absorption maximum at 275 nm, whereas its reduced form, ubiquinol (CoQH2 ), has a small maximum at 290 nm. The absorption of CoQ at 210 nm is six times higher than that at 275 nm, but absorption at 210 nm is not specific for CoQ; this reflects the double bonds of the polyisoprenoid moiety and is therefore unspecific. The two major features of the lipid are the quinone moiety and the side chain. The quinone moiety is the basis for the redox function of this coenzyme, allowing continuous oxidation reduction (Fig. 1) as a result of enzymatic actions. The long polyisoprenoid side chain gives the molecule its highly hydrophobic character and influences its physical properties and arrangement in membranes.

EXTRACTION AND ANALYSIS For analysis of the blood and tissue level of coenzyme Q, extraction is usually performed with organic solvents without previous acid or alkaline hydrolysis (1). The simplest procedure is using petroleum ether, hexane, or isopropyl alcohol and methanol. In this system, phase separation occurs, and the methanol phase retains all the phospholipids, which make up more than 90% of the total lipid in most tissues. The separated neutral lipids, among them coenzyme Q, are generally isolated and quantified by reversed phase high-performance liquid chromatography (HPLC) and UV detection. Both the sensitivity and the specificity of the method can be improved greatly by using electrochemical detection. In addition, this lat157

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Figure 1 Coenzyme Q10 , shown in its reduced ubiquinol-10 (top) and oxidized ubiquinone-10 (bottom) forms, consists of a long hydrophobic side chain and a substituted benzoquinone ring.

pyrophosphate to farnesyl pyrophosphate, resulting in alltrans polyprenyl pyrophosphate, giving the side chain of coenzyme Q. The chain length varies between different species, and in humans, the chain is mostly decaprenyl pyrophosphate, with some solanesyl pyrophosphate. The next step in the biosynthesis requires the precursor of the benzoquinone moiety, 4-hydroxybenzoate, which itself is produced from tyrosine and is present in excess amounts. After prenylation of 4-hydroxybenzoate, the ring is modified by C-hydroxylations, decarboxylation, O-methylations, and C-methylation. The final product of the biosynthetic process is reduced coenzyme Q, ready to serve as electron donor. The sequence of these reactions has been studied so far mainly in bacteria and yeast. In mammalian tissues, several genes have been identified through complementary recognition with yeast and the function for some of them was also established. Isolated enzymes are not available at present, although these will be required for the establishment of the details of coenzyme Q synthesis in animal tissues.

Enzymatic Reduction of CoQ A major function of coenzyme Q is to serve as a lipidsoluble antioxidant. This requires CoQ to be present in its reduced form, CoQH2 , raising the question of how CoQ is maintained in its reduced form, CoQH2 . Ascorbate readily reduces benzoquinone in a catalytic process controlled by molecular oxygen, although this reduction is not likely of biological importance, as the benzoquinone moiety of the lipid-soluble CoQ10 , when localized in biological membranes, is not accessible to the water-soluble vitamin C. Similarly, cytosolic DT-diaphorase, an enzyme proposed earlier for CoQ10 reduction, is not efficient in reducing benzoquinones containing long isoprene side chains. Based on studies with the inhibitors rotenone and dicoumarol, it is suggested that a cytosolic reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent CoQ reductase, different from the mitochondrial reductase and DT-diaphorase, is involved. More recently, the flavin adenine dinucleotide (FAD)-containing enzymes, lipoamide dehydrogenase and thioredoxin reductase, were found to reduce CoQ in vitro with high efficiency. These enzymes are homodimers, have a molecular weight of around 55 kDa, and belong to the family of pyridine nucleotide disulfide oxidoreductases.

Acetyl-CoA

Enzymatic Functions

Acetoacetyl-CoA HMG-CoA HMG-CoA reductase Mevalonate Mevalonate-P Mevalonate-PP Isopentenyl-PP Dimethylallyl-PP Tyrosine

Geranyl-PP

4-OH-Benzoate Decaprenyl-PP Decaprenyl-4-OHbenzoate transferase

Famesyl-PP

trans-Prenyltransferase

Squalene synthetase

Decaprenyl-4-OH-benzoate

Squalene

Coenzyme Q

Cholesterol

Protein isoprenylation Geranylgeranyl-PP cis-Prenyltransferase

Polyprenyl-PP Dolichol

Dolichyl-P

Figure 2 The mevalonate pathway leading to the biosynthesis of coenzyme Q, cholesterol, dolichol, and dolichyl phosphate.

The most thoroughly studied function of coenzyme Q is its participation in the mitochondrial electron transport chain. The lipid is essential in respiration as it shuttles electrons from nicotinamide nucleotide-linked (NADH) dehydrogenase and succinate dehydrogenase (complexes I and II) to the cytochrome system (complex III). During respiration, coenzyme Q is present in fully oxidized, fully reduced, and semiquinone forms. In the protonmotive Q cycle, there is a cyclic electron transfer pathway through complex III involving semiquinone that accounts for the energy conservation at coupling site 2 of the respiratory chain. An electron transport system is also present in the plasma membranes of cells for transferring electrons across the membrane (3). The system is composed of a quinone reductase located on the cytosolic side and is thought to reduce CoQ in the presence of NADH. The resulting CoQH2 then shuttles electrons to an NADH oxidase, located on the external surface of the plasma membrane, that reduces extracellular electron acceptors such as the ascorbyl radical, in this case to ascorbate. This oxidase is not related to the NADPH oxidase of phagocytes, which functions independent of coenzyme Q. The precise

Coenzyme Q10

function(s) of the NADH oxidase remain(s) to be elucidated, although it has been suggested to be involved in the control of cell growth and differentiation, the maintenance of extracellular ascorbic acid, the regulation of cytosolic NAD+ /NADH ratio, the induction of tyrosine kinase, and early gene expression. An electron transport system has also been proposed to be present in lysosomal membranes, transferring electrons from NADH to FAD, cytochrome b5 , CoQ, and molecular oxygen. This system could be involved in the translocation of protons into the lysosomal lumen.

Nonenzymatic Functions Modulation of Mitochondrial Pore Opening Ions and solutes may penetrate the inner mitochondrial membrane through specific transporters and ion channels. It has been observed in vitro, during the accumulation of Ca2+ , that a permeability transition occurs and macromolecules up to the size of 1500 Da cross the membrane as a result of opening of an inner mitochondrial complex, the membrane transition pore. A large number of different compounds can open or close the membrane transition pore. An opening in the inner mitochondrial membrane is highly deleterious as it leads to loss of pyridine nucleotides, hydrolysis of adenosine triphosphate (ATP), disruption of ionic status, and elimination of the protonmotive force. Opening of the membrane transition pore is suggested to be an early event in apoptosis, causing activation of the caspase cascade through release of cytochrome c. On the other hand, the membrane transition pore may also have a physiological function by acting as a fast Ca2+ release channel in mitochondria. Various coenzyme Q analogs that contain the benzoquinone moiety with or without a short saturated or unsaturated side chain are modulators of the membrane transition pore (4). They can inhibit, induce, or counteract the effects of inhibitors and inducers. Endogenous CoQ10 may play an important role in preventing the membrane transition pore from opening, as it counteracts several apoptotic events, such as DNA fragmentation, cytochrome c release, and membrane potential depolarization.

Uncoupling Protein Function It is well established that the inner mitochondrial membrane possesses uncoupling proteins that translocate protons from the outside to the inside of the mitochondria. As a result, the proton gradient established by the respiratory chain is uncoupled from oxidative phosphorylation and heat is produced instead of energy. In human tissues, five uncoupling proteins have been identified, but only uncoupling protein 1 has been studied in detail. It is present in brown adipose tissue and participates in thermogenesis. The content of uncoupling proteins in other tissues is low, since uncoupling is not a common event. Uncoupling protein 2 is found in most tissues, and uncoupling protein 3 is abundant in skeletal muscle. By overexpressing uncoupling proteins 1, 2, and 3 from Escherichia coli in liposomes, it was demonstrated that coenzyme Q is an obligatory cofactor for the functioning of uncoupling proteins, with the highest activity obtained with CoQ10 (5). Uncoupling proteins were able to transport protons only when CoQ10 was added to the

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membranes in the presence of fatty acids. Low concentration of ATP inhibited the activity. In this way, a proton is delivered from a fatty acid to the uncoupling protein with the assistance of CoQ10 in the inner mitochondrial membrane. This is followed by the translocation of a proton to the mitochondrial matrix by the uncoupling protein.

Antioxidant Activity Approximately 1% to 2% of the molecular oxygen consumed by mitochondria is converted to superoxide anion radical and hydrogen peroxide. In addition, reactive oxygen species are produced by other processes, including autoxidation reactions, and by the action of enzymes such as NADPH oxidases of phagocytes and other cells, mitochondrial monoamine oxidase, flavin oxidases in peroxisomes, and cytochromes P-450. Furthermore, nitric oxide, generated by nitric oxide synthases, can interact with superoxide and give rise to a number of reactive nitrogen species. These reactive species have the potential to damage lipids, proteins, and DNA, a process generally referred to as “oxidative damage.” Antioxidants are enzymes, proteins, or nonproteinaceous agents that prevent the formation of reactive oxygen and nitrogen species, or remove these species or biomolecules that have been oxidatively damaged. Coenzyme Q is the only lipid-soluble antioxidant synthesized endogenously (6). Its reduced form, CoQH2 , inhibits protein and DNA oxidation, but it is its effect on lipid peroxidation that has been studied in detail. Ubiquinol inhibits the peroxidation of cell membrane lipids and also that of lipoprotein lipids present in the circulation and in the walls of blood vessels. It has been suggested that CoQH2 is a more efficient antioxidant than vitamin E, for two reasons. First, its tissue (but not blood) concentration exceeds several fold that of vitamin E. Second, and similar to vitamin C, CoQH2 effectively reduces ␣-tocopheroxyl radical to ␣-tocopherol, and by doing so eliminates the potential pro-oxidant activities of vitamin E. In fact, CoQH2 has been suggested to act as the first line of nonenzymatic antioxidant defense against lipidderived radicals. In addition, CoQH2 can inhibit the initiation of lipid peroxidation by scavenging aqueous radical oxidants. As a result of its antioxidant action as a one-electron reductant, CoQH2 is oxidized initially to its semiquinone radical (CoQH• ), which itself may be oxidized further to CoQ, with the potential to generate the superoxide anion radical. Regeneration of CoQH2 is therefore required for coenzyme Q to maintain its antioxidant activity. The effectiveness of cellular reducing systems is suggested by the fact that in most human tissues, the bulk of coenzyme Q is recovered as CoQH2 .

Effects on Atherosclerosis Coenzyme Q10 can theoretically attenuate atherosclerosis by protecting low-density lipoprotein from oxidation. Ubiquinol-10 is present in human low-density lipoprotein and, at physiological concentrations, prevents its oxidation in vitro more efficiently than vitamin E. The antiatherogenic effects are demonstrated in apolipoprotein E-deficient mice fed a high-fat diet (7). Supplementation with pharmacological doses of CoQ10 not only increased aortic CoQ10 levels but also decreased the absolute

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concentration of lipoprotein-associated lipid hydroperoxides in atherosclerotic lesions. Most significantly, there was a clear decrease in the size of atherosclerotic lesions in the whole aorta. Whether these protective effects are solely due to the antioxidant actions of coenzyme Q remains to be established, as the tissue content of other markers of oxidative stress, such as hydroxylated cholesteryl esters and ␣-tocopherylquinone, did not decrease. Oral administration of CoQ10 to healthy humans results in increased concentrations of CoQ10 H2 in circulating lipoproteins (8), with reduction most likely taking place in the intestine. Administration of CoQ10 also results in uptake of the lipid into monocytes and lymphocytes but not into granulocytes, whereas this dietary treatment increases the vitamin E content in both mononuclear and polymorphonuclear cells (9). The phospholipid composition is modified selectively in mononuclear cells, which display elevated amounts of arachidonic acid. Basal and stimulated levels of ␤2 -integrin CD11b and complement receptor CD35, distributed on the surface of monocytes, are also decreased by CoQ10 supplementation. This may contribute to the antiatherogenic effect of dietary CoQ10 , since CD11b contributes to the recruitment of monocytes to the vessel wall during atherogenesis.

Effects on Blood Flow and Pressure It is uncertain whether or not CoQ10 reduces blood pressure in the long-term management of primary hypertension (10). It is possible that any blood pressure lowering effect is indirect—perhaps via improved diastolic and endothelial function. Endothelial dysfunction of the arteries has potentially serious consequences and is commonly seen in patients with established cardiovascular disease or elevated risk factors. Ubiquinone supplementation improves endothelial function measured as flow-mediated dilatation of the brachial artery in patients with uncomplicated type 2 diabetes and dyslipidemia but not in hypercholesterolemic subjects (11). In diabetic patients, CoQ10 administration has also been found to decrease systolic blood pressure and HbA1C , but not F2 -isoprostanes, suggesting that the protective effects may have been unrelated in the decrease of vascular oxidative stress.

Potential Anti-inflammatory Effects There is some evidence that pharmacological doses of CoQ10 may have anti-inflammatory effects in vivo under some conditions (12). This is an area worthy of further investigations, as inflammation is part of the etiology in many diseases, such as cardiovascular diseases, diabetes, and Alzheimer disease. An anti-inflammatory effect could help explain why positive health effects are reported in a number of investigations when uptake of the lipid into a specific organ was limited.

PHYSIOLOGY Tissue Distribution CoQ10 is present in all human tissues in highly variable amounts (Table 1). The amounts are dependent on several factors, the most important under normal physiological conditions is the age (see sect. “Aging”). The highest amount is found in the heart (114 ␮g/g wet weight) (13).

Table 1 Concentration of Coenzyme Q10 in Different Adult Human Tissues Tissue Brain Thyroid Lung Heart Stomach Small intestine Colon Liver Pancreas Spleen Kidney Testis Muscle

CoQ10 (␮g/g tissue) 13 25 8 114 12 12 11 55 33 25 67 11 40

In the kidney, liver, muscle, pancreas, spleen, and thyroid, the CoQ10 content is between 25 and 67 ␮g/g, and in the brain, lung, testis, intestine, colon, and ventricle, it is between 8 and 13 ␮g/g. This variation is explained by histological structure, and consequently there are great variations within the same organ. For example, in different regions of the bovine brain, the amount of CoQ10 varies between 25 ␮g/g (striatum) and 3 ␮g/g (white matter). Rapid extraction and direct measurement by HPLC show that the major part of coenzyme Q10 in tissues, with the exception of brain and lung, is the reduced form, CoQ10 H2 .

Intracellular Distribution In rat liver, the highest amount of coenzyme Q9 is found in the outer and inner mitochondrial membranes, lysosomes, and Golgi vesicles (1.9–2.6 g/mg protein); the concentration in plasma membranes is 0.7 ␮g/g, and it is 0.2 to 0.3 ␮g/g in the nuclear envelope, rough and smooth microsomes, and peroxisomes (Table 2) (13). The distribution pattern is quite different from that of other neutral lipids. For example, the major part of dolichol is localized in lysosomes, that of cholesterol in plasma membranes, and that of vitamin E in Golgi vesicles. Within membranes, coenzyme Q10 has a specific arrangement, with the decaprenoid side chain located in the central hydrophobic region, between the bilayer of phospholipid fatty acids. The functionally active group, the benzoquinone ring, is located on the outer or inner Table 2 Concentration of Coenzyme Q9 in Different Subcellular Organelles of Rat Liver Organelle Nuclear envelope Mitochondria Outer membrane Inner membrane Microsomes Rough microsomes Smooth microsomes Lysosomes Lysosomal membrane Golgi vesicles Peroxisomes Plasma membrane

CoQ9 (␮g/mg protein) 0.2 1.4 2.2 1.9 0.2 0.2 0.3 1.9 0.4 2.6 0.3 0.7

Coenzyme Q10

surface of the membrane depending on the functional requirement. Because of this central localization, coenzyme Q10 destabilizes membranes, decreases the order of phospholipid fatty acids, and increases permeability. These effects are in contrast to those of cholesterol, which is located adjacent to fatty acids on one side of the bilayer and that stabilizes the membrane, increases the order of its lipids, and decreases membrane permeability.

Transport While the mevalonate pathway from acetyl-CoA to farnesyl pyrophosphate is mainly cytoplasmic, the terminal parts of coenzyme Q biosynthesis are localized in the mitochondria and endoplasmic reticulum (ER)-Golgi system. The mitochondrial inner membrane probably receives its lipid from the biosynthetic system associated with the matrix–inner membrane space. Newly synthesized verylow-density lipoproteins assembled in the ER-Golgi system also contain de novo synthesized coenzyme Q, which has to be synthesized at this location, like the other lipid and protein components of the lipoproteins. It is most probable that the various other cellular membranes also receive their constitutive coenzyme Q from the ER-Golgi system, as is the case with other lipids. Judging by studies in plants in vivo and with reconstituted cell-free systems, intracellular transport of coenzyme Q is a vesiclemediated, ATP-dependent process, and cytosolic carrier proteins may also be involved. Due to its hydrophobicity, the existence of a binding/transfer protein for coenzyme Q seems plausible, and recently saposin B has been suggested to serve this function (14). Aqueous saposin B was reported to extract and bind coenzyme Q dissolved in hexane to form a saposin B-coenzyme Q complex, with the lipid-binding affinity decreasing in the order: CoQ10 >CoQ9 >CoQ7 ␣tocopherol cholesterol (no binding). Under normal conditions, all organs and tissues synthesize sufficient coenzyme Q, so that external supply is not required. Coenzyme Q present in small amounts in all circulating lipoproteins is derived from very-low-density lipoprotein newly synthesized and discharged by the liver. It likely functions as an antioxidant and protects lipoproteins, with restricted redistribution among them. In the case of dietary coenzyme Q, lipoproteins are the carriers in the circulation and interact with at least some types of tissues for cargo delivery. Thus, the situation differs from that of cholesterol, in which case several organs depend on external supply from the diet or the liver.

Bioavailability Plasma The uptake of coenzyme Q from the intestine occurs at a low rate, with only 2% to 4% of the dietary lipid appearing in the circulation. The uptake mechanism has not been studied so far but is probably similar to that of vitamin E and mediated by chylomicrons. In rats, dietary CoQ10 appears as CoQ10 H2 in mesenteric triacylglycerolrich lipoproteins, which enter the circulation and are converted by lipoprotein lipase to chylomicron remnants, which are then cleared rapidly by the liver. Some of this diet-derived coenzyme Q reappears in the circulation, perhaps as a result of hepatic synthesis and release of very-

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low-density lipoprotein. Depending on the diet, in healthy human controls the amounts of coenzyme Q in very-lowdensity, low-density, and high-density lipoproteins are 1.2, 1.0, and 0.1 nmol/mg protein, respectively. After dietary supplementation (3 × 100 mg CoQ10 /day for 11 days), the amounts are 3.2, 3.5, and 0.3 nmol/mg protein, respectively. These data are consistent with the notion that circulating coenzyme Q redistributes among lipoproteins to protect them against oxidation. For most tissues, the low bioavailability of CoQ limits the ability of supplements to restore normal tissue levels of CoQH2 where deficiency exist. There are several potential ways to approach this problem, including administration of the lipid in reduced form, and increasing bioavailability by either derivatization or administering CoQ in association with cyclodextrins. “Mitoquinone,” a cationic modified form of CoQ attained by coupling to triphenylphosphonium and targeted to mitochondria to improve mitochondrial function, has received much interest recently (15). However, it is important to point out that mitoquinone is not a form of CoQ naturally occurring in human tissue, and the increase in superoxide production observed after uptake of mitoquinone into mitochondria is of potential concern (16). A potential alternative approach to increase CoQ in blood and tissues may be via drugs that stimulate the endogenous synthesis. This would not only elevate the amount of the lipid but possibly also direct it to the appropriate location. Polyisoprenoid epoxides in tissue culture and peroxisome proliferator-activated receptor-␣ agonists in rodents increase CoQ synthesis and amounts; however, no drug for this purpose is presently available for humans.

Blood Cells Red blood cells contain very small amounts of coenzyme Q. In lymphocytes, the content of CoQ10 is doubled after one week of dietary supplementation with this lipid, and this enhances both the activity of DNA repair enzymes and the resistance of DNA to hydrogen peroxide-induced oxidation (17). Two months of CoQ10 supply to humans increases the ratio of T4/T8 lymphocytes (18), and an increase in the number of lymphocytes has been noted after three months of dietary supply of this lipid. Ten weeks of CoQ10 administration to healthy subjects elevated the lipid content by 50% in monocytes, but no increase was observed in polymorphonuclear cells.

Tissues There remains some controversy regarding the bioavailability of dietary coenzyme Q in different tissues. In rats, the liver, spleen, adrenals, ovaries, and arteries take up a sizeable amount of dietary coenzyme Q (19). Under normal physiological conditions, very limited uptake may also occur in the heart, pancreas, pituitary gland, testis, and thymus. No uptake is apparent in the kidney, muscle, brain, and thyroid gland. However, uptake into rat brain has been reported—possibly the outcome of the specific conditions employed. Similarly, in mice, some, but not all, investigators have reported uptake into tissues. Derivatization of coenzyme Q by succinylation and acetylation increases its uptake into blood but not into various organs. What is clear is that under normal conditions, the bioavailability of dietary coenzyme Q in most tissues is

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limited. This may be explained by its distribution and functional requirement. Under normal conditions, all cells synthesize sufficient lipid, so that external supply is not required. Exogenous coenzyme Q taken up by the liver does not appear in mitochondria, which house the bulk of this cellular lipid, but is found mainly in nonmembranous compartments, such as the lysosomal lumen. The situation is, however, different in states of severe coenzyme Q deficiency. Genetic modifications causing low levels of coenzyme Q have serious consequences for neuronal and muscular function (20). In children with genetic coenzyme Q deficiency, dietary supplementation greatly alleviates pathological conditions and reestablishes mitochondrial and other functions. Limited studies with biopsy samples from patients with cardiomyopathy also indicate that the cardiac levels of coenzyme Q are decreased and may be increased by dietary supplementation with the lipid. Thus, it appears that uptake and appropriate cellular distribution of coenzyme Q occur if there is a requirement for the lipid. Direct organ uptake of sizeable amounts is not necessarily the only way of action of coenzyme Q, as other redox-active substances can act by signaling, serving as primary ligands or secondary transducers. Thus, the presence of coenzyme Q in the blood may impact on the vascular system, the production of cytokines, the expression of adhesion molecules, and the production of prostaglandins and leukotrienes. The possibility that metabolites of coenzyme Q influence metabolic processes has not yet been investigated.

Catabolism The short half-life of coenzyme Q, ranging between 49 and 125 hours in various tissues (Table 3), indicates that the lipid is subject to rapid catabolism in all tissues. The main urinary metabolites identified have an unchanged and fully substituted aromatic ring with a short side chain containing five to seven carbon atoms and a carboxyl group at the ␻-end (21). Phosphorylated forms of these metabolites are also recovered from nonhepatic tissues. These water-soluble metabolites are transferred to the circulation and are excreted by the kidney through urine. In the liver, the coenzyme Q metabolites become conjugated to glucuronic acid for fecal removal via bile.

Table 3

Half-life of CoQ9 in Rat Tissues

Tissue Brain Thyroid Thymus Heart Stomach Small intestine Colon Liver Pancreas Spleen Kidney Testis Muscle

Half-life (hr) 90 49 104 59 72 54 54 79 94 64 125 50 50

Regulation of Tissue Coenzyme Q Content In contrast to cholesterol, coenzyme Q does not appear to be subject to dietary or diurnal variations. However, a number of treatments decrease the content of the lipid in experimental systems. Administration of thiouracil, which inhibits thyroid gland function, decreases liver coenzyme Q. Oral administration of vitamin A also lowers hepatic coenzyme Q. In selenium-deficient rats, the coenzyme Q content of the liver is decreased by 50%, and the amount of the lipid is also lowered in the heart and kidney (but not muscle). A protein-free diet for three weeks lowers coenzyme Q content in the liver and heart but not in the kidney, spleen, and brain. As indicated earlier, HMGCoA reductase controls cholesterol synthesis because the branch-point enzyme squalene synthase has a low affinity for farnesyl pyrophosphate, so that its pool size is the main regulatory factor (22). By contrast, the branch-point enzyme of coenzyme Q synthesis, trans-prenyltransferase, has a comparatively higher affinity for farnesyl pyrophosphate, so that a decrease in this substrate does not generally lower the rate of coenzyme Q synthesis. It appears, however, that the doses of statins employed for the treatment of hypercholesterolemia result in inhibition of synthesis, as the coenzyme Q concentration decreases in several tissues (23). As mentioned earlier, the bioavailability of dietary coenzyme Q is limited. For this reason, it would be advantageous to find compounds that elevate tissue concentrations of coenzyme Q by increasing its biosynthesis. In rats and mice, treatment with peroxisomal inducers, such as clofibrate, phthalates, and acetylsalicylic acid, induces coenzyme Q synthesis in most organs and elevates its concentration in all subcellular organelles (24). The upregulation takes place by interaction with a nuclear receptor: peroxisomal proliferator receptor-␣. This receptor interacts with a number of genes, resulting in the increased synthesis of several enzymes, many of them connected to lipid metabolism. However, peroxisomal proliferator receptor-␣ is poorly expressed in human tissue, and it is not known to what extent this transcription factor is involved in coenzyme Q metabolism. Agonists or antagonists to various nuclear receptors may be a future approach to the upregulation of coenzyme Q biosynthesis and its concentration in human tissues. Hormones control coenzyme Q metabolism, but their method of action is not known in detail. Growth hormone, thyroxin, dehydroepiandrosterone, and cortisone elevate coenzyme Q levels in rat liver to various extents. A liver-specific increase of coenzyme Q occurs in rat and mice after two to three weeks stay in the cold room (+4◦ C). Vitamin A deficiency more than doubles the coenzyme Q level in liver mitochondria and more than trebles that in liver microsomes. Squalestatin 1, an inhibitor of squalene synthase, greatly increases coenzyme Q synthesis by increasing the farnesyl pyrophosphate pool and saturating trans-prenyltransferase.

COENZYME Q10 DEFICIENCY Genetic Disorders Coenzyme Q deficiency is an autosomal recessive disorder that may present itself in the form of myopathy,

Coenzyme Q10

encephalopathy and renal disease, or ataxia (20). The myopathic form is characterized by substantial loss of muscle coenzyme Q, muscle weakness, myoglobinuria, ragged-red fibers, and lactic acidosis. Patients with encephalopathy and renal involvement possess a more general disease, with myopia, deafness, renal failure, ataxia, amyotrophy, and locomotor disability. In these cases, coenzyme Q is undetectable or present at very low levels in cultured fibroblasts. In the ataxic form of deficiency, weakness, cerebellar ataxia, cerebellar atrophy, seizures, and mental retardation dominate, and low levels of coenzyme Q are found in the skeletal muscle. Various types of mutations have been found to be responsible for decreased synthesis of CoQ (25). Most of the mutations are of the primary type, affecting proteins related to the biosynthesis of the lipid. COOQ1-PDSS1 and -PDSS2 (two subunits of decaprenyl diphosphate synthase), COOQ2 (decaprenyl-4-hydroxybenzoate transferase), COOQ8 (CABC1 or ADCK3, a putative protein kinase), and COOQ9 (nonidentified function) are genes established in this group. There are also secondary forms of deficiency caused by mutations in genes not involved in coenzyme Q biosynthesis. Mutations in APTX (encoding aprataxin) and ETFDH (multiple acyl-CoA dehydrogenase deficiency caused by defects in electron transfer flavoprotein or ETF-ubiquinone oxidoreductase) also result in CoQ deficiency. The cases described in the literature probably represent extreme forms of coenzyme Q deficiency, seriously affecting mitochondrial functions. Moderate coenzyme Q deficiency is probably more common, though this requires verification by appropriate analysis of tissue biopsy samples. Unfortunately, the coenzyme Q content in blood often does not mirror the tissue concentration of the lipid, and it is highly desirable to develop methods to estimate moderate degrees of coenzyme Q deficiency. At present, diagnosis depends on measuring the coenzyme Q content in muscle biopsy samples, cultured fibroblasts, and lymphoblasts, or analyzing mitochondrial respiration and enzymes that require coenzyme Q as intermediate. CoQ deficiency is of special interest since it is the only treatable mitochondrial disease and oral administration of CoQ re-establishes normal functions. Early diagnosis before development of clinical symptoms is of outmost importance since established kidney and brain damages may not be completely reversible. The treatment, however, stops the process and the improvement is dramatic as children leave the wheel-chair state and are able to perform various activities. The problem is that at present diagnosis requires a muscle biopsy and analysis of mitochondrial functions. This does not allow screening of larger populations. Therefore, development of simplified diagnostic procedures would be of great interest also for diagnosis of less severe cases, probably present in relatively high numbers.

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Table 4 Coenzyme Q10 Content (␮g/g) with Age in Human Organs and Human Brain Age Human organs Lung Heart Spleen Liver Kidney Pancreas Adrenal Human brain Nucleus caudatus Gray matter Hippocampus Pons Medulla oblongata White matter Cerebellum

2 days

2 yr

20 yr

41 yr

80 yr

2.2 36.7 20.7 13.9 17.4 9.2 17.5

6.4 78.5 30.2 45.1 53.4 38.2 57.9

6.0 110.0 32.8 61.2 98.0 21.0 16.1

6.5 75.0 28.6 58.3 71.1 19.3 12.2

3.1 47.2 13.1 50.8 64.0 6.5 8.5

34 yr

55 yr

70 yr

90 yr

11.6 16.4 14.5 11.6 11.1 5.0 13.2

11.7 16.2 13.8 11.7 10.8 5.0 13.0

10.5 16.0 12.6 10.5 10.0 4.9 12.9

6.6 13.5 8.0 6.6 4.7 2.0 11.0

it mainly takes place between 70 and 90 years, and its extent, between 20% and 60%, depends on the localization. This pattern is different from that seen for other lipids. In most tissues, the content of cholesterol and phospholipids remains unchanged during the whole life period, whereas the amounts of dolichyl phosphate and especially dolichol increase greatly with age. It is unclear whether the decrease in coenzyme Q content is caused by its lowering in all or some selected cellular membranes or, alternatively, by other changes such as decreased number of mitochondria.

Cardiomyopathy The uptake of dietary coenzyme Q into heart muscle is low in both rats and humans, but it may increase significantly in various forms of cardiomyopathy (27). A number of clinical trials performed during the last 30 years suggest that heart functional performance may be improved modestly by dietary coenzyme Q supplementation (28,29). In congestive heart failure, improvements have been reported for ejection fraction, stroke volume, and cardiac output. Patients with angina may respond with improved myocardial efficiency. Reperfusion injury, such as after heart valve replacement and coronary artery bypass graft surgery, includes oxidative damage, and treatment of patients with coenzyme Q prior to surgery may lead to decreased oxidative damage and functional improvement. However, the benefits reported have not been consistent, and despite the existence of a large body of literature, there remains a need for large, long-term, and well-designed trials to establish unambiguously whether CoQ10 supplements are beneficial in the setting of cardiomyopathy and the failing heart.

Neurological Disorders Aging In human organs, the coenzyme Q content increases threeto fivefold during the first 20 years after birth, followed by a continuous decrease, so that in some tissues the concentration may be lower at 80 years than at birth (Table 4) (26). The decrease is less pronounced in the brain, where

Judging by extensive animal studies, a number of neurological diseases involve mitochondrial dysfunction and oxidative stress. The positive effects obtained with coenzyme Q treatment in these models suggest that supplementation may also be beneficial in humans (30). Patients with early Parkinson disease were subjected to a trial

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in which the placebo group was compared with groups supplemented for 16 months with coenzyme Q up to daily doses of 1200 mg. It was found that coenzyme Q slowed the progressive functional deterioration, with the best results obtained with the highest dose. Platelets from these patients had decreased coenzyme Q content and also showed reduced activity of mitochondrial complex I and complex II/III. The ratio of CoQ10 H2 to CoQ10 was also decreased in these platelets, indicative of the presence of oxidative stress. Upon supplementation, the CoQ10 content in the platelets increased and complex I activity was also elevated. In Huntington disease, magnetic resonance spectroscopy detected increased lactate concentration in the cerebral cortex. Administration of CoQ10 caused a significant decrease in lactate that reversed upon discontinuation of the therapy. Deficiency of frataxin, a regulator of mitochondrial iron content, causes Friedreich ataxia. When patients with this disease were treated with coenzyme Q and vitamin E for six months, progression of their neurological deficits was slowed down, associated with an improvement in cardiac and skeletal muscle energy metabolism (31). Treatment of these patients with idebenone, an analog of coenzyme Q, reduced heart hypertrophy and improved heart muscle function. In several studies, patients with mitochondrial encephalopathy, lactic acidosis, and strokes (MELAS) displayed significant improvement after coenzyme Q or idebenone treatment (32). Several other trials were also performed during recent years with variable results. Since there are subtypes of individual neurodegenerative diseases, large numbers of patients are required to obtain reliable results, which is often difficult to accomplish.

Statin Therapy Statins are the drugs most commonly used for the treatment of hypercholesterolemia, and, in addition to efficient cholesterol lowering, they also have anti-inflammatory activities. The basis for their use is that inhibition of HMGCoA reductase decreases the farnesyl pyrophosphate pool to such an extent that squalene synthase, which catalyzes the terminal regulatory step in cholesterol synthesis, is no longer saturated, thereby inhibiting overall synthesis (22). It appears, however, that the extent to which the farnesyl pyrophosphate pool is decreased by therapeutic doses of the drug also affects the saturation of trans- and cisprenyltransferases in spite of the fact that these latter enzymes have a higher affinity for farnesyl pyrophosphate. Consequently, synthesis of both coenzyme Q and dolichol is inhibited. Rats treated with statins exhibit decreased levels of coenzyme Q, dolichol, and dolichyl phosphate in heart and muscle, and the same is probably also true in humans. In humans, statin treatment significantly decreases blood coenzyme Q concentration (33), although the clinical significance of this phenomenon remains to be established. Various degrees of myopathy, myalgia, and rhabdomyolysis have been reported in statin-treated patients, and it is possible, but not proven, that these conditions are related to decreased muscle coenzyme Q content. Initial trials of CoQ10 supplementation in patients with statin-induced myopathy have provided variable results (34). Given the widespread use of statins, it is important

that additional studies address a possible causal link between these side effects of statin treatment and altered tissue coenzyme Q content.

Exercise During endurance exercise training, the coenzyme Q concentration increases in rat muscle on a weight basis due to an increase in mitochondrial mass. After four days of high-intensity training, the coenzyme Q content in the exposed muscles of healthy persons is unchanged (35). Supplementation (120 mg/day) doubles the coenzyme Q concentration in the plasma, but there is no change in the muscle content as judged by HPLC analysis of the tissue homogenate and isolated mitochondrial fraction in both control and trained subjects.

Dosage So far, no toxic or unwanted side effects have been described for CoQ10 supplements in humans, not even after ingestion in gram quantities. In most studies, 100 to 200 mg has been given per day in two doses. In genetic disorders, in the case of adults, the dose may increase to 300 mg/day and in neurological diseases, up to 400 mg/ day or more. In the latter case, in the frame of large multicenter trials, doses up to 2400 mg have been supplied. A patent on the use of statins combined with coenzyme Q has expired recently, although this combined preparation has not been manufactured so far. Now it may be possible for the pharmaceutical industry to introduce capsules containing statins and coenzyme Q in order to decrease the potential for muscle damage. In this case, relatively low doses of CoQ10 (e.g., 50 or 100 mg/day) appear to be appropriate.

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Conjugated Linoleic Acid Kristina B. Martinez, Arion J. Kennedy, and Michael K. McIntosh

INTRODUCTION Conjugated linoleic acid (CLA) consists of a group of positional and geometric fatty acid (FA) isomers of linoleic acid (C18:2; cis-9, cis-12 octadecadienoic acid). CLA isomers are found naturally in ruminant meats and dairy products due to biohydrogenation of linoleic or linolenic acids in the rumen of these animals. Larger quantities of CLA are chemically synthesized for use in dietary supplements or fortified foods. Initially identified as a potential anticarcinogen, CLA has been reported to prevent obesity, diabetes, or atherosclerosis in different animal and cell models, depending on the doses, isomers, and models used. Potential mechanisms for preventing these diseases include inducing cancer cell apoptosis, increasing energy expenditure and delipidating adipocytes, increasing insulin sensitivity, or reducing aortic lesions. However, unequivocal evidence in human participants is still lacking. Ironically, potential side effects of CLA supplementation include chronic inflammation, insulin resistance, and lipodystrophy. Long-term, well-controlled clinical trials and more mechanistic studies are needed to better understand the true potential health benefits versus risks of consuming CLA isomers and their mechanisms of action.

CHEMISTRY AND SYNTHESIS OF CLA Natural Synthesis of CLA Isomers CLA isomers are produced naturally in the rumen of ruminant animals by fermentative bacteria Butyrovibrio fibrisolvens, which isomerize linoleic acid into CLA isomers (Fig. 1). A second pathway of CLA synthesis in ruminants is in the mammary gland via ␦-9-desaturase of trans-11, octadecanoic acid (1). Thus, natural food sources of CLA are dairy products including milk, cheese, butter, yogurt, and ice cream and ruminant meats such as beef, veal, lamb, and goat meat (2–4) (Table 1). The cis-9, trans-10 (9,11) isomer (i.e., rumenic acid) is the predominating CLA isomer in these products (∼80%), whereas the trans-10, cis-12 (10,12) isomer represents approximately 10%. Although several other isoforms of CLA have been identified, the 9,11 and 10,12 isomers appear to be the most biologically active (5). Levels of CLA isomers in ruminant meats or milk can be augmented by dietary manipulation, including feeding cattle on fresh pasture (6) or by adding oils rich in linoleic acid (e.g., safflower oil) or ingredients that alter biohydrogenation of linoleic acid (e.g., ionophores) to their diet (7).

Figure 1 Stuctures of linoleic acid, cis-9, trans-11 CLA, and trans-10, cis-12 CLA.

Chemical Synthesis of CLA Isomers Because of the relatively low levels of CLA isomers in naturally occurring foods that are high in fat content, the chemical synthesis of CLA has been developed for making supplements and for fortifying foods. CLA can be synthesized from linoleic acid found in safflower or sunflower oils under alkaline conditions, yielding a CLA mixture containing approximately 40% of the 9,11 isomer and 44% of the 10,12 isomer (reviewed in Ref. 8). Commercial preparations also contain approximately 4% to 10% trans9, trans-11 CLA and trans-10, trans-12 CLA, as well as trace amounts of other isomers.

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Table 1

CLA Content of Various Foods

Food Meats/fish Corned beef Lamb Fresh ground beef Salami Beef smoked sausage Knackwurst Smoked ham Veal Smoked turkey Fresh ground turkey Chicken Pork Egg yolk Salmon Vegetable oils Safflower oil Sunflower oil Peanut

mg/g fat Food 6.6 5.8 4.3 4.2 3.8 3.7 2.9 2.7 2.4 2.6 0.9 0.6 0.6 0.3 0.7 0.4 0.2

Dairy Condensed milk Colby cheese Butter fat Ricotta Homogenized milk Cultured buttermilk American processed cheese Mozzarella Plain yogurt Butter Sour cream Cottage cheese Low fat yogurt 2% milk Medium cheddar Ice cream Parmesan Frozen yogurt

mg/g fat 7.0 6.1 6.1 5.6 5.5 5.4 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.1 4.1 3.6 3.0 2.8

Sources: Based on values reported in Refs. 2–4; and the University of Wisconsin Food Research Institute (Dr. Pariza, Director).

PHARMACOKINETICS AND EFFICACY OF CLA Human and Animal Studies As with other long chain unsaturated fatty acids (FA)s, CLA is absorbed primarily in the small intestine, packaged into chylomicrons, and distributed to extrahepatic tissues having lipoprotein lipase (LPL) activity or returned to the liver via chylomicron remnants or other lipoproteins. The average daily intake of CLA is approximately 152 to 212 mg for nonvegetarian women and men, respectively (9), and human serum levels range from 10 to 70 ␮mol/L after supplementation (10,11). One major discrepancy between animal and human studies is the dose of CLA administered (i.e., equal levels of 9,11 and 10,12 isomers—referred to as a CLA mixture), when expressed per unit body weight. For example, most adult human studies provide 3 to 6 g/day of a CLA mixture, whereas rodent studies provide 0.5% to 1.5% of a CLA mixture (w/w) in the diet. When expressed per unit of body weight, humans receive approximately 0.05 g CLA/kg body weight, whereas mice received 1.07 g CLA/kg body weight, which is 20 times the human dose based on body weight. Thus, part of the discrepancy in results obtained from human and animal studies is likely due to this large difference in the dose of CLA administered. Supplementing humans with higher, or animals with lower, doses of CLA would address this issue. Other discrepancies in experimental designs include using CLA isomer mixtures versus single isomers, duration of CLA supplementation, and the age, weight, gender, and metabolic status of the subjects or animals.

Cell Studies In vitro studies have been conducted in a variety of cells types, primarily using an equal mixture of 9,11 and 10,12 CLA, or each isomer individually. Doses used in cell studies generally range between 1 to 100 ␮M, reflecting the concentration found in human participants follow-

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ing supplementation. Results from these studies suggest that these isomers are readily taken up by cells. For example, we found that 10,12 CLA is readily incorporated into neutral and phospholipid fractions of the primary human adipocyte cultures and reduced lipid and glucose metabolism (12). Similar to in vivo studies, 9,11 CLA acted more like the linoleic acid controls.

ANTICANCER PROPERTIES OF CLA CLA Reduces Tumor Growth Pariza’s group initially discovered that CLA isomers in fried ground beef acted as anticarcinogens (13). Subsequently, numerous investigators have shown that CLA mixtures or individual isomers decrease tumor cell growth or increase cancer cell death in in vitro and in vivo models of mammary, gastric, or skin cancer (reviewed in Ref. 14). For example, feeding 0.8% to 1.0% individual CLA isomers or mixtures block the initiation or progression of chemically induced carcinogenesis in several rodent models (15–17). A 5 ␮M CLA mixture prevented cell growth and cytokine production in transformed human keratinocytelike cells (18). Proposed anticarcinogenic mechanisms for CLA include decreasing nuclear factor (NF) ␬B and cyclooxygenase (COX) activity, thereby suppressing the levels of prostaglandin (PG)E2 , an inflammatory PG that promotes the progression of certain forms of cancer and induces human epidermal growth factor receptor 2 (HER2) oncogene expression (19).

CLA Induces Apoptosis of Cancer Cells Several groups have reported that CLA isomers cause apoptosis or programmed cell death in cancer cells (reviewed in Ref. 11). For example, 32 to 128 ␮M CLA mixture prevented rat mammary cancer cell growth through apoptosis and decreased DNA synthesis in rat mammary cancer cells (20). Moreover, 40 to 80 ␮M 10,12 CLA induces apoptosis in breast cancer cells (19,21,22). Proposed proapoptotic mechanisms of CLA include inducing atypical endoplasmic reticulum (ER) stress, leading to caspase12 activation (22). In contrast to the cell and animal studies cited in the preceding text, a recent prospective cohort study conducted in Sweden found no evidence to support a protective effect of CLA consumption on the development of breast cancer in women (23). Furthermore, some studies show that 10,12 CLA enhances the risk of developing certain types of cancer (24). Thus, clinical studies examining the effects of purified CLA isomers on preventing or treating cancer, and safety issues, are needed.

ANTIOBESITY ACTIONS OF CLA Due to the substantial rise in obesity over the past 30 years, there is a great deal of interest in CLA as a weight loss treatment, as it has been shown to decrease body weight and body fat mass (BFM). For example, supplementation with a CLA mixture (i.e., 10,12 + 9,11 isomers in equal concentrations) or the 10,12 isomer alone decreases BFM in many animal and some human studies (reviewed in Refs. 25 and 26). Of the two major isomers of CLA, the

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10,12 isomer is responsible for the antiobesity properties (27–31).

CLA Decreases Body Weight and Body Fat Mass Park et al. (32) were one of the first groups to demonstrate that CLA modulated body composition. Compared with controls, male and female mice supplemented with a 0.5% (w/w) CLA mixture had 57% and 60% less BFM, respectively. Since these findings, researchers have demonstrated that CLA supplementation consistently reduces BFM in mice, rats, and pigs (33–36). For example, dietary supplementation with 1% (w/w) CLA mixture for 28 days decreased body weight and periuterine white adipose tissue (WAT) mass in C57BL/6J mice (36). In humans, some studies show that CLA decreases BFM and increases lean body mass (LBM), whereas others show no such effects. For example, supplementation of 3 to 4 g/day of a CLA mixture for 24 weeks decreased BFM and increased LBM in overweight and obese people (37). On the other hand, supplementation of 3.76 g/day of a CLA mixture in yogurt for 14 weeks in healthy adults had no effect on body composition (38). Supplementation with 3.2 g/day of a CLA mixture decreased total BFM and trunk fat compared with placebo in overweight participants, but not obese participants (39). These contradictory findings among human studies may be due to the following differences in experimental design: (i) mixed versus individual CLA isomers, (ii) CLA dose and duration of treatment, and (iii) gender, weight, age and metabolic status of the participants. These antiobesity effects of CLA do not appear to be solely due to reductions in food intake in animals (40– 42) or humans (43,44). Several mechanisms by which CLA decreases BFM will now be examined.

CLA Increases LBM A recent meta-analysis of 18 human, placebo-controlled CLA studies found that consuming a CLA mixture increased fat-free mass (FFM) by 0.3 kg, regardless of the duration or dose (45). When these same 18 studies were examined for reductions in BFM, it was shown that CLA supplementation decreased BFM by 0.05 kg/week for up to one year (25). The average CLA mixture dose for these studies was 3.2 g/day. Collectively, these meta-analyses studies suggest that CLA supplementation of humans results in a rather small but rapid increase in FFM or LBM, and a much larger decrease in BFM over an extended period of time. The effects of CLA on FFM or LBM in humans may vary depending on baseline body mass index, gender, age, and exercise status of the participants. Two proposed mechanisms by which CLA increases LBM are via increasing bone or muscle mass. 10,12 CLA supplementation for 10 weeks with a 0.5% (w/w) CLA mixture increased bone mineral density (BMD) and muscle mass in C57BL/6 female mice (46). CLA supplementation has been proposed to increase BMD via increasing osteogenic gene expression and decreasing osteoclast activity (46,47). Furthermore, CLA supplementation alone or with exercise increased BMD compared with control mice (48). An alternative mechanism could be that CLA decreases adipogenesis of pluripotent mesenchymal stem cells (MSC) in bone marrow, and instead promotes their

commitment to become bone cells. Indeed, 10,12 CLA has been shown to decrease the differentiation of MSC into adipocytes and increase calcium deposition and markers of osteoblasts (49). In contrast, 9,11 CLA increased adipocyte differentiation and decreased osteoblast differentiation. Consistent with these in vitro data, CLA mixture supplementation of rats treated with corticosteroids prevented reductions in LBM, BMD, and bone mineral content (50). Increasing LBM is directly linked to an increase in basal metabolic rate (BMR). In addition to its effects on BMD, recent evidence supports a role of CLA in increasing endurance and muscle strength. For example, maximum swimming time until fatigue was higher in CLA fed versus control mice (51). Aging mice supplemented with a CLA mixture and 10,12 CLA had higher muscle weight compared with 9,11 CLA and corn oil controls (52). In addition, CLA isomers increased levels of antioxidant enzyme activity, ATP, and enhanced mitochondrial potential, indicating a protective effect against age-associated muscle loss (52). In humans, CLA increased bench-press strength in men supplemented with 5 g/day for seven weeks who underwent resistance training three days per week (53). Furthermore, supplementation with CLA combined with creatine monohydrate (C) and whey protein (P) led to greater increases in bench-press and leg-press strength than supplementation with C+P or P alone (54). Although preliminary, these data suggest that CLA may enhance exercise-induced muscle strength or prevent sarcopenia or age-related muscle loss.

CLA Increases Energy Expenditure CLA has been proposed to reduce adiposity by elevating energy expenditure via increasing BMR, thermogenesis, or lipid oxidation in animals (27,42,55). In BALB/c male mice fed mixed isomers of CLA for six weeks, body fat was decreased by 50% and was accompanied by increased BMR compared with controls (42). Enhanced thermogenesis may be associated with increased uncoupling of mitochondria via uncoupling protein (UCP)s, which facilitate proton transport over the inner mitochondrial membrane thereby leading to dissipation of energy as heat instead of ATP synthesis. UCP1 is highly expressed in brown adipose tissue (BAT), and in WAT at lower levels. UCP3 is expressed in muscle and in a number of other tissues, whereas UCP2 is the form expressed at the highest level across most tissues. Supplementation with a CLA mixture or 10,12 CLA in rodents induced UCP2 mRNA expression in WAT (29,56). Recently, it was demonstrated that CLA increased mRNA and protein expression of UCP1 in WAT (57). Similarly, CLA supplementation induced UCP gene expression and elevated ␤-oxidation in muscle and liver (58–62).

CLA Increases Fat Oxidation CLA has been shown to regulate the gene expression or activity of proteins associated with FA oxidation in adipose tissue, muscle, and liver. For example, CLA induced the expression of carnitine palmitoyl transferase 1 (CPT1) in WAT of obese Zucker fa/fa rats (63). Additionally, 10,12 CLA increased the expression of peroxisome proliferator-activated receptor (PPAR)␥ coactivator-1␣

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(PGC1␣ ) in WAT of mice (57). Consistent with these in vivo findings, 10,12 CLA increased ␤-oxidation in differentiating 3T3-L1 preadipocytes (64). Furthermore, 10,12 CLA treatment increased AMP kinase (AMPK) activity and increased phospho-acetyl-CoA carboxylase (ACC) levels in 3T3-L1 adipocytes, suggesting an increase in FA oxidation and a decrease in FA esterification to triglyceride (TG) (65). In muscle, 10,12 CLA increased CPT1 expression in hamsters fed an atherogenic diet (60). Supplementation of a CLA mixture in high fat fed hamsters led to increased CPT1 activity in muscle (66). A CLA mixture increased CPT1b, UCP3, acetyl-CoA oxidase (ACO) 2, and PPAR␣ mRNA levels in skeletal muscle of Zucker rats (67). Consistent with these data, 10,12 CLA increased mRNA levels (63) and activity (68) of CPT1 in the liver. Additionally, 10,12 CLA increased hepatic peroxisomal fatty ACO activity (68), suggesting increased peroxisomal ␤-oxidation in addition to mitochondrial oxidation. These findings suggest CLA may reduce adiposity through increased energy expenditure via increased mitochondrial uncoupling and FA oxidation in WAT, muscle, and liver. At least one report demonstrates that CLA increases FA oxidation in human participants (69). In this study, overweight adults supplemented with 4 g/day of a CLA mixture for six months had a lower respiratory quotient (RQ), indicating an increase in FA oxidation compared with placebo controls. However, others have shown no effect of CLA on energy expenditure or fat oxidation in humans (70,71). These discrepancies may be due to the length of treatment, time period of measurement, and time at which measurements are taken. For instance, CLA treatment for four to eight weeks had no effect on energy expenditure or FA oxidation, based on a 20-minute measurement during resting and walking (70). In contrast, the study by Close et al. (69) administered CLA for six months and measured FA oxidation over a 24-hour period and found that CLA increased FA oxidation and energy expenditure. Thus, discrepancies in this area may be due to insufficient duration of CLA treatment or measurements of energy expenditure or FA oxidation.

CLA Decreases A dipocyte Size Lipolysis is the process by which stored TG is mobilized, releasing free fatty acids (FFAs) and glycerol for use by metabolically active tissues. C57BL/6J mice fed 10,12 CLA for three days had increased mRNA levels of hormonesensitive lipase (HSL), a key enzyme for TG hydrolysis (56). Consistent with these data, acute treatment with CLA mixture or 10,12 CLA alone increased lipolysis in 3T3-L1 (32,72) and newly differentiated human adipocytes (73). In vitro, a CLA mixture and to a greater extent 10,12 CLA decreased TG content, adipocyte size, and lipid locule size in adipocytes (74). Similarly, mice fed 1% CLA displayed increased numbers of small adipocytes with a reduction in the number of large adipocytes (75). Furthermore, a CLA mixture reduced adipocyte size rather than cell number in Sprague Dawley (40) and fa/fa Zucker rats (76). Thus, CLA may reduce adipocyte size by increasing lipolysis.

CLA Decreases Adipocyte Differentiation The conversion of preadipocytes to adipocytes involves the activation of key transcription factors such as

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PPAR␥ and CAAT/enhancer-binding proteins (C/EBPs). There is much evidence showing that CLA suppresses preadipocyte differentiation in animal (77–79) and human (12,80) preadipocytes treated with a CLA mixture or 10,12 CLA alone. 10,12 CLA treatment has been reported to decrease the expression of PPAR␥ , C/EBP␤, sterol regulatory element-binding protein-1c (SREBP-1c), liver X receptor (LXR␣), and adipocyte FA-binding protein (aP2), thereby reducing adipogenesis and lipogenesis (12,29,79). In rodents, supplementation of 10,12 CLA decreased the expression of PPAR␥ and its target genes (79,81–83). In contrast, humans supplemented with a CLA mixture had higher mRNA levels of PPAR␥ in WAT, but no difference in body weight or BFM (38). In mature, in vitrodifferentiated primary human adipocytes or in mature 3T3-L1 adipocytes, 10,12 CLA treatment leads to a substantial decrease in the expression and activity of PPAR␥ (82,83), and a decrease in PPAR␥ target genes and lipid content (80). This shows that 10,12 is not only able to inhibit, but also to reverse the adipogenic process and indicates that this may be mediated by suppression of PPAR␥ activity. In addition to its effect on PPAR␥ , 10,12 CLA may also directly impact the activity of other transcription factors involved in adipogenesis and lipogenesis (i.e., LXR␣, C/EBPs, SREBP-1c), which could contribute to CLA’s antiobesity actions.

CLA Decreases Glucose and FA Uptake and TG Synthesis Conversion of glucose and FAs to TG is a major function of adipocytes. Genes involved in lipogenesis, such as a LPL, ACC, fatty acid synthase (FAS), and stearoylCoA desaturase (SCD), were decreased following supplementation with mixed isomers of CLA or 10,12 CLA alone (12,56,72,80). PPAR␥ is a major activator of many lipogenic genes including glycerol-3-phosphate dehydrogenase (GPDH), LPL, and lipin as well as many genes encoding lipid droplet-associated proteins, such as perilipin, adipocyte differentiation-related protein (ADRP), and cell death–inducing DNA fragmentation factor of apoptosislike effector c (CIDEC) (84). Thus, the antilipogenic action of 10,12 CLA may be explained by inhibition of PPAR␥ activity. In addition, CLA repression of expression of SREBP1 and its target genes may play an important role in delipidation. Finally, CLA suppression of insulin signaling may also impair insulin’s ability to activate or increase the abundance of a number of lipogenic proteins including LPL, ACC, FAS, SCD-1, and the insulin-dependent glucose transporter GLUT4.

CLA Decreases Adipocyte Number Apoptosis is another mechanism by which CLA may reduce BFM. Apoptosis can occur through activation of the death receptor pathway, ER stress, or the mitochondrial pathway. A number of in vivo and in vitro studies have reported apoptosis in adipocytes supplemented with a CLA mixture or 10,12 CLA alone (56,64,85,86). For example, supplementation of C57BL/6J mice with 1% (w/w) CLA mixture reduced BFM and increased apoptosis in WAT (75). Mice fed a high-fat diet containing 1.5% (w/w) CLA mixture had an increased ratio of BAX, an inducer of apoptosis relative to Bcl2, a suppressor of apoptosis (87).

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Figure 2 Reported mechanisms by which 10,12 CLA decreases adipose tissue mass and obesity.

Reported mechanisms by which CLA reduces adiposity are shown in Figure 2.

ANTIDIABETIC PROPERTIES OF CLA Feeding obese ob/ob C57BL/6 mice 0.6% 9,11 CLA for six weeks improved plasma levels of glucose, TG, and insulin and reduced the expression of markers of inflammation and insulin resistance in WAT (88). Furthermore, these authors demonstrated that 50 ␮M 9,11 CLA prevented tumor necrosis factor (TNF)␣-mediated insulin resistance in 3T3-L1 murine adipocytes. Their data suggest that 9,11 CLA improves insulin sensitivity by elevating GLUT4 levels or translocation to the plasma membrane, which are adversely affected by inflammation, thereby facilitating glucose disposal. Similarly, Wistar rats fed a high-fat diet supplemented with a 0.75% to 3.0% CLA mixture for 12 weeks had lower plasma levels of glucose, TG, and insulin compared with high-fat fed control rats (89). The CLA mixture enhanced the expression of PPAR␥ target genes in WAT, which was proposed to be responsible for the improvement in insulin sensitivity. Consistent with these data, adiponectin, a WAT-specific, PPAR␥ target gene that reduces blood glucose by enhancing its oxidation in liver and muscle, was increased in the plasma of Zucker diabetic fatty (ZDF) rats fed a 1% CLA mixture for eight weeks (55). Similarly, feeding 0.5% 9,11 CLA to insulin resistant C57BL/6J mice improved insulin sensitivity without affecting BFM (90). Conversely, these authors found that feeding 0.5% 10,12 CLA lowered BFM and increased LBM in these mice, but caused insulin resistance. Other studies have also reported that 10,12 CLA causes insulin resistance, especially in mice (81,99). Taken together, these data suggest that 9,11 and 10,12 CLA have opposite effects on insulin sensitivity, most likely due to their opposing effects on the activity of PPAR␥ , visa-vis 9,11 CLA activates PPAR␥ and 10,12 CLA inhibits PPAR␥ .

ANTIATHEROSCLEROTIC ACTIONS OF CLA CLA has been reported to decrease risk factors of atherosclerosis in several important animal models (reviewed in Ref. 91). For example, feeding 0.5% mixed or individual isomers of CLA to New Zealand White rabbits fed a high saturated fat and cholesterol-rich diet reduced blood lipids and atherosclerotic lesion area (92). Syrian Golden hamsters fed a high saturated fat and cholesterolrich diet containing 1.0% mixed CLA isomers (93), 0.9% 9,11 CLA (94) or 1.0% 10,12 CLA (95), had decreased aortic

lipid accumulation or fewer fatty aortic streaks compared with controls. In apoE−/− deficient mice, feeding a 1.0% CLA mixture decreased aortic lesion area, and reduced macrophage infiltration and inflammatory gene expression in the lesions (96). In contrast to these animal studies, other animal and clinical trials with CLA mixtures have yet to show beneficial effects on reducing risk factors for atherosclerosis (reviewed in Ref. 97).

SAFETY Adverse side effects have been reported for CLA supplementation such as elevated levels of inflammatory markers, lipodystrophy, steatosis, and insulin resistance. Most adverse side effects are due to the 10,12 CLA isomer.

CLA Increases Markers of Inflammation Treatment with 10,12 CLA increases the expression or secretion of inflammatory makers such as TNF␣, interleukin (IL)-1␤, IL-6, and IL-8 from adipocyte cultures (56,73,80,81,83). Moreover, CLA increases the expression of COX-2, an enzyme involved in the synthesis of PGs, and the secretion of PGF2␣ (79,98). These inflammatory proteins are known to antagonize PPAR␥ activity and insulin sensitivity (87,98–100). Consistent with these in vitro data, 10,12 CLA supplementation increases the levels of inflammatory cytokines and PGs in humans (101,102). For example, women supplemented with 5.5 g/day of a CLA mixture for 16 weeks had higher levels of C-reactive protein in serum and 8-iso-PGF2␣ in urine (44). 10,12 CLA supplementation in mice resulted in macrophage recruitment in WAT (81). In contrast, 9,11 CLA exhibits antiinflammatory actions (6).

CLA Causes Insulin Resistance Insulin resistance has been reported in vivo (56,102–104) and in vitro (12,73,79,98) following supplementation with a CLA mixture or 10,12 CLA alone. For example, 10,12 CLA supplementation of 3.4 g/day for 12 weeks in obese men with metabolic syndrome increased serum glucose and insulin levels and decreased insulin sensitivity (103). Supplementation with a CLA mixture in type-2 diabetics increased fasting plasma glucose levels and reduced insulin sensitivity (102). Mice fed 1% (w/w) 10,12 CLA displayed elevated fasted and feeding plasma insulin levels and had reduced insulin sensitivity (75). Consistent with these data, the mRNA levels of adiponectin, a key adipokine associated with insulin sensitivity, decrease following supplementation with 10,12 CLA in vivo (36,81,100) and in vitro (79,82,105,106).

CLA Causes Lipodystrophy The combination of inflammation and insulin resistance results in reduced FA and glucose uptake in WAT, leading to ectopic lipid accumulation in the blood (hyperlipidemia), liver (steatosis), or muscle. CLA-mediated hyperlipidemia and steatosis has been reported in several animal studies (36,76,107). For example, 1% (w/w) CLA time-dependently increased insulin levels and led

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REFERENCES

Figure 3 Reported mechanisms by which CLA reduces the risk of cancer, obesity, diabetes, and atherosclerosis.

to increased liver weight and liver lipid accumulation in C57BL/6J mice (36). Aging C57BL/6J mice fed 0.5% 10,12 CLA displayed increased insulin resistance and liver hypertrophy (107).

US Regulatory Status Recently, the FDA approved CLA as GRAS (generally recognized as safe) for use in foods and beverages (not to exceed 1.5 g/serving) due its potential favorable effects. However, the use of CLA as a dietary supplement or ingredient should be cautioned based on the aforementioned safety issues.

CONCLUSIONS There is an abundance of evidence in animals suggesting that CLA consumption may reduce the incidence or risk of developing cancer, obesity, diabetes, or atherosclerosis, depending on the type and abundance of CLA isomer consumed and the physiological status of the animal model (Fig. 3). Data on the antiobesity properties of 10,12 CLA in animals, especially mice, are the most reproducible. However, these potential benefits are not without risks, as the 10,12 isomer is associated with increased levels of inflammatory markers, lipodystrophy, and insulin resistance. More clinical studies are needed to determine the efficacy of CLA isomers in humans, and more mechanistic animal and cell studies are needed to determine the precise, isomer-specific mechanisms of action of CLA, and potential side effects.

ACKNOWLEDGMENTS This work was supported by NIH NIDDK R15 DK 059289, NIH NIDDK/ODS R01DK063070, USDA-NRI 199903513, and NCARS 06771 awards to Michael McIntosh, NRSA NIH Fellowships to Kristina Martinez (F31DK084812) and Arion Kennedy (F31DK076208), and a United Negro College Fund-Merck predoctoral Fellowship to Arion Kennedy.

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breast cancer cells: Possible role of COX2. PLoS One 2009; 4(4):1–9. Ip MM, Masso-Welch PA, Shoemaker SF, et al. Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture. Exp Cell Res 1999; 250(1):22–34. Wang L, Huang YW, Yan P, et al. Conjugated linoleic acid induces apoptosis through estrogen receptor alpha in human breast tissue. BMC Cancer 2008; 8:208–222. Ou L, Wu Y, Ip C, et al. Apoptosis induced by t10,c12 conjugated linoleic acid is mediated by an atypical endoplasmic reticulum stress response. J Lipid Res 2008; 49(5):985–994. Larsson S, Bergkvist L, Wolk, A. Conjugated linoleic acid intake and breast cancer risk in a prospective cohort study of Swedish women. Am J Clin Nutr 2009; 90(3):556–560. Meng X, Shoemaker S, McGee S, et al. t10,c12 conjugated linoleic acid stimulates mammary tumor progression in Her2/ErbB2 mice through activation of both proliferation and survival pathways. Carcinogenesis 2008; 29(5):1013– 1021. Whigham LD, Watras AC, Schoeller DA. Efficacy of conjugated linoleic acid for reducing fat mass: A meta-analysis in humans. Am J Clin Nutr 2007; 85(5):1203–1211. Wang YW, Jones PJ. Conjugated linoleic acid and obesity control: Efficacy and mechanisms. Int J Obes Relat Metab Disord 2004; 28(8):941–955. Park Y, Storkson J, Albright K, et al. Evidence that trans10, cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 1999; 34(3):235–241. Brown JM, Halverson YD, Lea-Currie R, et al. Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue. J Nutr 2001; 131(9):2316– 2321. House RL, Cassady JP, Eisen EJ, et al. Functional genomic characterization of delipidation elicited by trans-10, cis-12conjugated linoleic acid (t10c12-CLA) in a polygenic obese line of mice. Physiol Genomics 2005; 21(3):351–361. Brandebourg TD, Hu CY. Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids. J Anim Sci 2005; 83(9):2096–2105. Raff M, Tholstrup T, Toiubro S, et al. Conjugated linoleic acids reduce body fat in healthy postmenopausal women. J Nutr 2009; 139(7):1347–1352. Park Y, Albright KJ, Liu W, et al. Effect of conjugated linoleic acid on body composition in mice. Lipids 1997; 32(8):853– 858. Sisk MB, Hausman DB, Martin RJ, et al. Dietary conjugated linoleic acid reduces adiposity in lean but not obese Zucker rats. J Nutr 2001; 131(6):1668–1674. Cl´ement L, Poirier H, Niot I, et al. Dietary trans-10, cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse. J Lipid Res 2002; 43(9):1400–1409. Meadus WJ, MacInnis R, Dugan, M. Prolonged dietary treatment with conjugated linoleic acid stimulates porcine muscle peroxisome proliferator activated receptor gamma and glutamine–fructose aminotransferase gene expression in vivo. J Mol Endocrinol 2002; 28(2):79–86. Poirier H, Rouault C, Cl´ement L, et al. Hyperinsulinemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia 2005; 48(6):1059–1065. Gaullier JM, Halse J, Høivik HO, et al. Six months supplementation with conjugated linoleic acid induces regionalspecific fat mass decreases in overweight and obese. Br J Nutr 2007; 97(3):550–560. Nazare JA, de la Perri`ere AB, Bonnet F, et al. Daily intake of conjugated linoleic acid-enriched yoghurts: effects on

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Copper Leslie M. Klevay

INTRODUCTION

proved to be the active material in several foods that were curative and could prevent the condition. All the classic deficiency experiments with animals were performed with milk diets. Adequate copper permits normal utilization of dietary iron. In addition to preventing anemia, it assists in blood coagulation (8,9) and blood pressure control (10,11), crosslinking (2,3,12) of connective tissues of arteries, bones, and heart, defense against oxidative damage (1), energy transformations, myelination of brain and spinal cord, reproduction, and synthesis of hormones (13). Inadequate copper produces adverse effects (14–16) on the metabolism of cholesterol and glucose, on blood pressure control and heart function, on mineralization of bones, and on immunity. Isoprostanes are increased in deficiency (10). Hypercholesterolemia in copper deficiency has been found in at least 30 independent laboratories (11,17), most recently in studies by Galhardi et al., Kaya et al., and Rosario et al. (18–20) since the original observation (21). Glutathione is an effective regulator of 3-hydroxy3-methylglutaryl coenzyme A activity (22,23). Copper deficiency disrupts glutathione metabolism (24), leading to increased activity of this enzyme (25–27) and contributing to the hypercholesterolemia that occurs. In contrast, decreased activities of lecithin: cholesterol acyltransferase (28) and lipoprotein lipase (29) also contribute to the hypercholesterolemia of deficiency. Electrocardiograms of animals deficient in copper reveal human cardiovascular risk factors such as branch block and abnormalities of the ST segment (15); other heart blocks and wave pathologies are numerous (15). The heart blocks are probably caused by decreased activity of an ATPase isoform localized to the conduction system of the heart (30). Copper deficiency depresses vasodilation via alterations in nitric oxide physiology (31,32). The mechanism has been reviewed (24,33) and may involve, inter alia, guanylate cyclase, which contains copper (34). Paraoxonase, sometimes called PON1, is a homocysteine thiolactone hydrolase [activity (35) of which is decreased by copper deficiency] (36). The lactone accumulates when homocysteine is elevated and irreversibly inhibits lysyl oxidase (35), which depends on copper for crosslinking of connective tissue in arteries and bone. There seems to be little doubt that copper deficiency can affect desaturase (and elongase) enzymes, but agreement is lacking on the details and directions of all the changes. Some of the data have been reviewed (14,24,37,38). These enzymes can alter the number of

Since the discovery in 1928 that copper is an essential nutrient, hundreds of experiments to clarify its function have been performed with several species of animals and, under very controlled conditions, with adult human volunteers. People respond to copper depletion similar to animals (1). The earliest experiments involved hematology, which preoccupied nutritional scientists for decades. Gradually, evidence for the adverse effects of copper deficiency on the cardiovascular and skeletal systems accumulated. Cardiovascular research related to copper deficiency, including associated lipid metabolism and cardiovascular physiology, now exceeds that on hematology. Early work on bone structure and function is being collected and extended. Methods for assessing nutritional status for copper are poorly developed. However, there are a sufficient number of reports of low activities of enzymes dependent on copper and low copper values in important organs to suggest that a considerable number of people may be too low in this element. These data complement measurements of dietary copper suggesting that the Western diet, which is frequently low in copper, may be the source of this abnormal biochemistry. Some people with abnormal gastrointestinal physiology may absorb too little copper as well.

GENERAL DESCRIPTION Copper is an essential and versatile nutrient that operates as the active site in 10 to 15 enzymes (1–3). These proteins moderate the chemistry of this metallic element to enhance various metabolic processes related to oxidation. There also are several other copper-binding proteins of physiological importance (3) in addition to some newly discovered proteins called metallochaperones (4). The latter proteins act in the intracellular transport of metallic elements and help to ensure that free copper ion is nonexistent in the body (5,6).

ACTIONS, BIOCHEMISTRY, AND PHYSIOLOGY The essentiality of copper for mammals, including people, was discovered (7) when rats fed a milk diet with adequate iron became anemic and grew poorly. Copper

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double bonds in a fatty acid and can also increase its length. Prostaglandin metabolism is also affected (24).

Food Sources and Supplementation As far as is known, food source does not affect copper absorption, in marked contrast to iron and zinc, which are more easily absorbed from animal, than from plant, products. Higher concentrations of copper in many plant foods can compensate if fractional absorption is slightly lower. Vegetarian diets are high in copper (39,40). Phytates either have no inhibitory effect on copper, or have a markedly smaller effect than that on zinc (21,41). At intestinal pH, copper complexes with phytates are soluble whereas zinc complexes are not. Phytates can thus enhance the utilization of copper (42). Copper absorption at 55% to 75% is considerably higher than that of other trace elements; absorption occurs mainly in the upper small intestine, but stomach and colon may absorb the element as well (1). Thus, the concentration of copper in foods is an important characteristic that determines nutritional usefulness. In order of increasing concentration on a weight basis, fats and oils, dairy products, sugar, tuna, and lettuce are low in copper; legumes, mushrooms, chocolate, nuts and seeds, and liver are high in copper (43,44). Bread, potatoes, and tomatoes are consumed in sufficiently large amounts by U.S. adults for these foods to contribute substantially to copper intake, although they are not considered to be high-copper foods (45). Copper and magnesium are highly correlated in U.S. diets (46). Food groups high in folate tend to be high in copper (35). The Western diet typical of the United States, parts of Europe, and wealthy enclaves in the developing world is often low in copper. Approximately one-third of these diets are low in comparison with those used in successful depletion experiments of men and women (47–51) under controlled conditions and in comparison to the estimated average requirement (EAR) (52) and recommended dietary allowance (RDA) of the National Academy of Sciences (U.S.) (below). Estimations of dietary copper intakes based on calculations, for example, about Canadian octogenarians (53), from the amount of copper in individual foods are too high according to eight published comparisons (54) to chemical analysis of composite diets; the mean error from calculation is an excess of 77% (54). The calculated 25th, median, and 75th percentiles for intakes of 51- to 70-year-old men in a statistical sample of the U.S. population (Table C-15 in Ref. 55) are 1.19, 1.47, and 1.81 mg copper daily. Corrections based on the mean excess in copper found by calculation (54) decrease these estimates to 0.67, 0.83, and 1.02 mg daily. Although younger men seem to eat more copper, women eat less! Data from several publications on dietary intakes of copper based on chemical analyses were pooled (40,44) and a frequency distribution curve was derived for 849 analyzed diets; approximately one-third of the diets contained less than 1 mg of copper daily. Further analytical confirmation of diets low in copper is available from men and women randomly selected in Baltimore. Thirty-six percent and 62% of the diets were below the respective dietary reference intakes for copper (56).

Three approaches to supplementation are available. Diets below the EAR and the RDA can be improved by avoiding foods low in copper and by selecting foods high in copper (43). A copper-deficient salad (lettuce, mayonnaise, oil, tuna, etc.) can be improved by adding sunflower seeds, mushrooms, legumes, etc (44). Soy products are increasingly popular and are high in copper (57), as are nuts (58) and chocolate (59). Beer enhances the utilization of copper in rats fed a deficient diet, resulting in a sixfold increase in longevity, with less cardiac damage and lower plasma cholesterol (60). In contrast to iron, fortification of foods with copper is uncommon. Some new snacks and drinks promoted as products with exceptional nutritional properties are fortified with copper. A variety of tablets and capsules containing copper are available commercially. Copper gluconate is the only copper supplement listed by the United States Pharmacopeial Convention and probably is the best supplement for oral use (61). We have used copper sulfate effectively in experiments with animals (21,62) and human volunteers (47,49,51). Others have used copper salts of amino acids (63). Other compounds containing copper, such as the orotate, for which there are no data on bioavailability should not be used. It is not easy to identify the chemical form of copper in some of the available supplements. Cupric oxide is contained in some vitamin–mineral supplements; this form is no longer used in animal nutrition because the copper is utilized poorly (64). Cupric oxide is used in the preparations with many ingredients because of its high concentration of copper, not because of demonstrated efficacy. Deficient people should be supplemented with several times the EAR or RDA. Daily supplements of 3 to 7 mg of copper have been tolerated for long periods (65,66).

INDICATIONS AND USAGE The Western diet is associated with rapid growth in infancy, increasingly early sexual maturation, tall adults, and low rates of infection. This diet is also associated with common diseases of affluence such as cancer, heart disease, obesity, and osteoporosis etc. (67). Numerous anatomical, chemical, and physiological characteristics of people with some of these latter diseases have been found in several species of animals deficient in copper (15,16). No single indicator provides an adequate assessment of copper nutriture (nutritional status) (52). Indices useful in experiments with animals have sometimes been helpful in depletion studies of people, but most do not seem to be altered by marginal deficiency. Circulating copper may not reflect the actions of enzymes inside cells in various organs where the metabolic processes affected by copper take place. Liver copper, generally impossible to assess in people, is the best indicator in animal experiments (62). Experiments with animals reveal that plasma copper can be normal or increased even though copper in liver or other organs may be low (68–76). Thus, normal or high plasma copper values in people may not be an accurate reflection of copper nutriture.

Copper

According to the Oxford Textbook of Medicine (77), low nutrient intakes can reduce nutrient concentrations in tissues and compromise metabolic pathways. Diagnosis then is relatively straightforward upon measurement of the nutrient in suitable tissues or testing of metabolic pathways. Numerous medical publications (some of which are summarized here) reveal low copper concentrations and impaired enzymatic pathways dependent on copper in people. As “nutritional state often alters the expression and course” (77) of illness, extra copper should be provided if low measurements related to copper values are found whether or not they are the cause or the result of the pathology under consideration. Interpretation of copper or ceruloplasmin in serum or plasma in the assessment of nutriture may be difficult. Low values indicate impairment. Pepys (78) describes the acute phase response to acute and chronic inflammation: a number of plasma proteins, such as ceruloplasmin, are synthesized in liver under the influence of cytokines and are secreted into the circulation. Thus, any illness with a large inflammatory component may have falsely high values (78,79). Normal or high values cannot provide assurance that copper deficiency is not present. Clearly people with myelodysplasia and the new syndrome resembling the neurology of pernicious anemia (below) can be considered for supplementation. Possibly deficient people should be evaluated with some of the newer, potentially more sensitive, indices of copper status such as erythrocyte and extracellular superoxide dismutases, leukocyte copper, platelet cytochrome c oxidase or serum lysyl oxidase (80–84). Data on which to base dietary reference intakes for copper are elusive and, often, absent. Consequently, some of the values in Table 1 are rounded and values for males and females are combined. The adequate intake (AI) values are based on intakes of apparently healthy, full-term infants whose sole source of copper was human milk. Values for pregnancy are based on the amount of copper in the fetus and other products of conception. Those for lactation are the amounts needed to replace the average amount secreted in human milk. EARs are values estimated to meet the requirement of half of the healthy individuals of the group. Copper RDAs are based on the EAR plus an assumed coefficient of variation of 15%, which is larger than the 10% assumed for some other nutrients (57). In the United States, dietary reference intakes are median values with an assumed symmetry of distribution (85). However, there is virtually no information about the

Table 1 Daily Adequate Intake (AI), Estimated Average Requirement (EAR), and Recommended Dietary Allowance (RDA) for Copper (mg) Age

AI (mg)

0–6 mo 7–12 mo 1–3 yr 4–8 yr 9–13 yr 14–18 yr 19–70 yr Pregnancy Lactation

0.20 or 30 (␮g/kg) 0.22 or 24 (␮g/kg)

EAR (mg)

RDA (mg)

0.26 0.34 0.54 0.685 0.70 0.80 1.00

0.34 0.44 0.70 0.89 0.90 1.00 1.30

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shape of the copper distribution; distributions for most nutrients are skewed to the high end (85). People who are deficient in copper without obvious cause (below) probably have a personal requirement for copper considerably higher than the median requirements reflected in the RDAs. It seems clear that there is little or no copper deficiency in the industrialized world if one relies on traditional criteria of deficiency such as anemia with decreased plasma copper or ceruloplasmin. However, these markers are affected by the acute phase response and are easily increased by nondietary variables, such as inflammation, oral contraceptives, and pregnancy etc. Copper depletion experiments with men and women reveal unfavorable alterations in biochemistry and physiology with minimal or no changes in circulating copper and without anemia (above). Copper deficiency is the leading nutritional deficiency of agricultural animals worldwide; (86) can people be far behind? A 2001 report on dietary reference intakes (55) and its predecessors, for example in Ref. (87), summarize the reasons why people may decide to take (or avoid) nutrient supplements. Growth and function are improved when nutrients are increased above levels just sufficient to prevent deficiency. There is little evidence that small surpluses of nutrients are detrimental, while small deficits will lead to deficiency over time. There is no evidence of unique health benefits from the consumption of a large excess of any one nutrient. Meeting recommended intakes for nutrients will not provide for malnourished individuals. There seems to be little or no anemia responsive to copper in the United States, although this phenomenon does not seem to have been studied adequately in the last six decades. Copper deficiency can masquerade as the myelodysplastic syndrome, however (88–90). Supplementation of middle-aged Europeans with copper protected their red blood cells from oxidative hemolysis in vitro (63), indicating that extra copper improved the quality of the cells. Alzheimer’s disease is the leading cause of dementia in the elderly and is of unknown etiology. It is hypothesized that deficiency of dietary copper is the simplest and most general explanation for the etiology and pathophysiology of this disease because, inter alia, of numerous reports of low copper in the brain and low activity of enzymes dependent on copper in these patients (91). These findings are consonant with Golden’s criteria for diagnosing deficiency (77). Kumar (92) has reviewed and expanded upon a copper deficiency syndrome resembling the neuropathy of pernicious anemia (vitamin B12 deficiency). Supplementation with cyancobalamine is useless, but extra copper generally arrests the decline and sometimes reverses some of the signs. Several of the classical risk factors for ischemic heart disease have been produced in animals deficient in copper. Similar changes have been found in more than 30 men and women in successful copper depletion experiments using conventional foods and have been reversed by copper supplementation (47–51). Copper intakes of 0.65 to 1.02 mg daily in these experiments were insufficient. Criteria of depletion included abnormal electrocardiograms

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(47,48) and blood pressure regulation (51), dyslipidemia (50), glucose intolerance (49), and hypercholesterolemia (47). Two of these experiments were interrupted prematurely with early repletion with copper because of abnormal electrocardiography; all of the metabolic and physiological abnormalities disappeared with copper repletion. Low paraoxonase activity is found in conditions associated with increased risk of ischemic heart disease (36); isoprostanes are increased (93). In contrast is a balance experiment using a formula diet that failed to confirm these results (94). Applesauce, cheese, chicken, cornflakes, crackers, lettuce, margarine, milk, orange juice, and rice provided less than 31% to 34% of dietary energy (calculated at 2400 kcal/day) (95). As actual energy intake ranged from 2415 to 3553 kcal (94), the food part of the formula was probably about 26%. Because formula diets are known to lower serum cholesterol (96), the potential increase in cholesterolemia from the low copper intake may have been obscured. Activities of enzymes dependent on copper (97– 103) and organ copper concentrations (104–113) have been found to be decreased in people with cardiovascular (mostly ischemic) diseases. There is a positive correlation between cardiac output and copper in heart tissue of patients with coronary heart disease (112). Decreased copper in organs and decreased enzyme activities are evidence of impaired copper nutriture (77,114,115). No long-term copper supplementation has been performed in patients with cardiac arrhythmia, dyslipidemia, glucose intolerance, hypercholesterolemia, or hypertension. However, some dietary regimens found to alleviate some of these conditions may have included an increase in copper intake as a hidden variable: for example, the Lifestyle Trial (116), the protective effect of legumes on cholesterol, blood pressure, and diabetes (117) and the benefit of whole grain foods on coronary heart disease (118). Spencer (119) described two men and a woman whose premature ventricular beats, which had persisted for years, were thought to be due to coronary heart disease. These premature beats disappeared after they ingested 4 mg of copper (as copper gluconate) per day. Witte et al. (120) explain how deficiencies of micronutrients, copper among them, can contribute to cardiovascular disease. Patients with heart failure in their supplementation trial had improved ventricular function and quality of life (121); copper in the supplement may have contributed (122). Supplementation trials with vitamins to lower homocysteine may show clinical benefit if extra copper is included (123); copper supplementation (with zinc) improved survival in a long-term, doubleblind study of ocular disease (124). Copper-deficient people have osteoporosis that can be cured with extra copper (reviewed in Ref. 16). This phenomenon has been found mainly in young children. Adults may have skeletal pathology from low copper status as well. Copper is decreased in bone in both osteoarthritis and ischemic necrosis of the femoral head (125). Low serum copper in patients with fractures of the femoral neck (126) or decreased lumbar bone density (14,127,128) may indicate covert copper deficiency. Plasma copper and bone mineral density are correlated (129). Healthy men fed a diet low in copper (0.7 mg/day)

experienced increased bone resorption that returned to normal when copper was replaced (130). There can be no medical doubt that copper deficiency can cause osteoporosis in people. These references on osteoporosis from copper deficiency (131–141) have been found since the earlier review (16) of 17 articles. If copper deficiency turns out to be a major component of the osteoporosis of middle age, supplementation with copper alone is unlikely to be effective. If copper deficiency is corrected, another nutrient, particularly calcium and possibly zinc, may become limiting (77). Two double-blind, placebo-controlled trials have shown that trace element supplements including copper improved bone mineral density in postmenopausal women (65,142). Premature infants and people with extensive burns may need extra copper. The former (143) are sometimes born before their mothers can load them with copper in the last trimester (12). Premature infants have lower superoxide dismutase activity in erythrocytes and plasma copper after 100 days of life than term infants (144); premature placentas are low in copper and copper-dependent enzymes (145). In analogy to vitamin B12 deficiency, any disruption of the gastrointestinal tract has the potential to impair copper nutriture. Copper deficiency is being reported with increasing frequency in patients who have had bariatric surgery (90,92,146–149). Some people with cystic fibrosis or pancreatic insufficiency may need extra copper (150– 152). Copper-dependent enzyme activity and copper concentration have been found to be decreased in ulcerative colitis biopsies (153). Supplementation of people with these conditions should be performed under medical supervision. If adults have unmet needs for copper to provide cardiovascular, hematopoietic, or skeletal benefit, neither the dose nor the duration of therapy is clear. A potential role for copper supplements in the treatment of rheumatoid arthritis and psoriasis has not been proved. There is probably no reason to exceed the tolerable upper intake level (UL) of 10 mg daily (Table 2).

Potential Toxicity and Precautions All chemicals, including essential nutrients, are toxic if the dose is excessive. It seems that people have a 50- to 400-fold safety factor for copper considering usual dietary intakes and the tolerance level found with several species of experimental animals (154). The UL connotes an intake Table 2 Daily Tolerable Upper Intake Level (UL) for Copper (mg) Age group Children 1–3 yr 4–8 yr 9–13 yr Adolescents 14–18 yr Adults 19–70+ yr Pregnancy Lactation

UL (mg) 1.00 3.00 5.00 8.00 10 8.00 8.00–10.00

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that can, with high probability, be tolerated biologically by almost all individuals. Gastrointestinal signs and symptoms such as nausea are prominent in the setting of this limit. A small, double-blind study has revealed that adults are unaffected in 12 weeks by a daily supplement of 10 mg of copper (52). The UL values in Table 2 are based on this experiment; no value is available for infants less than 1-yearold. van Ravesteyn (155) administered 38 mg of copper daily to people for as long as 14 days; toxicity was not mentioned. Copper supplements should be taken with food (156,157) and should not be taken by people with biliary disease, liver disease, idiopathic copper toxicosis or Wilson’s disease, or by people taking penicillamine or trientine. Although copper can interfere with zinc utilization, this phenomenon does not seem to be of practical importance to people. In contrast, copper deficiency has been induced in people (and in numerous species of pets and animals in zoos) by the ingestion of recently minted pennies (United States), which are almost pure zinc (158). The dose of supplemental zinc that is excessive for adults is ill-defined, but the adult UL for zinc, 40 mg daily, is based on reduced copper nutriture from zinc in food, water, and supplements combined. A case of copperresponsive anemia has been reported in a patient with acrodermatitis enteropathica overtreated with zinc (159). This potential exists for patients with Wilson’s disease treated with zinc, particularly children (160). Demyelination of the central nervous system has been reported from overzealous treatment of Wilson’s disease with zinc (161). Denture creams high in zinc have led to copper deficiency (162,163). Vitamin C is known to interfere with the utilization of copper (131,164–168), but its UL of 2 g daily is not based on copper effects. Adverse effects on blood pressure regulation and copper utilization were found in women fed 1.5 g vitamin C daily (51). Simple sugars such as fructose, glucose, and sucrose interfere with the utilization of copper (169,170): High-fructose corn syrup is found in many processed foods and beverages. Excessive ingestion of soft drinks (171,172) has contributed to copper deficiency. High iron intakes can disrupt copper utilization (85,131,173–175). People with iron overload (176,177) and lead poisoning (178–180) may benefit from copper supplementation. Copper supplements should not be used as emetics.

CONCLUSIONS The Western diet often is low in copper. Statements to the contrary are based on dietary calculations, which are falsely high. The best way to ensure an AI of copper is to minimize the intake of foods low in copper and to increase that of foods high in it, such as cereals, grains, legumes, mushrooms, nuts, and seeds. Dietary copper can be increased by using the food pyramid as a guide. Only a few foods are fortified with copper. Copper gluconate is probably the best supplement. There seems to be little copper deficiency in Western society if one considers anemia as its only sign. However,

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adults with diseases of the cardiovascular, gastrointestinal, and skeletal systems have repeatedly been found to have low concentrations of copper in important organs and to have low activities of enzymes dependent on copper. These signs are consonant with deficiency. Premature infants may also be deficient in copper. Large intakes of vitamin C or zinc can impair the proper utilization of copper in people. People deficient in copper are being reported with increasing frequency. Although many circumstances seem without explanation and because the clinical signs differ from those traditionally associated with copper deficiency, the reports are often scattered in medical journals that do not have the word “nutrition” in their titles. Recognition of copper deficiency in the general population still seems rare enough to be published, but deficiency also is common enough that 10 cases are reported from one neurological clinic. The index of suspicion should be increased among those providing primary care. When obvious explanations such as bariatric surgery, dental adhesives high in zinc, hemochromatosis, lead poisoning, and soft drink excess are excluded one should consider the possibility that the patient has a dietary requirement higher than those mentioned among the dietary reference intakes. Some cases of myelodysplastic syndrome and heart failure respond to copper. Evaluations should not rely only on plasma copper or ceruloplasmin. Supplementation should be done with substantial doses of easily absorbed, copper salts under medical supervision. Some successful experiments in human copper depletion, particularly in relation to the cardiovascular system, are summarized here, as are some unsuccessful experiments. Others have been reviewed (181). Depletion experiments with positive results illustrate what is possible in the wider world, particularly when the potentially adverse effects were eliminated on repletion. Seemingly contrary or incongruous experiments should not promote denial or negativism; rather they should stimulate searches for explanations of differences. Individual animals and individual people fed the same depleted diets do not respond uniformly. Signs of deficiency are variable in experimental pellagra and in animal experiments on biotin, thiamin, or copper deficiencies. Our success rate in inducing copper depletion in people resembles that of Goldberger in inducing pellagra. One should not expect all people to respond uniformly to a diet low in copper (15). There is no information available on how copper requirements vary from person to person; dietary recommendations are based on assumptions of narrow variability. The amount of copper in the body and its variability are known, only inaccurately. Copper absorption is reasonably well defined, but data on copper losses are scant. Aside from some obvious causes of deficiency mentioned here, causes of human deficiency in some recent, medical reports are unknown. These reports have increased since the first edition and illustrate opportunities for research; it seems likely that people with unidentified high requirements for copper are eating too little. The field of copper nutrition is far from stagnant.

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181. Klevay LM, Medeiros DM. Deliberations and evaluations of the approaches, endpoints, and paradigms for dietary recommendations about copper. J Nutr 1996; 126:2419S– 2426S.

FURTHER READING 1. Kies, C. Copper bioavailability and metabolism. In: Advances in Experimental Medicine and Biology. 1st ed. New York: Plenum Press, 1989; 258. 2. Klevay LM, Medeiros DM. Deliberations and evaluations of the approaches, endpoints and paradigms for dietary recommendations about copper. J Nutr 1996; 126:2419S–2426S. 3. Lei KY, Carr TP. Role of Copper in Lipid Metabolism. Boca Raton, FL: CRC Press, 1990:287.

Cordyceps John Holliday, Matt Cleaver, Mojca Tajnik, Joseph M. Cerecedes, and Solomon P. Wasser

found in cultivated C. sinensis has lead many consumers to believe the wild collected variety is medicinally better than the cultivated type. But with new advances in biotechnology, this is rapidly changing (6).

ABBREVIATIONS ATP, adenosine triphosphate; GM-CSF, granulocyte macrophage colony-stimulating factor; GRAS, generally recognized as safe; HDL, high-density lipoprotein; IFN, interferon; Ig, immunoglobulin; IL, interleukin; iNOS, calcium-insensitive nitric oxide synthases; LD50 , median lethal dose; LDL, low-density lipoprotein; NK cell, natural killer cell; PAH, polyaromatic hydrocarbons; PAMP, pathogen-associated molecular patterns; TCM, traditional Chinese medicine; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Contamination and Adulteration of Cordyceps As found in its natural state, C. sinensis is attached to the mummified body of the caterpillar, from which it arose. It is harvested whole in this way, dried, and supplied into the market. Because C. sinensis is sold by weight and intact fruiting bodies fetch higher prices in traditional markets, collectors have historically inserted a small bit of twig into many of the caterpillars, resulting in an increase in weight and the appearance of intact fruiting bodies (5). This is probably a harmless practice, as long as the object inserted is from a nontoxic source. However, modern collectors have inserted lead or other metal in order to boost the weight, so anyone who chooses to use the wild collected C. sinensis, rather than the cultivated variety, would be well advised to break each one of the caterpillars in half before use, so that any bits of foreign matter can be readily discerned and removed.

INTRODUCTION Species of the genus Cordyceps (Fr.) Link (also known as Chinese caterpillar fungi, or Tochucaso in Japanese; Clavicipitaceae, Ascomycetes) are the fungi found growing on insect larvae (Fig. 1), mature insects, or fruiting bodies of truffles of genus Elaphomyces. Cordyceps has a long history as a rare and exotic medicinal fungus. It has been a highly regarded cornerstone of Chinese medicine for centuries; one that reportedly has a number of far reaching medicinal effects (1). Most people in the West have only come to know of Cordyceps within the last twenty years, during which time, modern scientific methods have been increasingly applied to the investigation of its seemingly copious range of medicinal applications, in an attempt to validate what Chinese practitioners have noted for centuries (2).

History and Traditional Uses The first written record of the Cordyceps mushroom comes from China, in the year AD 620, at the time of the Tang Dynasty, bringing substance to the once intangible allegorical narrative, which spoke of a creature, whose annual existence alluded to a transformation from animal to plant, in summer, and then again from plant to animal, in winter (1). Tibetan scholars wrote of the healing animal/plant through the 15th to 18th centuries, and in 1757, the earliest objective and scientifically reliable depiction of the Cordyceps mushroom was written by the author Wu-Yiluo in the Ben Cao Congxin (“New Compilation of Materia Medica”), during the Qing Dynasty (2–3). C. sinensis is found at high altitudes on the Himalayan Plateau, and thus, is difficult to harvest. Due to such difficulties, Cordyceps has always been one of the most expensive medicinal fungi known. Its high price had relegated it almost exclusively to members of the Emperor’s court and others among the Chinese nobility, historically beyond the reach of the average Chinese subject. Despite its cost and rarity, the unprecedented litany of medicinal possibilities for Cordyceps spp. has made it a highly valued staple of the TCM. The name Cordyceps comes from the Latin words, cord and ceps, respectively meaning, “club” and “head.” The Latin word conjunction accurately describes the appearance of these club fungi, whose stroma and fruit body

BACKGROUND Diversity and Artificial Cultivation There are currently more than 680 documented species of Cordyceps, found on all six inhabited continents and in many climatic zones and habitats, and occurring parasitically or commensally with a range of hosts (2–3). Due to the rarity and high prices of the wild collected variety, attempts have long been made to cultivate C. sinensis. By the mid 1980s, the majority of C. sinensis available in the world’s marketplace was artificially cultivated (4). Many companies now produce artificially cultivated C. sinensis products, both from the mycelium as well as from the fruit bodies. The increase in supply has given rise to variations in purity and quality, creating a situation in which there are a large number of counterfeit and adulterated products being sold (3). Recently, there have been introduced, new methods for assaying the quality of Cordyceps spp. products (5). The large variations in quality 185

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ering from a serious illness. Many also believe it to be a treatment for impotence, acting as an aphrodisiac in both men and women. C. sinensis is often prescribed for the elderly to ease general aches and pains. TCM practitioners also recommend the regular use of C. sinensis in order to strengthen the body’s resistance to infections, such as colds and flu, and to generally improve the homeostasis of the patient.

CHEMISTRY AND PREPARATION OF PRODUCTS Nutritional Components Cordyceps spp. contains a broad range of compounds, which are considered nutritional. C. sinensis contains all of the essential 18 amino acids. The content of amino acids after hydrolysis is mostly reported in the range of 20% to 25%. The highest contents are glutamate, arginine, and aspartic acid, and the major pharmacological components are arginine, glutamate, tryptophan, and tyrosine (7). Also found are vitamins E, K, and the water-soluble vitamins B1, B2, and B12. In addition, Cordyceps spp. contain many sugars, including mono-, di-, and oligosaccharides, and many complex polysaccharides, proteins, sterols, nucleosides, macro- and microelements (K, Na, Ca, Mg, Fe, Cu, Mn, Zn, Pi, Se, Al, Si, Ni, Sr, Ti, Cr, Ga, V, and Zr) (2,5).

Polysaccharides Figure 1

Cordyceps sinensis in natural habitat (4550 m in Tibet, China).

extend from the mummified carcasses of insect larvae, usually that of the Himalayan ghost Moth, Thitarodes armoricanus (Hepialis armoricanus). In historical and general usage, the term “Cordyceps” normally refers specifically to the species C. sinensis. However, the name “Cordyceps” has come to be used for a number of closely related species over the last few years, which have been found to be much easier to cultivate. While C. sinensis may be the most wellknown species, there are many other species in the genus Cordyceps, in which modern science may have uncovered potentially valuable medicinal properties. The medicinal values of Cordyceps spp. have been recognized since ancient times in China and the surrounding Orient; but knowledge of this only reached Western scientific audiences in 1726, when it was introduced at a scientific meeting in Paris. The first specimens were carried back to France by a Jesuit priest, who chronicled his experiences with the Cordyceps mushroom during his stay at the Chinese Emperor’s court (1,4). The range of therapeutic uses claimed for Cordyceps spp. is far reaching; although most of them have yet to be sufficiently investigated. In TCM, C. sinensis has been used to treat conditions including respiration and pulmonary diseases, renal, liver, and cardiovascular diseases, hyposexuality, and hyperlipidemia. It is also used in the treatment of immune disorders and as an adjunct to modern cancer therapies (chemotherapy, radiation treatment, and surgery) (1). C. sinensis is believed by many, particularly in and around Tibet, its place of origin, to be a remedy for weakness and fatigue; and it is often used as an overall rejuvenator for increased energy while recov-

C. sinensis contains a large amount of polysaccharides, which can be in the range of 3% to 8% of the total weight, and usually comes from the fruiting bodies, the mycelium of solid fermentation submerged cultures and the broth (7). Four ␤-D-glucan exopolysaccharides from C. militaris with different molecular masses ranging from 50 to 2260 kDa were reported by Kim et al. (8). In the case of C. sinensis, most of the heteropolysaccharides contained mannose, galactose, glucose, and mannose in higher levels with smaller amounts of arabinose, rhamnose, fructose, and xylose, respectively. The average molecular mass varies between 7 and 200 kDa. C. militaris polysaccharides consisted mostly of glucose, galactose, and mannose with traces of rhamnose and xylose and average molecular weight approximately 60 kDa (9).

Proteins and Nitrogenous Compounds Cordyceps spp. contain proteins, peptides, polyamines, all essential amino acids, some uncommon cyclic dipeptides, including cyclo-[Gly-Pro], cyclo-[Leu-Pro], cyclo-[ValPro], cyclo-[Ala-Leu], cyclo-[Ala-Val], and cyclo-[ThrLeu]. Small amounts of polyamines, such as 1,3-diamino propane, cadaverine, spermidine, spermine, and putrescine, have also been identified (4). Many nucleosides have been found in Cordyceps spp., including uridine, several unique deoxyuridines, adenosine, dideoxyadenosine, hydroxyethyladenosine, cordycepin [3 deoxyadenosine], cordycepin triphosphate, guanidine, deoxyguanidine, and other altered and deoxygenated nucleosides, many of which are found nowhere else in nature (Fig. 2). Chen and Chu (10) found cordycepin and 2 -deoxyadenosine in an extract of C. sinensis. Sugar-binding proteins named lectins were isolated from C. militaris. N-terminal amino acid sequence differed

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The widespread use of Cordyceps spp. in TCM has been discussed above in the section on History and Traditional Uses. One of the most significant proposed activities of medicinal mushrooms is their role as immunomodulators. Other activities ascribed to Cordyceps spp. are antitumor, antimetastatic, immunomodulatory, anti-oxidant, anti-inflammatory, insecticidal, antimicrobial, hypolipidaemic, hypoglycemic, anti-aging, neuroprotective and renoprotective activities (2).

HO N

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Figure 2

Various pure compounds, extracts, whole fungus, and other preparations have been used in preclinical and clinical studies, and several products are now available in the market, mostly used as food supplements. In TCM, hotwater extraction of whole fruiting bodies is traditionally used. Nowadays, extracts of polysaccharides are mainly obtained by hotwater extraction followed by ethanol precipitation (15). For pure compounds, different types of chromatography are used, mainly affinity, ion-exchange or size-exclusion chromatography. It should be noted that different types of extracts give different results in the studies mentioned, but all of them show positive medicinal value.

H H

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Some of the unique nucleosides found in C. sinensis.

Cancer greatly from other lectins (11). Production of the nonribosomal peptides cicapeptins I and II from C. heteropoda were reported by Krasnoff et al. (12).

Sterols A number of sterol-type compounds have been found in Cordyceps spp.: ergosterol, ␦-3 ergosterol, ergosterol peroxide, 3-sitosterol, daucosterol, and campeasterol, to name a few (1). Another compound, sterol H1-A was found by Chen et al. (13) and it was claimed to be effective in the treatment of autoimmune disorders.

Other Constituents Twenty-eight saturated and unsaturated fatty acids with the function of decreasing blood lipids and protecting against cardiovascular disease, and their derivatives, have been isolated from C. sinensis. The unsaturated fatty acid content includes Cl6:1, Cl7:1, Cl8:l, and Cl8:2 (7). Polar compounds of C. sinensis extracts include many alcohols and aldehydes (1). Particularly interesting is the range of polycyclic aromatic hydrocarbons produced by many C. sinensis strains, named PAH compounds, for which it was proposed to react with the polypropylene used in common mushroom culture bags, resulting in the production of byproducts toxic to C. sinensis and stunting growth as time progresses (5). Of particular note are various immunosuppressive compounds found in Cordyceps spp., including cyclosporin from C. subsessilis (anamorph: Tolypocladium inflatum) (14), and also compounds found in Isaria sinclairii, a species closely related to the genus of Cordyceps (3).

A possibly valuable therapeutic application of Cordyceps spp. is its potential as a treatment for cancer, and as an adjunct to chemotherapy, radiation, and other conventional and traditional cancer treatments (2,4). The mechanism by which Cordyceps inhibits the growth of various cancer cells might occur by one of several means: by enhancing immunological function and nonspecific immunity; by selectively inhibiting RNA synthesis, thereby affecting the protein synthesis; by restricting the sprouting of blood vessels (angiogenesis); by inducing tumor cell apoptosis; by regulation of signal pathways; anti-oxidation and antifree radical activity; anti-mutation effect; interfering with the replication of tumor-inducing viruses; and by inducing nucleic methylation (7). Growth inhibition of various cancer cells by enhancing immunological function and nonspecific immunity is usually linked to polysaccharides, especially ␤-D-glucans, which present major cell wall structural components in fungi and are also found in plants and some bacteria but not in animals. Consequently, they are considered to be classic pathogen-associated molecular patterns, called as PAMPs (16). PAMPs potently trigger inflammatory responses in a host, as if it was infected by a fungus. Studies have shown that ␤-D-glucans initiate biological response with binding to complement receptor 3 (CR3) located on the surface of the immune system effector cells, like macrophages, thereby setting up different intercellular activities of the immune system and leading to production of cytokines, such as TNF-␣, interleukins, interferons, and finally apoptosis of tumor cells (17). Toll-like receptors (especially TLR-2) and dectin-1 receptor play an important role in internalization and signaling responses to fungal ␤-D-glucans (18).

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The anti-tumor effect also has been related to the inhibition of DNA and RNA synthesis (19). Studies (20) have demonstrated that cordycepin can selectively inhibit mRNA synthesis, which affects protein synthesis by competing with adenosine nucleoside phosphatase. The inhibition may be blocked by adenosine. Cordycepin can also kill leukemia cells and extend the period of mitotic cells in the S and G phases. Nakamura et al. (21) found that, with respect to cancer cells and normal cells, cordycepin caused an inhibition rate of cell division on cancer cells of 55% while only 1.5% on normal cells. These results show that cordycepin may have a very slight effect on the human body while treating cancer. Another mechanism is inducing tumor cell apoptosis. Extracts of C. militaris inhibited cell growth of human leukemia cells in a dose-dependent manner (22), which was associated with morphological change and apoptotic cell death, such as formation of apoptotic bodies and DNA fragmentation. Results indicated that the antiproliferative effects were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products (23). In cancer research, there have been many studies made with Cordyceps spp. extracts using animal models. C. militaris inhibited the growth and metastasis of Lewis lung cancer cells and the growth of sarcoma S180 cells implanted in mice. In addition, the survival period of the mice was increased (24). A study using murine models verified that oral administration of a hot water extract of C. sinensis consequently resulted in the activation of macrophages, thereby increasing the production of GM-CSF and IL-6, which act on the systemic immune system (25). In another study (26), mice treated with cyclophosphamide, which suppresses immune function, and with C. sinensis hot water extract saw their immune function return to normal, as measured by the IgM and IgG response and macrophage activity.

Fatigue Trials in the mouse swim test, conducted using C. sinensis added to a standard diet compared with use of the untreated standard diet, have invariably shown the use of C. sinensis to significantly increase the time to exhaustion in laboratory animals over their control groups (4). The use of C. sinensis by athletes stems from publicity surrounding the performance exhibited by the Chinese Women’s Track and Field team at the Chinese National games in 1993. In this competition, nine world records were broken by substantial margins. The team’s coach attributed their success to C. sinensis (27). An increase in cellular ATP level results in an increase in useful energy, in contrast to the perceived increase in energy, which occurs from the use of other stimulants, such as caffeine, ephedrine, and amphetamines, ultimately resulting in an energy deficiency (28).

Hypoglycemic Effects In animal studies, isolated polysaccharides, have been shown to improve blood glucose metabolism and increase insulin sensitivity in normal animals, to lower blood sugar levels in genetically diabetic animals, and to positively effect blood sugar metabolism in animals with chemically

induced diabetes (29–31). The common thread throughout all these trials is the increase in insulin sensitivity and hepatic glucose-regulating enzymes, glucokinase and hexokinase.

Lung Ailments Mice treated with C. sinensis were able to survive up to three times longer than those left untreated, demonstrating a more efficient utilization of the available oxygen. Such efficacy alludes to the use of C. sinensis as an effective treatment for bronchitis, asthma, and chronic obstructive pulmonary disease (32). A study was conducted using in vivo mouse model-induced acute pulmonary edema, which causes systemic lack of oxygen, acidic body, and death. Research results show that animals taking C. sinensis had a significantly greater survival rate of 20% mortality in comparison with 80% mortality of the control group (33).

Male/Female Sexual Dysfunction C. sinensis has been used for centuries in TCM to treat male and female sexual dysfunction, such as hypolibidinism and impotence. Preclinical data on the effects of C. sinensis on mice showed sex steroid–like effects (4), and human clinical trials have demonstrated similarly the effectiveness of C. sinensis in combating decreased sex drive and virility (34). Treatment of rats on a diet supplemented with C. militaris mycelium resulted in an increase of serum cordycepin concentration, serum testosterone, and serum estradiol-17 concentrations. They proposed that supplementation with C. militaris improves sperm quality and quantity in rats (35).

Antiviral Activity The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells with many innate immunity receptors, such as Toll-like receptors, Ctype lectin receptors, and immunoglobulin-like receptors. Studies indicate that the immune modulating properties of C. sinensis could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity (36). It was shown that intranasal administration of an acidic polysaccharide, isolated from the extract of C. militaris grown on germinated soybeans, decreased virus titers in the bronchoalveolar lavage fluid and the lungs of mice infected with influenza A virus. Furthermore, it increased TNF-␣, IFN-␥ , IL-1, IL-6 and IL-10 levels, enhanced nitric oxide production, and induced iNOS mRNA expressions in murine macrophage cells (37).

CLINICAL STUDIES Due to the historically high cost of the fungus and the only recently developed methods for artificial cultivation, clinical trials of C. sinensis and its extracts are still relatively new endeavors. Earlier trials, although few in number, have set the precedent from which modern trials are building, expanding, and cementing our understanding of Cordyceps spp. The majority of clinical trials mentioned in this section used standard double-blind placebo-controlled protocols. Approval was granted in the

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countries where the trials were performed, but in most cases the trials were conducted in China.

Cancer The belief in the efficacy of C. sinensis against cancer is widespread in the Orient, and many cancer patients in Japan, Korea, and China are taking C. sinensis, or some other mushroom-derived immunomodulators [such as PSKTM , PSPTM , LentinanTM , AHCCTM , Immune AssistTM (a heteropolysaccharide complex formula), and arabinoxylanes (MGN3TM )], while undergoing conventional treatment (1). Clinical studies involving cancer patients have been conducted mostly in China and Japan (38,39). In one study of 50 patients with lung cancer, who were administered C. sinensis at 6 g/day, in conjunction with chemotherapy, tumors were reduced in size in 46% of the patients studied. A trial involving cancer patients with several different types of tumors found that C. sinensis, taken over a two-month period at 6 g/day, improved subjective symptoms in the majority of patients. White blood cell counts were kept at 3000/␮L, or higher; and even with radiation or chemotherapy, other immunological parameters showed no significant change, while tumor size was significantly reduced, indicating an improved tolerance for radiation and/or chemotherapy (1). In addition, natural C. sinensis has been shown to enhance the NK cell activity of normal patients by 74% and increased the NK activity of leukemia patients by 400% (39).

Fatigue In a placebo-controlled clinical study of elderly patients with chronic fatigue, results indicated that most of the participants treated with C. sinensis reported a significant clinical improvement in the areas of fatigue, cold intolerance, dizziness, frequent nocturia, tinnitus, hyposexuality, and amnesia, while no improvement was reported in the placebo group (4,40–42). Another study involving healthy elderly volunteers, with an average age of 65, tested the output performance and oxygen capacity of participants while exercising on stationary bicycles. A portion of the volunteers consumed C. sinensis for six weeks, while others consumed a placebo. The results demonstrated that the C. sinensis group had a significant increase in energy output and oxygen capacity over the placebo group after six weeks of the study (43). The presence of adenosine, cordycepin, D-mannitol, polysaccharides, vitamins, and trace elements may be, at least partially, the cause for such effects.

Kidney Ailments Traditional views of the Cordyceps spp. held that its consumption strengthened the kidneys. In a study of 51 patients suffering from chronic renal failure, it was found that C. sinensis significantly improved both the kidney function and overall immune function of treated patients, compared with the untreated control group (44). Patients with chronic renal failure or reduced kidney function often suffer from hypertension, proteinuria, and anemia. After a one-month treatment with C. sinensis, patients showed a 15% reduction in blood pressure, reduction in urinary protein, and increases in superoxide dismutase (44). Fiftyone percent improvement of chronic kidney diseases was

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shown only one month after taking C. sinensis supplement (45). In another clinical study, treatment with C. sinensis of patients having gentamicin-induced kidney damage resulted in the recovery of 89% of their normal kidney function after six days, compared with only 45% recovery by patients treated with more conventional methods (1).

Hypoglycemic Effects In a randomized trial, 95% of patients treated with C. sinensis showed improvement in their blood sugar profiles, while the control group showed only 54% improvement with treatment by other methods (46).

Lung Ailments There have been many trials in humans, using Cordyceps spp. to treat many respiratory illnesses, including asthma and bronchitis, either alone or as an adjunct to standard antibiotic therapy, and it appears to be useful for all of these conditions (47–50). Extracts of C. sinensis have been shown to inhibit tracheal contractions, especially important in asthma patients, since it allows for increased airflow to the lungs. In addition, its anti-inflammatory properties may prove to bring further relief to asthma patients, whose airways become obstructed, due to an allergic reaction resulting in the swelling of the bronchial pathways (1). In a double-blind placebo-controlled study with 30 elderly volunteers, C. sinensis significantly improved the maximum amount of oxygen these people were able to assimilate (51).

Heart Ailments It has been shown that C. sinensis, which often has a significant quantity of adenosine, along with adenosine-type nucleotides and nucleosides, has an effect on coronary and cerebral circulation (52,53). In studies of patients suffering from chronic heart failure, the long-term administration of C. sinensis, in conjunction with conventional treatments, promoted an increase in the overall quality of life (42). This included general physical condition, mental health, sexual drive, and cardiac function, compared with the control group. Studies have also shown the benefits of C. sinensis on heart rhythm disturbances, such as cardiac arrhythmias and chronic heart failure (54).

Liver Ailments In the Orient today, C. sinensis is commonly used as an adjunct in the treatment of chronic hepatitis B and C. In one study, C. sinensis extract was used in combination with several other medicinal mushroom extracts as an adjunct to lamivudine, for the treatment of hepatitis B. The group receiving C. sinensis along with other medicinal mushroom extracts had much better results in a shorter period of time than the control group, who received only lamivudine (55). Treatment of 22 patients, diagnosed with posthepatic cirrhosis, with C. sinensis (56), showed improvement in liver function tests, and in another trial on patients with hepatitis B and patients with cirrhosis taking C. sinensis supplement showed around 80% improvement of liver functions (57).

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Hypercholesterolemia

Drug Interactions

In both human and animal studies, administration of C. sinensis has been associated with cholesterol and triglyceride reduction and an increase in the ratio of HDL to LDL cholesterol (1). As such it may prevent, arrest, and even reverse coronary atherosclerosis (58). The studies have demonstrated that C. sinensis helps to lower total cholesterol up to 21% and triglycerides up to 26%. At the same time it helps to increase HDL cholesterol up to 30% (54).

There is observational evidence that the alteration of the body’s blood glucose metabolism in patients consuming Cordyceps spp. often results in the reduction of oral or injected anti-diabetic medications. It is also posited that the naturally occurring antiretroviral compounds found in C. sinensis (e.g., 2,3-dideoxyadenosine) are marketed as a major anti-HIV drug under the name Videx and Didanosine, as well as 3-deoxyadenosine (which has the same or at least similar activity); C. sinensis could result in increased effectiveness or decreased dosage requirements for patients undergoing concurrent therapy with other antiretroviral drugs. Caution should be exercised in these patients, especially considering the newer, more potent hybrid strains of Cordyceps spp. being developed, and the targeted medicinal compounds being selectively cultivated.

Antiviral Activity After three months of treatment of chronic hepatitis B patients using C. sinensis, CD4 and CD4/CD8 ratios increased significantly (59). The results suggest that beneficial effects might be obtained through adjustment of the T lymphocyte subsets level. Treatment of 65 cases (with 20 cases in the control group) of patients with posthepatic cirrhosis has shown similar results (60). Extracts of Cordyceps spp. are also effective against HIV infections. A C. sinensis containing formula named Immune Assist 24/7TM has recently been introduced throughout West Africa for use in treating HIV infections and other immune-deficient states (2), and is quite popular with both the doctors and the patients due to its low toxicity and cost when compared with other antiretroviral drug options.

Dosage Because clinical data on Cordyceps spp. is relatively new, and even more so in Western Countries, recommended dosage requirements may vary, depending on the source. In general, clinical trials have been conducted using 3 to 4.5 g of C. sinensis per day, except in cases of severe liver disease, where the dosage has usually been higher, in the range of 6 to 9 g per day (4). There are some practitioners known to these authors, who keep their cancer patients on 30 to 50 g of C. sinensis per day. While this may seem excessive, the clinical results seen with this treatment regimen are promising, and Cordyceps spp. related toxicity has never been reported. C. sinensis has been traditionally taken in tea or eaten whole, either by itself or cooked with a variety of meats. Today, in addition to the established traditional means of consumption, powdered mycelium and mycelial extracts are also available in capsulated and noncapsulated form. At present, there are no reliable standards by which to compare different brands, but in general, the quality of Cordyceps spp. is improving, as methods of more efficient cultivation are investigated; and as more clinical trials are conducted, a clearer picture of recommended dosages for a particular condition will become more standardized. Considering the quality of cultivated Cordyceps spp. available in the market today and the risk of lead exposure as well as the cost, such as with wild C. sinensis, the use of natural Cordyceps spp., over the artificially cultivated variety, is not recommended. Obtaining Cordyceps spp. from a reliable source, with complete analytical data provided, is the safest way to purchase species of Cordyceps.

Safety Profile None known contraindications.

Adverse Side Effects Very few toxic side effects have been demonstrated with Cordyceps spp. use, although a very small number of people may experience dry mouth, nausea, or diarrhea (1). One study reported that a patient had developed a systemic allergic reaction after taking a strain of cultivated C. sinensis called Cs-4 (61); however, this type of reaction is not common. There is little published data on the use of Cordyceps spp. in pregnant or lactating women, or in very young children, and appropriate precautions should be taken with these types of patients.

Toxicity No human toxicity has been reported, and animal models failed to find an LD50 (median lethal dose) injected IP in mice at up to 80 g/kg per day, with no fatalities after seven days (2). Given by mouth to rabbits for three months, at 10 g/kg per day (n = 6), no abnormalities were seen from blood tests or in kidney or liver function (62).

REGULATORY STATUS Cordyceps spp. remains, in many nations throughout the world, an unrecognized substance. Other than import/export taxes and restrictions, which vary from country to country (many of which ban the import of any such substance), most governments do not require a prescription to purchase or use Cordyceps spp. Among the few countries that do require a doctor’s prescription are Portugal, Romania, and Austria. Many governments require that vendors obtain a special license to distribute any product relating to human health. In the United States, Cordyceps spp. are marketed privately and considered by the FDA as a dietary supplement. GRAS applications referring to Cordyceps spp. status as a food additive are unavailable; however, a premarket notification to the FDA regarding species of Cordyceps, containing in-depth information relating to preclinical trials and toxicology studies, has been available to the public, via the FDA website.

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CONCLUSIONS When a natural product, such as C. sinensis, has such a long history of use, it seems logical that there is quite likely some truth behind the myths. Our challenge in the modern age is to scientifically unravel the many claims and conflicts. With C. sinensis this challenge has been greater than with many other herbals due to the enormous cost and scarcity of the material. We are fortunate that we live in an age of such rapidly expanding biotechnological progress. For now, we have ways at our disposal to produce Cordyceps spp. in large enough volume, and at a low enough cost, that research becomes possible to nearly anyone interested in looking at this unique organism. As time passes, we may find that this once rare fungal species may hold the key to controlling some of our more difficult to manage diseases. More research is needed into this and other species of medicinal mushrooms.

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Cranberry Marguerite A. Klein

juice by volume. As a dietary supplement, cranberry ranks among the top 10 selling herbal products in the U.S. market, with U.S. sales skyrocketing in 2007 by 15% (4). Also in 2007, cranberry supplements were among the top 20 supplements used by adults and children, who used nonvitamin, nonmineral, natural products for health reasons (5). Concurrent with increasing sales, publication of original scientific results, papers, and reviews almost doubled between 2004 and 2009 compared to the previous five-year period.

INTRODUCTION Cranberry (Vaccinium macrocarpon Aiton) is a native plant of North America. Today, it is one of the top selling herbal supplements in the U.S. market. Juice and dietary supplements derived from the berry reportedly exhibit various health benefits, including prevention and treatment of bacterial adhesion in urinary tract infections (UTIs) and stomach ulcers, prevention of dental caries, protection against lipoprotein oxidation, and anticancer activity. Some of these biologic effects have been linked to the presence of phenolic compounds. The composition of these compounds in cranberry is beginning to be assessed and quantified; however, their bioavailability and metabolism are for the most part not known. Interpretation of results from research on the efficacy/safety profile of cranberry is confounded by methodologic limitations. More research is needed to conclusively determine its health benefits.

CHEMISTRY AND PREPARATION OF PRODUCT The chemical composition for the nutrient constituents (Table 1) of cranberry has been well documented (6,7). Raw cranberries are relatively low in sugar content and minerals compared to other small fruits. They are a very good source of vitamin C, have a fair amount of vitamin A, but are relatively low in the B vitamins. Most of the biologic effects of cranberry have been linked to its high level of phenolic compounds (8–11), higher than 20 other fruits tested (12,13). The major phenolics in the berry are flavonoids and phenolic acids. Chen et al. (11) found a total of 400 mg of total flavonoids and phenolic compounds per liter of sample in freshly squeezed cranberry juice. About 44% were phenolic acids and 56% flavonoids. Phenolic acids include the cinnamic acids (C6–C3) and benzoic acids (C7). Cinnamic acids occur naturally in combination with other compounds, usually in the form of esters. The ester of caffeic with quinic acid is a classic example. On the contrary, benzoics usually occur as free acids. Benzoic acid is the major “phenolic” compound in cranberry (11). The fruits’ astringency is attributable to high levels of organic acids, primarily quinic, citric, malic, and benzoic. Cranberries contain three major subclasses of flavonoids: flavanols, flavonols, and anthocyanidins (Table 2) (8,9). Flavanols exist in the monomer form (catechin and epicatechin) and the oligomer or polymer form (proanthocyanidins). Proanthocyanidins, also known as condensed tannins, are polymeric compounds, the basic structural elements of which are polyhydroxyflavan-3-ol units linked together by carbon–carbon bonds (14). Unlike most fruits, cranberry contains a relatively high proportion of A-type proanthocyanidins (13,15). One subclass of proanthocyanidins is procyanidins. Cranberries contain a

BACKGROUND V. macrocarpon Aiton, the cultivated species, is a member of the heath family (Ericaceae), which includes blueberry, huckleberry, and bilberry. The wild plants are distributed over eastern United States and Canada. Cranberry was of great economic value to the Native Americans, especially since it was the only edible fruit available late in the season (September–November). Various parts of the plant were used as dyes, food, and medicines. They used the berries in poultices for treating wounds and blood poisoning; the leaves for urinary disorders, diarrhea, and diabetes; and infusion of branches for pleurisy (1). In addition, the European settlers applied cranberries therapeutically for the relief of blood disorders, stomach ailments, liver problems, vomiting, appetite loss, and cancer. Sailors took barrels of the fruit to sea to prevent scurvy. Over 100 years ago, women in Cape Cod were known to use it for the treatment of dysuria. About four decades back, consumption of the berry for treatment of UTI received attention and support within the medical community (2,3). Cranberry was first cultivated in the early 19th century. The principal areas of cultivation in North America are Wisconsin, Massachusetts, New Jersey, Oregon, and Washington, as well as parts of Canada. In the 1940s, cranberry juice cocktail became widely available and is the most common form of cranberry consumption today (1). This is a sweetened beverage of about 27% cranberry

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Table 1

Nutrient Content of Vaccinium macrocarpon

Source (100 g)

Water (g)

Energy (kcal)

Total sugars (g)

Ca (mg)

Mg (mg)

K (mg)

Vitamin C (mg)

Thiamin (mg)

Riboflavin (mg)

Vitamin A (IU)

Vitamin E (mg)

Cranberries, raw Cranberry juice cocktail

87.13 86.17

46 54

4.04 11.87

8 3

6 1

85 14

13.3 42.3

0.012 0.000

0.020 0.000

60 8

1.2 0.22

IU, International Units. Source: From Refs. 6, 7.

their achievable plasma concentration after ingestion as well as the possibility of conjugation and metabolism of bioactive components. In general, polyphenols reaching the colon are extensively metabolized by microflora into a wide array of low-molecular-weight phenolic acids. The concentration of intact polyphenols (parent compounds and their conjugated forms) in plasma rarely exceeds 1 ␮mol/L (1 ␮M) after consumption of a single compound. However, measurement of plasma antioxidant capacity suggests that more phenolic compounds are present, largely in the form of unknown metabolites, produced either in the tissues or by gut microflora. Their urinary recovery has been found in the range of 1% to 25% of ingested amount (21). The bioavailability of the major flavonoids from cranberry has not been studied. However, their bioavailability from other dietary sources (e.g., tea, cocoa or chocolate, red wine, onions, or fruits) has been analyzed (24–26). Less is known about absorption and metabolism of the proanthocyanidins than other flavanols, in part due to their complex structures and nonspecific analytic methods to detect them. Higher molecular weight polymers are considered to have poor absorption (27,28). Proanthocyanidins are degraded to low-molecular-weight metabolites by human colonic microflora (29). Although biologic activity is apparent after proanthocyanidin ingestion, only its metabolites have been measured in the urine and plasma (29).

variety of different procyanidins, mixtures of oligomers and polymers, with the last of these being the dominant procyanidins in cranberry (16). Procyanidins may contribute to organoleptic characteristics. Flavonols include the glycosides of quercetin, kaempferol, and myricetin (11,17). Quercetin is the major flavonol in cranberry and is glycosylated mainly at the 3-position with arabinose, galactose, rhamnose, and rhamnose–glucose. Myricetin also exists and has been identified as conjugates of both arabinose and galactose (18). The wide-ranging flavonol content of cranberry is high, exceeding 150 mg/kg (17). Anthocyanins, glycosylated anthocyanidins, are responsible for the fruit’s bright red color. Early studies found a somewhat higher content of anthocyanins than flavonols (19). The pigments present are cyanidin3-galactoside, -3-glucoside, and -3-arabinoside, as well as peonidin-3-galactoside, -3-glucoside, and -3-arabinoside (20). The major anthocyanins in cranberry are 3galactosides and 3-arabinosides of cyanidin and peonidin (21). Uniform requirements for the composition of cranberry products do not exist. In addition to the variable composition of the berries, processing and the product matrix contribute to product composition variability that may impact stability and bioactivity. Characterization and standardization of the bioactive constituents in cranberry products are needed to help in determining product stability and to allow comparison among studies. However, quantification is not always straightforward. A broad spectrum of methods is used to quantify the constituents, leading to differing results. Finally, no cranberry-standardized reference materials to which results of different analytic methods can be compared are available (22,23).

Urinary Acidification Cranberries contain quinic acid, which is excreted in the urine as hippuric acid. Early studies attributed the antibacterial nature of the fruit to the urinary acidifying activity due to the excretion of organic acids and increased concentration of hippuric acid (30–32). Other experiments showed no decreased pH, nor increased levels of hippuric acid or only a brief effect (33–35). Hippuric acid does have antibacterial effects if present in acidic urine (pH 5.0) and at concentrations of 0.02–0.04 M. However, cranberry juice rarely can achieve the bacteriostatic concentrations by itself without the addition of exogenous hippuric acid to the diet (36,37).

PRECLINICAL STUDIES Bioavailability The structural diversity of cranberry components has a major influence on their bioavailability, which in turn influences their biologic effects. Many studies have ignored

Table 2

Flavonoid Content of Vaccinium macrocarpon (mg/100 g) Anthocyanidins

Flavanols

Flavonols

Proanthocyanidins

Source

Cyanidin

Peonidin

(−)-Epicatechin

(+)-Catechin

Myricetin

Quercetin

Monomers

Polymers

Cranberries, raw Cranberry juice cocktail

41.81 0.38

42.10 NA

4.37 NA

0.39 0.19

6.78 0.51

15.09 1.27

7.26 0.56

233.48 8.33

NA, Not applicable. Source: From Refs. 8, 9.

Cranberry

Antiadhesion in Urogenital Infections In vitro and ex vivo studies indicate that cranberry products prevent adhesion of bacteria to the cell walls of the urinary tract, thus preventing UTIs. Emphasis has been on the role of components that act by interference with bacterial adherence of Escherichia coli to uroepithelial cells (38–40). Several ex vivo studies found antiadherence activity in mouse and human urine (15,38,41–43). Two compounds were identified that inhibited adherence. One was fructose and the other was a nondialyzed polymeric compound present only in cranberry. While fructose in vitro inhibits adherence (38,40), it is unlikely to contribute to in vivo antiadhesion activity in urine because it is metabolized before reaching the urinary tract. The nondialyzed polymeric compound proved to be A-type proanthocyanidins. This compound, but not B-type dimer or the (−)-epicatechin monomer, prevented uropathogenic E. coli from adhering to uroepithelial cells in vitro (14,39,44). Subsequently, isolated proanthocyanidins and whole cranberry products have been shown to inhibit E. coli adherence to model systems of primary cultured bladder and vaginal epithelial cells in a dose-dependent fashion, including clinically achievable doses (240 mL cranberry juice cocktail). However, only a very small portion of a dose may reach the bladder (45) and possibly not even excreted intact in the urine (46). A new group of urinary marker compounds, discovered by a robust antiadhesion assay, include two new coumaroyl iridoid glycosides and a depside (47). Furthermore, it is not known if any cranberry constituents reach vaginal tissues (45). In conclusion, to date no specific antiadherent cranberry constituents or metabolites, proanthocyanidin or otherwise, in the urine have been elucidated, and possible synergism among constituents needs to be considered.

Dental Plaque Cranberry compounds, alone or combined, may have the potential to inhibit the development of dental plaque (biofilm) and to prevent or reduce the severity of periodontal disease. Nondialyzable, high-molecular-weight cranberry compounds (anthocyanins and proanthocyanidins in combination) may limit extracellular matrix degradation and other pathologic processes leading to periodontal disease. In vitro studies of this test material showed it having a high capacity to inhibit proteolytic enzyme activity of specific metalloproteinases and elastase, as well as to inhibit production of metalloproteinases (48). These enzymes play a major role in gingival tissue destruction, connective tissue remodeling, and alveolar bone resorption. Their secretion from host cells may, in part, be stimulated by components of the dental biofilm. The pathogenesis of dental caries involves an interaction of diet constituents with microorganisms, which occurs within dental plaque. Streptococcus mutans is considered the primary microbial agent in this pathogenesis. It has two virulent traits: (i) synthesis of extracellular polysaccharides (glucans) through glucosyltransferases, and (ii) ability to produce and tolerate acids, both of which lead to cariogenic biofilms. Cranberry juice, crude extracts, and semipurified materials composed of low- and/or high-molecular-weight compounds have been shown in vitro to disrupt the virulent traits of S. mutans and

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Porphyromonas gingivalis (49–52). Eleven isolated, highly purified, low-molecular-weight cranberry constituents (including flavonols, phenolic acids, and proanthocyanidins) were tested alone and in combination to investigate which compounds influenced the virulence properties of S. mutans associated with glucan synthesis and acidogenicity (53). Phenolic acids showed little effect. However, specific flavonoids and proanthocyanidins resulted in moderate, statistically significant effects. Furthermore, certain combinations of these low-molecular-weight compounds appeared to have an additive effect.

Helicobacter pylori Infection Several mechanisms by which cranberry constituents may prevent or treat Helicobacter pylori infections have been examined and hypothesized, including (i) interference of bacterial adhesion, (ii) inhibition of cell growth and/or colonization, (iii) exerting bactericidal activities, (iv) induction of the bacteria to develop a coccoid (spheroid) form, and (v) neutralization of gastric pH. Adhesins mediate adhesion of H. pylori to epithelial cells. Because cranberry or its constituents have been shown to inhibit adherence of E. coli to uroepithelial cells in vitro, it has been hypothesized that it would prevent adhesion of H. pylori to gastric mucus and cells. A highmolecular-weight, nondialyzable material from cranberry juice was demonstrated to restrain the adhesion of twothirds of the tested strains of H. pylori to immobilized human gastric mucus and erythrocytes (54,55). Preliminary results indicate that cranberry phenolics may disrupt energy production and cause cell death (56). In addition, cranberry phenolics may inhibit urease activity. H. pylori releases the enzyme urease, which converts urea into ammonia in the stomach. This neutralizes the pH and protects H. pylori from stomach acid. Finally, the H. pylori inhibiting factor may not be unique to cranberry but common to all polyphenol-rich fruits (56,57). These in vitro effects have been demonstrated in animal models, with the administration of cranberry juice resulting in the eradication of the pathogen; however, mechanisms and specific cranberry constituents remain to be elucidated.

Antioxidant Antioxidant capacity is not restricted to a particular class of cranberry components but has been found in a wide range of fractions (58). Polyphenols are reducing agents, and together with others, such as vitamin C, they may protect the body’s tissues against oxidative stress. The antioxidant activity of the berry in vivo cannot be accounted for on the basis of increased vitamin C alone (59). Crude cranberry fruit extracts have significant antioxidant activity in vitro (60). The total antioxidant activity of 100 g of cranberry was estimated to be equivalent to that of 3120 mg of vitamin C (12). Isolated polyphenolic compounds from whole cranberries are comparable or superior to that of vitamin E in their activity (18). Cranberry ranks higher than apple, peach, lemon, pear, banana, orange, grapefruit, pineapple, avocado, cantaloupe, melon, nectarine, plum, and watermelon (13,61,62). However, cranberry juices ranked lower in antioxidant potency using a variety of antioxidant tests than many other leading U.S. brands of ready-to-drink, polyphenol-rich beverages,

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including pomegranate juice, red wine, Concord grape juice, blueberry juice, black cherry juice, and Acai juice (62). In comparing cranberry products to one another, it appears that processing decreases the quality of antioxidants. The quality is the result of changing the polyphenol composition and is independent of the quantity of antioxidants present (63). The contribution of individual phenolics to total antioxidant capacity is generally dependent on their structure and content in the berry. The highest antioxidant activity has been noted in peonidin-3-galactoside (21% of antioxidant capacity). Quercetin-3-galactoside, cyanidin3-galactoside, and peonidin-3-arabinoside each contribute about 10% to 11% (64). These four flavonoids have the most potent antioxidant activities compared to 16 other isolated compounds, including plant sterols, other flavonoids, derivatives of triterpenoids, and organic acids. The isolated compounds may have additive and synergistic effects (65). Animal model studies have shown wholebody antioxidant potential at clinically relevant doses and with dose-dependent responses (66,67). Different methods of assessment of antioxidant capacity, varying substrate systems, divergent ways of extraction, length of storage, and differential concentrations of active antioxidants confound the antioxidant activity– chemical structure relationship. Given the diversity and abundance of phenolic antioxidants in cranberry, considerable potential exists for cranberry products to prevent oxidative processes related to cardiovascular disease and cancer at the cellular level and in vivo.

Atherosclerosis Consumption of cranberry may decrease the risk of atherosclerosis (68). Possible mechanisms by which cranberry may reduce risk include: (i) inhibition of lowdensity-lipoprotein (LDL) oxidation (18,63), (ii) inhibition of platelet aggregation and adhesion, (iii) inhibition of the inflammatory response, (iv) induction of endotheliumdependent vasodilation, and (v) increase of reverse cholesterol transport and decrease of total and LDL cholesterol. Data supporting these mechanisms are preliminary and mostly from in vitro and animal model studies (69,70). In vitro studies suggest that molecules like quercetin, resveratrol, proanthocyanidin, anthocyanidin, hydroxycinnamic acid, and acetylsalicylic acid may contribute to an anti-inflammatory response. Human studies have shown that cranberry increases total antioxidant capacity, reduces plasma oxidized LDL (but not total LDL), and reduces cell adhesion molecules (71,72). It has been hypothesized that the potential effect of cranberry on atherosclerosis may result from additive or synergistic effects of multiple cranberry constituents due to various mechanisms and not just the antioxidant effect alone. The constituents contributing to the antioxidant effect were previously addressed.

Cancer The antioxidant capacity alone of cranberry constituents may not account for the observed effects (61,73,74). A soluble-free cranberry extract had the highest antiproliferative activity and maximum calculated bioactivity index for dietary cancer prevention compared to 10 other fruits

(12). Many of the cranberry compounds are likely contributors, including the flavonols, anthocyanins, proanthocyanidins, catechins, various phenolic acids, triterpenoids (e.g., ursolic acid), and even stilbenes (e.g., resveratrol) although these are present in lesser quantities than the other constituents (65,70). Cranberry’s effect on tumor initiation, growth, and metastases will depend largely on the bioavailability of its phytochemicals to the various target tissues. Given the diversity of molecular structures and bioactivity among the classes of phytochemicals in cranberry, it is likely that they may fight cancer individually, additively, or synergistically by several different mechanisms. In vitro evidence in a variety of cell lines exists for possible mechanisms, including (i) induction of apoptosis in a variety of cancer cells, (ii) reduction of invasion and metastasis by inhibition of matrix metalloproteinases, (iii) inhibition of ornithine decarboxylase expression and activity, (iv) inhibition of angiogenesis, (v) inhibition of inflammatory processes, and (vi) inhibition of H. pylori, a risk factor for gastric cancer (58,61,70,72,74–76). In vivo carcinogenesis studies will need to be performed to further confirm antitumor promotion activity and identify individual components and mixtures responsible for activity.

Safety Studies No animal toxicology studies of any cranberry products have been reported; however, two studies have reported on safety in animal models. A mouse model study of the effect of cranberry extract on cancer treatment reported weight loss indicative of toxicity (77). A safety study of a single oral dose of a proprietary multiberry supplement, including cranberry (66), did not cause any mortality and did not demonstrate any signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior in the treated rats. Similarly acute dermal toxicity, primary skin irritation, primary eye irritation via nonoral routes of administration caused no toxicity or harm in animal models.

CLINICAL STUDIES Efficacy Urinary Tract Infection The use of cranberry, particularly as a juice or juice cocktail, to prevent or treat UTI is common. The accumulating evidence from small, noncontrolled, and controlled clinical trials suggests that the berry may relieve symptoms associated with UTI and may reduce the need for antibiotics. The Cochrane Library conducted separate reviews of the fruit for the prevention (78) and treatment (79) of UTI. For treatment, no trials meeting the inclusion criteria were found; only a few uncontrolled trials were found. The Cochrane Library concluded that there was no good quality or reliable evidence of the effectiveness of cranberry juice or other cranberry products for the treatment of UTI. For both prevention and treatment, the review authors concluded that more research was needed. For prevention, 10 studies were included in the review, of which only four were of sufficient methodological quality to include in the meta-analysis. Juice, juice cocktail, or concentrate was investigated in seven studies and capsules or tablets

Cranberry

studied in four trials (one study investigated both juice cocktail and tablets). Intervention duration ranged from four weeks to one year and dosage was quite variable. Several studies reported a high number of withdrawals, and poor adherence to the intervention was also reported. Side effects were common in all studies. The authors concluded that cranberry products may decrease the number of symptomatic UTIs over 12 months. The National Institutes of Health (NIH) supported four, large Phase 2 clinical studies to investigate the effect of a research-grade, low-calorie cranberry juice cocktail on the prevention of UTI in men and nonpregnant women at high risk for UTI and in pregnant women. Subsequent to the Cochrane reviews, results of the cranberry juice cocktail study of asymptomatic bacteriuria in pregnancy have been reported (80). Similar to other studies, a high number of dropouts/withdrawals occurred and adherence to the intervention protocol was poor which led to a protocol change to reduce the dose of 240 mL (80 mg proanthocyanidin) from three to two times a day. Despite the limitation of the protocol change and problems with withdrawal, adherence, and intervention tolerability, the data suggest that cranberry juice cocktail may be protective of asymptomatic bacteriuria and symptomatic UTIs in pregnancy. Results from the other three NIH-supported trials will be reported. Many of the clinical study reports, with the exception of the NIH-sponsored studies, suffer from major limitations. Many trials have not been controlled or randomized, and randomization procedures have not always been described. Crossover designs used in some research may not be appropriate for studies of UTI. Other limitations include no blinding or failed blinding, lack of controlled diets or dietary assessment, use of convenience samples, and small numbers of subjects. Trials have been faulted for the large number of withdrawals. Intention-to-treat analyses were not often applied. Most studies have been conducted in older or elderly patients. Very few have been conducted in younger patients, with or without comorbidities, or in men. Primary outcomes have differed from study to study and have often included urinary pH, as well as rate of bacteriuria, biofilm load, and urinary white and red blood cell counts, rather than UTI. It is also not clear what the optimum dosage or type of product is. There is limited evidence of efficacy or safety for forms of cranberry product other than juice or juice cocktail. Finally, the published articles do not describe the quality and composition of the products tested.

H. pylori Infection A few randomized controlled studies of H. pylori infected male and female adults and children have been undertaken in China, Israel, and Chile with treatment outcomes determined by the C urea breath test as the gold standard to noninvasively detect active H. pylori infection (81–83). Although study limitations exist and generalizability is limited, results are encouraging and suggest that regular consumption of cranberry juice as a complement or alternative to standard triple therapy (a combination of antibiotics and a proton pump inhibitor) may suppress H. pylori infection. The studies suggest that females may be more responsive and that the effect may not persist when cranberry treatment is discontinued.

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Adverse Effects The U.S. Food and Drug Administration granted generally recognized as safe (GRAS) status to cranberry foods and beverages. This means that their safety is well established when consumed in food amounts. The safety or harm of dosages higher than food amounts cannot be confirmed without further high quality clinical studies. The safety of cranberry capsules, tablets, and concentrates, for example, in which doses could reach pharmacologic levels, has not been established. The Cochrane reviews of UTI prevention and treatment indicated that side effects were common in all cranberry juice cocktail studies included in the reviews (78,79). The reported side effects were primarily diarrhea or frequency of bowel movements and other gastrointestinal symptoms. A review of the safety of cranberry consumption by pregnant and lactating women indicated that there were no clinical studies in the evidence-based medicine literature of cranberry being either safe or contraindicated during pregnancy or lactation (84). Subsequent to the review, the first randomized, controlled trial of cranberry juice cocktail for the prevention of bacteriuria in pregnancy reported about 20% withdrawal due to gastrointestinal upset, including nausea, vomiting, diarrhea, at a dose of 240 mL three times a day (80). When the dose was reduced to two times a day, the juice cocktail was somewhat better tolerated. There were no differences between the active and placebo groups with regard to obstetric or neonatal outcomes.

Observed Drug Interactions and Contraindications There is insufficient reliable information available on cranberry dietary supplements or juice cocktail to assess their safety or their interaction with other dietary supplements, foods, medications, or laboratory tests. Because of its oxalate levels, cranberry may be a causative factor in nephrolithiasis. The results of two small studies of juice cocktail and tablets are equivocal, showing differences in urine acidification, calcium and oxalate excretion, and other promoters and inhibitors of stone formation (85,86). A third study (87) was designed to specifically assess the influence of diluted cranberry juice on urinary biochemical and physicochemical risk factors for calcium oxalate kidney stone formation. Three key urinary risk factors were favorably altered: (i) oxalate (reported to not be readily bioavailable from cranberry juice) excretion decreased, (ii) phosphate excretion decreased, and (iii) citrate (an inhibitor of stone formation) excretion increased. There is one report of an infant hospitalized for cranberry juice intoxication and acidosis (88). Theoretically, the juice could interfere with the copper-reduction glucose test because ascorbic acid (a reducing agent) and hippuric acid have each been reported to cause a false-positive reaction with the copperreduction glucose determination in vitro. However, the results of two small studies are equivocal and inconclusive indicating that interference may be variable and dependent on the type of reagent strip kit (89,90). Limited studies have evaluated the drug interaction potential of cranberry juice; no studies of cranberry supplements are reported. The present hypothesis exerts that constituents of cranberry and/or their metabolites may

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interact with liver CYP isoenzymes or with intestinal and renal drug transporters to alter the pharmacokinetics of drugs. Factors that alter the metabolism of drugs play an important role in dosing. Only one study to date has examined the interaction of cranberry and antibiotics commonly prescribed for recurrent UTIs, amoxicillin and cefaclor (91). This study of healthy women showed a modest delay in amoxicillin absorption and a slight delay in cefaclor absorption, neither delay being clinically significant. Their total absorption and renal clearance were not affected. Causal relationships cannot be proved by case reports; however, they often help in identifying adverse events and drug interactions. In 2003, the United Kingdom’s Committee on Safety of Medicines alerted health care professionals about the possibility of an interaction between cranberry juice and warfarin, the most commonly prescribed oral anticoagulation therapy (92). Five unsubstantiated reported cases suggested an interaction (92). By 2004, the Committee had received 12 anecdotal case reports of suspected interaction and concluded that there was sufficient evidence of interaction, even though the evidence was not credible (93). It now appears that reports of enhanced antithrombotic effects of warfarin associated with cranberry juice administration may be a coincidence; however, there is inconsistency among study findings. To address the effect of cranberry on CYP2C9, evaluations have been conducted in vitro and in vivo. In vitro studies have shown that cranberry juice potentially inhibits CYP3A and CYP2C9 (94,95). On the other hand, a number of in vivo human studies reported no alteration of warfarin pharmacokinetics (95–99). In addition, studies of coadministration of cranberry with other drugs primarily metabolized by CYP2C9 (95,99) or metabolized by CYP3A (100) or CYP1A2 (96) similarly show no pharmacokinetic change. There does remain, nevertheless, potential drug interaction liability with cranberry (101), because inconsistencies among studies may be due to participant characteristics, dosing, intervention duration, variability of cranberry test materials, physiochemical effects of cranberry on drug absorption, study design, and sample size. Worthy of further investigation is the new evidence of genotype-dependent interactions with warfarin (97). Because information concerning the influence of cranberry juice on the pharmacokinetics of CYP2C9 substrates is limited, it may be premature to reach a definite conclusion about the effect of cranberry juice on warfarin pharmacokinetics. Nevertheless, patients who are coadministered warfarin and especially large doses of cranberry (102) should be monitored for the most appropriate therapeutic range.

REGULATORY STATUS In the United States, cranberry is classified as a food when sold as juice, juice cocktail, and other conventional forms. Cranberry products, such as encapsulated powders, tablets, or tinctures, are regulated as “dietary supplements” in the United States. In Canada, conventional forms are sold as foods, whereas products promoting a health claim are sold as “natural health products.”

CONCLUSIONS There is a need for comprehensive chemical analyses of all classes of compounds present in cranberry. Individual structures and composition vary significantly among cranberry products and its isolated constituents. Composition varies by ripeness of the fruit, plant variety, growth conditions, extraction method, and processing. This suggests that bioactivities will also vary. However, quantitation of complex polyphenols has been and continues to be limited because of the lack of appropriate standardized analytical methods. Consequently, the precise estimation of cranberry constituent intake is hampered. Furthermore, the bioavailability, metabolism, stability, purity, and composition of cranberry products tested in clinical studies have not been established or published. Therefore, the ability to infer epidemiological relationships with health and disease can be confounded. Evidence for health benefit of cranberry is preliminary and inconclusive. Current evidence from in vitro and clinical studies has been conflicting. This could reflect differences among sources of cranberry or its constituents, form of product consumed, and level of intakes. In addition, clinical studies performed to date have had many methodological limitations and few have assessed safety. Nevertheless, results of clinical studies are encouraging for the relief of symptoms associated with and the prevention of UTI. The complex composition of cranberry creates problems in extrapolation of research results on dietary intake of individual constituents to intake of whole fruits or extracts of whole fruits. Synergistic effects of the whole may enhance the health benefits beyond what can be achieved by the individual constituents. The complex mixture of compounds could also protect against side effects. More research on potential synergistic and protective effects among the classes of compounds in cranberry and with other food constituents and pharmaceuticals is necessary. For these reasons, it is important to understand the composition of cranberry, determine the bioavailability and metabolism of its constituents in isolation and as part of the whole mixture, and rigorously examine the biologic effects of cranberry on disease conditions in order to establish its potential for being safe and providing health benefit.

REFERENCES 1. Henig YS, Leahy MM. Cranberry juice and urinary-tract health: Science supports folklore. Nutrition 2000; 16(7/8): 684–687. 2. Moen DV. Observations on the effectiveness of cranberry juice in urinary infections. Wis Med J 1962; 61:282–283. 3. Papas PN, Brusch CA, Ceresia GC. Cranberry juice in the treatment of urinary tract infections. Southwest Med 1966; 47(1):17–20. R 4. Nutrition Business Journal, NBJ’s Supplement Business Report, 2008. 5. Barnes PM, Bloom B, Nahin R. Complementary and alternative medicine use among adults and children: United States, 2007. National Health Statistics Reports; No 12. Hyattsville, MD; National Center for Health Statistics, 2008.

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6. U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 16. 2003. Nutrient Data Laboratory home page. http://www.nal.usda.gov/fnic/foodcomp. Accessed August 25, 2009. 7. U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 21. 2008. Nutrient Data Laboratory home page. http://www.ars.usda.gov/ba/bhnrc/ndl. Accessed August 25, 2009. 8. U.S. Department of Agriculture, Agricultural Research Service. USDA Database for the Flavonoid Content of Selected Foods. Release 2.1. 2007. http://www.ars.usda.gov/ Services/docs.htm?docid=6231. Accessed August 25, 2009. 9. U.S. Department of Agriculture, Agricultural Research Service. USDA Database for the Proanthocyanidin Content of Selected Foods. 2004. http://www.ars.usda.gov/ Services/docs.htm?docid=5843. Accessed August 25, 2009. 10. Zuo Y, Wang C, Zhan J. Separation, characterization, and quantitation of benzoic and phenolic antioxidants in American cranberry fruit by GC-MS. J Agric Food Chem 2002; 50:3789–3794. 11. Chen H, Zuo Y, Deng Y. Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J Chromatogr A 2001; 913:387–395. 12. Sun J, Chu Y, Wu X, et al. Antioxidant and antiproliferative activities of common fruits. J Agric Food Chem 2002; 50:7449–7454. 13. Vinson JA, Su X, Zubik L, et al. Phenol antioxidant quantity and quality in foods: Fruits. J Agric Food Chem 2001; 49:5315–5321. 14. Foo LY, Lu Y, Howell AB, et al. The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vivo. Phytochemistry 2000; 54:173–181. 15. Howell AB, Reed JD, Krueger CG, et al. A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 2005; 66:2281– 2291. 16. Gu L, Kelm M, Hammerstone JF, et al. Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normal-phase HPLC-MS fluorescent detection method. J Agric Food Chem 2002; 50:4852–4860. 17. Hakkinen SH, Karenlampi SO, Heinonen IM, et al. Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries. J Agric Food Chem 1999; 47:2274–2279. 18. Yan X, Murphy BT, Hammond GB, et al. Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J Agric Food Chem 2002; 50:5844– 5849. 19. Lees DH, Francis FJ. Quantitative methods for anthocyanins: 6. Flavonols and anthocyanins in cranberries. J Food Sci 1971; 36 (7):1056–1060. 20. Hong V, Wrolstad R. Use of HPLC separation/photodiode array detection for characterization of anthocyanins. J Agric Food Chem 1990; 38:527–530. 21. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr 2000; 130:2073S–2085S. 22. Howell AB. Bioactive compounds in cranberries and their role in prevention of urinary tract infections. Mol Nutr Food Res 2007; 51:732–737. 23. Krenn L, Steitz M, Schlicht C, et al. Anthocyaninand proanthocyanidin-rich extracts of berries in food supplements—Analysis problems. Pharmazie 2007; 62:803– 812. 24. Manach C, Williamson G, Morand C, et al. Bioavailability and bioefficacy of polyphenols in humans. I. Re-

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view of 97 bioavailability studies. Am J Clin Nutr 2005; 81(suppl):230S–242S. D’Archivio M, Filesi C, Di Benedetto R, et al. Polyphenols, dietary sources and bioavailability. Ann Ist Super Sanita 2007; 43:348–361. Williamson G, Manach C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am J Clin Nutr 2005; 81(suppl):243S–255S. Deprez S, Brezillon C, Rabot S, et al. Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids. J Nutr 2000; 130:2733–2738. Donovan JL, Manach C, Rios L, et al. Procyanidins are not bioavailable in rats fed a single meal containing a grape seed extract or the procyanidin dimer B3. Br J Nutr 2002; 87:299–306. Koga T, Moro K, Nakamori K, et al. Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds. J Agric Food Chem 1999; 47:1892–1897. Blatherwick NR, Long ML. Studies of urinary acidity. II. The increased acidity produced by eating prunes and cranberries. J Biol Chem 1923; 57:815–818. Fellers CR, Redmon BC, Parrott EM. Effect of cranberries on urinary acidity and blood alkali reserve. 1932; 6(5):455– 463. Jackson B, Hicks LE. Effect of cranberry juice on urinary pH in older adults. Home Healthc Nurse 1997; 15:199–202. Kahn HD, Panariello VA, Saeli J, et al. Effect of cranberry juice on urine. J Am Diet Assoc 1967; 51:251–254. Avorn J, Monane M, Gurwitz JH, et al. Reduction of bacteriuria and pyuria after ingestion of cranberry juice. JAMA 1994; 271:751–754. Nahata MC, Cummins BA, McLeod DC, et al. Effect of urinary acidifiers on formaldehyde concentration and efficacy with methenamine therapy. Eur J Clin Pharmacol 1982; 22:281–284. Bodel PR, Cotran R, Kass EH. Cranberry juice and the antibacterial action of hippuric acid. J Lab Clin Med 1959; 54(6):881–888. Tong H, Heong S, Chang S. Effect of ingesting cranberry juice on bacterial growth in urine. Am. J. Health-Syst Pharm. 2006; 63:1417–1419. Zafriri D, Ofek I, Adar R, et al. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Antimicrob Agents Chemother 1989; 33(1):92–98. Ahuja S, Kaack B, Roberts J. Loss of fimbrial adhesion with the addition of Vaccinium macrocarpon to the growth medium of P-fimbriated Escherichia coli. J Urol 1998; 159:559–562. Ofek I, Goldhar J, Zafriri D, et al. Anti-Escherichia coli adhesin activity of cranberry and blueberry juices (letter). N Engl J Med 1991; 324 (22):1599. Greenberg JA, Newmann SJ, Howell AB. Consumption of sweetened dried cranberries versus unsweetened raisins for inhibition of uropathogenic Escherichia coli adhesion in human urine: A pilot study. J Altern Complement Med 2005; 11:875–878. Di Martino P, Agniel R, David K, et al. Reduction of Escherichia coli adherence to uroepithelial bladder cells after consumption of cranberry juice: A double-blind randomized placebo-controlled cross-over trial. World J Urol 2006; 24:21–27. Valentova K, Stejskal D, Bedn´arˇ P, et al. Biosafety, antioxidant status, and metabolites in urine after consumption of dried cranberry juice in healthy women: A pilot doubleblind placebo-controlled trial. J Agric Food Chem 2007; 55:3217–3224.

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44. Howell AB, Der Marderosian A, Foo LY. Inhibition of the adherence of P-fimbriated Escherichia coli to uroepithelialcell surfaces by proanthocyanidin extracts from cranberries. N Engl J Med 1998; 339 (15):1085–1086. 45. Gupta K, Chou MY, Howell C, et al. Cranberry products inhibit adherence of P-fimbriated Escherichia coli to primary cultured bladder and vaginal epithelial cells. J Urol 2007; 177:2357–2360. 46. Turner A, Chen SN, Joike MK, et al. Inhibition of uropathogenic Escherichia coli by cranberry juice: a new antiadherence assay. J Agric Food Chem 2005; 53:8940–8947. 47. Turner A, Chen S-N, Nikolic D, et al. Coumaroyl; iridoids and a depside from cranberry (Vaccinium macrocarpon). J Nat Prod 2007; 70:253–258. 48. Bodet C, Chandad F, Grenier D. Inhibition of host extracellular matrix destructive enzyme production and activity by a high-molecular-weight cranberry fraction. J Periodontal Res 2007; 42:159–168. 49. Weiss EI, Lev-Dor, R, Kashamn Y, et al. Inhibiting interspecies coaggregation of plaque bacteria with a cranberry juice constituent. J Am Dent Assoc 1998; 129:1719–1723. 50. Koo H, de Guzman PN, Schobel BD, et al. Influence of cranberry juice on glucan-mediated processes involved in Streptococcus mutans biofilm development. Caries Res 2006; 40:20–27. 51. Duarte S, Gregoire S, Singh AP, et al. Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms. FEMS Microbiol Lett 2006; 257:50–56. 52. Yamanaka A, Kouchi T, Kasai K, et al. Inhibitory effect of cranberry polyphenol on biofilm formation and cysteine proteases of Porphyromonas gingivalis. J Periodontal Res 2007; 42:589–592. 53. Gregoire S, Singh AP, Vorsa N, et al. Influence of cranberry phenolics on glucan synthesis by glycosyltransferases and Streptococcus mutans acidogenicity. J Appl Microbiol 2007; 103:1960–1968. 54. Burger O, Weiss E, Sharon N, et al. Inhibition of Helicobacter pylori adhesion to human gastric mucus by a highmolecular-weight constituent of cranberry juice. Crit Rev Food Sci Nutr 2002; 42(suppl):279–284. 55. Shmuely H, Burger O, Neeman I, et al. Susceptibility of Helicobacter pylori isolates to the antiadhesion activity of a high-molecular-weight constituent of cranberry. Diagn Microbiol Infect Dis 2004; 50:231–235. 56. Lin YT, Kwon YI, Labbe RG, et al. Inhibition of Helicobacter pylori and associated urease by oregano and cranberry phytochemical synergies. Appl Enviorn Microbiol 2005; 71:8558–8564. 57. Matsushima M, Suzuki T, Masui A, et al. Growth inhibitory action of cranberry of Helicobacter pylori. J Gastroenterol Hepatol 2008; 23:S175–S180. 58. Kandil FE, Smith MAL, Rogers RB, et al. Composition of a chemopreventive proanthocyanidin-rich fraction from cranberry fruits responsible for the inhibition of 12-Otetradecanoyl phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity. J Agric Food Chem 2002; 50:1063–1069. 59. Pedersen CG, Kyle J, Jenkinson AM, et al. Effects of blueberry and cranberry juice consumption on the plasma antioxidant capacity of healthy female volunteers. Eur J Clin Nutr 2000; 54:405–408. 60. Wang SY, Stretch AW. Antioxidant capacity in cranberry is influenced by cultivar and storage temperature. J Agric Food Chem 2001; 49:969–974. 61. Roy S, Khanna S, Alessio HM, et al. Anti-angiogenic property of edible berries. Free Radic Res 2002; 36 (9):1023–1031. 62. Seeram NP, Aviram M, Zhang Y, et al. Comparison of antioxidant potency of commonly consumed polyphenol-rich

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Creatine G. S. Salomons, C. Jakobs, and M. Wyss

tion increases intelligence and memory performance tasks (13). The goal of this entry is to provide an overview on Cr and its metabolism in health and disease. The functions of Cr and PCr, Cr biosynthesis, its degradation, tissue distribution, transport and molecular aspects, as well as the benefits and risks of Cr supplementation are discussed. (For in-depth reviews, see Refs. 2, 3, 6 and references therein.)

INTRODUCTION Creatine (Cr)—methylguanidino acetic acid—is a naturally occurring compound that was first described by Chevreul in 1832. Its name is derived from the Greek word kreas (flesh). Creatine is found in abundance in skeletal muscle (red meat) and fish. It is essential in energy transmission and storage via creatine kinase (CK). The daily Cr dosage is obtained by both endogenous synthesis and via nutritional intake, followed by absorption in the intestine (1). Creatine supplementation is widespread among sportspersons because of its documented and/or presumed ergogenic effects (2–4). In addition, supplementation with Cr has proven to be instrumental for the treatment of rare inborn errors of metabolism due to defects in Cr biosynthesis enzymes (5–8). Creatine is stored in high concentrations in skeletal and heart muscles and to a lesser extent in the brain. It exists in both free and phosphorylated form [phosphocreatine (PCr)] and is important for maintaining high ratios between adenosine triphosphate (ATP) and adenosine diphosphate (ADP). Upon increases in workload, ATP hydrolysis is initially buffered by PCr via the CK reaction. During high-intensity exercise, PCr in muscle is depleted within several seconds. Whether de novo Cr biosynthesis occurs in the brain or whether Cr is taken up into the brain through the blood–brain barrier, is currently a matter of debate.

BIOCHEMISTRY AND FUNCTION Creatine Structure Creatine is a naturally occurring guanidino compound. Its chemical structure is depicted in Figure 1. Creatine is a hydrophilic, polar molecule. Phosphocreatine is zwitterionic, with negatively charged phosphate and carboxylate groups and a positively charged guanidino group.

Creatine Synthesis Biosynthesis The transfer of the amidino group of arginine to glycine yielding L-ornithine and guanidinoacetic acid (GAA) represents the first step in the biosynthesis of Cr and is performed by L-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1). This reaction is reversible and occurs in mitochondria, into which arginine has to be taken up for guanidinoacetate biosynthesis. The human AGAT mRNA encodes a 423-amino acid polypeptide including a 37-amino acid mitochondrial targeting sequence. The AGAT gene is located on chromosome 15q15.3, is approximately 17 kb long, and consists of 9 exons. The second step involves the methylation of GAA at the amidino group by (S)-adenosyl-L-methionine:Nguanidinoacetate methyltransferase (GAMT; EC 2.1.1.2), whereby Cr is formed. The methyl group is provided by (S)-adenosylmethionine. The human GAMT mRNA encodes a 236-amino acid polypeptide. The gene is located on chromosome 19p13.3, is approximately 12 kb long, and consists of 6 exons.

DEFICIENCY AND SUPPLEMENTATION Patients with Cr deficiency syndromes (CDS), that is, patients with a Cr biosynthesis defect or a Cr transporter defect, have developmental delay and mental retardation (MR), indicating that Cr is crucial for proper brain function. Surprisingly, however, CDS patients do not suffer from muscular or heart problems. Those with a Cr biosynthesis defect, in contrast to Cr transporter-deficient subjects, can partly restore their Cr pool in brain upon Cr treatment (5–10). Creatine supplementation, due to its ergogenic effects, has become a multimillion dollar business (3). In the Western world, Cr has received wide public interest. A simple search on “creatine” in the World Wide Web using common database search engines results in more than 500,000 entries. Besides the use by sportspersons, Cr supplementation is explored in several animal models of neuromuscular disease (i.e., Huntington and Parkinson disease, amyotrophic lateral sclerosis) and in human disease (3,6,11,12). A recent study suggests that Cr supplementa-

Chemical synthesis Creatine is produced by chemical synthesis, mostly from sarcosine and cyanamide. This reaction is prone to generation of contaminants such as dicyandiamide, dihydrotriazines, or Crn (14). Some manufacturers may fail to separate these contaminants from Cr. The toxicological profiles of these contaminants are often not known. Dicyandiamide liberates hydrocyanic acid (HCN) when exposed to strongly acidic conditions (such as in the stomach). For human consumption, only pure preparations of Cr should thus be allowed. Unfortunately, no generally accepted and 202

Creatine

COO−

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CH2 H 3C N H2N+

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O O O −O P O P O P O O− O− O−

N O

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N N

ATP

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NH2

CH2 O

H3C N

NH P O− + H2N O− PCr

O

−O P O P O O− O−

+

+H

N

+

N

O O

N

N

ADP OH OH

Figure 1 Schematic representation of the creatine kinase (CK) reaction, and chemical structures of creatine (Cr) and phosphocreatine (PCr).

meaningful quality labels are yet in place that would allow a consumer to judge the origin and quality of Cr in a given commercial product. Moreover, for most studies published so far, it is not possible to correlate the presence or lack of ergogenic, preventive, or adverse side effects with the quality of the many Cr preparations used.

Creatine Function (CK Reaction) Creatine is involved in ATP regeneration via the CK reaction. The phosphate group of PCr is transferred to ADP to yield Cr and ATP, the “universal energy currency” in all living cells. The CK reaction serves as an energy and pH buffer and has a transport/shuttle function for highenergy phosphates. Several CK subunits exist that are expressed in a tissue- and/or spatial-specific manner. In mammals, four CK isoforms exist: the cytosolic M-CK (M for muscle) and B-CK (B for brain) subunits form dimeric molecules, that is, the MM-, MB-, and BB-CK isoenzymes. The two mitochondrial CK isoforms, ubiquitous Mi-CK and sarcomeric Mi-CK, are located in the mitochondrial intermembrane space and form both homodimeric and homo-octameric interconvertible molecules. In fast-twitch skeletal muscles, a sizeable pool of PCr is available for immediate regeneration of ATP, which is hydrolyzed during short periods of intense work. In these muscles, the cytosolic CK activity is high and “buffers” the cytosolic phosphorylation potential that seems to be crucial for the proper functioning of a variety of reactions driven by ATP. Slow-twitch skeletal muscles, the heart, and spermatozoa depend on a more continuous delivery of high-energy phosphates to the sites of ATP utilization. In these tissues, distinct CK isoenzymes are associated with sites of ATP production (e.g., Mi-CK in the mitochondrial intermembrane space) and ATP consumption [e.g., cytosolic CK bound to the myofibrillar M line, the sarcoplasmic reticulum , or the plasma membrane] and fulfill the function of a “transport device” for high-energy phosphates. The ␥ -phosphate group of ATP, synthesized within the mitochondrial matrix, is transferred by Mi-CK

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in the mitochondrial intermembrane space to Cr to yield ADP and PCr. ADP may directly be transported back to the matrix where it is rephosphorylated to ATP. Phosphocreatine leaves the mitochondria and diffuses through the cytosol to the sites of ATP consumption. There, cytosolic CK isoenzymes locally regenerate ATP and thus warrant a high phosphorylation potential in the vicinity of the respective ATPases. Subsequently, Cr diffuses back to the mitochondria, thereby closing the cycle. According to this hypothesis, transport of high-energy phosphates between sites of ATP production and ATP consumption is achieved mainly by PCr and Cr. The CK system is required to allow most efficient high-energy phosphate transport, especially if diffusion of adenine nucleotides across the outer mitochondrial membrane is limited.

Physiology Tissue Distribution of Creatine and of Its Biosynthesis Enzymes In a 70-kg man, the total body creatine pool amounts to approximately 120 g (1). Creatine and PCr are found in tissues with high and fluctuating energy demands such as skeletal muscle, heart, brain, spermatozoa, and retina. In skeletal and cardiac muscle, approximately 95% of the total bodily Cr is stored, and the concentration of total creatine may reach up to 35 mM. Intermediate levels are present in brain, brown adipose tissue, intestine, seminal vesicles and fluid, endothelial cells, and macrophages. Low levels are found in lung, spleen, kidney, liver, white adipose tissue, blood cells, and serum (25–100 ␮M) (2). Until recently, GAA biosynthesis was presumed to occur mainly in the kidney (and pancreas), where AGAT is highly expressed, followed by its transport via the blood and uptake of GAA into the liver, the presumed major site of the second reaction, the methylation of GAA by GAMT. Current knowledge suggests that AGAT and GAMT expression is not limited to these organs. Synthesis outside of these organs may allow local supply of Cr (e.g., in brain; see creatine biosynthesis in mammalian brain) and may, to a minor extent, contribute to the total Cr content in the body.

Creatine Accumulation: Transporter-Mediated Creatine Uptake Cellular transport is of fundamental importance for creatine homeostasis in tissues devoid of Cr biosynthesis. Creatine needs to be taken up against a steep concentration gradient [muscle (mM), serum (␮M)]. The Cr transporter gene (SLC6A8) (MIM300036) has been mapped to chromosome Xq28. Northern blots indicated that this gene is expressed in most tissues, with the highest levels in skeletal muscle and kidney, and somewhat lower levels in colon, brain, heart, testis, and prostate. The SLC6A8 gene product is a member of a superfamily of proteins, which includes the Na+ -dependent and Cl− -dependent transporters responsible for uptake of certain neurotransmitters. The Cr transporter gene spans approximately 8.4 kb, consists of 13 exons, and encodes a protein of 635-amino acids.

Creatine/Creatinine Clearance Creatine can be cleared from the blood via either uptake into different organs by the Cr transporter or by excretion via the kidney. There is evidence that tissue uptake

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of Cr may be influenced by carbohydrates, insulin, caffeine, and exercise and that transporter molecules located in kidney are able to reabsorb Cr. Nevertheless, Cr is found under normal conditions in urine in various amounts. The main route for clearance of Cr is via creatinine excretion. Creatine and PCr are nonenzymatically converted to creatinine. The rate of creatinine formation, which mainly occurs intracellularly, is almost constant (∼1.7% per day of the Cr pool). Because muscle is the major site of creatinine production, the rate of creatinine formation is mostly a reflection of the total muscle mass. Creatinine enters the circulation most likely by passive transport or diffusion through the plasma membrane, followed by filtration in kidney glomeruli and excretion in urine.

erogeneous presentation, varying from very mild signs to severe MR, accompanied by self-injurious behavior.

AGAT Deficiency

Both AGAT and GAMT deficiencies are autosomal recessive inborn errors of metabolism. This is in contrast to the third disorder of Cr metabolism, which is an X-linked inborn error due to a defect in the Cr transporter (Table 1).

In 2001, the first family with AGAT deficiency (MIM602360) was identified. The two sisters, four and six years old presented with MR, developmental delay from the age of eight months, and speech delay. GAMT deficiency was ruled out because GAA was not increased in urine and plasma. Creatine supplementation (400 mg/kg body weight per day) increased the Cr content in the brain to 40% and 80% of controls within three and nine months, respectively. A homozygous nonsense mutation in the AGAT gene, predicting a truncated dysfunctional enzyme, was finally identified. Lymphoblasts and fibroblasts of the patients indicated impaired AGAT activity. A third related patient was identified with similar clinical presentation. The biochemical hints to detect this disorder are reduced levels of GAA (and creatinine) in plasma, cerebrospinal fluid (CSF) and possibly urine, together with reduced undetectable levels of Cr in the brain.

GAMT Deficiency

SLC6A8 Deficiency (Creatine Transporter Deficiency)

The first inborn error of Cr biosynthesis, GAMT deficiency (MIM601240), was identified in 1994. The absence of a Cr signal in the proton magnetic resonance spectroscopy (1 H-MR) spectrum of brain, the low amounts of urinary creatinine, and the increased levels of GAA in plasma and urine led to the diagnosis of this disease. In addition to creatinine, Cr is also reduced in body fluids. Clinical symptoms are usually noted within the first eights months of life. Possibly Cr is provided in high amounts in utero via the umbilical cord and in newborns via the mother’s milk, thereby delaying the clinical signs. All patients identified so far have developmental delay, MR to various degrees, expressive speech and language delay, epilepsy, autistiform behavior, and very mild-to-severe involuntary extrapyramidal movements. The disorder has a highly het-

Like AGAT deficiency, the X-linked Cr transporter defect was unraveled in 2001. An X-linked Cr transporter (MIM300352) defect was presumed because of: (i) the absence of Cr in the brain as indicated by proton magnetic resonance spectroscopy (MRS); (ii) elevated Cr levels in urine and normal GAA levels in plasma, ruling out a Cr biosynthesis defect; (iii) the absence of an improvement on Cr supplementation; and (iv) the fact that the pedigree suggested an X-linked disease. The hypothesis was proven by the presence of a hemizygous nonsense mutation in the male index patient and by impaired Cr uptake by cultured fibroblasts. The hallmarks of this disorder are MR, expressive speech and language delay, epilepsy, developmental delay, and autistiform behavior. The age at diagnosis of the affected males identified so far (>50) (9,15)

Creatine Deficiency Syndromes

Table 1

Overview of CDS Based on the Listed Number of Patients No. of patients

Trait

Clinical hallmarks

Metabolites

Treatment

References

3, related

AR

MR Dysphasia Autistiform behavior Epilepsy

Brain: Cr ↓ ↓ in H-MRS Plasma, CSF (urine?): GAA ↓, Cr ↓

Cr supplementation

(8)

GAMT (MIM601240)

20

AR

MR Dysphasia Autistiform behavior Extrapyramidal signs Epilepsy

Brain: Cr ↓ ↓ in H-MRS Urine, plasma, CSF: GAA ↑ ↑, Cr ↓

Cr and ornithine supplementation + arginine restriction

(7,8)

SLC6A8 (MIM300036)

> 50 (15 families)

X-linked

Males MR Dysphasia Autistiform behavior Epilepsy Female carriers 50%: learning and behavioral disabilities 50%: no clinical signs

Males Brain: Cr ↓ ↓ in H-MRS Urine: Cr/Crn ratio ↑ CSF: Crn ↓ ?

Cr supplementation: not successful in affected males

(9,10)

Deficiency AGAT

a MIM

(MIM602360)a

∼ 50% of female carriers Brain: Cr ↓ in H-MRS Urine: Cr/Crn ratio normal

Victor A. McKusick: Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov. Abbreviations: AR, autosomal recessive; Cr, creatine; Crn, creatinine; H-MRS, proton magnetic resonance spectroscopy; MR, mental retardation.

Creatine

varies from 2 to 66 years. In two cases, the disease-causing mutation had arisen de novo. In mothers and sisters who are carriers of the disease, learning and behavioral disabilities are noted in about 50% of the cases. Unfavorable skewed X-inactivation is likely the cause of the difference in severity of the clinical signs in females.

Intriguing Questions Linked to CDS Does a Muscle-Specific Creatine Transporter Exist? It is noteworthy that the SLC6A8-deficient patients do not seem to suffer from muscle and/or cardiac failure. This could indicate sufficient endogenous Cr biosynthesis in muscle. Alternatively, Cr uptake is taken over by other transporters, or a yet unknown Cr transporter exists that is specifically expressed in skeletal and cardiac muscle. Creatine Biosynthesis in Mammalian Brain It is a matter of debate whether Cr biosynthesis occurs in mammalian brain. The following findings suggest that it actually does: (i) In rat brain, AGAT and GAMT mRNA and protein were detected (16), (ii) The Cr content in brain of mice treated with guanidinopropionic acid, an inhibitor of the Cr transporter, was—in contrast to muscle tissues— hardly decreased. (iii) In contrast to skeletal muscle, Cr supplementation in AGAT- and GAMT-deficient patients requires months to result in an increment in Cr concentration in the brain. These findings make it unlikely that the brain is entirely dependent on Cr biosynthesis in the liver or on its nutritional intake, followed by transport through the blood–brain barrier into the brain. However, why do Cr transporter deficient patients also reveal Cr deficiency in the brain? One explanation could be that Cr synthesis in the brain, although present, is too low to be relevant physiologically. Alternatively, the expression of AGAT and GAMT may be separated spatially (i.e., AGAT and GAMT molecules may be found in the same or different cell types, but may not be expressed in one and the same cell). This is in line with data of Braissant et al. (17) showing such spatial separation in rat brain at both the mRNA and protein level. These findings suggest that GAA needs to be taken up into the appropriate cells prior to GAA methylation, which in case of the transporter defect is not feasible. This would explain the incapability to synthesize Cr in the brain of SLC6A8-deficient patients. Clearly, more thorough investigations are needed to study these discrepancies toward a better understanding of Cr metabolism in the human brain. Significance of CDS/relevance for Health Care Mental retardation occurs at a frequency of 2% to 3% in the Western population. In 25% of MR cases, a genetic cause is suspected, of which Down syndrome and fragile X syndrome are the most common. Mutations in the SLC6A8 gene may be, together with other X-linked MR genes, partly responsible for the skewed ratio in sex distribution in MR, autism, and individuals with learning disabilities. SLC6A8 deficiency appears to be a relatively common cause of X-linked MR, though not as common as fragile X. Creatine biosynthesis defects may be less common. Because the damage incurred in these three diseases is irreversible to a large part and an effective treatment

205

is available at least for the Cr biosynthesis defects, early diagnosis of these patients is highly important. To date, the clinical phenotype appears to be nonspecific and suggests that all MR patients should be tested in diagnostic centers by 1 H-MRS, metabolite screening, and/or sequence analysis of the SLC6A8 gene. In the case of X-linked MR or X-linked autism due to a genetic, but unknown, cause, the parents are confronted with a risk of recurrence (50% chance that the mother passes the mutant allele on to her child). The diagnosis of SLC6A8 deficiency or a Cr biosynthesis defect allows prenatal diagnosis for subsequent pregnancies.

Creatine Supplementation/Therapeutic Use Creatine Sources Creatine is present in high amounts in meat (4.5 g/kg in beef, 5 g/kg in pork) and fish (10 g/kg in herring, 4.5 g/kg in salmon), which are the main exogenous Cr sources in the human diet. Low amounts of Cr can be found in milk (0.1 g/kg) and cranberries (0.02 g/kg) (17). As discussed earlier, Cr is also synthesized endogenously, which supplies around 50% of the daily requirement of approximately 2 g. This suggests that in vegetarians, who have a low intake of Cr, the bodily Cr content is reduced, unless its endogenous biosynthesis is largely increased. Indeed, in vegetarians, the Cr concentration in muscle biopsies was reported to be reduced (18).

Dosing as an Ergogenic Aid Creatine can be obtained as nutritional supplement in the form of various over-the-counter creatine monohydrate products, which are supplied by many manufacturers. Commercial Cr is chemically produced. The majority of consumers are sportspersons, due to Cr’s documented and/or presumed ergogenic and muscle mass increasing effects. Usually, a loading phase of five to seven days of 20 g/day (in four portions of 5 g) is recommended, followed by a maintenance phase with 3–5 g Cr per day.

Benefits Benefits in Sportspersons Creatine supplementation is common among cyclists, mountain bikers, rowers, ski jumpers and tennis, handball, football, rugby, and ice hockey players. While there is a large body of evidence supporting the ergogenic effects of Cr in high-intensity, intermittent exercise, the situation is more controversial in sports involving single bouts of high-intensity exercise, such as sprint running or swimming (2,19). In endurance exercise, there is currently no reason to believe that Cr supplementation has any benefit. There is a widespread contention that Cr supplementation, by accelerating recovery between exercise bouts, may allow more intensive training sessions. Similarly, supplementation seems to enhance recovery after injury. In most studies, a significant weight gain has been noted upon Cr supplementation. The underlying basis for this weight gain is still not entirely clear, and may be due to stimulation of muscle protein synthesis or increased water retention. The proportion of fat tends to decrease. Most likely, the increase in body weight reflects a corresponding increase in actual muscle mass and/or volume. Therefore, it is not surprising that Cr use is popular among

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Salomons et al.

bodybuilders and wrestlers. On the other hand, in masssensitive sports like swimming and running, weight gain due to Cr supplementation may impede the performance, or may at least counteract the ergogenic effects of Cr. Creatine supplementation may improve muscle performance, especially during high-intensity, intermittent exercise, in four different ways by: (i) increasing PCr stores, which is the most important energy source for immediate regeneration of ATP in the first few seconds of intense exercise; (ii) accelerating PCr resynthesis during recovery periods; (iii) depressing the degradation of adenine nucleotides and possibly also the accumulation of lactate; and (iv) enhancing glycogen storage in skeletal muscle. Benefits in Neuromuscular Disease Besides its ergogenic effects, supplementary Cr has a neuroprotective function in several animal models of neurological disease, such as Huntington disease, Parkinson disease, and amyotrophic lateral sclerosis (ALS) (2,3,6,11). The rationale could be that these disorders, due to different causes, hamper cellular energy metabolism in the brain. In animal studies, Cr also protected against hypoxic and hypoxic-ischemic events. Therefore, Cr may be useful in the treatment of a number of diseases, for example, mitochondrial disorders, neuromuscular diseases, myopathies, and cardiopathies. Currently, the first clinical studies with Cr supplementation in neuromuscular disease are emerging. In two studies on patients with mitochondrial myopathies or other neuromuscular diseases, Tarnopolsky’s group showed increased muscle strength upon Cr supplementation (11). A randomized, doubleblind, placebo-controlled trial to determine the efficacy of creatine supplementation did not show a significant beneficial effect on survival and disease progression in a group of 175 ALS patients. These data are in contrast to what was suggested from animal models of ALS and tissue specimens of ALS patients (12). Studies on single subjects and small groups of neuromuscular disease patients have been reported to show both the presence and absence of beneficial effects of Cr supplementation. Recent publications on Cr supplementation in Huntington disease showed difficulty in proving the effect of Cr on the deterioration of cognitive function (20,21). In Duchenne muscular dystrophy, enhanced muscle strength upon treatment was shown; whereas, for example, in myotonic dystrophy type 2/proximal myotonic myopathy, no significant results were seen (22,23). Future studies with enough statistical power are warranted to unravel the relevance of Cr supplementation in these disorders. Clinical trials of patients with ALS, Parkinson, and other neurological diseases are currently ongoing (http://clinicaltrials.gov/). Benefits in Creatine Biosynthesis Disorders Oral supplementation with 350 mg to 2 g/kg body weight per day has been used in patients with GAMT and AGAT deficiencies. In these patients, the Cr concentration in their brains increased over a period of several months (5). In GAMT deficiency, the GAA concentration in plasma, urine, and CSF decreased with Cr supplementation, but still remained highly elevated. Guanidinoacetic acid was

found to be toxic in animals and may be partly responsible for some of the clinical signs (i.e., involuntary extrapyramidal movements). Combination therapy of Cr plus ornithine supplementation with protein (arginine) restriction reduced GAA in CSF, plasma, and urine, and almost completely suppressed epileptic seizures (7). In general, all patients with a Cr biosynthesis defect who were treated with Cr alone or in combination therapy showed improvements. Clearly, younger patients will experience the largest benefits, because less irreversible damage is to be expected. However, even older patients showed remarkable improvements (7).

Adverse Effects Weight gain is the only consistent side effect reported. Gastrointestinal distress, muscle cramps, dehydration, and heat intolerance have been reported repeatedly. Most of these complaints may be due to water retention in muscle during the loading phase of Cr supplementation. Although a causal relationship with fluid intake has not been proven yet, subjects should take care to hydrate properly to prevent these side effects. The French Agency of Medical Security of Food (www.afssa.fr/ftp/basedoc/2000sa0086.pdf) released a statement in January 2001 that the health risk associated with oral Cr supplementation is not sufficiently evaluated, and that Cr may be a potential carcinogen. Because at present there is no scientific basis for the assertion (both Cr and Cr analogs were actually reported to display anticancer activity), this in turn has resulted in a wave of protest from suppliers and defenders of oral Cr supplementation. In fact, based on the current scientific knowledge in healthy individuals, Cr supplementation at the recommended dosages (see dosing as an ergogenic aid) should be considered safe. Unfortunately, almost nothing is known about the use of Cr in pregnancy, nor are appropriate studies in children available. Furthermore, a potential health hazard is the possible presence of contaminants in some commercial Cr preparations (see chemical synthesis).

CONCLUSIONS Oral Cr supplementation is known or presumed to have a number of favorable effects. For example, it prevents or ameliorates clinical symptoms associated with inherited Cr biosynthesis defects, it may protect against neurological and atherosclerotic disease, (2,6) and it increases sports performance, particularly in high-intensity, intermittent exercise. Despite widespread use of Cr as an ergogenic aid and the significant public interest, the majority of studies on the properties, metabolism, and function of Cr have focused on physiological questions rather than on pharmacokinetics. As yet, the pharmacokinetics is difficult to interpret due to different (and incomplete) study designs. Currently, therefore, it is not adequately known whether Cr supplementation causes any long-term harmful effects. Some precaution is warranted based on the fact that the daily recommended dosage for ergogenic effects (i.e., 20 g during the loading phase, 3–5 g during the maintenance phase) cannot be met by normal food intake.

Creatine

REFERENCES 1. Walker J. Creatine: Biosynthesis, regulation, and function. Adv Enzymol Relat Areas Mol Biol 1979; 50:117–242. 2. Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev 2000; 80(3):1107–1213. 3. Persky AM, Brazeau GA. Clinical pharmacology of the dietary supplement creatine monohydrate. Pharmacol Rev 2001; 53(2):161–176. 4. Greenhaff P. The nutritional biochemistry of creatine. Nutr Biochem 1997; 8:610–618. ¨ 5. Stockler S, Hanefeld F, Frahm J. Creatine replacement therapy in guanidinoacetate methyltransferase deficiency, a novel inborn error of metabolism. Lancet 1996; 348(9030):789–790. 6. Wyss M, Schulze A. Health implications of creatine: Can oral creatine supplementation protect against neurological and atherosclerotic disease? Neuroscience 2002; 112(2):243– 260. 7. Schulze A, Bachert P, Schlemmer H, et al. Lack of creatine in muscle and brain in an adult with GAMT deficiency. Ann Neurol 2003; 53(2):248–251. 8. Stromberger C, Bodamer OA, Stckler-Ipsiroglu S. Clinical characteristics and diagnostic clues in inborn errors of creatine metabolism. J Inherit Metab Dis 2003; 26(2–3):299–308. 9. Salomons GS, van Dooren SJM, Verhoeven NM, et al. Xlinked creatine transporter defect: An overview. J Inherit Metab Dis 2003; 26(2–3):309–318. 10. deGrauw TJ, Salomons GS, Cecil K M, et al. Congenital creatine transporter deficiency. Neuropediatrics 2002; 33(5):232– 238. 11. Tarnopolsky MA, Beal MF. Potential for creatine and other therapies targeting cellular energy dysfunction in neurological disorders. Ann Neurol 2001; 49(5):561–574. 12. Groeneveld GJ, Veldink JH, van der Tweel I, et al. Randomized sequential trial of creatine in amyotrophic lateral sclerosis. Ann Neurol 2003; 53(4):437–445. 13. Rae C, Digney AL, McEwan SR, et al. Oral creatine monohydrate supplementation improves brain performance: A

14.

15.

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18.

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22.

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double-blind, placebo-controlled, cross-over trial. Proc Biol Sci 2003; 270(1529):2147–2150. Benzi G. Is there a rationale for the use of creatine either as nutritional supplementation or drug administration in humans participating in a sport? Pharmacol Rev 2000; 41(3):255–264. Stockler S, Schutz PW, Salomons GS. Cerebral creatine deficiency syndromes: clinical aspects, treatment and pathophysiology. Subcell Biochem 2007; 46:149–166. Braissant O, Henry H, Loup M, et al. Endogenous synthesis and transport of creatine in the rat brain: An in situ hybridization study. Brain Res Mol Brain Res 2001; 86(1– 2):193–201. ¨ Balsom PD, Soderlund K, Ekblom B. Creatine in humans with special reference to creatine supplementation. Sports Med 1994; 18(4):268–280. Braissant O, Villard A-M, Henry H, et al. Synthesis and transport of creatine in the central nervous system. Clinical and Molecular Aspects of Defects in Creatine and Polyol Metabolism, Symposia Proceedings; SPS Verlagsgesellschaft mbH: Heilbronn, Germany. In press. Burke DG, Chilibeck PD, Parise G, et al. Effect of creatine and weight training on muscle creatine and performance in vegetarians. Med Sci Sports Exerc 2003; 35(11):1946– 1955. Verbessem P, Lemiere J, Eijnde BO, et al. Creatine supplementation in Huntington’s disease: A placebo-controlled pilot trial. Neurology 2003; 61(7):925–930. Tabrizi SJ, Blamire AM, Manners DN, et al. Creatine therapy for Huntington’s disease: Clinical and MRS findings in a 1year pilot study. Neurology 2003; 61(1):141–142. Tarnopolsky MA, Mahoney DJ, Vajsar J, et al. Creatine monohydrate enhances strength and body composition in Duchenne muscular dystrophy. Neurology 2004; 62(10):1771–1777. Schneider-Gold C, Beck M, Wessig C, et al. Creatine monohydrate in DM2/PROMM: A double-blind placebocontrolled clinical study. Proximal myotonic myopathy. Neurology 2003; 60(3):500–502.

Dong Quai Roy Upton

INTRODUCTION

Chemistry and Preparation of Products The primary analytes of interest in dang gui are the Zalkylphthalides, most notably ligustilide (Fig. 2), low- and high-molecular-weight polysaccharides, and ferulic acid (Fig. 2). The alkylphthalides are present in the essential oil and are strongly aromatic. Both the crude extract and individual compounds have been correlated with biological activity (see preclinical studies; clinical studies). The crude extract has been associated with positive human clinical effects for the treatment of chronic obstructive pulmonary disease (COPD) and COPD with hypertension, increasing blood volume in postischemic patients, and decreasing platelet aggregation (3). The alkylphthalides and ferulic acid inhibit platelet aggregation and the formation of platelet thromboxane A2 (4–6) and elicit in vitro spasmolytic activity, increase coronary blood flow, slightly decrease myocardial contractility, and markedly prolong the effective refractory period (7). Total extracts have also been associated with hepatoprotective effects. Thus dang gui is utilized in portal hypertension and veno-occlusive disease. At least part of this activity is associated with the demonstrated antioxidant activity of ligustilide, ferulic acid, and polysaccharides (8–10), as well as the ability of dang gui to promote hepatic microcirculation. In human clinical trials both an aqueous extract and ligustilide have been found to be effective in treating dysmenorrhea (11). Dang gui polysaccharides stimulate hematopoiesis and, along with ferulic acid, elicit immunomodulatory activity (e.g., increased phagocytosis) (12,13). Ligustilide and ferulic acid elicit a strong uterine spasmolytic effect (14–17). All of these actions are consistent with the use of dang gui in traditional Chinese medicine. Investigations of polysaccharides derived from dang gui, specifically in conjunction with their potential immunomodulatory effects have been conducted. The polysaccharides, named A. sinensis polysaccharide fractions (APF 1, APF 2, APF 3) and crude angelica polysaccharide consist of rhamnose, galacturonic acid, glucose, galactose, mannose, and arabinose in various ratios (18,19). In TCM, the roots of dang gui are commonly prepared as a tea, extract, syrup, tablet, or capsule. In supplement form, dang gui occurs predominantly in tablets and capsules, and occasionally in tinctures. As with the majority of Chinese herbs, dang gui is most often used in combination with other botanicals and is predominantly featured in formulas for promoting healthy gynecological and cardiovascular systems and for a healthy liver. Different portions of the roots are used for different indications. The whole roots are said to “harmonize” the

The root of dang gui (Angelica sinensis; also known as dong quai; Fig. 1) is one of the primary botanicals used in traditional Chinese medicine (TCM) for the treatment of gynecological and circulatory conditions. In TCM its primary use is to both build and promote the movement of blood, and on the basis of these actions it is utilized for a myriad of conditions. Despite its widespread use among practitioners of TCM, there have been few clinical studies regarding its efficacy, although preclinical data support many of these traditional uses as well as suggest benefit for numerous other uses.

BACKGROUND Traditional and Modern Uses Dang gui grows at high altitudes in comparatively cold, damp, mountainous regions in China and other parts of East Asia. The plant is a fragrant perennial that has smooth purplish stems and bears umbrella-shaped clusters (umbels) of white flowers that grow to approximately 3 ft in height. Dang gui produces winged fruits in July and September. In the earliest known herbal text of China, the Divine Husbandman’s Classic of the Materia Medica (Shen Nong Ben Cao Jing), dang gui is described as a herb to “supplement nature” (1). In the monumental 52-volume Compendium of Materia Medica (Ben Cao Gang Mu), written by Li Shizhen in the 16th century, dozens of uses for dang gui were elaborated. These included the following: to tonify the five major viscera, especially the heart; to generate flesh; to stop headache, back pain, menstrual pain, toothache, and pain associated with the “belt channel” (dai mai); to treat a wide range of skin sores and rashes; and to correct menstrual problems such as irregular menstruation, amenorrhea, and dysmenorrhea (2). Modern research has focused on the use of dang gui for its ability to enhance circulation and oxygenation in hypoxic conditions specifically in regard to brain and cardiovascular effects. Despite the widespread popularity and use of dang gui in gynecology, there is a lack of research in modern English language journals regarding this use, though some data suggest estrogenic and both uterine relaxant and uterine stimulatory activity, depending on the fraction studied. A number of studies report on the ability of dang gui to promote the healing of tissues, specifically in ulcerative colitis and gastric ulcers, and other studies have focused on its anticancer and hepatoprotective effects, among others.

208

Dong Quai

209

model and suggest that ferulic acid is rapidly and almost completely absorbed from the intestinal tract (23). It has also been reported that ferulic acid crosses the blood–brain barrier, although in very low concentrations (24). The major metabolites of ferulic acid are nontoxic and water soluble, being excreted through the urine and bile as free acids and acid conjugates.

PRECLINICAL STUDIES Pharmacodynamics

Figure 1 Whole dang gui (Angelica sinensis) roots. Source: Courtesy of Roy Upton, Soquel, California. (View this figure in color at www.dekker.com)

blood; the dang gui root bodies (dang gui tou; ) are used to build and nourish the blood and are commonly included in soups for convalescence and blood deficiency; the tails (dang gui wei; ) are predominantly used to “break the blood” and prevent and treat abnormal blood stagnation.

Pharmacokinetics There are limited data on the pharmacokinetics of some of the compounds contained within dang gui. In a study of the bioavailability of ferulic acid in humans (n = 5), the peak time for maximal urinary excretion of ferulic acid following the consumption of 360 to 728 g tomatoes (providing approximately 21 to 44 mg ferulic acid) was 7 to 9 hours (20). A considerable proportion of ferulic acid was excreted as glucuronide in all subjects. The recovery of ferulic acid in the urine, on the basis of total free ferulic acid and feruloyl glucuronide excreted, was 11% to 25% of that ingested. The bioavailability of ferulic acid from beer is consistent with the uptake of ferulic acid from other dietary sources, such as tomatoes (21). Urinary and biliary metabolites of ferulic acid were primarily glucuronic acid and glycine conjugates of ferulic acid and vanillic acid (22). The pharmacokinetic parameters of ferulic acid following IV injection to mice fit a one-compartment open

CH3O

CO2H

O HO Z-Ligustilide

O

Figure 2

Ferulic acid

Major constituents of dang gui.

Dang gui is one of the most widely used of all Chinese botanicals. Historically and in modern Chinese medicine, it has been primarily used as a general blood tonic for the TCM diagnosis of blood deficiency, a syndrome closely related, but not exactly analogous or limited, to anemia. Dang gui has also been used for a myriad of gynecological indications, although there has been very little research done in this regard in English language journals. More recently, pharmacological research has focused on the potential of constituents of dang gui to elicit cardiovascular, hematopoietic, hepatoprotective, antioxidant, antispasmodic, and immunomodulatory effects. Chinese botanicals are most often used in multi-ingredient formulas rather than as single agents. Therefore, there are very few clinical trials on dang gui alone, although numerous preclinical studies exist. Due to the lack of primary English language literature, it is difficult to adequately access or adequately review the available data by non-Chinese language readers. Another difficulty in reviewing the available studies is that many of the investigations are of disease patterns that are unique to TCM and do not have well-defined corresponding Western diagnoses, or viceversa; studies are conducted for indications not synonymous with TCM indications. While the TCM findings are relevant to TCM practitioners, their importance may be ignored or even criticized by non-TCM practitioners. Study of non-TCM indications often is conducted for purposes of modern drug discovery and therefore can be criticized on different grounds. Lastly, it has been reported that up to 99% of studies presented in the Chinese medical literature show results favoring test intervention, suggesting the potential for a positive publication bias and hence the need for caution in interpreting the available data (25). Conversely, publication bias against dietary supplement research in the primary medical literature of the United States has been reported and may similarly limit a critical review of investigations of herbal products (26). The bioactive compounds most studied in dang gui are phthalides, polysaccharides, and ferulic acid. Studies using these compounds have reported a number of therapeutic effects, some of which are consistent with the use of dang gui in TCM and some of which are not. The contribution of ferulic acid to the therapeutic effect of dang gui is unlikely given its low concentration in crude dang gui (0.05–0.09%). The compounds used in pharmacological studies are often administered at doses exceeding those available from typical dosages of dang gui root preparations. While these data are presented, it is not possible to extrapolate results from such studies to clinical efficacy of orally administered crude drug products; hence, the reported findings must be evaluated critically.

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Cardiovascular and Hemorheological Effects Clinically, dang gui is widely used for the treatment of cardiovascular disease, specifically conditions that can benefit from enhanced circulation and a decrease in platelet aggregation. Preclinical studies using dang gui and some of its constituents suggest actions and mechanisms by which it may exert a cardiovascular effect. These include stimulation of circulation, platelet aggregation inhibition, decrease in myocardial oxygen consumption, and vasorelaxation (measured as a decrease in vascular resistance; see also effects on smooth muscle). A number of animal studies and in vitro assays support some of the putative cardiovascular effects of dang gui. These include an increase in myocardial perfusion, decrease in myocardial oxygen consumption, increase in blood flow, decrease in vascular resistance, and inhibition of platelet aggregation, ventricular fibrillation, and arrhythmias (3). However, a direct extrapolation of these findings to humans cannot be made without confirmatory human studies. A review of the activity of sodium ferulate reported that it both inhibits platelet aggregation and elicits a thrombolytic activity in vitro and in vivo. These effects were due to inhibition of cyclooxygenase and thromboxane A2 synthase with improvements in blood viscosity, reduction in the concentration of plasma fibrinogen, and increase in coronary perfusion. Additional cardiovascular effects reported include reduction of cholesterol biosynthesis and lowering of triglycerides, improvements in myocardial oxygen consumption, and antiarrhythmic, antioxidant, and antiatherogenic activity (24).

Hepatoprotective Effects A number of preclinical studies indicate that dang gui, dang gui polysaccharides, ferulic acid, and sodium ferulate have antioxidant effects that can protect the liver against damage due to chemically induced toxicity. Part of this action is due to the ability of dang gui polysaccharides to reduce the levels of nitric oxide (24.6%), serum alanine aminotransferase (40.8%), and serum glutathione S-transferase (18.4%) in animals with acetaminopheninduced or carbon tetrachloride-induced liver damage (8,9,27,28).

Gynecological Effects Dang gui is one of the most important herbal medicines in TCM for the treatment of menstrual disorders, especially when used in combination with other botanicals. It has traditionally been used to treat conditions associated with the TCM diagnosis of “blood stasis” and “blood vacuity,” which can be correlated with Western syndromes such as amenorrhea, dysmenorrhea, endometriosis, uterine fibroids, and certain forms of infertility. Its efficacy appears to have been demonstrated over the 750-year history of its use for these indications and its continued, and apparent, successful use by modern practitioners of TCM. However, there are few studies substantiating these effects (11,29), and those that are available lack methodological rigor. Using ELISA-type immunoassays of two steroidregulated proteins, presenelin-2 and prostate-specific antigen, in breast carcinoma cell line BT-474, researchers reported that dang gui extract showed “weak” estrogen and androgen antagonistic effects of 50% and 71% blocking activity, respectively, and no progestational activity (29). In

contrast to these findings, another group of researchers found no estrogen receptor binding, cell proliferation, or progestin activity of an aqueous-ethanol extract of dang gui (30). A study of ovariectomized rats showed that an extract of dang gui (300 mg/kg SC; 1% ligustilide) resulted in a thickening of the luminal epithelium suggesting a estrogenic activity, but one much lower than comparison with estradiol. The extract also suppressed luteinizing hormone secretion. The researchers considered ligustilide to be the active compound on the basis of previous in vitro research they conducted (31). Several preclinical studies have investigated the estrogenicity of dang gui or ferulic acid, with mixed, but largely negative, results. Some in vitro assays have reported that dang gui extract exhibited a significant dose-dependent inhibition of estrogen receptor binding, indicating that it competed with estradiol for receptor sites (32). In the same study, dang gui extract dose-dependently induced reporter gene expression in estrogen-sensitive rat uterine leiomyoma cells, suggesting a potentially proliferative effect on these cells. However, when tested in conjunction with the maximum stimulatory dose of estradiol, the extract inhibited estradiolinduced reporter gene expression, suggesting the possibility that dang gui may act as an estrogen antagonist when in the presence of physiological levels of estradiol. Another group of researchers reported similar findings (33).

Effects on Smooth Muscle Dang gui and its constituents have been shown to relax the smooth muscle tissue of the vascular system, trachea, intestines, and uterus. The spasmolytic effects of dang gui on trachea and uterine tissues are consistent with TCM indications. While the mechanism of the relaxant action has not been fully elucidated, preclinical studies suggest that it may be due, in part, to histamine receptor blocking activity, calcium ion channel effects, or modulation of cholinergic receptors. Both relaxing and stimulating effects on uterine tissue have been reported, with various constituents eliciting different actions. The therapeutic relevance of in vitro findings to humans is unknown given the lack of clinical evidence. Ex vivo studies demonstrate that ligustilide and butylidenephthalide isolated from the volatile oil of dang gui exhibit a strong spasmolytic effect on isolated uteri (34,35). Ligustilide was shown to relax early pregnant and nonpregnant uteri of experimental animals (34). Ligustilide and butylidenephthalide showed an inhibitory effect on prostaglandin F2 -, oxytocin-, or acetylcholine-induced contraction of nonpregnant rat uteri (14,36). This could explain the spasmolytic effect of the volatile oil. Other studies indicated that the observed spasmolytic effect may be due to an effect on calcium channels (14,37). Three of the available studies reviewed found that ferulic acid elicited a uterine spasmolytic effect. At oral doses of 300 to 1000 mg/kg and IV doses of 30 to 300 mg/kg, ferulic acid inhibited spontaneous uterine contraction in rats (16,17). The inhibitory effect of IV ferulic acid was not blocked by either propranolol or by cimetidine and it strongly inhibited the uterine contraction induced by oxytocin (0.3 unit/kg), but not that induced by acetylcholine (0.1 mg/kg) or serotonin (10 ␮g/kg). It was suggested that the uterine relaxant effect of ferulic acid is partially due to the oxytocin receptor

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system rather than its inhibitory effect on prostaglandin biosynthesis (38). Another study, however, suggested that ferulic acid may not be responsible for the spasmolytic effect of dang gui, since its content in raw material is low (approximately 0.03% to 0.06%) (39).

Hematopoietic Effects One of the traditional applications of dang gui in TCM is its use in the treatment of “blood vacuity,” which closely, but not completely, corresponds to a Western medical diagnosis of anemia. Limited clinical and preclinical data support this use. One proposed mechanism of action is its reported effect in stimulating hematopoiesis. These actions appear to be primarily associated with the polysaccharide fraction (13,40). One study demonstrated the hematopoietic effects to at least partially be associated with proliferation of bone marrow mononuclear cells through signal transduction pathways (e.g., MAPK/ERK pathway) (41).

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doxorubicin-induced cardiotoxicity, without decreasing the antitumor activity of the drug (53).

Wound-Healing Effects In addition to the beneficial effects of dang gui’s antioxidant activity on tissues noted earlier, specific wound-healing properties have been reported. One group of researchers found that a crude extract of dang gui (characterization and dosage not available) significantly accelerated epithelial cell proliferation in wounds (27,46,54). The activity was reportedly associated with an increase in DNA synthesis and epidermal growth factor mRNA expression. The same researchers observed direct wound-healing effects of dang gui crude extract, with activity associated with increased ornithine carboxylase activity and increased c-Myc expression. Another study found that dang gui prevented bleomycin-induced acute injury to rat lungs. Alveolitis and the production of malondialdehyde were all reduced (P < 0.01 or P < 0.001), suggesting immunomodulatory and antioxidant effects (55).

Antioxidant Effects There have been numerous studies demonstrating an antioxidant effect of dang gui and its constituents. Much of these have focused on the antioxidant activity of ferulic acid, which is well known for its ability to prevent lipid peroxidation, inhibit superoxide anion radical formation, scavenge free radicals, and protect against radiation damage (42–44). Dang gui contains only trace amounts of ferulic acid, so these in vitro findings cannot be extrapolated to the use of crude dang gui preparations. There are, however, animal studies showing that dang gui polysaccharides have a protective effect against chemically induced ulcerative colitis and inflammation. In one study, dang gui polysaccharides elicited anti-inflammatory effects in the gastrointestinal mucosa through inhibition of neutrophil infiltration in the stomach (45). In another study, dang gui polysaccharides (5 mg and 10 mg/mL in drinking water) attenuated colonic lesions caused by oxidative damage induced by 2,4-dinitrobenzene sulfonic acid in rats in a dose-dependent manner. This action was associated with a preservation of endogenous glutathione levels. Other studies reported tissue-healing effects of dang gui to be associated with ornithine carboxylase activity, c-Myc protein expression, and epidermal growth factormediated pathway (27,46). A follow-up study by the same group of researchers showed that crude extract of dang gui (50 mg/kg PO) significantly accelerated the healing of gastric ulcers in animals and showed an anti-angiogenic activity and a quicker restoration of mucosal synthesis and mucosal cell proliferation (47). Studies suggest that the antioxidant activity of dang gui may reduce ischemia–reperfusion induced injury (48,49), ameliorate cognitive dysfunction associated with postischemic brain damage (49), and inhibit the damage associated with aggregation of amyloid-␤ peptide, suggesting a possible use of dang gui in Alzheimer’s. These effects were reported to be correlated with both ferulic acid and Z-ligustilide (50). Other studies show that dang gui provides antioxidant protection against free radical induction of rat adrenal medulla (PC12) cell lines (51) and suppressed radiation-induced expression of tumor necrosis factor␣ and tumor growth factor-␤-1 (52), and prevented

Immunomodulatory Effects and Potential Anticancer Activity Limited animal and in vitro studies have reported on specific immunomodulatory effects of dang gui, including stimulation of phagocytic activity and interleukin-2 production, and an anti-inflammatory effect. There is evidence to suggest that the polysaccharide fraction of dang gui may contribute to these effects. However, there is no clinical evidence supporting these effects, and there appears to be no direct correlation between TCM use of dang gui and immunomodulatory activity (56–58). A new direction in investigation of the use of dang gui is for its potential anticancer activity. Ligustilide has been shown to have direct cytotoxic activity against several human and animal cell lines (59–61). In the absence of clinical and directly applicable toxicological investigation, little emphasis should be placed on these in vitro findings. There have, however, been a number of animal studies suggesting immunomodulatory and anticancer activity. In one study, n-butylidenephthalide suppressed growth of subcutaneous rat and human brain tumors, reduced tumor volume, and significantly prolonged survival in treated rats. This activity was reported to be due to an induction of cell cycle arrest and apoptosis (62). Polysaccharides have similarly been shown to inhibit growth of murine tumors (S180, EAC, L1210) in vivo, resulting in a prolonged survival of treated animals. In vitro, dang gui polysaccharides were shown to inhibit the metastasis of human hepatocellular cancer cell lines (63). A variety of immunomodulatory activities have been reported for dang gui polysaccharides, including enhanced macrophage and T cell numbers, increased production of interleukin and interferon, improved CD4/CD8 ratios, and a general regulation of Th1- and Th2-related cytokines (18). Other actions reported for polysaccharides include release of nitric oxide from peritoneal macrophages and enhanced cellular lysosomal enzyme activity (64,65).

Effects on Bone Cells Dang gui is traditionally used in formulas for bone and tendon injuries. A recent study investigated the

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pharmacology behind this indication by testing the in vitro effects of a 1% aqueous extract of dang gui on human osteoprecursor cells. Cells were incubated for five days in medium with (12.5–1000 ␮g/mL) and without the extract. Compared to untreated control cell cultures, cell proliferation was enhanced at extract concentrations less than 125 ␮g/mL (P < 0.05), whereas it was inhibited at concentrations greater than 250 ␮g/mL (P < 0.05 at 1 mg/mL). Protein secretion in osteoprecursor cells and type-I collagen synthesis were significantly increased (P < 0.05) (66).

Hormonal Effects and Effects on Menopausal Symptoms Because of the putative effects of dang gui in gynecological imbalances, various studies have investigated its potential for eliciting hormonal effects. In one human study (72), one of the few double-blind, placebo-controlled trials with dang gui, no statistically significant differences in endometrial thickness, vaginal cell maturation, or menopausal symptoms were observed between subjects taking dang gui and those taking placebo. This contrasts with a study of ovariectomized rats which showed that an extract of dang gui (300 mg/kg SC; 1% ligustilide) resulted in a thickening of the luminal epithelium suggesting estrogenic activity, but one much lower than comparison with estradiol (see gynecological effects).

CLINICAL STUDIES The clinical data regarding the use of dang gui alone are scarce and of poor methodological quality.

Cardiovascular and Hemorheological Effects One study reported that 0.08 g/day/IV of sodium ferulate relieved symptoms of angina pectoris after three to seven days of treatment (24). Limited clinical studies have investigated the use of dang gui for the treatment of patients with acute ischemic stroke or COPD with pulmonary hypertension. Results provide fairly weak evidence that dang gui exerts hypotensive and cardioprotective effects. In general, the study design of the available reports was poor and the patient populations extremely limited. One study looked at the effects of dang gui in 60 patients with COPD (67). In the dang gui group, levels of blood endothelin-1, angiotensin II, endogenous digitalislike factor, mean pulmonary arterial pressure, and pulmonary vascular resistance were decreased significantly (P < 0.05 or P < 0.01) compared to those in the controls (20 ± 6%, 36 ± 9%, 38 ± 11%, 17 ± 5%, and 27 ± 8%, respectively). Another study showed that dang gui decreased the mean pulmonary arterial pressure in patients with COPD without changing blood pressure and heart rate, suggesting a vasodilatory effect on pulmonary vessels without effect on systemic circulation (3). In another study, it was suggested that dang gui and dextran exhibited positive effects on neurological and hemorheological symptoms in patients recovering from stroke (68). However, no control group was included, and so any claimed effects are questionable. Other clinical studies with very small numbers of patients (11,69) have reported on an ability of dang gui to decrease blood viscosity, an effect consistent with its traditional use. While this effect may be real, the mechanisms by which this may occur and the constituents involved have not been well articulated.

Hepatoprotective Effects There is some evidence to suggest that dang gui and its constituents can decrease portal hypertension in patients with liver cirrhosis without affecting systemic hemodynamics. This use is consistent with the traditional actions of dang gui in improving circulation, because portal hypertension is thought to be due to the obstruction of hepatic microcirculation (70,71).

Analgesic Effects Two uncontrolled clinical trials were found that addressed the traditional Chinese use of dang gui as an analgesic for pain due to “blood stasis”; both used injectable preparations. In one, an ethanol extract was administered (intramuscularly) on alternate days for a total of 20 doses into the pterygoideus externus of 50 patients with temporomandibular joint syndrome. A 90% cure rate was claimed (73). Thirty cases of refractory interspinal ligament injury were treated by local injection of 2 mL of 5% or 10% dang gui twice weekly for two to three weeks. Twenty-four (80%) of these patients reported a disappearance of pain, no tenderness, and the ability to work as usual; four (13%) patients reported alleviation of pain; two (7%) reported no improvement (74). These uses are consistent with the traditional use of dang gui in TCM. However, the effects of injectable preparations cannot be extrapolated to oral use of dang gui.

Dosages r Crude herb: 6 to 12 g daily to be prepared as a decoction. r Fluid extract (1:1): 3 to 5 mL three times daily (75).

SAFETY PROFILE Side Effects On the basis of a review of the available traditional and scientific data, dang gui is a very safe herb with a low probability of side effects when used within its normal dosage range. One review article that claimed to cover 200 reports on dang gui pharmacology stated that dang gui had no major side effects (35). Individual case reports regarding the potential of dang gui to promote bleeding have been prepared.

Contraindications On the basis of a review of the available literature and the experience of practitioners, dang gui is contraindicated prior to surgery and, generally speaking, in those with bleeding disorders.

Precautions Precautions regarding the use of dang gui and other botanicals used in traditional systems of medicine must be differentiated between those recognized in the scientific literature and those recognized traditionally. There is evidence suggesting an anticoagulant effect for dang gui, and there

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are two published reports on its ability to enhance the effects of chronic treatment with warfarin (see interactions). A few unpublished case reports suggest that high doses or chronic administration of dang gui alone during pregnancy may be associated with miscarriage. There are also anecdotal reports of administration of dang gui alone causing increased blood flow during menses (R.U., personal communication). Therefore, patients should consult with a qualified health care professional prior to using dang gui if they have bleeding disorders, are using anticoagulant medications, or wish to use it during menses or in the first trimester of pregnancy. It must, however, be noted that in TCM, dang gui is specifically indicated for certain bleeding disorders that are due to an underlying diagnosis of blood stasis and in certain cases of threatened miscarriage. For such uses, dang gui must be used according to TCM principles under the guidance of a qualified TCM practitioner.

Interactions Two reports are available suggesting that dang gui can enhance the effects of the anticoagulant warfarin. According to one of these, a 46-year-old woman with atrial fibrillation who had been stabilized on warfarin for almost two years (5 mg daily) consumed a dang gui product concurrently for four weeks (565–1130 mg daily). She experienced a greater than twofold elevation in prothrombin time (from 16.2 to 27 sec) and international normalized ratio (from 2.3 to 4.9). No other cause for this increase could be determined. Within one month of discontinuing dang gui use, coagulation values returned to acceptable levels (76). An animal study investigated the interaction of dang gui and a single dose or a steady-state dose of warfarin (77). Six rabbits were administered a single dose of warfarin (2 mg/kg SC). Seven days later, the same animals were given an aqueous extract of dang gui (2 g/kg PO, twice of a 2 g/mL extract daily) for three days, after which they were again given a single dose of warfarin. Plasma warfarin concentrations were measured at intervals up to 72 hour after each warfarin dose, and prothrombin time was measured daily during dang gui treatment and after the warfarin doses. Mean prothrombin time did not change significantly during the dang gui treatment period. However, when measured after coadministration of dang gui and warfarin, prothrombin time was significantly lowered at 24, 36, and 48 hours compared to that with warfarin treatment alone (P < 0.05 or P < 0.01). No significant variations in the single dose pharmacokinetic parameters of warfarin were observed after treatment with dang gui. Hence, the mechanism of decrease in prothrombin time could not be correlated to the pharmacokinetics of warfarin. Another group of six rabbits was given 0.6 mg/kg of warfarin SC daily for seven days; a steadystate plasma concentration was achieved after day 4. On days 4, 5, and 6, the rabbits were treated as above with dang gui. Mean prothrombin time was again significantly increased after coadministration with dang gui and two rabbits died at days 6 and 7 after the dang gui treatment had begun. Plasma warfarin levels did not change after dang gui treatment. The authors suggested that these results indicate that precautionary advice should be given to patients who medicate with dang gui or its prod-

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ucts while on chronic treatment with warfarin. Another study reported that dang gui acted synergistically with aspirin (24). General enhancement of cytochrome P450 isoforms has been reported for both water (CYP2D6 AND 3A) and ethanol extracts (CYP2D6) of dang gui in animal models (78). One study reported that dang gui might enhance the antitumor effect of cyclophosphamide in mice with transplanted tumors (79).

Pregnancy, Mutagenicity, and Reproductive Toxicity Because of its blood-moving properties, dang gui should be used in pregnancy only under the supervised care of a qualified health professional. According to TCM practice, dang gui is used in combination with other herbs in various stages of pregnancy (29). Formula traditionally used in pregnancy are prescribed within the context of specific diagnoses in which the use of dang gui in pregnancy is clearly indicated. In the West, dang gui is often used alone out of this traditional medical context. Because of this, several Western sources consider dang gui to be contraindicated in pregnancy. Data regarding the effect of dang gui preparations on the fetus are lacking.

Lactation There are three unpublished case reports of a rash in infants of lactating mothers who were taking dang gui. The rashes reportedly resolved upon discontinuation of the preparation by the mother. Specific details regarding the preparations used were lacking (Romm, August 1, 2002, oral communication to AHP). Dang gui is a member of the botanical family Apiaceae, a group of plants that contain many types of photoreactive compounds known to cause rashes.

Carcinogenicity Data regarding the effects of dang gui in relationship to carcinogenicity are mixed with both tumorigenic and antitumorigenic activity reported. Antitumor activity due to an induction of cell cycle arrest and apoptosis has been reported for ligustilide (62). Polysaccharides have been shown to inhibit growth of murine tumors (S180, EAC, L1210) in vivo. This was accompanied by a prolonged survival of animals and an inhibition of metastasis in vitro (63). Another animal study identified a possible antitumor effect of dang gui applied to mice with Ehrlich ascites tumors (80). Regarding the potential effects of dang gui on estrogen-positive tumors the data are mixed. One in vitro assay found that dang gui stimulated the growth of MCF7 breast cancer cell lines 16-fold, with no measurable effect on estrogen receptors (81), while another found a possible antitumor effect in T-47D and MCF-7 cell lines (82). Data regarding the potential estrogenic effects of dang gui have been mixed.

Influence on Driving On the basis of the experience of modern herbal practitioners, no negative effects are to be expected.

Overdose On the basis of the available literature, its use as a “food” ingredient in soups, and the experience of modern herbal

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practitioners, dang gui appears to be safe when used at recommended doses.

Treatment of Overdose No data available.

is most likely due to its antioxidant activity. Assays for an estrogenic effect of dang gui have had mixed, but largely negative, results. The relevance of many of these actions to the therapeutic use of dang gui in humans has not yet been demonstrated.

Toxicology The following lethal dose (LD50 ) values have been reported for dang gui extract (8:1 or 16:1), 100 g/kg PO in rats (83,84); dang gui aqueous extract, 100 g/kg IV in mice (85); dang gui 50% ethanol extract, greater than 40 g/kg PO in mice (86); dang gui total acids, 1.05 ± 0.49 g/kg IP in mice (87). The LD50 of ferulic acid IV in mice was reported to be 856.6 mg/kg, (16) and that of ligustilide, approximately 410 mg/kg IP (88). In a review of the toxicology literature on dang gui, it was reported that IV injection of the volatile fraction of dang gui could cause kidney degeneration (76).

REGULATORY STATUS Regulated as a dietary supplement (USC 1994).

CONCLUSIONS Dang gui is one of the most important herbal drugs in TCM, primarily being used for blood tonification and the treatment of gynecological disorders. More recently, interest has focused on dang gui’s possible cardiovascular, hepatoprotective, hematopoietic, antioxidant, antispasmodic, and immunomodulatory effects. Despite its long tradition of use and current widespread clinical utility, there has been very little clinical work verifying the therapeutic efficacy of dang gui when used alone, primarily due to the fact that in TCM, botanicals are generally used in combinations rather than as single agents. On the basis of the literature available and keeping many of its limitations for an English readership in mind, there is limited clinical support for the use of dang gui alone for the following indications: pulmonary artery and portal hypertension, acute ischemic stroke, dysmenorrhea, infertility, and pain due to injury or trauma. The use of dang gui for most of these indications is consistent with TCM. One trial on menopausal symptoms found no effect of dang gui on hormonal activity. Most of the trials available are of poor methodological quality. Clinical and preclinical studies provide some support for a wide variety of actions of dang gui. These include the promotion of circulation, vasodilation/relaxation, and the inhibition of platelet aggregation, all of which are consistent with the “blood quickening” properties ascribed to dang gui in TCM. Similarly, the hematopoietic effect of dang gui is consistent with its use in TCM to “nourish blood.” Its smooth muscle (uterus, vessels, trachea) relaxant effects are consistent with its use for dysmenorrhea, asthma, and coughing. Dang gui may relax or stimulate the uterus depending on a variety of factors. In general, the volatile oil fraction appears to be a uterine relaxant, while the nonvolatile constituents appear to stimulate contractions. There is some support for the traditional use of dang gui as an analgesic and vulnerary. The radiation protective effect of dang gui in animals

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61. Kan WLT, Cho CH, Rudd JA, et al. Study of the antiproliferative effects and synergy of phthalides from Angelica sinensis on colon cancer cells. J Ethnopharmacol 2008; 120:36–43. 62. Tsai NM, Chen YL, Lee CC, et al. The natural compound nbutylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem 2006; 99:1251–1262. 63. Shang P, Qian AR, Yang TH, et al. Experimental study of anti-tumor effects of polysaccharides from Angelica sinensis. World J Gastroenterol 2003; 9(9):1963–1967. 64. Yang XB, Zhao Y, Lv Y. In vivo macrophage activation and physicochemical property of the different polysaccharide fractions purified from Angelica sinensis. Carbohydr Polym 2008; 71:372–379. 65. Yang XB, Zhao Y, Wang ZZ, et al. Chemical composition and immuno-stimulating properties of polysaccharide biological response modifier isolated from Radix Angelica sinensis. Food Chem 2008; 106:269–276. 66. Yang Q, Populo SM, Zhang JY, et al. Effect of Angelica sinensis on the proliferation of human bone cells. Clin Chim Acta 2002; 324:89–97. 67. Xu JY, Li BX, Cheng SY. Short-term effects of Angelica sinensis and nifedipine on chronic obstructive pulmonary disease in patients with pulmonary hypertension. Zhongguo Zhong Xi Yi Jie He Za Zhi 1992; 12(12):707, 716–718. 68. Tu J, Huang H. Effects of Radix Angelicae sinensis on hemorrheology in patients with acute ischemic stroke. Gong Zazhi 1984; 4(3):225–228. 69. Terasawa K, Imadaya A, Tosa A, et al. Chemical and clinical evaluation of crude drugs derived from Angelica acutiloba and Angelica sinensis. Fitoterapia 1985; 56(4):201– 208. 70. Huang ZP, Liang KH. Effect of Radix Angelicae sinensis on serum gastrin levels in patients with cirrhosis. Zhonghua Nei Ke Za Zhi 1994; 33(6):373–375. 71. Huang ZP, Guo B, Yuan SY, et al. Effects of Radix Angelicae sinensis on systemic and portal hemodynamics in cirrhotics with portal hypertension. Zhonghua Nei Ke Za Zhi 1996; 35(1):15–18. 72. Hirata JD, Swiersz LM, Zell B, et al. Does dong quai have estrogenic effects in postmenopausal women? A doubleblind, placebo-controlled trial. Fertil Steril 1997; 68(6):981– 986.

73. Tong YF. Treatment of temporomandibular joint dysfunctional syndrome by injection of Angelica sinensis extract into pterygoideus externus: Clinical analysis of 50 cases. Zhongyi Za Zhi 1991; 12(5):293. 74. Cui LX. Treatment of interspinal ligament injury with danggui injection. Shanghai J Acupunct Moxibust 1989; 8(1):22. 75. Pharmacopoeia of the People’s Republic of China; Chemistry and Industry Press: Beijing 2000. 76. Page RL, Lawrence JD. Potentiation of warfarin by dong quai. Pharmacotherapy 1999; 19(7):870–876. 77. Lo ACT, Chan K, Woo KS. Danggui (Angelica sinensis) affects the pharmacodynamics but not the pharmacokinetics of warfarin in rabbits. Eur J Drug Metab Pharmacokinet 1997; 20(1):55–60. 78. Tang JC, Zhang JN, Wu YT, et al. Effect of the water extract and the ethanol extract from traditional Chinese medicines Angelica sinensis (Oliv.) Diels, Ligusticum chuanxiong Hort., and Rheum palmatum L. on rat liver cytochrome P450 activity. Phytother Res 2006; 20:1046–1051. 79. Gao G, Yang J. Synergistic effect of Angelica sinensis on cyclophosphamide in treating transplanted tumors of mice. Zhongguo Yiyuan Yaoxue Zazhi 1997; 17(7):304–305. 80. Choy YM, Leung N, Cho CS, et al. Immunopharmacological studies of low molecular weight polysaccharide from Angelica sinensis. Am J Chin Med 1989; 22(2):137–145. 81. Amato P, Christophe S, Mellon PL. Estrogenic activity of herbs commonly used as remedies for menopausal symptoms. Menopause 2002; 9(2):145–150. 82. Dixon-Shanies D, Shaikh N. Growth inhibition of human breast cancer cells by herbs and phytoestrogens. Oncol Rep 1999; 6(6):1383–1387. 83. Mills S, Bone K. Principles and Practice of Phytotherapy. Edinburgh, UK: Churchill Livingstone, 2000. 84. Zhu DPQ. Dong quai. Am J Chin Med 1987; 15(3, 4):117–125. 85. Wei ZM. Pharmacological effects of Angelica sinensis on the cardiovascular system. Xinjiang Zhongyiyao 1987; 3:43–46. 86. Yang HY, Chen CF. Acute toxicity and bioactivity evaluation of commonly used Chinese drugs: Analgesic Chinese drugs. J Chin Med 1992; 3(2):41–59. 87. Zhu YZ, Yang QL, Zhang PY. Antiarrhythmic effect of the total acid of Angelica sinensis. Lanzhou Med Coll 1989; 15(3):125–128. 88. Xie FX, Tao JY. Central inhibitory effect of ligustilide of Angelica sinensis. Shanxi Zazhi 1985; 14(8):59–62.

Dehydroepiandrosterone Salvatore Alesci, Irini Manoli, and Marc R. Blackman

INTRODUCTION

DHEA, is derived from cholesterol through the action of the cytochrome P450 side-chain cleavage enzyme (CYPscc). It is converted into DHEA by cytochrome P450 17␣hydroxylase (CYP17), while hydroxysteroid sulfotransferase (DHEAST) catalyzes the transformation of DHEA into its 3-sulfated metabolite DHEAS (Fig. 2). This can be converted back to DHEA by the action of sulfohydrolases (DHEASH), located in the adrenal gland and peripheral tissues. Human plasma contains DHEA-fatty esters (DHEAFA), which are formed from DHEA by the enzyme lecithincholesterol acyltransferase. Newly formed DHEA-FA are incorporated into very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) and may be used as substrates for the synthesis of active oxidized and hydroxylated metabolites in the periphery, such as 7␣/␤hydroxy-DHEA in the brain, and androstenedione, androstenediol, and androstenetriol in the skin and immune organs (3,4).

DHEA is the acronym used to designate the hormone “dehydroepiandrosterone,” also referred to as “prasterone.” The chemical name for DHEA is 5-androsten-3␤-ol-17-one (Fig. 1). DHEA is available as an over-the-counter dietary supplement in the United States, as the Food and Drug Administration did not include it in the 2004 Anabolic Steroid Control Act, which reclassified other related agents as anabolic steroids and hence, controlled substances. As a dietary supplement, it is marketed under different trade names (e.g., Nature’s Blend DHEA, Nature’s Bounty DHEA, DHEA Max, DHEA Fuel, etc.). A pharmaceuticalgrade preparation, currently available only for experimental use, has been assigned the trade name Prestara (previously known as Aslera or GL701).

HISTORICAL OVERVIEW AND GENERAL DESCRIPTION Regulation of DHEA Production

Discovered in 1934, DHEA is the most abundant steroid hormone, and is produced by the adrenal glands in humans and other primates. It acts as a weak androgen and serves as a precursor of other steroids including more potent androgens and estrogens. To date, however, the exact functions of this hormone remain unknown. DHEA is broadly traded on the Internet, under the claim of being a “marvel hormone.” Despite the growing popularity of its use, there is insufficient scientific evidence supporting the purported potential health benefits, and little information regarding the potential short- and long-term adverse risks of consuming exogenous DHEA. Moreover, variations in quality control and manufacturing practices of dietary supplements result in differences in concentrations and purity of the marketed compounds, and insufficient surveillance for side effects.

Release of DHEA by the adrenals is mostly synchronous with that of cortisol, under the stimulus of the hypothalamic corticotropic-releasing hormone (CRH) and pituitary adrenocorticotropic hormone (ACTH). However, the finding of dissociation between DHEA and cortisol secretion during several physiologic and pathophysiologic states suggests that other non-ACTH-mediated mechanisms may be involved in the modulation of DHEA secretion (Table 1). Estrogens, growth hormone, insulin, and prolactin stimulate DHEA secretion by human adrenal cells. However, these findings have not always been replicated in animal or clinical studies. A complex intra-adrenal network involving vascular and nervous systems, local growth and immune factors, and a “cross talk” between cells of the cortex and medulla, the other component of the adrenal gland, also regulate DHEA secretion. The existence of a specific “adrenal androgen-stimulating hormone” has also been postulated, but remains controversial (5).

BIOCHEMISTRY AND PHYSIOLOGY Biosynthesis and Metabolism

Adrenarche and Adrenopause

DHEA is primarily produced in the zona reticularis of the adrenal cortex. In healthy women, the adrenal gland is the principal source of this steroid, whereas in men, 10% to 25% of the circulating DHEA is secreted by the testes (1). It can also be synthesized within the central nervous system (CNS), and can be considered a “neurosteroid” (2). Pregnenolone, the immediate precursor of

At birth, DHEAS is the predominant circulating steroid. However, a dramatic involution of the fetal adrenal zone, starting in the first postnatal month and continuing through the first year of life, is paralleled by a sudden decrease in DHEA/DHEAS, which remains unchanged for the next six years. By the age of six to eight years, the adrenal gland matures, culminating in the creation of 217

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Table 1 Dissociation of Cortisol and DHEA/DHEAS Secretion During Physiological and Pathological Conditions

O DHEA

Condition H

H

H

HO

(DHEA) Figure 1

Chemical structure of dehydroepiandrosterone (DHEA).

the zona reticularis, followed by an abrupt elevation in DHEA and DHEAS concentrations, termed the “adrenarche” (6). Peak concentrations of DHEA (180–800 ng/dL) and DHEAS (45–450 ␮g/dL) are reached during the third decade of life. Subsequently, there is a progressive 2% decline per year in DHEA and DHEAS secretion and excretion, with concentrations equal to 20% of the peak by the age of 80, and values lower in women than in men (5,7). This marked decline has been termed “adrenopause.” DHEA and DHEAS levels are higher in men than in women at all ages.

Mechanisms of Action Despite the identification of high-affinity binding sites for DHEA in rat liver, T-lymphocytes, and endothelial cells, the search for a specific, cognate DHEA receptor has been unsuccessful. Multiple mechanisms of action have been proposed for DHEA. Most important among these are that DHEA can be metabolized into more potent androgens [testosterone and dihydrotestosterone (T and DHT)] and estrogens (estradiol and estrone) in the periphery, which

Cholesterol CYPSCC

Pregnenolone CYP17

17α−Hydroxypregnenolone CYP17

Androstenedione

3β-HSD

DHEA

17β-HSD

Testosterone

5α-R 3α-HSD

DHEASH DHEAST

CYParo

Androsterone

DHEAS

Estradiol

Figure 2 Schematic diagram of DHEA/DHEAS biosynthesis and metabolism. Abbreviations: CYPscc, cytochrome P450 side-chain cleavage enzyme; CYP17, cytochrome P450 17 ␣-hydroxylase; 3␤/17␤/3␣-HSD, 3␤/17-␤/3␣ hydroxysteroid dehydrogenase; 5␣-R, 5-␣ reductase; DHEASH, DHEA sulfohydrolase; DHEAST, DHEA sulfotransferase; CYParo, cytochrome P450 aromatase.

Physiological (age-related) Fetal stage Birth Infancy and childhood Adrenarche (6–8 yr) Puberty Adrenopause (50–60 yr) Pathological Anorexia nervosa Chronic/severe illness Burn trauma Cushing disease Congenital adrenal hyperplasia Ectopic ACTH syndrome Idiopathic hirsutism Partial hypopituitarism without ACTH deficiency End-stage renal diseases Stress

DHEA

DHEAS

Cortisol

↑ ↑ ↓

↑ ↑ ↓ ↑ ↑ ↓

N N N N N N or ↑

↓

↓ ↓ ↓ N ↑ N, ↑, or ↓ ↑ ↓

↑ ↑ ↑ ↑ ↓ ↑ N N

↓

↓ ↓

↑ ↑

↓

N N, ↑, or ↓

N = normal serum levels; ↑ = increased serum levels; ↓ = decreased serum levels. Source: From Ref. 5.

can interact with specific androgen and estrogen receptors. DHEA itself can bind to the androgen and estrogen receptors, but its affinity is extremely low compared with those of the native ligands. It has been estimated that DHEA and DHEAS function as precursors of 50% of androgens in men, 75% of active estrogens in premenopausal women, and 100% of active estrogens in postmenopausal women (8). Lipophilic DHEA, but not hydrophobic DHEAS, can be converted into both androgens and estrogens intracellularly in target tissues by “intracrine” processes (4). This conversion depends on the levels of different steroidogenic and metabolizing enzymes, and on the hormonal milieu. For example, DHEAS and sulfatase are present in high concentrations in the prostate, and the resultant metabolism of DHEA to DHT accounts for up to one-sixth of the intraprostatic DHT (9). DHEA can also function as a neurosteroid, by modulating neuronal growth, development, and excitability, the latter via interaction with ␥ -aminobutyric acid (GABAA ), N-methyl-D-aspartate (NMDA), and sigma receptors (10). It is known to be a potent inhibitor of glucose-6-phosphate dehydrogenase (G6DPH), thus interfering with the formation of mitochondrial nicotinamide adenine dinucleotide phosphate [NADP(H)] and ribose-6-phosphate and inhibiting DNA synthesis and cell proliferation (11). The steroid hormone has also been proposed to exert antiglucocorticoid, cytokine modulatory, potassium channel and cyclic guanyl monophosphate (cGMP) stimulatory, and thermogenic effects.

PHARMACOKINETICS Absorption and Tissue Distribution While DHEA is marketed as an oral product, it has also been shown to be absorbed when administered by the transdermal, intravenous, subcutaneous, and vaginal

Dehydroepiandrosterone

routes. Crystalline and micronized formulations result in higher DHEAS serum concentrations, possibly due to an enhanced rate of absorption (12). After absorption in the small intestine, DHEA is mainly sulfated in the liver. The nonoral route averts first pass liver degradation, resulting in higher serum levels. DHEA concentrations are high in the brain, with a brain-to-plasma ratio of 4–6.5:1 (13) and plasma, spleen, kidney, and liver concentrations follow in descending order. Cerebrospinal, salivary, and joint fluid levels are directly related to those of serum.

Bioavailability, Metabolism, and Clearance Pharmacokinetic studies on DHEA reveal a clearance rate compatible with a two-compartment model. The initial volume of distribution is 17.0 ± 3 L. DHEA disappears from the first compartment in 17.2 ± 6.2 minutes and from the second in 60.2 ± 12.3 minutes (14). DHEAS follows a one-compartment model of disappearance. Its volume of distribution is 4.6 ± 0.9 L, while the half-life from that compartment is 13.7 hours (15). In men, 77.8 ± 17.3% of the DHEAS that enters the circulation will reappear as DHEA, while in women, it is 60.5 ± 8.2%. The opposite conversion of DHEA to DHEAS is much smaller, 5.2% ± 0.7% in men, and 6.25% ± 0.54% in women. Mean metabolic clearance rates (MCRs) were calculated using the constant infusion technique: The DHEA MCR is 2050 ± 160 L/day in men and 2040 ± 160 L/day in women, whereas the MCR for DHEAS is 13.8 ± 2.7 L/day in men and 12.5 ± 1.0 L/day in women. The differences in the clearance rates of DHEA and DHEAS are partly explained by different binding efficiencies with albumin. Circulating DHEA is primarily bound to albumin, with only minimal binding to sex hormone-binding globulin (SHBG); the remaining small amount is free. There is no known specific DHEA-binding protein. In comparison, DHEAS is strongly bound to albumin but not to SHBG, and an even smaller amount is protein free. Obesity results in increased MCR for DHEA from 2000 to 4000 L/day in women. A rise in MCR is also caused by insulin infusion in men (16).

Supplementation Considerations related to the metabolism of DHEA become more complicated when it is administered as a dietary supplement (in the United States) or as a drug (in some other countries). The steroid is usually given orally in a single morning dose, as its constant interconversion to DHEAS and the long half-life of DHEAS make multiple dosing unnecessary. In addition, morning dosing mimics the natural rhythm of DHEA secretion. Doses ranging from 25 to 1600 mg/day have been used in different studies. After an oral dose, the half-lives of DHEA/DHEAS are longer (24 hours) than those reported in intravenous tracer studies, which may reflect the conversion of DHEAS to DHEA (17). Oral administration of 20 to 50 mg of DHEA in patients with primary or secondary adrenal insufficiency restores serum DHEA and DHEAS concentrations to the range observed in normal young individuals, while a dose of 100 to 200 mg/day results in supraphysiological concentrations.

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Different metabolic pathways for exogenous DHEA in relation to gender and age have been reported. DHEA levels after oral administration of 25 or 50 mg DHEA for eight days were persistently higher in women versus men (17). Similarly, oral administration of 200 mg of micronized DHEA in single or multiple doses for 15 days in healthy adult men and women resulted in higher serum concentrations and bioavailability (measured by DHEA Cmax and AUC) in women. The net increase in DHEAS levels was 21-fold in women and 5-fold in men (18). The metabolic fate of exogenous DHEA also differs by gender and age. While in pre- and postmenopausal women, DHEA is mostly transformed into androgens, in men it is preferentially metabolized into estrogens. Higher serum concentrations of DHEA, testosterone, and estradiol are achieved in elderly subjects (19).

THERAPEUTIC APPLICATIONS DHEA Replacement in Adrenal Insufficiency Patients with primary (Addison disease) and secondary adrenal insufficiency, typically exhibit very low, often undetectable, serum concentrations of DHEA(S) compared with values in age-matched control subjects. Despite optimal glucocorticoid and mineralocorticoid replacement, however, these patients often experience chronic fatigue, reduced sense of well-being, and lack of sexual interest. Some studies have suggested that DHEA replacement exerts a positive effect on mood, vitality, sexuality, and overall well-being mostly in women and, to a lesser extent, in men, with chronic adrenal insufficiency (20–23). For example, oral administration of 50 mg/day of DHEA for four months to 24 women with adrenal insufficiency increased serum levels of DHEAS, androstenedione, testosterone, and androstenediol glucuronide, and improved overall well-being, mood, and sexual activity (20). In another study of 15 men and 24 women with Addison disease, administration of 50 mg/day of DHEA for three months corrected the hormonal deficiency and improved self-esteem, while it tended to enhance overall well-being, mood, and energy (21). However, other studies have failed to show similar effects (24–26). Moreover, a meta-analysis of 10 randomized placebo-controlled studies concluded that DHEA may improve health-related quality of life and depression in women with adrenal insufficiency, though the effect size (0.21; 95% confidence interval, 0.08–0.33; inconsistency (I2 ) = 32%) was too small to support the routine use of DHEA in this patient group (27). In the earlier studies, a single oral dose of DHEA (25–50 mg) was sufficient to keep serum concentrations of DHEA within the normal range, while signs of overreplacement included acne, hirsutism, or alopecia. Moreover, concerns have been raised about the effects of DHEA supplementation on the lipid profile and specifically on high-density lipoproteins (HDL) levels in hypoadrenal women (28), though it has been reported to improve their insulin resistance (29). Because a pharmaceutically controlled DHEA preparation, as well as proof of effectiveness and safety in long-term phase III trials, are still lacking, replacement of DHEA in patients with adrenal insufficiency should be

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restricted to those individuals whose well-being or libido is severely impaired despite adequate glucocorticoid and mineralocorticoid replacement.

DHEA Replacement in Adrenopause and Age-Related Disorders As noted, aging is accompanied by profound decreases in circulating concentrations of DHEA and DHEAS in both sexes (5,7). Epidemiological studies suggest an association between the DHEA and DHEAS declines and the adverse effects of aging, albeit with gender differences. One large, prospective observational study reported a small, but significant, inverse correlation between serum DHEAS concentrations and risk of cardiovascular mortality in men at 19-year follow-up, whereas in women, high DHEAS levels were associated with an increased risk of cardiovascular death at 12-year follow-up, and this trend lost significance at 19-year follow-up (30). Similar results were reported in another study of 963 men and 1171 women aged >65 years: all-cause and cardiovascular mortality were highest in men with DHEAS levels in the lowest quartile, whereas no significant association between circulating DHEAS and mortality was found in women (31). Other studies failed to demonstrate this inverse relationship in men (32). Positive correlations between low circulating DHEAS concentrations and depressed mood and bone loss have been reported in aged women (33). In comparison, DHEAS levels are reduced in men, with noninsulin-dependent diabetes mellitus (NIDDM) (34). Reports of an association between low DHEA levels and Alzheimer disease are conflicting. It remains uncertain as to whether the DHEAS decline is simply a biomarker of aging, or is causally related to morbidity and mortality in the elderly. One study of the effects of oral administration of 50 mg/day of DHEA for six months to 13 men and 17 women aged 40 to 70 years showed restoration of DHEA and DHEAS levels to young–adult values, with improvement in physical and psychological sense of well-being, but not sexual interest, in both genders using a questionnaire for self-assessment. These effects were accompanied by increased serum levels of insulinlike growth factor 1 (IGF-I), reduced IGF-I binding protein-1 (IGFBP-1), and a significant decrease in apolipoprotein A1 and HDL-cholesterol in women, but no change in insulin sensitivity and body composition (35). Another study using 100 mg/day of DHEA for six months in 9 men and 10 women aged 50 to 65 years reported decreased fat mass and enhanced muscle strength in men, whereas increased levels of downstream androgens were detected in women (36). In a third study of 39 elderly men treated for three months with 100 mg/day of oral DHEA, no treatment effect on body composition or subjective well-being was found, whereas a significant reduction in HDL-cholesterol was reported (37). In the largest study to date, 280 men and women aged 60 to 79 years were treated for 12 months with 50 mg/day of oral DHEA. No improvement in wellbeing was detected using a variety of validated tools. In addition, there were no significant changes in body composition, metabolic parameters, or muscle strength. However, a slight but significant increase in bone mineral density (BMD) at the femoral neck and the radius,

and an increase in serum testosterone, libido, and sexual function were observed in women >70 years old (38). Similar changes were reported in 14 women aged 60–70 years who were treated for 12 months with a 10% DHEA skin cream. In addition to a 10-fold increase in DHEA levels, the authors described increased BMD at the hip, and decreased osteoclastic and increased osteoblastic bone markers. Other changes included improved well-being, a reduced skinfold thickness, and lower blood glucose and insulin levels, with no adverse change in lipid profile (39). DHEA replacement did not affect BMD in other studies (36). Little information is available regarding the effects of DHEA therapy on cardiovascular function and insulin sensitivity from interventional studies, other than that provided in the preceding text. The effects of DHEA treatment on lipid profile were evaluated in more than a dozen studies performed in men and women with various doses and routes of administration. The majority of these studies showed no effect of DHEA on plasma lipids (40). In one study of 24 middle-aged men, administration of 25 mg/day of oral DHEA for 12 weeks decreased the plasma levels of plasminogen activator inhibitor type 1 (PAI-1), and increased dilatation of the brachial artery after transient occlusion (41). In rodent models of NIDDM, dietary administration of DHEA consistently induced remission of hyperglycemia and increased insulin sensitivity. Some clinical studies in aged men and women have shown improved insulin sensitivity after DHEA replacement (39,42), whereas others have not confirmed those findings in women (35,43). The effects of DHEA supplementation on cognitive function in healthy elderly people were recently reviewed in depth by Grimley et al. All randomized placebo-controlled trials enrolling people aged >50 without dementia to whom DHEA had been administered for more than one day were considered for inclusion. Five studies provided results from adequate parallel-group data. The authors concluded that findings from these controlled studies did not support any beneficial effect of DHEA supplementation on cognitive function in nondemented elderly people (44). Moreover, DHEA replacement did not affect memory in any of the controlled studies in healthy elderly, as previously discussed. Therefore, the use of DHEA supplementation to improve cognitive function should not be recommended in the elderly population at this time.

Potential Beneficial Effects of DHEA Supplementation DHEA administration, often in large doses, has been proposed in the management of numerous disorders, including obesity, cancer, autoimmune diseases, AIDS, mood disorders, as well as in enhancing physical performance. The scientific evidence supporting the benefits of DHEA therapy in these conditions is, however, very limited. Pharmacologic treatment with DHEA in mice genetically predisposed to become obese reduced weight gain and fat cell size. In obese rats, the steroid decreased food intake by 50%. In humans, some observational studies indicate a relationship between circulating DHEA and

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DHEAS levels, body mass index (BMI) and weight loss, whereas others do not confirm this (45). Similar inconsistencies are encountered in interventional studies. Administration of a high oral dose of DHEA (1600 mg/ day) decreased body fat and increased muscle mass, with no net change in body weight, in a small group of healthy young men. As mentioned earlier, a reduction in fat mass was also reported in healthy elderly men after six months administration of 100 mg/day of DHEA (36), whereas topical application of a 10% DHEA cream for one year decreased femoral fat and skinfold thickness in postmenopausal women (39). Other studies in healthy elderly individuals and in obese men failed to demonstrate changes in body fat after DHEA treatment (37,43). DHEA has been reported to exhibit chemopreventive activity in mouse and rat models, although it has also been found to be hepatocarcinogenic in rats. Epidemiological research has revealed increased DHEA levels to be associated with a rise in risk of ovarian cancer and breast cancer in postmenopausal women, whereas decreased levels are linked with increased risk of bladder, gastric, and breast cancer in premenopausal women. To date, we are unaware of clinical studies documenting the effects of DHEA intervention on cancer initiation or propagation. Fluorinated DHEA analogs that cannot be converted into androgens or estrogens appear to have antiproliferative effects and have been tested in several preclinical cancers including bowel polyposis (46) and used without side effects in doses up to 200 mg/day orally for four weeks (47). Increased antibody production in response to bacterial infections and decreased mortality from endotoxic shock were reported in DHEA-treated mice. In a study of 71 aged individuals, however, DHEA administration did not enhance antibody response to influenza vaccine (48). In contrast, 50 mg/day of oral DHEA increased the activity of natural killer cells by twofold, with a concomitant decrease in T-helper cells in postmenopausal women (49). In 28 women with mild-to-moderate systemic lupus erythematosus (SLE), oral treatment with 200 mg/day of DHEA for three to six months improved well-being and decreased disease activity and prednisone dosage requirements. The same DHEA dose administered orally for 56 days to patients with Crohn disease or ulcerative colitis decreased disease activity in a small, uncontrolled study (50). DHEA did not separate itself from placebo in a randomized controlled trial in patients ¨ with Sjogren syndrome (51). The effect of DHEA supplementation in SLE was recently reviewed more systematically. Analysis of the results from seven randomized placebo-controlled trials of at least three months duration revealed a modest but clinically significant beneficial effect of DHEA on measures of quality of life in SLE patients, whereas effects on measures of disease activity were inconsistent (52). Serum DHEA and DHEAS levels in patients infected with HIV are directly related to CD4 cell counts and disease stage and progression. This observation has stimulated self-administration of DHEA as an adjunct to antiviral treatment by AIDS patients. In the only published placebo-controlled trial of DHEA in patients with advanced HIV disease, treatment with 50 mg/day orally

for four months resulted in increased DHEAS levels and improved mental function, with no change in CD4 cell count (53). These results were consistent with those from a previous open label study in 32 HIV-positive patients treated with DHEA doses of 200 to 500 mg/day for eight weeks (54). Studies in adults and adolescents with major depressive disorders have revealed a blunted DHEA circadian variation, with low DHEA and high cortisol/DHEA ratio at 8:00 AM. In 22 patients with medication-free or stable major depression, supplementation with 30 to 90 mg/day of oral DHEA for six weeks decreased Hamilton depression scale scores as much as 50% (55). Similar results were reported in a well-controlled study in 15 patients aged 45 to 63 years with midlife-onset dysthymia who, after a three-week administration of 90 mg of DHEA, reported improvements in depressive symptoms (56). In a recent double-masked trial in schizophrenic patients with predominant negative symptoms, supplementation with 100 mg/day of DHEA also led to improvement in depression and anxiety (57). DHEA administration improves memory test results and decreases serum levels of ␤-amyloid in aging mice. In contrast, treatment with 100 mg of oral DHEA did not improve cognitive performance in patients with Alzheimer disease, similar to the findings reported in healthy elderly subjects (58). DHEA is a popular dietary supplement among athletes. Nevertheless, at 150 mg/day orally, it did not improve body mass or strength in young male athletes and weight lifters (59,60). In contrast, increased quadriceps and lumbar strength were reported in healthy elderly men on DHEA replacement (36), and an increase in lean body mass of 4.5 kg was observed in healthy young men taking 1600 mg/day of DHEA for four weeks (61). A summary of the various potential therapeutic applications of DHEA replacement/supplementation is presented in Table 2.

Table 2

Clinical Conditions for Which DHEA Use Has Been Proposed

Condition

Effect

References

Adrenal insufficiency

Improved general well-being, mood, sexual function

20–23

Aging

Autoimmune disease

Body composition

Insulin resistance

No significant effects

24–27

Improved physical well-being, bone mineral density or sexual function Decrease in HDL or no effects

35,38,39

Improved well-being, fatigue, disease activity in SLE patients, enhanced immune response

52

36,37

No effect in HIV patients

53,54

Decreased fat and increased muscle mass

36,61

No changes

37,43

Improved insulin sensitivity

39

No change

35

Depression Alzheimer disease

Improved mood No effect on cognitive function

55–57 58

Cardiovascular

Improved endothelial function No effect on lipids

38 40

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CLINICAL PHARMACOLOGY AND TOXICOLOGY Dietary/Nondietary Sources and Available Preparations There are no known dietary sources of DHEA, although it was suggested that the supplement chromium picolinate could stimulate endogenous DHEA secretion. The Mexican plant “wild yam” contains some natural DHEA precursors, which cannot be converted into DHEA. However, sterol extracts of “wild yam” (such as diosgenin and dioscorea) are used to produce various forms of synthetic DHEA, including tablets, capsules, injectable esters (Gynodian Depot, Schering), sublingual and vaginal preparations, topical creams, lozenges, and herbal teas. DHEA is usually sold in tablets of 5 to 50 mg. A pharmaceutical-grade preparation (Prestara) has been developed for potential use as a prescription drug. During the manufacture of DHEA, other steroid-like compounds, like androstenedione, may be produced and could contaminate DHEA. Moreover, the real steroid content of the DHEA preparation sold over the counter may vary from 0% to 150% of the amount claimed (62). In addition, there is a lack of information about comparability or bioequivalence among the many products on the market or information about lot-to-lot variability of any particular product in terms of characterization (content) and standardization (contaminants).

oral DHEA conducted in 31 HIV-positive men revealed no serious dose-limiting toxicity (63). Because of its potent androgenic and estrogenic effects, it would appear prudent to avoid DHEA replacement/supplementation in individuals with a personal or family history of breast, ovarian, or prostate cancer. This would seem especially important for postmenopausal women, considering the demonstrated direct correlation between circulating concentrations of DHEA and breast and ovarian cancers in this group (46), and the reported increase with DHEA supplementation in free IGF-1 (64). Caution may also be appropriate in HIV patients, since high DHEA levels have been implicated in the pathogenesis of Kaposi’s sarcoma. Moreover, increased insulin resistance and decreased cholesterol and HDL after administration of a high dose 1600 mg/day of DHEA for four weeks have been reported in aged women (43). DHEA administration is not recommended for people younger than 40 years of age, unless there is a documented deficiency state. It should be avoided during pregnancy, lactation, and in persons younger than 18 years, as dosage and safety of the treatment under these conditions have not been evaluated.

Known Drug Interactions Dosage and Administration There is no consensus on a recommended dietary allowance (RDA) or optimal treatment dose for DHEA. Replacement with oral doses of 20 to 50 mg/day for men and 10 to 30 mg/day for women appears adequate to achieve DHEA/DHEAS levels similar to those in young adults, as suggested by most studies in subjects with adrenal or age-related DHEA/DHEAS deficiency, though oral doses as low as 5 mg/day have been reported to be effective. Higher DHEA doses may be necessary for patients with very low endogenous DHEA levels secondary to glucocorticoid administration or chronic disease. Doses of 200 to 500 mg and 200 mg/day have been used in patients with HIV and SLE, respectively. Serum DHEAS levels and its androgenic and estrogenic metabolites must be closely monitored during replacement to enable appropriate dose adjustments. Rigorous dose ranging studies are needed to determine the optimal doses to achieve a beneficial effect.

Adverse Reactions, Long-Term Effects, and Contraindications DHEA appears to elicit few short-term side effects when used in the recommended doses. Women may experience mild hirsutism, increased facial sebum production, and acneiform dermatitis. Circulating concentrations of downstream androgens rise above young–adult values in healthy elderly women treated with 100 mg/day DHEA; however, the long-term consequences of this increase are unknown. No significant changes in complete blood count, urinalysis, hepatic, and thyroid indices were found in women after 28 days of treatment with 1600 mg DHEA/day (43). A dose escalation study of 750 to 2250 mg

Drugs known to interfere with DHEA and/or DHEAS include various hormone preparations, drugs acting on the CNS, cardiovascular drugs, adrenergic and adrenergic blocking agents, and anti-infective agents. Synthetic glucocorticoids such as dexamethasone are the most potent suppressors of DHEA and DHEAS. DHEA and/or DHEAS levels are known to be decreased in patients taking aromatase inhibitors, oral contraceptives, dopaminergic receptor blockers, insulin, troglitazone, and multivitamins, or fish oil. They are also decreased due to the induction of the cytochrome P450 enzymes, by carbamazepin, phenytoin, or rifampicin. Metformin (a biguanide antihyperglycemic drug) and calcium channel blockers are shown to increase DHEA and DHEAS levels. The effect of alcohol is controversial (65).

Compendial/Regulatory Status In the early 1980s, DHEA was widely advertised and sold in U.S. health food stores as an “anti-aging,” “anti-obesity,” and “anti-cancer” nonprescription drug. However, on the basis of unknown potential long-term risks, and following the ban by the International Olympic Committee, in 1985 the U.S. Food and Drug Administration reclassified DHEA as a prescription drug. In October 1994, the U.S. Dietary Supplement Health and Education Act allowed DHEA to be sold again as an over-the-counter dietary supplement, as long as no claims are made regarding therapeutic efficacy. DHEA remains available over the counter in the United States, as it was not reclassified as an anabolic steroid, and hence a controlled substance, in the 2004 Anabolic Steroid Control Act.

Dehydroepiandrosterone

CONCLUSIONS Despite a considerable amount of research related to DHEA, and its alleged utility to sustain “eternal youth,” several key questions remain unanswered. For example, the physiologic function(s), regulation, and mechanisms of actions of DHEA remain unknown, and a causal link between age-related declines in DHEA and adverse effects of aging has not been proven. Results from clinical studies suggest that DHEA may be beneficial in some patients with adrenal insufficiency, whereas data from intervention studies in healthy older people have been inconclusive, save for some modest gender differences in selected outcome measures. Well-characterized and standardized products need to be evaluated for safety and efficacy, starting with dose ranging to determine an optimal dose. Because DHEA can be converted to estrogen and testosterone, its potential adverse effects in patients with breast or prostate cancer need to be determined. Longterm, well-designed clinical studies, with clear end points, will be necessary before the beneficial and/or detrimental effects of “replacement” or “pharmacological” DHEA therapies in human aging and disease can be firmly established.

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28. Srinivasan M, Irving BA, Dhatariya K, et al. Effect of dehydroepiandrosterone replacement on lipoprotein profile in hypoadrenal women. J Clin Endocrinol Metab 2009; 94(3):761– 764. 29. Dhatariya K, Bigelow ML, Nair KS. Effect of dehydroepiandrosterone replacement on insulin sensitivity. Diabetes 2005; 54(3):765–769. 30. Barrett-Connor E, Goodman-Gruen D. The epidemiology of DHEAS and cardiovascular disease. Ann N Y Acad Sci 1995; 774:259–270. 31. Trivedi DP, Khaw KT. Dehydroepiandrosterone sulfate and mortality in elderly men and women. J Clin Endocrinol Metab 2001; 86(9):4171–4177. 32. LaCroix AZ, Yano K, Reed DM. Dehydroepiandrosterone sulfate, incidence of myocardial infarction, and extent of atherosclerosis in men. Circulation 1992; 86(5):1529– 1535. 33. Greendale GA, Edelstein S, Barrett-Connor E. Endogenous sex steroids and bone mineral density in older women and men: The Rancho Bernardo study. J Bone Miner Res 1997; 12(11):1833–1843. 34. Barrett-Connor E. Lower endogenous androgen levels and dyslipidemia in men with non-insulin-dependent diabetes mellitus. Ann Intern Med 1992; 117(10):807–811. 35. Morales AJ, Nolan JJ, Nelson JC, et al. Effects of replacement dose of dehydroepiandrosterone in men and women of advancing age. J Clin Endocrinol Metab 1994; 78(6):1360– 1367. 36. Morales AJ, Haubrich RH, Hwang JY, et al. The effect of six months treatment with a 100 mg daily dose of dehydroepiandrosterone (DHEA) on circulating sex steroids, body composition and muscle strength in age-advanced men and women. Clin Endocrinol (Oxf) 1998; 49(4):421– 432. 37. Flynn MA, Weaver-Osterholtz D, Sharpe-Timms KL, et al. Dehydroepiandrosterone replacement in aging humans. J Clin Endocrinol Metab 1999; 84(5):1527–1533. 38. Baulieu EE, Thomas G, Legrain S, et al. Dehydroepiandrosterone (DHEA), DHEA sulfate, and aging: Contribution of the DHEAge Study to a sociobiomedical issue. Proc Natl Acad Sci U S A 2000; 97(8):4279–4284. 39. Labrie F, Diamond P, Cusan L, et al. Effect of 12-month dehydroepiandrosterone replacement therapy on bone, vagina, and endometrium in postmenopausal women. J Clin Endocrinol Metab 1997; 82(10):3498–3505. 40. Tchernof A, Labrie F. Dehydroepiandrosterone, obesity and cardiovascular disease risk: A review of human studies. Eur J Endocrinol 2004; 151:1–14. 41. Kawano H, Yasue H, Kitagawa A, et al. Dehydroepiandrosterone supplementation improves endothelial function and insulin sensitivity in men. J Clin Endocrinol Metab 2003; 88(7):3190–3195. 42. Lasco A, Frisina N, Morabito N, et al. Metabolic effects of dehydroepiandrosterone replacement therapy in postmenopausal women. Eur J Endocrinol 2001; 145(4):457– 461. 43. Mortola JF, Yen SS. The effects of oral dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women. J Clin Endocrinol Metab 1990; 71(3):696–704. 44. Grimley Evans J, Malouf R, Huppert F, et al. Dehydroepiandrosterone (DHEA) supplementation for cognitive function in healthy elderly people. Cochrane Database Syst Rev 2006; (4):CD006221. 45. Williams JR. The effects of dehydroepiandrosterone on carcinogenesis, obesity, the immune system, and aging. Lipids 2000; 35(3):325–331.

46. Johnson MD, Bebb RA, Sirrs SM. Uses of DHEA in aging and other disease states. Ageing Res Rev 2002; 1(1): 29–41. 47. Davidson M, Marwah A, Sawchuk RJ, et al. Safety and pharmacokinetic study with escalating doses of 3-acetyl-7-oxodehydroepiandrosterone in healthy male volunteers. Clin Invest Med 2000; 23(5):300–310. 48. Danenberg HD, Ben-Yehuda A, Zakay-Rones Z, et al. Dehydroepiandrosterone treatment is not beneficial to the immune response to influenza in elderly subjects. J Clin Endocrinol Metab 1997; 82(9):2911–2914. 49. Casson PR, Andersen RN, Herrod HG, et al. Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women. Am J Obstet Gynecol 1993; 169(6):1536–1539. 50. Andus T, Klebl F, Rogler G, et al. Patients with refractory Crohn’s disease or ulcerative colitis respond to dehydroepiandrosterone: A pilot study. Aliment Pharmacol Ther 2003; 17(3):409–414. 51. Hartkamp A, Geenen R, Godaert GL, et al. Effect of dehydroepiandrosterone administration on fatigue, well-being, and functioning in women with primary Sjogren syndrome: A randomised controlled trial. Ann Rheum Dis 2008; 67:91– 97. 52. Crosbie D, Black C, McIntyre L, et al. Dehydroepiandrosterone for systemic lupus erythematosus. Cochrane Database Syst Rev 2007; (4):CD005114. 53. Piketty C, Jayle D, Leplege A, et al. Double-blind placebocontrolled trial of oral dehydroepiandrosterone in patients with advanced HIV disease. Clin Endocrinol (Oxf) 2001; 55(3):325–330. 54. Rabkin JG, Ferrando SJ, Wagner GJ, et al. DHEA treatment for HIV + patients: Effects on mood, androgenic and anabolic parameters. Psychoneuroendocrinology 2000; 25(1): 53–68. 55. Wolkowitz OM, Reus VI, Keebler A, et al. Double-blind treatment of major depression with dehydroepiandrosterone. Am J Psychiatry 1999; 156(4):646–649. 56. Bloch M, Schmidt PJ, Danaceau MA, et al. Dehydroepiandrosterone treatment of midlife dysthymia. Biol Psychiatry 1999; 45(12):1533–1541. 57. Strous RD, Maayan R, Lapidus R, et al. Dehydroepiandrosterone augmentation in the management of negative, depressive and anxiety symptoms in schizophrenia. Arch Gen Psychiatry 2003; 60:133–141. 58. Wolkowitz OM, Kramer JH, Reus VI, et al. DHEA treatment of Alzheimer’s disease: A randomized, doubleblind, placebo-controlled study. Neurology 2003; 60(7):1071– 1076. 59. Wallace MB, Lim J, Cutler A, et al. Effects of dehydroepiandrosterone vs. androstenedione supplementation in men. Med Sci Sports Exerc 1999; 31(12):1788– 1792. 60. Brown GA, Vukovich MD, Sharp RL, et al. Effect of oral DHEA on serum testosterone and adaptations to resistance training in young men. J Appl Physiol 1999; 87(6):2274– 2283. 61. Nestler JE, Barlascini CO, Clore JN, et al. Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men. J Clin Endocrinol Metab 1988; 66(1): 57–61. 62. Parasrampuria J, Schwartz K, Petesch R. Quality control of dehydroepiandrosterone dietary supplement products. JAMA 1998; 280(18):1565. 63. Dyner TS, Lang W, Geaga J, et al. An open-label doseescalation trial of oral dehydroepiandrosterone tolerance and

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Echinacea Species Rudolf Bauer and Karin Woelkart

ing the incidence, severity, or duration of acute URIs have been published. The majority of trials investigated whether Echinacea preparations shorten the duration or decrease the severity of symptoms of the common cold (for reviews see 5,8–12). A recent Cochrane review (10) concluded that especially preparations based on the aerial parts of E. purpurea might be effective for the early treatment of colds in adults, but results are not fully consistent. Beneficial effects of other Echinacea preparations for preventative purposes might exist, but have not been shown in independently replicated rigorously randomized trials. Therefore, further clinical studies are needed to evaluate the therapeutic role of Echinacea preparations. Many of the in vivo studies performed so far used phytochemically insufficiently characterized Echinacea preparations. The regulatory status of Echinacea products is variable. In the United States, they are considered as dietary supplements, and manufacturers can therefore produce, sell, and market herbs without demonstrating safety and efficacy first, as is required for pharmaceutical drugs. In Canada, they are regulated as Natural Health Products (NHPs), and in several European countries they have drug status. The EMEA (European Medicines Agency) Committee on Herbal Medicinal Products (HMPCs) has published the following guidance documents for Echinacea products (www.emea.europa.eu):

INTRODUCTION Echinacea is a herbal medicine that has been used for centuries, customarily as a treatment for common cold, coughs, bronchitis, upper respiratory infections (URIs), and inflammatory conditions. It belongs to Heliantheae tribe of the Asteraceae (Compositae) family, and the nine species of these perennial North American wildflowers have a widespread distribution over prairies, plains, and wooded areas.

BACKGROUND Three species of Echinacea are used medicinally: E. purpurea (Fig. 1), E. pallida (Fig. 2), and E. angustifolia (Fig. 3). In the United States of America, Echinacea preparations have been the best selling herbal products in health food stores. However, an analysis reveals that completely different preparations are sold under the name Echinacea using different plant parts and different extraction solvents (1). The most investigated preparation, which is mainly available on the German market, contains the expressed juice of E. purpurea aerial parts. Besides this, hydroalcoholic tinctures of E. purpurea aerial parts and roots, as well as from E. pallida and E. angustifolia roots, can be found (2,3). In North America, especially, it is also common to sell encapsulated powders from aerial parts and roots of these three species. Preclinical studies indicate that Echinacea constituents modulate immune mechanisms and there is increasing evidence that Echinacea preparations containing alkamides can suppress stress-related cellular immune responses. As reviewed by Chicca et al. (4), the interaction of alkamides with the cannabinoid receptor type-2 (CB2 ), which is a modulator of inflammation, may provide a mechanistic basis for the anti-inflammatory and immunomodulatory effects exerted by purple coneflower. Echinacea alkamides are now considered as a new class of cannabinomimetics (5,6). Recent findings suggest that Echinacea has a modulatory role on the innate immune system, able to both stimulate and inhibit the immune response. Since Echinacea-derived alkamides significantly suppressed T-lymphocytes (7), it is apparent that the multiplicity and diversity of parts of various plants, methods of extraction, and solvents used, as well as the components on which the extracts have been standardized, have hampered clear recommendations regarding Echinacea usage. Several reviews on the evidence regarding the effectiveness of orally ingested Echinacea extracts in reduc-

r Community Herbal Monograph on Echinacea purpurea (L.) Moench, herba recens (13) r Community Herbal Monograph on Echinacea purpurea (L.) Moench, radix (14) r Community Herbal Monograph on Echinacea pallida (Nutt.) Nutt., radix (15) r Community List Entry for Echinacea purpurea (L.) Moench, herba recens (16) For HMPCs published as a Community Herbal Monograph, the industries in general have still more possibilities in change. In contrast, entries in the Community List are legally binding to applicants and competent authorities.

ACTIVE PRINCIPLES, PHARMACOLOGICAL EFFECTS, AND STANDARDIZATION The constituents of Echinacea, as in any other plant, cover a wide range of polarity, ranging from the polar polysaccharides and glycoproteins, via the moderately polar caffeic acid derivatives, to the rather lipophilic polyacetylenes 226

Echinacea Species

Figure 1

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Echinacea purpurea.

and alkamides. This makes it necessary to study separately the activity of different polar extracts of Echinacea, such as aqueous preparations, alcoholic tinctures, and hexane or chloroform extracts.

Polysaccharides and Glycoproteins Systematic fractionation and subsequent pharmacological testing of the aqueous extracts of the aerial parts of E. purpurea led to the isolation of two polysaccharides (PS I and PS II) with immunostimulatory properties. They were shown to stimulate phagocytosis in vitro and in vivo, and enhance the production of oxygen radicals by macrophages in a dose-dependent way. Structural analysis showed PS I to be a 4-O-methylglucuronoarabinoxylan with an average MW of 35,000

Figure 2

Echinacea pallida.

Figure 3

Echinacea angustifolia.

Da, while PS II was demonstrated to be an acidic arabinorhamnogalactan of MW 45,000 Da. A xyloglucan, MW 79,500 Da, was isolated from the leaves and stems of E. purpurea (17), and highly water-soluble fructans have been recently isolated from E. purpurea (L.) Moench roots (18). Polysaccharides from E. angustifolia have also been found to possess anti-inflammatory activity (19). In a Phase-I clinical trial, a polysaccharide fraction (EPO VIIa), isolated from E. purpurea tissue culture and injected at doses of 1 and 5 mg, caused an increase in the number of leukocytes, segmented granulocytes, and tumor necrosis factor-alpha (TNF-␣) (20). Three glycoproteins, MW 17,000, 21,000, and 30,000 Da, have been isolated from E. angustifolia and E. purpurea roots. The dominant sugars were found to be arabinose (64–84%), galactose (1.9–5.3%), and glucosamines (6%). The protein moiety contained high amounts of aspartate, glycine, glutamate, and alanine (21). An enzyme-linked immunosorbent assay (ELISA) method has been developed for the detection and determination of these glycoproteins in Echinacea preparations (22). In 2005, an arabinogalactan protein (AGP) and an arabinan were isolated from the roots of E. pallida (Nutt.) Nutt (23). Most AGPs contain mostly carbohydrate moieties (>90%) along with a small amount of protein (58%) as the glucuronidated form. Treatment of rats with a green tea polyphenol preparation (0.6% wt/vol) in the drinking fluid has been shown to result in increasing plasma levels over a 14-day period with levels of EGC and EC being higher than those of

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EGCG (60). Plasma levels then decrease over the subsequent 14 days suggesting an adaptive effect. EGCG levels have been found to be highest in the rat esophagus, intestine, and colon, which have direct contact with tea catechins, whereas EGCG levels are lower in the bladder, kidney, lung, and prostate, which depend on systemic bioavailable EGCG. When the same polyphenol preparation is given to mice, the EGCG levels in the plasma, lung, and liver are much higher than in rats (60). These levels appear to peak on day 4 and then decrease to less than 20% of the peak values in days 8–10. In mice, the absolute bioavailability of EGCG has been found to be higher than in rats (26.5% vs. 0.1%) (61). This may be one of the reasons that inhibitory effects of green tea and EGCG on tumorigenesis in mice, but not in rats, have been consistently observed in different laboratories. Concentrations of EGCG in the small intestine and colon are 45 and 7.9 nmol/g following IG administration of 75 mg/kg EGCG (61). The levels in other tissues are less than 0.1 nmol/g. Following IV administration of EGCG, levels are highest in the liver (3.6 nmol/g), lung (2.7 nmol/g), and small intestine (2.4 nmol/g). Greater than 50% of plasma EGCG is present as the glucuronide, whereas EGCG is present mainly as the free form in the tissues (61). The dose-dependent study of EGCG plasma and tissue levels in mice (50–2000 mg/kg, IG) indicated that there was a linear dose relationship in the plasma, prostate, and liver but not in the small intestine and colon, which showed a plateau between 500 and 2000 mg/kg, IG (62). These results may suggest that absorption of EGCG from the small intestine is largely via passive diffusion; however, at high concentrations, the small intestine and colonic tissues become saturated.

POTENTIAL HEPATOTOXICITY OF GREEN TEA POLYPHENOLS There have been 34 case studies linking consumption of green tea–based supplements to hepatotoxicity (reviewed in Ref. 63). In most cases, elevations in serum transaminase levels, as well as increased serum bilirubin, were observed. Histological examination revealed inflammatory, cholestatic, or necrotic liver damage depending on the subject. In a subset of cases, additional liver damage following rechallenge with the same preparation was observed, suggesting a causal relationship between hepatotoxicity and green tea. Laboratory studies of green tea–derived preparations in rodents and dogs have generally supported the potential toxicity of those preparations at high doses (64,65). Oral administration of a green tea extract containing 91.8% EGCG for 13 weeks to Beagle dogs resulted in dose-dependent toxicity and death (65). Toxicity included vomiting, diarrhea, proximal tubule necrosis, and elevated serum bilirubin. Oral administration of this extract to rats resulted in lethality in 80% of animals treated (65). Histological analysis revealed hemorrhagic lesions in the stomach and intestine. Intraperitoneal administration of EGCG to CD-1 mice resulted in dose-dependent increases in alanine aminotransferase and lethality beginning at 0.33 mmol/kg (64). These findings suggest that caution should be exercised in the

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use of green tea–based dietary supplements and that further studies are needed to determine the upper limit of safety for bolus dosing with tea polyphenols as well as the underlying mechanisms of toxicity.

CONCLUSIONS Despite the demonstration of cancer prevention by tea in many animal studies, epidemiological trials have yielded mixed results concerning its effectiveness as a cancer chemopreventive agent in humans. This may be due to several factors: (i) the dose of the chemopreventive agent is generally higher in animal studies than is typically consumed by humans; (ii) the model of carcinogenesis, especially certain chemical carcinogens, may not be relevant to human carcinogenesis; (iii) interindividual variation in metabolism of tea constituents as well as other confounding factors may mask the effects of tea consumption on cancer. A clearer understanding of the bioavailability of tea polyphenols may resolve some of these confounding factors. These same limitations apply to the current knowledge of the beneficial effects of tea against other chronic diseases. Definitive conclusions on the effectiveness of tea as a preventive agent for chronic human disease will require well-designed intervention and prospective epidemiological studies.

ACKNOWLEDGMENTS This work was supported by NIH grants CA121390 (to SS), AT004678 (to JDL), CA120915, CA122474, and CA133021 (to CSY).

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Hawthorn Egon Koch, Werner R. Busse, Wiltrud Juretzek, and Vitali Chevts

INTRODUCTION

ration should contain not less than 0.6% C-glycosylated flavones, expressed as vitexin (C21 H20 O12 ), and not less than 0.45% O-glycosylated flavones, expressed as hyperoside (C21 H20 O12 ), calculated on a dry basis (United States Pharmacopoeia–National Formulary, USP-NF).

Hawthorn is one of the most popular herbal medicines for the heart worldwide. In Europe, particularly in Germany, Austria, and Switzerland, hawthorn preparations are eligible for marketing authorization as drugs for the treatment of mild forms of heart insufficiency. In the United States, hawthorn products are regulated as dietary supplements. Numerous preparations are available, including teas, homeopathic preparations, and tinctures as well as simple and standardized extracts. Different starting materials are used. Extracts are prepared from leaves, flowers, or fruits. A substantial number of hawthorn products in the United States consist of comminuted hawthorn leaves, flowers, or fruits. Over the last decades, the pharmacodynamic action of standardized extracts from leaves and flowers as well as different fractions thereof has been extensively evaluated in in vitro studies and animal experiments. Similarly, the clinical efficacy of hawthorn extracts has been assessed in more than a dozen double-blind, placebo-controlled clinical trials. The results of these studies are reviewed in this entry.

Fruits (Berries) Hawthorn berries consist of the dried false fruits of C. monogyna Jacq. (Lindm.) or C. laevigata (Poir.) DC., or their hybrids, or a mixture of these (EP). They should contain not less than 1.0% procyanidins, calculated as cyanidin chloride (C12 H11 ClO6 ; Mr 322.7) with reference to the dried product (EP).

Constituents of the Starting Material Leaves and Flowers The major constituents are flavonoids (up to 2%) such as vitexin-2 -O-␣-L-rhamnoside, hyperoside, rutin, and vitexin as well as procyanidins formed by the condensation of catechin and/or epicatechin with varying degrees of polymerization (Fig. 1). The most important are oligomeric procyanidins (OPCs) containing 2–8 monomeric units, for example, the dimeric procyanidin B2 (Fig. 2). The content of OPCs is approximately 3% (1). Further constituents are triterpenoid acids (approximately 0.6%), for example, ursolic, oleanolic, and crataegolic acid and phenol carboxylic acids such as chlorogenic and caffeic acid, as well as various amines (2).

GENERAL DESCRIPTION Plant Description Hawthorn species (Crataegus L.; family Rosaceae) grow as shrubs or trees with hardwood and generally thorny twigs throughout the temperate zones of the world. Leaves are more or less lobed, with margins typically slightly serrated. Flowers are arranged in clusters and are mostly white and sometimes red. Small false fruits (berries) are formed and are red, black, or yellow and mealy.

Fruits (Berries) The fruits contain relatively low levels of flavonoids. The procyanidins contained in the fruits reportedly have a higher degree of polymerization than those in the leaves and flowers. Total procyanidins amount up to 3% of which about 1.9% is OPCs. Triterpenoid acids are also present in the fruit (approximately 0.45%) (3).

Starting Material The starting material consists of collected wild plant parts.

Preparations

Leaves and Flowers

Hawthorn extracts from leaves, flowers, and fruits are characterized by different quantitative flavonoid patterns (4).

Hawthorn leaves and flowers consist of the whole or cut, dried, flower-bearing branches of C. monogyna Jacq. (Lindm.), C. laevigata (Poir.) DC. (C. oxyacanthoides Thuill.), or their hybrids or, more rarely, other European Crataegus species, including C. pentagyna Waldst. et Kit ex Willd., C. nigra Waldst. et Kit, and C. azarolus L. The preparation should contain not less than 1.5% flavonoids, calculated as hyperoside (C21 H20 O12 ; Mr 464.4), with reference to the dried substance (European Pharmacopoeia, EP). The United States Pharmacopoeia (USP) only recognizes the first two species. According to the USP, the prepa-

Leaves and Flowers Extracts are produced from the herbal product by a suitable procedure using either water or a hydroalcoholic solvent equivalent in strength to a minimum of 45% ethanol. Aqueous extracts contain a minimum of 2.5% flavonoids and hydroalcoholic extracts contain a minimum of 6.0% flavonoids expressed as hyperoside (dried extracts) (EP). 411

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Fruits (Berries)

(A) OH HO

OH OH

O

HO

O

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Extracts are produced either from dried fruits, in compliance with the European Pharmacopoeia using alcohol (25–60%, vol/vol), or from fresh fruits. To date, no official monograph is available advocating the use of preparations from hawthorn fruits. Water extracts, water–alcohol extracts, wine infusions, and fresh juice from hawthorn fruits have been utilized traditionally to strengthen and invigorate heart and circulatory function (6).

O

(B)

ACTION AND PHARMACOLOGY OH O

HO

OH O

OH

O OH

O HO

OH OH

(C) OH O

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Pharmacological investigations with Crataegus preparations have been reported in a great number of publications. Unfortunately, many of these studies have been performed with insufficiently characterized extracts: Information on plant species, plant parts, and solvent and production conditions, for example, is not provided. Moreover, the applied pharmacological models and experimental details are frequently ill defined. Thus, formation of a clear judgment on the pharmacological activities of many of these products is not possible. Almost all clinical studies reported until now have been performed with two different extracts, prepared from leaves and flowers of selected Crataegus species with either 70% methanol (LI 132) or 45% ethanol (WS 1442). The present entry will mainly review the pharmacological actions of these well-defined preparations.

OH

Figure 1 (A) Vitexin [␤-D-glucopyranosyl-5,7-dihydroxy-2-(4hydroxyphenyl)-4H-1-benzopyran-4-one]; (B) hyperoside [2-(3,4dihydroxyphenyl)-3-(␤-D-galactopyranosyloxy)-5,7-dihydroxy-4H-1benzopyran-4-one]; (C) L-epicatechin [(2R-cis)-2-(3,4-dihydroxyphenyl)3,4-dihydro-2H-1-benzopyran-3,5,7-triol].

Standardized dry extracts are adjusted to 18.75% R OPCs (WS 1442; extraction solvent 45% ethanol) or 2.2% flavonoids (LI 132; extraction solvent 70% methanol), with a ratio of starting material to genuine extract (DER) of 4–7:1. The daily recommended dose is currently set at 160–900 mg in two or three divided doses (1,5).

OH O

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OH OH OH

OH

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Figure 2

Procyanidin B2 .

Positive Inotropic Action At concentrations between 30 and 180 ␮g/mL, LI 132 was found to raise the contraction amplitude of isolated cardiomyocytes of rats by up to 53% (7) and to improve oxygen utilization in comparison to ␤-adrenergic agonists or the cardiac glycoside ouabain. It is supposed that the inotropic action of LI 132 may be due to enhanced intracellular Ca2+ sensitivity. An increase of contraction amplitude was also observed in electrically stimulated canine papillary muscles (8). In isolated, electrically stimulated left ventricular muscle strips of human failing myocardium, WS 1442 significantly augmented force of contraction by about 30% (50 ␮g/mL) and improved the frequency-dependent force generation (9). In normal human myocardial tissue, WS 1442 raised the Ca2+ gradient as well as the force generation and displaced bound 3 H-ouabain from cell membranes. As the extract did not influence the activity of adenylate cyclase, the pharmacological mechanism of WS 1442 is suggested to be similar to the cAMP-independent positive inotropic action of cardiac glycosides. However, this conclusion is weakened by the fact that an extract fraction enriched for water-soluble, low-molecular-weight constituents displaced 3 H-ouabain but did not elicit any inotropic effect. Likewise, a significant dose-dependent effect of WS 1442 on the shortening of isolated and electrically stimulated myocytes isolated from right atria and left ventricles (LVs) of failing human hearts has been reported (10). Using an isolated guinea pig heart preparation, two independent research groups (11,12) observed a maximal

Hawthorn

increase of contraction force between about 10% and 20% at concentrations of 10 ␮g/mL LI 132.

Increase of Coronary Flow and Vasorelaxing Effects An increase of coronary flow has repeatedly been reported after perfusion of isolated hearts with mediumcontaining, ill-defined hawthorn extracts. These earlier observations were confirmed by a comprehensive study investigating the influence of LI 132 on different functional parameters in isolated guinea pig hearts. At a concentration of 3 ␮g/mL, LI 132 maximally enhanced coronary flow by 64%. A similar effect was brought about by amrinone and milrinone, while epinephrine had only a marginal effect, and digoxin concentration-dependently reduced coronary perfusion (11). Addition of the nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine (L-NNA) and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) completely abolished the increase in coronary flow induced by WS 1442 in the isolated rat heart, while addition of indomethacin (a cyclooxygenase inhibitor) or aminoguanidine (a selective inhibitor of inducible NOS) had no effect on the coronary flow. Furthermore, in the presence and absence of L-NNA, WS 1442 significantly enhanced the relaxant effect of the NO donor nitroprusside. Thus, it has been concluded that Crataegus extracts increase the endothelial release of NO and may also inhibit NO metabolism possibly due to its antioxidative properties (13). In a recent investigation, an endothelium-dependent vasorelaxing effect of WS 1442 was confirmed using rat aorta and human mammary artery. This effect was mainly mediated by the OPC fraction. It was found that WS 1442 induced an enhanced NO liberation from human coronary artery endothelial cells following activation of endothelial NO synthase (eNOS) by phosphorylation at serine 1177, while no eNOS translocation or phosphorylation at serine 114 or threonine 495 was observed (14). Further studies on the molecular mechanism(s) involved in the vasorelaxing action of WS 1442 were performed by Anselm and coworkers (15). Vascular reactivity was assessed in porcine coronary artery rings, whereas reactive oxygen species (ROS) formation in artery sections was judged by microscopy, and phosphorylation of Akt and eNOS in endothelial cells was determined by Western blot analysis. The effect of hawthorn on endothelium-dependent relaxation was reduced by L-NNA and by charybdotoxin plus apamin (two inhibitors of endothelium-derived hyperpolarizing factormediated responses). Relaxation to WS 1442 was also inhibited by intracellular ROS scavengers and inhibitors of Src and PI3-kinase but not by an estrogen-receptor antagonist. Thus, the authors conclude that WS 1442 stimulates the endothelial formation of ROS in artery sections and subsequently induces endothelium-dependent, NOmediated relaxations of coronary artery rings through the redox-sensitive Src/PI3-kinase/Akt-dependent phosphorylation of eNOS. However, based on these investigations, endothelium-derived hyperpolarizing factor, besides release of NO, also appears to contribute to the vasorelaxing activity of WS 1442. Endothelium-dependent, NO-mediated relaxation has also been observed by an extract from hawthorn fruits

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in rat mesenteric arteries (16) and a procyanidin-enriched fraction in the rat aorta (17). Therefore, relaxation of the noradrenalin-precontracted rat aorta has been proposed as a bioassay to investigate the pharmacological equivalence of different hawthorn extracts (18). In unanesthetized dogs, the effect of oral (PO) treatment with WS 1442 on local blood flow in the myocardium of the LV was measured by means of chronically implanted heat-conduction probes. WS 1442 led to a dosedependent temporary rise in blood flow, and repeated application caused a sustained increase of basal blood flow (19). In a pilot study, the effect of LI 132 on the microcirculation in the mesenteric vessels of rats was compared with those of ␤-acetyldigoxin by intravital microscopy. Compared with digitalis, the Crataegus extract improved the erythrocyte flow rate in all investigated vessel types and reduced both leukocyte adhesion to the endothelium and leukocyte diapedesis (20).

Antiarrhythmic Effects Evidence for an antiarrhythmic potential of LI 132 was provided by Poepping et al. (7), who observed a prolongation of the refractory period in isolated rat cardiac myocytes. Similarly, in isolated guinea pig hearts, an increase in left ventricular pressure and coronary flow was obtained, while at the same time the duration of the refractory period was prolonged (11). This combination of effects was unique among inotropic drugs, as epinephrine, amrinone, milrinone, and digoxin shortened the effective refractory period in a concentration-dependent manner. Using cultured unpaced neonatal murine cardiomyocyte, several hawthorn preparations were found to have negative chronotropic effects (21). As compared with conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extracts had a unique activity profile. They appeared to be antiarrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extracts did not cause ␤-adrenergicreceptor blockade at concentrations that caused negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries, had similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple-cardiac activities and revealed that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging. In guinea pig papillary muscles, LI 132 was observed to significantly increase action potential duration and time required for recovery of the maximum upstroke velocity of the action potential. These effects indicate class III and class I antiarrhythmic effects, respectively (12). Using patch-clamp techniques, the researchers obtained evidence that the prolongation of the action potential duration in isolated guinea pig ventricular myocytes is due to a weak blockade of both delayed and inward rectifier potassium currents (12). These investigators also attempted to get information on the mechanism responsible for the positive inotropic action of LI 132. As no influence on the L-type calcium current was detected, an inhibition of

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phosphodiesterase or a ␤-sympathomimetic action, which had previously been proposed to account for the cardiotonic action of hawthorn extracts, can be excluded. In vivo, antiarrhythmic effects of Crataegus extract WS 1442 were investigated in a rat model of ischemia/ reperfusion-induced arrhythmia. Oral treatment for 7 days (100 mg/kg/day) effectively protected animals from reperfusion-induced arrhythmias, mortality, and hypotensive crisis following 7 minutes of occlusion of the left coronary artery. Treatment with the extract, however, did not modify the elevated plasma creatine kinase concentrations during reperfusion (22). The influence of intake of a diet containing 2% LI 132 for 3 months on the incidence of reperfusion arrhythmias was also studied ex vivo in isolated rat hearts after global no-flow ischemia. Depending on the duration of ischemia, the average prevalence of malignant arrhythmias was significantly reduced by up to 83% in hearts of treated animals (23). However, in a recent publication, no protection against reperfusion-induced arrhythmias in isolated hearts was reported after 8 weeks’ treatment (0.5 g/kg/day) of Wistar rats with LI 132 (24).

Cardioprotective Effects Besides protecting against arrhythmias, hawthorn extracts have also been shown to prevent the leakage of intracellular enzymes upon ischemic injury. For this investigation, male Wistar rats were fed for 3 months with a diet containing 2% LI 132. As an index of myocardial cell damage, the concentration of lactate dehydrogenase (LDH) was determined in the perfusate of the isolated heart. LDH activity increased slightly during occlusion of the left coronary artery and was elevated dramatically after reperfusion. However, in treated animals, LDH release was suppressed significantly (control: 3795 ± 512 mU/min; LI 132: 1777 ± 452 mU/min) (25). Fractionation of WS 1442 established that its cardioprotective effect is almost exclusively due to its standardized content of 18.75% OPC. A subfraction of WS 1442 enriched for OPCs was found to exert potent antioxidative action and to inhibit the enzymatic activity of neutrophil elastase (26). Since restoration of blood flow into a previously ischemic tissue is associated with the formation of oxygen free radicals as well as the accumulation and activation of leukocytes, it has been suggested that these activities may contribute to protection against reperfusion injury. As ischemia lasting for more than 20–30 minutes causes irreversible tissue damage and cell death, Veveris et al. (2004) (27) evaluated whether treatment of rats with WS 1442 also improves cardiac function and prevents myocardial infarction during prolonged ischemia and reperfusion lasting for 240 and 15 minutes, respectively. Oral administration of WS 1442 (10 or 100 mg/kg/day) for 7 days before ligation of the left coronary artery dosedependently suppressed the decrease of the pressure rate product. Treatment also attenuated the elevation of the ST segment in the ECG, diminished the incidence of ventricular fibrillations, and reduced the mortality rate. Furthermore, the area of myocardial infarction within the ischemic zone was significantly smaller in treated rats when compared with controls. It is suggested that these

pharmacological effects are accounted for by the combined antioxidative, leukocyte elastase-inhibiting, and endothelial NO synthesis-enhancing properties of WS 1442. A protection against ischemia/reperfusion-induced brain damage was also described after administration of Crataegus flavonoids in a Mongolian gerbil stroke model (28). Likewise, an alcoholic extract of Crataegus oxycantha was shown to preserve mitochondrial function during isoproterenol-induced myocardial infarction in rats (29). Cardiac hypertrophy (CH) is an adaptive enlargement of the myocardium in response to diverse pathophysiological stimuli such as hypertension, valvular disease, or myocardial infarction. Although this process is generally a beneficial response that temporarily augments cardiac output, sustained hypertrophy often becomes maladaptive and is a leading cause for the development of heart insufficiency. Activation of the protein phosphatase calcineurin (PP2B) is discussed as a major intracellular signaling pathway that contributes to the growth of cardiomyocytes. Using an in vitro test system, it was observed that WS 1442 inhibits the enzymatic activity of calcineurin. Thus, the effect of WS 1442 on the development of CH in animal models of hypertension was investigated. Hypertension and subsequent CH were induced in rats by aortic constriction (AC) or administration of deoxycorticosterone acetate (DOCA) in combination with NaCl/KCl-substituted drinking water, respectively. Animals were treated orally for a period of 14 (AC) or 28 days (DOCA-salt) with vehicle (0.2% agar suspension) or WS 1442 (100 and 300 mg/kg/day). In both experimental models, a marked increase in blood pressure (BP) and enlargement of the heart and the LV were observed. Treatment with WS 1442 dose-dependently lowered the pathologically increased BP but had no effect on the BP in normal control animals. In parallel with the reduction in the BP, development of CH was inhibited. This study demonstrates that oral treatment of rats with WS 1442 prevents development of CH induced by primary or secondary hypertension and thus supports its therapeutic use in the treatment of mild forms of heart failure (30). The effects of WS 1442 treatment on remodeling and function of the LV was observed after 1 month of pressure overload–induced CH in male Sprague–Dawley rats (31). Animals were subjected to sham operation or AC for 4 weeks and treated orally with WS 1442 (1.3, 13, and 130 mg/kg/day) for 3 weeks after surgery. AC increased the LV/body weight ratio by 34% in vehicle- and WS 1442treated rats, but WS 1442 markedly reduced LV chamber volumes and augmented relative wall thickness. In addition, WS 1442 attenuated the AC-induced reduction of velocity of circumferential shortening. The authors conclude that WS 1442 treatment modifies left ventricular remodeling and counteracts myocardial dysfunction in early pressure overload–induced CH. In a subsequent study, the effect of WS 1442 (1.3, 13, or 130 mg/kg, PO) on left ventricular remodeling and function in pressure overload–induced heart failure was observed over a period of up to 5 months (32). AC increased the LV/body weight ratio by 53% in vehicletreated rats, and administration of WS 1442 did not significantly affect this ratio. LV volumes and dimensions at systole and diastole significantly increased 5 months after AC compared with baseline in rats given vehicle (>20%

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increase) but not in those given WS 1442 at 130 mg/kg (6.4 mg lycopene/day) was compared with the lowest quartile (< 2.3 mg lycopene/day, RR = 0.79, 95% CI = 0.64–0.99, P = 0.04 for trend). A few years later, a case-control study of 797 men in New Zealand found a weak, nonsignificant trend between lycopene intake and prostate cancer incidence when comparing the lowest quartile (1994 ␮g/day) [OR = 0.76, 95% CI = 0.53–1.26, P = 0.30 for trend; (90)]. Interestingly, in this study, the estimated median intake of lycopene was less than half of the median in the HPFS cohort (1.2 vs. 3.4–4.6 mg lycopene/day, respectively). Recently, updated data from the HPFS for the period from 1992 through 1998 confirmed the earlier findings. Lycopene intake was associated with a decreased risk for prostate cancer [RR = 0.84 for high compared to low quintiles; 95% CI = 0.73–0.96; P = 0.003 for trend; (96)]. Several reports have investigated the relationship between blood concentrations of lycopene and prostate cancer risk. In a study conducted at the Memorial SloanKettering Cancer Center from 1993 to 1997, Lu et al. (88) showed that when plasma carotenoid levels from men in the highest and lowest quartiles were compared, inverse associations for prostate cancer risk were statistically significant for plasma lycopene, zeaxanthin, lutein, and ␤-cryptoxanthin levels. Hsing et al. (97) evaluated serum obtained in 1974 from 25,802 persons in Washington County, Maryland, and reported lower mean serum lycopene concentrations in prostate cancer cases compared with controls. A 50% reduction in the relative risk for prostate cancer was observed when cases in the highest serum lycopene quartile were compared

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to more in the lowest quartile. A study conducted by Nomura et al. (98) in a cohort of 6860 Japanese–American men examined from 1971 to 1975, however, showed no association between several plasma micronutrients and carotenoids, and prostate cancer risk. A nested casecontrol investigation was undertaken, which involved the analysis of carotenoids in blood samples from men enrolled in the Physicians’ Health Study (a randomized, placebo-controlled trial of aspirin and ␤-carotene). In this study, subjects in the highest quintile (>580.1 ng/mL) of serum lycopene levels had a significantly lower risk of prostate cancer compared with those in the lowest quintile (≤261.7 ng/mL, OR = 0.56, 95% CI = 0.34–0.92, P = 0.05). The inverse association between serum lycopene and aggressive prostate cancer was particularly significant for men who were not consuming ␤-carotene supplements [OR = 0.40 for highest quintile vs. lowest quintile, 95% CI = 0.19–0.84, P = 0.006 for trend; (99)]. Several laboratories have conducted studies on lycopene and prostate carcinogenesis in rodents. An investigation using the DMAB and PhIP-induced rat prostate cancer models failed to detect a chemopreventive effect of lycopene provided as an extract of 99.9% purity from LycoRed (100). In another study, two different doses of a lycopene-rich tomato oleoresin were fed to lacZ mice to study the effects on short-term benzo[a]pyrene (BaP)induced and long-term spontaneous in vivo mutagenesis in the colon, prostate, and lungs (101). Spontaneous mutagenesis was inhibited in prostate and colon tissue at the higher dose of tomato oleoresin. In addition, BaP-induced mutagenesis in the prostate was also slightly inhibited in mice fed with tomato oleoresin. Boileau et al. (102) completed a large rat study evaluating the ability of lycopene or freeze-dried tomato powder to inhibit survival in the N-nitrosomethylurea-androgen-induced prostate cancer model. In this system, a very small beneficial trend for lycopene and a significant benefit of tomato powder were reported, suggesting that tomato consumption may provide additional benefits. There are few human intervention studies investigating the role of lycopene on processes that are related to the development of prostate cancer. The most provocative observations have been published by Kucuk et al. (76). The study involved 26 men diagnosed with presumed localized prostate cancer who were scheduled to undergo a radical prostatectomy. The subjects were randomized to consume 30 mg of lycopene/day from two tomato oleoresin capsules (Lyc-O-Mato; LycoRed Natural Products Industries) or to continue their normal diet for 3 weeks prior to surgery. Postsurgical prostate tissue specimens were then compared between the two groups. Men consuming the lycopene supplement had 47% higher prostatic tissue lycopene levels than the control group (0.53 ± 0.03 ng/g vs. 0.36 ± 0.06 ng/g, P = 0.02). However, plasma lycopene levels were not significantly different between the groups and they did not change significantly within each group. Those who consumed the lycopene supplement were less likely to have involvement of surgical margins (73% vs. 18% of subjects, P = 0.02). In addition, they were less frequently found to have highgrade prostatic intraepithelial neoplasia in the prostatectomy specimen (67% vs. 100%, P = 0.05). Furthermore, the

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intervention group was found to have smaller tumors, a greater reduction in prostate-specific antigen (PSA) over the 3-week study period, and a higher expression of connexin 43. However, none of these differences were statistically significant. A case-control study by Chen et al. (140) investigated the effects of daily tomato sauce consumption (30 mg/day lycopene) for 3 weeks in 32 prostate cancer patients before a radical prostatectomy. Analysis of the prostate tissue after surgery revealed a decrease in DNA damage in the treatment group as compared to controls, as measured by the ratio of 8-hydroxy-2 -deoxyguanosine (a marker of oxidative DNA damage) to 2 -deoxyguanosine A ratio = 0.76 was observed in the treatment group, as compared to 1.06 in the controls (P = 0.03) (140). In addition, a 20% decrease in serum PSA levels was observed in the treatment group (P < 0.001). Another recent study examined the effects of tomato and tomato product consumption with and without soy protein isolate in men (n = 41) with recurrent, asymptomatic prostate cancer (141). Subjects were split into two groups, with one group asked to consume tomato-based products for the entire 8-week study and the other group asked to consume tomato-based products for the final 4 weeks of the study. Subjects were asked to consume enough tomatoes and tomato-based products to meet a target of at least 25 mg/day lycopene. On the completion of the study, serum PSA levels were reduced in 34% of the subjects (141).

CARDIOVASCULAR DISEASE Epidemiologic studies investigating the relationship between lycopene exposure and the risk for vascular diseases are beginning to emerge. Consumption of tomatoes and tomato-based products has been associated with a reduced risk of cardiovascular disease. In one study of 38,445 women, evaluation of highest and lowest quintiles of intake revealed that higher levels of tomato-based product intake were associated with a reduced risk of cardiovascular disease (RR = 0.71, Ptrend = 0.029) and myocardial infarction (RR = 0.39, Ptrend = 0.033) (7). Tissue and serum concentrations of lycopene have also been found to be correlated with a reduced risk for coronary heart disease (CHD) in several case-control studies. A multicenter case-control study was conducted to evaluate the relationship between adipose tissue concentration of antioxidants (i.e., ␣- and ␤-carotene and lycopene) and acute myocardial infarction (103). Cases and control subjects were recruited from 10 European countries to ensure maximum variability in exposure. Upon simultaneous analyses of the carotenoids and adjustment for other variables, lycopene was the only carotenoid associated with protection against acute myocardial infarction (OR = 0.52 when the 10th and 90th percentiles were compared, 95% CI = 0.33–0.82, P = 0.005 for trend). Similarly, lower serum lycopene concentrations were found to be related to an increased risk of and mortality from cardiovascular disease (CVD) in a concomitant cross-sectional study evaluating Swedish and Lithuanian populations displaying diverging mortality rates from CHD (n = 210) (104). Klipstein-Grobusch et al. (105) investigated the

relationship between serum concentrations of the major carotenoids (i.e., ␣-carotene, ␤-carotene, ␤-cryptoxanthin, lutein, lycopene, and zeaxanthin) and aortic atherosclerosis as determined by the presence of calcified plaques of the abdominal aorta. A subsample of the elderly population of the Rotterdam Study consisting of 108 subjects with aortic atherosclerosis and controls was used for the case-control analysis. A 45% reduction (OR = 0.55, 95% CI = 0.25–1.22, P = 0.13 for trend) in the risk of atherosclerosis was observed for the highest versus the lowest quartile of serum lycopene. When adjustments for smoking status were made, the inverse association was greatest for current and former smokers (OR = 0.35, 95% CI = 0.13–0.94, P = 0.04 for trend). No associations were observed with any of the other serum carotenoids studied. A report on men (aged 46–64 years; n = 725) from the Kuopio Ischaemic Heart Disease Risk Factor Study indicated that those in the lowest serum lycopene quartile had a 3.3-fold (95% CI = 1.7–6.4; P < 0.001) increased risk of acute coronary events or stroke when compared to the others (106). In addition, subjects in the lowest quartile of serum lycopene had a significant increment in both mean intima-media thickness of common carotid artery wall (CCA-IMT) (P < 0.006 for difference) and maximal CCA-IMT (P = 0.002) as compared with others. In a cross-sectional analysis of 520 men and women from the Antioxidant Supplementation in Atherosclerosis Prevention Study, low plasma lycopene levels were associated with an 18% increase in IMT in men when compared with those with plasma lycopene levels higher than the median [P = 0.003 for difference; (107)]. Short-term dietary intervention studies have also supported a relationship between tomato-based products and tomato extract supplements on positive improvements in lipid biomarkers and a reduction in biomarker oxidation (142–143).

SAFETY AND ADVERSE EFFECTS Safety assessment of phytochemicals from fruits and vegetables or supplements is necessary to ensure efficacy without toxicity in future trials. In rats, the consumption of 3 g lycopene/kg body weight/day for up to 13 weeks was reported to have no adverse physiologic effects or abnormalities in the animals (145). The safety of multiple acute doses of lycopene in humans was studied by DiwadkarNavsariwala et al. (108). A Phase I study in healthy male subjects, using a physiological pharmacokinetic model, was conducted to study the disposition of lycopene, administered as a tomato beverage in five graded doses (10, 30, 60, 90, or 120 mg). The subjects reported no signs of toxicity at any level of intake. However, long-term consumption of these doses was not evaluated. Consumption of extreme amounts of lycopene or lycopene-containing tomatoes and/or tomato-based products over an extended period of time can have adverse effects. La Placa, Pazzaglia, and Tosti (109) described a case study of a 19-year-old Italian girl who had consumed four to five large red tomatoes and pasta with tomato sauce daily for 3 years. She displayed yelloworange discoloration of the skin and abdominal pain.

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Upon investigation of the abdominal pain, a hepatic ultrasound revealed a digitate area that was relatively hypoechogenic, measuring 2 cm in diameter, in the upper portion of the parenchyma, consisting of deposits of lycopene. These clinical features and dietary history suggested the diagnosis of lycopenemia. Additional studies are required to assess the safety of varying levels of lycopene from multiple sources for long periods of time. These experiments will enable future scientists to identify the optimum combination of intake and time to maximize the benefits without adverse effects. Therefore, caution should be exercised when recommending sources and amounts of this carotenoid.

WHOLE FOODS VERSUS SUPPLEMENTS Although lycopene has received a great deal of attention as an important phytochemical from tomatoes and tomato-based foods, it is premature to suggest that lycopene alone is responsible for the reported beneficial health effects of these foods. As briefly discussed earlier, a study was conducted by Boileau et al. (102) to evaluate the effects of tomato powder or lycopene beadlet consumption on prostate carcinogenesis in N-methyl-Nnitrosourea (NMU) and testosterone-treated rats. This investigation showed that consumption of tomato powder but not lycopene alone inhibited prostate carcinogenesis, suggesting that tomato-based products contain compounds in addition to lycopene that modify prostate cancer development and/or progression. In addition, the study by Aust et al. (129) indicated greater photoprotection conferred from the consumption of a tomato extract or tomato beverage versus synthetic lycopene. Care should be taken not to make the assumption that all the health benefits brought about by fruit and vegetable consumption are attributed to a single component such as lycopene. Because tomato and tomato-based product consumption is the primary source of lycopene in the North American diet, other compounds in tomatoes and tomato-based products may be responsible for positive effects observed in epidemiological studies. When the Food and Drug Administration (FDA) reviewed a proposed health claim on tomatoes, lycopene, and cancer, they concluded that there is “no credible evidence to support an association between lycopene intake and a reduced risk of prostate, lung, colorectal, gastric, breast, ovarian, endometrial or pancreatic cancer,”(146). However, the FDA found, “very limited evidence to support an association between tomato consumption and reduced risks of prostate, ovarian, gastric, and pancreatic cancers” (146). The limited number of high-quality clinical studies in the literature was one of the main reasons cited in their decision. Additional studies are needed to determine the differences between lycopene supplementation and lycopene-containing diets on biologic processes related to chronic disease.

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Maca Ilias Muhammad, Jianping Zhao, and Ikhlas A. Khan

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Maca is a dietary supplement derived from the processed tuberous root of Lepidium meyenii Walpers (family Brassicaceae; common names: Peruvian ginseng, maka, mace, maca-maca, maino, ayak chichira, ayuk willku, pepperweed) (1). The genus Lepidium contains approximately 150 to 175 species (2). This plant was first described by Gerhard Walpers in 1843 and domesticated in the Andean mountain at altitudes from 3500 to 4450 m above sea level in the puna and suni ecosystems (3). It is arguably the highest altitude plant in cultivation. The genus probably originated in the Mediterranean region, where most of the diploid species are found (2,4); information about its origin and distribution are sketchy. Maca is an important staple for the Andean Indians and indigenous peoples and was domesticated during the pre-Inca Arcaica period sometime around 3800 B.C. It is the only species cultivated as a starch crop (5) and is rich in sugars, protein, starches, and essential minerals, especially iodine and iron. Based on a long history of traditional use of maca in Peru and elsewhere, a wide array of commercial maca products have gained popularity as dietary supplements throughout the world for aphrodisiac purposes and to increase fertility and stamina. Limited research has been carried out during the past two to three decades by academia and the industry, including isolation and identification of several potentially bioactive constituents, as well as evaluation of biological activities, mainly focused on its aphrodisiac and nutritional properties. Here, we present a comprehensive review of the published literatures on maca, which includes morphological descriptions, traditional uses, nutritional status, chemical constituents, biological activities, cosmetic uses, and standardization.

Figure 1 Dried tuberous root and above-ground parts of maca (Lepidium meyenii). (View this art in color at www.dekker.com.)

Cultivation Unlike many other tuberous plants, L. meyenii is propagated by seed, and 7 to 9 months is required to produce the harvested root. It is cultivated on rocky soil on rough Andean terrain under intense sunlight, high wind, and fluctuating temperatures between –20◦ C and 20◦ C. The soil used for cultivation is acidic clay or limestone with a relative humidity of approximately 70%, and the plant can grow without shade or in semishade. Maca is sown from September to October at the beginning of the rainy season, and harvesting starts from May to July after a vegetative phase of 260 to 280 days. The yield is variable, from 2 to 16 ton/ha depending on the cultivation practices, fertilization, and pest control. Well-formed hypocotyls are selected and transplanted to fertilized seed beds for seed production. After a 100- to 120-day generative phase, seeds are harvested (5). Maca seeds represent centuries of cumulative selection by indigenous farmers, but it is only recently that scientists and governments have been growing out, testing, and saving them. The plantation area for maca has expanded drastically because of the increased demand, both domestically and for export. In 1994, less than 50 ha

BACKGROUND Classification L. meyenii (Fig. 1) is a herb or subshrub belonging to the Brassicaceae family (4,5). Chacon recommended changing its name to L. peruvianum because herbarium specimens from Bolivia and Argentina were classified as L. meyenii but had no resemblance in shape to maca in many cases (6). It has been suggested that the cultivated maca of today is not L. meyenii but the new species, L. peruvianum. Although most maca sold in commerce is still referred to as L. meyenii, it is L. peruvianum.

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of maca was cultivated in Peru; by 1999, production had increased 24-fold to 1200 ha, and it now stands at more than 2000 ha.

Traditional and Medicinal Uses Maca was grown for food by the Pumpush, Yaros, and Ayarmaca Indians. Conquistadors fed the baked or boiled root powder to animals for fertility problems at high altitudes, and the Chinchaycochas Indians used it in bartering. Maca was also used to make beverages, to which hallucinogenic products were also added, which were consumed during dances and religious ceremonies. The tuberous root of maca is generally consumed fresh or dried and has a tangy taste and an aroma similar to butterscotch. Dried roots are brown, soft, and sweet, with a musky flavor, and retain their flavor for at least 2 years, and a 7-yearold root still has 9% to 10% protein. In South America, the sweet aromatic porridge of dried maca is consumed under the name mazamorra. In Huancayo, Peru, maca jam and pudding are popular, and maca is often made into a sweet, fragrant, fermented drink called maca chichi. According to folk belief, maca can enhance male sexual drive and female fertility in humans and domestic animals. The Spanish conquerors found “well-fed babies and tall adults” in the high Andes, which they attributed to a diet based on maca (3). It is also reputed to regulate hormonal secretion, stimulate metabolism, and improve memory, and is touted for antidepressant and anticancer properties, as well as for curing anemia, leukemia, and AIDS. However, these properties have not been substantiated by scientific research. Due to its wide spectrum of putative qualities, maca is also known as Peruvian ginseng (3). In Peruvian herbal medicine, it has been used as an immunostimulant, for anemia, tuberculosis, menstrual disorders, menopause symptoms, stomach cancer, and sterility, and for other reproductive and sexual disorders, as well as to enhance memory.

CHEMISTRY Nutritional Constituents Maca is very nutritious, with 60% to 75% carbohydrates, 10% to 14% protein, 8.5% fiber, and 2.2% lipids (7,8). The dried root contains approximately 13% to 16% protein and is rich in essential amino acids, whereas the fresh root is unusually high in iodine and iron. It contains approximately 250 mg of calcium, 2 g of potassium, and 15 mg of iron in 100 g of dried root, and sterols (0.05– 0.1%), minerals, and vitamins. Maca contains 3.72% fatty acids, including caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid (9). In addition, a new acyclic keto acid, 5-oxo-6E,8E-octadecadienoic acid [1] has been isolated from the tubers (10). Yellow maca has been found to have higher lipid and carbohydrate content compared to the red and black varieties (8). Evaluation of the nutritional property of maca in albino Swiss mice has shown that the serum values for content of total proteins and albumin are statistically higher for mice eating cooked maca than for those consuming raw maca, with no sign of malnutrition or overweight in any of the groups (11).

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Secondary Metabolites The major secondary metabolites present in L. meyenii can be classified into four groups: (a) essential oils; (b) glucosinolates; (c) alkaloids; and (d) macamides. In addition, the presence of malic acid and its benzoate ester [2] (12) as well as five sterols (7) and catechins, are also reported (Fig. 2)

Essential oils A total of 53 essential oil components have been identified, using retention indexes and mass spectral data (13) Among the constituents, phenylacetonitrile (85.9%), benzaldehyde (3.1%), and 3-methoxyphenylacetonitrile (2.1%) are the major components of the steam-distilled oil.

Glucosinolates The glucosinolates are a class of secondary metabolites found in 15 botanical families of dicotyledonous plants, notably including the Brassicaceae. Over 100 have been reported to date from plant sources. Glucosinolates are present at approximately 1% in fresh L. meyenii root, but no novel ones have been reported so far in maca. The presence of two main glucosinolates, glucotropeolin [3] and mmethoxybenzylglucosinolate [4], have been reported from maca (12,14,15), and their combined presence in L. meyenii may be used as a chemotaxonomic marker, because the combination of 3 and 4 does not occur in other members of the Brassicaceae (14). Glucosinolates and their derived products have received increasing attention due to their biological activities; examination of glucosinolate degradation products in the hexane extract has revealed the presence of benzyl isothiocyanate [5] and its m-methoxy derivative [6] (15). the former reported to be present in the range of 0.1% to 0.15% in standardized maca product (9). Several maca products derived from processed hypocotyls of L. peruvianum and other organs have been assessed by high-performance liquid chromatography (HPLC) for glucosinolate content. The most abundant glucosinolates were found to be 3 and 4 in fresh and dry hypocotyls and leaves. The richest sources of glucosinolates are seeds, fresh hypocotyls, and sprouts, in that order. Maca seeds and sprouts differ in profile from hypocotyls and leaves due to the presence of several modified benzylglucosinolates, including 5-methylsulfinylpentylglucosinolate [7], indolyl-3-methylglucosinolate [8], pent-4-enylglucosinolate, 4-methoxyindolyl-3-methylglucosinolate, glucolepigramin, and 4-hydroxybenzylglucosinolate, whereas the liquor and tonic contain sinigrin [9] (12). A HPLC method was reported for the quantification of benzyl isothiocyanate [5] released by the action of the thioglucosidase enzyme on the substrate [3], the predominant glucosinolate of maca hypocotyls (16).

Alkaloids Qualitative detection of alkaloid like compounds in L. meyenii was first reported by Dini et al. (7) and a further detailed chemical analysis of the tubers by Muhammad et al. (10) reported the benzylated derivative of 1,2-dihydroN-hydroxypyridine, named macaridine [10]. From the methanol extract of the tuber, (1R,3S)-1-methyltetrahydro␤-carboline-3-carboxylic acid [11], and uridine [12] and its

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benzoyl derivative have been isolated (15). Two new imidazole alkaloids, lepidiline A [13] and lepidiline B [14], were isolated from root extracts (17) (Fig. 2) and their 1,3dibenzylimidazolium chloride derivatives were patented for treating proliferative diseases (18).

Macamides Maca contains novel polyunsaturated acids and their amides, called macaene and macamide as reported by Zheng et al. (9). From purified standardized products of maca, three new macamides, N-benzyloctamide, N-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide, and N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide, have been isolated and identified by HPLC. (9) In addition, 17 other analog of macamide and macaene have been reported, but their chemical identity has not been disclosed. From maca tubers, seven additional

alkamides, N-benzyl-5-oxo-6E,8E-octadecadienamide [15], N-benzylhexadecanamide [16], N-benzyl-9-oxo-12Zoctadecenamide [17], N-benzyl-9-oxo-12Z,15Z-octadecadienamide [18], N-benzyl-13-oxo-9E,11E-octadecadienamide [19], N-benzyl-15Z-tetracosenamide [20], and N-(m-methoxybenzyl)hexadecanamide [21] have been isolated (10,19). In addition, N-benzylhexadecanamide, Nbenzyl-(9Z)-octadecenamide, N-benzyl-(9Z,12Z)-octadecadienamide, N-benzyl-(9Z,12Z,15Z)-octadecatrienamide and N-benzyloctadecanamide were identified by using HPLC–UV–MS/MS (20).

COMMERCIAL PREPARATIONS AND STANDARDIZATION A wide array of commercial products, including soft drinks, pills, and capsules, are currently processed and

Maca

CHO

H N NH

COOH

N OH

10

11 R

OH

HO O

N OH HN

N

+

+Cl– N

O

O

13 R=H 14 R=Me

12 O

O

O

N H

N H

(CH2)8

15 O

16 O

N H 17 O

O

N H 18 O

O

N H 19 O N H 20 O MeO N H 21

Figure 2

(Continued)

(CH2)14

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distributed by various companies throughout the world. These products are sold in markets and drug stores in South America, including Peru, and many of these are exported abroad. Today, maca is advertised as an aphrodisiac, stamina builder, and fertility promoter in the world market and is available for purchase through the World Wide Web as a dietary supplement. Ganzera et al. (21) have reported an analytical method for the determination of the main macamides and macaenes of L. meyenii. The analysis of several commercially available maca products reveals a similar qualitative pattern for macamides and macaenes, but significant differences in the quantitative composition. The purified standardized product of maca has been analyzed by HPLC (9), and three new macamides and 17 other analogs of macamide and macaene have been reported. Several products (such as pills, capsules, flour, liquor, tonic, and mayonnaise) derived from processed maca (L. peruvianum) have also been analyzed and profiled by HPLC for glucosinolate content (12). Total macamides have been quantified by HPLC–UV in plant material from different vendors using n-benzylhexadecanamide as an external standard. The amount of macamides in the dried plant material ranged from 0.0016% to 0.0123% (20). Through chromatographic techniques, ␤-sitosterol was identified and quantificated (22). Chen et al. investigated the polysaccharide in maca, and a colorimetric method was developed for its determination (23,24). FT-IR, TLC, and GC/MS techniques were employed for the identification of maca or maca products in the market (25).

PRECLINICAL STUDIES Fertility-Enhancing and Aphrodisiac Activities The aphrodisiac activities of maca have been reported by several research groups. Oral administration of the purified lipid extract decreased the latent period of erection in male rats with erectile dysfunction, as well as enhanced the sexual function of mice and rats by increasing the number of complete intromissions and the number of spermpositive females in normal mice (9). The effects of maca on mouse sex steroid hormones and embryo implantation were investigated. Progesterone and testosterone levels increased significantly in mice that were treated with maca. However, there were no marked changes in blood levels of 17␤-estradiol or the rate of embryo implantation (26). Bogani et al. conducted an investigation to test whether maca contains testosterone-like compounds, is able to bind the human androgen receptor, and can promote transcription pathways regulated by steroid hormone signaling. The results showed that the maca extracts (obtained with different solvents, such as methanol, ethanol, hexane and chloroform) were not able to regulate GRE (glucocorticoid response element) activation (27). The root of maca has been used to help alleviate the symptoms of menopause. The effect of ethanol extract of maca on osteoporosis in ovariectomized rat was studied. The findings derived from the basis of bone mineral density, biomechanical, biochemical and histopathological parameters indicated that higher dose of ethanol extract of maca was effective in the prevention of estrogen-deficient

bone loss (28). Maca has also been traditionally used to increase fertility. A study on the effects of maca on several fertility parameters of female mice at reproductive age showed that administration of aqueous extract of yellow maca to adult female mice could increase the litter size. Moreover, this treatment could increase the uterine weight in ovariectomized animals (29). Oral administration of an aqueous extract of maca roots resulted in an increase in the weights of the testis and epididymis, but not seminal vesicle weight, and the root invigorated spermatogenesis in male rats by acting on the initial portions of the seminiferous tubules, where mitosis occurs (30). To determine the acute and chronic effects of maca on male sexual behavior and to examine chronic administration of maca on anxiety, maca (25 and 100 mg/kg) was orally administered to male rats for 30 days. Ejaculatory and mounting behavior and postejaculatory interval were monitored. An elevated plus maze, locomotion, and social interaction with another male were used for anxiety tests. The investigation showed that maca treatment did not produce large changes in male sexual behavior. However, an increase in ejaculation latency and postejaculatory interval was observed after both acute and 7 days of treatment. After 21 days of treatment maca had no effect on sexual behavior. Chronic administration of maca did not increase locomotion or anxiety (31). Antagonistic effect of red maca (RM) on prostatic hyperplasia induced with testosterone enanthate (TE) in adult mice was investigated (32). Testosterone and oestradiol levels, as well as prostatic stroma, epithelium, and acini were measured. It was found that RM reduced prostate weight at 21 days of treatment. Weights of seminal vesicles, testis, and epididymis were not affected by RM treatment. Cicero et al. reported that the subacute oral administration of a lipophilic hexane extract improved sexual performance parameters most effectively in sexually inexperienced male rats (33,34). Effect of aqueous extract of maca on spermatogenesis in male rats was investigated to test the hypothesis that maca can prevent high altitude–induced testicular disturbances. The data showed that altitude reduced spermatiation (stage VIII) to half and the onset of spermatogenesis (stages IX–XI) to a quarter on days 7 and 14 but treatment with maca (666.6 mg/day) prevented these changes (35). The same group also investigated the effects of maca extracts on spermatogenesis in rodents, following spermatogenic damages induced by lead acetate and organophosphorous pesticide malathion (36,37). Assessment of the relative length of stages of the seminiferous epithelium showed that maca treatment resulted in rapid recovery of the effect of malathion. Administration of maca to rats treated with lead acetate resulted in higher lengths of stages VIII and IX–XI with respect to lead acetate–treated rats. Moreover, treatment with maca to lead acetate–treated rats resulted in lengths of stages VIII and IX–XI similar to the control group. Maca has different ecotypes described according to the color of its hypocotyls (38). Gonzales et al. studied the different biological properties among different varieties of maca. They reported that black maca presented the greatest effect on sperm production in male rats and

Maca

on latent learning in ovariectomized female mice in comparison with yellow and red (39–41); whereas red maca reduced ventral prostate size in normal and TE treated rats (42,43). However, the difference of active secondary metabolites present in different maca varieties is still unknown.

Cytotoxic and Chemopreventive Activities Glucosinolates appear to have little biological impact by themselves. However, release of biologically active products such as isothiocyanates, organic cyanides, oxazolidinethiones, and ionic thiocyanate (SCN− ) upon enzymatic degradation by myrosinase, which is typically present in cruciferous plants as well as in the gut microflora of mammals (44), is responsible for the observed activities. Natural isothiocyanates derived from glucosinolate are effective chemoprotective agents that detoxify carcinogens and prevent several types of cancer in rodent models. Isothiocyanates apparently induce mammalian Phase 1 and 2 drug-metabolizing enzymes and their coding genes, resulting in decreased carcinogen–DNA interactions (45). Benzyl isothiocyanates, most importantly, have been reported to be potent cancer inhibitors of mammary gland and stomach cancers (46) and of liver cancer (47) in rats treated with carcinogens. The abovementioned work suggests that the type of glucosinolate and total concentration have important implications with respect to overall biological activity, including chemoprevention, in both human and animal nutrition. However, no tests on the chemopreventive activity of maca itself have been reported so far. Valentova et al. studied the biological activity of methanolic and aqueous extracts of maca on rat hepatocytes and human breast cancer MCF-7 cells (48). Cytotoxicity in hepatocyte primary cultures was not observed up to 10 mg/mL of the extract concentration as measured by the MTT [3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazolium bromide] viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 hours, extracts inhibited LDH and AST leakage from the hepatocytes. Both methanolic and aqueous extracts showed estrogenic activity comparable with that of silymarin in MCF-7 cell line, but weak antioxidant activity in the ␣,␣-diphenyl-2picrylhydrazyl (DPPH)-radical scavenging test with IC50 values of 3.46 ± 0.16 and 0.71 ± 0.10 mg/mL, for aqueous and methanolic extracts, respectively.

Other Biological Activities Maca has the capacity to scavenge free radicals and protect cells from oxidative stress. The antioxidant activity of maca was assessed, and the IC50 values for scavenging DPPH and peroxy radicals were found to be 0.61 and 0.43 mg/mL, respectively (49). Deoxyribose protection by maca (1–3 mg/mL) against hydroxyl radicals was of the order of 57% to 74%. Maca (1 mg/mL) protected RAW 264.7 cells against peroxynitrite-induced apoptosis and increased ATP production in cells treated with H2 O2 (1 mM). The oil of L. meyenii was selectively toxic toward the cyanobacterium Oscillatoria perornata, a blue-green alga

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that causes off-flavor in commercial catfish production, compared with the green alga Selenastrum capricornutum, with complete growth inhibition at 100 ␮g/mL (13). Mortality of the Formosan subterranean termite, Coptotermes formosanus, was numerically, but not significantly, higher in tests conducted on filter paper treated with maca oil. At 1% (wt/wt), maca oil appeared to act as a feeding deterrent to termites. Several minor components of the essential oil, including 3-methoxyphenylacetonitrile and benzyl thiocyanate, were significantly active against the termite (13). Rainbow trout fry fish were fed with maca and different extracts obtained from four different solvents to study their effects on growth performance, feed utilization, and survival of the fish (50). The fish were fed eight casein-based semipurified isonitrogenous and isocaloric diets containing 15% wheat flour (control, diet 1), 15% maca meal (diet 2), 12.5% maca meal residue after extraction (diet 3), mixture of 4 maca meal extracts (diet 4), hexane extract (diet 5), dichloromethane extract (diet 6), ethyl acetate extract (diet 7), and methanol extract (diet 8). After 14-week feeding, the fish fed diet 2 had the highest growth rate among all the dietary treatments. Fish fed diets 2, 3, and 8 had higher growth than the fish fed with the other diets. Feed intake was higher in fish fed with diets 2, 3, and 8 than in fish fed with diets 1 and 5. Feed conversion ratio and protein efficiency ratio were also improved in fish fed with diets 2 and 3 versus fish fed with diets 1, 5, 6, and 7. Survival was higher in fish fed with diet 2 versus 1, 5, and 6. The effects of maca on lipid, antioxidative, and glucose parameters in hereditary hypertriglyceridemic rats were investigated (51). Maca was administered to rats as a part of a high-sucrose diet for 2 weeks. Rosiglitazone (0.02%) was used as a positive control. Maca significantly decreased the levels of VLDL (very-low-density lipoproteins), LDL (low-density lipoproteins), and total cholesterol, and also the level of TAG (triacylglycerols) in the plasma, VLDL, and liver. Maca, as well as rosiglitazone, significantly improved glucose tolerance, as the decrease of AUC (area under the curve) of glucose showed, and lowered levels of glucose in blood. The activity of SOD (superoxide dismutase) in the liver, the GPX (glutathione peroxidase) in the blood, and the level of GSH (glutathione) in liver increased in all cases significantly.

COSMETIC USES There are patent claims that compositions containing papain-treated papaya (Carica papaya) powders, papaintreated maca (L. meyenii) powders, papain, and substantially water-free powders or oils are useful as face cleansers, packs, and bath preparations that show skinconditioning effects (52). A face cleanser has been prepared from mannitol 50.0, soap 30.0, kaolin 10.0, talc 3.0, olive oil 1.0, papain 2.0, papain-treated papaya powder 2.0, and papain-treated maca powder 2.0 wt.%. Addition of polyols, mucopolysaccharides, sugars, and/or amino acids to the extract is claimed to improve the skin-moisturizing effect (53). Water-extracted maca is a desirable hygroscopic material, probably because it exhibits relatively good hygroscopic properties under

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conditions of varying humidity and has high-moisture retention capacity even in dry silica gel desiccators (53) Interestingly, a water extract of L. meyenii inhibited tyrosinase, a key enzyme in the production of the skin pigment melanin, with an IC50 of 150 ␮g/mL (54). Two maca extracts (0.13 mg/mL), one obtained after boiling and the other without boiling, were administered on the dorsal surface of male Holtzman rats to study their protecting function against ultraviolet radiation (UVR) (55). The rats were exposed to UVR once a week during 3 consecutive weeks. A commercial sunscreen was used as a positive control. The results showed that UVR caused significant increase in skin epidermal thickness. The epidermal height in animals treated with maca was similar to those who did not receive UVR. The aqueous extract of maca after a boiling process had better effect than maca extract without a boiling process. A dose–response effect was observed with increasing doses of aqueous extract of maca after a boiling process.

CLINICAL STUDIES Fertility-Enhancing and Aphrodisiac Activities Gonzales et al. reported that maca does not affect serum reproductive hormone levels in adult men, but rather improves sperm motility and sperm production in a dosedependent manner (56). In a similar protocol, Gonzales et al. (57). demonstrated the improvement of sexual desire after 8 weeks of treatment. Maca demonstrated an effect on sexual desire at 8 and 12 weeks of treatment, and this effect was independent of Hamilton depression and anxiety scores as well as serum testosterone and estradiol levels (57). A randomized, double-blind, placebo-controlled, crossover trial was performed over 14 postmenopausal women. The women completed the Greene Climacteric Scale to assess the severity of menopausal symptoms, and the blood samples were collected for the measurement of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex-hormone-binding globulin. In addition, aqueous and methanolic maca extracts were tested for androgenic and estrogenic activity by using a yeastbased hormone-dependent reporter assay. No differences in serum concentrations of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex-hormone-binding globulin between baseline, maca treatment, and placebo (P > 0.05) were observed. The findings showed that maca reduced psychological symptoms, including anxiety and depression, and lowered measures of sexual dysfunction in postmenopausal women independent of estrogenic and androgenic activity (58). A double-blind, randomized, pilot dose-finding study was carried out to determine whether maca is effective for selective-serotonin reuptake inhibitor (SSRI)-induced sexual dysfunction, comparing a low-dose (1.5 g/day) to a high-dose (3.0 g/day) maca regimen in 20 remitted depressed outpatients (mean age 36±13 years; 17 women) with SSRI-induced sexual dysfunction. The results demonstrated that maca root may alleviate SSRIinduced sexual dysfunction, and there may be a doserelated effect. Maca may also have a beneficial effect on libido (59).

A pilot investigation into the effect of maca supplementation on physical activity and sexual desire in trained male cyclists was conducted. Eight participants each completed a 40 km cycling time trial before and after 14 days supplementation with both maca extract and placebo, in a randomized crossover design. Subjects also completed a sexual desire inventory during each visit. The result showed that maca extract administration significantly improved 40 km cycling time performance compared with the baseline test (P = 0.01), but not compared with the placebo trial after supplementation (P > 0.05). Maca extract administration significantly improved the self-rated sexual desire score compared with the baseline test (P = 0.01), and compared with the placebo trial after supplementation (P = 0.03). Long-term clinical studies involving more volunteers are needed to further evaluate the efficacy of maca extract in athletes and normal individuals and to explore its possible mechanisms of action (60). A small but significant effect of maca supplementation on subjective perception of general and sexual wellbeing in adult patients with mild erectile dysfunction (ED) was observed through a double-blind clinical trial on 50 Caucasian men affected by mild ED, randomized to treatment with maca dry extract, 2400 mg, or placebo (61). The treatment effect on ED and subjective well-being was tested administrating the International Index of Erectile Function (IIEF-5) and the Satisfaction Profile (SAT-P) before and after 12 weeks. After 12 weeks of treatment, both maca- and placebo-treated patients experienced a significant increase in IIEF-5 score (P < 0.05 for both). However, patients taking maca experienced a more significant increase in IIEF-5 score than those taking placebo (1.6 ± 1.1 vs. 0.5 ± 0.6, P < 0.001). Both maca- and placebotreated subjects experienced a significant improvement in psychological performance-related SAT-P score, but the maca group higher than that of placebo group (+9 ± 6 vs. +6 ± 5, P < 0.05). However, only maca-treated patients experienced a significant improvement in physical- and social-performance-related SAT-P score compared with the baseline (+7 ± 6 and +7 ± 6, both P < 0.05). Although the “Preclinical Studies” section as well the “Clinical Studies” section suggests potential beneficial effects of maca, demonstration of efficacy in humans requires the conduct of clinical trials using randomized, double-blind, placebo-controlled protocols, and administering standardized maca extracts.

CONCLUSIONS Maca has been established as a nutritionally valuable food and food supplement through decades of research. The commercial activity of maca has grown explosively with the passing of the Dietary Supplement Health and Education Act in 1994. As with other herbal dietary products, quality, safety, and efficacy have been the critical concern for the consumer and industry. Furthermore, the rapid expansion of demand and diversity of products has created critical problems, as the scientific base of the industry has failed to keep pace. Further studies are required for the accurate authentication of raw plant material, including L. meyenii (maca), prior to commercial use. The quality of the tuberous root may depend upon the cultivation of

Maca

maca by using good agricultural practice. This includes selection of maca-specific habitat areas (typically highlands), soil and climatic conditions, seed stock, and correct storage of tubers. The presence of herbicides, pesticides, and heavy-metal residues needs to be analyzed during the quality control of raw material to insure the safety of the products. Second, extraction, preparation, and standardization of commercial maca products should be carried out using validated analytical methods, including the chemical profiling of marker compounds (21). The secondary metabolites of maca, including alkaloids, glucosinolates, macamides, and sterols, are just some of the marker constituents that may provide desirable nutritional, biological, and therapeutic (such as fertility-enhancing, aphrodisiac, and chemopreventive) leads. Future research efforts should be directed toward the isolation of the active constituents and the study of their mechanisms of action. Finally, more clinical studies related to specific disease areas should be directed to ensure safety, including from side effects and toxicity, and efficacy.

ACKNOWLEDGMENTS We are indebted to Dr. Hala ElSohly and Dr. D. Chuck Dunbar, NCNPR, School of Pharmacy, University of Mississippi, for critical reading of the original manuscript, and to Dr. Vaishali C. Joshi for assistance in preparing the graphic of maca tuber and editorial assistance on its morphological description. This work was supported in part by the USDA Agricultural Research Service Specific Cooperative Agreement No. 58–6408-2–0009.

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34. Cicero AF, Bandieri E, Arletti R. Lepidium meyenii Walp. improves sexual behaviour in male rats independently from its action on spontaneous locomotor activity. J Ethnopharmacol 2001; 75(2–3):225–229. 35. Gonzales GF, Gasco M, Cordova A, et al. Effect of Lepidium meyenii (Maca) on spermatogenesis in male rats acutely exposed to high altitude (4340 m). J Endocrinol 2004; 180(1):87– 95. 36. Bustos-Obregon E, Yucra S, Gonzales G.F. Lepidium meyenii (Maca) reduces spermatogenic damage induced by a single dose of malathion in mice. Asian J Androl 2005; 7(1): 71–76. 37. Rubio J, Riqueros MI, Gasco M, et al. Lepidium meyenii (Maca) reversed the lead acetate induced—damage on reproductive function in male rats. Food Chem Toxicol 2006; 44(7):1114– 1122. 38. Gonzales C, Rubio J, Gasco M, et al. Effect of short-term and long-term treatments with three ecotypes of Lepidium meyenii (MACA) on spermatogenesis in rats. J Ethnopharmacol 2006; 103(3):448–454. 39. Yucra S, Gasco M, Rubio J, et al. Effect of different fractions from hydroalcoholic extract of Black Maca (Lepidium meyenii) on testicular function in adult male rats. Fertil Steril 2008; 89(suppl 5):1461–1467. 40. Gasco M, Aguilar J, Gonzales GF. Effect of chronic treatment with three varieties of Lepidium meyenii (Maca) on reproductive parameters and DNA quantification in adult male rats. Andrologia 2007; 39(4):151–158. 41. Rubio J, Dang H, Gong M, et al. Aqueous and hydroalcoholic extracts of Black Maca (Lepidium meyenii) improve scopolamine-induced memory impairment in mice. Food Chem Toxicol 2007; 45(10):1882–1890. 42. Gonzales GF, Vasquez V, Rodriguez D, et al. Effect of two different extracts of red maca in male rats with testosteroneinduced prostatic hyperplasia. Asian J Androl 2007; 9(2):245– 251. 43. Gasco M, Villegas L, Yucra S, et al. Dose–response effect of Red Maca (Lepidium meyenii) on benign prostatic hyperplasia induced by testosterone enanthate. Phytomedicine 2007; 14(7–8):460–464. 44. Farnham MW, Stephenson WW, Fahey JW. The capacity of broccoli to induce a mammalian chemoprotective enzyme varies among inbred lines. J Am Soc Hortic Sci 2000; 125(4):482–488. 45. Gross HB, Dalebout T, Grubb CD, et al. Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana. Plant Sci 2000; 159(2):265–272. 46. Wattenberg LW. Inhibition of carcinogen-induced neoplasia by sodium cyanate, tert-butyl isocyanate, and benzyl isothiocyanate administered subsequent to carcinogen exposure. Cancer Res 1981; 41(8):2991–2994. 47. Sugie S, Okamoto K, Okumura A, et al. Inhibitory effects of benzyl thiocyanate and benzyl isothiocyanate on methylazoxymethanol acetate-induced intestinal carcinogenesis in rats. Carcinogenesis 1994; 15(8):1555–1560. 48. Valentova K, Buckiova D, Kren V, et al. The in vitro biological activity of Lepidium meyenii extracts. Cell Biol Toxicol 2006; 22(2):91–99. 49. Sandoval M, Okuhama NN, Angeles FM, et al. Antioxidant activity of the cruciferous vegetable Maca (Lepidium meyenii). Food Chem. 2002; 79(2):207–213. 50. Lee KJ, Dabrowski K, Sandoval M, et al. Activity-guided fractionation of phytochemicals of maca meal, their antioxidant activities and effects on growth, feed utilization, and survival in rainbow trout (Oncorhynchus mykiss) juveniles. Aquaculture 2005; 244(1–4):293–301.

51. Vecera R, Orolin J, Skottova N, et al. The influence of maca (Lepidium meyenii) on antioxidant status, lipid and glucose metabolism in rat. Plant Foods Hum Nutr 2007; 62(2):59–63. 52. Arita J, Hirao K. Cosmetic Compositions Containing PapainTreated Papaya and Maca Powders. JP Patent, 2,003,155,213, May 27, 2003. 53. Shimofuruya H, Suzuki I, Kunieda Y. Studies on hygroscopic abilities of the water-extracted maca. Memoirs Suzuka Coll Technol 2003; 36:131–134. 54. Mitsuma T, Hirao K. Skin-Lightening, Rough Skin-Treating, and Moisturizing Cosmetics Containing Extract of Lepidium Plant (Cruciferae). JP Patent, 2,001,039,854, February 13, 2001. 55. Gonzales-Castaneda C, Gonzales GF. Hypocotyls of Lepidium meyenii (maca), a plant of the Peruvian highlands, prevent ultraviolet A-, B-, and C-induced skin damage in rats. Photodermatol Photoimmunol Photomed 2008; 24(1):24–31. 56. Gonzales GF, Cordova A, Vega K, et al. Effect of Lepidium meyenii (Maca), a root with aphrodisiac and fertilityenhancing properties, on serum reproductive hormone levels in adult healthy men. J Endocrinol 2003; 176(1):163– 168. 57. Gonzales GF, Cordova A, Vega K, et al. Effect of Lepidium meyenii (MACA) on sexual desire and its absent relationship with serum testosterone levels in adult healthy men. Andrologia 2002; 34(6):367–372. 58. Brooks NA, Wilcox G, Walker KZ, et al. Beneficial effects of Lepidium meyenii (Maca) on psychological symptoms and measures of sexual dysfunction in postmenopausal women are not related to estrogen or androgen content. Menopause 2008; 15(6):1157–1162. 59. Dording CM, Fisher L, Papakostas G, et al. A doubleblind, randomized, pilot dose-finding study of maca root (L. meyenii) for the management of SSRI-induced sexual dysfunction. CNS Neurosci Ther 2008; 14(3):182–191. 60. Stone M, Ibarra A, Roller M, et al. A pilot investigation into the effect of maca supplementation on physical activity and sexual desire in sportsmen. J Ethnopharmacol 2009; doi: 10.1016/j.jep.2009.09.012. 61. Zenico T, Cicero AF, Valmorri L, et al. Subjective effects of Lepidium meyenii (Maca) extract on well-being and sexual performances in patients with mild erectile dysfunction: a randomised, double-blind clinical trial. Andrologia 2009; 41(2):95–99.

FURTHER READING 1. Leon J. The “maca” (Lepidium meyenii), a little-known food plant of Peru. Econ Bot 1964; 18(2):122–127. 2. USDA Plants Database: http://plants.usda.gov/java/profile? symbol=LEME19. Accessed October 2009. 3. National Research Council. Lost Crops of the Incas: LittleKnown Plants of the Andes with Promise for Worldwide Cultivation, Report of an Ad Hoc Panel of the Advisory Committee on Technical Innovation, Board on Science and Technology for International Development. Washington, DC: National Academy Press, 1989. ´ el caso 4. de Leon M. Castro Un cultivo Andino en extinction: de la maca. Pero Indig 1990; 12;85–94. 5. http://www.rain-tree.com/maca.htm. Accessed October 2009. 6. http://www.cfsn.com/maca.html. Accessed October 2009. 7. Valentov´a K, Ulrichov´a J. Smallanthus sonchifolius and Lepidium meyenii—prospective andean crops for the prevention of chronic diseases. Biomed Papers 2003; 147:119–130.

Magnesium Robert K. Rude

INTRODUCTION

Structural Modification of Nucleic Acids and Membranes Another important role of Mg is its ability to form complexes with nucleic acids. The negatively charged ribose phosphate structure of nucleic acids has a high affinity for Mg2+ ; the resulting stabilization of numerous ribonucleotides and deoxyribonucleotides induces important physicochemical changes that affect DNA maintenance, duplication, and transcription (2). Mg, calcium, and some other cations react with hydrophilic polyanionic carboxylates and phosphates of the various membrane components to stabilize the membrane and thereby affect fluidity and permeability. This thereby influences ion channels, transporters, and signal transducers.

Magnesium is an essential nutrient and is vital for numerous biologic processes in the body. This entry reviews the biochemistry, physiology, and homeostasis of magnesium. Dietary magnesium intake and requirements as well as current dietary recommendations are discussed. Causes of and risk factors for magnesium deficiency are reviewed along with the clinical manifestations of moderateto-severe magnesium depletion. As dietary magnesium intake falls below the recommended daily allowance, possible complications of this nutrition deficiency such as hypertension, cardiovascular disease, and osteoporosis are discussed.

Ion Channels Ion channels constitute a class of proteins across the cell membrane, which allow passage of ions in or out of cells when the channels are open. Ion channels are classified according to the type of ion they allow to pass such as sodium (Na+ ), potassium (K+ ), or calcium (Ca2+ ) (3). Mg2+ plays an important role in the function of a number of ion channels. A deficit of Mg results in cellular potassium depletion (4). Mg2+ is necessary for the active transport of K+ out of cells by Na+ , K+ ATPase. Another mechanism for the K+ loss is an increased efflux of K+ from cells via other Mg2+ -sensitive K+ channels as has been seen in skeletal muscle and in heart muscle. Therefore, a deficiency in Mg2+ leads to a reduced amount of intracellular K+ . The arrhythmogenic effect of Mg deficiency, as discussed later, may therefore be related to its effect on maintenance of intracellular K+ . Mg has been called nature’s physiological calcium channel blocker (3). During Mg depletion, intracellular calcium rises. This may be due to both an increase from extracellular calcium and release from intracellular calcium stores. Mg2+ has been demonstrated to decrease the inward Ca2+ flux through slow calcium channels. In addition Mg2+ will decrease the transport of Ca2+ out of the sarcoplasmic reticulum into the cell cytosol. There is an inverse ability of inositoltriphosphate to release Ca2+ from intracellular stores in response to changes in Mg2+ concentrations, which would also allow greater rise in intracellular Ca2+ during a fall in Mg2+ .

BIOCHEMISTRY AND PHYSIOLOGY Magnesium (Mg) is widely distributed in nature being the eight most abundant element on earth and the second most abundant cation in sea water (1). It has therefore been incorporated widely in biology and is the fourth most abundant cation in the body and the second most prevalent intracellular cation. Due to its positive charge, Mg binds to negatively charged molecules. Most intracellular Mg binds to ribosomes, membranes, and other marcomolecules in the cytosol and nucleus. Mg provides specific structure and catalytic activity for enzymes as discussed later.

Enzyme Interactions Mg is involved in more than 300 essential metabolic reactions (2). Mg2+ is essential for many enzymatic reactions and has two general interactions: (a) Mg2+ binds to the substrate, thereby forming a complex with which the enzyme interacts, as in the reaction of kinases with MgATP and (b) Mg2+ binds directly to the enzyme and alters its structure and/or serves a catalytic role. Overall, the predominant action of Mg is related to adenosine triphosphate (ATP) utilization. ATP provides highenergy phosphate and exists in all cells primarily as Mg ATP2− (MgATP). Mg therefore is essential for the function of many pathways and enzymes including the glycolytic cycle, citric acid cycle, protein kinases, RNA and DNA polymerases, lipid metabolism, and amino acid activation, as well as playing a critical role in the cyclic adenosine monophosphate and phospholipase C second messenger systems.

BODY COMPOSITION AND HOMEOSTASIS Composition The distribution of Mg in various body compartments of apparently healthy adult individuals is summarized in 527

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Table 1 Adulta

Distribution and Concentrations of Magnesium (Mg) in a Healthy

Site Bone Muscle Soft tissue Adipose tissue Erythrocytes Serum % Free % Complexed % Bound Mononuclear Blood cells

Percentage of total body Mg 53 27 19 0.012 0.5 0.3 65 8 27

Platelets [Mg2+ ] Cerebrospinal fluid free 55% complexed 45% Secretions Saliva, gastric, bile Sweat

Concentration/content 0.5% of bone ash 9 mmol/kg wet weight 9 mmol/kg wet weight 0.8 mmol/kg wet weight 1.65–2.73 mmol/L 0.88 ± 0.06 mmol/L 0.56 ± 0.05 mmol/L 2.91 ± 0.6 fmol/cell 2.79 ± 0.6 fmol/cell 3.00 ± 0.4 fmol/cell 2.26 ± 0.29 mmol/Lj 0.5–1.0 mmol/L 1.25 mmol/L

0.3–0.7 mmol/L 0.3 mmol/L (38◦ C) 0.09 mmol/hr

Note: 1 mmol = 2 mequiv = 24.3 mg. a Total body: 833–1170 mmol or 20–28 g.

Table 1. Approximately 60% of Mg is in the skeleton of which two-thirds of it is within the hydration shell and one-third on the crystal surface (5), which may serve as a reservoir for maintaining extracellular and intracellular Mg. Only 1% of Mg in the extracellular fluid and the rest is intracellular.

Cellular Mg Homeostasis As already stated, Mg is compartmentalized within the cell and most of it is bound to proteins and negatively charged molecules. Significant amounts of Mg are found in the nucleus, mitochondria, the endoplasmic and sarcoplasmic reticulum, and the cytoplasm (1,6). Total cell Mg concentration has been reported to range between 5 and 20 mM. Ninety to ninety-five percent of that in the cytosol is bound to ligands such as ATP, ADP, citrate, proteins, and nucleic acids. The remainder is free Mg2+ , constituting 1% to 5% of the total cellular Mg. The concentration of free ionized Mg2+ in the cytoplasm of mammalian cells has ranged from 0.5 to 1.0 mM similar to circulating ionized Mg2+ . The Mg2+ concentration in the cell cytoplasm is maintained relatively constant even when the Mg2+ concentration in the extracellular fluid is experimentally varied to either high or low nonphysiological levels. The relative constancy of the Mg2+ in the intracellular milieu is attributed to the limited permeability of the plasma membrane to Mg and to the operation of specific Mg transport proteins, which regulate the rates at which Mg is taken up or extruded from cells. Maintenance of a normal intracellular concentration of Mg2+ requires that Mg be actively transported out of the cell. Mg transport in or out of cells appears to require the presence of carrier-mediated transport systems. The efflux of Mg from the cell appears to be coupled to Na transport and requires extrusion of sodium by Na+ , K+ -ATPase. There is also ev-

idence for a Na-independent efflux of Mg (7). Mg influx appears to be linked to Na transport but by a different mechanism than efflux. It has been reported that at least seven transmembrane Mg2+ channels have been cloned (7). These include NIPA2 (8) and MagT1 and TUSC3 (9). Studies of human hereditary diseases (see later) have identified paracellin-1 (claudin 16) and two transient receptor potential channel family members, TRPM6 and TRPM7 (10). TRPM6 is expressed in the kidney and TRPM7 is constitutively expressed. Studies have demonstrated that tissues vary with respect to the rates at which Mg exchange occurs and the percentage of total Mg that is readily exchangeable. The rate of Mg exchange in heart, liver, and kidney exceeded that in skeletal muscle, lymphocytes, red blood cells, brain, and testis. The processes that maintain or modify the relationships between total and ionized internal and external Mg are not completely understood. Changes in cytosolic Mg2+ regulate some channels (TRPM6 and TRPM7) (7). Mg transport in mammalian cells may be influenced by hormonal and pharmacological factors. Mg2+ efflux was stimulated after short-term acute exposure of isolated perfused rat heart and liver or thymocytes to ␣- and ␤agonists and permeant cAMP. Activation of protein kinase C by diacyl-glycerol or by phorbol esters stimulates Mg2+ influx and does not alter efflux. Epidermal growth factor has been shown to increase Mg2+ transport into a vascular smooth muscle cell line. Insulin and dextrose were found to increase 28 Mg uptake by a number of tissues, including skeletal and cardiac muscle. The mechanism of insulininduced Mg transport is likely due to an effect on protein kinase C. An insulin-induced transport of Mg into cells could be one factor responsible for the fall in the serum Mg concentration observed during insulin therapy of diabetic ketoacidosis. It is hypothesized that this hormonally regulated Mg uptake system controls intracellular Mg2+ concentration in cellular subcytoplasmic compartments. The Mg2+ concentration in these compartments would then serve to regulate the activity of Mg-sensitive enzymes.

BODY HOMEOSTASIS Homeostasis of the individual with respect to a mineral depends on the amounts ingested, the efficiency of intestinal and renal absorption and excretion, and all other factors affecting them. A schema for Mg balance is given in Figure 1.

Dietary Intake Mg is widely distributed in plant and animal sources but in differing concentrations. In terms of major food sources (11), vegetables, fruits, grains, and animal products account for approximately 16% each; dairy product contributes 20% in adolescents and 10% beyond the third decade. The 1994 U.S. Department of Agriculture Continuing Survey of Food Intakes by Individuals (CSFII) indicated that the mean daily Mg intake was 323 mg in males and 228 mg in females, which was similar to the NHANES III survey. These values fall below the current Recommended Daily Allowance (RDA) recommendation of approximately 420 mg for males and 320 mg for females (12). Indeed, it has been suggested that 75% of

Magnesium

of dietary fiber have been reported to decrease Mg utilization in humans, presumably by decreasing absorption. High dietary zinc intake decreased Mg absorption and balance whereas vitamin B6 depletion was associated with negative Mg balance. The presence of excessive amounts of free fatty acids and oxalate may also impair Mg absorption.

300 mg 2300 mg 100 mg INTESTINE

PLASMA 12 mg

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2400 mg

12 mg KIDNEY

BONE 200 mg 100 mg

Figure 1 Mg homeostasis in man. A schematic representation of magnesium metabolism indicating (a) its absorption from the alimentary tract, (b) its distribution into bone, and (c) its dependence on the kidney for excretion. Homeostasis depends upon the integrity of intestinal and renal absorptive processes.

Absorbability of Mg Salts Multiple salts of Mg are available as dietary supplements including oxide, hydroxide, citrate, chloride, gluconate, lactate, and aspartate. The fractional absorption of a salt depends on its solubility in intestinal fluids and the amounts ingested (15). Absorption of enteric-coated Mg chloride is 67% less than that of the acetate in gelatin capsules. Mg citrate was found to have high solubility whereas Mg oxide was poorly soluble; better absorption of the citrate salt was demonstrated in humans. Little difference in absorption has been demonstrated among other salts however.

Regulation of Intestinal Mg Absorption subjects in the United States have dietary Mg intake that falls below the recommended intake (see “Mg Requirements” section).

Intestinal Absorption In humans, the primary site of intestinal Mg absorption is the jejunum and ileum although absorption can occur at other sites including the colon (13). Under a normal dietary Mg intake, 30% to 40% is absorbed. There exists both a passive paracellular mechanism and an active transport process for Mg absorption. The paracellullar mechanism is dependent on transcellular potential difference generated by sodium transport and accounts for approximately 90% of intestinal Mg absorption (14). There exists a Mg-specific transport protein/channel, TRPM6, which accounts for the remainder of Mg absorption and may be influenced by a number of hormones (14). Net Mg absorption increases with increasing Mg intake however fractional Mg absorption falls; absorption fell progressively from approximately 65% to 70% with intake of 7 to 36 mg down to 11 to 14% with intake of 960 to 1000 mg (15).

Bioavailability: Influence of Other Dietary Factors The fractional absorption of ingested Mg by healthy humans is influenced by its dietary concentration as discussed earlier as well as the presence of dietary components inhibiting or promoting Mg absorption (12). Longterm balance studies in healthy individuals, for the most part, indicate that increasing oral calcium intake does not significantly affect Mg absorption or retention. Increased amounts of Mg in the diet have been associated with either decreased calcium absorption or no effect. Some reports indicate decreased Mg absorption at high levels of dietary phosphate, whereas others found no consistent effect. Increased amounts of absorbable oral Mg have been noted to decrease phosphate absorption, perhaps secondary to formation of insoluble Mg phosphate. Increased intakes

No hormone or factor has been described that regulates intestinal Mg absorption although several hormones may influence the TRPM6 channel as already discussed. Vitamin D and its active metabolites have been shown to increase intestinal Mg absorption in a number of studies (13). 1,25(OH)2 -vitamin D increases intestinal absorption in normal human subjects and patients with chronic renal failure. In balance studies, vitamin D increased intestinal Mg absorption but much less than calcium and mean Mg balance was not affected. In patients with impaired calcium absorption due to intestinal disease given vitamin D, only small increases in Mg absorption were observed compared with calcium. Mg was absorbed by individuals with no detectable plasma 1,25(OH)2 -vitamin D and, in contrast to calcium absorption, there is no significant correlation between plasma 1,25(OH)2 -vitamin D and Mg absorption.

Renal Mg Regulation The kidney is the critical organ regulating Mg homeostasis. Mg handling is a filtration/reabsorption process (16). Approximately 2400 mg of Mg is normally filtered daily through the glomeruli in the healthy adult; of this only approximately 5% is excreted in the urine. The fractional absorption of the filtered load in the various segments of the nephron is summarized in Figure 2. Approximately 15% to 20% of filtered Mg is reabsorbed in the proximal convoluted tubule presumably by a paracellular mechanism. The majority, 65% to 75%, is reclaimed in the cortical thick ascending limb of Henle. The mechanism also appears to be paracellular transport. Paracellin-1 (claudin-16) and claudin-19 appear to mediate this transport (10). The distal convoluted tubule reabsorbs 5% to 10% of filtered Mg via an active transcellular pathway. Several proteins may be involved including the sodium chloride cotransporter (10). TMPR6 is also expressed in the distal tubule. Mutations of TRPM6 results in decrease intestinal Mg absorption and renal Mg wasting (10).

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Table 2 Proximal convoluted tubule

Loop of Henle Distal convoluted tubule 5 -10%

15 - 20%

Thick ascending limb Cortical

Collecting duct

65 - 75% 100% Mg Thin decending limb

Medullary

Recommendations for Daily Intakes of Mg (mg)

Age (years)

Male

0–0.5 0.5–1.0 1–3 4–8 9–13 14–18 19–30 31–50 51–70 >70 Pregnancy

30a 75b

3 - 5%

Figure 2 Fractional segmental reabsorption of filtered Mg in the nephron. The percentage absorption of filtered Mg2+ has been determined by micropuncture techniques in various laboratory animals as the Mg proceeds through the nephron. Approximately 15% to 20% of the Mg2+ is reabsorbed in the proximal convoluted tubule. The major site for Mg2+ reabsorption is the thick ascending limb of the loop of Henle, primarily in its cortical portion. Here, 65% to 75% of Mg leaves the lumen. In the distal convoluted 5% to 10% of Mg is reabsorbed. Source: From Cole DE, Quamme GA. Inherited disorders of renal magnesium handling. J Am Soc Nephrol 2000;11:1937–1947, with permission.

Hormonal and Other Regulatory Influences on Renal Mg Reabsorption No hormone or factor has been described, which regulates renal Mg homeostasis. Micropuncture studies in rodents show that arginine, vasopressin, glucagon, calcitonin, and PTH, when added individually to the bath of mouse segments of the cortical thick ascending limb of loop of Henle and/or the distal convoluted tubule, significantly increased Mg absorption (16). The physiological significance of these observations however is unclear. A number of conditions affect absorption, principally in the ascending thick limb. Inhibition occurs with hypermagnesemia and hypercalcemia (16). This is thought to occur because these cations binding to a calcium-sensitive receptor on the basolateral aspect of these tubular cells and decreasing transepithelial voltage and thereby decreasing the paracellular absorption of both Mg and calcium. Decreased Mg intake in experimental animals and humans rapidly decreases Mg excretion, even before serum/plasma Mg levels fall below the normal range, suggesting an adaption of the kidney to Mg insufficiency.

Female

80 130 240 410 400 420 420 420

30a 75b 80 130 240 360 310 320 320 320

≤18 19–30 31–50

400 350 360

≤18 19–30 31–50

360 310 320

Lactating

a Intake from human milk by healthy b Human milk plus solid food.

breast-fed infants.

has been the dietary balance study. For infants and young children, the figures are based primarily on estimates of Mg intakes of milk and other foods that allow good development. Such data have been transmuted into the dietary reference intakes in the United States (12). The U.S. reference intakes for Mg are given in Table 2. These are estimates of intake that meet the needs of 97% to 98% of healthy individuals. There are some problems with the study of balance. Laboratory analysis of foods revealed a Mg content 115% to 124% greater than those calculated by tables of food composition (18). A critique of previous RDAs for Mg noted that most of the published human balance data referenced in its various editions often did not meet the criteria for acceptable methodology (18). The balance studies (usually short term) were done mostly in adolescents and younger adults, and balance data presented for pregnant women were less than adequate. Published data about the elderly were meager. The need was pointed out for improved definition of acceptable standards, evaluation of the optimum base (i.e. weight, fat-free mass, or lean body mass), documentation of the accepted data, and more awareness of the ways in which homeostatic mechanisms conserve body Mg. Although the database of the 1997 reference values (Table 2) has eliminated the poorer studies, a question remains as to the accuracy of many balance studies in terms of adherence to acceptable methodology.

Tissue Sources Extracellular, intracellular, and bone Mg fall during Mg depletion. Bone may serve as an important reservoir for Mg as human iliac crest Mg content fell an average of 18% with depletion (5). In young Mg-deficient rats and mice, approximately 30% of bone is lost (17).

MG REQUIREMENTS Assessment For healthy older children, adolescents, and adults, the primary approach for assessing dietary Mg requirement

Dietary Intake Estimates of Mg intakes in NHANES III (1988–1991) indicated that children 2 to 11 years grouped by gender, age, and race/ethnicity had median intakes well above their RDA. Those ages 1 to 5 years in the lower fifth percentile took in approximately 90% of the RDA. On the other hand, males and females from 12 to more than 60 years, grouped by race and ethnicity, with the exception of nonHispanic white males, had low median intakes in terms of the RDA. The Third Report on Nutrition Monitoring in the United States (1995) analyzed intake in relation to the

Magnesium

RDA for age and gender; it concluded that Mg presents a potential public health issue requiring further study. One reason given was that the medium intakes of Mg from food were lower than the RDAs in various population groups. Assessment of Mg status at various dietary Mg intakes has not been performed. It is therefore impossible to estimate what level of intake would place one at risk for a Mg-deficient-associated problem.

ASSESSING MG STATUS Analytic Procedures As Mg is mostly within cells or in bone, assessment of Mg status is most difficult. A number of laboratory techniques are used in clinical and research investigations (19). Atomic absorption spectrophotometry (AAS) has been widely used to determine total Mg in many sources and still remains the reference method as it provides greatest accuracy and precision although a number of metallochromic indicators and dyes are commonly used in automated methods. Ion-selective electrodes (ISEs) can measure ionized Mg (70% of total Mg) in serum, plasma, and whole blood. However, calcium and lipophilic cations interfere with the determination of ionized Mg. Literature indicates that ISEs from various manufacturers differ in accuracy from each other and from AAS and may give misleading results in sera with low Mg concentrations. Also, in critically ill patients there is a poor correlation between total and ionized serum Mg levels (20). Other techniques have been developed to assess intracellular Mg concentration, which includes nuclear magnetic resonance spectroscopy and fluorescent indicators (19). These methods are reserved as research tools. Mg isotopes have been used as biologic tracers to follow the absorption, distribution, and excretion of the Mg ion. The radioisotope 28 Mg has been used in human studies. Its value is limited by its radioactivity, its short half-life of 21.3 hours, and its short supply.

Assessment Tests Total serum Mg is the only test available to clinicians to assess Mg status (21). There are a number of reports of normal serum/plasma levels associated with a variety of illnesses but with low values in various blood cells and other organs. Consequently, total serum/plasma Mg values in such situations may be considered unreliable indicators of depletion. The level of ionized Mg may be more relevant under certain circumstances than that of total Mg. As already discussed, it should be noted that there exist intermethod differences for ionized Mg and therefore reference ranges must exist for each analyzer and may not be comparable to a different manufacturer. Erythrocytes and blood mononuclear cell Mg content have been measured in experimental human Mg deficiency and patient populations and suggest that these measurements are more accurate than the serum Mg in assessing Mg status. These are not commercially available and technical issues however appears to limit its use in assessing Mg status in any given individual. Assessing urine Mg excretion may be of use. When there is a reduction in the amounts of Mg ingested, there is a fairly rapid reduction in urinary Mg excretion. Serum Mg

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may still be within normal limits, but urine levels low. This would not indicate whether the Mg deficits were acute or chronic however. In situations in which renal Mg wasting occurs, the resulting hypomagnesemia is associated with excessive urinary Mg excretion (>1 mmol/day). Such a relationship would suggest renal tubular dysfunction as the cause of the hypomagnesemia. The IV Mg retention test provides an estimate of the proportion of infused Mg that is retained over a given period. Persons retaining more than the percentage retained by Mg-replete individuals (e.g., 20–25%) are considered to have some body depletion. A suggested clinical protocol that has been tested in a relatively large number of hypomagnesemic patients, chronic alcoholics, and animal controls has been published (22). It is an invasive, time-consuming, nonstandardized, and expensive test, requiring hospitalization or other close supervision for the partial or full 24 hours after infusion, with careful urine collection for laboratory analysis.

RISK FACTORS/CAUSES OF DEFICIENCY Prevalence The many risk factors for Mg depletion (Table 3) suggest that this condition may not be a rare occurrence. Up to 11% of hospitalized patients having routine Mg determinations were hypomagnesemic (13). The true prevalence of hypomagnesemia is not known because this ion is not included in routine electrolyte testing in many clinics or hospitals. Similar high rates of depletion have been reported in studies of ICU patients.

Gastrointestinal Disorders As already discussed, dietary Mg intake falls below the recommended intake in a large proportion of the population (12). Therefore, nutritional Mg deficiency can be observed and it contributes to Mg depletion when other conditions exist, which impair Mg balance. Gastrointestinal disorders (Table 3) may lead to Mg depletion in various ways (13). The Mg content of upper intestinal tract fluids is approximately 1 mEq/L. Vomiting and nasogastric suction therefore may contribute to Mg depletion. The Mg content of diarrheal fluids and fistulous drainage are much higher (up to 15 mEq/L), and consequently Mg depletion is common in acute and chronic diarrhea, regional enteritis, ulcerative colitis, and intestinal and biliary fistulas. Malabsorption syndromes may also result in Mg deficiency. Steatorrhea and resection or bypass of the small bowel, particularly the ileum, often results in intestinal Mg loss or malabsorption. Acute severe pancreatitis is associated with hypomagnesemia, which may be due to the clinical problem causing the pancreatitis, such as alcoholism, or to saponification of Mg in necrotic parapancreatic fat. Recently, proton pump inhibitors have been reported to cause hypomagnesemia in some patients (23). The evidence suggests that it is due to intestinal Mg malabsorption. A primary defect in intestinal Mg absorption, which presents early in life with hypomagnesemia, hypocalcemia, and seizures, has been described as an autosomal recessive disorder linked to chromosome 9q22. This disorder appears to be caused by mutations in

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Table 3

Causes of Mg Deficiency

1. Gastrointestinal disorders a. Nutritional deficiency b. Prolonged nasogastric suction/vomiting c. Acute and chronic diarrhea d. Intestinal and biliary fistulas e. Malabsorption syndromes f. Extensive bowel resection or bypass g. Acute hemorrhagic pancreatitis h. Primary intestinal hypomagnesemia (mutation of TRPM6 channel) i. Proton-pump inhibitors 2. Renal loss a. Chronic parenteral fluid therapy b. Osmotic diuresis (glucose, urea, manitol) c. Hypercalcemia d. Polyuric phase of acute renal failure, renal transplant, post renal-obstruction e. Nondrug-associated tubulointerstial nephropathy f. Alcohol g. Diuretics (furosemide, hydrochlorothiazide) h. Epidermal growth factor blockers (cetuximab, panitumumab) i. Renal tubular nephrotoxins (aminoglycosides, cisplatin, amphotericin B, pentamidine) j. Calcineurin inhibitors (cyclosporin, tacrolimus) k. Genetic mutations of Mg transport channels l. Activating mutation of the calcium-sensing receptor 3. Endocrine and metabolic disorders a. Diabetes mellitus (glycosuria–osmotic diuresis) b. Phosphate depletion c. Primary hyperparathyroidism d. Hypoparathyroidism e. Primary aldosteronism f. Excessive lactation 4. Cutaneous loss a. Sweat–athletics b. Burns 5. Redistribution of Mg to bone/soft tissues a. Hungry bone syndrome b. Parenteral nutrition/refeeding syndrome

TRPM6, which expresses a protein involved with active intestinal Mg transport (14).

Renal Disorders Excessive excretion of Mg into the urine may be the basis of Mg depletion (Table 3) (16,24). Renal Mg reabsorption is proportional to tubular fluid flow as well as to sodium and calcium excretion. Therefore, chronic parenteral fluid therapy, particularly with saline, and volume expansion states such as primary aldosteronism and hypercalciuric states, may result in Mg depletion. Hypercalcemia has been shown to decrease renal Mg reabsorption probably mediated by calcium binding to the calcium-sensing receptor in the thick ascending limb of Henle and decreasing transepithelial voltage. Osmotic diuresis due to glucosuria will result in urinary Mg wasting. Hypermagnesuria also occurs during the polyuric phase of recovery from acute renal failure in a native kidney, during recovery from ischemic injury in a transplanted kidney, and in postobstructive diuresis. In such cases, it is likely that residual tubule reabsorptive defects persisting from the primary renal injury play as important a role as polyuria itself in inducing renal Mg2+ wasting (25) Renal Mg2+ wasting has occasionally been reported in patients with acute or chronic tubulointerstitial nephritis

not caused by nephrotoxic drugs, for example, in chronic pyelonephritis and acute renal allograft rejection (25). Alcohol ingestion may also cause renal Mg wasting and is one cause of the high prevalence of Mg deficiency in chronic alcoholics. Many pharmaceutical drugs may cause renal Mg wasting and Mg depletion. The major site of renal Mg reabsorption is at the loop of Henle, therefore diuretics such as furosemide result in Mg wasting (26). Hypomagnesemia is common in patients receiving the epidermal growth factor (EGF) receptor blockers, cetuximab and panitumuma (27), which are monoclonal-blocking antibodies of the EGF receptor that are used in the treatment of metastatic colorectal cancer. Renal tubular nephrotoxins (aminoglycosides, amphotericin B, cisplatin, and pentamidine) have been shown to cause a renal lesions that results in hypermagnesuria and hypomagnesemia (25,28– 30). Similarly, calcineurin inhibitors (cyclosporine and tacrolimus) has been reported to result in renal Mg wasting in patients after organ transplantation due to a downregulation of the distal tubule Mg channel, TRPM6 (31). Several renal Mg-wasting disorders have been described, which may be genetic or sporadic (32). One form, which is autosomal recessive, results from mutations in the paracellin-1 gene on chromosome 3 (Claudin 16). This disorder is characterized by low-serum Mg as well as hypercalciuria and nephrocalcinosis. Another autosomal dominant form of isolated renal Mg wasting and hypomagnesemia has been linked to chromosome 11q23 and identified as a mutation on the Na+ ,K+ -ATPase ␥ -subunit of gene FXYD2. More recently, a mutation of the Mg channel, TRPM6 may result in Mg wasting. Gitelman’s syndrome (familial hypokalemia–hypomagnesemia syndrome) is an autosomal recessive disorder due to a genetic defect of the thiazide-sensitive NaCl cotransporter gene on chromosome 16. Other undefined genetic defects also exist (32).

Diabetes Mellitus Special consideration must be given to diabetes mellitus. It is the most common disorder associated with magnesium deficiency (33). It is generally thought that the mechanism for magnesium depletion in diabetics is due to renal magnesium wasting secondary to osmotic diuresis generated by hyperglycosuria. Dietary magnesium intake however falls below the RDA in diabetics therefore nutritional deprivation may be a factor. Magnesium deficiency has been reported to result in impaired insulin secretion as well as insulin resistance (34,35), which may contribute to hypertension (36). The mechanism is unclear but may be due to abnormal glucose metabolism as magnesium is a cofactor in several enzymes in this cycle. In addition, magnesium depletion may decrease tyrosine kinase activity at the insulin receptor and magnesium may influence insulin secretion by the ␤ cell. Diabetics given magnesium therapy appear to have improved diabetes control. Two studies have reported that the incidence of type 2 diabetes is significantly greater in people on a lower magnesium diet (34,35). Genetic variants of TRPM6 and TRPM7 have been reported to increase the risk of type 2 diabetes in women when they are on a diet of less than 250 mg/day of Mg (37). Magnesium status should

Magnesium

therefore be assessed in patients with diabetes mellitus as a vicious cycle may occur: diabetes out of control leading to magnesium loss and the subsequent magnesium deficiency resulting in impaired insulin secretion and action and worsening diabetes control.

predisposing and complicating disease states. The clinical presentation of Mg deficiency in disease states may coexist or be masked by the signs and symptoms of the primary disorder.

Moderate-to-Severe Mg Deficiency

Other Hypomagnesemia may accompany a number of other disorders (13). Phosphate depletion has been shown experimentally to result in urinary Mg wasting and hypomagnesemia. Hypomagnesemia may also accompany the “hungry bone” syndrome, a phase of rapid bone mineral accretion in subjects with hyperparathyroidism or hyperthyroidism following surgical treatment. Mg may also shift into soft tissue during the refeeding syndrome resulting in a fall inserum Mg (38,39). Mg loss may occur from the skin in sweat and in burn patients (40,41)

CLINICAL PRESENTATION OF MAGNESIUM DEFICIENCY As Mg plays an essential role in a wide range of fundamental biologic reactions, it is not surprising that Mg deficiency may lead to serious clinical symptoms. Human subjects have been studied in the course of Mg deficiency induced by diets low in this element (13) and these observations, along with those in humans who have Mg deficiency due to secondary causes, identify the manifestations of this deficit. Symptoms and signs of deficiency are given in Table 4. Mg deficiency occurs in a number of Table 4

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Manifestations of Mg Depletion

I. Bone and mineral metabolism a. Hypocalcemia 1. Impaired PTH secretion 2. Renal and skeletal resistance to PTH 3. Impaired formation and resistance to 1,25(OH)2 -vitamin D b. Osteoporosis II. Neuromuscular a. Positive Chvostek’s and Trousseau’s sign b. Spontaneous carpal-pedal spasm c. Seizures d. Vertigo, ataxia, nystagmus, athetoid, and chorioform movements e. Muscular weakness, tremor, fasciculation, and wasting f. Psychiatric: depression, psychosis III. Potassium homeostasis a. Hypokalemia 1. Renal potassium wasting 2. Decreased intracellular potassium IV. Cardiovascular a. Cardiac arrhythmia 1. EKG: prolonged P-R interval and Q-T interval, U waves 2. Atrial tachycardia, premature contractions and fibrillation 3. Junctional arrhythmias 4. Ventricular premature contractions, tachycardia, fibrillation 5. Sensitivity to digitalis intoxication 6. Torsades de pointes b. Myocardial ischemia/infarction (putative) c. Hypertension d. Atherosclerotic vascular disease (putative) V. Other a. Migraine b. Asthma c. Colon cancer

When Mg deficiency is recognized in the clinical setting, it is usually of moderate-to-severe depletion. Biochemical, neuromuscular, and cardiac complication are the most prevalent findings in the Mg-deficient patient.

Hypocalcemia Calcium is the major regulator of parathyroid hormone (PTH) secretion. Mg however modulates PTH secretion via the Ca2+ -sensing receptor in a manner similar to calcium (42). Although acute changes in the extracellular Mg concentrations will influence PTH secretion qualitatively similar to calcium, Mg deficiency perturbs mineral homeostasis (42,43). Hypocalcemia is a prominent manifestation of Mg deficiency. Mg deficiency must become moderate to severe before symptomatic hypocalcemia develops. Mg therapy alone restores serum calcium concentrations to normal. Calcium and/or vitamin D therapy will not correct the hypocalcemia. One major cause for the hypocalcemia is impaired parathyroid gland function. The majority of patients with hypocalcemia due to Mg deficiency have low or inappropriately normal serum PTH levels. The administration of Mg will result in an immediate rise in the serum PTH level. The presence of normal or elevated serum concentrations of PTH in the face of hypocalcemia suggests that there may also be end-organ resistance to PTH action. Skeletal resistance to exogenous PTH in hypocalcemic Mg-deficient patients has been reported. Similarly, urinary excretion of cyclic AMP and/or phosphate in response to PTH in such patients has been observed (42,43). The mechanism for impaired PTH secretion and action in Mg deficiency remains unclear. It has been suggested that there may be a defect in the second messenger systems in Mg depletion. Adenylate cyclase has been universally found to require Mg for cyclic AMP generation both as a component of the substrate (Mg-ATP) and as an obligatory activator of enzyme activity. PTH has also been shown to activate the phospholipase C second messenger system. Mg depletion could perturb this system via several mechanisms as an Mg2+ -dependent guanine nucleotide regulating protein is involved in activation of phospholipase C and Mg2+ has also been shown to be a noncompetitive inhibitor of IP3-induced Ca2+ release (43). Mg is also important in vitamin D metabolism and/or action (42,43). Patients with hypocalcemia and Mg deficiency have also been reported to be resistant to pharmacological doses of vitamin D, 1␣ hydroxy vitamin D and 1,25-dihydroxy-vitamin D. The exact nature of altered vitamin D metabolism and/or action in Mg deficiency is unclear. Serum concentrations of 1,25-dihydroxyvitamin D have been found to be low or low normal in most hypocalcemic Mg-deficient patients. Because PTH is a major trophic for 1,25-dihydroxy vitamin D formation, the low serum PTH concentrations could explain the low 1,25-dihydroxy vitamin D levels suggesting that Mg deficiency in man impairs the ability of the kidney to

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synthesize 1,25-dihydroxy-vitamin D. Mg is known to support the 25-hydroxy-1␣-hydroxylase in vitro.

Hypokalemia A common feature of Mg depletion is hypokalemia (44,45). Experimental human Mg deficiency demonstrated a negative potassium balance resulting from increased urinary loss. During Mg depletion there is also loss of intracellular potassium. Attempts to replete the potassium deficit with potassium therapy alone are not successful without simultaneous Mg therapy. The reason for this disrupted potassium metabolism may be related to Mg dependence of the Na, K, ATPase. During Mg depletion, intracellular sodium and calcium rise, and Mg and potassium fall. Mg also appears to be important in regulation of potassium channels in cardiac cells that are characterized by inward rectification. This biochemical feature may be a contributing cause of the electrocardiographic findings and cardiac dysrhythmias discussed later.

Neuromuscular Manifestations Neuromuscular hyperexcitability is a common presenting complaint of a patient with Mg deficiency (13). Latent tetany, as elicited by a positive Chvostek’s and Trousseau’s sign, or spontaneous carpal-pedal spasm may be present. Seizures may also occur. Although hypocalcemia contributes to the neurologic signs, Mg deficiency without hypocalcemia has been reported to result in neuromuscular hyperexcitability. Other signs occasionally seen include vertigo, ataxia, nystagmus and athetoid, and choreiform movements. Muscular tremor, fasciculation, wasting, and weakness may be present. Reversible psychiatric aberrations also have been reported. There may be several mechanisms for these neuromuscular problems. Mg has been shown to stabilize the nerve axon. Lowering the serum Mg concentration decreases the threshold of axonal stimulation and increases nerve conduction velocity. Mg also has been shown to influence the release of neurotransmitters, such as glutamate, at the neuromuscular junction by competitively inhibiting the entry of calcium into the presynaptic nerve terminal. It is likely that a decrease of extracellular Mg would allow a greater influx of calcium into the presynaptic nerves and the subsequent release of a greater quantity of neurotransmitters, resulting in hyperresponsive neuromuscular activity.

dysrhythmias may be resistant to usual therapy. As intracellular Mg depletion may be present despite a normal serum Mg concentration, Mg deficiency always must be considered as a potential factor in cardiac dysrhythmias.

Cardiovascular: Acute Myocardial Infarction Acute myocardial infarction (AMI) is the leading cause of death in the United States. Mg deficiency may be a risk factor as it has been shown to play a role in systemic and coronary vascular tone (see later), cardiac dysrhythmias as mentioned earlier, and by inhibiting platelet aggregation. Over the past decade, debate arose over the clinical utility of adjunctive Mg therapy for AMI. Although several small controlled trials suggested that adjunctive Mg therapy reduced mortality from AMI by 50%, three major trials define our understanding regarding Mg therapy in AMI (47). LIMIT-2 was the first study involving large numbers of participants. Over a 6-year period, 2316 participants with suspected AMI were randomized to receive adjunctive Mg therapy or placebo. The Mg-treated group showed an approximately 25% lower mortality rate (7.8% vs. 10.3%; P < 0.04). The ISIS-4 study randomized over 58,000 participants over a 3-year period to examine the effects of captopril, nitrates, and Mg on AMI. Unlike LIMIT-2, the mortality rate in the Mg-treated group was not significantly different from the control group (7.64% vs. 7.24%). The conclusion was that Mg therapy was not indicated in suspected AMI. Despite the null result, some suggested that the ISIS-4 design masked the benefits of Mg therapy. Two major criticisms involved the timing of the Mg therapy and the severity of patient illness. ISIS-4 randomized participants up to 24 hours after presentation. The leading theory regarding the role of Mg therapy in AMI involves the prevention of ischemia-reperfusion injury. The recently published MAGIC Trial was designed to address the issues regarding ISIS-4 study design; namely, early intervention in higher risk patients would more likely show the benefit of Mg therapy (47). Over a 3-year period, 6213 participants were studied. The Mg-treated group mortality at 30 days was not significantly different from the placebo group mortality (15.3% vs.15.2%). Unless there is a high suspicion of Mg deficiency, the overall evidence from clinical trials does not support the routine application of adjunctive Mg therapy in patients with AMI (48)

CHRONIC LATENT MAGNESIUM DEFICIENCY Cardiovascular Manifestations: Dysrhythmias Cardiac dysrhythmias are an important consequence of Mg deficiency. Electrocardiographic abnormalities of Mg deficiency in humans include prolonged P-R interval and Q-T interval. Intracellular potassium depletion and hypokalemia are complicating features of Mg deficiency and may contribute to these electrocardiographic abnormalities. Mg-deficient patients with cardiac dysrhythmias have been treated successfully by Mg administration (46). Supraventricular dysrhythmias including premature atrial complexes, atrial tachycardia, atrial fibrillation, and junctional arrhythmias have been described. Ventricular premature complexes, ventricular tachycardia, and ventricular fibrillation are more serious complications. Such

Although the diets ordinarily consumed by healthy Americans fall below the RDA (12), they do not appear to lead to symptomatic Mg depletion. A number of clinical disorders however have been associated with a low-Mg diet. It has been suggested that more mild degrees of Mg deficiency present over time may contribute to disease states such as hypertension, coronary artery disease, preeclampsia, and osteoporosis.

Hypertension A number of studies have demonstrated an inverse relationship between populations that have low dietary intake of Mg and blood pressure (7,49). Hypomagnesemia

Magnesium

and/or reduction of intracellular Mg have also been inversely correlated with blood pressure. Patients with essential hypertension were found to have reduced free Mg2+ concentrations in red blood cells. The Mg2+ levels were inversely related to both systolic and diastolic blood pressure. Intervention studies with Mg therapy in hypertension have led to conflicting results. Several studies have shown a positive blood-pressure lowering effect of Mg supplements whereas others have not. Other dietary factors may also play a role. A diet of fruits and vegetables, which increased Mg intake from 176 to 423 mg/day (along with an increase in potassium), significantly lowered blood pressure (50). The addition of nonfat dairy products that increased calcium intake as well further lowered blood pressure. The mechanism by which Mg deficiency may affect blood pressure is not clear but may involve decreased production of prostacyclin, increased production of thromboxane A2, and enhanced vasoconstrictive effect of angiotensin II and norepinephrine. Recently, it has been suggested that vascular TRPM7 Mg channel may be altered in hypertension (7).

Atherosclerotic Vascular Disease Another potential cardiovascular complication of Mg deficiency is the development of atheromatous disease (51). Lipid alterations have been reported in hypomagnesemic human subjects; however, they are often complicated by factors related to underlying lipoprotein abnormalities occurring in diabetes, coronary artery disease, myocardial infarction, and other diseases. Epidemiologic studies have related water hardness (calcium and Mg content) inversely to cardiovascular death rates. Platelet hyperactivity is a recognized risk factor in the development of cardiovascular diseases. Mg has been shown to inhibit platelet aggregation against a number of aggregation agents. Diabetic patients with Mg depletion have been shown to have increased platelet aggregation. Mg therapy in these subjects returned the response toward normal. The antiplatelet effect of Mg may be related to the finding that Mg inhibits the synthesis of thromboxane A2 and 12-HETE, eicosanoids thought to be involved in platelet aggregation. Mg also inhibits the thrombin-induced Ca influx in platelets as well as stimulates synthesis of PGI2 , the potent antiaggregatory eicosanoid.

Preeclampsia and Eclampsia Preeclampsia complicates 1 in 2000 pregnancies in developed countries and is responsible for over a 50,000 maternal deaths per year. Mg therapy has been used for decades in both preeclampsia and eclampsia and contributes to the very low mortality rate in developed countries (52). Despite decades of use, no large randomized trial examining the efficacy of Mg therapy had been performed until the MAGPIE Trial in 2002. This trial, which compared women with preeclampsia treated with MgSO4 to nimodipine, a specific cerebral arterial vasodilator, showed a lower risk (0.8% vs. 2.6%) of eclampsia in the Mg therapy group (52). The Mg status of women with preeclampsia has been difficult to establish. No difference was found in the plasma Mg levels of women with preeclampsia and those of healthy pregnant women; however, in women

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with preeclampsia there was a decreased RBC Mg level. Women with preeclampsia and women pr-term labor had no differences in ionized or total serum Mg levels. Although subtle deficits in total body Mg may contribute to hypertension during pregnancy, the role of Mg may relate more to its stabilizing neuronal and vascular effects rather than the correction of an electrolyte deficit. Mg therapy is clearly indicated for women with preeclampsia. It has been shown to decrease the incidence of eclampsia and likely to decrease overall mortality.

Osteoporosis Dietary Mg deficiency in animals results in a decrease in growth of the skeleton (17,43). Osteoblastic bone formation has been found reduced. Markers of bone formation have been reduced, suggesting a decease in osteoblastic function. An increase in the number and activity of osteoclasts in the Mg-deficient rats and mice has been reported. Bone from Mg-deficient rats has been described as brittle and fragile. Biomechanical testing has directly demonstrated skeletal fragility in both rats and pigs. In humans, epidemiologic studies have demonstrated a correlation between bone mass and dietary Mg intake in appendicular and axial skeleton (43). Few studies have been conducted assessing Mg status in patients with osteoporosis. Low serum and red blood cell Mg concentrations as well as high retention of parenterally administered Mg has suggested a Mg deficit, however these results are not consistent from one study to another. Similarly, whereas low skeletal Mg content has been observed in some studies, others have found normal or even high Mg content. The effect of dietary Mg supplementation on bone mass in patients with osteoporosis has not been extensively studied. The effect of Mg supplements on bone mass has generally led to an increase in bone mineral density, although study design limits useful information. Larger longterm placebo-controlled double-blind investigations are required. There are several potential mechanisms that may account for a decrease in bone mass in Mg deficiency. Mg is mitogenic for bone cell growth that may directly result in a decrease in bone formation. Mg also affects crystal formation; a lack of Mg results in a larger, more perfect crystal, which may affect bone strength. Mg deficiency results in a fall in both serum PTH and 1,25(OH)2 D as discussed earlier. Because both hormones are trophic for bone, impaired secretion or skeletal resistance may result in osteoporosis. An increased release of inflammatory cytokines may result in activation of osteoclasts and increased bone resorption in rodents (17,43).

OTHER DISORDERS Mg deficiency has been associated with migraine headache and Mg therapy has been reported to be effective in the treatment of migraine (53). Because Mg deficiency results in smooth muscle spasm, it has also been implicated in asthma and Mg therapy has been effective in asthma in some studies (54). Lastly, a high dietary Mg intake has been associated with reduced risk of colon cancer (55).

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MANAGEMENT OF DEPLETION Seizures, acute arrhythmias, and severe generalized spasticity require immediate IV infusion. One to two grams MgSO4 ·7H2 O (8.2–16.4 mequiv Mg2+ ) is usually infused over 5 to 10 minutes, followed by continuous infusion of 6 g over 24 hours or until the condition is controlled. Correction of electrolyte (especially potassium) and acid– base imbalances should accompany the Mg therapy. In addition, levels of serum Mg and other electrolytes should be determined at least twice daily in such patients. Less severe manifestations (e.g., paresthesias with latent or active tetany) are likewise best treated by the IV route, again in conjunction with appropriate therapy for the underlying condition and with correction of other electrolyte and acid–base abnormalities. When renal function is good, 6 g (48 mequiv) of Mg sulfate may be given intravenously over 24 hours in saline or dextrose solutions, with other nutrients as required. This may be continued for 3 to 5 days until the signs and symptoms and/or electrolytes abnormalities are corrected. When the IV route cannot be used, IM injections can be given, although these are painful. This regimen is continued for 2 or more days, and the situation is then reassessed. The dosages given must always exceed the daily losses as indicated by serum levels and urinary excretion. The return to the normal or slightly higher range of serum Mg levels with any of these schedules is relatively rapid. However, repletion of Mg lost from bone and other tissues requires more prolonged Mg therapy. When intestinal absorption is normal and renal Mg wasting is present, supplements should be added to the usual diet to tolerance (onset of diarrhea) to maintain normal serum levels. In some instances, oral Mg may not be sufficient and IM and/or IV Mg may be required. Those with continuing severe Mg and potassium losses in the urine (as in cisplatin nephrotoxicity or hereditary renal defects) may require long-term supplements by IV infusion via an indwelling central catheter for home administration. When depletion is modest and persistent, initial efforts should be directed to increased intake of Mg-rich foods. When necessary and feasible, supplementary oral Mg may be taken. Three hundred to six hundred milligrams may be given in divided doses three to six times per day, with a full glass of water to prevent or minimize Mg-related diarrhea and to ensure solubilization. For the individual on enteral feeding, one of these salts may be dissolved in the formula. Improvement of existing steatorrhea by dietary or other medical means will decrease fecal Mg losses. Again, treatment of underlying disease and replacement of potassium deficits are essential.

CONCLUSIONS In conclusion, Mg is a vital nutrient necessary for essential biologic processes. Despite its rigid homeostasis by the body, Mg deficiency is not uncommon due to numerous diseases, disorders, and medications, which impair the normal metabolism of magnesium. The relatively low dietary Mg intake compounds this problem in many patients. Indeed, even the reduction in suggested dietary

Mg may contribute to chronic disease states in otherwise healthy individuals. It is clear that education is necessary for the general population to increase awareness of the importance of Mg in maintaining health. In addition, further research efforts in both basic science and clinical science are needed to clarify the role of Mg deficiency in disease states.

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FURTHER READING 1. Cowan JA. Introduction to the Biological Chemistry of Magnesium. In: The Biological Chemistry of Magnesium: Cowan JA, ed. New York: VCH Publishers, 1995:1–24. 2. Rude RK, Shils ME. Magnesium. In: Shils ME, ed. Modern Nutrition in Health and Disease. Philadelphia, PA: Lippincott Williams and Wilkins, 2006:223–247.

Melatonin Amnon Brzezinski and Richard J. Wurtman

identifying the active compound, using assays based on the ability of purified extracts to aggregate the melanin granules in the frog’s pigment cells. In 1958, Lerner et al. (2) discovered the compound’s chemical structure to be 5-methoxy-N-acetyltryptamine and named it melatonin. Around that time, scientists made four seemingly unrelated discoveries, which became coherent, like a partly completed crossword puzzle, once melatonin was identified. In chronologic sequence, these were (i) the demonstration, by Kitay and Altschule, that surgical removal of the rat’s pineal accelerated the growth of the ovaries, whereas administration of bovine pineal extracts had the opposite effect (3); (ii) Fiske’s observation that housing rats in a continuously lit environment led to a decrease in the weights of their pineals (4); (iii) Ariens– Kappers’ discovery (5) that, though the pineal gland originates embryologically as part of the brain, it loses most or all of its CNS connections by birth, and instead receives its innervation from peripheral sympathetic nerves; and (iv) the demonstration that both pinealectomy and prolonged light exposure accelerate the growth of the rat’s ovaries to an equal extent, and that both responses are blocked by administering pineal extracts (6). In 1963–1964, it was shown that melatonin is a true hormone in rats, that it is the gonad-inhibiting substance previously described in pineal extracts (7), and that its synthesis in the pineal gland is suppressed when rats are exposed continuously to light, the light acting not directly, as on a “third eye,” but indirectly, via the animal’s eyes and sympathetic nerves (8). [The chemical that mediates the sympathetic nervous signals was shown to be norepinephrine (9), which stimulates pineal beta-receptors and increases cyclic-AMP production (10).] The rates at which the rat’s pineal synthesizes serotonin and melatonin were soon shown to vary with circadian rhythms, and the melatonin rhythm was ultimately found to be generated by intrinsic circadian signals emanating from the suprachiasmatic nucleus (SCN) of the brain (11), the phasing of which was controlled primarily by the light–dark cycle. Finally, in 1975, it was shown that melatonin production in humans also exhibits a pronounced circadian rhythm (12), causing nocturnal plasma melatonin levels to be at least 10-fold higher than those observed in the daytime. Moreover, this rhythm was not simply a response to the environmental light–dark cycle, because if people were suddenly placed in an environment that was dark between 11 a.m. and 7 p.m. (instead of the usual 11 p.m. to 7 a.m.), it took their melatonin rhythms 5 to 7 days to re-entrain. The view thus became canonized that the

INTRODUCTION Melatonin is a hormone, like the estrogens and testosterone: It is synthesized in the pineal gland and secreted into the blood and cerebrospinal fluid. It conveys signals to distant organs, principally the brain, which affect the synthesis of second messengers and, ultimately, sleep and circadian rhythms. However, unlike the estrogens and testosterone, melatonin is marketed in the United States as a dietary supplement, which implies that people normally obtain this compound from the diet and that melatonin pills simply supplement that which the diet provides. No food has ever been found to elevate plasma melatonin levels nor is there acceptable evidence that any food actually contains more than trace amounts of the hormone. This entry describes the history of our knowledge of melatonin; the hormone’s synthesis, metabolism, and physiologic regulation; the factors that affect plasma melatonin levels; the known effects of endogenous and exogenous (oral) melatonin; and the present usage of melatonin and some synthetic analogs.

HISTORY OF MELATONIN Few people would now doubt that the human pineal gland is an important structure, and that it transmits signals to the brain and other organs by secreting a unique hormone, melatonin. However, this consensus is only a few decades old. For most of the twentieth century, the pineal was generally dismissed as a “vestige”—a “third eye” in certain lower vertebrates, which, in humans, died and became calcified early in life. Tumors of the pineal gland were known sometimes to be associated with a reproductive disorder—precocious puberty, especially in boys—and some scientists attributed this phenomenon to the destruction of functioning pineal tissue. However, most concluded that the accelerated sexual maturation simply resulted from increased intracranial pressure or from the secretion of gonadotropins from tumor tissue. The modern history of the pineal gland probably began with the discovery in 1917 (1) that extracts of cow pineals could lighten the skin of frogs. The physiologic significance of this relationship seemed obscure, inasmuch as bovine pineal extracts had no effect on pigmentation in bovines (or humans), and frog pineals lacked detectable skin-lightening ability. However, the finding did indicate that the pineal contained a compound with at least some biological activity, and it provided a way of 538

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pineal is a “neuroendocrine transducer” (13) that tells all mammals when it is dark outside by raising plasma melatonin levels. The uses to which the body puts this information vary considerably among species: In diurnal, but not nocturnal, animals, melatonin promotes sleep onset and maintenance; in animals that breed seasonally, melatonin affects the choice of breeding season (i.e., spring or fall); and in those like humans and rats, which breed throughout the year, melatonin’s reproductive effects can be minimal. Much subsequent pineal research has concerned the human brain’s responses to melatonin. The most compelling evidence now available supports two such uses that are discussed below: the involvement of nocturnal melatonin secretion in initiating and maintaining sleep, and control by the day/night melatonin rhythm of the timing of other 24-hour rhythms. It is melatonin’s effect on sleep that underlies most of its current use as a “dietary supplement.” Some additional possible benefits of melatonin supplementation have been proposed (e.g., as an antioxidant, or to slow aging, or to suppress cancer growth and hypertension). However, evidence supporting these effects is sparse. Evidence is even more sparse that there is any rational basis for calling melatonin a “dietary supplement.” For melatonin to earn this appellation, it would have to be shown that at least some of the melatonin in human plasma derives from food sources, and that “supplementary” exogenous melatonin simply adds to what the foods provide. But as described later, there is no satisfactory evidence, on the basis of contemporary analytic techniques, that any actual foods contain more than trace amounts of melatonin—if that—and no evidence at all that eating any food elevates human plasma melatonin levels. Melatonin is a hormone, such as thyroxine and estrogens, and should be labeled and regulated as such. Only its extraordinary lack of overt toxicity apparently keeps the Food and Drug administration (FDA) from insisting that it undergo such regulation.

Almost all the melatonin formed in mammals is synthesized within the pineal gland, starting with the uptake of the amino acid tryptophan from the plasma. Because the pineal lies outside the blood–brain barrier, this process—in contrast to tryptophan’s uptake into the brain—is not subject to competition from other circulating neutral amino acids and is not enhanced by carbohydrate consumption and insulin secretion. The tryptophan is first 5-hydroxylated (by the enzyme tryptophan hydroxylase) and then decarboxylated (by the enzyme aromatic Lamino acid decarboxylase) to form 5-hydroxytryptamine or serotonin (Fig. 1) (9). During daylight hours, the serotonin in pinealocytes tends to be stored and is unavailable to enzymes (monoamine oxidase and the melatonin-forming enzymes) that would otherwise act on it. With the onset of darkness, postganglionic sympathetic outflow to the pineal increases and the consequent release of norepinephrine to pinealocytes causes stored serotonin to become accessible for intracellular metabolism. At the same time, the norepinephrine activates the enzymes [especially serotonin-N-acetyltransferase (SNAT), but also hydroxyindole-O-methyltransferase (HIOMT)] that convert serotonin to melatonin (Fig. 1) (9,11). Consequently, pineal melatonin levels rise manyfold. (Pineal levels of 5-methoxytryptophol, the corresponding deaminated and O-methylated metabolite of serotonin, also rise (14) even though formation of this compound is independent of SNAT.) The melatonin then diffuses out of the pineal gland into the blood stream and cerebrospinal fluid (15), rapidly raising human plasma melatonin levels from approximately 2–10 to 100–200 pg/mL (12). Melatonin is highly lipid soluble, because both the ionizable groups in serotonin—the hydroxyl and the amine—have been blocked by its O-methylation and N-acetylation (Fig. 1).

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Thus, it diffuses freely across cell membranes into all tissues, and travels in the blood largely bound to albumin. Most of the melatonin in the circulation is inactivated in the liver, where it is first oxidized to 6-OHmelatonin by a P450-dependent microsomal oxidase and then largely conjugated to sulfate or glucuronide before being excreted into the urine or feces (16). Approximately 2% to 3% is excreted unchanged into the urine or saliva, enabling measurements of urinary or salivary melatonin to be used as rough estimates of plasma melatonin levels. (Salivary melatonin apparently corresponds to the 25–30% of blood melatonin that is not bound to albumin.) Studies using radioactively labeled melatonin of high specific activity have identified three probable melatonin receptors, two of which have been cloned by using human sources (17). These macromolecules are concentrated, respectively, within the suprachiasmatic nucleus of the hypothalamus, the pars tuberalis of the pituitary and cardiac blood vessels (mt1 ), in the retina and hippocampus (MT2 ), and in kidney, brain, and various peripheral organs (MT3 ). Their affinities for melatonin are enhanced by several G-proteins. Activation of the mt1 and MT2 receptors by melatonin suppresses cAMP production. The MT3 site shares 95% homology with a detoxifying enzyme, quinone reductase 2; its effects on specific signal transduction pathways await identification. Because of melatonin’s unusual lipid solubility, its receptors could be located intracellularly, in contrast to the plasma membrane receptors characteristic of neurotransmitters; indeed, a nuclearbinding site has been identified. The mt1 receptors in the SCN allow melatonin to inhibit the firing of SCN neurons during the night—an action that might contribute to melatonin’s sleep-promoting effects. The SCN’s MT2 receptors apparently mediate melatonin’s effects on the SCN’s own circadian rhythms, as well as on other rhythms that this brain region controls. In all species examined thus far, melatonin secretion manifests a characteristic circadian rhythm, causing plasma levels to be low during the daylight hours, ascend after the onset of darkness, peak in the middle of the night between 11 p.m. and 3 a.m., and then fall sharply before the time of light onset. (It is interesting that high nocturnal plasma melatonin levels characterize both diurnally active species, in which these levels promote sleep onset and maintenance, and nocturnally active ones, in which melatonin has no obvious relationship to sleep.) Although this rhythm is normally tightly entrained to the environmental light cycle, it does persist when people are placed for a few days in a dark room, and it does not immediately phase-shift when the light schedule is altered, indicating that it is not simply generated by the light–dark cycle but also by cyclic endogenous signals, probably from the SCN (11). These reach the pineal via a retinohypothalamic tract, the superior cervical ganglia, and postganglionic sympathetic fibers that re-enter the cranial cavity (5). In certain fish, birds, and reptiles, pineal glands also contain true photoreceptors, and denervated (or even cultured) glands can sustain circadian rhythms in melatonin synthesis that can be entrained by the light– dark cycle; in contrast, light has no known direct effects on melatonin synthesis in human or other mammalian pineals.

PLASMA MELATONIN LEVELS Plasma melatonin normally reflects the amounts secreted by the pineal gland, the flux of melatonin in and out of tissues, melatonin’s destruction in the liver, and its secretion into urine and saliva. Because melatonin is now also available as a dietary supplement, plasma levels can reflect consumption of the exogenous compound as well. Available evidence does not support the view that humans derive any plasma melatonin from foods. Several laboratories have described a compound in dietary fruits or vegetables [e.g., tomato (18,19)] that they concluded was melatonin. But in only one of these studies (19) was the identity of the melatonin unambiguously confirmed by gas chromatography-spectrometry (GCMS), and in that study, the melatonin concentrations determined by GCMS were very low (less than 20 ng/kg of fruit), and the “. . . concentrations . . . indicated by RIA were 6–100fold higher than . . . by GCMS for the same extracts, suggesting . . . contamination by an immunological interference . . .” Of perhaps greater relevance, no investigator has ever presented evidence that feeding any amount of any food to humans can raise plasma melatonin levels. Usually, the principal factor affecting plasma melatonin levels is its rate of secretion, which varies with the circadian rhythm described above and as a function of age (Fig. 2). Nocturnal melatonin levels are also affected by drugs that interfere with the transmission of neurotransmitter signals to pineal cells (like propranolol, a beta-blocking agent (20), those that inhibit melatonin’s metabolism (like 8-methoxypsoralen (21), and a few drugs that lack clear links to melatonin’s synthesis or metabolism (e.g., caffeine, ethanol (22), ibuprofen, and indomethacin, which decrease melatonin). Nocturnal melatonin secretion is also suppressed by exposure to environmental lighting (23), even by a relatively dim 100-200 lx, when pupils are dilated. Melatonin secretion by the human pineal gland exhibits a pronounced age dependence (Fig. 2). Secretion is minimal in newborns, starts during the third or fourth months of life (coincident with the consolidation

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of sleeping at night (24)), increases rapidly at ages 1–3 year, and then declines slightly to a plateau that persists through early adulthood. Nocturnal melatonin secretion then starts a marked continuing decline in most people, with peak nocturnal levels in most 70-year-olds being only a quarter or less of what they are in young adults (25). This decline may reflect the progressive unexplained but ubiquitous calcification of the pineal gland and resulting loss of secretory tissue. Obviously, one strategy in using supplemental melatonin is to administer to older people doses that are just sufficient to compensate for this age-related decline. The first person to examine the effects of exogenous melatonin was the scientist who discovered it, Aaron Lerner; he explored its actions (and possible toxicities) by giving himself 200 mg IV/day for 5 consecutive days. Lerner described feeling “relaxed.” Neither he nor the investigators who subsequently gave it (in doses of 10 mg to 6.6 g) to 96 other subjects prior to 1977 measured its effects on plasma melatonin levels. However because most of them administered doses in excess of 1 g, it can be assumed that massive increases in plasma melatonin ensued. When Waldhauser et al. (26) administered 80-mg doses to two male volunteers in 1987, plasma levels increased more than 1000-fold, and serum prolactin levels rose significantly—an effect not observed with physiologic melatonin doses. In 1993, Dollins et al. examined the effects of 10, 20, 40, or 80 mg melatonin on various behavioral indices (auditory vigilance; self-reported fatigue, confusion, and sleepiness; reaction times), body temperature, and plasma melatonin levels. All the doses tested produced similar changes in the behavioral assays and in body temperature. And all raised plasma melatonin levels to at least 5000 pg/mL—well beyond the normal nocturnal range of 100 to 200 pg/mL (27). Hence, the study was repeated using much lower doses (0.1–10 mg orally) (28). The authors found that oral doses as low as 0.1 to 0.3 mg caused dose-related decreases in sleep latency and increases in sleep duration and self-reported sleepiness and fatigue, but without reducing body temperature or elevating plasma melatonin levels beyond their normal nocturnal range (Fig. 3). This suggested that nocturnal melatonin secretion—which produces plasma melatonin levels similar to those seen after the 0.3-mg dose—has a physiologic effect on sleep. It also identified the dosage range that investigators needed to use if they wanted to examine melatonin’s physiologic effects. It should be noted that there is considerable personto-person variability in the bioavailability of melatonin: In one study using single 80-mg doses, there were 25fold variations in areas under the curve (AUCs) in the five subjects studied. In another, using 0.5 mg oral doses, peak plasma melatonin levels among four subjects varied from 480 to 9200 ng/L (29). Melatonin’s bioavailability was relatively poor—10% to 56%—which the authors attributed to person-to-person differences in firstpass hepatic extraction, perhaps reflecting such differences in hepatic function. Older subjects given a 0.3-mg oral dose of melatonin exhibit considerably greater increments in plasma melatonin levels, with correspondingly greater variability, than young adults receiving that dose.

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These findings all suggest that although a 0.3-mg dose given to young subjects during the daytime, or to older insomniacs at night, can, on average, produce normal nocturnal plasma melatonin levels, some individuals may need a little more, or a lot less, melatonin to attain this effect. The pharmacokinetic properties of any oral dosage of melatonin can also vary depending on the lipid solubility of the inert ingredients that accompany it. A preparation containing corn oil plus 0.05 mg melatonin elevated plasma melatonin levels to as high a peak [from 4 to 118 pg/mL (30), though for a shorter period, as one containing 0.3 mg melatonin plus microcrystalline cellulose (15–105 pg/mL (28)].

EFFECTS OF MELATONIN Because melatonin is available as a dietary supplement and is relatively nontoxic, physicians, researchers, and even consumers are able to administer or consume doses that elevate its plasma levels to hundreds or even thousands of times those ever occurring normally (26,31). Indeed, even the 1 to 10 mg doses most commonly marketed raise these levels to 3 to 60 times their normal peaks (Fig. 3) (28). Not surprisingly, such concentrations produce biological effects, for example, sleepiness, which might or might not also occur physiologically. Does the demonstration that a pharmacologic dose of melatonin produces such an effect indicate that the effect also occurs at normal nighttime plasma melatonin levels? Or, by extension, that a deficiency in melatonin (e.g., in older people) can contribute to a related disease process? Alas, no; enormous melatonin concentrations inhibit the aggregation of A-beta peptides to form amyloid in vitro (32); however, this no more means that the age-related decline in plasma melatonin causes Alzheimer’s disease than that poison ivy dermatitis—which can be treated with cortisone—is a sign of adrenocortical insufficiency.

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What evidence must be adduced before one can propose that some effect of a melatonin megadose also occurs in response to secreted melatonin? First, that the effect occurs when plasma melatonin levels rise or fall within their normal range. Second, that administering melatonin in the daytime, in doses that increase plasma melatonin concentrations to—but not beyond—peak nighttime levels, also produces the effect. This type of study can sometimes be done in vitro. If melatonin were found to suppress ␤-amyloid aggregation at concentrations found nocturnally in plasmas of young people (up to approximately 1 nM), but not in concentrations more typical of many older people (less than 0.3 nM), this would indeed be suggestive. By using these criteria, two probable physiologic effects have been associated with melatonin administration—the promotion of sleep onset and maintenance (28), and the phase-shifting of circadian rhythms, including the rhythm in melatonin itself (33). Both are produced by physiologic doses, that is, 0.1 to 0.3 mg for sleep and 0.5 mg for phase-shifting. Melatonin’s actions on sleep include both a direct action (which decreases sleep latency, increases sleep efficiency, and increases total sleep time) and an indirect effect on the daily rhythm in the phasing of sleep onset.

Sleep A 1997 review (34) on melatonin’s hypnotic effects listed 24 papers, almost all of which described sedation, fatigue, decreased alertness, increased reaction time, shortened sleep latency (i.e., number of minutes needed to fall asleep), increased sleep efficiency (i.e., percentage of the total sleep period actually spent sleeping), and/or increased total sleep time. A recent (2005) meta-analysis (35) of all the 17 studies (36–43), involving 284 subjects, which satisfied inclusion criteria demonstrated a significant decrease in sleep latency and significant increases in sleep efficiency and total sleep duration. The inclusion criteria were that a study include at least six subjects, all adults, be randomized and double-blinded, involve placebo-controlled clinical trials, and use objective measures of sleep evaluation. Studies could use cross-over or parallel group designs; however, case reports were excluded. Statistical significance was obtained in spite of considerable variations among the studies in melatonin doses and routes of administration, the general health of the subjects, and the measures used to evaluate sleep. The effects of exogenous melatonin on sleep have been examined under three types of experimental conditions in relation to the onset or offset of endogenous melatonin secretion. In some studies, the hormone was administered during the daily light period, such that blood melatonin levels would be transiently elevated but would then return to baseline before the initiation of nocturnal melatonin secretion. Such experiments were used to demonstrate that melatonin decreases sleep latency at any time in the afternoon or evening, and that this effect is independent of an action on sleep rhythms (as no treatment can immediately shift the phase of a circadian rhythm by 8–10 hour). In others, the hormone was given close enough to the onset of darkness for blood melatonin levels to still be elevated when nocturnal melatonin secretion started.

The period during which plasma melatonin levels were continuously elevated would thus be prolonged. Such experiments reflected the use of melatonin to decrease sleep latency and maintain continuous sleep in, for example, a shift worker or eastbound world traveler who needed to start sleeping earlier. In yet others, the hormone was given at the end of the light period to older insomniacs with low nighttime plasma melatonin levels. The intent was to prolong the portion of the night during which their plasma melatonin concentrations would be in the same range as those of noninsomniac young adults. In all these situations, oral melatonin decreased sleep latency and, when tested, increased sleep duration and sleep efficiency. A 0.3-mg dose was either as effective as, or more effective than (44), higher doses, particularly when the hormone was administered for several days. This dose had no effect on body temperature, affirming that, although pharmacologic doses can cause hypothermia, melatonin’s ability to promote sleep is not mediated by such a change, as had been suggested. The hormone had no consistent effect on sleep architecture (e.g., REM time). Its effects differed from those of most hypnotic drugs, as after receiving melatonin, subjects could readily keep from falling asleep if they so chose and their cognitive abilities the next morning were unchanged or improved. In a relatively large (N = 30) study (44) on people who were 50 years old or older and did or did not suffer from clinically significant insomnia (i.e., sleep efficiencies of 70–80% in the insomniacs vs. 92% in controls), melatonin was found to produce statistically and clinically significant improvements in sleep efficiency among insomniacs (Fig. 4). A 0.3-mg dose caused the greatest effect (P < 0.0001), particularly during the middle portion of the nocturnal sleep period (Fig. 5). No effects were noted in subjects without insomnia, or in latency to sleep onset (which is not abnormal in this population). Dose-related increases in plasma melatonin levels were observed (Fig. 6), the 0.3-mg dose causing peak levels in the range usually observed nocturnally among young adults. When subjects received a higher dose (3.0 mg) but not 0.3 mg, plasma melatonin levels remained significantly elevated during much of the following day, and the subjects exhibited hypothermia (Fig. 7).

Circadian Rhythms: Phase-Shifting and Jet Lag The ability of exogenous melatonin to synchronize and shift the phases of various human circadian rhythms is generally accepted. As little as 0.5 mg of pure melatonin (33), or 0.05 mg of melatonin in corn oil (30) (which causes earlier peaks in plasma melatonin levels), advanced the onset of nocturnal melatonin secretion when administered at 5 p.m., (30) and larger doses caused greater phase advances. [The hormone was also able to shift the core body temperature rhythm. However, a statistically significant effect was found only after a dose that elevated plasma melatonin levels well beyond their normal range, i.e., to 1327 pg/mL (30).] As previously described, melatonin can also control the timing of sleep and sleepiness rhythms—an effect readily demonstrated among blind people with free-running melatonin and sleep rhythms (45) but also among sighted individuals.

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Melatonin’s ability to phase-shift circadian rhythms underlies its common use to prevent or treat “jet lag”— particularly that which is associated with eastbound travel (possibly because the melatonin can be taken while the traveler is still awake). A 1999 review (46) cited nine placebo-controlled field studies on this use; in seven, subjective measures of sleep and alertness improved. Adequate data are not available on the relationship between the ability of a particular melatonin dose to treat jet lag

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and to raise plasma melatonin levels. Some investigators recommend taking the melatonin at a specific time (e.g., at 2 a.m. in the traveler’s new geographic environment); others simply propose “. . . a . . . pre-flight early evening treatment before an eastbound flight, followed by treatment at bedtime for four days after arrival. . . .” (46). Westbound, the traveler is advised to take the melatonin late in the evening, to sustain nocturnal plasma melatonin levels for as long into the night as possible.

Reproduction Melatonin affects reproductive performance in a wide variety of species. In 1963, one of us (RJW) first reported that exogenous administration of melatonin reduces the weight of the ovaries of female rats (47). Since then, abundant evidence has been adduced that the pineal gland, acting via melatonin, affects reproductive performance in a broad range of animals (48). There is mounting evidence that the pattern of melatonin secretion, coupled to photoperiod, directly affects reproductive function. The major physiologic role of melatonin is to encode the daily light–dark cycle. The onset and offset of pineal melatonin secretion synchronize to dusk and dawn, respectively, and therefore the duration of the melatonin signal varies in proportion to the length of the night. In seasonal mammals, this variation in melatonin signal duration is used to synchronize neuroendocrine rhythms with the annual variation in day-length. In addition, fetal and newborn animals use the maternal melatonin signal to entrain endogenous circadian rhythms before direct photic information is available. The efficacy of exogenous melatonin in modifying particular reproductive functions has been found to vary markedly among species, depending on the age of the animal, the time at which melatonin is administered relative to the prevailing light–dark cycle, or phase of the estrus cycle (49). In most, but not all, animals, melatonin has an antigonadotrophic effect. Species that exhibit major seasonal shifts in gonadal function also tend to exhibit the greatest responses to exogenous melatonin. Seasonal changes in the number of hours per day that melatonin is secreted mediate the temporal coupling of reproductive activity to seasonal changes in day-length. In animals with short life cycles or a duration of gestation and time to weaning of approximately 1 year, circadian rhythms can provide a sense of time of the year. Thus day-to-day changes in the time of the re-entraining stimulus of light may be associated with the tightly controlled seasonal onset of puberty and adult infertility/fertility cycles. For example, the hamster reproductive system is inhibited by short photoperiod, leading to testicular regression in males and to anestrus in females (48). In rodents, puberty onset is altered by day-length so that long durations of darkness inhibit sexual maturation. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. The impact of short day-length is obvious in animals such as hamsters, which, outside the laboratory setting, live in higher latitudes and/or environments where food availability is highly seasonal. In

longer-lived species such as sheep, the time of puberty of animals born late in the season may even be delayed until the following year (48). These observations stimulated a search for a role for the pineal gland and melatonin in human reproduction. The clinical experience has yielded inconclusive and sometimes conflicting results (50). The following is a brief review of currently available information about the effects of melatonin on human reproductive processes (e.g., puberty, ovulation, fertility). Although humans are obviously not a seasonally breeding species, a seasonal distribution in human natural conception and birth rates has been reported by epidemiological studies in several geographical areas (51). Suppressed pituitary–ovarian activity (52) and reduction in the conception rate have been reported to occur during the dark winter near the Arctic Circle (53). These observations and the data from studies in mammals stimulated reproductive physiologists to search for a role for the pineal gland and melatonin in human sexual maturation (i.e. puberty) and reproduction (54,55). Puberty results from withdrawal of the “gonadostat” mechanisms and from increased gonadotropin sensitivity to GnRH. It has been hypothesized that GnRH release may be modulated by a nonsteroid-mediated mechanism. Modifications of neuropeptides, neurotransmitters, and neurosteroids may underlie the onset of pubertal processes (56). Melatonin may be an important factor in the complex process that occurs in the awakening of hypothalamic–pituitary–ovarian axis. The origin of the hypothesis that the human pineal affects puberty dates back to 1898 when Heubner (57) described a 4.5-yearold boy who exhibited both precocious puberty and a nonparenchymal tumor that destroyed the pineal. Many other similar cases were later described, mostly in boys (58). There are data showing an association between endogenous melatonin levels and the onset of puberty. It was hypothesized that melatonin has an antigonadotrophic effect on sexual maturation and that the timing of the onset of puberty is related to the observed statistical reduction in circulating melatonin concentrations with pubescence (59). It remains to be clarified, using longitudinal studies, whether plasma melatonin levels do, in fact, progressively decline in individuals undergoing puberty. Early studies reported conflicting observations on circulating melatonin levels during the normal menstrual cycle. More recent studies (60,61) failed to detect any fluctuations in melatonin secretion throughout the menstrual cycle. In fact, a remarkable consistency of the circadian pattern of melatonin secretion was observed, independent of the changes in plasma estrogen and progesterone levels. No significant effect on melatonin’s rhythm was noted in response to varying endogenous (ovarian stimulation (62) or exogenous [oral contraceptives (63)] sex steroids. These observations suggest that in humans, unlike some other species (64), melatonin secretion is not significantly modulated by sex hormones. The lack of effect of sex steroids on human melatonin secretion does not necessarily rule out a role for melatonin in human reproduction. In female rats, for example, which like humans are not seasonal breeders, large doses of exogenous melatonin completely inhibited ovulation and prevented the LH surge when administered during

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the critical period of proestrus. There are indications that abnormally elevated endogenous melatonin levels and pharmacologic doses of melatonin have antigonadal effects in humans as well. Increased plasma concentrations of melatonin were repeatedly found in women suffering of functional (“hypothalamic”) amenorrhea (60,65,66). Similar results were also reported for hypogonadotrophic men (67) and melatonin administration apparently alters semen quality in healthy men (68). Pharmacologic doses of exogenous melatonin, given to healthy young women (daily oral 300 mg for 4 months) altered ovarian activity and partially inhibited ovulation (69). A synergistic inhibitory effect on ovulation by a melatonin–progestin combination was reported by the same group. Observations of elevated melatonin levels in both men and women with hypogonadism and/or infertility are consistent with a hypothesis that melatonin is antigonadal in humans. The significant increase in circulating melatonin levels in women with functional amenorrhea raised the possibility of a causal relationship between high melatonin concentrations and suppressed hypothalamic–pituitary–gonadal axis in humans. Acute elevations of melatonin occur in response to fasting and sustained exercise (70). Both of these events, if prolonged, may cause amenorrhea. However, the hypersecretion of melatonin may merely be coincidental and reflect adrenergic, dopaminergic, or opiodergic secretions that are characteristic of these conditions. Another possible explanation lies in the fact that melatonin stimulates the production of prolactin in humans (71). Hyperprolactinemia in turn is known to interfere with the ovulatory process in humans. It has also been suggested that melatonin could exert an effect on human reproduction by directly modulating ovarian function (72) and spermatogenesis (68). Substantial amounts of melatonin are present in the ovarian follicular fluids of both stimulated (73) and spontaneous (74) menstrual cycles. The follicular concentrations of the hormone markedly exceeded those of serum samples obtained concurrently. Receptors to melatonin are highly concentrated in the human ovary (75) and have been observed on human granulosa cell membranes (76). Physiologic nighttime concentrations of melatonin-stimulated progesterone synthesis by human granulosa lutein cells in vitro (77) and increased the stimulatory effect of hCG on progesterone production by these cells (78). All theses findings strongly suggest that melatonin may play a role in the intraovarian regulation of steroidogenesis and thus abnormally high concentrations of the hormone might interfere with normal ovarian function. Pharmacologic high doses of melatonin have been tried as a contraceptive agent but the preliminary efficacy results did not justify further development (69). In conclusion, the data presented earlier demonstrate that the antigonadal effects of melatonin in humans are apparently much less significant than in some seasonally breeding mammalian species. This is not surprising as humans are clearly not “seasonally breeding” species. Currently, the balance of evidence from clinical studies suggests that the effect of melatonin on human reproductive processes such as ovulation and fertility is attenuated. Its role in the timing of the onset of puberty is substantial but it is difficult to differentiate its effect from the

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complex interplay among neuropeptides, neurotransmitters, and neurosteroids, which occurs in the awakening of hypothalamic–pituitary–ovarian axis. The genes that drive the circadian rhythm are emerging as central players in gene regulation throughout the organism, particularly for cell-cycle regulatory genes and the genes of apoptosis. The biological adaptation of humans probably includes development of some degree of photorefractoriness, which is correlated with a change in circadian expression of clock genes in the SCN (circadian pacemaker) and the pars tuberalis (PT, a melatonin target tissue) (79). Melatonin may have some modulatory effects on human diseases that are related to the reproductive system. For example, lighting during the night of sufficient intensity apparently reduces circulating melatonin levels and resets the circadian pacemaker of the suprachiasmatic nuclei. This phenomenon has been related to increased risk of breast cancer (80) (perhaps by downregulating gonadal synthesis of steroids, by acting on receptor sites within the neuroendocrine reproductive axis, or by altering estrogen receptor function). Therefore, in the right circumstances, it is possible that by reinforcing and optimizing our temporal organization, melatonin may have substantial benefits for reproductive health.

Other Reported Effects It has been suggested (81) that melatonin is a potent antioxidant, and that supplements of the hormone may protect against such age-related diseases as atherosclerosis, cancer, and Alzheimer’s disease. None of these proposed uses has been tested in a controlled clinical trial and all remain controversial because of lack of confirmation, the enormousness of the melatonin concentrations or doses needed to produce the effect, the failure of the investigators to provide data on actual blood or tissue melatonin concentrations after treatment, and the lack of studies comparing melatonin’s effects with those of known antioxidants such as vitamins C or E (31,82). It has usually been possible to demonstrate antioxidant or free radical scavenger effects in vitro; however, these have generally required melatonin concentrations 1000 to 100,000 times those ever occurring in vivo (31). Similarly, although high doses of melatonin (10–450 mg/kg body weight parenterally) have sometimes elicited antioxidant effects in experimental animals in vivo, neither their long-term safety nor their effects on the animals’ blood melatonin levels have been characterized. In humans—if not in nocturnally active laboratory rodents— such megadoses might ultimately impair sleep or various circadian rhythms, perhaps by downregulating melatonin receptors. Only one study (31) has described careful dose– response studies on the ability of melatonin to protect against autoxidation and compared melatonin, with known antioxidants. That study, by Duell et al. (31), examined the cell-mediated (by human macrophages) and cell-free (by copper sulfate) oxidation of low-density lipoproteins (LDL), a process believed to contribute to atherosclerosis. Melatonin did exhibit weak antioxidant activity, but only at 10,000- to 100,000-fold physiologic concentrations. In contrast, a vitamin E preparation

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(alpha-tocopherol) was 50- to 100-fold more potent than melatonin and was efficacious at physiologic concentrations. Similarly, vitamin C (ascorbic acid) and tryptophan, melatonin’s indolic circulating precursor (Fig. 1) were significantly more potent than melatonin and were active at physiologic concentrations. Some investigators suggest—on the basis of small studies on laboratory rodents—that melatonin “maintains juvenile conditions” and is a “geroprotector.” There is no evidence that melatonin has any “antiaging” actions in humans. In several small studies, melatonin was found to reduce blood pressure when given to normotensive men or women in daytime or the early evening, or to patients with essential hypertension. This possible effect should be explored further. Recent studies of gene loci associated with elevated fasting plasma glucose concentrations, involving samples from more than 50,000 European subjects, report that variants in the gene encoding a melatonin receptor (1B; MTNR1B) are consistently associated with such elevations, and also with an increased risk of type 2 diabetes (83,84,85). As this receptor is known to be transcribed in human islets (25,86) and melatonin can inhibit insulin secretion (26,87), melatonin may have largely unexplored but important effects on metabolic homeostasis.

PRESENT USAGE OF MELATONIN In the United States, the hormone melatonin is sold, without regulation by the FDA, as a dietary supplement. In most of the rest of the world, it is not sold at all, because it is regulated as a drug and no pharmaceutical company has presented an appropriate regulatory body with a successful new drug application (NDA) for its use. Some countries allow very low doses—less than 100 mg—to be sold without regulatory approval. Why is melatonin not subject to FDA approval and oversight, whereas other hormones are subject to such regulation? This is a consequence of the way the Dietary Supplement Health and Education Act of 1994 (Public Law 103-147) has been implemented. That act exempts from FDA regulation a product that is “. . . intended to supplement the diet that . . . contains one or more of the following dietary ingredients . . . ,” a list that includes “(D) an amino acid” (e.g., tryptophan) and “(F) . . . a metabolite . . . of any ingredient described in clause . . . (D)” (e.g., melatonin). Not exempted are products like L-dopa that have been “. . . approved as a new drug . . .” or “. . . authorized for investigation as a new drug . . .” Thyroxine, estrogens, and testosterone had also been approved as drugs prior to passage of the 1994 Dietary Supplement Act, whereas melatonin had not; thus, melatonin is treated as a dietary supplement, even though there is virtually no “dietary melatonin” for the “dietary supplement” to supplement. What have been the consequences of melatonin not being regulated by the FDA? Apparently no deaths to date have been reported; if melatonin-related deaths had occurred, the 1994 Act would have allowed the FDA to investigate, and then perhaps to start regulating it. In fact, few serious side effects have been described. A 2001 ar-

ticle described a 35-year search (1966–2000) of reports on melatonin toxicity by using the Medline database. Nine articles were found to describe adverse effects of melatonin; in all cases, the doses administered were in the pharmacologic range (1–36 mg). Individual patients exhibited, autoimmune hepatitis, confusion, optic neuropathy, a psychotic episode, headache, or nystagmus. Four suffered fragmented sleep, four described seizures, and two exhibited skin eruptions. Obviously, no clear pattern of side effects emerges from this review. In the absence of FDA regulation, companies are able to sell melatonin of uncertain purity, at dosages that are many times those needed for promoting sleep or shifting rhythms, or for restoring normal nocturnal plasma melatonin levels in older people. These dosages can elevate plasma melatonin to levels thousands of times greater than those that ever occur normally, and produce mild but not benign side effects like hypothermia and “hangovers.” Paradoxically, they also may, through receptor downregulation, exacerbate the insomnia that the consumer was trying to treat. Several synthetic melatonin analogs have recently been approved in the United States and/or the European Union for treating insomnia [ramelteon (88) tasunekteib (89)] or depression [agomelatine (90)]. All require doses substantially higher than sleep-promoting doses of melatonin (44). Moreover, there is no compelling evidence that melatonin itself has antidepressant activity, so the possibility arises that the antidepressant effect of agomelatine results from a different mechanism, not involving melatonin receptors.

CONCLUSIONS This entry describes melatonin, a hormone that is presently marketed as a dietary supplement. Melatonin is synthesized at night in the human pineal gland and released into the blood and cerebrospinal fluid. It acts on the brains of humans to promote sleep, and also affects the phasing of sleep and various other circadian rhythms. During the day, plasma melatonin levels are low; at night, they rise 10- to 100-fold or more in young adults, but by considerably less in older people—who often may have frequent nocturnal awakenings as a consequence. Very small oral doses of melatonin—approximately 0.3 mg or less—raise daytime plasma melatonin to night-time levels, thus making it easier for people to fall asleep in the afternoon or evening. Such doses can also help older people remain asleep during the night. Melatonin has also occasionally been claimed to confer other medical benefits—for example, preventing such age-related diseases as atherosclerosis, cancer, and Alzheimer’s disease. The evidence in support of such claims is sparse.

ACKNOWLEDGMENTS The author acknowledges with thanks the contributions of Dr. Irina Zhdanova, which included discussing this article before it was written and making useful suggestions for the finished product. Some of the data in this article were obtained with support from an NIH grant to

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MIT-MGH Clinical Center (5MO1RR01066-26) and a grant from the Center for Brain Sciences and Metabolism Charitable Trust.

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Milk Thistle Elena Ladas, David J. Kroll, and Kara M. Kelly

INTRODUCTION

HISTORY Use in Historical Times

Milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae); also Carduus marianus L.] (Fig. 1) is a herb widely used in Europe for the treatment of liver and biliary disorders. Although milk thistle is the most commonly used name for the herb, other names include silymarin, holy thistle, St. Mary thistle, Mary thistle, Marian thistle, Mariendistel, and lady’s thistle. The plant is indigenous to Europe but can be found in the western and southwestern United States. In ancient times, the leaves of milk thistle were used as part of the European diet. The medicinal properties of the herb reside in its seeds (Fig. 2). The primary active component, silymarin, is a potent antioxidant mixture composed of several related flavonolignans.

Milk thistle has been used medicinally for over 2000 years, primarily as a treatment for liver dysfunction. In ancient Greece, Dioscorides recommended the herb as a treatment for serpent bites (1). Subsequently, Pliny the Elder (A.D. 23– 79) prescribed milk thistle for “carrying off bile” (1,2). In the Middle Ages, Culpepper reported it to be effective for relieving obstructions of the liver (1,2). In 1898, the eclectic physicians Felter and Lloyd stated that the herb was good for congestion of the liver, spleen, and kidneys (1,2).

Current Use Native Americans use milk thistle to treat boils and other skin diseases. Homeopathic practitioners use preparations from the seeds in the treatment of jaundice, gallstones, peritonitis, hemorrhage, bronchitis, and varicose veins (1). Currently, the German Commission E recommends

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10

11

Figure 2 Milk thistle seeds. The seeds of the thistle are relatively large (5–6 mm in length) and, when hulled, should contain 1% to 2% (w/w) silybins. The most common crude extract, silymarin, is off-white to yellow powder composed primarily of seven flavonolignans and the flavonoid taxifolin (dihydroquercetin). Selective extraction of silymarin yields silibinin, once thought to be a pure compound but now known to be a mixture of two silybins (see Table 1 for comparison).

Figure 1 Milk thistle (Silybum marianum). Herbal supplements from this plant are most commonly made from organic extracts of the fruits/seeds present at the base of the spiny tuft.

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Milk Thistle

its use for dyspeptic complaints, toxin-induced liver damage, and hepatic cirrhosis, and as a supportive therapy for chronic inflammatory liver conditions (3).

Table 1

Compounds Present in Silymarin and Silibinin

Silibinina

Silymarinb

Silybin A Silybin B

Silybin A Silybin B Isosilybin A Isosilybin B

CHEMISTRY Although the chemical composition of milk thistle seed extracts has been studied since the 1950s, a precise nomenclature for the biologically active constituents has been evasive until very recently, Historically, the terms “silymarin” and “silibinin,” or “silybinin,” have been used interchangeably (4) to denote the content of standardized milk thistle extracts. However, none of these terms refers to a single pure compound (Table 1). Many of the primary active compounds in milk thistle extracts are classified as flavonolignans, each derived from the biosynthetic condensation of taxifolin, a flavonoid, and coniferyl alcohol, a precursor of lignins and lignans. The terms silymarin, silymarin group and silymarin complex have all been used to refer to the group of flavonolignans present in organic extracts of dried milk thistle seeds. The primary flavonolignan present in silymarin is silibinin, a 1:1 diastereomeric mixture of silybin A and silybin B (Fig. 3) (6). Silymarin also contains several other flavonolignans, each with a formula weight of 482. Isosilybin is a 1:1 diastereomeric mixture of isosilybin A and isosilybin B, each of which differs from its corresponding silybin only in the interchange of substituents at the C-10 and C-11 positions (Fig. 3) (6). The other flavonoid compounds found in the seed of milk thistle are shown in Table 1, and these remaining structures have been known for quite some time. The resolution of these eight compounds was

H 16

H 8

HO 7

8a

2 3

B

5

4

4a

OH

14

13

12a

mixture of these eight compounds; 20% to 35% is accounted for by other polyphenolics and undefined compounds. Some milk thistle extracts are incorrectly labeled as standardized for 65% to 80% silymarin. With the exception of the small percentage of taxifolin, a more accurate term for these extracts would be 65% to 80% silibinin equivalents, because the other seven compounds share the same chemical formula. Milk thistle products, especially those used in clinical trials, should be analyzed for the composition of each of these compounds and manufacturers should be encouraged to provide these data to consumers for each lot (5).

accomplished in 2003 (6), and the stereochemical assignment of the silybins and isosilybins was confirmed thereafter (Fig. 4) (7). The relative biological importance of each of these flavonolignans likely depends on the therapeutic indication, For example, in preclinical studies of human prostate cancer, silibinin is comparable in its growth-inhibitory and antitumor effects to silymarin (8). In contrast, silymarin is eightfold more potent than silibinin in scavenging free radicals in vitro (9). Consistent with this observation, silydianin and silychristin (present only in silymarin) are 2- to 10-fold more potent than silibinin (9). Therefore, future biological studies will be aided by the recent advances in milk thistle flavonolignan chemistry in determining whether different product formulations are better suited for specific indications.

H

10 11

17

O

18

OCH3

H OH

21

OH

O

Silybin B 21

H 8

HO 7

8a

A

6 5

OH

4a

C

O B 4

O

2 3

15a

14

D 12a

13

O 10 11

O H

E 17

OH

OH

22

H 19

H

20

O 19

18

OCH3 O

HO

O

CH2OH

H

23

CH2OH

OH OH

Isosilybin A

OCH3

H

OH H

OH

H

Silybin A

15

OCH3

O OH

H

O

O

HO

20

22

OH

H

19

E

H

CH2OH

O

CH2OH

D

Silychristin Isosilychristin Silydianin Taxifolin

a Approximately a 1:1 mixture of silybin A and B. b The major part (65–80%), of silymarin is a variable

23

O

C

O

A

6

15

16a

551

O

H Isosilybin B

Figure 3 Structures of silybins and isosilybins. Silibinin is a 1:1 diastereomeric maxiture of the two enantiomers silybin A and silybin B but contains no other flavonolignans. In contrast, silymarin is composed of the two silybins as well as varying concentrations of isosilybins A and B and other compounds. Source: From Ref. 6.

552

Ladas et al.

OH

OH 3'

H 8

HO

5

4

10

OH

O

4' 5'

1'

2

6

3'

1'

O

9 7

O

4'

2'

2'

6'

6'

5'

OH

9

O

5

10

4

OH

O

3

OH H

2'

9

5

10

OH

O

2'

6'

5'

OH

4'

O

3

9

O

OH

2

1'

OH 4'

H

8

HO 7

3'

O 4

6'

H O 1'

2'

6

5'

H

H

7

Isosilychristin

1'

6'

HO

5'

3'

5'

8

6'

OH

OCH3

4'

H

OCH3

CH2OH

Silychristin HO

6'

2

7

3'

4'

H

8

HO

2'

O 1'

2'

6

H

5'

3'

OH

CH2OH

3

4'

OCH3

3' 2'

OH

3

6 5

10

OH

4

OH

O

H

H Silidianin

Taxifolin

Figure 4 Structures of silychristin, isosilychristin, silydianin, and taxifolin. The three flavonolignans and the flavonoid, taxifolin, are present only in silymarin. Silychristin, isosilychristin, and silidianin share the same chemical formula and formula weight (482) as the silybins and isosilybins shown in Figure 3. Despite this similarity, silidianin is the most potent free-radical scavenger of the class. Source: From Ref. 6.

ABSORPTION AND TRANSPORT Silymarin is not water soluble; therefore it is used primarily in capsule rather than in tea (aqueous decoction) form. To facilitate absorption, silibinin bound to a phosphatidylcholine complex is used in most human trials, The bioavailability of this complex is on average 4.6 times higher than that of free silibinin (10), and in patients post cholecystectomy, biliary bioavailability of silibinin– phosphatidylcholine was 4.2-fold greater than that of a similar dose of silymarin (11). Studies suggest that silibinin is absorbed directly by the portal pathway from the intestinal tract. It then undergoes extensive metabolism, as evidenced by the presence of sulfate and/or glucuronide conjugates in the blood (10,12). Once in the liver, silibinin may be packaged into lipid micelles and transported to extrahepatic tissues, a hypothesis supported by the observation of radiolabeled silibinin in micelles in increasing quantities according to their lipophilicity, with the highest concentrations in triglycerides and verylow-density lipoproteins (VLDL). Approximately 80% of silibinin is excreted in bile, with only 3% excreted in urine (13). Pharmacokinetic studies in humans have found absorption to be rapid, with peak plasma levels occurring within 2 hours after a single dose and within 1 hour after multiple doses (10,12). The pharmacokinetics of single dose and multiple doses exhibit superimposable curves, although secondary peaks have been observed in multiple-dose studies (10,12). This may be due to extensive enterohepatic cycling (13). Following a sin-

gle dose of silibinin–phosphatidylcholine (80 mg silibinin equivalents), conjugated silybins are observed at 2.5-fold greater concentrations than free silybins, with a mean residence time of nearly 7 hours (14). Pharmacokinetic studies in humans have shown that steady-state levels are achieved within 4 days of dosing with the complex (10,12). The median peak plasma level of unconjugated flavonolignans (usually expressed as silibinin equivalents, with molar values relative to flavonolignan, which has a formula weight of 482.1) found in multiple human studies is 185 ng/mL, or 0.38 ␮M (range 67–3787 ng/mL; 0.14– 7.8 ␮M), with high interindividual variability (10,12,15). However, the interpretation of existing pharmacokinetic studies is hampered by varying dosing regimens with numerous products, and only one study has attempted to examine the differential pharmacokinetics of individual flavonolignan isomers (16). Adverse effects have not correlated with higher plasma levels of free or free and conjugated silibinin (17). This is possibly due to its short half-1ife (approximately 2–6 hours). Human studies have found similar pharmacokinetic patterns in studies of patients with cirrhosis of the liver (18,19). Studies have found that the silibinin–phosphatidylcholine form and silymarin are more concentrated in the bile relative to plasma. In nine cholecystectomy patients administered a single oral dose of either silibinin– phosphatidylcholine or silymarin (120 mg silibinin equivalents), peak biliary concentrations reached 116 and 29 ␮g/ mL, or 240 and 60 ␮M, respectively (11). Mean residence time for both preparations was in excess of 10 hours (11).

Milk Thistle

ACTION AND PHARMACOLOGY Milk Thistle as an Antioxidant Silibinin demonstrates potent antioxidant effects in vitro and in vivo. As with other flavonolignans, this mixture has been found to be a free-radical scavenger, to mildly chelate metals, and to inhibit lipid peroxidation (20–23). Silibinin is an effective scavenger of OH and HOCI species, but has not exhibited any affinity toward H2 O2 and O− 2 radicals (24). It inhibits lipid peroxidation induced by ADP/Fe+ 2 (20), Fe111 (23), tert-butyl hydroperoxide (25), and phenylhydrazine (26), as measured by effects on malondialdehyde (MDA) production. Silymarin has also been found to be effective in decreasing lipid peroxidation in human platelets in a dose-dependent manner (27). Despite their common chemical formulas, the flavonolignan isomers vary substantially in their in vitro potency for sequestering 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (9). Silydianin and silychristin are 2- to 10-fold more potent than the silybins or isosilybins, and this may account for the eightfold greater potency of silymarin compared with the silybins (9). In vitro data suggest that silibinin affects enzymes involved with phase II detoxification through its effects on intracellular glutathione concentrations and glutathioneS-transferase enzymes. It stimulates enzyme transcription and the activity of glutathione-S-transferase enzymes and has been found to prevent a decrease in glutathione in the liver cells of rats treated with acetaminophen (28–30). This mechanism is agent specific, as treatment with buthionine sulfoximine, a glutathione-depleting agent, failed to prevent the depletion of intracellular glutathione in rat hepatocytes (21). R In addition, pretreatment with silymarin [Legalon (10 ␮g/mL, 0.1 mg/mL, and 15 mg/mL)] had no effect on MDA production or glutathione or glutathione peroxidase activity in human erythrocytes exposed to doxorubicin or acetaminophen (31). The significance of these antioxidant effects in the intact organism is uncertain.

Effects on Liver Cells By acting on protein synthesis, silymarin may accelerate liver regeneration and production of hepatocytes through its actions on DNA-dependent RNA polymerase I and 5.8S, 18S, and 28S ribosomal RNA (32,33). These hypotheses were later supported in one clinical trial that found improvements in liver histology in patients with acute and subacute liver disease (34). Silymarin has also been found to protect the liver from toxic substances, presumably through alteration of hepatocyte membrane permeability. In vitro studies demonstrate that silymarin exerts a protective effect in hepatocytes exposed to tert-butyl hydroperoxide, phenylhydrazine, acetaminophen, and carbon tetrachloride (23,25,26,35). This protective effect has been supported by human case reports as well (36). Silymarin may also affect phospholipid metabolism in the liver. Silibinin has been shown to inhibit alcohol-induced phospholipid synthesis (37), by decreasing the rate of glycerol incorporation into phospholipids (38). Investigations in rat liver Kupffer cells indicate that it inhibits leukotriene B4 formation with an IC50 of 15 ␮M. This significant inhibition is also observed with as little as 5 ␮M, a concentration achievable in the liver (32). The relevance of this

553

effect in the prevention of fibrotic liver disease continues to be studied. Silymarin has been found to inhibit signals that promote fibrosis of the liver tumor necrosis factor (TNF)-␣ and nuclear factor (NF)-␬B involved in the development of cirrhosis (39,40). Silibinin inhibited intrahepatic activation of NF-␬B and inhibited intrahepatic expression of TNF, interferon-␥ , interleukin (IL)-4, IL-2, and inducible nitric oxide synthase (iNOS) in mice at doses of 25 mg/kg (41). IL-2 and -4 were expressed in mice fed 10 mg/kg of silymarin, but significant increases were not observed at doses of 50 or 250 mg/kg. Expression of TNF-␣ and proinflammatory cytokines (1 L-6, IL-1␤, iNOS) was stimulated in mice treated with 50 and 250 mg/kg of silymarin. The antioxidant effects of silymarin may also account for its ability to prevent or slow the progression of liver disease.

Antilipidemic Effects Silymarin may be an effective hypocholesterolemic drug (Table 2) (42). Preliminary research suggests that silymarin on its own or in combination with other polyphenolic compounds found in milk thistle may inhibit absorption of lipids from the gastrointestinal tract, decrease synthesis of lipids in the liver, inhibit enzymes involved in lipid neogenesis, and prevent oxidation of low-density lipoprotein (LDL) vesicles. Silibinin and silibinin–phosphatidylcholine complex do not prevent the accumulation of cholesterol in the liver (46,47). However, silymarin in combination with other milk thistle flavonoids decreased cholesterol absorption from the small intestine in rats fed a high-fat diet, through an action similar to bile-acid sequestrants. Taxifolin, a flavonoid compound found in milk thistle, has also been found to inhibit cholesterol absorption (48). This suggests that other components of the herb besides silibinin are responsible for its anticholesterolemic effects. Silymarin inhibits key enzymes involved in cholesterol biosynthesis. Silibinin inhibits 3-hydroxy-3methylglutaryl-CoA (HMG-COA) reductase activity in a dose-dependent manner in cell lines, but this has not been observed in rat liver microsomes (44). Silymarin decreases the concentration of cholesterol in VLDL (42,47,48), but its effect on plasma cholesterol levels is uncertain. An inverse relationship between plasma cholesterol levels and silymarin was seen in one study (46), but another study has not found an association (48,49). The effects of silymarin on plasma triglycerides, VLDL, LDL, and high-density lipoproteins (HDL) has been evaluated. It lowers plasma VLDL (46–48) but has no effect on plasma LDL (46,48). Silymarin and silibinin increase HDL (46,48). The mechanisms through which silymarin may exert these effects is unknown, but its antioxidant properties may be responsible for inhibition of LDL oxidation (47).

Anticancer Effects The effects of silymarin and silibinin have been investigated in various cancer models (Table 3). The two mixtures have been evaluated for their ability to exert direct cytotoxic effects, mitigate the toxicity of certain anticancer agents, and enhance the efficacy of chemotherapeutic agents. These effects have been most

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Ladas et al.

Table 2

Summary of Laboratory Studies: Antilipidemic Effects

Reference

Model

Treatment (concentration)

Results

(43) (44)

Rat liver homogenates Rats

Silibinin (7.5 × mol/L) Silibinin (100 mg/kg; 50 mg/kg)

(45)

Rat liver microsomes

(37) (38)

Rats Rat hepatocytes

Silymarin and silibinin (100 mg/kg) Silibinin (100 mg/kg IV) Silibinin (1 or 0.1 mM)

(46)

Rat liver removed after IP administration (liver homogenates) Hypercholesterolemic rats

Silymarin

(47)

Rats

Silibinin Silymarin

(48)

Rats

IdB 1016 Silymarin

Inhibition of precursors of cholesterol synthesis ↓Biliary CHO and phospholipid concentration at higher dose only No effect on liver CHO Inhibition of HMG-CoA reductase activity ↓Turnover of phospholipids only in vitro (findings not confirmed in vivo) Inhibition of EtOH-induced phosphosynthesis ↓Incorporation of glycerol in TG synthesis ↓TG production in the liver Stimulation of phosphatidylcholine synthesis ↑Choline phosphate cytidyltransferase ↓Liver and plasma CHO, VLDL, phospholipids ↑HDL No effect on TG No effect ↓Liver and Plasma VLDL ↑HDL Not effective ↓Liver CHO, TG ↓Plasma VLDL, TG ↓Concentration of CHO in VLDL ↑HDL No effect on plasma CHO and LDL ↓CHO in liver homogenates ↓CHO in liver microsomes No significant effect

Rabbits

10–6

Silibinin (7.5 × 10–6 to 7.5 × 10–4 mol/L)

Silymarin–phospholipid complex Silymarin alone

Abbreviations: CHO, cholesterol; EtOH, ethanol; HDL, high density lipoprotein; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase; IP, intraparenteral; IV, intravenous; TG, triglyceride; VLDL, very low density lipoproteins; ↑ increase; ↓ decrease.

extensively investigated in prostate cancer cell lines (DU 145, LNCaP, PC-3) (53–56,70) and a mouse skin cancer model (64,65,72–75). Other in vitro studies have investigated their properties in breast cancer (MDA-MB 468, MCF-7) (8,51,52) hepatic cancer (HepG2) (58,76), epidermoid cancer (A431) (58), colon cancer (Caco-2) (77), ovarian cancer (OVCA 433, A2780) (78), histiocytic lymphoma (U-937) (61), and leukemia (HL-60) (50,79) cells. Silymarin and silibinin have been investigated in animal tumor models of skin (64,65,72–74) tongue cancer (65,71–75) bladder cancer (69), and adenocarcinoma of the colon (62,63) and small intestine (62). Silymarin and silibinin have activity against prostate cancer. They can inhibit growth factors and cell-to-cell signaling that stimulate cell growth (53,55,56,70), promote cell cycle arrest in G1 (54,55), and inhibit antiapoptotic activity (57), Silymarin (75 ␮g/ mL of medium) inhibits epidermal growth factor B1 and subsequent signaling processes leading to growth inhibition of DU 145 cells (54). In LNCaP cells, the G1 arrest caused by silibinin appears to be mediated by an increase in complex formation between the retinoblastoma gene product, Rb, and members of the E2F transcription factor family. Administration of silibinin in the diet to nude mice significantly lowered tumor volume and wet tumor weight (70). These effects on human prostate cancer xenografts correlated with plasma levels of 14 to 27 ␮M. Importantly, the investigators monitored food consumption and found that mice ingested 1.8 to 3.5 mg silibinin/day. By using typical allometric scaling to a 70-kg human, this correlates to a daily silibinin dose of 650 to 1300 mg. Although this is higher than

the dose recommended for hepatic protection, the low toxicity of silibinin and silymarin should make it possible to increase the dose to achieve a therapeutically relevant anticancer concentration of the flavonolignans. Silymarin and silibinin have also been extensively investigated in the SENCAR mouse nonmelanoma skin cancer model (64–67,73–75,80). Silymarin treatment significantly reduced tumor incidence, multiplicity, and volume in cells treated with ultraviolet (UV) B to induce tumor promotion, but not in cells treated with UVB to induce tumor initiation (65). Silymarin (6 mg dose in 0.2 mL of acetone) inhibits TNF-␣ mRNA in the mouse epidermis, possibly by inhibiting tumor promotion (68). Silymarin and silibinin have chemopreventive effects in breast (MDA-MB-468) and cervical (A43l) cancer cell lines (8). In male F344 rats with azoxymethaneinduced colon cancer, supplementation with silymarin (100, 500, and 1000 ppm in the diet) resulted in a reduction of colon tumorigenesis and a decrease in multiplicity of tumor growth (63). Pretreatment of human promyelocytic leukemia (HL-60) cells with silibinin resulted in inhibition of cell growth and differentiation. Silymarin interferes with cell-to-cell signaling in breast cancer cell lines (MDAMB-468) (52), histiocytic lymphoma (U-937) (61), and hepatoma cell lines (76). It may also have a chemopreventive role in tongue and bladder carcinogenesis (69,71). Silymarin has also been investigated as a possible adjunctive agent in mitigating some of the toxicity associated with chemotherapeutic agents. Silibinin and silychristin exerted a protective effect on monkey kidney cells and rats exposed to vincristine or cisplatin chemotherapy

Milk Thistle

Table 3

Summary of Laboratory Studies: Anticancer Effects

Cancer type Cell lines Leukemia (50) (HL-60) Breast (51,52) (MCF-7, MDA-MB-468)

Prostate (43,45,51, 53–57) (DU145, PC-3, LNCaP)

Hepatic (58) (HepG2) Other Testicular (59) (H12.1, 577LM, 1777NR CI-A) Gynecologic (60) (A2780, OVCA 433) Histiocytic lymphoma (61) (U-937) Animal models Colon (62,63) (male F344 rats; Sprague-Dawley rats)

Skin (64–68) (SENCAR mice)

Other Bladder (69) (male ICR mice) Prostate (DU145) (70) (athymic male nude mice) Tongue (71) (F344 rats)

Main findings/target of action Inhibited cell proliferation; induced cell differentiation Inhibited VEGF (25, 50, 100 ␮g/ mL) Exerted antiangiogenic properties by inhibiting VEGF Inhibited cell growth, cell proliferation, and CDK expression Inhibited VEGF Inhibits erbB1; induced CDKIs; induced cell cycle arrest Stimulated IGFBP-3 Inhibited intracellular PSA and cell growth Inhibited EGF, erbB1, ERK1/2 Inhibited NF-␬B, p65, p50; Increased l␬B␣ levels; sensitized cells of TNF␣ Inhibited Rb phosphorylation; induced G1 arrest Exerted antiangiogenic prosperities by inhibiting VEGF secretion Inhibited binding and expression of NF-␬B Decreased APAP-induced toxicity No interference in antitumor effects in cisplatin- and ifosfamide-treated cells Enhanced efficacy of cisplatin; no stimulation of tumor growth by IdB 1016 Inhibited transcription of NF-␬B. Inhibited phosphorylation and degradation of 1␬B␣ no effect on AP-1 Reduced incidence and multiplicity of chemical-induced colon cancer of (dietary silymarin: 100, 500, 1000 ppm in diet) Decrease frequency of adenocarcinoma (silymarin flavonolignans; 0.10%) Inhibited activity and mRNA of ODC Inhibited mRNA of TNA␣ Inhibited tumor promotion; no effect on tumor initiation Inhibited ligand binding of EGFR, cell cycle arrest, DNA synthesis Inhibited lipid peroxidation and proinflammatory cytokines; prevented depletion of antioxidant enzymes Inhibited MAPK/ERK 1/2, stimulated SAPK/JNK 1/2 and p38 MAPK Inhibited initiation and proliferation of tumor cells Inhibited tumor volume; induced IGFBP-3

Inhibited tumor initiation and promotion

Abbreviations: APAP, acetaminophen; CDKI, cyclin-dependent kinase inhibitor; EGF, epidermal growth factor; EGFR and erbB1, epidermal growth factor receptor; IGFBP-3, insulin-like growth factor binding protein 3; I␬B␣, inhibitory subunit of NF-␬B; MAPK/ERK1/2, mitogen-activated protein kinase/extracellular signal-regulated protein kinase; NF-␬B, nuclear factor kappa B; ODC, ornithine deoxycarboxylase; PSA, prostate specific antigen; Rb, retinoblastoma; SAPK/JNK1/2, stress-activated protein kinase/jun NH2 terminal kinase; TNF-␣, tumor necrosis factor ␣; VEGF, vascular endothelial growth factor.

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(59,81,82). In germ cell tumors, silibinin did not interfere with the antineoplastic effects of cisplatin or ifosfamide. It potentiated the cytotoxic effect of cisplatin and doxorubicin in breast cancer and ovarian cell lines (60,78). The flavonolignan mixture may increase the chemosensitivity of DUI45 prostate cancer cells resistant to chemotherapy (57). The clinical significance of these investigations in humans as well as the effective dose, timing, and duration of treatment with silymarin in humans with cancer needs further investigation.

INDICATIONS AND USAGE Liver Disorders Although research has been conducted in humans with silymarin and a variety of diseases of the liver, its mechanisms of action are largely unknown. Some human studies suggest that silymarin may be more effective in the earlier stages of liver disease (34). This may be explained by its ability to prevent toxins from entering the hepatocyte, thereby preventing initial damage to the cell (Table 4). The studies investigating silymarin in humans with liver disease are described later.

Cirrhosis Three double-blind, randomized, controlled trials using similar dosing regimens have investigated milk thistle in the treatment of alcohol-induced cirrhosis. The studies have reported mixed results (Table 4). Ferenci et al. followed patients with alcohol- and non-alcohol-induced cirrhosis for 2 years and found increased survival in patients supplemented with 420 mg/day of silymarin. However, no effects on measurements of liver function were found. A later study using a similar dosing regimen and duration found that silymarin had no effect on survival (87). In a smaller study, patients with alcohol-induced cirrhosis were supplemented with 150 mg/day of silymarin. Significant increases in glutathione levels and decreased malondialdehyde levels were observed (88). No significant effects on measurements of liver function tests were found.

Alcoholic or Virus-Induced Hepatitis Five studies have investigated the effect of silymarin in the treatment or prevention of progression of alcohol- or virus-induced hepatitis (17,83–86). Variations in the form and dosage of silymarin make comparisons difficult. In the only double-blind placebo-controlled trial, 59 subjects were randomly assigned to Legalon or placebo (89). Substantial increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin were noted, but P values were not reported.

Subacute or Acute Liver Disease Two double-blind, randomized, controlled trials and one observational study have investigated silymarin in the treatment of patients with liver disease (34,89,90). In a large observational study (n = 2637), subjects with liver disease of various etiologies and severity were supplemented for 8 weeks with a mean Legalon dose of 267 + 103.6 mg. Considerable decreases in ALT, AST, and ␥ -glutamyl transferase (GGT) and a decrease in the

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Table 4

Summary of Human Studies

Reference

Duration of study

Results

2 weeks

↓ALT, GGT

5 weeks 21–28 days

No significant findings Improvements in bilirubin, AST, ALT, (significance NR)

NR

“Recuperation” was faster among patients with “normal” evolution of disease

Silymarin/140 mg t.i.d Silymarin/150 mg t.i.d Silymarin (MZ-80)/ 150 mg t.i.d

2 yr 2 yr 6 mo

↑Survival No significant findings Significant increases in erythrocyte glutathione ↓Platelet MDA values No significant differences in liver function tests

Legalon/420 mg

4 weeks

Silymarin/267 mg (±103 mg)

8 weeks

Liver damage (unspecified etiology)

NR

28 days

↓LFTs Improved histology ↓ALT, AST, GGT (significance NR) ↓Number of patients with enlarged liver (significance NR) ↓AST (P < 0.1) ALT (P < 0.05), GGT (P < 0.05)

Primary biliary cirrhosis

Silymarin/140 mg t.i.d

1 yr

No significant findings

Hyperlipoproteinemia type II

Legalon/420 mg

3 mo

↓CHO, HDL, Apo Al/AII No effect on TG, CHO

Type of study/sample size

Type of disease

Formulation/dosage

Viral or alcoholic hepatitis

(83) (84)

Phase II randomized open trial/60 Controlled, randomized trial/52 Double-blind trial/59

Viral hepatitis B Acute viral hepatitis

IdB 1016/80–120 mg b.i.d./t.i.d Silymarin/210 mg Legalon/70 mg

(85) (86)

DBRCT/45 DBRCT/77

Chronic hepatitis Acute viral hepatitis

Silymarin/ Legalon/420 mg

DBRCT/170 DBRCT/200 DBRCT/60

Cirrhosis Alcohol-induced cirrhosis Alcohol-induced cirrhosis

Acute and subacute liver disease Toxic liver pathology (unspecified)

Hepatitis (17)

Cirrhosis (87) (88)

Other liver diseases (34) DBRCT/106 (89)

Observational study/2637

(90)

DBRCT/66

Primary biliary cirrhosis (91) Noncontrolled, open trial/27 Lipid (92) Nonrandomized/14

Abbreviations: ALT, alanine aminotransferase; Apo A1, apolipoprotein A1; Apo Al1, apolipoprotein A11; AST, aspartate transaminase; CHO, cholesterol; GGT, ␥-glutamyl transpeptidase; HDL, high density lipoprotein; LFT, liver function test; MDA, malondialdehyde; NR, not reported; TG, triglycerides; ↑ increase; ↓ decrease.

number of patients with hepatomegaly were noted, but P values were not reported. In the two double-blind, placebo-controlled trials, significant decreases in ALT, AST, and GGT were observed.

Primary Biliary Cirrhosis In a nonrandomized pilot study (n = 27), silymarin (140 mg three times daily) was administered to patients with primary biliary cirrhosis nonresponsive to standard medical care (91). No noteworthy changes were observed.

Lipidemia One study suggests that silymarin may be a potential therapeutic agent in the prevention of atherosclerosis (92). Oxidation of low-density lipoprotein (LDL) particles plays an important role in the development of atherosclerosis. Silymarin may reduce atherosclerosis through its effects on apolipoproteins A-I and A-II. Apolipoproteins reside on the surface of HDL and activate lecithin/cholesterol acyltransferase (LCAT), thereby clearing cholesterol from extrahepatic tissues. Apolipoprotein A-I facilitates uptake of cholesterol into cells. A small trial of 12 weeks’ silymarin supplementation in 14 adults with Type-II hyperlipidemia resulted in significant reduction in apolipoprotein A-I levels, thereby showing that silymarin is not beneficial in this condition. Significant decreases in apolipoprotein A-II lev-

els were also observed. However, the role of apolipoprotein A-II in atherosclerosis is not well defined (93). As apolipoprotein A-II is inversely associated with insulin resistance and plasma triglycerides, silymarin may be useful in both atherosclerosis and diabetes. Further study is needed.

Cancer Silymarin may have a therapeutic role in the treatment of certain malignancies. However, no clinical trials have reported the safety or efficacy of silymarin in combination with cancer treatment. Two case studies have reported the use of silymarin in humans with cancer in combination with cancer therapy. Silymarin was used as an adjunctive treatment in a 34-year-old woman with elevated liver enzyme content undergoing chemotherapy. A daily dose of 800 mg was associated with reductions in AST and ALT and thereby enabled the patient to receive the prescribed chemotherapy (94). Spontaneous regression of hepatocelluar carcinoma has been reported in a 52-year-old man taking silymarin (95).

Other Amanita phalloides is a mushroom that, upon ingestion, causes amatoxin (a-amanitin) poisoning. This toxin damages both liver and kidney by irreversibly binding to RNA

Milk Thistle

polymerase II. This leads to hepatic failure and often results in death. IV administration of silymarin is the only treatment (36). Histochemical investigations in rat hepatocytes demonstrate that silymarin stabilizes cell membranes (96). Silibinin may also inhibit absorption of the toxin from the gut through its extensive enterohepatic cycling (96). The effectiveness of silibinin depends on the dose and timing of administration after amatoxin exposure (96). Silymarin has been administered to pregnant women with cholestasis without any teratogenic effects on the fetus (97). Milk thistle has also been used as a lactogogue. Herbalists have also used milk thistle for the treatment of psoriasis. No clinical studies have been reported for these indications.

AVAILABLE DOSAGES/FORMS Capsules and extracts of milk thistle are usually quantified as 65% to 80% silibinin or silibinin equivalents, with the remaining 20% to 35% consisting of less-defined polyphenolic compounds and fatty acids. Most of the clinical trials have used capsules standardized to silibinin content (Table 5). However, the composition of the capsules can vary. The relatively straightforward selective precipitation of the silybins from milk thistle extracts has led to the widespread marketing of silibinin as the purified active principle component of silymarin. Analysis of a representative lot of Legalon (sold in Germany as Legalon and R imported into the United States as Thisilyn ) revealed 66.1% flavonolignans in the proportions of 30.1% silybin, 9.1% isosilybin, 14.9% silychristin, and 12% silydiR R anin (4,9). Another preparation, Siliphos or Silipide (1dB 1016), is a silibinin preparation sold by Indena S.p.A. and is a patented mixture of 33% silibinin and 66% soy lecithin (phosphatidylcholine). An IV preparation of milk thistle extract is sold in Europe as silibinin hemisuccinate in aqueous solution to be given for acute A. phalloides poisoning (20 mg/kg total silibinin per day given in four 5 mg/kg infusions of 2 hours each). Silibinin (S-0417) as sold by Sigma-Aldrich (St. Louis, MO) is the most commonly used source of milk thistle extract for preclinical Table 5

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studies and comprises nearly identical proportions of silybin A and silybin B. An average dose of 200 to 400 mg/day in divided doses has been used in most of the studies investigating silymarin for hepatic disorders and antilipidemic effects. Teas made from the crushed seed are used for mild gastrointestinal disorders (98); however, due to its lipophilic properties, only a small percentage of silymarin is found in aqueous solution (2,98). A list of formulations and suppliers is described in Table 5.

CONTRAINDICATIONS/ADVERSE REACTIONS Few side effects are reported when silymarin and silibinin are used within the recommended dose ranges (3,98). Rare cases of a mild laxative effect have been described. One human dose escalation study reported nausea, heartburn, and dyspepsia in subjects treated with 160 mg/day, dyspepsia in patients treated with 240 mg/day, and postprandial nausea and meteorism in patients treated with 360 mg/day. None of these side effects were dose related. At doses greater than 1500 mg/day, mild allergic reactions have been reported. Episodes of sweating and gastrointestinal distress have been associated with the use of milk thistle (99). The symptoms resolved upon discontinuation of the supplement, but it is unknown whether these effects were due to milk thistle or contamination of the capsule.

DRUG INTERACTIONS Interactions between milk thistle (silymarin) and medications or other herbal remedies are largely unknown. Silymarin inhibits CYP3A4 and UDP-glucuronyltransferase UGT1A6/9 in cultured human hepatocytes, and silibinin inhibits CYP2C9 and some activities of CYP3A4 in isolated human liver microsomes (100,101). However, the concentration at which inhibition is observed is high (100–500 ␮M in hepatocyte studies) and not achievable with oral intake of silymarin (102). However, the biliary concentrations of flavonolignans can be greater than plasma levels by an order of magnitude or more: Single 120 mg doses of silymarin or silibinin–phosphatidylcholine result in

Formulations and Suppliers of Silymarin and Silibinin

Brand name

Formulationa

Manufacturer

Legalon MZ-80

One tablet contains 35, 70, or 140 mg of silymarin. Standardized to silymarin content One capsule contains 175 mg of 80% silymarin (140 mg silymarin)

Madaus A.G., Ostmerheimer Strasse 198, Cologne, Germany

Thisilyn, Thisilyn ProTM

IdB 1016 Silipide R Siliphos

One capsule contains 150 mg of a 1:2 ratio of silibinin complexed with soy-derived phosphatidylcholine

Manufactured in Germany by Madaus A.G. for US distribution by Nature’s Way Products, Inc, Springville, Utah, [also doing business as Murdock Madaus Schwabe Professional Products (MMS Pro), Inc, 10 Mountain Springs Parkway, Springville, Utah 84663) Indena S.p.A. Viale Ortles 12, Milan 20139, Italy

R

Note: The regulatory status of herbal medicine varies between countries. For more information on the regulatory status of herbal therapies in selected countries, refer to Legal Status of Traditional Medicine and Complementary/Alternative Medicine: A Worldwide Review; WHO/EDM/TRM/2001.2: WHO, Geneva, 2001; 189 p. (ISBN 92–4–154548–8; Swiss Fr. 35). a The information on formulations was supplied by the manufacturers of the product and has not been subject to confirmation by an outside agency.

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maximum mean biliary concentrations of 60 and 240 ␮M, respectively (11). The observation that patients with HIV often use milk thistle to prevent or manage hepatitis or protect the liver from hepatotoxic drugs led to clinical trials investigating the potential for interactions with the HIV protease inhibitor indinavir and CYP3A4. Two independent trials of coadministration of milk thistle and indinavir within the recommended dosages in healthy individuals found no interference with indinavir pharmacokinetics (103,104). These findings are also consistent with the observation that a 2-day exposure of isolated human hepatocytes to 10 ␮M silymarin has no effect on CYP3A4 gene expression (105). Theoretically, its antioxidant and free-radical scavenging properties suggest that silymarin may interact with any free-radical-generating medication, such as the anthracycline chemotherapy agent doxorubicin. This has not yet been investigated in human or laboratory studies.

OVERDOSAGE No reports of overdosage have been documented. Silymarin has been well tolerated in high doses. IV administration of silibinin (20–50 mg/kg body weight) in the treatment of humans with A. phalloides poisoning resulted in no adverse effects. No toxicity has been observed in rats and mice when it is given in doses as high as 5000 mg/kg. Rats and dogs have received silymarin at doses of 50 to 2500 mg/kg for a 12-month period. Investigations including postmortem analyses showed no evidence of toxicity. The Merck Index lists no LD50 value in any species for silibinin or silymarin.

REGULATORY STATUS Milk thistle is classified in the United States as a dietary supplement under the Dietary Supplement Health and Education Act (DSHEA) 1994 and cannot be marketed for the treatment of any disease. In Germany, the Commission E approves the internal use of crude milk thistle fruit preparations for dyspeptic complaints. Formulations consisting of an extract of 70% to 80% silymarin are also approved for toxic liver damage and for supportive treatment in chronic inflammatory liver disease and hepatic cirrhosis. The U.S. Pharmacopoeia and National Formulary (USP-NF) monographs dictate that milk thistle seeds (with pappus removed) used for extraction contain not less than 2% (w/w) silymarin, expressed as silybin, using a USP spectrophotometric assay method and botanical identification confirmed by thin-layer chromatography and macroscopic and microscopic examinations (USP 24NF 19, 1999), German pharmacopeial-grade milk thistle should also contain not less than 1.5% (Deutsches Arzneibuch/DAB, 1997).

CONCLUSIONS Milk thistle is a herbal plant that has a long history of use in the treatment of a variety of illnesses. Laboratory and

clinical research suggests that silymarin, a complex of active components from milk thistle, may be a possible agent in the prevention or treatment of cancer, atherosclerosis, hepatitis, and cirrhosis. The use of milk thistle for these indications has been further reviewed elsewhere (1–3,97). The low-toxicity profile of silymarin makes it an attractive agent for further studies. Future investigations are needed to determine the effective dose, duration, and formulation so that standardized recommendations can be developed.

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95. Grossman M, Hoerman R, Weiss M, et al. 52-year old man with biopsy-confirmed hepatocellular carcinoma resolved spontaneously. Am J. Gastroenterol 1995; 90(9):1500–1503. 96. Enjalbert F, Rapior S, Nouguier-Soule J, et al. Treatment of amatoxin poisoning: 20-year retrospective analysis. J Toxicol Clin Toxicol 2002; 40(6):715–757. 97. Hernandez R, Nazar E. Effect of silymarin in intrahepatic cholestasis of pregnancy. Rev Chil Obstet Ginecol 1982; 47(1):22–29. 98. Blumenthal M. Milk thistle. The ABC Clinical Guide to Herbs. Austin, TX: Thieme New York, 287–295. 99. Adverse Drug Reactions Advisory Committee. Med J Aust 1999; 170:218–219. 100. Beckmann-Knopp S, Rietbrock S, Weyhenmeyer R, et al. Inhibitory effects of silibinin on cytochrome P-450 enzymes in human liver microsomes. Pharmacol Toxicol 2000; 86(6):250–256. 101. Venkataraman R, Ramachandran V, Komoroski BJ, et al. Milk thistle, a herbal supplement decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures. Drug Metab Dispos 2000; 28(11):1270–1273. 102. Zuber R, Modriansky M, Dvorak Z, et al. Effect of silybin and its congeners on human liver microsomal cytochrome P450 activities. Phytother Res 2002; 16(7):632–638. 103. Piscitelli SC, Formentini E, Burstein AH, et al. Effect of milk thistle on the pharmacokinetics of indinavir in healthy volunteers. Pharmacotherapy 2002; 22(5):551–556. 104. DiCenzo R, Shelton M, Jordan K, et al. Coadministration of milk thistle and indinavir in healthy subjects. Pharmacotherapy 2003; 23(7):866–870. 105. Raucy JL. Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products. Drug Metab Dispos 2003; 31(5):533–539.

Niacin Christelle Bourgeois and Joel Moss

ABBREVIATIONS ADPR/P, adenosine diphosphate ribose/phosphate; ART, mono-ADP-ribosyltransferase; NAAD/NAADP, nicotinic acid adenine dinucleotide/nicotinic acid adenine dinucleotide phosphate; NE, niacin equivalents; NMNAT1–3/NaMNAT, nicotinamide/nicotinic acid mononucleotide adenosine 5 -triphosphate adenylyltransferase; Nrk, nicotinamide riboside kinase; PARP, poly(adenosine diphosphate ribose) polymerase; PBEF, pre-B; colony-enhancing factor; PRPP, phosphoribosyl pyrophosphate.

Figure 1 riboside.

Structure of nicotinic acid, nicotinamide, and nicotinamide

INTRODUCTION its association with poor nutrition, inadequate meat and milk intake, and use of corn as the principal constituent of the diet. In 1922, they suggested that pellagra was an amino acid deficiency. Five years later, it was demonstrated that nicotinic acid cured pellagra and in 1949, that tryptophan reversed the symptoms. In 1961, Goldsmith quantified the conversion of tryptophan to nicotinic acid by monitoring such nicotinic acid metabolites as N1 -methyl-5-carboxamide-2-pyridone. Elucidation of the biochemical pathway for conversion of tryptophan to nicotinic acid mononucleotide (Fig. 2) took more than 10 years—from 1950, when Knox and Mehler showed that the first step in the biodegradation of tryptophan to Nformylkynurenine was catalyzed by tryptophan pyrrolase, to 1963, when work by Nishizuka and Hayaishi revealed that quinolinic acid reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinic acid mononucleotide, a reaction catalyzed by the enzyme quinolinic acid phosphoribosyltransferase.

Niacin, also designated vitamin B3, is found mostly in meat, grains, milk, and eggs. In the United States, “niacin” means nicotinic acid, and the amide form, nicotinamide, is called niacinamide. Elsewhere, “niacin” denotes nicotinic acid and/or nicotinamide. Deficiency of this vitamin causes pellagra, a disease characterized by dermatitis, diarrhea, and dementia that is endemic today in parts of India and China, and may result in death in severe cases. As a precursor of pyridine nucleotides [nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)], niacin participates in the function of numerous enzymatic pathways, which are critical for normal cell metabolism, involving, for example, redox reactions and those that consume NAD. Discovery of the antihyperlipidemic properties of pharmacological doses of nicotinic acid and of the importance of NAD metabolism for the maintenance of genome stability has renewed interest in this vitamin in developed countries.

GENERAL DESCRIPTION ACTIONS AND PHARMACOLOGY

The structures of nicotinic acid, nicotinamide, and nicotinamide riboside are shown in Figure 1. They consist of a pyridine ring substituted in position 3 with a carboxylic group in nicotinic acid and with a carboxamide group in nicotinamide. Niacin was initially studied because of its association with pellagra, a nutritional deficiency disease, symptoms of which are dermatitis, diarrhea, and dementia, with death as the eventual outcome. Pellagra was first documented by Casal as “mal de la rosa” in 1735, but was linked to niacin deficiency only about two centuries later, during an epidemic in the southern United States (reviewed in Ref. 1). The disease was initially thought to be of infectious origin, until Goldberg and Tanner observed

The major dietary sources of niacin are meats, poultry, and fish, followed by dairy and grain products (2). Preformed niacin exists in foods as nicotinamide, nicotinic acid, nicotinamide riboside, or the pyridine nucleotide coenzymes, NAD and NADP (Fig. 3). Nicotinamide riboside, a recently discovered salvageable precursor of NAD, is particularly abundant in cow milk (3). L-Tryptophan, the in vivo precursor of nicotinamide (Fig. 2), also contributes to the total niacin-equivalent (NE) content of foods and should be taken into account when calculating the vitamin intake. Eggs and milk, for instance, with their high tryptophan content, are a significant source of NE. Niacin 562

Niacin

CH2 CH COOH Tryptophan

NH2

NH

Tryptophan dioxygenase

O C CH2 CH COOH

N-Formylkynurenine

NH CH NH2 O

Formyl kynurenine formamidase

O C

Kynurenine

CH2 CH COOH NH2

NH2

Kynurenine hydroxylase (Vit B2-dependent)

O C

CH2 CH COOH

3-Hydroxy-kynurenine

NH2

NH2 OH

Kynureninase (Vit B6-dependent)

O C OH

3-Hydroxyanthranilic acid

NH2 OH

3-hydroxyanthranilic acid oxygenase

O C α-Amino-β-carboxy muconic-ε-semialdehyde

OH O:CH NH2 O=C OH O C

Non enzymatic

OH OH

Quinolinic acid

O

N H

Quinolinic acid Phosphoribosyltransferase

C O C

Nicotinic acid mononucleotide

–O3HP

+

O

CH N2 O

HO Figure 2

OH

OH

Conversion of tryptophan to nicotinic acid mononucleotide; “de novo” synthesis pathway.

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O C NH2 O– O–

Nicotinamide +

Tryptophan

NH2 N

“De novo” pathway

N

H 2C-O-P-O-P-O-CH 2 N N O OH HO OX Ribose Adenosine

N

Nicotinic acid

HO O HO OH

1

2 Nicotinamide riboside

X = H(NAD) X = PO3(NADP) Figure 3

Structure of the pyridine coenzymes.

Nicotinic acid mononucleotide

6

4

Nicotinic acid adenine dinucleotide

Nicotinamide mononucleotide

3

NAD

Nicotinamide

intake is, therefore, generally expressed in NE. It is estimated that 60 g of the amino acid is converted to 1 g of the vitamin, with a variation of approximately 30% (standard deviation) among individuals. The efficiency of tryptophan conversion to nicotinic acid depends on nutritional history and hormonal factors (4). Quantitatively, tryptophan is primarily used for protein biosynthesis, even in conditions of niacin deficiency. Estimation of dietary niacin content should also consider another factor: bioavailability. Indeed, in certain cereal grains, such as corn, niacin is largely present as niacytin, a polysaccharide/glycopeptide/polypeptidebound form, most of which is unavailable for intestinal absorption. In maize, for instance, 70% of niacin is in a biologically unavailable form (5). However, niacin availability can be improved by specific processes such as the alkali treatment of corn used in the preparation of tortillas (4). Otherwise, absorption of nicotinic acid and its amide by the gastric and intestinal mucosa is very efficient, proceeding via sodium ion-dependent facilitated diffusion at low concentrations, and passive diffusion at high concentrations. In the gut, NAD and NADP are degraded by glycohydrolase and pyrophosphatase activities into nicotinamide and nicotinamide ribonucleotide, respectively, which are bioavailable sources of the vitamin. Tissues absorb free nicotinic acid as well as nicotinic acid bound to proteins. Metabolic trapping, in which nicotinic acid and nicotinamide are converted to NAD, accounts for retention of these vitamins (4). Nicotinic acid and, to a lesser extent, nicotinamide are lipid-soluble molecules, and adipose tissue is responsible for the rapid clearance of nicotinic acid after an IV dose. In addition, receptor-mediated uptake has been reported for nicotinamide (4). A transporter for nicotinamide riboside has been identified in yeast (6). In liver, nicotinic acid and nicotinamide can be converted to NAD and NADP or metabolized for clearance. Nicotinic acid is eliminated as a glycine conjugate, nicotinuric acid, whereas the main metabolites of nicotinamide are N1 -methylnicotinamide and its oxidized products, 2- and 4-pyridones (7). Nicotinic acid and nicotinamide metabolites are then excreted in urine; quantification of this excretion is useful in evaluating niacin nutritional status. Nicotinic acid, nicotinamide, nicotinamide riboside, and tryptophan are precursors of NAD and NADP (Fig. 4).

2

5

NADP

“Salvage” pathway

NAD consuming enzymes (e.g., ARTs, PARPs, SIRTs, CD38, CD157)

Figure 4 General pathways of NAD metabolism. In liver, NAD may be synthesized from dietary nicotinic acid, nicotinamide, nicotinamide riboside, and tryptophan (“de novo” pathway) or may be readily absorbed from foods. NAD may also be synthesized through the “salvage pathway,” which is fueled by the nicotinamide resulting from the activity of NAD-consuming enzymes. Abbreviations: PARPs, poly(adenosine diphosphate ribose) polymerases; ARTs, monoADP-ribosyltransferases; SIRTs, Sir2-like protein deacetylases; NAD, nicotinamide adenine dinucleotide. 1: Nicotinic acid phosphoribosyltransferase; 2: (nicotinamide/nicotinic acid) mononucleotide adenosine-5 -triphosphate adenylyltransferase (NMNAT/NaMNAT); 3: NAD synthase; 4: nicotinamide phosphoribosyltransferase (or PBEF); 5: NAD kinase; 6: nicotinamide riboside kinases (Nrks).

These nucleotides can be synthesized “de novo,” using tryptophan from the diet to generate nicotinic acid mononucleotide (Fig. 1) or through the “salvage pathway” (Fig. 4), using nicotinic acid, nicotinamide, and nicotinamide riboside absorbed from nutrients, or through nicotinamide recycled from signaling reactions that involve NAD catabolism [for review see Ref. (8)]. Tryptophan metabolism, initiated by tryptophan-2,3dioxygenase, a tryptophan-inducible enzyme, occurs primarily in liver. Because quinolinic acid phosphoribosyltransferase activity in mammals was detected only in liver and kidney, other tissues rely mostly on an exogenous supply of nicotinic acid/nicotinamide/nicotinamide riboside for NAD biosynthesis, hence their role as essential nutrients. The rate-limiting step of the salvage pathway is catalyzed by a nicotinamide phosphoribosyltransferase, also known as pre-B colony-enhancing factor (PBEF), an inflammatory cytokine (9). The last step of NAD synthesis is catalyzed by nicotinamide/nicotinic acid mononucleotide adenylyltransferases (NMNAT1–3, in humans) (10–12), which use both nicotinamide mononucleotide and nicotinic acid mononucleotide as targets for the adenylyl-transfer reaction. In yeast, genes of the “de novo” synthesis pathway are silenced by an NAD-dependent histone deacetylase, which functions as a sensor of levels in

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nuclear NAD pools (13). NADP is formed directly from NAD by phosphorylation catalyzed by a specific kinase present in most tissues except skeletal muscle (14). These pyridine nucleotides are involved in numerous reactions, ranging from energy metabolism to cell signaling. As coenzymes, they are required in most of the metabolic redox processes of the cell, in which dehydrogenases use NAD/P(H) as coenzymes to oxidize or reduce substrates. NADP dehydrogenases are preferentially involved in anabolic reactions (e.g., synthesis of fatty acids and cholesterol) (15). In contrast, NAD is used in catabolic reactions to transfer the potential free energy stored in macronutrients such as carbohydrates, lipids, and proteins to NADH, which is then used to form ATP, the primary energy currency of the cell. Besides its well-known role in energy transduction, NAD is also substrate for four other classes of enzymes, mono-ADP-ribosyltransferases (ARTs), poly (adenosine diphosphate ribose) polymerases (PARPs), ADP-ribosylcyclases (e.g., CD38, CD157), and Sir2-like protein deacetylases (SIRTs). ARTs and PARPs catalyze the activation of the ␤-N-glycosylic bond of NAD and transfer of the ADP-ribose moiety to acceptor proteins or another ADP-ribose in the case of PARPs. Many of the ARTs also demonstrate NAD glycohydrolase activities, in which water is the ADP-ribose acceptor, as do ADPribosylcyclases. The latter, in addition, catalyze the formation of potent calcium-mobilizing second messengers, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide (NAAD) from NAD, as well as cyclic ADPribose phosphate (cADPRP), and nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP. In contrast, most SIRTs use NAD as an acceptor of the acetyl group that is removed from proteins, thereby generating 2 -O-acetyl-ADP-ribose although SIRT4 was recently identified as a mitochondrial ART (16). ARTs transfer a single ADPR moiety per specific acceptor amino acid (e.g., arginine, cysteine, asparagine, histidine). In general, the purpose of this covalent modification is to alter the biological activity of the acceptor protein (reviewed in Ref. 17). The known vertebrate ARTs (ART1–7) are secreted or glycosylphosphatidylinositolanchored proteins. Their enzymatic activities have been implicated in the regulation of diverse cell processes including myocyte differentiation and modulation of immune cell functions (e.g., T-lymphocyte cytotoxicity and neutrophil chemotaxis) (17). The well-established modulatory properties of mono-ADP-ribosylation on intracellular targets (e.g., G proteins, chaperone proteins, cytoskeleton, Golgi components) suggest that additional ARTs are yet to be identified. The first described PARPs were recognized for their ability to synthesize highly negatively charged ADPribose polymers on themselves and/or target proteins, thereby affecting protein folding and, hence, protein function (for review, see Ref. 18,19). However more recently, other proteins carrying the PARP signature domain have been shown to transfer one single ADPR moiety similarly to ARTs (20). This posttranslational modification is transient as the polymers or monomers are rapidly metabolized by three enzymes, poly-ADP-ribose glycohydrolase, pyrophosphatases, and lyase. Poly-ADP-ribosylation is involved in the regulation of many vital cellular events,

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for example, DNA replication and repair, chromatin structure, transcription, apoptosis, and regulation of telomere length (18,19). In general, members of the PARP family are nuclear DNA-binding proteins that catalyze the polymerization and branching of ADPR chains on target proteins. The most extensively studied member, PARP1, is markedly activated at sites of single-strand DNA breaks. Overactivation of PARP1 after extensive DNA damage leads to rapid depletion of NAD and ATP and ultimately cell death. Because niacin contributes to maintaining the NAD levels for PARP, niacin deficiency could compromise DNA repair and increase the risk of cancer. Alternatively, the inhibitory effect of niacin on PARP1 activity should also be taken into account; an excess of niacin could also impair DNA repair. However, there is a growing body of experimental and epidemiological evidence for a relationship between niacin status and genomic stability (18,19). Several PARP1 inhibitors are in clinical trial as potential antitumor drugs (21–24). The third group of enzymes, the NADglycohydrolase/ADP-ribosylcyclases, includes CD38 and CD157 in mammals (25). These membrane proteins use NAD and NADP to generate signaling molecules, cADPR and cADPRP, by cyclization and NAADP by transglycosidation. These pyridine derivatives have critical signaling functions in the mobilization of intracellular calcium stores via modulation of the Ca2+ -releasing channel ryanodine receptors. Thanks to its very strong NADase activity, CD38 may also control NAD levels in cells and thus NAD-dependent physiologic processes (25). As shown by targeted gene inactivation in mice, CD38 is required for appropriate cell-dependent antibody and innate immune responses to bacterial pathogens (26), whereas CD157 participates in the regulation of the humoral T-cell-independent immune and mucosal thymus-dependent responses (27). Both cyclases have been implicated in the development of autoimmune disorders, although their role(s) is (are) not well established. CD38 has been proposed as a mediator of glucoseinduced insulin secretion from pancreatic ␤-cells via the increase of intracellular Ca2+ concentration and may be involved in the pathogenesis of autoimmune diabetes (28). It has been postulated that upregulation of CD157 expression in some patients with rheumatoid arthritis may contribute to the development of this autoimmune disease (26). Sir2-like proteins are related to the yeast silent information regulator (Sir2), an enzyme required for lifespan expansion in conditions of nutrient scarcity in many organisms (29,30). In humans, SIRT1, the most closely related to yeast Sir2, deacetylates p53, thereby inhibiting apoptosis in response to DNA damage. In mice, absence of SIRT1 leads to p53 hyperacetylation, impaired development, a shortened lifespan, and sterility. Thus, in several species, SIRT1 and homologs appear to regulate diverse pathways that have one common feature, that is, their impact on aging (31). Because nicotinamide is a potent inhibitor of SIRT activity, it has been proposed to serve as a physiologic regulator, whose level would be controlled by the rate of its conversion to nicotinic acid through the NAD+ salvage pathway, and/or to N-methyl nicotinamide, by the excretion pathway. Whether a decrease in nicotinamide, or an increase in NAD levels, is responsible

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for the increased activity of Sir2 during caloric restriction is still debated (29,32). Thus, NAD-consuming enzymes, by their activities, link the nutritional and metabolic status of the cell to the regulation of essential cell functions, such as gene silencing, maintenance of genome integrity, and innate immunity. Many of these reactions yield nicotinamide in addition to other molecules, thus fueling the NAD “salvage pathway” for NAD resynthesis.

INDICATIONS AND USAGE Supplementation to Achieve Recommended Intake Levels The recommended dietary allowance (RDA), as defined in the report of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Niacin (5), is summarized in Table 1. These values were established according to the doses required to prevent pellagra (11.3–13.3 mg of NE/day). The tryptophan content of a reasonable dietary protein intake is itself likely to provide at least 13.2 mg/day NE (based on a 2000 kcal/day diet). There are no relevant data concerning niacin requirements in pregnancy and lactation. Thus, requirements were estimated based on an average increase in energy expenditure of 300 kcal/day during pregnancy and an average daily secretion of 1.4 mg of NE during lactation (5). As numerous NAD-dependent enzymes (e.g., PARPs, SIRTs) can affect genomic stability, insufficient nicotinic acid intake is likely to increase the risk of cancer and other diseases attributable to increased DNA damage (33). It has been suggested that maintenance of adequate NAD levels would, in the long term, prevent or retard the multistage Table 1

Recommended Dietary Allowances of Niacina

Age (yr) Infants 0–0.5 0.5–1.0 Children 1–3 4–8 Males 9–13 14–18 19 and above Females 9–13 14–18 19 and above Pregnancy 14–50 Lactation 14–50 a RDA,

process of carcinogenesis and age-related diseases. Several lines of evidence in yeast and in rodents support this hypothesis (34). Furthermore, studies in rats suggest that niacin supplementation could decrease the risk for development of chemotherapy-related malignancies in cancer patients with compromised nutritional status (35,36). According to preliminary experimental data linking niacin status and genomic stability, the doses required to prevent pellagra would not be sufficient to promote genomic stability (37). Niacin nutriture has been assessed in several ways by a variety of methods (for review, see Ref 38). Dowex1 formate chromatography is used to separate pyridine nucleotides and N1 -methylnicotinamide. Measurement of the latter and its 2-pyridone derivative in urine is most commonly used. Excretion of N1 -methylnicotinamide below 0.8 mg/day indicates niacin deficiency (38). A ratio of N1 -methyl-5-carboxamide-2-pyridone to N1 methylnicotinamide of 1.3 to 2.0 is considered normal; in niacin deficiency, it is less than 1.0. Niacin status can also be assessed by measuring its physiologically active forms, NAD(H)/NADP(H). A method devised by Lowry et al. in 1961, and modified by Slater and Sawyer in 1962 and Nisselbaum and Green in 1969, uses appropriate dehydrogenases specific for either NAD or NADP and thiazolyl blue, which, when reduced by NADH and NADPH, forms purple formazan in an amount proportional to the concentration of the coenzymes (oxidized and reduced). This assay is used to measure pyridine nucleotides in tissue and blood. Assays of NAD/NADP in erythrocytes and cultured cells suggest that the intracellular NAD level may reflect niacin status, whereas NADP levels do not. Measurement of NAD/NADP content and tryptophan level in erythrocytes has been proposed to evaluate niacin deficiency (4).

RDA (mg)b 2c 4c 6d 8d 12d 16d 12 12d 14d 14 17 18

1998 United States Food and Nutrition Board of the Institute of Medicine. b One milligram niacin = 60 mg tryptophan = niacin equivalent (NE). c For infants, given values correspond to the adequate intake (AI) level, which is based on the observed mean intake of preformed niacin by infants fed with human milk. d No data being available for these ranges of age, RDAs were estimated by extrapolation from adult values.

Treatment of Niacin Deficiency In humans, the combination of inadequate intakes of tryptophan and niacin leads to pellagra. This name was given by Frapolli in 1771, from the Italian words “pelle” for “skin” and “agra” for “rough” to describe the roughened, sunburned-like appearance of the skin of niacin-deficient patients exposed to sunlight. Other symptoms include diarrhea and neuropathy (39). In its most acute form, deficiency can lead to death. Maize-based diets predispose to pellagra because of the limited bioavailability of the niacin contained in this grain and its low tryptophan level. Niacin bioavailability can be improved, however, by alkaline treatment. In Central America, where corn used for the preparation of tortillas is first soaked in lime solution, the incidence of pellagra is very low, despite the corn-based staple diet (4). Today, this disease seems to be endemic mostly in parts of India, China, and Africa (39). Advanced stages of pellagra can be cured with nicotinamide in IM doses of 50 to 100 mg three times a day for 3 to 4 days, followed by similar quantities orally, supplemented with 100 g of proteins daily. Other factors such as alcoholism or AIDS may promote the appearance of pellagra (reviewed in Ref 40). Vitamins B6 and B2 (riboflavin) are coenzymes required for the efficient conversion of tryptophan to niacin (Fig. 2). Hence, an inadequate intake of these vitamins is likely to

Niacin

predispose to pellagra. An excess of leucine also impairs tryptophan bioconversion by competing for transport and by inhibiting kynureninase, resulting in decreased NAD formation. There are several reports of a higher incidence of pellagra in women than in men. This difference may have several causes, including cultural factors that determine food intake, metabolic stresses due to repeated pregnancies, and lactation. In addition, estrogen metabolites can inhibit kynureninase and kynurenine hydroxylase activity. When the intake of preformed niacin and tryptophan is low, inhibitory effects of estrogens on tryptophan bioconversion could contribute to a greater susceptibility of women to pellagra. Inborn disorders of tryptophan metabolism can cause nondietary pellagra (reviewed in Ref 41). In Hartnup’s syndrome (an autosomal recessive disorder), decreased absorption and/or increased excretion of tryptophan lead to inadequate conversion of this essential amino acid to niacin. The symptoms of niacin deficiency can be alleviated by large doses of the vitamin (40–250 mg/day).

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which are consistent with PARP1 inhibition, nicotinamide exhibits PARP1-independent actions (19) that may be attributable to its inhibition of other signaling pathways (e.g., SIRTs) and its function as a precursor of pyridine nucleotides (46). Nicotinamide has been proposed as a possible means of increasing the survival of pancreatic ␤-cells after diagnosis of Type I diabetes (insulin-dependent diabetes mellitus, IDDM), or to prevent onset of the disease in high-risk individuals (reviewed in Ref 19). This latter notion was not confirmed, however, by the recently published European Nicotinamide Diabetes Intervention Trial (ENDIT), a large-scale evaluation of nicotinamide benefits in first-degree relatives of Type I diabetic patients (47).

Adverse Effect of Drugs on Niacin Status Isoniazid, which is commonly used to treat tuberculosis, causes vitamin B6 depletion and hence may lower the efficiency of the “de novo” synthesis pathway that converts tryptophan into nicotinic acid, thereby predisposing to pellagra (4).

Treatment of Hyperlipidemia In pharmacological doses (2–6 g/day), nicotinic acid, but not nicotinamide, significantly reduces atherosclerotic cardiovascular disease and mortality (42). The benefits of nicotinic acid treatment are due to its antihyperlipidemic effects at high doses. It decreases the levels of plasma lowdensity lipoproteins (LDLs), very low-density lipoproteins (VLDLs), and triglycerides (TGs), and increases the high-density lipoproteins (HDLs), thus reducing the LDL/HDL ratio. The mechanism of action of nicotinic acid on lipoprotein metabolism has not been completely elucidated (reviewed in Ref 43). Available data suggest that nicotinic acid decreases the formation of LDL and VLDL by inhibiting the lipolysis of TG in adipose tissue and TG synthesis in liver. In adipose tissue, the antilipolytic effect is mediated by niacin activation of a recently characterized Gi/o protein-coupled high-affinity receptor that inhibits cAMP-stimulated lipolysis (44,45). On the other hand, nicotinic acid promotes the synthesis of HDL by preventing the catabolism of a major protein component of HDL apolipoprotein A-I (apoA-I), but not of cholesterol esters from HDL. It has been proposed that an increase in the amount of apoAI available for HDL synthesis would augment reverse cholesterol transport, facilitating the removal of excess cholesterol from peripheral tissues and thereby lowering the risk of atherosclerotic cardiovascular disease. When nicotinic acid monotherapy does not lower the blood cholesterol level sufficiently, it is administrated in combination with other lipid-lowering drugs that act through different mechanisms (e.g., bile-acid-binding resins, statins). This strategy has proved successful in several clinical trials (46).

Prevention of Oxidant-Induced Cell Injury in Pathological Conditions At high doses (up to 3.5 g/day), nicotinamide is protective against cell death and inhibits the production of inflammatory mediators in animal and in “in vitro” models of oxidant-induced cell injury. In addition to these effects,

CONTRAINDICATIONS Because of its potential side effects, antihyperlipidemic treatment with nicotinic acid is contraindicated in patients with active peptic ulcer or frequent gout attacks. Until recently, those with Type II diabetes mellitus were also considered at risk, but new clinical data seem to indicate that nicotinic acid can be used safely to treat diabetic dyslipidemia (48).

PRECAUTIONS AND ADVERSE REACTIONS Prostaglandin-mediated flushing is the major specific side effect experienced by users of pharmacological doses of nicotinic acid in the initial days of treatment. Symptoms can be reduced by ingestion of the drug with food and/or by gradually increasing the dose. Tolerance develops with continued use in most patients. Flushing has been documented in patients using immediate-release nicotinic acid (IR-nicotinic acid) and sustained-release forms, as well as by some subjects on extended-release nicotinic acid (ERnicotinic acid). However, in general, the extended-release formulation achieves the efficacy of the immediate-release form with a reduced incidence of flushing and without the hepatic problems caused by slow-release nicotinic acid (49). Other reported adverse effects of nicotinic acid treatment include pruritis, nausea, gastrointestinal upset, hypotension, tachycardia, and elevated serum blood glucose and uric acid levels. Because of potential hepatic toxicity, liver enzymes (aminotransferases and/or alkaline phosphatase) should be monitored before the initiation of therapy, 6 weeks after initiation and/or any change of dose, and two or three times a year thereafter. If liver enzymes exceed three times the upper limit of normal, treatment should be discontinued. To avoid liver toxicity, it is recommended that the starting dose should not exceed 250 to 300 mg/day with monthly increments not greater than 250 to 300 mg/day until a maximum of 3 g/day is reached for IR-nicotinic acid and 1.5 to 2 g/day for the sustained-release form.

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Nicotinic acid may cause insulin resistance, which requires compensatory insulin secretion, and, in patients with dysfunctional pancreatic ␤-cells, it may trigger hyperglycemia. Those with diabetes mellitus, therefore, require special monitoring during niacin treatment (19). No adverse effects of the pharmacological doses of nicotinamide used during the ENDIT study were reported (47). However, there has been concern that saturation of the nicotinamide excretion pathway may divert methylation equivalents required for anabolic pathways to nicotinamide methylation and lead to growth retardation in children. Furthermore, as a strong inhibitor of SIRTs, nicotinamide might interfere with cell survival (34). Thus, more data are needed on the long-term effects of therapeutic doses of nicotinamide.

OVERDOSAGE No adverse events of the consumption of naturally occurring niacin in food have been reported. Side effects have been widely recognized in patients treated for hyperlipidemia with high doses (3–9 g/day) of pharmaceutical preparations of nicotinic acid for periods of months to years (48). Symptoms of nausea and vomiting and signs of liver toxicity with intake of more than 3000 mg/day of nicotinamide or 1500 mg/day of nicotinic acid have been reported. Most frequently, patients develop jaundice and increased levels of serum transaminases. In the most severe cases, liver dysfunction and fulminant hepatitis can result (49).

CONCLUSIONS Meat, cereals, eggs, and milk are the main sources of vitamin B3, the general term to designate niacin (nicotinic acid and nicotinamide) and NE (tryptophan). Deficiency, which may be caused by poor dietary intake or inherited disorders (e.g., Hartnup’s syndrome), results in pellagra. The diversity of pellagra symptoms is representative of the wide spectrum of pathways that require adequate niacin intake to function. The molecular mechanisms by which insufficient niacin uptake causes these symptoms are poorly understood. Some may reflect primarily the role of the vitamin as a precursor of NAD and NADP, others to its requirement, as coenzyme or substrate in many enzymatic reactions. Niacin nutritional status may have consequences for cellular functions as diverse as immunity, genomic stability, and energy supply. In addition to its role as a niacin source, nicotinic acid is well established, and widely employed therapeutically, because of its efficacy as an antihyperlipidemic agent. Risks of hepatotoxicity and other side effects have decreased with the development of new niacin formulations that improve drug delivery and are better tolerated by patients. The recent characterization of different types of nicotinic acid receptors may help in the development of more specific agonists with fewer side effects. Nicotinamide is the niacin source of choice to treat pellagra. Other pharmacological applications of nicotinamide have produced mixed results. It did not seem effective in preventing the development of autoimmune diabetes or for protection against

oxidant-induced cell death. This may be due to the roles of nicotinamide as both an NAD precursor and an inhibitor of several relevant enzymes (e.g., SIRTs, PARPs). Recent studies suggest that, by sustaining adequate NAD levels, pharmacological doses of niacin could contribute to the prevention or delay of age-related diseases in healthy individuals and protect cancer patients against secondary effects of anticancer therapy. However, because of their multiple functions, more studies are necessary to evaluate the benefits and consequences of pharmacological doses of nicotinamide and nicotinic acid.

ACKNOWLEDGMENTS We thank Dr. Martha Vaughan and Dr. Vincent Manganiello for helpful discussions and critical review of the manuscript.

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32. Lin SJ, Guarente L. Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and disease. Curr Opin Cell Biol 2003; 15(2):241–246. 33. Kirkland JB. Niacin status and treatment–related leukemogenesis. Mol Cancer Ther 2009; 8(4):725–732. 34. Anderson RM, Bitterman KJ, Wood JG, et al. Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J Biol Chem 2002; 277(21):18881–18890. 35. Spronck JC, Kirkland JB. Niacin deficiency increases spontaneous and etoposide-induced chromosomal instability in rat bone marrow cells in vivo. Mutat Res 2002; 508(1–2):83–97. 36. Bartleman AP, Jacobs R, Kirkland JB. Niacin supplementation decreases the incidence of alkylation-induced nonlymphocytic leukemia in Long-Evans rats. Nutr Cancer 2008; 60(2):251–258. 37. Fenech M. Micronutrients and genomic stability: a new paradigm for recommended dietary allowances (RDAs). Food Chem Toxicol 2002; 40(8):1113–1117. 38. Sauberlich HE. Niacin. Laboratory Tests for the Assessment of Nutritional Status. 2nd ed. Boca Raton, FL: CRC Press, 1999:161–174. 39. Karthikeyan K, Thappa DM. Pellagra and skin. Int J Dermatol 2002; 41(8):476–481. 40. Sauve AA. NAD+ and vitamin B3: from metabolism to therapies. J Pharmacol Exp Ther 2008; 324(3):883–893. 41. Levy HL. Chapter 193: Hartnup disorder. The Metabolic and Molecular Basis of Inherited Diseases. New York: McGrawHill, 2003. Available at http://genetics.accessmedicine. com/index.html. 42. Brown BG, Zhao XQ. Nicotinic acid, alone and in combinations, for reduction of cardiovascular risk. Am J Cardiol 2008; 101(8A):58B–62B. 43. Digby JE, Lee JM, Choudhury RP. Nicotinic acid and the prevention of coronary artery disease. Curr Opin Lipidol 2009; 20(4):321–326. 44. Tunaru S, Kero J, Schaub A, et al. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect. Nat Med 2003; 9(3):352–355. 45. Wise A, Foord SM, Fraser NJ, et al. Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 2003; 278(11):9869–9874. 46. Klaidman L, Morales M, Kem S, et al. Nicotinamide offers multiple protective mechanisms in stroke as a precursor for NAD+, as a PARP inhibitor and by partial restoration of mitochondrial function. Pharmacology 2003; 69(3):150–157. 47. Gale EA, Bingley PJ, Emmett CL, et al. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Lancet 2004; 363(9413):925–931. 48. Guyton JR, Bays HE. Safety considerations with niacin therapy. Am J Cardiol 2007; 99(6 A):22C–31C. 49. Alsheikh-Ali AA, Karas RH. The safety of niacin in the US Food and Drug Administration adverse event reporting database. Am J Cardiol 2008; 101(8 A):9B–13B.

Noni Alison D. Pawlus, Bao-Ning Su, Ye Deng, and A. Douglas Kinghorn

INTRODUCTION Morinda citrifolia L. (Rubiaceae) (noni) is an important medicinal plant of South Asian origin. All parts of this species have been used traditionally in regions such as South and Southeast Asia, Polynesia, Northeastern Australia, and the Caribbean for a wide range of ailments, primarily topical diseases. The fruits, and more rarely the leaves, are currently being marketed in the United States as a botanical dietary supplement for several chronic ailments and to promote overall general health. Several limited in vitro and in vivo studies in mice have suggested that noni may help treat or prevent cancer, pain, and cardiovascular disease. These studies are limited in scope and only a few pure compounds from noni have been tested for biological activity, including against targets related to cancer and inflammation. The biological activity primarily ascribed to noni in books and other promotional information written for a general audience is based on a hypothesis developed by Ralph M. Heinicke. This idea, centered on the mythical compound “xeronine,” has not been scientifically validated. A phase I clinical trial on a freeze-dried noni fruit extract, sponsored by the National Center for Complementary and Alternative Medicine (NCCAM), National Institutes of Health, Bethesda, MD, was conducted in Hawaii in cancer patients. There is an active scientific literature on aspects of the phytochemistry, biological activity testing, and potential safety of the constituents of the various plant parts of M. citrifolia, with such reports found as peer-reviewed research articles, meeting abstracts, and patents.

Figure 1 Drawing of Morinda citrifolia L. (calibration bar = 1 cm). Source: From Singh YN, Ikahihifo T, Panuve M, et al. Folk medicine in Tonga. A study on the use of herbal medicines for the obstetric and gynaecological conditions and disorders. J Ethnopharmacol 1984; 12:305–529, with permission from Elsevier.

Background M. citrifolia L. (Rubiaceae) has a host of common names, including ach, awl tree, canary wood, doleur, gardenia hedionda, gogu atogi, great morinda, hog apple, Indian mulberry, kura, lada, mengkoedoe, mengkudu, morinda, mulberry, nen, nh`au, nino, and noni (Fig. 1) (1,2).

a waxy, semitranslucent skin, and the surface is covered with four- to six-sided outlines with a central “eye.” The ripe fruit is reported to vary in scent, with some trees containing nonscented to slightly scented fruits to other trees with a strong, unpleasant butyric acid odor (2). The seeds include an air chamber, which makes them buoyant, and can remain viable after months of floating in water. This attribute led to the hypothesis that noni was spread among the Polynesian islands by floating from island to island (1,3,4). There are two recognized varieties of noni, namely, M. citrifolia var. citrifolia and M. citrifolia var. bracteata and one cultivar, M. citrifolia cultivar Potteri. Of these, M. citrifolia var. citrifolia is the most commonly found and has the greatest importance economically (3,4).

Botanical Description M. citrifolia L. (noni) is a small evergreen tree or shrub, 3 to 10 m in height, which grows throughout the Pacific and other regions. Noni is known to be very adaptive and can easily colonize new islands and terrains, as it grows in a wide range of soil types and in both wet and dry areas. The leaves of noni are opposite, pinnately veined, glossy, elliptic to elliptic-ovate in shape, and are 20 to 45 cm long and 7 to 25 cm wide. The flowers are perfect with fivelobed, small, white corollas. The fruits are syncarpous, yellowish white, fleshy, and 5 to 10 cm long and 3 to 4 cm in diameter at maturity. The lumpy fruits are soft with 570

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Noni is currently cultivated for commercial use in the Pacific islands such as Tahiti and Hawaii and in Australia. The fruits are harvested year-round and can be picked just prior to fully ripening to be shipped or can be gathered when ripe for local processing (1–4).

Ethnomedical Use of Noni The leaves and fruits are eaten as food in times of famine, although it has been suggested in several botanical reports on noni that some cultures have ingested this plant as a regular food (1,2). Medicinally, various parts of M. citrifolia are used both externally and internally, alone or in combination with other plants. Externally, the fruits and leaves have been used to treat boils, pain and inflammation, ringworm, scabies, and wounds (5–7). In Fiji, the leaves and flowers are heated and the juice squeezed into ulcers, and the leaves are then used as a bandage (6). Internally, the different parts of the noni plant have been used to stimulate the appetite in patients with wasting diseases and also as an antiemetic (8). The juice of the fruit is also applied to aching teeth or sore gums and to treat halitosis. A concoction of the root with coconut is taken internally for scabies and skin eruptions and to treat intestinal infections (6). In Vietnam, the root-bark is used to treat hypertension and a decoction made from the leaves is used for diarrhea and fever. The fruit is also used to treat dysentery, neuralgia, and a number of other common ailments (9). Also, noni fruits have been used in combination with kava (Piper methysticum Forst. f.) (Piperaceae) to treat tuberculosis (1).

Current Use of Noni in the United States The use of noni fruit in the United States as a botanical dietary supplement is becoming more widespread, and noni products are widely available in health food stores, pharmacies, chain grocery stores specializing in natural foods, and on the Internet. The current rise in popularity of noni may be due to the increase in publicity as a general “cure-all” or panacea for chronic conditions. This is exemplified by a number of books devoted entirely to the subject of noni, which provide anecdotal evidence of the use of noni to alleviate problems such as cancer, chronic pain, depression, drug addiction, hypertension, and obesity (10,11). Cancer patients, in particular, are using noni as an alternative method to treat or to complement their anticancer drug regimens. A small number of scientific studies (summarized later), along with ethnobotanical use, has suggested that noni juice may help alleviate symptoms and/or increase efficacy of cancer chemotherapy. The disparity between traditional ethnobotanical uses and currently marketed uses of noni has recently been expounded upon in a review by McClatchey (2). Briefly, his ethnobotanical research in Fiji, Hawaii, Rotuma, and Soma found that expert healers primarily use the leaves, followed by the young green fruit and the root bark and inner stem for topical ailments. The ripe fruit, which is the predominant formulation used in the United States is used in these same areas as home remedies by nonhealers.

Chemistry and Preparation of Products Chemistry Investigation of the chemical constituents of this valuable medicinal plant began over 150 years ago. In

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1848, Anderson crystallized a compound described as light yellow needles of faintly bitter taste, which was named morindin, from the root bark of noni (12). A few other structurally similar compounds (anthraquinone glycosides) were also obtained by extraction and crystallization from other Morinda species (12,13), and all of these isolates were also named “morindin.” The structure of morindin isolated from the root bark of noni was determined to be 1,5-dihydroxy-2-methyl-6-␤primverosyloxyathraquinone on the basis of the chemical conversion evidence and analysis of its physical and spectroscopic data. In addition to anthraquinones, other major compounds isolated from this plant are flavonol glycosides (14), iridoid glycosides (14–16), lignans (17,18), lipid glycosides (19,20), and triterpenoids (21–23). So far, over 130 compounds have been isolated from different anatomical parts of noni and the structures of a few selected components are given in Figure 2. It is noteworthy that most of the iridoid glycosides isolated from noni are oxygenated at C-10 (Fig. 2). Also, an alkaloid (21), fatty acids (23,24), monoterpenoids (24), and steroids (22,23) have also been isolated and characterized. Recently, the composition of an ethanol-insoluble polysaccharide constituent has been determined for a noni fruit sample obtained from Vietnam (25). Despite these phytochemical studies, the precursor of a compound called “xeronine” has been touted to be the major biologically active constituent of noni in books and other promotional materials oriented toward the lay public. This stems from a 1985 paper by Ralph Heinicke entitled “The Pharmacologically Active Ingredient of Noni,” where he proposed a hypothesis as to how noni is able to help cure a wide range of diseases (26). Briefly, he stated that “xeronine” is required by all cells and that a deficiency can lead to a number of ailments that can be prevented or cured by noni supplementation. However, Heinicke’s paper, along with all subsequent documentation, lack any supportive evidence for the presence, structure, or biological requirement of “xeronine” and, therefore, his theory is questionable at best (2).

Preparation of Products Although noni fruit, and occasionally the leaves, is sold as tablets and in the form of herbal teas, it is encountered most commonly as a juice derived from the fruits. These noni fruit juices are frequently prepared by diluting the dried powdered fruit with other juices such as grape juice to increase palatability. A few noni products are available, which claim to be standardized to a given percentage of polysaccharides. This is presumed to stem from the studies demonstrating the potential anticancer activity of the polysaccharide-rich partition (summarized later). The main delineation between the currently marketed noni juice products in the United States is the plant source, with most either of Tahitian or Hawaiian origin. Two recent reports have pointed to various marker molecules that may be used in the quality control of commercial noni juice and/or capsule products by using chromatographic methods, including substances of the anthraquinone, coumarin, fatty acid glucoside, flavonoid, and iridoid classes (27,28).

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O

O HO HO HO

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OMe

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Figure 2

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The structures of selected constituents of M. citrifolia (noni).

Stability

D-glucopyranose, isolated from noni fruits, suppressed 12-

No information is available.

O-tetradecanoylphorbol-13-acetate (TPA)- and epidermal growth factor-induced AP-1 transactivation in JB6 mouse epidermal cells (31). Furthermore, an anthraquinone isolated from noni roots, damnacanthal, is a potent tyrosine kinase inhibitor. Although damnacanthal has been shown to be an inhibitor of specific tyrosine kinases (32), the activity disappeared in one study when the authors switched from a cell-free to a whole-cell system (33). Damnacanthal and its structural analog, morindone, have demonstrated strong topoisomerase-II inhibition in a cell-free test system (34). Damnacanthal was also found to induce normal morphology in cells expressing the ras oncogene, termed K-rasts -NRK cells, but not in cells expressing the src oncogene (35). Several recent reports have correlated constituents of M. citrifolia with cancer chemoprevention. The anthraquinone, 2-methoxy-1,3,6-trihydroxyanthraquinone, a trace component of noni fruits, was demonstrated as a very potent inducer of quinone reductase (QR), a phaseII-metabolizing enzyme considered protective at the

Preclinical Studies Cancer A number of in vitro bioassay evaluations have been performed on isolated compounds from noni, which suggest possible cancer preventative or therapeutic activities. For example, several compounds have demonstrated inhibition of activator protein-1 (AP-1) in different cell systems. AP-1 is a transcription factor involved in the tumor promotion stage of carcinogenesis and its inhibition may be an important mechanism in a variety of cancers at this stage. The iridoids, citrifolinin A and citrifolinoside, isolated from the leaves of noni prevented ultraviolet B (UV-B)-induced AP-1 activity in cell culture. UV-B irradiation acts as both an initiator and a tumor promoter in the development of skin cancers (29,30). Two other compounds, asperulosidic acid and the glycoside 6-O-(␤-D-glucopyranosyl)-1-O-octanoyl-␤-

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initiation stage of carcinogenesis. Furthermore, this compound was not found to be cytotoxic for the Hepa 1c1c7 murine hepatoma host cells (36). Four structurally related anthraquinones from noni roots were also found to be QR-inducing agents (37). Several constituents (anthraquinones, saccharide fatty acid esters, the iridoid glycoside, asperulosidic acid, and the flavonoid glycoside, rutin) of noni fruits were reported as inhibitors of the activation of Epstein–Barr virus early antigen induced by TPA (12-O-tetradecanoylphorbol 13-acetate) (38). However, none of these in vitro results have been followed up by a relevant in vivo bioassay germane to cancer chemoprevention, such as a two-stage carcinogenesis inhibition test.

Angiogenesis Inhibition In order for solid tumors to grow, they must be able to develop new blood vessels through a process termed angiogenesis. Without angiogenesis a tumor would cease growing due to metabolic deprivation. Therefore, there has been an interest in finding antiangiogenic agents that can prevent the development of tumors or target their recently formed vasculature. In an in vitro angiogenesis model using placental vein explants, noni juice at a concentration of 5% in the growth media was able to effectively suppress angiogenic initiation. In a similar experiment where the placental explants were allowed to grow for 7 days and then treated with 10% noni juice, there was an observed inhibition of new growth and a breakdown in the recently developed vasculature. The same experiments were carried out using human breast cancer explants where new blood vessel development was also suppressed with media supplemented with noni juice. No positive controls appear to have been used for these studies (39).

Immunostimulation Several studies performed at the University of Hawaii have demonstrated potential anticancer activity of the polysaccharide-rich, ethanol-insoluble precipitate (EtOHppt) of noni fruits. Initially, the juice from the noni fruits significantly prolonged the lifespan of mice injected IP with Lewis lung carcinoma cells. Further studies to compare the EtOH-soluble precipitate and EtOHppt demonstrated the life-prolonging activity to be in the EtOH-ppt. This antitumor activity was abrogated by concurrent administration of the immunosuppressants, 2-chloroadenosine or cyclosporin, indicating that the activity may be due to an immune-stimulating effect of the EtOH-ppt (40). Furthermore, there was an increase in survival by the concurrent administration of the EtOH-ppt with the chemotherapeutic agents vincristine, 5-fluorouracil, cisplatin, and adriamycin when compared with each of these agents alone, suggesting a potential therapeutic complementary treatment or synergistic effect of noni (41). In a more recent study, this same research group performed similar studies using a sarcoma 180 tumor system in mice that is particularly responsive to immune system modulation. The EtOH-ppt, obtained from noni grown in both Hawaii and Tahiti, was similarly injected and demonstrated an increase in survival rate of mice, particularly when given prior to tumor cell inoculation. The number of mice surviving up to 40 to 50 days

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without signs of tumor growth was increased by 45% and 53% when given prophylactic treatment with noni from Hawaii and Tahiti, respectively. This increase in survival in both the prophylactic and the therapeutic treatments with EtOH-ppt was inhibited using 2-chloroadenosine, antiasialo GM1 antibody, and cyclosporin. These are specific inhibitors of macrophages, natural killer cells, and T-cells, respectively, which further support the theory of immune system involvement. Furthermore, similar increases in the survival of test mice were seen when the EtOH-ppt was administered along with certain anticancer agents, but not all (41). It should be emphasized that these studies, while promising, involve the IP injection of EtOH-ppt physically on top of the IP cancer cells, and no studies appear to have been published where the EtOH-ppt was administered orally with analogous efficacy. Recently, it has been shown that noni fruit juice and fruit juice concentrates (dose 1.5 mg/mL) from Tahiti activated cannabinoid 2 (CB2 ) and inhibited cannabinoid 1 (CB1 ) receptors, in a concentration-dependent manner. Coupled with this observation, in the same study administration of the fruit juice to mice for 16 days (ad libitum) decreased the production of interleukin-2 and increased that of interferon-␥ cytokines, suggesting a modulation of the immune system (42). The agents responsible for these effects were not structurally defined.

Cardiovascular Disease A methanol-soluble extract of noni leaf was tested for its ability to prevent oxidation of low-density lipoprotein (LDL) and upregulation of LDL receptors (LDLr) to determine if noni can prevent or slow down the processes involved in cardiovascular disease. The oxidation of LDL, referred to colloquially as “bad cholesterol,” is considered a risk factor for atherosclerosis. The upregulation of LDLr in liver cells is believed to decrease one’s risk for atherosclerosis by decreasing the overall LDL levels in the blood stream. By using these two in vitro bioassays, of 12 plants tested, noni was one of the two that did not demonstrate inhibition of LDL oxidation, yet it was one of the four that caused a significant increase in LDLr in liver cells. In fact, the noni extract was more effective than the positive control, green tea, at upregulating LDLr (43). In another study, noni fruit extract inhibited copperinduced LDL oxidation. Bioassay-guided fractionation of the ethyl acetate partition led to the isolation of six lignans, four of which, 3,3 -bisdemethylpinoresinol, americanol A, morindolin, and isoprincepin, demonstrated potent activity similar to or stronger than the positive control, butylated hydroxytoluene (17). In a recent report on noni produced in Okinawa, Japan, ad libitum intake of 10% noni juice given in the diet to mice for 7 days resulted in a protective effect on neuronal damage after ischemic stress, whereas a lower dose of 3% noni juice in the diet was not effective in this regard (44).

Antioxidant Activity In a study funded by Morinda Inc. (Orem, Utah, USA), their product, Tahitian Noni Juice (TNJ), demonstrated antioxidant activity in both lipid hydroperoxide and tetrazolium nitroblue assays. TNJ exhibited a dose-dependent antioxidant activity that was compared with vitamin C, pycnogenol, and grape seed powder using the current

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recommended daily allowance or manufacturer recommendations for testing concentrations. TNJ had a greater free-radical scavenging activity than the positive controls, but because varying doses were used, a direct comparison is difficult. Animal studies using 10% TNJ in the drinking water of female SD mice and male C57 Bl-6 mice were performed to determine if TNJ can prevent 7,12dimethylbenzanthracene (DMBA)-DNA adduction formation. After 7 days of administering TNJ, intragastric administration of the carcinogen, DMBA, was done and the animals were sacrificed after 24 hours. In both animal models, TNJ was able to decrease the amount of DMBADNA adducts in the heart, lung, liver, and kidney compared with negative controls (45). In a further study, using the leaf, root, and fruit methanol extracts and ethyl acetate partitions, antioxidant activity was measured using the ferric thiocyanate method and thiobarbituric acid test. The root methanol extract had comparable antioxidant activity to the positive controls, ␣-tocopherol and butylated hydroxytoluene. The ethyl acetate partitions of all parts of the plant tested were similar to the positive controls (46). In another study to determine possible antioxidant constituents, diphenylpicrylhydrazyl and peroxynitrite freeradical scavenging assays were used to test compounds isolated from a powdered noni fruit extract. Of the 19 compounds isolated from the n-butanol-soluble partition, the neolignan, americanin A, was found to exhibit potent free-radical scavenging activity in these bioassays (18).

Anti-inflammatory and Analgesic Activity The ability of ethanol extracts of the bark and leaves, the fresh fruit juice, and the fruit powder of noni to inhibit cyclooxygenase I (COX-1) was determined using an in vitro bioassay that measured the arachidonic acid metabolites, PGE2 and PGD2 . Using 3.4 mg/mL as test concentration, the fruit powder had a “high” inhibition of COX-1 whereas the leaf extract had a “moderate” inhibition, as defined by the authors. The IC50 value of the noni fruit powder was 163.3 ␮g/mL and the positive controls, aspirin and indomethacin, had IC50 values of 241.15 and 1.19 ␮g/mL, respectively (47). It has been suggested that TNJ has a higher selectivity for COX-2 than for COX-1, with a COX2/COX-1 IC50 ratio of 0.76, with celecoxib (CelebrexTM ), indomethacin, and aspirin having a ratio of 0.34, 40, and 119, respectively (48). Three lignans and two common flavonoids (quercetin and kaempferol) isolated from noni fruits collected in Tahiti were found to exhibit inhibitory effects (IC50 values PUFA > monounsaturated acids) (41,42); however, cPLA2 hydrolyzes DHA only slowly, likely because of steric hindrance by the nearby 4 double bond as shown decades earlier for pancreatic lipase. Nonetheless, phospholipids with ␻-3 DHA bind well to the enzyme and interfere with enzymatic release of the alternate substrate, ␻-6 AA (41). DHA and AA are likely released from phospholipids of astrocytes by Ca2+ -independent iPLA2 and Ca2+ -sensitive cPLA2 , respectively (43). There are different variants of iPLA2 but the splice variants of the Group VI enzymes range in molecular mass from 85 to 88 kDa (39). The known iPLA2 variants are regulated by cellular levels of cAMP and cAMP-activated kinases in ways that differ from cPLA2 (39).

TRANSFORMATION OF ω-6 EFAs INTO EICOSANOIDS Free AA can be transformed to three types of active hormone-like compounds collectively known as eicosanoids including prostanoids (PGs), leukotrienes (LTs), epoxides (EETs), and related hydroxyl fatty acids (22). Also, LA can be converted to some 18-carbon oxygenated products by the same synthetic enzymes as eicosanoids. AA may exert some of its actions as a free acid. For example, it will activate NADPH oxidase (44) and serve as an activating ligand for peroxisomal proliferatoractivated receptors [PPARs; (45), activate ion channels such as a two-pore domain Kfl channel (44), and stimulate apoptosis (47)]. [␻-Hydroxylated epoxy fatty acids and certain prostanoids may also activate PPARs (48).] However, selectivity or specificity claimed for various actions of nonesterified PUFAs and HUFAs is often modest. As noted earlier, ␻-6 AA synthesized from LA is currently the most abundant ␻-6 HUFA in tissue phospholipids, and some attributions of AA-selective events (such as cPLA2 catalyzed cleavage from phospholipids) result from the availability of AA rather than its selective interaction with proteins (49). PGs, leukotrienes, and epoxides are made by many cells and tissues, where they act locally before being rapidly inactivated by further metabolism. Hence, their biology is difficult to study. However, because ␻-6 eicosanoid overproduction occurs in important pathologies, potent enzyme inhibitors and receptor antagonists have been developed for many therapeutic applications. The relatively recent availability of specific knockout mice for most of the various biosynthetic enzymes and prostanoid receptors, plus the use of specific enzyme inhibitors, and specific receptor agonists and antagonists has greatly enhanced our understanding of eicosanoid physiology and pathology (22).

Biosynthesis and Actions of (PGs) The structures and biosynthetic interrelationships of the most important prostanoids are shown in Figure 4. Letters after PG and TX (thromboxane) denote the nature and location of the oxygen-containing substituents present in the cyclopentane ring. Prostanoids with a subscript ‘‘2’’ are

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Figure 4 Pathways for the synthesis and actions of prostanoids. Selective events in the biosynthesis and action of prostaglandins. Abbreviations: PG, prostaglandin; PGHS, prostaglandin H synthase; COX, cyclooxygenase; POX, peroxidase; DP, receptor for PGD; EP, receptor for PGE; FP, receptor for PGF; IP, receptor for PGI; TP, receptor for TXA; PGDS, PGD synthase; PGES, PGE synthase, PGFS, PGF synthase(s); TxAS, TxA synthase; PGIS; PGI synthase. Numbers shown in gray adjacent to various enzymes and receptors are the ratio of activity with ␻-3 EPA versus ␻-6 AA or the corresponding products formed from these precursors.

formed from ␻-6 AA and those with subscript “3” are from ␻-3 EPA. The PGs are synthesized and released rapidly by cells in response to certain hormones and physical stimuli (22). The dynamics of stimulus-induced prostanoid formation and action involves four distinct stages (Fig. 4). Each has counteracting forces that can cause modest differences in the forward rates to amplify into important physiologic differences: 1. Mobilization of free AA from membrane phospholipids (22) through the activation of Ca2+ -dependent cPLA2 and sPLA2 . This is augmented by phosphorylation of cPLA2 by various kinases and counteracted by the actions of acyl-CoA synthetases that rapidly mediate reesterification of HUFAs. Figure 4 notes similar rates of

hydrolysis for ␻-6 and ␻-3 structures by cPLA2 and sPLA2 . 2. Conversion of AA to PG endoperoxide H2 (PGH2 ) by a PG endoperoxide H synthase (PGHS; also known as cyclooxygenase or COX). The oxygenation reaction can counteracted by removal of obligatory hydroperoxide activators (50) or changes in the levels of nonesterified, nonsubstrate fatty acids (51). PGHS activity is also diminished by “suicide” inactivation of the enzyme (52). 3. Conversion of PGH2 to one of the major types of active prostanoids by a specific synthase (e.g., TXA synthase). Formation of active prostanoid is counteracted by rapid inactivation by a 15-hydroxy dehydrogenase (53) and other metabolic reactions (22). Frequently, specific prostanoids are formed by specific cells types; for

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example, TXA2 is almost exclusively made by platelets, whereas PGI2 is synthesized by vascular endothelial cells. 4. Binding of active prostanoids to specific G-proteinlinked receptors (54,55). Activated receptors interact with downstream effectors such as adenylate cyclase and phospholipase C to modulate the formation and action of second messengers such as cAMP, Ca2+ , and diacylglyceol to affect physiologic processes. The efficacy of prostanoids formed from ␻-6 versus ␻-3 fatty acids (i.e., AA vs. EPA) is an understudied area. A unique, comprehensive report (56) compares the relative specificities of AA versus EPA and their metabolites with enzymes and receptors of the PG pathways. Products derived from ␻-6 AA tend to be more active on prostanoid receptors than those from ␻-3 EPA (56).

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sary to provide sufficient active ligand bound to nearby cellular receptors. When synthase rates are less than inactivation rates, little signaling occurs even though appreciable hormone may be formed. Nine distinct prostanoid receptors are G-proteinlinked receptors (54,55)—two for PGD, four for PGE, and one each for PGF, PGI, and TXA. Figure 4 notes a fivefold greater action of ␻-6 PGF2␣ compared with ␻-3 PGF3␣ at the FP receptor (56). This may provide more effective parturition and more intense dysmenorrhea. The greater action of ␻-6 than ␻-3 forms of PGE at the four EP receptors (Fig. 4) may have important consequences. In addition, the greater action of ␻-3 PGD3 than ␻-6 PGD2 at the DP receptor may enhance observed antiplatelet actions of ␻-3 HUFA (56).

Biosynthesis and Actions of Leukotrienes (LTs) Two isoforms of PGHSs are known as PGHS-1 and -2 or COX-1 and -2 (57). The enzymes are encoded by separate genes. In general, PGHS-1 is expressed constitutively, and its ␻-6 products are essential in parturition, platelet aggregation, and crypt stem cell survival (20,58). In contrast, PGHS-2 is essential for ovulation, implantation, resolution of inflammation, perinatal kidney development, ductus arteriosis remodeling, and ulcer healing (58). PGHS-2 is induced in response to growth factors, cytokines, and inflammatory stimuli, and thus it can be suppressed by steroidal anti-inflammatory glucocorticoidslike dexamethasone (22,59). Figure 4 notes much lower rates of formation for ␻-3 than ␻-6 structures with both PGHS-1 and PGHS-2. Both enzymes are inhibited by nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, and naprosyn. Examples of COX-2-specific inhibitors are celecoxib and rofecoxib. PGHS-2 can convert 2-arachidonoyl-glycerol to 2-PGH2 -glycerol efficiently, and this intermediate can be converted to 2-prostanoylglycerol esters (with the exception of TXA2 ) (57). 2Arachidonoyl-glycerol has hormonal actions on cannabinoid receptors, but the importance of 2-prostanoylglycerol derivatives from PGHS-2 action is unknown. The substrate specificities of PGHS-1 and -2 have been examined in detail (56,60). AA is the most efficient substrate for both PGHS-1 and -2. EPA is a particularly poor one for PGHS-1 and DHA is inactive. Indeed, EPA and DHA can inhibit AA oxygenation by PGHS-1 (61,62). EPA is a substrate for PGHS-2, and DHA can also be oxygenated by PGHS-2. However, neither of these fatty acids inhibit AA oxygenation by PGHS-2 as effectively as PGHS-1 (41). LA can be converted to 9- or 13-HODE by PGHS-1 and -2 at about one-fourth the efficiency of AA. The degree to which oxygenation of LA by PGHS occurs and has biological importance is not known. 13-HODE formed via a 15- LO has been implicated as an effector of cell growth (63). Formation of active prostanoids from PGH2 (and PGH3 ) is catalyzed by two different PGD synthases, at least three PGE synthases, two forms of PGF synthase, a PGI synthase, and a TXA synthase (22). Not all cells express all of these synthases, making prostanoid formation somewhat cell specific. Transcellular formation occurs when the PGH2 formed by platelets diffuses to endothelial cells where PGI2 can be formed. The active eicosanoids rapidly disappear, making a fast rate of formation neces-

The biosynthetic pathway for the formation of LTs is shown in Figure 5 (22). Like prostanoids, LTs have four stages in their formation and action when they are formed in response to cellular stimuli that mobilize HUFA from phospholipids by activating cPLA2 . The LTs are produced from ␻-6 AA (or its ␻-3 analog) by the action of 5lipoxygenase (5-LO), which both forms 5-hydroperoxy6,8,11,14- (E,Z,Z,Z)-eicosatetraenoic acid (5-HPETE) and then dehydrates this product to form LTA4 . The enzyme acts at the nuclear membrane and requires several cofactors for activity, including Ca2+ , ATP, hydroperoxides, and phosphatidylcholine (22). The activity is regulated by a suicide inactivation mechanism, making continued

Figure 5

Pathways for the biosynthesis of leukotrienes.

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synthesis of the enzyme important for sustained formation of active hormone. During the search for pharmacological antagonists of LT biosynthesis, a protein called 5-lipoxygenase-activating protein was discovered. This protein is important for the efficient production of LTs by cells, but it is not clear how it functions. It may serve as a protein that transfers HUFA to 5-LO (22). A methyleneinterrupted cis double bond system is the major determinant in the action of various lipoxygenases toward C18 and C-20 fatty acids (64). Most lipoxygenases use the HUFA, AA, in preference to the PUFA LA, although some attributions of selective actions with AA may be explained by a high availability of AA under many experimental conditions (49). This is notably the case in 5-lipoxygenase action during LT formation when little ␻-3 EPA is provided to the oxygenase. Two different types of potent leukotriene hormones are formed from LTA4 . LTA hydrolase hydrolyses LTA4 to produce LTB4 whereas LTC synthase conjugates glutathione to carbon-6 of LTA4 to create the peptidoleukotriene called LTC4 . Cellular locations for forming LTB and LTC from LTA can be limited and cell specific (22). For example, human neutrophils produce LTB4, whereas mast cells and eosinophils form LTC4. Unequivocal evidence for transcellular formation in vivo showed transfer of more than half the total LTA4 produced moved efficiently between cells (65). LTB4 is a very potent chemotactic and chemokinetic agent for human neutrophils. LTC4 constricts bronchial smooth muscle and mediates leakage of vascular fluid during edema. Metabolism and inactivation of leukotrienes is rapid, making their action evanescent without a continued availability of LTA4 . The peptidoleukotriene, LTC4 , can rapidly form LTD4 , which also activates receptors. However, peptidase cleavage to LTE4 gives a less active leukotriene that is excreted in the urine as the N-acetyl derivative. The biological activities of both LTB4 and LTC4 are mediated by specific G-protein-coupled receptors (22,66). Two LTB receptors, BLT1 and BLT2, mediate chemotactic effects, and three receptors, including CysLT1 and CysLT2, mediate cysteinyl leukotriene actions (22). Even before BLT receptors were recognized, the much greater chemotactic proinflammatory action of the ␻-6 LTB4 over the ␻-3 LTB5 (67) indicated that the receptors discriminate between ␻-6 and ␻-3 structures with important consequences. LTC4 receptor antagonists (e.g., montelukast, pranlukast, and zafirlukast) and a 5-LO inhibitor zileuton are commercially available to treat asthma. There are lipoxygenases other than 5-LO, including 8-, 12- and 15lipoxygenases that introduce oxygen at different positions

in the AA chain (68). Certain of these lipoxygenases (e.g., 15-LO) will use LA as a substrate such as in the formation of 13-HODE, which, as already noted, may be able to ameliorate the scaly skin of EFA-deficient animals (27).

Biosynthesis and Actions of Related Oxygenated Acids—P450 Hydroxylase and Epoxygenase Pathways AA can be hydroxylated by many different cytochrome P450 isoforms (CYP1 A, CYP2B, CYP2 C, CYP2D, CYP2G, CYP2 J, CYP2 N, CYP4 A, CYP), leading to epoxyeicosatrienoic acids [EETs; Fig. 6; (22,69)]. Some epoxides have potent roles as an endothelium-derived hyperpolarizing factor regulating renal vascular tone and fluid/electrolyte transport (22). The epoxide availability can be counteracted by enzymatic hydrolysis to dihydroxy acids, conjugation with glutathione, activation to acyl-CoA esters followed by esterification into lipids or ␤-oxidation. Because of desirable antihypertensive actions of certain EETs, researchers are exploring epoxy hydrolase inhibitors to increase EET availability at its receptors. The AA esterified at the sn2 position of any phospholipids can also be oxidized nonenzymatically to yield a complex racemic mixture of esterified ‘‘isoprostanes,’’ which are then mobilized presumably through the actions of phospholipase A2 (70). Isoprostanes are formed in abundance, particularly under conditions where tissuefree radical damage occurs and some isoprostanes have potent biological activities (71).

PATHOLOGIES ASSOCIATED WITH ω-6 EFA ACTIONS Although an absence of ␻-6 LA or ␻-6 AA in the diet can cause EFA deficiency, intakes as low as 0.3% of daily calories prevent it. Current diets in the United States, Europe, and highly developed countries in the Far East (e.g., Japan) contain levels of LA 10- to 20-fold greater. The proportions of ␻-6 in tissue HUFAs show a correspondence with mortality from cardiovascular deaths in various human populations (2). One ␻-6 eicosanoid—TXA2 —is an important causal mediator in cardiovascular mortality (72,73). There are correlations showing that COX-2 is overexpressed in colon cancers (74), that knock out of microsomal PGE synthase-1 in mice diminishes tumor development and that nonsteroidal anti-inflammatory drugs may decrease mortality from colon cancer. This suggests that overproduction of AA-derived eicosanoids that could

Figure 6 Representative eicosanoids formed via various cytochrome P450s. Abbreviation: EET, epoxy eicosatetraenoic acid.

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result from excessive intake of ␻-6 fatty acids can lead to serious health consequences. Actions of ␻-6 eicosanoids continue to be intensively studied by pharmaceutical researchers looking for new therapies to treat ␻-6 eicosanoid-mediated pathologies. The intensity of interest in developing agents to decrease excessive actions of ␻-6 metabolites in the “arachidonate cascade” is reflected in the fact that 38,360 articles retrieved by the term “arachidonic” in a recent PubMed search contained 21,420 articles retrieved by the combined terms “arachidonic” and “drug”, and 1579 of those were reviews. The present review offers readers insight to some of the established evidence about selective and nonselective competitions that occur during the metabolism of ␻-6 and ␻-3 molecules. These competitions accompany daily personal food choices that have profound influence on physiologic states and clinical status. Knowing the established basic biochemistry and physiology of ␻-6 fatty acids will allow readers to interpret more fully the relevance of data gathered and discussed in less-detailed epidemiologic and clinical studies in which intakes of ␻-6 and ␻-3 fats have been altered.

CONCLUSIONS The major ␻-6 EFAs in human tissues are LA and AA. Relatively small amounts of LA are required in the diets of humans for growth, health, and reproduction. The basis for the requirement is partially explained by tissue AA being converted to ␻-6 eicosanoids essential for reproduction and other important physiologic actions. However, overproduction of ␻-6 derived eicosanoids can amplify cellular responses to pathological levels. This may be prevented by decreasing the upstream dietary intake of ␻-6 LA and increasing the dietary intake of ␻-3 EFAs such as EPA and DHA.

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Pancreatic Enzymes Naresh Sundaresan, Unwanaobong Nseyo, and Joel Moss

␣-amylase cleaves internal ␣-1,4-glucose linkages, yielding glucose and dextrins. It cannot cleave terminal glucose residues or ␣-1,6-glucose linkages. The dextrins produced by ␣-amylase digestion are further digested into glucose by intestinal brush border enzymes. Lipase hydrolyzes fats into fatty acids and glycerol. The pancreatic lipases are responsible for the majority of fat digestion, with minor contributions from lingual and gastric lipases. Pancreatic triglyceride lipase (PTL) is a carboxyl esterase, catalyzing the hydrolytic cleavage of acylglycerides into glycerol and free fatty acids. The pancreas secretes excess PTL, meaning that a large decrease in PTL secretion must occur before signs of fat malabsorption are apparent. Many digestion products present in the duodenum have inhibitory effects on PTL activity. There are also molecules that complement PTL, such as colipase, which is a coenzyme required to maintain PTL activity. Colipase, which stabilizes the active conformation of PTL, has itself no enzymatic activity. It is secreted as a procolipase, which is active after cleavage of its N-terminus by trypsin. Other major pancreatic contributors to lipid digestion include phospholipase A2, which catalyzes hydrolysis of the acyl ester bond in phospholipids and carboxyl ester lipase, which hydrolyzes triglycerides, cholesterol esters, phospholipids, lysophospholipids, ceramides, vitamin esters, and galactolipids. Three important serine proteases are produced by the pancreas: trypsin, chymotrypsin, and elastase. Trypsin is the most abundant of the pancreatic digestive enzymes. It hydrolyzes peptide bonds at the C-termini of arginine and lysine residues, except those in a proline linkage. Trypsin is secreted from the pancreas as a zymogen trypsinogen, which is activated by cleavage of its activation peptide catalyzed by enterokinase, a duodenal enzyme. Autoactivation of trypsin can also be mediated by calcium and pH, specifically pH between 7.5 and 8.5. Trypsin is inactivated through autolysis, which is inhibited by calcium. Another contributor to pancreatic enzyme–catalyzed digestion is chymotrypsin, which cleaves peptide bonds at the C-terminus of an aromatic amino acid (i.e., tyrosine, phenylalanine, tryptophan). The zymogen chymotrypsinogen is secreted from the pancreas and then activated through autolysis. Elastase also hydrolyzes peptides at the C-terminus of amino acids alanine, glycine, and serine. It is secreted as proelastase and is both activated and inactivated by trypsin (2). Several additional molecules that are present in pancreatic secretions contribute to nutrient digestion and absorption, including proteases, such as carboxypeptidase, nucleases, and enzyme cofactors (1).

INTRODUCTION Pancreatic enzymes are critical for the normal physiological digestion of fats, proteins, and carbohydrates. Many additional conditions and molecules throughout the digestive system complement and assist in the digestion of essential nutrients. As occurs often in diseases involving the pancreas such as cystic fibrosis (CF), deficiencies of pancreatic enzymes can result in significant malabsorption and nutritional deficiencies. This chapter briefly outlines the normal functions of the pancreas and pancreatic enzymes. Enzymes that are primary components of pancreatic supplements are defined and the function of each is described. We then consider conditions in which the use of pancreatic enzyme supplements is recommended. We review also clinical studies in which pancreatic enzyme supplements were used to improve digestion or as a supplement to augment the natural age-related decrease in pancreatic exocrine output and explore the uses of pancreatic enzymes in cases of disease, for example, cystic fibrosis for which pancreatic enzyme supplementation may be beneficial. The chapter concludes with a summary of recommendations for pancreatic enzyme supplementation.

THE PANCREAS The pancreas has both endocrine and exocrine functions. The exocrine pancreas secretes enzymes crucial to digestion. Cells responsible for exocrine functions comprise the largest part of the pancreas. Clusters of acinar cells constitute the primary functional unit and release between 6 and 20 g of digestive enzymes and zymogens each day in approximately 2.5 L of fluid. These products are secreted into the duodenum of the small intestine via the pancreatic ducts, in which they are mixed with sodium bicarbonate secretions produced by pancreatic ductal cells. Pancreatic enzymes reduce complex nutrients into simple molecules that can be absorbed by the small intestine; sodium bicarbonate secretions neutralize the acidic chyme as it moves from the stomach to the duodenum (1).

PANCREATIC ENZYMES AND FUNCTIONS Amylase, lipase, and protease are three main categories of pancreatic enzymes. Each type serves a specific digestive function. Amylase, which is also produced in less quantity by salivary glands, acts on carbohydrates. Pancreatic 598

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NORMAL DIGESTION AND PANCREATIC FUNCTION Digestion serves to extract necessary nutrients from ingested complex food sources such as proteins, carbohydrates, and fats. Protein digestion begins within the stomach, where acid and proteases hydrolyze proteins into peptides. This continues within the small intestine via pancreatic proteases and intestinal brush border proteases. Carbohydrates and starches are substrates for salivary amylase within the mouth and further digested by pancreatic amylase and intestinal brush border oligosaccharidases within the small intestine. Fat digestion begins in the stomach and is catalyzed by the lingual and gastric lipases, yielding glycerol and long-chain fatty acids. Gastric hydrolysis accounts for only about 10% of lipid digestion. Products of intragastric lipolysis are transferred to the duodenum for additional hydrolysis by pancreatic lipases, a process requiring the neutralization of acidic chyme by pancreatic bicarbonate secretions to facilitate the pH-determined functioning of pancreatic lipases. Products of completely digested lipids are absorbed by the intestinal mucosa, after which the fatty acids are reincorporated into triglycerides to be packaged as chylomicrons for delivery to the bloodstream via lymphatic vessels (3). Regulation of exocrine pancreatic secretions is influenced significantly by nutrients acting in the distal bowel. Digestive products, especially free fatty acids, stimulate release of the hormone cholecystokinin from the small intestine. This in turn stimulates the pancreas to release its enzymatic secretions. In normal, healthy individuals, the pancreas releases between 10 and 20 times the amount of prandial enzyme required for digestion (4). When considering activities of pancreatic enzymes, it is important to assess the stability of enzymatic activity during intestinal transit. Amylase is the most stable of all pancreatic enzymes as a majority of that released reaches the terminal ileum in its active state. Proteases are less resistant to degradation, and only 20% to 30% of protease activity is retained. Lipases are the most sensitive to inactivation during intestinal transit. Although the presence of lipid substrates does enhance lipase stability, only a small fraction of released lipase is active on reaching the terminal ileum (4). The coexistence of numerous digestive processes that overlap pancreatic enzyme activities allows maintenance of protein and carbohydrate digestion even in the face of severe pancreatic insufficiency. Fat digestion is not, however, appreciably augmented by nonpancreatic mechanisms, thus critical fat malabsorption can be associated with pancreatic insufficiency.

ENZYME DEFICIENCIES Pancreatic insufficiency (PI) is caused by a significant deficit in pancreatic enzyme output and differs widely in severity. Symptoms of PI usually manifest after a 90% reduction in pancreatic enzyme output. Although protein and starch digestion are relatively easily corrected by pancreatic enzyme supplementation, fat malabsorption is more difficult to treat effectively because of the instability of lipase molecules (3).

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Deficiency in the production of any of the pancreatic enzymes can impair gastrointestinal (GI) function. Decreased lipase production and subsequent fat malabsorption is associated with steatorrhea, or fatty stool. A secondary consequence of pancreatic insufficiency is an inadequate uptake of fat-soluble vitamins, notably vitamins A, D, E, and K. Fat malabsorption occurs earlier in pancreatic insufficiency than does malabsorption of other nutrients for several reasons: fat digestion is not adequately compensated by other digestive mechanisms; declines in the synthesis and secretion of pancreatic lipases are more rapid than they are for other pancreatic enzymes and are readily inactivated in an environment of high pH caused by the lack of pancreatic bicarbonate secretions; and lipases are more sensitive than other digestive enzymes to protease degradation (5). Protein malabsorption can also occur, with evidence of creatorrhea or protein in the stool. Both protein and carbohydrate malabsorption are usually seen only late in the course of severe PI, and sometimes not at all, because of supplementation of digestive function by intragastric proteolysis or intestinal brush-border peptidases and salivary amylase plus intestinal oligosaccharidases for starches (4). As a consequence of impaired enzyme output in PI, the site of maximal absorption within the small intestine becomes more distal (3). Further reduced nutrient digestion and absorption increases gastric and intestinal motility (6). Impaired protein digestion has been linked to food allergies (7), and proteolytic enzymes are necessary for preservation of a healthy intestinal microbial flora (8).

PANCREATIC ENZYME REPLACEMENT THERAPY Pancreatic enzyme replacement therapy (PERT) is intended to correct insufficient pancreatic enzyme levels in the proximal small intestine through the ingestion of exogenous enzymes. Delivery of exogenous pancreatic enzymes with each meal can restore enzymatic activity to the duodenum thereby reducing many of the symptoms associated with pancreatic insufficiency. To accomplish this, PERT needs to address effectively the need for exogenous enzymes to survive the acidic and proteolytic environment of the stomach and to be in a form that will mix and efficiently enter the duodenum simultaneously with food. A third requirement is the temporal and geographical localization of enzymes with substrates to ensure an optimal absorption of necessary nutrients. Two types of enzyme formulations are generally used: unprotected, conventional preparations and acid-resistant enteric-coated preparations. Because gastric acids and proteases can cause significant inactivation of lipases, effective doses of conventional preparations will be large. Protected preparations contain pancreatin, which is enteric-coated as protection from enzymatic action in the stomach, but allowing release in environments of pH > 5, such as the duodenum, which facilitates its administration in doses lower than those of uncoated preparations (9). Protected preparations and brands differ in methods of delivery. Enzyme packaging is very important to achieve adequate mixing with food and transit from stomach to duodenum. Two predominant forms of

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enteric-coated pancreatin preparations are tablets and microspheres, which have proven superior to tablets with regard to the three aforementioned criteria. Enteric-coated particles >2 mm in diameter cannot be adequately emptied from the stomach and thus, lead to increased gastric retention (5) so that enzymes and food are not simultaneously traversing the digestive system. In a direct comparison, delivery of lipase via microspheres was significantly superior to that by tablets for increasing body weight and reducing abdominal pain as well as fecal fat excretion. Through their ability to enter the duodenum simultaneously with food (10), microspheres appeared to prevent the problem of pyloric retention. Timing of ingestion of enzymes supplements is critical. Patients should swallow supplemental capsules just after beginning a meal, during the meal, and immediately after. This allows for protection of supplemental enzymes from gastric acid and proteases and ensures optimal mixing with food (11). If with standard dosages symptoms of steatorrhea persist, dosages should be increased (9). Impaired bicarbonate secretion in patients with PI and the resulting low duodenal pH can hinder enzyme release from enteric-coated particles. Administration of medication to reduce gastric acidity, such as famotidine, an H2 -receptor antagonist, or omeprazole, a proton pump inhibitor, has been shown to reduce fecal fat content, especially when given in combination with high doses of pancreatin (12,13). When supplemental enzymes were used in combination with antacids, both duodenal pH and enzyme activities were increased. Patients with the lowest responses to PERT experienced the greatest benefits of a regimen of antacids during meals (14). Increasing gastric pH because of antacid treatment also increased the efficacy of gastric lipase, which has its highest enzymatic activity at pH 5.4 (15).

PANCREATIC INSUFFICIENCY IN CYSTIC FIBROSIS CF is highly associated with PI. Approximately 95% of patients with cystic fibrosis will develop steatorrhea by adulthood due to PI (16). CF is an autosomal recessive disorder caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cyclic AMP-regulated chloride channel, and is expressed on the luminal surface of pancreatic ductal cells. Normal function and water secretion involves a chloride/bicarbonate anion exchange, which is disrupted by the malfunctioning chloride channel, resulting in pancreatic secretions that contain lower amounts of bicarbonate, lower pH, and lower volumes (13). This results in hyperconcentrated, viscous pancreatic secretions that obstruct pancreatic ducts and lead to the destruction of acinar cells with pancreatic fibrosis (17). CF patients with different genetic defects have different probabilities of developing pancreatic symptoms. A F508 mutation, which is almost always associated with PI, is present in the majority of Caucasian CF patients (18). Pancreatic dysfunction and its severity are highly correlated with mutations in the CFTR gene, regardless of clinical manifestations of CF (19). Early diagnosis of CF is important for children to ensure proper growth and development to allow for recog-

nition of the need for and the prompt initiation of enzyme replacement therapy. The majority of CF patients suffering from PI present with symptoms within the first year of life, although in some patients PI may develop with age. Symptoms in the latter group are usually first recognized as acute pancreatitis following ingestion of high-fat meals (20). Maintenance of proper nutrition through modified diet and pancreatic enzyme replacement therapy is of primary importance as it both enables normal development and improves respiratory function (21). Children within normal weight ranges have a higher FEV1 and better survival rates than those who are underweight (22). It is recommended that children with CF have 110% to 200% of the energy intake of healthy children of similar age, sex, and size to compensate for malabsorption. Diets high in fat and protein are also recommended and have been shown to maximize fat digestion and absorption (23).

SUPPLEMENTATION OF NORMAL DIGESTION Although pancreatic enzyme supplements are traditionally prescribed for individuals with pancreatic dysfunction, supplements may also improve digestion for those with normal pancreatic function. Studies have shown that pancreatic secretions change in response to diet, specifically depending on its composition (24). This feedback mechanism, however, begins to alter pancreatic enzyme secretions only after a sustained change in diet. For more immediate effects on digestion, the use of pancreatic enzymes supplementation has proven useful. Microencapsulated pancreatic enzymes were shown to reduce postprandial symptoms in normal individuals. In a study analyzing the use of pancreatic enzyme supplementation in normal digestive function, it was found that healthy volunteers experienced a significant reduction in abdominal symptoms associated with indigestion after eating a high-calorie, high-fat meal. Healthy volunteers reported reductions in bloating, gas, and a feeling of fullness (25). Pancreatic enzyme supplements have also been recommended for idiopathic digestive disorders with symptoms of excessive gas and bloating. It is thought that exogenous enzymes can break down undigested food material that is responsible for abdominal discomfort after meals, although more research is needed to define the exact mechanism of action and the utility of pancreatic enzyme supplementation for treatment of common GI symptoms (26).

AGING Earlier studies showed a significant decrease in pancreatic output of bicarbonate and digestive enzymes with increasing age (27,28). Also, pancreatic enzyme output was less in the elderly than in the younger individuals (29). With age physiological changes in pancreatic structure and function are seen that affect the entrance of pancreatic secretions to the duodenum. These include increased caliber of the main pancreatic duct, vascular calcification, fibrosis, accumulation of fat in the pancreas, defective protein synthesis, and decreased pancreatic cell mass (30). In

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a study of pancreatic secretions during stimulation by IV infusion of secretin and cerulein in healthy volunteers, pancreatic bicarbonate and enzyme output were significantly lower in the elderly than in the younger groups (31). Given the importance of pancreatic function in nutrient absorption, malnutrition in the elderly could potentially be prevented through pancreatic enzyme supplementation. Treatment of aged endotoxemic rats with pancreatic enzymes improved their nutritional status and recovery from infection (30). This evidence supports the possibility that use of pancreatic enzymes to supplement normal pancreatic function is beneficial for digestive health, as malabsorption and nutritional deficiencies due to impaired digestion tend to increase in frequency with age.

FOOD ALLERGIES When malabsorption of nutrients occurs due to pancreatic insufficiency, complex proteins and carbohydrates remain undigested. With an accumulation of undigested materials, proteins begin to leak into the circulation where they may be received by components of the immune system as foreign and subjected to immunological attack. In food allergies, the body treats innocuous substances as if they were harmful, triggering a hypersensitive immune response (7). It is hypothesized that malabsorption can contribute to food allergies by allowing certain undigested food materials to pass into the circulation and elicit an immune response. Symptoms associated with food allergies are typically gastrointestinal, and include diarrhea, abdominal pain, and dyspepsia. Treatment with pancreatic enzymes has been shown to reduce the clinical symptoms of food allergies and also limit the allergen-induced inflammation (in studies with patients with confirmed food allergies). Pancreatic enzymes cannot only reduce GI discomfort, but also prevent allergen activation of the immune system, potentially through degradation of the allergen (32).

ALTERNATIVE CANCER THERAPIES Pancreatic enzyme supplementation is used in alternative cancer therapies. The Gonzalez therapy arose from the similar Gerson Therapy, which also involved pancreatic enzyme supplementation (33); it is based on the belief that toxins from environmental and processed food sources collect in tissues and gradually lead to imbalances in the autonomic nervous system, weaken immunity, and lead to cell damage that ultimately gives rise to cancer. Proponents of the Gonzalez therapy believe that removing these toxins will help in combating cancers. To detoxify the body, a regimen of orally ingested pancreatic enzymes, an organic diet, nutritional supplements, and coffee enemas are prescribed. Pancreatic enzymes are thought to play a role in detoxification by helping eliminate abnormal cells, toxins, and waste material, and help repair cell damage. Therefore, pancreatic enzyme supplements are the main anticancer component of the Gonzalez therapy (34). A case study was conducted by the originators of the therapy in which 11 patients diagnosed with

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inoperable stage II to IV pancreatic adenocarcinoma underwent the Gonzalez therapy. This group reported a oneyear survival of 81%, a two-year survival of 45%, and a three-year survival of 36%. This exceeded the National Cancer Data Base Report on Pancreatic Cancer from 1995, in which the overall one and two-year survival rates were 25% and 10%, respectively (35). These findings led to a National Cancer Institute-funded nonrandomized study comparing the Gonzalez therapy to gemcitabine-based chemotherapy among pancreatic cancer patients. Patients who chose the chemotherapy lived 14 months, compared to those who chose the Gonzalez therapy, who survived 4.3 months (36). Pancreatic enzymes are also used in the WobeMugos E treatment, which is a mixture of calf thymus extracts and enzymes from papaya plants, cow pancreas, and pig pancreas (37). This treatment has been shown to reduce the side effects of radiation, and is thought to function by eliminating toxic metabolic and inflammatory substances, as well as disintegrating microthrombi to improve microcirculation (36).

NEW TREATMENT APPROACHES As an alternative to conventional and enteric-coated pancreatic enzyme supplements for patients with PI, a new pancreatic enzyme replacement product, TheraCLECTotal is being developed. The formulation substitutes bacterial lipase and fungal protease and amylase for the porcine enzymes currently used in pancreatic enzyme supplementation (38). Notable in this new formulation is bacterial lipase, which has been shown to be more resistant than porcine lipases to acid and protease degradation. In dogs, bacterial lipases proved more effective for correction of steatorrhea and required only 1/75 the amount of porcine lipase usually used (240 mg bacterial lipase vs. 18 g porcine lipase) (39). To provide additional stability, lipases and proteases in TheraCLEC-Total are crystallized, and lipases are cross-linked. The crystallization and crosslinking of lipase also provides greater protection and maximizes lipolytic activity, thereby enabling the use of lower doses, which should aid in reducing the risks of fibrosing colonopathy seen with high doses of supplemental pancreatic enzymes in CF patients (38).

STANDARD DOSAGE To improve digestion and decrease malabsorption, enzyme activity must be delivered to the duodenum with each meal. Mean lipase activity in the duodenal chyme must be between 40 and 60 units/mL, which requires a timely intraduodenal delivery of 25,000 to 40,000 units of lipase per meal. Dosages can be increased if additional lipolytic action is needed, but should not exceed 75,000 units of lipase per meal. If the need exceeds this amount, alternatives should be considered. Recommended doses for infants are 400 to 800 units of lipase/g of dietary fat (18). For children and young adults, recommended dosage is 500 to 2000 units of lipase/kg/meal, or 500 to 4000 units of lipase/g of fat ingested. Dosages more than 2500 units

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of lipase/kg/meal or 10,000 units of lipase/kg/day are not recommended (3).

CONTRAINDICATIONS AND OVERDOSING Ingestion of very large doses of pancreatin has been associated with a dose-dependent risk of fibrosing colonopathy in cystic fibrosis patients. Generally, dosages in excess of 75,000 units of lipase per meal are not recommended (5). In children younger than 12 years, dosages in excess of 6000 units of lipase/kg/bw/meal have been associated with colonic strictures (3). Diminished folate absorption has also been seen with the use of exogenous pancreatic extracts, and therefore should be monitored during the use of pancreatic enzyme supplements (40).

FDA REGULATION The FDA has recently required that all pancreatic enzyme products gain FDA approval because of variations in activity and release rate due to different formulations, dosages, and manufacturing methods (41). Creon (pancrelipase) has thus far been the only delayed-release pancreatic enzyme product to be approved; it was approved in May 2009. All unapproved pancreatic enzyme products can remain on the market until the approval deadline, April 28, 2010, but must gain FDA approval to be sold after the deadline (42).

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chemotherapy for the treatment of pancreatic cancer. J Clin Oncol 2009, Aug 17. DOI: 10.1200/JCO.2009.22.8429. 36. Dorr W. Herrmann T. Efficacy of Wobe-Mugos E for reduction of oral mucositis after radiotherapy: Results of a prospective, randomized, placebo-controlled, tripleblind phase III multicenter study. Strahlenther Onkol 2007; 183(3):121–712. 37. Wobe-Mugos E. Description of Cancer Terms. http://www. nci.nih.gov/dictionary/?CdrID = 45007. Accessed December 18, 2009. 38. Borowitz D, et al. Safety and preliminary clinical activity of a novel pancreatic enzyme preparation in pancreatic insufficient cystic fibrosis patients. Pancreas 2006; 32(3):258–263.

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39. Suzuki A, et al. Effect of bacterial or porcine lipase with low- or high-fat diets on nutrient absorption in pancreaticinsufficient dogs. Gastroenterology 1999; 116(2):431–437. 40. Russell RM, et al. Impairment of folic acid absorption by oral pancreatic extracts. Dig Dis Sci 1980; 25(5):369– 373. 41. FDA requires pancreatic extract manufacturers to submit marketing applications. http://www.medicalnewstoday. com/articles/7723.php. Accessed December 18, 2009. 42. FDA approves pancreatic enzyme creon (pancrelipase). http://www.gluvsnap.com/news/FDA Approves Pancreatic Enzyme Creon %28pancrelipase%29 180.html. Accessed December 18, 2009.

Pantothenic Acid Lawrence Sweetman

INTRODUCTION

inhibited by acetyl CoA and malonyl CoA and activated by free CoA (3) It is also inhibited by the intermediates 4 -phosphopantothenate and dephospho-CoA as well as CoA in other studies (4). Carnitine protects from the inhibition by CoA and acyl CoA by competing with them for their binding to pantothenate kinase. It has been shown that palmitoylcarnitine can prevent and reverse the strong inhibition of the human mitochondrial pantothenate kinase 2 isoform, and enable the synthesis of CoA, which is needed for mitochondrial beta oxidation of palmitate and other long-chain fatty acids (5). The human genes for the last four enzymes of CoA synthesis have been cloned, expressed, purified, and reconstituted in vitro to synthesize CoA (6). 4 -Phosphopantothenate is the substrate for 4 -phosphopantothenoylcysteine synthetase, which couples ATP hydrolysis with the formation of an amide bond between the carboxyl of 4 -phosphopantothenate and the amino group of the sulfur amino acid, cysteine. The product, 4 -phosphopantothenoylcysteine, is decarboxylated by 4 -phosphopantothenoylcysteine decarboxylase to form 4 -phosphopantetheine. A bifunctional protein with adenyltransferase activity adds the 5 -AMP group of ATP to the 4 -phospho group of 4 -phosphopantetheine to form dephospho-CoA, and a kinase activity catalyzes the final step in the synthesis of CoA, the phosphorylation of the 3 -hydroxyl of dephospho-CoA utilizing ATP. The adenyltransferase may be a secondary point of control of the biosynthesis of CoA. All the enzymes in the CoA biosynthetic pathway are present in the cytosol, but the last two enzymes can also be found in mitochondria. Notable features of the structure of CoA are the 3 -phosphoAMP moiety linked with the pantoate portion, and the reactive sulfhydryl group at the end of the long flexible chain derived from ␤-alanine and cysteine. CoA is often abbreviated as CoASH to illustrate this reactive sulfhydryl group, while thioesters of organic acids with CoASH are often referred to as acyl-SCoA.

Pantothenic acid is a water-soluble B vitamin (vitamin B5 ) that is not synthesized by animals but is widely available in the diet. Pantothenic acid is metabolized to two important cofactors for enzymes: coenzyme A (CoA) and acyl carrier protein (ACP). Both cofactors contain a sulfhydryl group (-SH), which reacts with carboxylic acids to form thioesters. ACP has a central role in the synthesis of fatty acids. CoA forms thioesters with a very wide range of metabolic intermediates and has been estimated to be a cofactor for about 4% of all known enzymes (1). It is also involved with fatty acid synthesis but has broader functions in fatty acid oxidation, ketone body metabolism, oxidative metabolism of pyruvate via pyruvate dehydrogenase and the citric acid cycle, and in the metabolism of a wide variety of organic acids, including those in catabolic pathways of amino acid metabolism.

MICROBIAL SYNTHESIS Micro-organisms synthesize pantoic acid (pantoate) from ␣-ketoisovaleric acid, the keto acid derived from the amino acid valine (1). A hydroxymethyl group is attached to ␣-ketoisovaleric acid, and the keto group is reduced to a hydroxy group to form pantoic acid. Beta-alanine produced by the decarboxylation of the amino acid aspartate is condensed with pantoic acid to form pantothenic acid (pantothenate) (Fig. 1). This synthesis does not occur in humans or in other animals, which must obtain pantothenic acid from the diet. Pantothenic acid is quite widely distributed in foods, giving rise to its name from the Greek word pan- (also panto-) meaning all or every. Liver, meats, milk, whole grain cereals, and legumes are good sources. It is contained in foods in various bound forms, including CoA and CoA esters, ACP, and as a glucoside in tomatoes.

Coenzyme A Subcellular Location

COFACTOR SYNTHESIS Coenzyme A Synthesis

The majority of the CoA in cells is found within the mitochondria, with about 75% of liver CoA in mitochondria and 95% of heart CoA in mitochondria. This is consistent with the mitochondria being the major cellular organelle involved in fatty acid oxidation and in the final oxidative steps in the catabolism of all fuels; CoA plays a major role in these processes. Because the mitochondria represent only a small fraction of the cellular volume, the concentration of CoA here (2.2 mM) is 40 to 150 times that in the cytosol (0.015–0.05 mM). This large difference is

Within cells, pantothenic acid is metabolized to CoA in the cytosol (Fig. 2). The initial reaction is phosphorylation of the hydroxyl group of the pantoic acid portion of pantothenic acid with ATP, catalyzed by pantothenate kinase, to form 4 -phosphopantothenate. This is the rate-limiting step for synthesis of CoA, and regulation of pantothenate kinase activity is the primary control of the rate of CoA synthesis. The activity of pantothenate kinase is strongly 604

Pantothenic Acid

5,10-metTHF

O

THF

KPHMT CO2H α-ketoisovalerate

NADP+

NADPH

O

OH

KPR

CO2H

panB

CO2H

panE

OH

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ketopantoate

pantoate ATP PS panC

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PPi

β-alanine OH

H N

CO2H

O OH pantothenate

Figure 1

Pantothenic acid structure and biosynthetic pathway in micro-organisms. Source: From Ref. 2.

maintained by the transport of the negatively charged CoA into the mitochondria, which is driven by the membrane electrical gradient (8). CoA is also involved in the oxidation of very-long-chain fatty acids in peroxisomes, but little is known about how it enters these organelles. In the cytoplasm, CoA is also utilized for the synthesis of the ACP domain of the fatty acid synthase enzyme, which catalyzes fatty acid synthesis.

Acyl Carrier Protein Synthesis There are several ACPs known in yeast and bacteria, but the ACP domain of fatty acid synthase is the most important and best studied. Fatty acid synthase is the only mammalian enzyme complex containing the ACP domain that has been well characterized. It is a single, homodimeric, multifunctional protein with seven enzymatic activities required for fatty acid synthesis (9). The synthase is synthesized with the ACP domain as an enzymatically inactive apoprotein lacking the prosthetic group. But after covalent attachment of the phosphopantetheine group, it becomes the enzymatically active holo-acyl carrier protein (holo-ACP) (10). This reaction, catalyzed by 4 -phosphopantetheinyl transferase, utilizes CoA to form a phosphoester bond between the 4 -phosphopantetheine portion of CoA and a specific serine residue of the ACP, with the release of the 3 ,5 -ADP moiety of CoA (Fig. 3). Note that as in CoA, the reactive sulfhydryl group of ACP is at the end of the long chain derived from ␤-alanine and cysteine.

COFACTOR DEGRADATION The intermediates in the degradation of CoA are the reverse of those in the synthesis but involve different

enzymes. CoA does not appear to be degraded in the mitochondria, but in the lysosomes, the 3 -phosphate group is removed by nonspecific phosphatases to form dephospho-CoA. This is degraded to 4 phosphopantetheine and 5 -AMP by a nucleotide pyrophosphatase located in the plasma membrane fraction. CoA is also degraded by this enzyme but at a much lower rate and with a much higher Km . Surprisingly, acyl CoAs are also readily degraded to 4 -phosphopantetheine. There is an ACP hydrolase that releases 4 -phosphopantetheine from holo-ACP to reform apo-ACP. Interestingly, the combined action of the ACP hydrolase and synthetase results in the rapid turnover of the 4 -phosphopantetheine of ACP with a half-life measured in hours compared to that of the fatty acid synthase, which is measured in days in rat tissues (11). Whether 4 -phosphopantetheine is derived from the degradation of CoA or from the turnover of ACP, the phosphate is removed by phosphatases to give pantetheine. This is hydrolyzed to pantothenic acid and cysteamine by pantetheinase, which is found in both the microsomal and lysosomal fractions of rat liver and kidney. The pantothenic acid can be excreted or used for resynthesis of CoA. The cysteamine is oxidized to hypotaurine and further oxidized to taurine, which may be excreted in the urine.

METABOLIC ROLE CoA has many functions in metabolism including its role in the formation of ACP. Both CoA and ACP are used to form thioesters with carboxylic acid groups of fatty acids and other compounds. Much of the metabolism of fatty acids and certain amino acid derivatives, as well as a number of amphibolic steps in metabolism, occurs

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CH3 HO

C

CH

H3C

OH

CH2

NH

C

CH2

COO–

CH2

O Pantothenate

ATP Pantothenate Kinase ADP CH3

O –O

P

CH2

O

O–

C

CH

C

H3C

OH

O

NH

CH2

COO–

CH2

4´-Phosphopantothenate + H3N

ATP ADP +P1

P

O

C

CH2

O–

CH

H3C

C

NH

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COO–

CH2

C

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4´-Phosphopantothenoylcysteine Decarboxylase

CH3

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CO2

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CH2

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ATP Adenylytransferase PP1 O CH2

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CH2

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O Adenine H H H H

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O O

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NH

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CH2

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CH2

CH2

SH

NH

CH2

CH2

SH

O

Dephosphecoenzyme A

OH OH

CH2

C

CH3

O O

P O–

Dephosphocoenzyme A Kinase

O

CH2

C

CH

C

NH

H3C OH O Coenzyme A (CoA or CoASH)

CH2

CH2

C O

O–

Figure 2

CoA synthesis and structure: CoA is synthesized from pantothenic acid, the amino acid cysteine, and ATP in mammalian cells. Source: From Ref. 7.

Pantothenic Acid

C

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O

HC

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NH Seryl Residue in Apoacyl Carrier Protein O CH2

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3959-ADP

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Holoacyl Carrier Protein

Figure 3 Acyl carrier protein synthesis: CoA is cleaved to form 3 ,5 -ADP and attach 4 -phosphopantetheine as a phosphate ester of the hydroxyl of a serine residue in apo-ACP to form holo-ACP, a component of fatty acid synthetase. Source: From Ref. 7.

using CoA thioester substrates and producing CoA thioester products.

the concentration of malonyl CoA, which is determined by regulation of the activity of acetyl CoA carboxylase.

Acyl Carrier Protein and Coenzyme A in Fatty Acid Synthesis

Coenzyme A in Oxidative Decarboxylation

Both CoA and ACP are essential for the synthesis of fatty acids in the cytosol. Acetyl CoA, the substrate for fatty acid synthesis, is generated from citrate and CoA by citrate lyase in the cytosol. The citrate is transported out of the mitochondria where it was formed in the tricarboxylic acid cycle from acetyl CoA produced by the oxidation of pyruvate. Acetyl transacylase transfers the acetyl group from acetyl CoA to the pantetheine sulfhydryl of ACP, releasing free CoA in the process. These two carbons from acetyl CoA form the methyl end of the fatty acid that will be synthesized. A biotin-containing enzyme, acetyl CoA carboxylase, utilizes bicarbonate and ATP to convert acetyl CoA to malonyl CoA. Fatty acid synthetase utilizes this malonyl CoA to sequentially add two carbon units to the acetyl or acyl ACP, with the liberation of the third carbon of malonyl CoA as CO2 . This process results in the synthesis of even-numbered fatty acids of 16 or 18 carbons. When the synthesis of a fatty acid is complete, a thioesterase hydrolyzes the ACP-fatty acid thioester, releasing the fatty acid and regenerating the ACP sulfhydryl. The rate of fatty acid synthesis is primarily regulated by

A key role for CoA in fuel metabolism is its function in ␣-keto acid dehydrogenase complexes that catalyze the oxidative decarboxylation of keto acids. In the metabolism of carbohydrates, the end product of the glycolytic pathway for glucose is the simple three-carbon␣-keto acid, pyruvate. In order for pyruvate to be completely oxidized via the tricarboxylic acid cycle and oxidative phosphorylation, it is oxidatively decarboxylated to acetyl CoA [with release of CO2 and reduction of nicotinamide adenine dinucleotide (NAD)] by the pyruvate dehydrogenase complex. This complex reaction involves five coenzymes (four of them derived from vitamins): thiamine pyrophosphate, NAD, flavine adenine dinucleotide, lipoate, and CoA. In the decarboxylation of pyruvate, the two-carbon aldehyde unit is attached to thiamine pyrophosphate, oxidized, transferred to a lipoyl enzyme, and then to CoA to form acetyl CoA. Acetyl CoA is a central compound in metabolism, having several catabolic as well as anabolic fates. The CoA is eventually released as free CoA as further metabolism of acetyl CoA progresses. Two other enzyme complexes catalyze the oxidative decarboxylation of keto acids with the formation of acyl CoA products.

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The ␣-ketoglutarate dehydrogenase in the tricarboxylic acid cycle converts ␣-ketoglutarate to succinyl CoA. The CoA is released from succinyl CoA in the next step of the tricarboxylic acid cycle. The branched-chain ␣-keto acid dehydrogenase complex, again in a series of reactions analogous to those of pyruvate dehydrogenase, catalyzes the first committed step in the catabolic pathway for the branched-chain amino acids. The ␣-keto acids from transamination of valine, isoleucine, and leucine are oxidatively decarboxylated to form branched-chain acyl CoA products with one less carbon in the chain. These are metabolized in a number of different steps as CoA esters and ultimately yield simple acyl CoA products, such as acetyl CoA and propionyl CoA, which enter general metabolism.

Coenzyme A in Fatty Acid β-Oxidation

CoA plays a major role in the ␤-oxidation of fatty acids in the mitochondria, which may result in the complete degradation of fatty acids to acetyl CoA that can be further oxidized in the tricarboxylic acid cycle. Most of the fatty acids consumed in dietary triglycerides (fat or oils) or obtained from adipose tissue stores have chains of 16 or 18 carbons. These long-chain fatty acids require a carrier system for their transport from the cytosol into the mitochondria. In the cytosol, the free long-chain fatty acids are activated to CoA thioesters by acyl CoA synthetases that couple ATP hydrolysis with thioester formation. These fatty acyl CoAs are transesterified to carnitine to form “energy-equivalent” acyl carnitines, which can be transported across the mitochondrial inner membrane. On the outer mitochondrial membrane, the enzyme carnitine palmitoyl transferase I (CPT I) converts the fatty acyl CoA to acyl carnitine and free CoA. A carnitine/acyl carnitine translocase moves the acyl carnitines into the mitochondria and free carnitine out of the mitochondria. Carnitine palmitoyl transferase II (CPT II) on the inner mitochondrial membrane regenerates fatty acyl CoA in the mitochondria, freeing up carnitine for transport out of the mitochondria. In the ␤-oxidation, two-carbon segments of the fatty acyl CoA are sequentially removed as acetyl CoA. The series of reactions for each cycle are dehydrogenation to the unsaturated acyl CoA, hydration to 3-hydroxyacyl CoA, dehydrogenation to the 3-ketoacyl CoA, and thiolytic cleavage by CoA to release acetyl CoA and a fatty acyl CoA with two less carbons. There are multiple dehydrogenases with overlapping chain length specificities that favor acyl CoAs with very long, long, medium, or short chains. Reducing equivalents generated in the various dehydrogenation steps are funneled into the electron transport chain. Although most tissues other than the brain can use fatty acids as fuel, cardiac muscle and skeletal muscle are especially dependent on fatty acid oxidation for energy. The rate of fatty acid oxidation is controlled by the rate of transport of fatty acids into the mitochondria. The rate of transport is controlled largely by the activity of CPT I, which is strongly inhibited by malonyl CoA. When fatty acid synthesis is increased by insulin activation of acetyl CoA carboxylase to produce more malonyl CoA as substrate for fatty acid synthetase, the increased malonyl CoA inhibits CPT I, decreasing fatty acid transport into the mitochondria, and thus preventing

the reoxidation of newly synthesized fatty acids. Increased glucagon enhances fatty acid ␤-oxidation indirectly by inhibiting acetyl CoA carboxylase, decreasing the synthesis of malonyl CoA and fatty acids, and reducing malonyl CoA inhibition of CPT I so that fatty acids enter the mitochondria for ␤-oxidation.

Coenzyme A in Ketone Body Metabolism Ketone bodies are an important source of fuel derived from fat metabolism when glucose is limiting as in starvation. Acetoacetate and its reduction product, 3hydroxybutyrate, were called ketone bodies because some acetoacetate is spontaneously decarboxylated to acetone, a ketone. Ketone bodies are synthesized in the liver from acetoacetyl CoA and acetyl CoA produced via ␤-oxidation of fatty acids. Acetoacetyl CoA is condensed with acetyl CoA to form 3-hydroxy-3-methylglutaryl CoA and free CoA by mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthetase. This is then cleaved by HMG CoA lyase to form free acetoacetate and acetyl CoA. The net result of this cycle is the conversion of acetoacetyl CoA to acetoacetate and free CoA, but there is no enzyme that directly catalyzes this hydrolysis. Acetoacetate and 3-hydroxybutyrate are interconverted by 3hydroxybutyrate dehydrogenase with NAD and NADH, with 3-hydroxybutyrate being the major form. Acetoacetate and 3-hydroxybutyrate are released from the liver into the blood and are then taken up by other tissues that are able to use them as fuels. In the extrahepatic tissues, the acetoacetate is converted to a CoA ester using succinyl CoA as the CoA donor. The acetoacetyl CoA can then be metabolized to acetyl CoA (last step of ␤-oxidation) and further oxidized by the tricarboxylic acid cycle and oxidative phosphorylation. The brain, which cannot utilize fatty acids for energy, can use the ketone bodies produced from fatty acids by the liver.

Coenzyme A in Organic Acid Metabolism CoA is also involved in the mitochondrial metabolism of a large number of other carboxylic acids as CoA thioesters. The catabolism of many amino acids involves the removal of the amino group, leaving a carboxyl group that can be esterified to CoA for further metabolism. As described earlier, the branched-chain ␣-keto acids derived from valine, isoleucine, and leucine are oxidatively decarboxylated to form acyl CoA derivatives. Leucine is catabolized to HMG CoA, which is cleaved to acetoacetate (a ketone body) and acetyl CoA by the lyase involved in ketone body synthesis. Valine and isoleucine are metabolized via pathways involving acyl CoAs to form propionyl CoA and propionyl CoA plus acetyl CoA. The amino acids threonine and methionine are also metabolized to propionyl CoA. The propionyl CoA is converted to succinyl CoA and enters the tricarboxylic acid cycle. The amino acids lysine, hydroxylysine, and tryptophan are catabolized to acetoacetyl CoA. In addition to catabolic pathways, acyl CoAs are involved in many synthetic reactions. The CoA ester of 3-hydroxy-3-methylglutarate (HMG CoA), formed in the cytosol, is the starting material for the synthesis of isoprenoids, cholesterol, and steroids. Acetyl CoA is a substrate for the acetylation of amino and hydroxyl groups of many compounds. Another role for CoA is the

Pantothenic Acid

detoxification of drugs and other exogenous compounds. An example is the conversion of aspirin to a CoA ester, then transfer to the amino group of glycine to form salicylurate for excretion.

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acid (15). The transport across the blood–brain barrier is also saturable but does not appear to be Na+ dependent (4).

Excretion

Carnitine interrelations The esters of CoA and carnitine have very similar energy contents. They are maintained in equilibrium by carnitine acyl CoA transferases. The carnitine palmitoyl CoA transferases and their role in transporting long-chain fatty acids into mitochondria for fatty acid ␤-oxidation have already been described. In addition, carnitine acetyl CoA transferase catalyzes the interconversion of a number of short-chain carnitine esters and CoA thioesters. Additional transferases act on medium-chain length acids. Free carnitine and carnitine esters act as a buffer to maintain free CoA and acyl CoA levels. If acyl CoAs accumulate as that occurring in inherited disorders of fatty acid oxidation or metabolism of some organic acids, free CoA could be depleted below the levels needed for its essential roles in metabolism. The conversion of some acyl CoAs to acyl carnitines frees up CoA and maintains a more normal ratio of free-to-esterified CoA. In addition, acyl CoAs are inhibitors of a number of enzymes, and decreasing their concentration by converting them to acyl carnitines reduces this inhibition. The acyl carnitines can also be translocated out of the mitochondria, enter the blood circulation, and be excreted by the kidneys as a means of removing accumulated esters of CoA that may be toxic. A side effect of this is that in inherited disorders, in which acyl carnitines are excreted in large amounts, carnitine itself may become depleted in tissues and this, in turn, will decrease the transport of fatty acids into the mitochondria.

In the kidney tubules, pantothenic acid is largely reabsorbed at physiological concentrations by a Na+ dependent process (4). At higher concentrations, there is tubular secretion of pantothenic acid (excretion of a higher concentration in the urine than is present in the plasma). As a result, there is a positive correlation between dietary intake of pantothenic acid and its excretion in the urine. There are no known catabolites of pantothenic acid; only pantothenic acid is excreted in urine.

DIETARY SOURCES Pantothenic acid is widely distributed in plant and animal sources, existing both free and bound as ACP and CoA. Total pantothenic acid in foods is determined by hydrolysis of the bound forms to free pantothenic acid and quantitation of the released pantothenic acid by microbiological growth assays, radioimmune assays, or more recently, stable isotope dilution mass spectrometric assays (16). There is considerable loss of pantothenic acid in highly processed foods (4). The average dietary intake of pantothenic acid in the composite Canadian diet is about 5 to 6 mg/day, with somewhat lower intake in the elderly and young children (17). Another study of mixed total diet composites of young adults in the United States found a mean pantothenic acid intake of 5.88 mg ± 0.50 standard deviation (18). There is limited information about the bioavailability of pantothenic acid (19). From studies of dietary intake and urinary excretion, it is estimated that only about 50% of dietary pantothenic acid is available.

PHYSIOLOGY Absorption CoA and ACP from the diet are enzymatically degraded in the intestine to release free pantothenic acid (12). CoA, dephospho-CoA, and phosphopantetheine are not absorbed by the intestine and must be digested to pantothenic acid before absorption. Uptake of pantothenic acid is mediated by a saturable Na+ -dependent transporter utilizing the Na+ electrochemical gradient for active transport with the highest rate of transport in the jejunum (13). This multivitamin transporter, which also transports biotin and lipoic acid, has been cloned from human intestinal cells (14). Pantetheine is also absorbed by the intestine but is hydrolyzed to pantothenic acid in the intestinal cells. The absorbed pantothenic acid is transported by the blood, primarily as bound forms in red blood cells. How this is made available to tissues is unclear, and it may be that the low concentration of free pantothenic acid in plasma (0.06–0.08 mg/L as compared with 1.0–1.8 mg/L in whole blood) is the form taken up by tissues (4).

Transport Pantothenic acid (pantothenate) is transported into mammalian cells by the saturable Na+ -dependent multivitamin transporter, which also transports biotin and lipoic

RECOMMENDED INTAKES No recommended daily allowance has been determined for pantothenic acid. Ingestion of 1.7 to 7 mg/day, depending on age, is considered adequate dietary reference intake (Table 1). Pantothenic acid is included in most multivitamin supplements, generally in the amount of 10 mg. Table 1

Adequate Intakes of Pantothenic Acid

Group Infants 0–6 mo 7–12 mo Children 1–3 yr 4–8 yr 9–13 yr Males >13 yr Females >13 yr Pregnancy Lactation Source: From Ref. 20.

Amount (mg/day) 1.7 1.8 2 3 4 5 5 6 7

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DEFICIENCY Because of the wide distribution of pantothenic acid in foods, no spontaneous deficiency has been reported. Deficiency has been induced in a small number of human volunteers with a pantothenic acid–free diet. There were no clinical symptoms at nine weeks, even though urinary excretion of pantothenic acid had decreased by 75%, but the volunteers appeared listless and complained of fatigue (21). Others fed a diet deficient in pantothenic acid together with an antagonist (␻-methylpantothenic acid) to block pantothenic acid utilization developed headaches, fatigue, a sensation of weakness and numbness, and burning sensations in hands and feet (22,23). Additional symptoms included personality changes, sleep disturbances, impaired motor coordination, and gastrointestinal disturbances. All symptoms were reversed by stopping the antagonist and giving pantothenic acid.

SUPPLEMENTATION The effect of supplementation with very high levels of pantothenic acid and thiamin derivatives on physiology and performance of trained cyclists was compared to placebo in a randomized double-blind study (24). There was no difference in any of the physiological parameters or in time trials. Pantothenic acid together with ascorbic acid may improve wound healing, giving more solid and resistant scars, by affecting the trace metal concentrations (25). Exposure of rats to gamma radiation lowers liver CoA, glutathione, cholesterol, and phospholipid levels and causes lipid peroxidation. Administration of pantothenol, which is readily converted to pantothenic acid, prevented these effects, presumably by maintaining CoA levels (26). Patients with fatty liver and hypertriglyceridemia treated with large amounts of pantethine show decreased fat in liver and the viscera, but increased subcutaneous fat (27). In the dietary supplement marketplace, there are many more claims for a wide range of beneficial health effects of very large doses of 500 to 1000 mg of pantothenic acid. These amounts are hundreds of times the adequate daily intake, considered to be about 5 mg/day for adults. No data on toxicity of pantothenic acid in humans at these or higher doses have been reported, and only minor gastrointestinal effects occur at even higher doses. Studies in rats showed that the extremely high dose of 3% calcium pantothenic acid by weight in the diet for 29 days caused enlargement of the testes, diarrhea, hair damage, reduced food intake, and reduced weight gain (28). There were no ill effects from calcium pantothenate at 1% by weight of the diet for 29 days, or the control (normal) diet with calcium pantothenate at 0.0016% by weight of the diet. Although there appears to be no risk of toxicity with gram quantities of pantothenic acid, there is very little evidence to support the health claims for clinical benefits of pantothenic acid. The broad health claims include increased energy and athletic ability, a cure for acne, decreased symptoms in arthritis, increased immunity, prevention of hair loss and graying, anti-aging, activation of the adrenal glands, synthesis of the neurotransmitter acetylcholine, lowering cholesterol and triglyceride levels, and improved wound

healing. The claims are often based on a single or a very few old studies with a small number of subjects, and wellcontrolled double-blind clinical studies with a larger numbers of subjects have not been done to validate the claims.

INHERITED DISORDER An inherited disorder, pantothenate kinase-associated neurodegeneration (previously called HallervordenSpatz syndrome), has been shown to be due to mutations in a pantothenate kinase gene, PANK2 (29). This disorder is an autosomal recessive neurodegenerative disorder with iron accumulation in the basal ganglia of the brain, onset in childhood, and a progressive course with early death. There are four different PANK genes in humans, with different expressions in different tissues. PANK1 is most expressed in heart, liver, and kidney; PANK3 in liver; PANK4 in muscle; and PANK2, the mitochondrial isoform, in most tissues, including basal ganglia. A mutation in PANK2, which resulted in low activity of pantothenate kinase in those tissues, where it is the major expressed pantothenate kinase, would be expected to affect CoA levels since this enzyme is rate limiting for the synthesis of CoA. In a large study of patients with PANK, all those with the classic syndrome showing early onset with rapid progression had mutations in PANK2, most often resulting in protein truncation (30). Only about a third of patients with atypical disease (often with prominent speech-related and psychiatric symptoms) had PANK2 mutations, and these generally caused an amino acid change. Some of these patients with residual activity of pantothenate kinase may benefit from treatment with large doses of pantothenic acid. The classic and atypical disease due to PANK2 abnormalities is now generally referred to as pantothenate kinaseassociated neurodegeneration. A recent review compares pantothenate kinase-associated neurodegeneration with other inherited disorders that cause neurodegeneration with brain iron accumulation (31). A common mutation of the PANK2 gene accounts for 25% of the alleles in affected individuals. A mouse model lacking PANK2 does not show the neurological phenotype of PANK but does show growth retardation, azoospermia, and retinal degeneration. When fed a pantothenic acid–deficient diet, these mice died suddenly without discernable neurological problems (32). Wild type mice on the pantothenic acid– deficient diet developed azoospermia and a movement disorder, which were reversible on restoring use of pantothenic acid.

REFERENCES 1. Begley TP, Kinsland C, Strauss E. The biosynthesis of coenzyme A in bacteria. Vitam Horm 2001; 61:157–171. 2. Jones CE, Brook JM, Buck D, et al. Cloning and sequencing of the Escherichia coli panB gene, which encodes ketopantoate hydroxymethyltransferase, and overexpression of the enzyme. J Bacteriol 1993; 175:2125–2130. 3. Rock CO, Calder RB, Karim MA, et al. Pantothenate kinase regulation of the intracellular concentration of coenzyme A. J Biol Chem 2000; 275:1377–1383. 4. Tahiliani AG, Beinlich CJ. Pantothenic acid in health and disease. Vitam Horm 1991; 46:165–228.

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5. Leonardi R, Rock CO, Jackowski S, et al. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine. Proc Natl Acad Sci U S A 2007; 104:1494–1499. 6. Daugherty M, Polanuyer B, Farrell M, et al. Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics. J Biol Chem 2002; 277:21, 431– 421, 439. 7. Sweetman L. Pantothenic acid and biotin. In: Stipanuk MH, ed. Biochemical and Physiological Aspects of Human Nutrition. Philadelphia, PA: W.B. Saunders Co., 2000:519–540. 8. Tahiliani AG. Dependence of mitochondrial coenzyme A uptake and the membrane electrical gradient. J Biol Chem 1989; 264:18, 426–18,432. 9. Smith S, Witkowski A, Joshi AK. Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 2003; 42:289–317. 10. Joshi AK, Zhang L, Rangan VS, et al. Cloning, expression, and characterization of human 4 -phosphopantetheinyl transferase with broad substrate specificity. J Biol Chem 2003; 278:33, 142–133, 149. 11. Tweto J, Liberati M, Larrabee AR. Protein turnover and 4-phosphopantetheine exchange in rat liver fatty acid synthetase. J Biol Chem 1971; 246:2468–2471. 12. Shibata K, Gross CJ, Henderson LM. Hydrolysis and absorption of pantothenate and its coenzymes in the rat small intestine. J Nutr 1983; 113:2107–2115. 13. Fenstermacher DK, Rose RC. Absorption of pantothenic acid in rat and chick intestine. Am J Physiol 1986; 250:G155–G160. 14. Prasad PD, Wang H, Huang W, et al. Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter. Arch Biochem Biophys 1999; 366: 95–106. 15. Prasad PD, Ganapathy V. Structure and function of mammalian sodium-dependent multivitamin transporter. Curr Opin Clin Nutr Metab Care 2000; 3:263–266. 16. Rychlik M. Pantothenic acid quantification by stable isotope dilution assay based on liquid chromatography-tandem mass spectrometry. Analyst 2003; 128:831–837. 17. Hoppner K, Lampi B, Smith DC. An appraisal of the daily intakes of vitamin B12, pantothenic acid and biotin from a composite Canadian diet. Can Inst Food Sci Technol J 1978; 11:71–74. 18. Iyengar GV, Wolf WR, Tanner JT, et al. Content of minor and trace elements, and organic nutrients in representative mixed total diet composites from the USA. Sci Total Environ 2000; 256:215–226. 19. Berg H van den. Bioavailability of pantothenic acid. Eur J Clin Nutr 1997; 51(suppl 1):S62–S63.

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20. The Institute of Medicine. Dietary Reference Intakes for Thiamine, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, D.C.: National Academy Press; 1998. 21. Fry PC, Fox HM, Tao HG. Metabolic response to a pantothenic acid deficient diet in humans. J Nutr Sci Vitaminol 1976; 22:339–346. 22. Bean WB, Hodges RE. Pantothenic acid deficiency induced in human subjects. Proc Soc Exp Biol Med 1954; 86: 693–698. 23. Hodges RE, Ohlson MA, Bean WB. Pantothenic acid deficiency in man. J Clin Invest 1958; 37:1642–1657. 24. Webster MJ. Physiological and performance responses to supplementation with thiamin and pantothenic acid derivatives. Eur J Appl Physiol 1998; 77:486–491. 25. Vaxman F, Olender S, Lambert A, et al. Can the wound healing process be improved by vitamin supplementation? Eur Surg Res 1996; 28:306–314. 26. Slyshenkov VS, Omelyanchik SN, Moiseenok AG, et al. Pantothenol protects rats against some deleterious effects of gamma radiation. Free Radical Biol Med 1998; 24:894–899. 27. Osono Y, Hirose N, Nakajima K, et al. The effects of pantethine on fatty liver and fat distribution. J Atheroscler Thromb 2000; 7:55–58. 28. Shibata K, Takahashi C, Fukuwatari T. et al. Effects of excess pantothenic acid administration on the other water-soluble vitamin metabolism in rats. J Nutr Sci Vitaminol (Tokyo) 2005; 51:385–391. 29. Zhou B, Westaway SK, Levinson B, et al. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet 2001; 28:345–349. 30. Hayflick SJ, Westaway SK, Levinson B, et al. Genetic, clinical and radiographic delineation of Hallervorden-Spatz syndrome. N Eng J Med 2003; 348:33–40. 31. Gregory A, Polster BJ, Hayflick SJ. Clinical and genetic delineation of neurodegeneration with brain iron accumulation. J Med Genet 2009; 46:73–80. 32. Kuo YM, Hayflick SJ, Gitschier J. Deprivation of pantothenic acid elicits a movement disorder and azoospermia in a mouse model of pantothenate kinase-associated neurodegeneration. J Inherit Metab Dis 2007; 30:310–317.

FURTHER READING 1. Tahiliani AG, Beinlich CJ. Pantothenic acid in health and disease. Vitam Horm 1991; 46:165–228.

Pau d’Arco Memory P. F. Elvin-Lewis and Walter H. Lewis

INTRODUCTION Pau d’arco and lapachol are the Portuguese and Spanish names used to identify about 26 species of shrubs and trees of the genus Tabebuia (Bignoniaceae). These species are indigenous to the American tropics from Mexico to southern South America, with the majority of species found in Brazil and neighboring states. They possess numerous bioactive compounds, with core activity in the naphthoquinones, particularly lapachol and ␣- and ␤-lapachones. Other classes of compounds include the anthraquinones, flavonoids, iridoids, lignans, and terpenoids, all less well known or active than the more prevalent naphthoquinones. The stem bark and trunk or heartwood of T. impetiginosa (Mart. ex DC.) Standl. (synonym T. avellanedae Lor. ex Griseb.), sometimes referred to as the Ipe Roxo tree, T. rosea (Bertol.) DC., and T. serratifolia (Vahl) Nichols. are the materials and species most commonly used in the preparation of botanicals and traditional and herbal medicines, and for research and clinical purposes. The inner bark of T. impetiginosa and possibly other related species are the source of taheebo, a phytomedicine also referred to as ipˆe-cavat˜a, ipˆe-comum, ipˆe-reto, ipˆe-rosa, ´ ipˆe-roxo-damata, lapacho negro, pau d’arco-roxo, peuva ´ or piuva. Historical uses of pau d’arco species are most commonly reported for use as a tonic, for the treatment of syphilis, fevers, malaria, cutaneous infections, backache, toothache, and stomach and bladder disorders. With a research impetus starting in the 1960s in Brazil, which led to preliminary clinical claims of efficacy for treating cancers, fresh interest in the significance of pau d’arco and its bioreactive components has arisen, both regarding basic and clinical research among the general public.

Figure 1 Flowering branch of Tabebuia impetiginosa, a 30-m tree common in tropical South America. Source: Courtesy of Al Gentry, Missouri Botanical Garden.

its use until distributors prove their products are safe and effective. One of the most important sources of pau d’arco inner bark and heartwood is T. impetiginosa (Fig. 1), a large tree up to 30 m tall, with deep pink to purple flowers, found from Mexico and Central America to tropical South America, south to northern Argentina, and Bolivia. Significant also are the large trees T. rosea, with pink to purple flowers, found from Mexico to northern South America, and T. serratifolia (Fig. 2), having yellow flowers, which occurs from Colombia to the Guianas south to Brazil and Bolivia. These species and a majority of the remaining 23 species of Tabebuia tested contain lapachol or related compounds, each with varying concentrations in their inner stem bark, heartwood, and leaves. For medicinal purposes, indigenous people prefer the inner bark, although the heartwood is considered more potent. Leaves and flowers are less frequently used, and the roots rarely so. Usually these phytomedicines are used as infusions or decoctions. Reports of traditional medicinal uses of Tabebuia species can be found on herbarium labels and in the literature, and they provide useful anecdotal evidence of prior use. Pau d’arco is most commonly reported for treatment of syphilis, fevers, malaria, cutaneous infections, tooth and back pain, and bladder and stomach disorders. Some examples are:

BACKGROUND Widely used historically and currently in South America to treat a variety of infections and diseases, pau d’arco became known in North America and Europe in the early 1980s. Acceptance of this botanical has been rapid, however, for the bark or wood prepared as an infusion or decoction is ingested regularly by at least one million people (1). According to Information Resources Inc. (IRI), in 2009 pau d’arco was ranked 46th in single herb supplement sales in the UnitedStates with dollar sales amounting to $35, 636. This was an increase of 11.68% over the 2008 sales (C. Cavaliere, written communication, 2010). However, in Canada the Federal Health Protection Branch has banned

r T. impetiginosa to treat impetigo in Brazil around 1843 (2). 612

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Figure 2 Flowering branch of Tabebuia serratifolia, a 30-m tree common in Amazonian Brazil. Source: Courtesy of Al Gentry, Missouri Botanical Garden.

r T. rosea bark drunk to treat malaria (Steyermark 51372, F); bark to treat rabies in Guatemala (Ruano 425, US); decoction of flowers, leaves, and roots taken internally and also applied externally for treating snakebites in Costa Rica; bark decoction prepared as a remedy for fevers, colds, and headaches (3); bark infusion or decoction as a gargle in Colombia to treat throat ailments and fevers, and as an astringent (4). r T. serratifolia bark as a medicinal for the stomach among the Panar´e of Venezuela (Boom and Grillo 6209, MO). Other examples of pau d’arco’s early ethnopharmacological uses are cited in current reviews of T. impetiginosa (5) and lapachol (6). Sometimes such information is difficult to separate from neo-Western herbalism (7) when those practicing domestic medicine adopt new uses reported in popular press releases on the basis of limited research. This is particularly evident in reports involving anticancer uses conducted in Brazil since 1960; no primary herbarium material or literature reference of traditional antineoplastic use could be found prior to this date. Thus, the citation of Schunke 14259 (MO) in 1998 reporting that bark infusions of T. impetiginosa are drunk by Peruvian natives to cure diabetes, malignant tumors, leukemia, other cancers, anemia, and Parkinson’s disease, is undoubtedly an instance of recent incorporation into indigenous pharmacopeia. Use of T. rosea bark by the Maya of Mexico against cancer can likewise be traced to the year 1985 (8). Similarly, the utilization of T. serratifolia bark in Colombia as an anticancer infusion or decoction can be attributed to Brazilian research of the 1960s and 1970s (4). All species known as pau d’arco should not be considered equivalent in toxicity or efficacy. The purpleflowered species are considered less toxic than the yellowflowered ones, and the former are preferred. However, the bark and heartwood are routinely collected and used without regard to taxonomic identification, chemical composition, or biological activity.

Of the 26 species of Tabebuia known as pau d’arco, the secondary metabolic chemistry of about half has been well documented. Most contain naphthoquinone derivatives, but only T. impetiginosa also contains anthraquinones. In addition, some species possess flavonoids, iridoids, lignans, triterpenes, and other classes of compounds. The naphthoquinones (Fig. 3) are the most prevalent class of compounds in Tabebuia. While a few species lack lapachol (2), it is commonly found in the three most important species, T. impetiginosa, T. rosea, and T. serratifolia. The derivatives prenylnaphthoquinone (1) and lapachol methyl ether (3) are also found in T. impetiginosa. Other prenylnaphthoquinones found in these species include dehydro-␣-lapachone (4), dihydro-␣-lapachone (5), and ␤-lapachone (LAPA, ARQ 501) (6). In addition, the furanonaphthoquinone derivatives 7–18 are common in T. impetiginosa. The majority of these (2, 4–7, 10–12, and 17 and 18) have been found in the inner bark (9). The anthraquinone-naphthaquinol dimer, tabeuin 22, has also been reported for this species (10). In addition to lapachol, 4, 7, 11, and 14 (11) have been isolated from the root, bark, and heartwood of T. rosea, along with tabeuin 22 (12), dehdyrotectol 19, and the tecomaquinones 20, 21, 23, and 24 from its heartwood (Fig. 4) (13). T. serratifolia also contains the constituents 2, 4, 5, 11–13, and 15, obtained from extracts from the trunk’s heartwood (14). Studies on

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Figure 3

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Naphthaquinones from Tabebuia spp.

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tion of a contaminant, its isomer ␣-lapachone (21). The compound has also been synthesized (22). Mechanisms by which synthetic and biosynthetic pathways have been utilized to produce these compounds are known. Although these techniques are important in verifying these and other related structures, the abundance of the natural product as a metabolite in many tropical trees species negates this approach as an alternate source unless such species become threatened by overexploitation (6).

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pau d’arco’s compounds and synthetic derivatives are also discussed in other current reviews (5,6). Initially pharmacological research focused on lapachol; however, this is only one of the N-factors (naphthoquinones) responsible for pharmacological activities of pau d’ arco (15). Lapachol is obtained in gram quantities from these species ranging from 2% to 7% from the heartwood (16), a level confirmed by Linardi (17) who obtained 3.2% of the compound from petroleum and methanol extracts of finely powdered wood of T. impetiginosa. It was also obtained as a minor constituent from nonaqueous extracts of its inner bark (9), but not detectable in aqueous extracts. In an early publication (1914), T. serratifolia wood was reported to have 7.64% lapachol (18). Lapachol and ␤-lapachone are readily synthesized from 2-hydroxy-1, 4-naphthoquinone in high yields (19). A number of current mechanistic studies and patents have also cited the use of the naturally occurring o-naphthoquinone derivative of lapachol, namely 3, 4-Dihydro-2,2-dimethyl-2H-naphtho(1,2-b)pyran-5, 6-dion or ␤-lapachone, which is also referred to as LAPA, and ARQ 501. Because of its potential pharmaceutical value as an anticancer and anti-infective agent (20) addressing sourcing issues, physiochemistry and pharmacotech characterization has identified suitable purifying and storage processes in addition to the isola-

BOTANICAL PRODUCTS, USES, AND ADVERSE REACTIONS Products Pau d’arco is available as capsules, tablets, skin salves, extracts, and tea bags. Products with lapacho as a component are sold under such names as Advance Defense System Tablets, Brazilian Herbal Tea, Candistroy, Cat’s Claw Defense Complex, Cellguard Coq 10 Nac, Healthgard with Echinacea, Immuno-Nourish, Ipe Roxo, Lapacho, Pau d’Arco, Pau d’Arco Inner Bark, Taheebo, and many others. Powdered inner bark and/or heartwood are often prepared in the United States as decoctions, with one cup taken two to eight times per day. A decoction is prepared by boiling one teaspoon of powdered bark/heartwood for each cup of water for five to ten minutes (23). In other examples two to three teaspoons of inner bark are simmered in 500 mL of water for 15 minutes and taken three times per day (24). In a more specific example using 460 mg capsules of inner bark/heartwood orally, 1 to 2 capsules are ingested at meals with water twice daily, or 3 to 4 capsules 3 times daily for not more than 7 days, depending on use. In a study of 15 commercial products of pau d’arco obtained in Canada, naphthoquinones were detected in all samples except two, although no naphthoquinones were found in the three concentrates examined. Lapachol was detected in only two of these products. However, in two Brazilian products studied, one wood and the other a concentrate, both contained lapachol and related compounds (25).

Antioxidant Use Lapacho or T. impetiginosa bark extracts are used to stabilize compositions that contain oxygen-labile active agents particularly for cosmetic use (26), to whiten skin with limited irritation (27), and in an antioxidant Japanese carbonated drink known as Purple Ipe (28).

Antifungal Use As an example of herbal medicinal use, pau d’arco tea is drunk or applied vaginally as a douche to treat Candida, or an extract-soaked tampon is used to treat this and similar infections, often with associated inflammations (29,30). Such extracts are also part of a patented nail varnish formulation for the treatment of human onychomycosis and paronychia (31).

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Anticancer Activity

Drinking extracts of yellow-flowered T. umbellata or T. pedicellata caused abnormal swellings similar to burns and skin pustules to form. Only weak teas made from 1 part bark to 10 parts water should be drunk (1). Pau d’arco should be avoided when pregnant, breast-feeding, and taking anticoagulants and by persons having severe liver disease, von Willebrand’s disease, or thrombocytopenia. Its use may cause anemia, nausea, pinkish urine, unusual or excessive bleeding or vomiting, and diarrhea (5,32,33).

The use of pau d’arco in Brazilian remedies to treat cancers has led to the identification of pau d’arco’s active components, the production of some derivatives, partial elucidation of their mechanisms of action, and the conduct of preclinical and clinical evaluations in the treatment of carcinomas and leukemias (42,43). Current in vitro studies have provided a better understanding of the antiproliferative and immunosuppressive effects observed originally (44). For example, in an attempt to identify a selective estrogen receptor for breast cancer therapy that did not elicit adverse effects, human breast carcinoma ER+ MCF-7 cells were treated with aqueous extracts of pau d’arco (taheboo). This preparation elicited time-dependant growth inhibition of the S phase and the initiation of apoptosis associated with a downregulation of the cell cycle regulation and estrogen responsive genes and an upregulation of apoptosis specific and xenobiotic metabolic specific genes (45). The naphthoquinones inhibit enzymes critical to cellular DNA replication, and other cellular functions, resulting in cell death. Their effects occur at macromolecular levels and result in the selective killing of certain cancer cell lines (colon, lung, prostate, breast, ovary) in unique ways that suggest that lapachol derivatives have the potential for use in therapies for specific cancer types, for example, breast and prostate (46). Some also have increased cytotoxicity (47) and others are effective in drug-resistant cell lines (48). Also, in lower doses, the napthoquinones are effective radiosensitizers. They act by specifically and synergistically enhancing the cytotoxic effects of DNA-damaging agents and the effects of X-rays following prolonged drug exposures. For example, ␤-lapachone may elicit DNA conformational changes, which can inhibit potentially lethal damage repair or during this process, enhance lethality by converting single- into double-stranded DNA breaks (49). The mechanism by which ␤-lapachone radiosensitizes cancer cells with elevated NQO1 levels is related to its ability to inhibit error-prone nonhomologous end joining needed in the repair of DNA double-stranded breaks (50). Among these the isomer of lapachol, ␤-lapachone appears to be the most promising and has replaced lapachol in the US National Cancer Institute’s (NCI) anticancer chemotherapeutic studies (5). This is because this molecule and other potent ␤-cycled-pyran-1, 2-naphthoquinones [0.1 ␤1/4 M < IC50 < 0.6 ␤1/4 M] produce hydrogen bond interactions that are able to affect hydrophobic areas of a receptor more completely than those of the moderately active 1,4-naphthoquinone derivatives (51). These molecules have currently been studied extensively to understand the molecular basis for their anticancer activities and their potential uses in cancer chemotherapy. While not affecting the regulatory systems of normal cells, ␤-lapachone can affect unchecked and persistent expression patterns of unscheduled checkpoint molecules present in regulatory-defective precancer and cancer cells (52). It exerts its antineoplastic activity by inducing either apoptotic or necrotic cell death within the range of 1 to 10 ␮M (IC50 ). Cytotoxicity has been elicited in a wide variety of transformed cell lines including those derived

Allergic Reactions and Irritation Exposure to wood dusts may cause skin and mucosal symptoms associated with allergic dermatitis. In the timber trade, allergic reactions to pau d’arco sawdust are common (1). Both lapachol and deoxylapachol are considered allergens (34).

PRECLINICAL STUDIES In Vitro Studies Antibacterial Activity Both gram-positive and -negative microorganisms are affected by certain naphthoquinones through the generation of superoxide anions and hydrogen peroxide (35), by the uncoupling of oxidative phosphorylation, and through electron transfer inhibition (36). Of relevance to traditional uses to treat skin and gastrointestinal infections are inhibitory activities of lapachol, ␤-lapachone, ␣-xyloidone, and related compounds against Staphylococcus aureus (36), including those with methicillin resistance (37), and Salmonella (29,30). Susceptibility of other organisms has also been demonstrated (38). Using the disc technique (100 ␮g/disc), lapachol’s effects on intestinal bacteria can vary with greater sensitivity being demonstrated for Clostridium paraputrificum, and less so for C. perfringens and Escherichia coli. Unaffected, even at at high concentrations of 1000 mg/disc were Bifidobacterium adolescentis, B. bifidum, B. infantis, and Lactobacillus acidophilus, and L. casei. When the chemical components of the dried inner bark of T. impetiginosa were tested against Helicobacter pylori by disc diffusion and minimum inhibitory concentration (MIC) bioassays it was found that 2-(hydroxymethyl) anthroquinone exhibited strong activity at 0.01 mg/disc, whereas 2-carboxylic acid, lapachol and the control metronidazole only exhibited moderate effects at 0.1 mg/disc (39). Overall these compounds were more effective than metronidazole but less effective than amoxicillin and tetracycline. The basis for lapachol and its derivatives’ antimicrobial activities are related to the presence of a methyl group in the C-2 position of the 1,4-naphthoquinone (40). Additional studies with an analog of lapachol, “furanonapthoquinone” show that significant antibiotic effects on five strains of H. pylori can occur at MIC levels of 0.1 ␮g/mL. Also, when furanonapthoquinone was combined with several antibiotics such as ampicillin, cefacior, levofloxacin, minocycline, and vancomycin their MIC values against H. pylori were reduced two- to eightfold (41).

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from patients with promyelocytic leukemia (53), prostate (54), malignant glioma (55,56), hepatoma (57), colon (58), breast (59), ovarian (60), pancreatic (50,61–65), retinoblastoma (66), and multiple myeloma cell lines (67), including drug-resistant lines (48). The mechanisms by which anticancer activities are elicited are complex. Shortly after exposure to ␤-lapachone, malignant cells rapidly release cytochrome c. This activity causes a decrease in mitochondrial transmembrane potential (delta psi), which is followed by the activation of caspase-3 in apoptotic cell death but not in necrotic cell death (68). Studies indicate that its antitumor effect occurs indirectly by inducing p53-independent apoptosis and the arrest of cell cycles through the altered activities of cell cycle control regulatory proteins such as the downregulation of retinoblastoma protein (pRB), a transcriptional repressor target at transcription factor E2F-1 and the induction of the expression of cyclin dependent kinase inhibitor 1A (CDKN1A or p21). During the cell cycle, G1/S-phase transition requires both E2F-1 and p2, and several studies indicate that ␤-lapachone affects the E2F1 checkpoint pathway and induces cell death in cancer cells from a variety of tissues without affecting normal cells from these tissues. Also, cytotoxicity occurs through the induction of reactive oxygen species in vivo (69). In addition, apoptosis elicited by ␤-lapachone may also be due to the loss of reduced NADH or NAD(P)H. This is because NAD(P)H:quinone oxidoreductase (NQO1) causes a reduction of ␤-lapachone causing “futile cycling” between the quinine and hydroquinone forms with a concomitant loss of these reduced enzyme cofactors (70). Its potential use as a chemopreventive agent for liver cancer is also associated with its ability to induce apoptosis in hepatocarcinoma HepG2 cells. The mechanisms involved relate to the formation of apoptotic bodies and DNA fragmentation. These activities are linked to the downregulation of antiapoptotic Bcl-2 and Bcl-X (L) and upregulation of proapoptotic Bax expression with the proteolytic activation of caspase-3 and -9 and degradation of poly (ADP-ribose) polymerase (PARP) protein without affecting either the Fas/FasL system or the inhibitor of apoptosis family proteins (71). ␤-lapachone inhibits cell viability and migration of human hepatocarcinoma cell lines, HepG2, and Hep3 in a dose-dependant manner. Western blot analysis indicates that at an early point in time, amplified levels of protein, as well as mRNA expression of early growth response gene1 (Egr-1) and thrombospondin-1 (TSP-1) occurs and then decreases in a time-dependent manner. Also, in the matrigel invasion assay a decrease in invasive ability takes place indicating a downregulation of Snail and upregulation of E-cadherin expression. The viability of HepG2 cells is affected through the induction of apoptosis and the formation of apoptotic bodies and DNA fragmentation. This activity is associated with a proteolytic activation of caspase-3 and -9 and degradation of PARP protein, downregulation of antiapoptotic Bcl-2 and Bcl-XL and upregulation of proapoptotic Bax expression. However, the family of inhibitor proteins and the FAS/FasL system are not affected. These studies indicate that this compound is potentially a chemopreventive agent for liver cancer (71,72). Similar results have been observed using the T24 line of bladder cancer cells (73), cultured human prostate carci-

noma DU145 cells (74), human colon cancer cells (HCT116 (75), SW480, SW620, and DLD1), (58) and breast cancer cells. (59,76). The role of the ubiquitous enzyme, NAD(P)H: quinone oxidoreductase-1 (NQO1) is to detoxify cells affected by xenobiotics containing quinone moieties by catalyzing a two-electron reduction in quinones, using NADH or NADPH as the electron donor. Unlike other reductases, NQO1 produces a highly stable intermediate in the presence of ␤-lapachone treatment. In certain tumors of the breast, cancer, and lung NQO1 is overexpressed (77) and treatment with this compound causes cell death due to the catalytic action of NQO1 by eliciting futile oxidoreductions of ␤-lapachone leading to reactive oxygen species’ generation, a novel caspase independent of p53, DNA breaks, ␤-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1 (5,48). Unlike normal cells, the vulnerability of cancer cells to topoisomerase inhibition is linked to their ability to overcome certain restrictive mechanisms enabling them to continually divide. Initial studies on the effects of ␤-lapachone indicated that its mode of action on irradiated cells differs from campothecin, which causes chromosome damage and strand breaks (43). It was proposed that enhanced cytotoxicity was due to inhibition of topoisomerase I activity and the modification and inhibition of lesion repair (49,54,78,79). ␤-lapachone blocks the binding of human DNA topoisomerase I to DNA (80,81), and by preventing DNA repair sensitizes cells to DNA-damaging agents including radiation (82,83). By interacting directly with these enzymes ␤-lapachone prevents catalysis and blocks the formation of a cleavable complex (80) or with the complex itself, causing religation of DNA breaks and dissociation of the enzyme from DNA (84). However, inhibition of either topoisomerase I or topoisomerase II (81,84) activities is no longer considered the primary way in which cell lethality is induced by ␤-lapachone but rather that NAD(P)H:quinine oxidoreductase activity is the principal determinant of ␤-lapachone cytotoxicity (43,70). Prevention of neovascularization is an important factor in inhibiting tumor growth. Studies on the effect of ␤-lapachone on endothelial cell death, indicate that intracellular cGMP levels and the mitochondria membrane potential (MMP) are decreased, and calpain and caspases are activated. Addition of nitric oxide (NO), which is an important factor in mediating vascular cell growth and migration, downregulates the ␤-lapachone-induced cGMP depletion and protects the cells from apoptosis by blocking the MMP decrease and increases of calcium. The fact that antiangiogenic effects are not affected in this process suggests that ␤-lapachone may have a potential as an antiangiogenic drug (85) The apoptic synergistic action of ␤-lapachone and taxol on cancer cells is based upon their ability to affect cell arrest at conflicting checkpoint signals. By exploiting these cell death “collisions” this combination has the potential of treating cancers such as multiple myeloma (43). It is recommended that the combination of a G1 or S phase drug, such as a ␤-lapachone should be given first, with a G2/M drug, such as a taxane derivative added either simultaneously or after ␤-lapachone. In this way ␤-lapachone causes cell-cycle delays in late G1 and S phase and taxol arrests cells at G2/M, resulting in multiple checkpoint delays

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before committing to apoptosis (60). Additional studies with human retinoblastoma Y79 cells combining ␤-lapachone with paclitaxel suggest that the basis for this synergistic apoptotic action is related to phospho-Akt lowering of inhibitor apoptosis proteins and by activation of Bid and caspases-3 and -6 with lamin B and PARP breakdown. Phospho-Akt is a serine/threonine protein kinase, which acts as a powerful promoter of cell survival as it antagonizes and inactivates various components of the apoptotic cascade, and PARP is a protein involved in a number of cellular processes involving mainly DNA repair and programmed cell death) (86).

Antifungal Activity Pau d’arco extracts and their components lapachol and ␤-lapachone are active against more than one known infection of the skin (ringworm) and nails (36), and pathogenic yeasts such as Candida and Cryptococcus neoformans (36). Activities of these naphthoquinones differ in that lapachol activity is comparable to that of ketaconazole, while ␤-lapachone is superior (36). In vitro tests using lapachol show it is an effective antifungal agent against the human yeast Pityrosporum ovale, which causes the chronic skin infection called Tinea versicolor in addition to the wood rotting basidiomycete, Gloeophyllum trabeum (87). Tabebuia caraiba extracts are also effective against the dermatophyte, Trichophyton rubrum (88). In a U.S. patent application 2003-674145, 2005-049207, T. impetiginosa is cited for use against cancers at antifungal doses (89). Modest antifungal activity has also been associated with synthesized napthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (-)-5-hydroxy-2-(1 -hydoxyethyl)naphtho[2,3-b]furan-4, 9-dione and its positional isomer, (-)-8-hydroxy-2(1 -hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (90). Lapachol’s significant antifungal activities against Candida albicans, C. tropicalis, C. elegans, and Cryptococcus neoformans is considered because of its interaction with their cellular membranes (91).

Anti-inflammatory activity Studies on the affects of various solvent fractions of pau d’arco (taheebo) on washed rabbit platelets and cultured rat aortic vascular smooth muscle cells (VSMCs) suggest that platelet aggregation induced by collagen and arachidonic acid is inhibited in a dose dependant way by nHexane, chloroform, and ethyl acetate fractions. Of these, the chloroform fractions were the most active by significantly suppressing arachidonic acid liberation induced by collagen in [(3)H]AA-labeled rabbit platelets, effectively inhibiting cell proliferation and DNA synthesis induced by platelet derived growth factor (PDGF)-BB, in addition to inhibiting the levels of phosphorylated extracellular signal regulated kinase (ERK1/2) mitogen-activated protein kinase stimulated by PDGF-BB, in the same concentration range that inhibits VSMC proliferation and DNA synthesis (92). Aqueous and ethanolic extracts of pau d’arco (taheebo) studied for their anti-inflammatory potentials indicated that the aqueous extract possessed the ability to negatively modulate macrophage-mediated inflammatory responses by suppressing prostaglandin E(2) production. This activity was evident by treating LPS-stimulated RAW264.7 macrophages where significant suppression of

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NO production and blockage of COX-2 and inducible NO synthase (iNOS) occurred as did the phosphorylation of extracellular signal-related kinase (ERK) and U0126, a selective ERK inhibitor. In vivo blockage of COX-2 was also evident when mouse ear edema was diminished in arachidonic acid treated mice given 100 mg/kg of pau d’arco (taheebo), but not those whose edema was evoked by croton oil, an activator of lipoxygenase (93). Using the carrageen inflammatory paw model in rats, lapachol at doses of 100 and 500 mg/kg was found to significantly inhibit the production of edema and abscess formation (20,94). A number of studies indicate that ␤-lapachone (LAPA) has the potential of being a useful antiinflammatory agent. It is not only able to inhibit the expression of NO and iNOS in alveolar macrophages but when studied in lipopolysaccharide-stimulated BV2 microglial cells it inhibits NO and the fever inducing, prostaglandin, dinoprostone (PGE2). A blockage at transcriptional levels is suggested by inhibition of iNOS and COX-2. In addition, the expression of mRNA and proteins of proinflammatory cytokines, such as interleukin IL1B, IL-6, and tumor necrosis factor (TNF-alpha) are affected in a dose-dependant manner. ␤-lapachone further elicits its anti-inflammatory activity by suppressing the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-␬B) by blocking IkappaBalpha degradation and downregulating the ERK, p38 mitogen-activated protein kinase and the Akt pathway associated with cellular survival (95). Furthermore as a topoisomerase inhibitor, ␤-lapachone has the potential of being useful in the treatment of inflammatory disorders (96) and as antiarthritic agents (97). Likewise, two lapachol derivatives 3-iodo-␣-lapachone (I) and 3-iodo-␤-lapachone (II) are considered useful as probable immunomodulatory, antimicrobial and anti-inflammatory agents (98).

Antileishmania Activity ␤-lapachone and its derivatives have also been synthesized and tested as antiparasitic agents. A number of ␤-lapachone derivatives obtained via the Prins reaction of lapachols possess Leishmanicidal activities (99). Also 3-allyl- ␤-lapachone can elicit negative affects on the developmental cycle of Trypanasoma cruzi by inhibiting the growth and functions of epimastigotes as well as decreasing or causing the total disappearance of trypomastigotes from mouse-infected blood (100). Additional studies on the inhibition of epimastigote growth and trypomastigote viability with lapachol, ␣- and ␤-lapachone derivatives indicate the substitution of the benzene ring by a pyridine moiety enhances these activities (101).

Antimalarial Activity Early antimalarial studies claimed that lapachol was almost as active as quinine (102). However, when tested, its activity against Plasmodium falciparum proved disappointing (103), although several analogs of lapachol show enhanced bioreactivity (104,105). In addition several benzo[a]phenazines synthesized from 1, 2-naphthoquinone, lapachol, and ␤-lapachone proved effective against P. falciparum. (106) as have the dihydroxyfuranonaphthoquinones, 5 and 8-hydroxy-2(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-diones isolated from T. incana bark infusions (107).

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The antimalarial activity of lapachol and other napthoquinones is linked to their ability to interfere with electron transport and thus to inhibit the respiratory chain (43,108). A study with Plasmodium knowlesi treated with 100 mg/mL of lapachol indicated that absorption of oxygen was inhibited by 74% and succinate oxidase by 26%. This inhibition is possibly related to lapachol’s ability to inhibit the interaction between cytochromes b and c. (6,109).

Anti-Giardia Activity Giardia lamblia is a diplomonad protozoan that parasitizes the small intestine of vertebrates and is responsible for millions of people acquiring intestinal infections and diarrhea. In vitro studies with ␤-lapachone indicate it can elicit apoptosis by cell shrinkage, chromatin condensation, membrane blebbing, and vacuolization. Drug treatment also can alter lipid rafts, co-localized with regions containing membrane blebbing. By inducing encystation it may also affect mechanisms of parasite resistance (110).

Antimetastatic Activity Alterations in the protein profile and inhibition of cellular invasiveness was demonstrated in a HeLa cell assay using lapachol at a nontoxic concentration of 400 ␮g/mL (corresponding to 1012 molecules of the drug/cell). These activities correspond to a significant antimetastatic potential of this compound (111).

Antioxidant Activity The antioxidant activity of volatile constituents of the dried inner bark of T. impetiginosa is comparable to that of the antioxidants ␣-tocopherol and butylated hydroxytoluene (112). Antioxidant activities have also been identified in a number of T. heptaphylla bark compounds (113).

Antipsoriatic Activity Evaluations of several naturally occurring pau d’arco (lapocho) compounds from the inner bark of T. impetiginosa and a number of synthetic analogs on the growth of the human keratinocyte cell line HaCaT indicate that ␤-lapachone is comparable to the antipsoriatic drug anthralin with an IC50 value of 0.7 ␮M. Other active constituents of lapacho inhibited keratinocyte growth, with IC50 values in the range of 0.5 to 3.0 ␮M with the most potent synthetic analaog 2-acetyl-8-hydroxynaphtho[2, 3-b]furan-4,9-dione eliciting an IC50 value of 0.35 ␮M. Like anthralin, treatment of HaCaT cells with these potent lapacho compounds caused damage to the plasma membrane (9). Pharmaceutical compositions containing several synthetic lapacho derivatives have been proposed for the treatment of cell proliferative disorders including psoriasis, cancer, or precancerous conditions (114).

Antiviral Activity Studies indicate that lapachol, ␤-lapachone, and certain of their derivatives possess broad-spectrum antiviral capacities in vitro. Of those DNA viruses affected, lapachol inhibits replication of members of the herpesvirus group including HHV1, 2 (115,116), and HHV4 (Epstein Barr Virus or EBV) (117). Inhibitory effects against representative RNA viruses, (118) including HIV-1 (119), and HIV

reverse transcriptase (120) have been demonstrated as has its ability to inhibit reverse transcriptases from myeloblastosis virus and Rauscher murine leukemia virus in addition to eukaryotic DNA polymerase-alpha (121). The ability of ␤-lapachone to inhibit regulatory proteins, including Tat, which affects the viral switch from latency to active replication, is the subject of several current patents (119). Acute and chronically infected HIV-1 infected cells treated with ␤-lapachone are unable to produce P 24 since the compound is a potent inhibitor of 1 LTR-directed gene expression of HIV-1 (119). A polyherbal invention containing Tabebuia combines a redox active polyphenol, an oxidizing agent and a redox-active transition metal ion and is claimed to be antimicrobial and antiviral (HIV) (122).

Antitrypanosomal Activity Chagas’ disease, or American trypanosomiasis, is a devastating disease in South America, and transmission through blood transfusions is a serious concern. In vitro and in vivo inhibitory effects on Trypanosma cruzi of lapachol, ␤-lapachone, and several 1,2-naphthoquinone derivatives (100,123) have led to the development of oxazolic, imidazolic (124), and phenazine derivatives (125), which have the potential to replace crystal violet as blood sterilants. Activity against T. cruzii is associated with a three bond-distance from nitrogen to the imidazole ring, of the semi-synthetic pyran[b-4,3]naphtho[1,2-d]imidazoles from ␤-lapachone (126). The production of oxidation radicals leading to cytotoxic activities seen with Trypanosomatids Crithidia fasciculata and Leptomonas seymouri treated with ␤-lapachone and structurally related lipophilic o-naphthoquinones and mansonones are also related to their tricyclic structures, including the presence of a naphthalene ring, a 1,2b or 1,8bc pyran ring, and two orthocarbonyl groups (127).

Neurodegenerative Effects Lapachol and other naphthoquinones modulate the Tau aggregation of proteins, and thus are assumed to effect the treatment or prophylaxis of neurodegenerative diseases and/or clinical dementias, such as Alzheimer’s disease (128).

Snakebites Tabebuia rosea bark demonstrated 100% neutralization against the minimum hemorrhagic dose of Bothrops atrox venum in vitro. This correlates with traditional healers’ statements from northwestern Colombia that T. rosea has antihemorrhagic properties (129).

Spasmolytic Activity. When tested in the guinea-pig ileum model, Lapachol, ␣-lapachone and ␤-lapachone (or LAPA) can elicit nonselective spasmolytic activities. ␤-lapachone was also found to exert this effect by blocking voltage-gated calcium channels (L-type Cav channels) (130).

In Vivo Studies Anticancer Activity (rodents) Crude extracts and lapachol were tested in implanted rodents against Walker 256 carcinosarcoma. Lapachol showed highly significant antitumor activity especially when administered orally, with relatively little effect on

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host body weight (131). In this model, a 92% reduction in tumor growth occurred, and lapachol’s tetra-acetylglucoside derivative increased the life span by 80% in mice with lymphocytic leukemia P-388 (17). Treatment with ␤-lapachol following exposure to irradiation optimized the effects of delayed tumor growth in mice with RKO-induced (colorectal cancer cells) tumors (132). In human ovarian and prostate tumor prexenografted mouse models, a synergistic cytotoxic effect was demonstrated using taxol and ␤-lapachone (60).

Antileishmania (hampsters) Lapachol and its derivatives can generate oxygen free radicals and through the induction of nitric oxide synthetase by IFN-␥ , NO. Together they are considered capable of killing intracellular amastigotes in murine macrophages, with NO playing the major role in killing Leishmania brasiliensis. However, when hampsters were given oral doses of lapachol of 300 mg/kg/day for 42 days the development of LVb-induced lesions was not prevented. The rationale for this lack of clinical success was considered due to leishmanicidal activity being lost in macrophages, the formation of inactive metabolites or the inability to achieve sufficient plasma levels of the drug (133).

Antimalarial (mice) Several benzo[a]phenazines synthesized from 1,2naphthoquinone, lapachol, ␤-lapachone containing polar (-Br,-I) and ionizable (-SO3H, -OH) groups were compared with the activities of lapachol and ␤-lapachone in the Plasmodium berghei mouse model. The best candidate was 3-sulfonic acid-␤-lapachone-derived phenazine which elicted 98% inhibition of parasitemia in long term treatment (7 doses) subcutaneously whereas the phenazine from 3-bromo- ␤-lapachone was inactive. Those compounds with antimalarial activities are considered as potential prototypes for use against chloroquine resistant strains (106).

Molluscicidal Activity In tropical South America schistosomiasis is caused by Schistosoma mansoni, with other species known elsewhere. This fluke is endemic to parts of Brazil, Suriname, and Venezuela but it is also prevalent in parts of the Caribbean, and tropical areas of Asia and Africa. Control of the disease is based upon eliminating freshwater snails such as Biomphalaria, which can serve as its intermediate host and primary reservoir. They release cercariae into lakes, ponds, and other water sources, which can penetrate the skin of those wading or swimming. In humans they become shistosomulae which develop into adult worms that reside in the mesenteric or rectal veins shedding eggs, which circulate in the liver and are shed in the stools. In water these eggs develop into miracidia that infect the snails (134). In various parts of the world wherever schistomosiasis is prevalent snail-erradication programs for waterways can vary and are dependant upon the availability and costs of suitable methods. For this reason, much research has gone into identifying local plant species and their compounds that possess molluscicial activity (135), which can be succesfully and practically applied, for example, the Ethiopian Gopo berry (Phytolacca dodecandra) (136).

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Species within the Bignoniaceae including Tabebuia aurea have been investigated for this purpose (137). Earlier research with lapachol (124,138) and a number of its derivatives (138,139) indicated that these compounds exhibited important molluscicidal activities against Biomphalaria glabrata, with potassium salts of lapachol and isolapachol eliciting significant molluscicidal activity against the adult snail (LC90 < 7 ppm) and snail egg masses (LC90 < 3 ppm) (140). An additional study with a number of 2-hydroxy-3-alkyl-1,4-naphthoquinones indicated that those easily reduced were the most active whereas lowto-moderate activity was elicited by new amino derivatives of lapachol and another which was partially hydrogenated (141). The basis for this lethal activity is because lapachol is bioactivated by P450 reductase into reactive species through redox cycling and the eventual generation of superoxide anion radicals, which cause DNA scissions to occur (140).

Antischistosomiasis Activity (mice and rats) South American schistosomiasis is asymptomatic at first, except for a rash or itchy skin, and may not present with constitutional symptoms of fever, chills, cough, and muscle aches until one to two months after infection. Children who are repeatedly infected may be subclinically symptomatic with symptoms such as mild anemia, malnutrition, and learning difficulties. As the disease progresses into its chronic state it can cause granulomatous reactions and fibrosis, resulting in colonic polyposis with diarrhea, and portal and pulmonary hypertension. Only rarely is the brain and spinal cord involved causing seizures, paralysis, and spinal cord inflammation. Mortality rates are generally low (142). Preclinical studies with lapachol and other napthoqinones address preventing cercariae infection. Oral administration of lapachol protected mice from topical infection with Schistosoma mansoni cercariae and also significantly reduced the trematode burden in infected mice (143). Topical application of other naphthoquinones also prevented cercariae penetration with highest activities found when lapachol and its 0-alkyl and 9-acetyl derivatives or ␤-lapachone were used (144).

Cancer Chemopreventive Agents (mice) 1,4-Furanonapthoquinones with an OH group on the dihydrofuran-ring, such as avicequinone-A and avicenolA, showed the highest bioreactivities in the EBV early activation model and in a chemoprovocative tumor-inducing mouse model (145). ␤-lapachone and several of its derivatives can prolong the lives of mice with Raucher leukemia (146). Several derivatives and analogs of ␤-lapachone are cited in a number of patents with a variety of substituent’s at the 3-position as well as in place of the methyl groups attached at the 2-position (147–149), with substituents at the 2-, 3-, and 4-positions and 2-, 3-, 8-, and/or 9-positions (146), and sustituents at the 2-, 8-, and 9-positions (150). Another publication describes sulfur-containing hetero-rings in the “␣” and “␤” positions of lapachone (151).

Antiulcerogenic Activity (rats) Studies using an ethnolic extract of the bark of T. impetiginosa (T. avellanedae) in in rats with chemically induced

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acute and chronic ulcerations indicate that its gastroprotective capacity involves the maintenance of protective factors such as mucus and prostaglandin, as well as reducing total gastric acidity (152).

Toxicity in vivo (rodents, dogs, monkeys) Lapachol from the bark of T. ochracea was tested during rat fetogenesis from days 17 to 20 of pregnancy. While lapachol was not toxic to rat mothers, it was fetotoxic, leading to fetal intrauterine growth retardation of pups compared to untreated controls (P < 0.01). There was also significant weight reduction (P < 0.01) in lungs, livers, and kidneys of treated pups. Putative effects in women cannot be ignored (153,154). In oral toxicity tests using lapachol, rodents, dogs, and monkeys developed moderate-to-severe anemia during the first two weeks of treatment, but recovery was evident at four weeks of treatment. Monkeys receiving up to 0.25 g/kg/day completed the treatment and recovery periods, with those receiving higher doses developing infrequent emesis, anorexia, pallor of mucous membranes, and periods of diarrhea. Dogs and rats were able to tolerate much larger doses of lapachol than either monkeys or mice. A gender difference was also evident in mice: males tolerated less lapachol than females (155). The napthoquinones, ␤-lapachone, 3-hydroxy␤- N lapachone, and ␣-lapachone may have promise as topical antibiotics used to treat methicillin-resistant Staphylococcus infections since they do not elicit dermal toxicity on the skin of healthy rabbits (156).

CLINICAL TRIALS Anticancer Efficacy Promising anticancer reports for the use of pau d’arco in humans were widely disseminated in South America in the popular press and the medical literature in the 1960s (29,30). Even as late as 1993, analysis of data from trials evaluating whole plant extracts of pau d’arco versus their bioreactive compounds indicated that potency is diminished as plant extracts are fractionated into their component parts. These studies implied that the totality of bioreactive compounds including lapachol and other napthoquinones in crude extracts act in a synergistic manner to elicit positive clinical effects (42). In Brazil pau d’arco extracts are used to treat cancers and leukemias. While not meeting the rigorous standards of current clinical evaluations, claims of efficacy have been made in a number of Brazilian and Argentinan studies (5). These types of assertions likely contribute to its use as an herbal remedy for every kind of medical complaint including cancer. Anecdotal reports of the efficacy of crude extracts continue to be lauded in the lay literature and internet fueling their value for these purposes (15). Lapachol was the first component of pau d’arco to be evaluated in a phase I clinical trial by the U.S. National Cancer Institutes (NCI). Patients with nonleukemic tumors or chronic myelocytic leukemia were given lapachol at dose ranges of 250 to 3750 mg daily for five days and up to 3000 mg daily for 21 days. The trial was terminated because at critical plasma levels of 30 mg/mL, toxicity occurred causing nausea, vomiting, and reversible prolongation of prothrombin times due to its anti-vitamin K action.

According to NCI this study was disappointing and did not fulfill the criteria for further development (157). Because lapachol targets vitamin K-dependent reactions, such as the reversible activation of ligand for the Axl receptor tyrosine kinase, this compound, like warfarin, may have value in cases where Axl is overexpressed. Consequently, lapachol use may be possible for the treatment of small cell carcinoma, metastatic colon cancer, and adenocarcinomas of the colon (158). ␤-lapachone is considered a broad spectrum anticancer agent, which has promise as an anticancer agent, alone or in combination with radiation therapy. However, before these applications can be realized there is a need to overcome the low solubility of ␤-lapachone in common solvent systems needed for topical and parenteral administration such as enhancing its solubility in aqueous solution by providing either water- or oil-based solubilizing carrier molecules (159,160). Under development by Arquile Inc. and Roche is the fully synthetic ␤-lapachone called ARQ-501, which activates E2F1-mediated checkpoint pathways leading to selective apoptosis of cancer cells. A number of phase trials have already been completed. These include two Phase 1 trials in subjects with cancer (NCT0075933), in combination with Docetaxel in patients with cancer (NCT00099190), and a Phase 1/11 trial involving an extension study for patients previously treated with ARQ 501 (NCT00622063). Also finished are three, Phase 11 trials with ARQ 501 in combination with Gemcitabine in subjects with pancreatic cancer (NCT00102700), with hydroxypropyl-␤ -cyclodextrin for safety and efficacy in adult patients with leiomyosarcoma (NCT00310518), and in patients with squamous cell carcinoma of the head and neck (NCT00358930) (161). An additional Phase 1 trial involving an exploratory biomarker study of ARQ 501 in patients with advanced solid tumors (NCT00524524) is ongoing and is no longer recruiting participants (162). Of relevance, studies on one of its metabolites (glycosylsulfate conjugate (m/z 241) with ARQ 501) found in the plasma of treated (nu/nu) mice, rats, and human subjects suggest that this is the first time glycosyl conjugates have been found in mammals (22). While none have been clinically evaluated, several derivatives show promise for the treatment of prostate cancer particularly ␤-lapachone (i.e., R and R1, both being hydrogen), allyl-␤-lapachone, particularly 3-allyl␤-lapachone (i.e. R being allyl and R1 being hydrogen), and 3-bromo-␤-lapachone (i.e. R being bromo and R1 being hydrogen). 3-allyl-␤-lapachone is considered less toxic (163,164). ␤-lapachone is also proposed for use with kinetin riboside and glucocorticosteroid to inhibit cyclin D in cancers such as multiple myeloma, non-Hodgkins lymphoma, breast cancer, and other cancers dependent on cylin D (165). Also, for future clinical assessment in the treatment of breast, non-small cell lung, pancreatic, colon, and prostate cancers are a number of napthoquinone prodrugs with the ability to convert beta-lapachone with cancers having elevated NAD(P)H:quinone oxidoreductase 1 levels (64).

Anticervicitis/Antivaginitis Twenty Brazilian patients suffering from cervicitis and cervicovaginitis caused by Trichomonas vaginalis or Candida

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albicans were evaluated for the effectiveness of daily changes of tampons soaked with extracts of T. impetiginosa heartwood. After 5 to 29 days, successful treatment was considered complete, with re-epithelialization of inflamed areas. No patient reported adverse side effects (166).

REGULATORY STATUS The availability of pau d’arco varies from country to country. In Germany its use is limited to some registered herbal medicines used as general stimulants but not for serious medications (M. Heinrich, written communication, 2010). It is not listed in either The Complete German Commission E Monographs (1998) or the Herbal Medicine Supplement (2000), which provides the standards for use of botanicals and phytomedicines in Germany (167). In the United Kingdom it is not registered or licensed, and in Canada, classified as a “new drug” its sale is prohibited throughout the country. In Brazil, lapachol is cited as being commercially available for antitumor therapy (168). There are no specific regulations for the sale of pau d’arco in the United States. It is “generally regarded as safe” (GRAS) by the Federal Drug Administration and is available in a variety of formulations defined as dietary supplements. It is probable that much of the material from South America is poorly identified, adulterated, or harvested incorrectly. Many shipments may represent other genera, other Tabebuia species, or mixtures of several plants. There is little guarantee of quality control at the source and thus a product labeled as pau d’arco bark may be incorrect (5,169).

CONSERVATION Since it became popular as a medicinal in the 1960s, many populations of Tabebuia species have been destroyed indiscriminately. Timber use is also impacting the availability of T. angustatus and T. heterophylla in the West Indies, T heptaphylla and T. rosea wherever native, and T. billbergii and T. chrysantha in Ecuador (1). A program to conserve these and other members of the Bignoniaceae is sorely needed throughout the Neotropics.

ACKNOWLEDGMENTS We appreciate the personal communications from Dr. ´ Lucia Lohmann, Brazilian researcher of the Bignoniaceae at the Missouri Botanical Garden, St. Lous, Missouri, and thanks to Gayle Engels of the American Botanical Council, Austin, Texas, and Natasha Y. Hall of the American Herbal Products Association, Silver Spring, Maryland for documents on pau d’arco. This review is an updated version of an original paper published in 2005 (170).

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Phosphorus John J. B. Anderson and Sanford C. Garner

used by the food industry to preserve moisture or color, as emulsifiers and sequestrants, or to enhance and stabilize frozen foods. Such processing of foods, now commonplace in the United Sates, adds significant amounts of phosphate to daily intakes—an estimated minimum of 200 to 300 mg/day. Approximately 125 phosphate additives on the generally accepted as safe (GRAS) list are commonly used; those with up-to-date toxicology information are listed in Table 1. Common foods that contain phosphate additives are soft drinks, processed cheese, luncheon meat, products with leavening agents (like waffles), frozen foods (like pizza with added flavorings), and fast food items. As more and more phosphate-containing additives enter the food supply, largely unnoticed, the effects of lower calcium to phosphorus ratios in the diet need to be considered as potentially negative to the retention of skeletal mass (see actions of phosphate). Many dietary supplements now contain phosphorus. One such supplement commonly used by athletes is creatine phosphate. This substance, found naturally in muscle fibers, can be used to generate adenosine triphosphate (ATP) and serves as a “quick energy” source. Creatine phosphate, promoted as a way to increase muscle strength during workouts, is commonly used by athletes and body builders. Health professionals do not promote the product, because research has failed to show any real beneficial effects. Many other “muscle-building” formulas are also high in phosphorus because they contain large amounts of animal protein. Complete nutritional supplements, nutritionally balanced in macronutrients and micronutrients and consumed predominately by older individuals, contain

COMMON AND SCIENTIFIC NAME Phosphorus is the name of the element (number 32 of the periodic table), but phosphorus does not exist in biological tissues or foods as such because of its chemical reactivity; rather it exists almost exclusively as phosphate anions. Most of these anions are inorganic, but some are derived from pre-existing organic molecules that contain phosphate groups (see general description). In the biological sciences, the term phosphate is used rather than phosphorus.

GENERAL DESCRIPTION The two major anionic forms are HPO4 2− (metaphosphate) and H2 PO4 − (orthophosphate), which are interconvertible by the addition or removal of a hydrogen ion. A third form, PO43− , exists but it is quite rare in biological tissues: it is the anion of phosphoric acid (H3 PO4 ). In human body fluids (pH 7.4), the usual ratio is 4 HPO4 2− ions to 1 H2 PO4 − ion. The structures of the two major biological phosphate anions and their relationship at equilibrium are as follows: 2− + H2 PO− 4 < - - - - > HPO4 + H [equilibrium favoring HPO2− 4 ]

Phosphorus (P), primarily in the form of phosphates, is found in three major dietary sources: (i) foods containing natural phosphates; (ii) foods containing phosphate additives; and (iii) supplements containing phosphates. Although some amount of phosphorus is present in all foods, foods high in protein are typically also high in phosphorus. Milk, eggs, meat, poultry, and fish contain the highest amounts of phosphorus, whereas fruits and vegetables have relatively less amounts. Sixty percent (60%) of the daily phosphorus intake of North Americans comes from milk and meat against only 10% from fruits and fruit juices (1). Legumes, cereals, and grains are also good sources of phosphorus and contribute almost 20% of the dietary intake. Phosphorus consumption from foodstuffs is increasing, despite an overall decline in the consumption of red meat and milk, because of the steadily increasing consumption of cheese (especially processed types), poultry, and fish (2). Phosphate additives, the most rapidly growing source of phosphorus in the U.S. diet, may contribute to as much as 30% of overall phosphorus intake (3). This source of the mineral remains largely unnoticed by consumers because the phosphate content of a food product is not required on the label. Many salts containing phosphorus are

Table 1 Commonly Used Phosphate-containing Food Additives Ammonium phosphate Monoglyceride/diglyceride derivatives Sodium aluminum pyrophosphate Calcium phosphate Phosphoric acid Sodium acid pyrophosphate Dipotassium phosphate Potassium phosphate Sodium phosphate Ferric phosphate Potassium pyrophosphate Sodium tripolyphosphate Magnesium phosphate Potassium tripolyphosphate Modified food starches, distarch phosphate

626

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627

PHOSPHATE HOMEOSTASIS

Table 2 Dietary Reference Intake (DRI) for Phosphorus (mg/day) Life stage group

Males

Females

9–18 yr 19–>70 yr

1250 700

1250 700

Source: From Ref. 4.

calcium and phosphorus at a ratio of approximately 1:1. Infant formulas have a ratio of greater than 1:1. These products are not likely to contribute to excessive phosphorus intakes. The recommended intakes of phosphorus for U.S. and Canadian citizens have recently been revised (4). The Dietary Reference Intakes of phosphorus for men and women older than 19 years is 700 mg/day (Table 2). Phosphorus is usually consumed with the protein fraction of food. Generally, every gram of protein consumed is accompanied by 15 mg of phosphorus. The rate of intestinal phosphorus absorption, 50% to 70% on average, is high in relation to the rates of other minerals.

OVER-THE-COUNTER PHOSPHATE SUPPLEMENTS The Physicians’ Desk Reference (5) includes a few entries on phosphate salts, but these are typically combined with other nutrients, particularly calcium. Common overthe-counter supplements generally contain little or no phosphate.

INTESTINAL ABSORPTION OF PHOSPHORUS AS PHOSPHATE IONS The intestinal absorption of phosphorus as an inorganic phosphate (Pi ) is highly efficient, particularly in infants where up to 80% to 90% of Pi may be absorbed. The absorption efficiency in adults is lower but may still be in the range of 50% to 60% or even higher. In contrast, the intestinal absorption of calcium is usually considered to be between 25% and 30% in adults. The absorption of organic phosphorus in phospholipids and other molecules may occur, but phosphate groups are typically split out in the gut lumen or on cell surfaces by phosphatases and phospholipases, which are either secreted by the pancreas or exist on the surface of intestinal absorbing cells.

BLOOD CONCENTRATIONS OF PHOSPHATE IONS Phosphorus circulates in the blood both as a component of organic molecules, primarily phospholipids, and as inorganic phosphate. Inorganic phosphate can exist in several different ionization states, including PO4 3− , HPO4 2− , and H2 PO4− . Because of the relative solubility of the different forms of Pi, approximately 44% of total Pi is in the form of free H2 PO4 − , whereas 10% is present as free HPO4 2− . The remaining 46% is bound to either serum proteins (12%) or complexed with cations (34%), primarily calcium.

The serum Pi concentration is regulated by most of the same processes that regulate serum ionized calcium. However, the homeostasis of serum Pi is not as rigorous as that of calcium. The hormonal regulation of serum Pi primarily involves parathyroid hormone (PTH), FGF-23 (a phosphatonin, i.e., a hormone that acts to increase renal excretion), and 1,25-dihydroxyvitamin D, but many other hormones, including calcitonin, insulin, glucagons, growth hormone, estrogens, adrenaline, and adrenal corticosteroids, also may affect Pi homeostasis. A meal rich in phosphate or a phosphate supplement, results in increases of serum PTH and FGF-23 that reduce renal phosphate ion reabsorption. Although a direct feedback mechanism has been proposed for Pi concentration in the regulation of 1,25-dihydroxyvitamin D synthesis, most of the regulatory feedback for PTH is believed to involve the concentration of serum ionized calcium. The use of calcium ion concentration in regulation is understandable given the well-known tendency of serum phosphate and ionized calcium to move in opposite directions. Because phosphate and calcium ions readily form a complex with each other, an increase in phosphate will decrease the concentration of ionized calcium, while a decrease in the phosphate concentration will allow more calcium to circulate in its free or ionized form. Thus, regulation of Pi in serum is mediated through changes in ionized calcium resulting from renal hormonal action. Phosphate homeostatic mechanisms primarily involve renal regulation. If the kidneys decline in function, as in chronic renal failure, phosphate cannot be efficiently excreted and the serum phosphate ion concentration increases, perhaps even to levels twice as high as the serum calcium concentration. Dysregulation of calcium may have several deleterious effects, including arterial and heart valve calcification.

URINARY PHOSPHATE EXCRETION A major regulatory mechanism for control of the serum Pi concentration is renal excretion of Pi . Free Pi from serum passes freely through the glomeruli as part of the urinary filtrate. The reabsorption process, which is under the control of PTH, can return most of the filtered Pi to the serum. PTH reduces the efficiency of the reabsorption and increases the excretion of Pi , thus lowering the circulating serum Pi concentration even when the efflux of Pi from bone is increased. Because renal excretion of Pi is the major regulatory mechanism to control the concentration of this ion, a decrease in glomerular filtration rate during the development of renal failure results in a characteristic increase in serum Pi concentration. As the increased serum Pi complexes more ionized calcium in the serum, the resulting hypocalcemia stimulates secretion of PTH, contributing to an increased movement of calcium and phosphate from bone into serum. The increased load of Pi acts to worsen the hyperphosphatemia. Eventually the chronic elevation of PTH can cause a high-bone-turnover lesion known as osteitis fibrosa. The formation of such bone lesions and others

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(a) Purine or Pyrimidine Base

Purine or Pyrimidine Base N

Purine or Pyrimidine Base N

N

CH2 CH2

CH2 O

O

5′

P

O O O

P

P

O

O O

H

O

H

O

H

O′

O′

O′ O

3′ O

O O O

(b) Purine or Pyrimidine Base

Purine or Pyrimidine Base N

Purine or Pyrimidine Base N

N

CH2 CH2 CH2

O

O

5′ OH

P

O′

O′ O

O′ O

3′

O

O O OH

O

P

P

O

O OH

O

O

O O O

Figure 1 Nucleic acids. Phosphate groups (in the shaded boxes) serve to link the deoxyribose and ribose sugar molecules in (A) deoxyribonucleic acid (DNA) and (B) ribonucleic acid (RNA).

resulting from chronic renal failure is described as renal osteodystrophy. An important aspect of treating chronic renal failure is the control of the elevated serum Pi . The usual approaches to this control are decreased dietary Pi intake and treatment with phosphate binders, such as calcium carbonate. Aluminum-based phosphate binders, such as aluminum hydroxide, were often used in the past, but the toxic effects of aluminum on bone and the nervous system have greatly decreased their use. Newer phosphate binders, such as sevelamer, are more effective and less toxic.

ACTIONS OF PHOSPHATES The widespread biological use of phosphate groups makes these anions essential for both organic and inorganic components within cells and in extracellular structural tissues such as bones and teeth. About 600 g (19.4 mol) of phosphorus is present in the adult human body: 85% of which is in the skeleton, 14% in the soft tissues, and 1% in the extracellular fluids, intracellular structures, and cell membranes. The small amount of phosphate ions in extra- and intracellular fluids serves as the compartment to which dietary phosphorus is first added and from which the kidneys clear phosphate ions. Excretion of phosphate ions permits additional hydrogen ions to be secreted by renal tubules, which acidifies the urine. Phosphate ions that are

resorbed from bone also enter this fluid compartment. The concentration of phosphorus in adult plasma ranges from 2.5 to 4.5 mg/dL (0.81–1.45 mmol/L), but this concentration gradually declines with age (6). Phosphate anions participate in numerous cellular reactions and physiological processes and they are key components of essential molecules such as the phospholipids, ATP, and nucleic acids. Phosphate ions interact with calcium ions in the body and thereby influence the secretion of PTH. Excessive absorption of dietary phosphate lowers the serum calcium ion concentration, which in turn signals the parathyroid glands to increase PTH secretion. If PTH secretion remains elevated continuously because of a low dietary calcium-to-phosphorus ratio, bone resorption may also be continuously upregulated, which may lead, over a period of months to years, to a significant reduction in bone mass and density. This potential scenario of a low calcium–high phosphate ratio has only been observed experimentally for short periods, up to as long as a month, with continuous elevation of PTH in young healthy adult women (7). High phosphate intakes contribute to acid generation and to an acidic urine. Such an increase in dietary acid load may require buffering by bone (8), which may result in the loss of bone mass and density. Phosphate ions are essential to life for both their cellular roles and their extracellular uses such as the mineralization of bones and teeth. Excessive amounts of

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Phosphorus

dietary phosphorus plus a low calcium intake may have adverse effects on skeletal retention of mineral and, therefore, strength.

(a)

NH2

N

PHOSPHATE IN BONE MINERALIZATION Phosphate ions move in and out of the bone fluid compartment from the extracellular fluid, including blood, in large amounts over a 24-hour period. These bi-directional fluxes relate to bone formation and resorption. In the growth phases of life, especially in children, a net gain of phosphate occurs as bone mass increases; in late life when resorption predominates over formation, the phosphate flux out of bone is greater. Both phosphate ions and calcium ions are required for the mineralization of matrix, and the skeletal ratio between the two remains constant throughout life.

N −

O−

O −O

P

O

O

P

The phosphorylation of specific intracellular protein molecules plays a large role in the cellular regulation of many functions, including transcription, translation, and cell signalling. The amino acids commonly phosphorylated by phosphorylating enzymes (protein kinases) are serine and threonine because of their side-chain hydroxyl groups. Tyrosine kinases are especially important in the transfer of phosphate groups from ATP to the regulatory proteins. The same amino acids may have phosphate groups removed by phosphatase enzymes. Thus, the on and off states involving phosphates are critical for many cellular regulatory activities. Besides proteins, a number of other molecules incorporate phosphate groups in their structures. These molecules include nucleotides and nucleic acids (DNA and RNA), ATP, phospholipids, creatine phosphate, and others. A few of these molecules are illustrated in Fig. 1–3.

INDICATIONS AND USAGE Limited therapeutic uses of phosphates exist. Treatment with phosphate salts is not recommended except for a few clinical situations. Premature babies or failure-to-thrive infants who are deficient in phosphorus, as measured by serum inorganic phosphate, will need phosphate salts to survive. [A single plasma Pi measurement of less than 6.0 mg/dL would require a confirmatory measurement to establish deficiency (the acceptable lower limit for newborns and infants within six months of age is 7.0 mg/dL).] Management of any type of adult phosphate depletion, e.g., abuse of aluminum-containing antacids or vitamin D-resistant hypophosphatemic rickets or osteomalacia, would also require oral phosphate supplementation or possibly intravenous therapy (9). The same may be stated for a patient who is hypercalcemic; phosphate salt administration and plasma calcium concentrations need to be carefully monitored.

N

O− O

P

O

O

OCH2

O

OH

HO (b)

PHOSPHORYLATION REACTIONS INVOLVED IN CELL REGULATION

N

(c) −

−O

P

H N

O H3C

O−

HOOC

O

C C

NH

H2C

O

P

O−

O

N CH2 COOH

Figure 2 Energy-storing molecules. Phosphate groups (in the shaded boxes) provide the high-energy bonds in (A) adenosine triphosphate (ATP), (B) creatine phosphate, and (C) phosphoenol pyruvate, which are used to store energy in a bioavailable form.

Although phosphate deficiency remains rare in the United States, it may be present in approximately 5% of the elderly (10) who may be truly undernourished with respect to protein, energy, and most micronutrients. The need for additional phosphates is complicated by the needs for practically all macronutrients and micronutrients, so that these individuals should be provided increased amounts of nutrient-rich foods before considering phosphate supplementation, much as undernourished prisoners of war have been rehabilitated in the past. Elderly may also be losing phosphate ions because of a renal “leakage.” A postulated scenario of low phosphate dietary status leading to renal phosphate leakage and bone loss is illustrated in Fig. 4.

CONTRAINDICATIONS Phosphate supplementation as phosphate salts is not typically recommended because of concern about the calcium to phosphorus ratio of the diet and the potential increase in PTH. With the exception of appropriate medical use of supplementary phosphate, this statement applies across the life cycle.

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Anderson and Garner

(a)

Insufficient Diet: Low Energy Intake and Low Phosphorus Intake

O C

HO

Increase in Gluconeogenesis via Increase in Glucagon and Increase in Ketogenesis and Acidosis

+ N

O

OCH2

NH2

NH2

OH

N

N

N O− P

Bone Loss

N

O− O

P

Figure 4 The postulated sequence of events leading to the renal leakage of phosphate ions. OCH2

O

O

O

HO

O−

(b) O

Increase in Renal Phosphate Excretion and Hypophosphatemia

H

O

P

O−

C O

O− CH2

O

P

level without taking supplements, such as creatine phosphate. The total daily intakes for adult males from foods, both naturally occurring phosphates and phosphate additives, approximate 1300 to 1800 mg in males and 900 to 1300 mg in females (Table 3). Therefore, adult intakes without supplements should not come anywhere near the upper limit of 4000 mg.

O−

O N

Figure 3 Enzyme cofactors. The enzyme cofactors (A) nicotinamide adenine dinucleotide phosphate (NADP) and (B) pyridoxal phosphate each contain one or more phosphate groups (in the shaded boxes).

PRECAUTIONS AND ADVERSE REACTIONS Subjects supplementing with creatine phosphate, used as an ergogenic aid by athletes, may consume excessive amounts of phosphorus in a day over a considerable time period. Reports of adverse reactions from high phosphorus intakes have been very few, but the FDA has been concerned about potential deleterious actions of creatine phosphate. So, creatine phosphate is currently on the “watch” list for potential adverse effects.

OVER-DOSAGE The upper limit for phosphorus is 4 g (4000 mg) per day for adults up to 70 years, but it is very difficult to achieve this

COMPENDIAL/REGULATORY STATUS Phosphorus, as phosphate salts, is on the GRAS list of FDA additives. Because of the longstanding safe use of phosphate additives, they appropriately belong on this list, but concern about excessively low calcium-to-phosphorus intake ratios makes it desirable for the FDA and other federal agencies to review the status of phosphate additive use vis-a-vis low calcium intakes. If the calcium:phosphate ratio goes below 1:4 (0.25) on a chronic dietary pattern, a chronic increase in PTH will certainly follow and contribute to an increase in bone resorption and the loss of bone mass and density.

CONCLUSIONS In general, phosphate supplements are not needed because the diet provides sufficient amounts of phosphate anions; to the contrary, healthy individuals may be put at risk by taking phosphate supplements because of the downward regression of the calcium:phosphate ratio. When the ratio of a typical dietary pattern is reduced to 1:4 (0.25), the excessive parathyroid secretion may lead to sufficient bone loss and may compromise skeletal integrity.

Phosphorus

1994 USDA CSFII Data∗

2005–2006 USDA Data

bone loss. So, phosphate supplementation, while rare, should only result from a clinical diagnosis of established deficiency.

Calcium (mg) Phosphorus (mg) Ca:P Ratio

Calcium (mg) Phosphorus (mg) Ca:P Ratio

REFERENCES

457 322 1.42:1 703 612 1.15:1 766 926 0.83:1 808 1059 0.76:1

––– ––– 947 1034 0.92:1 961 1145 0.84:1

980 1359 0.72:1 1094 1582 0.69:1 954 1613 0.59:1 857 1484 0.58:1 708 1274 0.55:1 702 1176 0.60:1

1023 1321 0.77:1 1256 1681 0.75:1 1141 1656 0.69:1 1145 1727 0.66:1 991 1492 0.66:1 878 1270 0.69:1

889 1178 0.75:1 713 1097 0.65:1 612 1005 0.61:1 606 990 0.61:1 571 966 0.59:1 517 859 0.60:1 1154 1581 0.73:1

942 1176 0.80:1 843 1067 0.79:1 851 1120 0.76:1 886 1197 0.74:1 795 1106 0.72:1 759 985 0.77:1 1237 1484 0.83:1

Table 3 Mean Calcium (Ca) and Phosphorus (P) Intakes, with Means of Calcium:Phosphorus Ratio

Life stage Males and Females 0–6 months 7–12 months 1–3 years 4–8 years Males 9–13 years 14–18 years 19–30 years 31–50 years 51–70 years >70 years Females 9–13 years 14–18 years 19–30 years 31–50 years 51–70 years >70 years Pregnancy ∗ Source:

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From Ref. 10.

If phosphate supplements are deemed by a physician to be essential to correct for phosphate deficiency, such supplements are truly indicated. Such supplementation is clearly rare and a physician’s diagnosis of phosphate deficiency must be documented. Selfsupplementation by consumers may place them at risk because of the potential for chronic elevation of PTH and

1. Anderson JJB, Garner SC, eds. Calcium and Phosphorus in Health and Disease. Boca Raton, FL: CRC Press, 1996. 2. Anderson JJB, Sell ML, Garner SC, et al. Phosphorus. In: Russell RM, Bowman BR, eds. Present Knowledge in Nutrition. 8th ed. Washington, DC: ILSI Press, 2002. 3. Calvo MS, Park YK. Changing phosphorus content of the U.S. Diet: Potential for adverse effects on bone. J Nutr 126: 1168S–1180S. 4. Institute of Medicine (IOM) Committee on Dietary Reference Intakes, Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, D.C.: National Academy Press, 1997. 5. Physician’s Desk Reference. 63rd ed. Oradell, NJ: Medical Economics, 2009. 6. Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, PA: Saunders, 1999. 7. Calvo MS, Kumar R, Heath H, III. Persistently elevated parathyroid hormone secretion and action in young woman after four weeks of ingesting high phosphorus, low calcium diets. J Clin Endocrinol Metab 1990; 70:1334–1340. 8. Barzel US. The skeleton as an ion exchange system: Implications for the role of acid-base imbalance in the genesis of osteoporosis. J Bone Miner Res 1995; 10:1431–1434. 9. Hardman JG, Limbird LE, eds. Goodman & Gilman’s The Pharmacologic Basis of Therapeutics. 9th ed. New York, NY: McGraw-Hill, 1996. 10. Moshfegh A, Goldman J, Ahuja J, et al. What We Eat in America, NHANES 2005-2006: Usual Nutrient Intakes from Food and Water Compared to 1997 Dietary Reference Intakes for Vitamin D, Calcium, Phosphorus, and Magnesium. Washington, D.C.: U.S. Department of Agriculture, Agricultural Research Services, 2009.

Polyphenols Overview Navindra P. Seeram

According to chemical nomenclature, a “phenolic” compound contains at least one aromatic ring bearing a hydroxyl (-OH) group and thus, a “polyphenol” contains multiple aromatic rings and hydroxyl groups (1–3). Phenolics/polyphenols are secondary metabolites (or natural products) of widespread occurrence in the plant kingdom. Over 8000 different polyphenolic structures have been reported (1–3). Most are derived from intermediates of the shikimic acid pathway which gives rise to a large number of aromatic compounds related to phenylalanine and tyrosine (4). Several types are of interest for their potential biological effects ranging from the lower molecular weight phenolic acids to larger complex polymers such as the condensed and hydrolyzable tannins (5–7). Polyphenols can be classified by their number and arrangement of carbon atoms and many are found naturally in conjugated forms (1–3). Although the most common conjugates are sugars/glycosides, it is not unusual to encounter polyphenols that are acylated with aliphatic and organic acids. In addition, polyphenol monomers can conjugate with themselves to form oligomers and polymers as well as undergo chemical and enzymatic changes when they are extracted and/or processed. Therefore, the possible combinations of polyphenolic structures are limitless which explains their vast structural diversity. Because of this wide diversity, and for simplification of discussion, polyphenols are grouped in this overview into two chemical subclasses, namely flavonoid type and nonflavonoid type. Figure 1 shows representative chemical structures of some of these polyphenols and they are individually discussed below. Polyphenols discussed in separate chapters elsewhere in this encyclopedia include “Aloe Vera,” “Cascara Sagrada,” “Cranberry,” “Echinacea Species,” “Elderberry,” “Feverfew,” “French Maritime Pine,” “Ginger,” “Ginkgo,” “Grape Seed Extract,” “Green Tea Polyphenols,” “Hawthorn,” “Isoflavones,” “Milk Thistle,” “Pau d’Arco,” “Proanthocyanidins,” “Quercetin,” “Red Clover,” “St. John’s “Wort,” and “Valerian.”

skeleton starts from the oxygen atom, proceeds to the A ring, and is followed by the B ring. The basic flavonoid skeleton, as described, can have a wide variety of substituents at any number of different positions and many exist naturally in healthy plant tissues as glycosides. It is common to find hydroxyl groups at the 5, 7, and 4 positions. Whereas substituents such as hydroxyls and glycosides cause flavonoids to be more water-soluble than their corresponding aglycones, others, for example, methoxyls (OCH3; OMe), impart lipophilic properties. On the basis of several types of chemical modifications of the central C ring, including the presence and/or absence of carbonyl (C=O), hydroxyl, and unsaturation (carbon–carbon double bonds; C=C), flavonoids can be further categorized into: flavonols (2), flavones (3), isoflavones (4), flavanones (5), flavanols (also, flavan-3-ols or catechins; 6), and anthocyanidins (7). These are the major polyphenol constituents in human diet; ubiquitous in fruits, berries, vegetables, herbs, spices, and many other plant-derived products; and beverages including cocoa, chocolate, cider, coffee, fruit juices, red wine, and tea. A short description of each of these flavonoid subgroups follows. The first, flavonols, is the most extensively distributed and abundant of dietary flavonoids. They occur with immense structural variations and are commonly substituted with hydroxyl and carbonyl groups at the 3and 4-positions, respectively. Further, the 3-position is often conjugated with O-glycosides although, substitutions are common at other positions including the 5, 7, 3 , 4 , and 5 -carbons. As can be imagined, this gives rise to tremendous structural diversity depending on what substituents are present and the particular substitution pattern. Despite this inherent structural diversity, several common flavonol aglycones are known as quercetin (most ubiquitous), kaempferol, and myricetin. Although the number of flavonol aglycones may be limited, the number of possible conjugates is enormous. For example, over two hundred structural variations of sugar conjugates of quercetin alone have been reported. The second subgroup, the flavones, may be regarded as flavonols that lack hydroxyl substitution at position 3. These polyphenols also have a large variation in substitution pattern including hydroxylations, methoxylations, and O- and C- alkylations and glycosylations. It is also common to find glycosylation at position 7. Well-known flavones include apigenin and luteolin, present in parsley and celery, and the polymethoxylated derivatives, tangeretin and nobiletin, common to citrus. As their name suggests, the third subgroup, isoflavones, may be regarded as isomers of flavones

FLAVONOID-TYPE POLYPHENOLS As a subclass, flavonoids constitute the vast majority of polyphenols (1,3–7). Their structure is based on fifteen carbons consisting of two aromatic (C6 ) rings connected by a three-carbon (C3 ) bridge (1). Thus, flavonoids are commonly referred to as having a C6 –C3 –C6 (= 6-carbon ring or A ring; 3-carbon ring or C ring; 6-carbon ring or B ring) skeleton. The central C3 ring in the majority of flavonoids is an oxygen heterocycle. Numbering of the fifteen carbon 632

Polyphenols Overview

633

3' 1

8

A

5'

1'

C

10

5

B

O

4

Flavonoid skeleton (1) O O

O O

OH O

O

Flavonol (2)

Flavone (3)

O

O

O

O

Isoflavone (4)

O+ OH

OH

Flavanol or Catechin (6)

Flavanone (5) R1

Anthocyanidin (7)

COOH OH

HO

O

R2

8 3

OH

OH

OH

n

Stilbenoid (11)

Phenolic acid (9)

R1= R2 = H, Propelargonidins R1= H, R2= OH, Procyanidins R1, R 2 = OH, Prodelphinidins Proanthocyanidins (8)

HO

CH2OH

HO

CH2OH

OH

Lignan (12)

HO OH HO HO HO HO

O C O C O O O OH HO HO

O C O HO C HO O O O HOHO O O O O HO C O OH HO O OH OH OH OH OH

OH O O O

OH O

C

OH

O OH OH OH

OH OH OH

Hydrolyzable Tannin (Ellagitannin;10)

Sanguiin H-6

wherein the B ring is located at the 3-position instead of at the 2-position, as observed for the majority of the other flavonoids. The occurrence of isoflavones is restricted to leguminous (Fabaceae) plants and soya (Glycine max) is a recognized source, as is the Japanese Kudzu plant (Pueraria lobata). Depending on the absence or presence of a hydroxyl group at the 5-position, two common isoflavone aglycones encountered are daidzein and genistein, respectively. Glucosylation at the 7-position of these aglycones forms daidzein and genistein, respectively. The fourth subgroup, flavanones, differs from the majority of other flavonoids in that while ring B is similarly attached to position 2 of the C ring (excluding isoflavones), it is in an ␣-orientation thus forming a chiral center. Among

Figure 1 Examples of chemical structures of some polyphenols.

common plant foods, flavanones and their glycosides are predominantly found in citrus fruits and include hesperidin and naringin. The fifth subgroup, flavanols (also known as flavan3-ols or catechins), are the only flavonoid subclass which are not found naturally in glycosylated forms. They lack unsaturation at C2 –C3 , and immense structural diversity results because of this nonplanarity, chirality, and stereoisomerism. In fact, flavanols are arguably the most structurally complex of the flavonoid subclasses. They may be found occurring naturally as simple monomers such as the isomers, (+)-catechin and (−)-epicatechin, hydroxylated to form gallocatechins or esterified with gallic acid to form larger polyphenols such as epigallocatechin

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gallate (all found in green tea). Importantly, flavanol monomers may condense and link to form oligomeric and polymeric proanthocyanidins (PACs) (8), which are also known as condensed tannins. Here, it should be noted that although PACs are tannins, they are grouped within the flavonoid type of polyphenols. This separates PACs from the other class of tannins, i.e., hydrolyzable tannins, which are grouped within the nonflavonoid type of polyphenols (discussed later). As previously mentioned, the inherent chirality within flavanol monomer units of a PAC structure leads to enormous structural variability due to stereoisomerism/enantiomerism in these molecules. In addition, connections between different positions and/or types of linkages (for e.g., oxidative C–C couplings between C4 and C6 /C8 , ether linkages between C2- -O5 /O7 , etc.) further diversify PACs. Thus, PACs with ether linkages are referred to as A-type PACs (common in cranberries and cinnamon), while those with C–C linkages are B-type PACs (common in grape skin and seeds, blueberries, green tea, and cacao/chocolate). PACs may occur as polymers in excess of 50 monomer units reaching molecular weights exceeding 1000 Daltons. PACs may also be grouped into constituent units that are produced, after acid hydrolysis, on the basis of the nomenclature system established for anthocyanidins (discussed later). For example, those PACs which release cyanidin on acid hydrolysis are called procyanidins. These are the most common PACs in plants and are based exclusively on (epi)catechin units. Similarly, PACs based on (epi)afzelechin or (epi)gallocatechin units, are known as propelargonidins and prodelphinidins, respectively. Finally, flavanol monomers may be extensively transformed because of chemical and enzymic changes encountered during processing and extraction of plantderived foods. These are also referred to as derived polyphenols and examples include those complex polyphenolics found in aged wines and black tea (contains thearubigins and theaflavins). The last major subclass of flavonoids discussed here is the anthocyanidins, which can be regarded as aglycones of anthocyanins (i.e., glycosylated anthocyanidins). In fact, anthocyanins are the naturally occurring forms of anthocyanidins found in plant tissues. They are water-soluble pigments and are notorious for imparting the blue, red, and purple colors to berries, many other fruits, and vegetables. They are also the only flavonoid subclass that bears a positive charge (i.e., found as oxonium ions). Despite several hundred anthocyanin structures reported, most are based on the skeletons of six common anthocyanidins, namely cyanidin (most ubiquitous), delphinidin, pelargonidin, malvidin, petunidin, and peonidin. These anthocyanidins are distinguished by different numbers and substitutions of hydroxyl and/or methoxyl groups on the B ring. Although sugar conjugation at the 3-position is very common, glycosylation may also be observed at positions 5, 7, 3 , and 5 . In addition, further diversification in anthocyanin structure may be achieved by conjugation with phenolic and organic acids. Finally, apart from the aforementioned flavonoid/ bioflavonoid-type compounds, other biologically important polyphenols, albeit minor constituents in plant foods, include coumarins, chalcones and dihydrochalcones, aurones, napthoquinones, anthraquinones, and xanthones.

These polyphenols may also be present in botanical dietary supplements depending on the plant source.

NONFLAVONOID-TYPE POLYPHENOLS These compounds include phenolic acids (9), of which a notable one is gallic acid, the biosynthetic precursor of hydrolyzable tannins (10), as well as stilbenoids (11), and lignans (12). These are briefly described below. Phenolic acids have a C6 –C1 skeleton and may be regarded as derivatives of hydroxybenzoic acid. Common phenolic acids include gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid. They may also be esterified with organic acids, such as tartaric or quinic acids, to form chlorogenic acid derivatives. Hydrolyzable tannins, similar to the other class of tannins, namely condensed tannins or PACs (discussed earlier), constitute a large class of phenolic polymers. They are found as glucose esters of gallic acids, called gallotannins, or of hexahydroxydiphenic acid, which on hydrolysis form ellagic acid (hence referred to as ellagitannins). The latter group, as aptly named, hydrolyzes during processing and extraction of plant materials to release ellagic acid, a bioactive polyphenol. Stilbenes which are based on a C6 –C2 –C6 skeleton include the popular bioactive polyphenol, resveratrol (3,4 ,5-trihydroxystilbene) present in grape and red wine. Because of the carbon–carbon double bond connecting the two C6 rings, geometric isomerism that is cis and trans isomerism results. Further substitution by hydroxyl, methoxyl, and glycosides on the aromatic rings forms a number of stilbene derivatives. In addition, stilbene monomer units may oxidize to form dimers and polymers such as the viniferins. Lignans (distinct from lignins, the constituent of plant cell walls) are polyphenols formed from phenylpropanoid units linked by the central carbon atoms of their side chains. They are commonly found in plants as dimers such as secoisolariciresinol and matairesinol in flax and sesame. However, more complex lignan oligomers may also be present in plants.

REFERENCES 1. Gotham J. In: Harborne JB, ed. Plant Phenolics. London, UK: Academic Press, 1989:159–196. Methods in Plant Biochemistry; vol 1. 2. Harborne JB. In: Harborne JB, ed. Plant Phenolics. London, UK: Academic Press, 1989:1–28. Methods in Plant Biochemistry; vol 1. 3. Harborne JB, Mabry TJ. The Flavonoids. London, UK: Chapman and Hall, 1982. 4. Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 2001; 126:485–493. 5. Rice-Evans CA, Packer L. Flavonoids in Health and Disease. New York, USA: Marcel Dekker, 1997. 6. Harborne JB. The Flavonoids: Advances in Research Since 1986. London, UK: Chapman and Hall, 1993. 7. Andersen O, Markham KR. Flavonoids: Chemistry, Biochemistry and Applications. Boca Raton, Florida, USA: CRC Press, 2006.

Proanthocyanidins Catherine Kwik-Uribe, Rebecca Robbins, and Gary Beecher

The unique polyhydroxy phenolic nature of proanthocyanidins and the resulting electronic configuration allows relatively easy release of protons and hydrogen radicals, and as a result, they have been shown to have substantial antioxidant activity in vitro. Employing many antioxidant systems, investigators have shown that proanthocyanidins have high antioxidant and radical scavenging activity in vitro (3–7), usually greater than vitamins C and E, the “gold standards.” In addition, these unique chemical structures bind divalent cations, such as iron and copper, reducing the availability of these prooxidant metals. In doing this, proanthocyanidins may work indirectly to reduce the oxidative stress and damage caused by such redox active metals. The antioxidant activity is indirect because both iron and copper stimulate oxidative type reactions (Fenton reaction), but by effectively reducing the concentration of these cations (through binding), the extent of oxidative activity can be greatly reduced by the proanthocyanidins. Conversely, the role that proanthocyanidin–cation binding has on the bioavailability of such minerals as copper, iron, or aluminum is uncertain (2). The biological relevance of these antioxidant effects of proanthocyanidins will be discussed later in this chapter.

INTRODUCTION Name and General Description Proanthocyanidins, also named condensed tannins, are oligomers and polymers of monomeric flavonoids. More specifically, they are polyflavans: condensed molecules of those flavonoids with a saturated “C” ring (Fig. 1A). Fifteen subclasses of proanthocyanidins have been identified (1), however, only a few of these are prominent in foods and supplements that are generally consumed. The various subclasses are named on the basis of the conversion of the “interior” monomeric units (M) to the corresponding anthocyanidin during acid-catalyzed depolymerization; hence, this broad class of polymers is named proanthocyanidins. Examples include conversion of (epi)catechin monomers to cyanidin (procyanidins) and (epi)gallocatechin monomers to delephinidin (prodelphinidins). In these tannins, the monomeric units are primarily linked through single 4→6 or 4→8 carbon–carbon bonds (B linkages), or through 4→8 carbon–carbon and 2→7 ether bonds (A linkages) (Fig. 1). Other linkages have also been identified, but have been isolated from nonfood plants or they constitute minor compounds of foods such as cocoa (1). Proanthocyanidins range in size from dimers (Degree of Polymerization, DP = 2) through very large polymers (DP > 10) and are found in many plant-based foods and dietary supplements.

ANALYSIS Content analysis research for proanthocyanidins, although often not in the forefront, is directly connected with clinical and health investigations. To gather accurate data for food composition analysis and dietary intake levels, robust, reproducible quantitative methods are a necessity. The natural diversity of proanthocyanidins in foods and the inherent complexity of food matrices cause great difficulties in the accurate analysis of proanthocyanidins. In plant materials, these compounds are known to exist in free aglycone and conjugated forms with sugars and organic acids, as well as in a diverse array of oligomeric and polymeric forms (and typically also accompanied by flavanols, the monomeric building blocks of proanthocyanidins). Additionally, proanthocyanidins can occur in soluble, suspended, colloidal, or in covalent combinations within cell wall components. This structural diversity, solubility, and interaction with the matrix (plant or food matrix) impose a significant challenge in extraction, isolation, and analysis in foods and dietary supplements (8). Further difficulties arise because of the fact that proanthocyanidins are highly reactive and demonstrate general

CHEMISTRY Proanthocyanidins are secondary metabolites of plants, that is, they are not required for the structural or metabolic integrity of the organism. Proanthocyanidins, however, do serve important biological functions for plants. Specifically, proanthocyanidins help in the protection of plants from invasion and predation by microbes, fungi, and animals. One of the earliest biochemical properties of proanthocyanidins to be realized was their ability to bind to and denature proteins. Their use in the conversion of animal hides into leather, a process called tanning (protein denaturation), led to the generic name of tannins for these compounds. The interaction between proanthocyanidins and proline-/hydroxyproline-rich proteins and other polymers is very strong (2). As collagen, a prominent protein in animal skin and hides, is rich in proline and hydroxyproline, the interaction of proanthocyanidins with these hydroxyl-containing amino acids serves as the basis for the tanning effect of these natural plant constituents. 635

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(A)

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Figure 1 Representative linkages within proanthocyanidin molecules. (A) Monomeric representation with carbon 4 and 8 shown as potential linkages. Structure of (−)-epicatechin shown as example. Letters within rings identify individual phenolic or heterocyclic ring. n may equal 2 (dimer) to ∼50. (B) Example of B type (4→8) linkage. Specific compound is procyanidin B2 (dimer), epicatechin-(4␤→8)-epicatechin. (C) Example of B type (4→6) linkage. Specific compound is procyanidin B5 (dimer), epicatechin-(4␤→6)-epicatechin. (D) Example of A-type (2␤→O7; 4␤→8) linkage. Specific compound is procyanidin A2 (dimer), epicatechin-(2␤→O7; 4␤→8)-epicatechin.

instability. These molecules are subject to enzymatic oxidation, electrophilic, nucleophilic, and single electron– mediated chemical reactions. All these challenges mean that extraction and sample preparation for analysis are investigations in their own right and contribute to the very limited availability of commercially available reference standards necessary for quantification (9,10). Yet, even with these many challenges, there exist a number of techniques that have been developed for the quantification of proanthocyanidins, each providing different levels of content information (11 and references therein). Two broad categories of analysis include colorimetric and chromatographic methodologies. Common colorimetric assays for phenolics and proanthocyanidins are the Folin Ciocalteu (FC) method, Vanillin, 4-(dimethylamino)-cinnamaldehyde (DMAC) and hydrochloric acid–butanol assays. Generally speaking, colorimetric methods yield results that are empirical and not specific. For example, the FC reagent reacts broadly with the phenol functional group, rather than specifically with proanthocyanidins; therefore, the measurement is for all phenolics. The vanillin assay is more specific to flavanols, but still does not distinguish between monomeric, oligomeric, or polymeric compounds (12).

Although these colorimetric approaches are rapid and relatively easy, they provide gross, nonspecific estimates rather than the detailed information required for research focused on the composition of foodstuffs (including native foods, food ingredients, and supplements) and studies on health effects of specific chemical constituents. Colorimetric measurements do not physically separate compounds, that is, assays are performed on mixtures. The quantitative results are most often stated as catechin equivalents (sometimes gallic acid or epicatechin equivalents), making comparisons of content levels and data interpretations between studies confusing. A wide variety of chromatographic analytical procedures have been employed for the bulk measurement of proanthocyanidins (13). Both reversed-phase and normalphase chromatographic separations exist for proanthocyanidins in a variety of foods, food parts and dietary supplements. Individual dimers and trimers traditionally have been quantified with reversed-phase high performance liquid chromatography systems (HPLC) (14– 16). Reversed-phase separations, however cannot separate and measure the larger polymeric proanthocyanidins. Normal-phase chromatography can separate proanthocyanidins based on DP. Quantification of individual

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proanthocyanidin oligomers (DP ≤ 12) have been achieved using normal-phase HPLC techniques coupled with sophisticated detection instrumentation (17–19). Higher molecular weight proanthocyanidins (DP > 12) are not chromatographically resolved from one another and have been quantified together as a single chromatographic peak (19). More recently, an alternate HPLC method employing environmentally friendly normal-phase solvents and a diol stationary phase was developed and expanded to a multilaboratory assessment (20,21). Using a diol stationary phase with nonhalogenated mobile phases, flavanols and procyanidins in cocoa and chocolate can be quantified. In this method, polymeric materials greater than DP 10 were not measured. Often, to obtain higher levels of characterization of proanthocyanidins in complex materials, more than one type of technique or method is employed on the same sample (22). In a more comprehensive analysis of grape seed and pine bark extracts, Weber et al. (23) employed reversed-phase liquid chromatography with UV detection as a tool to fingerprint (profile) components, and also used Atmospheric Pressure Chemical Ionization liquid chromatography/mass spectrometry for further identification of monomers, dimers, and trimers. Gel permeation chromatography (GPC) has also been used to generate a molecular weight profile, along with gas chromatography/mass spectrometry for analysis of volatile components and, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identification of the polymeric species (23). Such comprehensive analyses are often conducted to capture detailed information about the content of a broad range of molecules in complex food extracts.

PHYSIOLOGY Foods or supplements containing high levels of proanthocyanidins are characteristically recognized as being astringent. This is due in part to the binding of these dietary constituents to proline-rich salivary proteins (2). As a result, formulation of palatable foods and supplements containing substantial levels of proanthocyanidins has been a challenge for food technologists and supplement formulation experts. Although binding of proanthocyanidins to digestive enzymes has been a concern in animal nutrition where dietary concentrations of these components may be as high as a few percent, human foods contain much lower levels and as a result, interference with digestive enzymes is of little concern (2). Despite the low natural proanthocyanidin content of the human diet, in recent years there has been some research interest in exploring the potential weight loss benefits of exaggerated concentrations of proanthocyanidin-rich plant extracts. In vitro and in vivo studies, including a small probe trial in healthy adults, have demonstrated the ability of these proanthocyanidinrich extracts to alter lipase activity and in turn, fat absorption and metabolism (24–26). Though these data suggest that plant-derived proanthocyanidin-containing extracts may be effective agents in reducing the energy density of the diet by reducing fat absorption, larger and more conclusive studies are warranted.

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Early findings regarding the absorption of intact proanthocyanidins were mixed, with some animal studies reporting proanthocyanidin absorption (reviewed in (5) and others failing to demonstrate absorption of these food components in rats (27,28), chicken, or sheep (5). In recent years, the absorption of both intact A-type and intact B-type proanthocyanidins (see chapter “Polyphenols Overview”) has been reported. Shoji et al. (29), detected intact procyanidin dimers and trimers in rat plasma following the acute oral administration of apple-derived procyanidin oligomers. Interestingly, molecular size as well as stereochemical structure appeared to influence bioavailability. In the study, apple-derived dimers were more bioavailable than the larger trimeric oligomers, and the B2 dimer (epicatechin(4␤→8)-epicatechin) appeared more bioavailable than the structurally related B1 dimer (epicatechin-(4␤→8)catechin). Work by Appeldoorn et al. (30) further explored the influence of stereochemistry on bioavailability by comparing the absorption and metabolism of B-type and A-type procyanidin dimers. Using in situ perfusion of the rat small intestine, the absorption of individual procyanidin dimers [A1: epicatechin-(2␤→O7; 4␤→8)catechin; A2: epicatechin-(2␤→O7; 4␤→8)-epicatechin; B2: epicatechin-(4-8)-epicatechin] or a mixture of A-type, these and other larger oligomers was compared. Like the work reported by Shoji (29), stereochemical configuration did influence bioavailability. Appeldoorn et al. (30) showed that the A-type dimers were found to be more readily absorbed than dimer B2 and that A-type trimers were not absorbed. Interestingly, when administered as the pure chemical, dimer B2 was not detected in rat plasma; however, when administered in combination with A1 and an enriched A-type tetrameric fraction, B2 could be detected in rat plasma, suggesting that the co-consumption of different procyanidin oligomers may influence bioavailability. Information on the biotransformation, specifically glucuronidation, methylation, and sulfation, are sparse, yet some evidence of biotransformation has been reported (29). In contrast to the large number of animal studies, studies on the bioavailability of proanthocyanidins in humans have been limited. Similar to what has been reported in animal models, intact procyanidin dimers have been detected in human plasma. Following the consumption of proanthocyanidin-containing grape seed extract (31) and cocoa powder (32), procyanidin dimer B1 (grape seed extract) and B2 (cocoa) were detected in plasma within two hours of consumption. In these studies, plasma procyanidin dimer concentrations were reported to average in the range of 10 to 40 nM, markedly less than ␮M concentrations that have been reported for the monomeric procyanidin subunits, that is, epicatechin and catechin. To date, no human studies have reported the absorption of intact procyanidins larger than dimers. In addition to investigations into the absorption of proanthocyanidins, there has been research into the catabolism of these compounds by gut microflora. Metabolism of monomeric polyphenols by microflora of the lower gastrointestinal (GI) tract has been recognized for many years (33). In vitro experiments employing human colonic microflora demonstrated that purified (34) and semipurified proanthocyanidins (primarily

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hexamers and heptamers, but free of monomers, dimers, and trimers); (35) were readily catabolized. The primary products of these experiments were monohydroxylated derivatives (meta and para isomers) of phenylacetic, phenylpropionic, and phenylvaleric acids, which are similar to those resulting from the metabolism of monomeric flavonoids (33). Studies in rats support the extensive catabolism of proanthocyanidins by gut microflora, with a variety of phenolic acid metabolites identified in urine, indicating that these catabolites are readily absorbed (34). Though only a limited number of studies exist, there is research to support similar gut effects in humans. Studies utilizing cocoa (36), apple (37), and grape seed (38) proanthocyanidins demonstrate that following consumption, a variety of phenolic acids can be detected in urine. As suggested by in vitro and in vivo animal models, the microflora within the human GI tract are capable of extensive catabolism of the proanthocyanidins, generating a number of low-molecular-weight phenolic acids which are readily absorbed and are thus possible contributors to the putative health benefits ascribed to diets rich in proanthocyanidin-containing foods. The lack of availability of purified individual oligomers and polymers limits the feasibility of experiments with well-characterized materials in the variety of plant extracts that are readily utilized in studies. Nonetheless, investigations are repeatedly undertaken in which natural plant extracts are administered (in vitro and in vivo) and thus great care must be taken in the interpretation of these published findings as the experimental repeatability and relevance to human health must be questioned when the analytical characterization of these materials is missing or incomplete.

ALTERATION OF BIOLOGICAL MARKERS ASSOCIATED WITH CHRONIC AND OTHER DISEASES Proanthocyanidins as Antioxidants Many life processes generate free radicals. The resulting reactive oxygen and nitrogen species (ROS, RNS), if left unchecked, have the potential to cause oxidative damage to DNA, lipids, and proteins, resulting in a cascade of degradative effects that may contribute to human disease pathophysiology (39). Free radical scavengers and/or antioxidants may protect cells against oxidative damage (40). Proanthocyanidins and their purported digestion products, hydroxylated phenolic acids, have high antioxidant activity in vitro (41,42). In the case of proanthocyanidins, major contributions to this activity are the presence of a catechol group (hydroxyl groups adjacent to one another) on the B-ring, and the stability of the reduction products, semiquinones and quinones (41). Though in vitro studies have demonstrated the direct antioxidant activity of proanthocyanidins, the relevance of these findings to humans is questionable. As noted previously, the absorption of intact proanthocyanidins is limited, with nothing larger than dimers having been detected in human plasma following the consumption of proanthocyanidin-containing cocoa or a grape seed extract. Though there is limited understanding of the metabolism of these compounds in humans, animal work suggests that methylation and glucuronidation of

absorbed proanthocyanidins are possible. Assuming that similar processes may occur in humans, such chemical modifications would markedly reduce the hydrogendonating capacity of a given proanthocyanidin. Furthermore, given that only low nM concentrations of dimers have been detected in plasma (31,32), the ability of plasma proanthocyanidins to effectively compete with conventional plasma antioxidants such as vitamin C, urate, and glutathione, which are present as micromolar /millimolar concentrations in plasma, is highly unlikely (43). Finally, adding to the limited support for direct antioxidant benefits associated with proanthocyanidins are the recent results of multiple human intervention studies with proanthocyanidin-containing foods which fail to demonstrate any improvements in markers of oxidative stress and damage (44–47). Given that such low concentrations of proanthocyanidins, specifically procyanidin dimers, have been reported in the circulation, it seems unlikely that these compounds have any direct antioxidant effect in vivo. One exception to this may, however, be in the lumen of the gastrointestinal tract. From the dietary constituents themselves and as a consequence of digestive processes, the gastrointestinal tract is exposed to a variety of reactive oxygen and nitrogen species (48–50). As a consequence of their limited bioavailability and reported stability, at least through the initial phase of digestion (31,32), it is possible that micromolar concentrations of these compounds can be achieved within the GI tract following the consumption of proanthocyanidin-rich foods. Thus, the direct hydrogen-donating and metal-chelating capacities of proanthocyanidins may allow native proanthocyanidins to serve an important role in protecting the GI tract from oxidative stress, particularly through the digestive process. Human intervention studies with grape (48) and cocoa (51) products provide some evidence that postprandial oxidative stress may be reduced as a consequence of the consumption of these proanthocyanidin-containing food components.

Cancer Many in vitro and in vivo systems have been employed to investigate the effects of proanthocyanidins on cancer processes, with a number of these studies suggesting that these compounds may offer preventative, and even potential therapeutic benefits in the management of cancer. Work with a variety of proanthocyanidin-enriched materials as well as highly purified proanthocyanidins demonstrate the ability of these compounds to inhibit cell growth and promote cell death (52–55) in vitro. One challenge in the interpretation of these (and other) studies is the stability of these compounds in vitro under the environmental conditions and the duration of incubation (hours to days) that are commonly employed. Proanthocyanidins have been shown to be unstable under commonly employed cell culture conditions, resulting not only in the loss of the intact proanthocyanidin (56,57), but also in the generation of hydrogen peroxide, semiquinone, and quinone species that may be the actual mediators of the observed response (58). The anticancer potential of proanthocyanidins, however, cannot be completely discounted, because there is evidence that when efforts are taken to minimize artifact

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formation, select proanthocyanidins can be cytotoxic to a number of human cancer cell lines (55). The influence of proanthocyanidins has been investigated on biological markers for cancer in several animal models. Proanthocyanidins fed as a condensed tannin extract of red alder bark or as grape seed extract significantly inhibited the multiplicity, size, and distribution of chemically induced colonic aberrant crypt foci in mice and rats (59,60). Experiments with proanthocyanidins isolated from cacao liquor and fed to Sprague-Dawley rats showed substantial inhibition of the initiation of 2-aminomethyl-6phenylimidazo[4,5-b]pyridine (PhIP)-induced pancreatic carcinogenesis (61). In vitro studies suggested that proanthocyanidins also directly inhibited the mutagenic activity of PhIP, perhaps through nonspecific binding. Feeding proanthocyanidins extracted from grape seeds to SKH-1 hairless mice also decreased both UVB-induced skin carcinogenesis and malignant transformation in terms of incidence, multiplicity, and size (62). A suggested mechanism for the inhibition of carcinogenesis is the antioxidant activity conferred by the dietary proanthocyanidins. Grape seed proanthocyanidins fed to mice or rats, however, were not effective in curtailing chemically induced mammary tumorigenesis (60,61). Several foods also contain proanthocyanidins; however, there is a paucity of observations on their effect on carcinogenic processes. Although black and green teas have been extensively investigated for their anticancer activity, green teas contain only limited proanthocyanidins (63) whereas black teas have substantial concentrations of derived tannins (theaflavins, thearubigins, and others), which are a heterogeneous mixture of oxidation products of monomeric flavonoids and structurally different from proanthocyanidins (64). To date, no human intervention trials investigating the potential preventative or therapeutic benefits of proanthocyanidins have yielded conclusive evidence (65); however, multiple trials are currently registered which examine the potential application of various plantderived polyphenols and flavonoids, including grape seed proanthocyanidins, in the prevention and management of specific cancers (the reader may search http:// clinicaltrials.gov/ for details on these registered trials). Interestingly, epidemiological studies do support the notion that the intake of proanthocyanidin-rich foods is inversely associated with the risk for development of non-Hodgkins lymphoma (66) and colorectal cancer (67). Though not causal, these studies suggest that the regular inclusion of proanthocyanidin-rich foods in the diet may offer protection from the development of certain types of cancer.

Atherosclerosis Atherosclerosis is an inflammatory disease process (68,69), and today it is well accepted that a very early event in the atherosclerotic process is a disruption in the proper functioning of the cells that comprise the lining of blood vessels—the endothelium. The importance of blood vessels in the regulation of vascular homeostasis is well recognized today (reviewed in (70). Furthermore, it is well recognized that a disruption in the function of the endothelium is an early indicator of the health of the vascular system, and there is growing evidence that assessing the

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function of these endothelial cells may provide prognostic data and serve as an independent predictor of cardiovascular disease risk (reviewed in (70,71). As a result of both in vitro and in vivo studies, there is evidence to suggest that exposure to proanthocyanidins may positively influence various steps in this complex disease process.

Inhibition of LDL Oxidation Experiments investigating the interaction between synthetic liposomes and cocoa proanthocyanidins revealed that liposome oxidation originating in the aqueous phase was inhibited most effectively by flavan-3-ol monomers, as well as proanthocyanidin dimers and trimers (72). Conversely, protection was greatest with higher polymers (DP 3–6) when oxidation was initiated in the lipid phase (73,74). In vitro studies with isolated LDL (low density lipoprotein) particles have shown that individual isolated procyanidins (monomer through hexamer) or several natural products rich in proanthocyanidins (cranberry extract, grape seed extract) inhibited chemically induced oxidation of LDL (72,75–77). In a coppercatalyzed LDL system, equimolar concentrations of individual proanthocyanidins indicated antioxidation activity that was proportional to the DP of the procyanidins (75). Employing a similar system, isolated fractions from cranberries rich in proanthocyanidin oligomers (DP 3–9) and containing one to three A-type linkages were also effective in delaying LDL oxidation (76). When results from an 2,2 -azobis (2-amidino-propane) dihydrochloride (AAPH)-induced LDL conjugated diene formation system were expressed on a monomer equivalent basis, inhibitory activity of the various polymers was similar, suggesting that antioxidant capacity was a function of the number of available catechol groups (75). Studies with fractions containing mixed oligomers gave similar results in terms of antioxidant capacity, but higher polymers (DP 5–9) appeared to have greater affinity for LDL particles than oligomers with a lower DP (77). Similar studies in a cellular system (endothelial cell-mediated LDL oxidation) changed preference of antioxidant to monomeric catechin and dimers rather than higher polymers of proanthocyanidin (75). It is difficult to interpret results with polymeric proanthocyanidins and chemically induced LDL oxidation in terms of biological activity, because studies to date suggest oligomers larger than DP 2 are only minimally absorbed and circulated in the blood stream. A controlled, double-blind, randomized, crossover human study demonstrated that the daily inclusion of proanthocyanidin-containing cocoa powder and chocolate in the context of an average American diet was shown to be effective in decreasing LDL oxidation susceptibility and slightly increasing serum total antioxidant capacity as well as high density lipoprotein cholesterol (HDL-C) levels (78). Similar results of cocoa ingestion on LDL oxidation susceptibility were observed in additional studies for which dietary control was less rigorous (79) and referR ences therein). Pycnogenol brand pine bark extract (150 mg/day) fed to healthy individuals for six weeks did not alter LDL oxidizability, but reduced LDL-cholesterol and increased HDL-C levels in the plasma of two-thirds of the subjects (80). However, the same extract (360 mg/day) given to patients with chronic venous insufficiency decreased total cholesterol and LDL-C values, but did not

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alter HDL-C levels (81). Addition of grape seed extract to the diet of hypercholesterolemic subjects for eight weeks substantially reduced the level of antibodies to oxidized LDL (measure of oxidized LDL), compared to results of the placebo control group (82). Results from in vitro studies suggested that isolated cocoa-proanthocyanidins were inhibitors of mammalian 15-lipoxygenase-1, an enzyme that oxygenates LDL to an atherogenic form (83). Studies with red wine or red wine polyphenol-containing diets (rich in proanthocyanidins) gave mixed results in terms of plasma antioxidant capacity and resistance to ex vivo LDL oxidation (5,79).

Inhibition of Inflammatory Response Studies with isolated or purified cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase demonstrated that their activities were inhibited by flavanols and oligomeric proanthocyanidins from cocoa at concentrations similar to drugs used for the same purpose, for example, indomethacin (83–85). Short-term (6 hr) in vivo experiments with human subjects fed proanthocyanidin-rich chocolate resulted in increased plasma levels of prostacyclin, decreased concentrations of leukotrienes, and a decreased leukotriene/prostacyclin ratio, all measures of the proinflammatory/anti-inflammatory eicosanoid balance (86). Similar results were observed with treated aortic endothelial cells in vitro. Longer-term studies (4 and 6 wk) with subjects consuming a daily combination of cocoa powder and dark chocolate plus an average American or a low-flavonoid diet failed to alter the urinary excretion of F2 isoprostane, thromboxane B2 , 6-keto-prostaglandin F1␣ , or their ratio (78,87). Consumption of proanthocyanidincontaining purple grape juice, but not several other juices or coffee devoid of proanthocyanidins, significantly increased 6-keto-prostagladin F1␣ at two hours postconsumption (88). Pycnogenol (200 mg/day) consumption reduced thromboxane B2 levels in smokers but did not alter levels in nonsmokers (89). Results from these experiments suggest proanthocyanidins and proanthocyanidincontaining foods and supplements may alter eicosanoid metabolism in favor of an anti-inflammatory environment. However, environmental interactions as well as time course and magnitude of this response require further investigation. Endothelial injury causes increased expression of cellular adhesion molecules (CAMs) (i.e., ICAM1 [intracellular CAM], VCAM-1 [vascular CAM], Eselectin) that mediate recruitment of monocytes and their subsequent differentiation into phagocytic macrophages (77). Employing HaCaT cells (human keratinocyte), Pycnogenol pretreatment inhibited IFN gamma-induced adherence of these cells to Jurkat T cells and expression of ICAM-1 (90). Pycnogenol also inhibited NF␬B activation and VCAM-1 and ICAM-1 expression in tumor necrosis factor ␣ (TNF-␣) treated human umbilical vein endothelial cells (HUVECs) (91). A gene that codes for an oxidized LDL receptor directly linked to foam cells and atherosclerosis, CD36, was found to be downregulated by grape seed proanthocyanidin extract (GSPE) in TNF-␣induced HUVECs (82). In vitro studies with peripheral blood mononuclear cells (PBMC), isolated from human subjects that had low production of transforming growth factor (TGF)-beta-1, showed that TGF␤-1 production was

greatly stimulated by dimeric and tetrameric proanthocyanidins isolated from cocoa, compared to higher polymers (DP > 5) (92). In contrast, TGF␤-1 secretion from high producing PBMC at baseline was inhibited by all cocoa proanthocyanidin fractions tested (DP 2–10). A study with cultured vascular smooth muscle cells demonstrated that exposure to red wine polyphenolic compounds inhibited both the mRNA expression of vascular endothelial growth factor, as well as the release of vascular endothelial growth factor in response to platelet-derived growth factor AB, TGF␤-1, or thrombin (93). Elucidation of the mechanism suggested that the redox-sensitive activation of the p38 mitogen-activated protein kinase had been inhibited. Though such in vitro studies appear promising, the limited-to-overt lack of bioavailability of most of these proanthocyanidins calls into question the physiological relevance of these findings in the context of foods and supplements. Human intervention trials examining the impact of proanthocyanidin-rich foods on various circulating markers of inflammation and adhesion have been completed. Overall, the findings have been mixed. Some studies using wine (94,95) and cocoa (96,97) reported significant improvements in these endpoints, while other human intervention trials reported no significant effects (78,87,98). This variability in results may be attributable to a number of elements such as the health status of the study participants (i.e., healthy and disease-free versus those with known CV disease), small sample size, and duration of the studies. As such, further studies are needed in order to draw firm conclusions regarding the potential antiinflammatory effects in vivo.

Decreased Platelet Aggregation In vitro experiments with whole blood showed that cocoa procyanidin trimers and pentamers as well as dealcoholized red wine increased expression of platelet activation markers (fibrogen binding conformation of GPIIbIIIa and P-selectin) in unstimulated platelets but suppressed platelet activation response to epinephrine (99). Both short-term (2–6 hr) studies and a long-term (28 day) study with human subjects demonstrated that consumption of proanthocyanidin-rich cocoa beverage lowered P-selectin expression and platelet aggregation (ADP-, collagen-, epinephrine-induced) in ex vivo experiments (47,75,99–101). The effects observed were qualitatively similar to aspirin, but less profound (75). There is also evidence that in addition to a direct action on platelets, the consumption of proanthocyanidin-containing foods can modulate the function of leukocytes (101). Other food sources of proanthocyanidins (and minor constituents) such as purple grape juice, combined extracts of grape seeds and grape skins, but not citrus juices, also were active in the reduction of platelet aggregation when administered to dogs, monkeys, or humans beings (77,102). Extract of Ginkgo biloba (120 mg/day for 3 mo) fed to healthy volunteers modulated collagen-, but not PAF-mediated platelet aggregation (103). However, on giving the same extract to subjects with type 2 diabetes decreased platelet aggregation stimulated by both systems. An in vivo model based on cyclic flow reductions caused by platelet aggregation in the partially occluded circumflex coronary artery of anesthetized dogs has been

Proanthocyanidins

employed to test platelet activity and platelet–vessel wall interactions (102) and references therein). Several of the same dietary sources of proanthocyanidins (red wine, purple grape juice) that were active in vitro, were also active in preventing thrombus formation in this model. A similar model, based on experimental venous thrombosis in spontaneously normolipidemic rats fed a cholesterol-rich diet, demonstrated that dealcoholized red wine added to their diet reversed the prothrombotic effect of the hyperlipidemic factors (104).

Animal Models Two animal models have been developed to study dietary and other effects on progression of atherosclerosis. Golden Syrian hamsters, when fed diets of high cholesterol and coconut oil for ten weeks, have a lipid profile similar to hypercholesterolemic humans beings. This treatment also results in the formation of foam cells on aorta walls, the extent of which has been used as a biomarker of the early stages of atherosclerosis (atherosclerotic index) (82). Addition of grape seed extract to hypercholesterolemic hamster diets (50 mg or 100 mg/ kg body weight) resulted in a substantial and significant reduction of the atherosclerotic index. In addition, total plasma cholesterol and triglyceride levels also were significantly reduced in the GSPE-fed animals. New Zealand White rabbits fed hypercholesterolemic diets respond with high plasma total cholesterol levels (400+ mg/dL) and the formation of Sudanpositive stained lesions (fatty streaks) on the walls of their aorta (biomarker of atherosclerosis potential) (105). AddiR tion of a grape seed extract (Leucoselect Phytosome ) to hypercholesterolemic diets of a group of rabbits reduced aortic arch lesions to nearly control levels (3%), whereas atherosclerotic diets alone resulted in lesions that covered 18% of the vessel wall. Currently, there are limited data available to support the translation of these effects from animal models into humans. Studies with human beings who consumed a combination of cocoa powder and dark chocolate for relatively long periods (4 and 6 wk) only slightly, but significantly, increased HDL levels in one experiment (78), but did not significantly alter plasma cholesterol, triglyceride, or other lipoprotein concentrations (78,87). In contrast cinnamon, which contains a series of unique trimeric and tetrameric procyanidins with A-type linkages (106), significantly decreased plasma levels of triglycerides as well as total and LDL-C, when administered (1–6 g/day) for only 20 days (107). Grape seed proanthocyanidins fed to rats along with high-cholesterol diets also reduced serum cholesterol levels compared to nonproanthocyanidin-fed controls (5). Studies with proanthocyanidin-rich cranberry juice powder fed to familial hypercholesterolemic pigs significantly lowered plasma total cholesterol and LDL, and slightly raised HDL (77). However, the same powder fed to normocholesterolemic pigs did not alter levels of circulating cholesterol fractions.

Nitric Oxide-Dependent Vasodilation The enzyme nitric oxide synthase (NOS) uses L-arginine and oxygen as substrates to produce NO, which interacts with smooth muscle cells to cause vasorelaxation. A common inhibitor of NOS, NG -nitro-L-arginine methyl ester,

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when infused, nullified vasodilation observed with treatments that stimulate NO production (45,104,108) thereby validating the action of NOS and role of NO in vasodilation. Three distinct NOS isozymes have been identified: endothelial, the critical isoform relative to maintenance of vascular function; neuronal; and an inducible form found in a number of cell types, including macrophages and vascular smooth muscle cells (109). In vitro studies demonstrated that red wine and Pycnogenol, but not white wine, improved vasodilation and simultaneously increased endothelial NO production (102). Further characterization of proanthocyanidin fractions isolated from red wine showed that vasodilation activity was greatest in the presence of low-molecularweight oligomers (DP 2–3), whereas higher polymers were inactive. Examination of the mechanism of increased NO production with rat aorta ring strips and G. biloba extract suggested inhibition of Ca2+ influx through Ca2+ channels, thereby activating NO release (110). Contrary to the above findings, proanthocyanidins isolated from female inflorescences of hops (Humulus lupulus), a common ingredient of beer, were strong inhibitors of neuronal nitric oxide synthase activity, with procyanidin dimer B2 having the highest inhibitory activity (111). Procyanidin dimer B3, an isomer of B2, was noninhibitory in this system. An explanation for the differential action of these isomers on two isoforms of NOS is not apparent at this time. Two noninvasive in situ systems have been developed to test the efficacy of various dietary components, drugs, and environmental conditions on vasodilation. A study in patients with coronary artery disease showed improved flow-mediated vasodilation of the brachial artery when purple grape juice was consumed compared to beverages that did not contain proanthocyanidins (77). Similar studies have been done utilizing proanthocyanidinrich cocoa product, supporting both acute (45,108,112,113) as well as sustained improvements (46,114,115) in endothelial function. In several of these studies, the positive effects on endothelial function were linked to the production of nitric oxide, a potent vasoactive molecule key to regulation of vascular tone (44,45,114).

Vasoconstriction Angiotensin II is a vasoconstrictor that is produced in the pulmonary capillaries by angiotensin converting enzyme (ACE) and can be involved in the development of hypertension and atherosclerosis (77). Several proanthocyanidins and preparations containing them inhibited angiotensin converting enzyme activity in both in vitro and in vivo experiments. These included Pycnogenol, proanthocyanidins isolated from red grapes, and extracts of Erythroxylum laurifolium (endemic species on Reunion Island in the Indian Ocean) and fruits of Cupressus sempervirens L. (Italian cypress).

Reperfusion Induced ischemia–reperfusion studies in hearts isolated from laboratory animals simulate myocardial infarction and recovery in human beings. This model permits investigation of various dietary interventions and other environmental and circulatory alterations on recovery of hearts postischemia. Hearts from grape seed extract, red wine-, or red wine proanthocyanidin-fed rats were more

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resistant to ischemia–reperfusion injury than hearts from control animals (82,116). Blood flow parameters were improved, whereas infarct size, formation of hydroxyl radicals, and malondialdehyde levels of heart perfusate were all modulated as a result of feeding animals proanthocyanidins or proanthocyanidin-containing ingredients to animals. These same dietary treatments also reduced the levels of proapoptotic factors JNK and c-Jun, as well as the proportion of apoptotic cardiomyocytes. Similar studies with a short-term recovery (12 min) showed opposite effects of G. biloba extract (EGb 761) pretreatment in terms of decreased inducible nitric oxide synthase mRNA expression and NO production (117).

Other Metabolic Alterations Bacterial Antiadhesion Anecdotal observations and recent critical evaluation of the scientific literature provides some evidence that consumption of cranberries or its products is effective in the prevention of urinary tract infections (reviewed in 118). Although the therapeutic effect was long thought to be increased urinary acidity due to hippuric acid excretion (119), it is now attributed to a family of unique proanthocyanidins and/or their catabolites, which have been characterized as containing a high proportion of A-type linkages (76,120,121). In the case of urinary tract infection, the primary effect is inhibition of cellular adherence of P-type (mannose-resistant) uropathogenic strains of Escherichia coli (119,122,123). In addition, evidence has been presented to the effectiveness of cranberries for similar responses with Helicobacter pylori to gastric epithelial cells (124) and a host of organisms commonly found in the oral cavity (125). Proanthocyanidin extracts from cocoa and cranberries have also been reported to have bacteriostatic properties (126,127).

Diabetes, Glucose, and Insulin Metabolism Impaired glucose uptake and insulin resistance are subtle but common metabolic alterations that may be general etiologies for several age-related disorders and chronic diseases (128). Thus, identification of dietary components and natural products that have the potential to maintain these metabolisms throughout life has a highly favorable risk/benefit ratio. Several foods, biological materials, and synthetic preparations, such as tea, several spices, GSPE, and niacin-bound chromium, have been found to be effective (128–130). However, the chromium content of natural materials (long associated with insulin potentiating activity) was not associated with improved insulin action or glucose metabolism (131), which suggested that other biologically active components were responsible. Relative to proanthocyanidins, an extract of cinnamon, which contained a series of two trimers and a tetramer of flavan-3-ols, each with an A-type linkage (106), was effective in significantly reducing fasting blood glucose in a group of type 2 diabetic patients (107). Several studies with proanthocyanidin-containing cocoa products also support the potential for these types of cocoa products to positively influence insulin metabolism (115,132). Longer term intervention studies, and mechanistic investigations are still needed to verify these findings and determine what role proanthocyanidins may play in these processes.

Immune Function Nonspecific or innate immune response of the immune system is one of the first lines of defense of the body to a host of environmental challenges. Many dietary components and drugs stimulate this system to an elevated level of preparedness. Besides those components of the immune system associated with atherosclerosis, the effect of proanthocyanidins also has been tested, in vitro, in PBMC. In a series of experiments investigating the effects of isolated individual proanthocyanidins from cocoa on resting PBMC, higher molecular weight fractions (DP 5–10) stimulated interleukins (IL)-1␤ (proportional to DP), IL-4 production, and IL-1␤ gene expression (135, 136), whereas intermediate-sized polymers (DP 4–8) were most active in the stimulation of TNF-␣ release (133,134). Employing a similar system, IL-2 and IL-5 secretion was unresponsive to isolated proanthocyanidin treatment (133–136). The influence of Pycnogenol has been studied on some of the components of the immune system in cell culture. In RAW 264.7 macrophages, Pycnogenol treatment of LPS-stimulated cells reduced production of IL-1␤ and its mRNA levels in a dose-dependent manner (137). In the same cell line, Pycnogenol blocked the activation of NF␬B and activator protein-1, two transcription factors involved in IL-1␤ gene expression, and abolished LPSinduced I␬B degradation. Collectively these results suggest Pycnogenol treatment of this cell line can inhibit expression of proinflammatory cytokine IL-1 through the regulation of redox-sensitive transcription factors. When individual proanthocyanidins were investigated in the same cells induced by interferon gamma, monomers and dimers repressed NO production, TNF-␣ secretion, and NF␬B-dependent gene expression, whereas procyanidin C2 (trimer) and Pycnogenol enhanced these parameters (138). These latter two treatments also increased TNF-␣ secretion in unstimulated RAW 264.7 macrophages. Studies with stimulated Jurkat E6.1 cells indicated that Pycnogenol depressed IL-2 mRNA expression, but that the mechanism of transcriptional regulation was different from regulation of IL-1␤ (137). Using isolated proanthocyanidin fractions from Ecdysanthera utilis Hayata & Kawak. (du zhong teng, a Chinese medicinal plant) and a PHA-stimulated PBMC system, procyanidin A1 (dimer with A-type linkage) inhibited IL-2 and interferon-gamma production, which may have caused suppression of PBMC proliferation (139). Two newly identified trimers, each with an A-type linkage, failed to alter the response of cytokines or factors from PBMC. A polyphenol-rich fraction isolated from cocoa liquor inhibited mitogen-stimulated proliferation of T cells and polyclonal Ig production by B cells (140). In addition, this cocoa–liquor fraction also inhibited IL-2 mRNA expression and IL-2 secretion by T cells. Specific to cranberries, potential viral antiadhesion properties have also been suggested; a high molecular weight-containing fraction from cranberry inhibited hemagglutination of A (H1N1) and B (H3N2) virus strains as well as decreased viral infectivity (141), suggesting a potential therapeutic potential of these plant compounds. Given the limited bioavailability of proanthocyanidins, these in vitro findings are likely primarily limited in their application to the mucosal immune system of the gut. To date, a limited number of in vitro studies have

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been conducted with colonic or intestinal epithelial cell lines and animal models of intestinal inflammation; however, extracts of proanthocyanidin-containing plants including French maritime pine bark, cocoa, grape seeds, and pomegranate fruit peel were found to modulate various markers of intestinal inflammation (142) and references therein). Human studies are needed to provide clear evidence of the potential benefits of dietary proanthocyanidins in the gut immune response.

DIETARY SOURCES AND INTAKE Dietary Sources Foods

Dietary Intake Foods

A wide variety of analytical procedures have been employed for the measurement of “total” proanthocyanidins (see analysis section). Employing normal-phase HPLC procedures, a large number of food samples, selected on the basis of market share and demographics within the United States (143), were analyzed for proanthocyanidin content (19). These data and others have been combined into a database of values for foods available online from the USDA Nutrient Data Laboratory at http://www.nal.usda.gov/fnic/foodcomp. Data for the proanthocyanidin content of selected foods containing substantial amounts are tabulated in Table 1. The data for red grapes reported in Table 1 are for seedless “eating” grapes, whereas cultivars of red-wine grapes and their wines have higher proanthocyanidin contents (144,145). This is reflected in the data for several red wines common in Spain, which contained dimers through polymers DP 13 and represented 77% to 84% of total flavanols (144). In general, a large number of vegetables, many spices, and some fruits (particularly citrus) had undetectable levels of proanthocyanidins (19,146). Fifty-six different kinds of common Spanish foods have been analyzed for flavanols, including dimers and trimers, but not higher oligomers (147). Results indicated procyanidin B2 was the most abundant dimer or trimer, and flavanols were very low or not detected in most vegetables.

Supplements There are a number of commercially available proanthocyanidin-containing dietary supplements in the market. However, rigorous qualitative and quantitative data on the proanthocyanidin content of these diTable 1

etary supplements is less precise than for foods because these dietary components have not been subjected to the same rigorous sampling and analysis programs. Though rigorous assessments of most of the commercially available proanthocyanidin-based dietary supplements are lacking, there are some exceptions. Some commercially available extracts of Maritime pine bark, grape R R R extracts (Meganatural , Activin , Gravinol ), apples R R   (Applephenon ), and lychee (Oligonol ) have been characterized, and putative health benefits and aspects of safety investigated.

Based on proanthocyanidin content for over 60 U.S. foods and daily food intake data [USDA Continuing Survey of Food Intakes by Individuals (CSFII) for 1994–1996], consumption by individuals in the United States was calculated for the first time in 2004 (146). The mean intake for all ages ( > 2 years old) was estimated at 54 mg/day/person for all proanthocyanidins with DP of 2 or more. Detailed examination of intakes for age/sex groups indicated a bimodal high intake phenomenon for children (2–5 yr and 6–11 yr) and older males (40–59 yr and > 60 yr) each of whom consumed 59 mg/day or more. Proanthocyanidin consumption among adults ranged from 46 mg/day (20–39 yr, female) to 66 mg/day ( > 60 yr, male). As outlined earlier, these data do not include proanthocyanidins that might be included in the consumption of red wines or other commonly consumed foods that have substantial polymer content but were not analyzed. Nonetheless, these results provided the scientific community with the first estimates of proanthocyanidin consumption. With the release in 2004 of the USDA database for the proanthocyanidin content of selected foods, it is now more easily possible to gain better estimates of intakes within populations and to examine the relationship between proanthocyanidin intakes and health and disease endpoints. In 2007, the largest survey of flavonoid intake, including proanthocyanidins, and the relationship of intake to cardiovascular disease mortality was published (148). A survey of nearly 35,000 postmenopausal women in the Iowa’s Women’s Health study revealed much wider range of intakes from what had been reported in 2004. In this population, the average proanthocyanidin intake in the lowest quintile was estimated to be 62 mg/day

Proanthocyanidin Content of Selected Foods (mg/100 g food)

Food/Spice

Dimersa

DP 3–10a

DP >10a

Total

Typeb

Apples, red delicious, with peel Blueberries Chocolate, baking Chocolate, milk Cinnamon, ground Cranberries Grape seed (dry) Grapes, red Pecans Plums, black

14 7 207 26 256 26 417 2 42 16

64 40 680 105 5319 152 1354 19 211 100

38 129 551 33 2509 234 1100 59 223 115

116 176 1438 164 8084 412 2817 80 476 231

B, PC B, PC B, PC B, PC A, B, PC, PP A, B, PC B, PC B, PC B, PC, PD A, B, PC

a Dimers, DP 2; DP 3–10, trimers through decamers summed; DP > 10 indicates values for polymers larger than decamers which eluted as a single chromatographic

peak. Linkage type (A, B) and proanthocyanidin subclasses (PC, procyanidin; PD, prodelphinidin; PP, propelargonidin) identified.

b

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while the top quintile reported an average intake of 524 mg/day. Amazingly, daily intakes of over 3 g of proanthocyanidins were reported in this study. Though there was no statistically significant association between proanthocyanidin intake and cardiovascular disease mortality, the intake of several proanthocyanidin-containing foods including chocolate, red wine, pears, apples, and strawberries was found to be inversely associated with a reduction in cardiovascular disease mortality. Two additional studies examining the relationship between the intake of specific flavonoids, including proanthocyanidins, and the risk of esophageal cancer (149) and non-Hodgkin lymphoma (66), reported intakes in the range of 3 mg to ∼350 mg/day. Though intakes were variable, these data provided invaluable information regarding proanthocyanidin intakes among various population groups.

Supplements Because of the dearth of analytical data for proanthocyanidin content in supplements, botanicals and herbals, comprehensive intakes from these dietary sources are not available.

ADVERSE BIOLOGICAL EFFECTS Traditionally, condensed tannins (proanthocyanidins) have been considered antinutrients in animal nutrition due to their astringency (reduced feed intake) and ability to bind several macronutrients, thus reducing their digestion and absorption (2,150). Although Pycnogenol has been shown to bind selected purified intracellular enzymes (151), the precise role of these polymers in the alteration of the metabolic equilibrium in the gastrointestinal tract of human beings is unknown. Toxicological studies on long-term (90 days) oral administration of grape seed extract to rats established a no-observed-adverse effect of 1.4g/kg of body weight per day for males and 1.5g/kg of body weight per day for females (152). Similarly, the LD50 of a single oral dose of grape seed extract IH636 was greater than 5g/kg of body weight for both male and female rats (153). Feeding IH636 at the rate of 100 mg/kg/day to male B6C3F1 mice for a year or 500 mg/kg/day to female mice for six months had no detectable adverse effects on the pathologies of vital organs or on serum chemistries (153). In terms of dermal irritation, IH636 was rated as moderately irritating and the no-observed-effect level for systemic toxicity was set at 2 g/kg for male and female albino rats (153). Observations in both rats and human subjects consuming FastOne, a herbal supplement containing extracts of kola nut, grape, green tea, and G. biloba, suggested an increased risk of colorectal cancers as substantiated by induced activity of CYP1A2 (154). In a review of potential drug–dietary supplement interactions, about one-half of patients taking prescription medication and at least one dietary supplement had potential for an “interaction of significance” (155). Of these patients, only 6% had the potential of a severe interaction. Investigation into the impact of various commercial supplements on P450-CYP3A4 activity revealed that specific plant extracts could alter enzyme activity (156,157), suggesting that the coconsumption of some

herbal supplements with certain medications could alter drug metabolism and thus drug effectiveness. Given the concerns that herbal supplements may antagonize or enhance the effects of medications (158), the reporting of herbal/botanical usage to family physicians and other health care professionals is encouraged.

RESEARCH NEEDED Although there are many areas of research on proanthocyanidins that can be identified for emphasis, three are critically important for substantial advancement of the association of these dietary components with human health: 1. Identify biologically active compounds that are absorbed and their tissue distribution. Studies with human subjects demonstrate that in addition to the absorption of monomeric flavanols, humans can absorb dimeric proanthocyanidins. To date, there is no evidence that higher oligomeric proanthocyanidin species are capable of being absorbed intact. Even though dimeric proanthocyanidins can be absorbed, current evidence demonstrates that circulating levels are in the low nanomolar concentration range and thus questions remain as to the biological relevance of these findings. In contrast to the intact molecules, there is considerable evidence of the catabolism of proanthocyanidins in the lower GI tract to many different phenolic acids (35,36) and evidence that these phenolic acids are readily absorbed and metabolized. A phenolic acid, 3, 4-dihydroxyphenylacetic acid, whose concentration was raised in plasma after consumption of diets rich in fruits and vegetables, significantly modulated platelet activity at concentrations observed in plasma (69), supporting the concept that catabolites of proanthocyanidins are putative bioactives that may contribute to the physiological improvement noted following the consumption of proanthocyanidin-containing foods. Characterization of metabolites and catabolites and their concentrations in various tissues will be of great advantage in terms of designing in vitro studies for the elucidation of mechanisms of action of these dietary constituents. 2. Assess intake of proanthocyanidins from dietary supplements. The recent development of robust analytical techniques for the measurement of proanthocyanidins resulted in the analysis of a large number of foods, the development of a database of values for foods, and estimates of intakes of these components from foods (17– 19,146,159). Similar efforts must be applied to those botanicals and herbals known to contain proanthocyanidins. In addition, accurate estimates of supplement consumption (especially botanicals and herbals) must be included in National Nutrition Surveys so that the contribution of these dietary sources can be calculated (160,161). 3. In order to truly establish the health benefits of proanthocyanidins, larger, longer, and more robust human intervention trials are required. Ideally, these studies should be randomized, double-blind, controlled investigations that examine the impact of multiple levels of proanthocyanidins. Foundational to these studies is the

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use of analytically well-characterized food products, as well as the use of study endpoints that are biologically relevant within the context of human health. It is only with such a methodical approach will progress be made towards establishing clear recommendations to the public regarding the consumption of specific proanthocyanidin-containing foods.

CONCLUSIONS There is emerging science to support a range of potential health benefits associated with proanthocyanidincontaining foodstuffs. In vitro and in vivo studies using animal models provide evidence in support of a range of biological effects. Efforts are still needed in understanding how these effects translate to humans, and to understand the overall impact of these effects to human health. Importantly, critical research is still needed to clearly identify the relevant proanthocyanidins with biological activity so that mechanisms of action at the tissue, cellular, and subcellular level can be elucidated. Fundamental to this biological research is the detailed analysis of proanthocyanidins, because the accurate and reliable measurement and characterization of these components in the materials used in research is necessary for understanding and substantiating the purported health effects.

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140. Sanbongi C, Suzuki N, Sakane T. Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in humans in vitro. Cell Immunol 1997; 177(2):129– 136. 141. Weiss EI, Houri-Haddad Y, Greenbaum E, et al. Cranberry juice constituents affect influenza virus adhesion and infectivity. Antiviral Res 2005; 66(1):9–12. 142. Romier B, Schneider YJ, Larondelle Y, et al. Dietary polyphenols can modulate the intestinal inflammatory response. Nutr Rev 2009; 67(7):363–378. 143. Pehrsson PR, Haytowitz DB, Holden JM, et al. USDA’s National Food and Nutrient Analysis Program: Food sampling. J Food Compost Anal 2000; 13:379–389. 144. Monagas M, Gomez-Cordoves C, Bartolome B, et al. Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. Cv. Graciano, Tempranillo, and Cabernet Sauvignon. J Agric Food Chem 2003; 51(22):6475–6481. 145. Sanchez-Moreno C, Cao G, Ou B, et al. Anthocyanin and proanthocyanidin content in selected white and red wines. Oxygen radical absorbance capacity comparison with nontraditional wines obtained from highbush blueberry. J Agric Food Chem 2003; 51(17):4889–4896. 146. Gu L, Kelm MA, Hammerstone JF, et al. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J Nutr 2004; 134(3):613–617. 147. De Pascual-Teresa S, Santos-Buelga C, Rivas-Gonzalo JC. Quantitative analysis of flavan-3-ols in Spanish foodstuffs and beverages. J Agric Food Chem 2000; 48(11):5331–5337. 148. Mink PJ, Scrafford CG, Barraj LM, et al. Flavonoid intake and cardiovascular disease mortality: A prospective study in postmenopausal women. Am J Clin Nutr 2007; 85(3):895– 909. 149. Bobe G, Peterson JJ, Gridley G, et al. Flavonoid consumption and esophageal cancer among black and white men in the United States. Int J Cancer 2009; 125(5):1147–1154. 150. Reed JD. Nutritional toxicology of tannins and related polyphenols in forage legumes. J Anim Sci 1995; 73(5):1516– 1528.

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151. Moini H, Guo Q, Packer L. Enzyme inhibition and proteinbinding action of the procyanidin-rich french maritime pine bark extract, pycnogenol: Effect on xanthine oxidase. J Agric Food Chem 2000; 48(11):5630–5639. 152. Yamakoshi J, Saito M, Kataoka S, et al. Safety evaluation of proanthocyanidin-rich extract from grape seeds. Food Chem Toxicol 2002; 40(5):599–607. 153. Ray S, Bagchi D, Lim PM, et al. Acute and long-term safety evaluation of a novel IH636 grape seed proanthocyanidin extract. Res Commun Mol Pathol Pharmacol, 2001. 109(34):165–197. 154. Ryu SD, Chung WG. Induction of the procarcinogenactivating CYP1A2 by a herbal dietary supplement in rats and humans. Food Chem Toxicol 2003; 41(6):861–866. 155. Peng CC, Glassman PA, Trilli LE, et al. Incidence and severity of potential drug-dietary supplement interactions in primary care patients: An exploratory study of 2 outpatient practices. Arch Intern Med 2004; 164(6):630–636. 156. Wanwimolruk S, Wong K, Wanwimolruk P. Variable inhibitory effect of different brands of commercial herbal supplements on human cytochrome P-450 CYP3A4. Drug Metabol Drug Interact 2009; 24(1):17–35. 157. Ulbricht C, Chao W, Costa D, et al. Clinical evidence of herb-drug interactions: A systematic review by the natural standard research collaboration. Curr Drug Metab 2008; 9(10):1063–1120. 158. Gardiner P, Phillips R, Shaughnessy AF. Herbal and dietary supplement–drug interactions in patients with chronic illnesses. Am Fam Physician 2008; 77(1):73–78. 159. Gu L, Kelm MA, Hammerstone JF, et al. Liquid chromatographic/electrospray ionization mass spectrometric studies of proanthocyanidins in foods. J Mass Spectrom 2003; 38(12):1272–1280. 160. Dwyer J, Picciano MF, Raiten DJ. Collection of food and dietary supplement intake data: What We Eat in AmericaNHANES. J Nutr 2003; 133(2):590S-600S. 161. Dwyer J, Picciano MF, Raiten DJ. Food and dietary supplement databases for What We Eat in America-NHANES. J Nutr 2003; 133(2):624S–634S.

Pygeum Franc¸ois G. Brackman and Alan Edgar with Paul M. Coates

INTRODUCTION

This extract is obtained by a process of maceration and solubilization of the P. africanum bark in organic solvent. The solvent is eliminated and the extract is purified. The extract has a soft-to-hard consistency, a dark brown color, and a very strong aromatic odor. It is freely soluble in chloroform but is insoluble in water. The P. africanum bark extract contains numerous constituents, including saturated and unsaturated fatty acids (C12–C22), phytosterols (␤-sitosterol, ␤-sitosteryl glucoside, and ␤-sitosterone), pentacyclic triterpenoids (ursolic acid, 2 a-hydroxyursolic acid, and oleanolic acid), alcohols (n-tetracosanol and n-docosanol), and carbohydrates (triacontane and nonacosane). The pharmaceutical properties are documented in the European Pharmacopoeia (Monograph no. 07/2002:1986). The American Pharmacopoeia mentions P. africanum bark, extract, and capsules in its Pharmacopeial Forum 29 (4) July–August 2003.

Pygeum africanum (also called Prunus africana) is a tree belonging to the Rosaceae family. It grows in tropical and humid equatorial mountain zones, at altitudes between 1000 and 2400 m. The tree is commonly found in countries such as Cameroon, Kenya, Madagascar, Congolese Democratic Republic, Equatorial Guinea, Uganda, Tanzania, Angola, South Africa, Ethiopia, Burundi, Rwanda, Malawi, and Nigeria. The origin of the use of P. africanum bark is documented back to at least the early 19th century. The ground bark was used in a water, tea, or milk mixture as a drug, the use being triggered by its flavoring effects (hydrocyanic acid). The bark was used by the Zulus, who had observed beneficial effects on urinary symptoms. Other tribes from Africa and Madagascar used it for the relief of symptoms such as gastric pain, urinary disorders, and also for its aphrodisiac properties. Such uses are, however, poorly documented, and are based on extracts whose contents and properties might differ both between modes of extraction and from the pharmaceutical standardized extract. The standardized P. africanum extract is used to alleviate lower urinary tract symptoms (LUTS) including those accompanying benign prostatic hyperplasia (BPH). Recent pharmacological and clinical studies have demonstrated that P. africanum extract quantitatively and qualitatively improves bladder as well as prostate-related parameters and symptoms causing urinary disorders. P. africanum is featured in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) II list of endangered species, imposing strict regulations on its harvest and trade. The exploitation of the bark is done within the framework of durable development. Only adult trees, with a diameter of at least 30 cm, undergo partial bark harvest (opposite quarters) that does not compromise the tree’s survival. Reforestation and forest enrichment could contribute to conservation and sustainability.

CLINICAL STUDIES Benign Prostatic Hyperplasia BPH is a very common finding in aging men. Its prevalence above the age of 50 varies from 50% to 75% in most cases. Transurethral and open surgical adenomectomy are the most widely used treatments for BPH patients who have severe symptoms. However, because of the clinically significant incidence of complications associated with surgery, such as blood loss, urinary tract infections, urethral stenosis, incontinence, impotence, and the need for reintervention after prostatic surgery (1), the management of BPH and LUTS has been rapidly changing. In addition, a large proportion of patients with LUTS do not have prostate enlargement and do not need surgical intervention. Medical approaches used in the treatment of BPH include ␣-adrenoceptor-blockers, 5-␣-reductase inhibitors, and plant extracts. ␣-Blockers partially alleviate symptoms of BPH (2) by reducing the ␣-adrenergic tone of the smooth muscle. However, they may have significant cardiovascular side effects that may limit their therapeutic application. The 5-␣-reductase inhibitors were developed from an elegant series of medicinal chemistry studies. Through a reduction in prostate size, they are supposed to act on symptoms by a direct effect on the mechanical component of obstruction. However, there is certain delay in the onset of the improvement (6–12 mo) and an important incidence of side effects, in particular, on sexual function (3). The limitations of 5-␣-reductase inhibitors and ␣-blockers in treating bladder outlet obstruction might be related to the

PHARMACEUTICAL DESCRIPTION Because the origin of the P. africanum bark and the extraction and standardization processes play a role in the final quality and content of the extract, results obtained with a given preparation might not necessarily apply to others. Most investigations performed and published were done using a standardized extract, which allows easier comparison of results. 650

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functional and structural remodeling of the bladder that also develops as the disease progresses. BPH is largely considered to be pathologically a disease of the prostate, while being symptomatically a disease of the bladder. The analogy of an enlarged prostate impinging upon bladder function still holds true in general, but numerous individuals see no improvement in urinary function in spite of objective reduction in prostate size. Still others see clear improvement in LUTS independent of any discernable change in prostate size (4). Modifications in the prostate do not necessarily evolve in parallel with bladder remodeling. The efficacy of the various treatment options depends, in part, on the judicious use of the appropriate treatment corresponding to the stage of advancement of the disease. Treatment directed at improving bladder function may be more efficacious in older individuals, while that designed to solely affect the prostate may be more beneficial in relatively younger patients. BPH results from progressive enlargement of the transition zone of the prostate and involves both glandular and stromal prostate tissue. Stromal elements contain smooth muscle, and contraction is mediated by ␣-1-adrenergic receptors. Hyperplasia of the transition zone is responsible for the organ enlargement. Prostate enlargement also mechanically and physically affects bladder dysfunction by a progressive denervation via damage to intramural nerves and synapses, as seen in animal models and in obstructive dysfunction in men (5–7). The involvement of specific bladder components indicates that effective treatment should not solely target the prostate, but must also be directed at the bladder. Still, the origin of the modification of bladder function in BPH remains the prostate, as witnessed by the paucity of symptoms in castrated individuals (8). More than 2000 patients were enrolled in clinical trials with P. africanum extract (9). These studies were conducted either in a double-blind placebo-controlled manner (10–12) or as open-labeled studies (13). There are 18 published randomized controlled trials comparing preparations of P. africanum with placebo or medical therapy for more than 30 days in men with symptomatic BPH. Thirteen trials included comparison of P. africanum extract with placebo (14,15). Most often, the treatment regimen was 100 mg/day (50 mg t.i.d.) for one to two months. The persistence of the effect and the long-term safety profile over more than five years has been investigated in an observational study (16). The effects observed in those studies have demonstrated that:

r More than 67% of patients reported ‘‘excellent,’’ ’’very good,’’ or ‘‘good’’ results. r Mean maximum urinary flow rate was improved in all studies in which it was measured. r Nocturia was improved in 50% to 100% of the patients in whom it was measured. r Daytime frequency was improved in 50% to 95% of patients in whom it was measured. r Hesitancy, urgency, weak stream, and dysuria were improved in the majority of studies. r Quality of life scores were improved. Standardized assessment scores such as International Prostate Symptoms Score (IPSS) confirmed these results.

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r Both clinical and urodynamic improvements were maintained during long-term treatment in a high proportion of patients. r Both clinical and urodynamic improvements were maintained after 12 months of treatment. The most frequently used dosage of P. africanum extract is 50 mg b.i.d. One prospective clinical trial (12,17) demonstrated the equivalence of effects of the extract 100 mg/day given either as 50 mg t.i.d., or as a single daily dose of 100 mg in men with moderate-to-severe urinary symptoms associated with BPH. Both daily dosage modalities of P. africanum extract (100 mg/day and 50 mg t.i.d.) decreased the total IPSS according to an equivalent pattern and to a similar extent (41% and 37.5%, respectively). Mean maximum flow rate increased after two months of treatment by 16% in the 50-mg b.i.d. group and by 18.5% in the 100-mg q.d. group. Similar effects were observed on all components of the condition and on quality of life scores.

MECHANISM OF ACTION Effects on In Vitro and In Vivo Growth Factor-Mediated Prostate Growth Normal prostate growth is controlled by an orchestration of growth factor-mediated autocrine and paracrine communication acting on epithelial and stromal prostatic tissue. Mutually distinct biochemical and suborganelle perturbations within the prostate can predispose toward BPH or prostatic carcinoma (18). The BPH affects predominantly stromal and glandular cell growth, where basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are major regulators along with insulin-like growth factor. Keratinocyte growth factor is expressed in epithelial tissue and has a paracrine effect on stromal cells, whereas transforming growth factor-␤1 has been shown to inhibit prostatic fibroblast proliferation. Finally, testosterone via dihydrotestosterone (DHT) has a causal role in controlling prostate growth and affects the expression of the aforementioned growth factors and their receptors indirectly. In advanced BPH, the role of androgens may be no more than permissive, whereas in prostatic carcinoma, androgen receptor signaling is not strictly a function of DHT levels. Furthermore, in men 50 years old or more, the role of testosterone relative to estrogen appears to diminish (19). Following an initial observation of an effect of P. africanum extract on bFGF- and EGF-mediated proliferation of 3T3 fibroblasts in vitro (20), a detailed series of experiments were performed comparing the potential of the extract to affect prostatic stromal cell proliferation mediated by various prostate-derived growth factors in rats (21). At concentrations devoid of cytotoxicity, P. africanum extract was shown in vitro to affect stromal fibroblast cell proliferation induced by EGF, bFGF, and insulin growth factor-I as well as with protein kinase C activators, but not by keratinocyte growth factor. Furthermore, the inhibition of cell proliferation was observed with IC50 values in the range of 10 mg/mL. The similarity of the individual IC50 values of P. africanum extract on the various growth factors suggests that a locus common to the three growth

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factors was being affected (22). Furthermore, the in vitro studies demonstrated that stromal fibroblast proliferation was inhibited via a site that converged near the level of protein kinase C. The results were not limited to rodent prostatic cells, as P. africanum extract was also shown to inhibit proliferation of fibroblasts from human hyperplastic prostate and bladder as well (23). The precise molecular component(s) of P. africanum extract responsible for the in vitro effects is not known. Therefore, these results cannot infer in vivo activity per se, as it is not possible to establish a correlation between the in vitro IC50 value and a Cmax value for the same metabolite following oral administration. Therefore, in an attempt to confront this problem, subsequent in vivo studies (24) focusing on the rat ventral prostate, which is approximately equivalent to the transition zone of the human prostate, showed that P. africanum extract affected adenyl cyclase-mediated cross-talk in cell signaling pathways also at the level of protein kinase C. Finally, ventral prostate hyperplasia induced by DHT treatment in vivo in rats was reversed by oral administration of the extract, while the latter had no effect on dorsal prostate enlargement (25). The molecular mechanism(s) underlying the in vitro and the in vivo effects of P. africanum extract are similar. The extract exhibited an effect on in vitro fibroblast proliferation of both rodent as well as human cells, which correlated with a growth factor profile signature corresponding to stromal cell proliferation, and this was paralleled by a preferential in vivo effect of P. africanum extract on ventral (stromal) prostate hyperplasia in rodents.

Effects on Bladder Function In man as well as in selected animal models, bladder obstruction induced by enlargement of the prostate or partial outlet obstruction of the urethra leads to a progressive increase in urethral resistance. The latter is initially compensated by an increase in bladder wall thickness, increased pressure generation, and alterations in flow parameters that limit the immediate deterioration in bladder function. This phase of compensated bladder function is slowly, though inexorably, followed by a decompensated bladder with a marked loss of contractility. The initial phase displays impaired detrusor smooth muscle function characterized by neuronal degeneration leading to reduced postsynaptic innervation, mitochondrial dysfunction, and loss of intracellular Ca2+ homeostasis that collectively compromise myosin contractility. The preceding section described in vitro data corroborated by in vivo results that demonstrated an effect of P. africanum extract on molecular aspects of prostate growth mediated by prostate-derived growth factors. Growth factor-mediated fibroblast proliferation is common to both the hyperplastic prostate as well as the hypertrophic bladder secondary to (26,27) similarly involving bFGF, EGF, and transforming growth factor-␤. This common link was the rationale for investigating the effect of P. africanum extract in various models of impaired bladder function (28). Indeed, initial results showed that the extract inhibited bFGF-stimulated fibroblast proliferation (23) and modestly reduced bladder weight in vivo in a preliminary

study that was confirmed in larger subsequent studies. Pretreatment of rabbits with P. africanum extract prior to partial outlet obstruction reduced contractile dysfunction in both a time- and dose-dependent fashion, determined by measuring the contractile responses to field stimulation, bethanechol and KCl. At a dose of 100 mg/kg, it improved the response to field stimulation by 50% and to KCl by 70%, and completely normalized the response to bethanechol when subsequently determined three weeks following partial outlet obstruction. Of the three parameters measured, partial outlet obstruction affects field stimulation to a greater extent, reflecting marked deterioration of synaptic function. Therefore, the initial experiment was complemented by a series of investigations that first determined the time course of the effects of P. africanum extract and correlated this to specific perturbations in synaptic and postsynaptic membranes coupled to alterations in key mitochondrial enzymes and calcium homeostasis. The results of the time-course study indicated that P. africanum extract was able to normalize the response to field stimulation after two weeks of treatment. These results were superior to those obtained after three weeks and may indicate a reduced efficacy of P. africanum extract after prolonged obstruction or an irreversible deterioration of synaptic function over time in this model. In an attempt to mimic the clinical situation, wherein bladder function is already compromised when treatment is initiated, it was then shown that the efficacy of P. africanum extract was maintained when administered only after the application of partial outlet obstruction. Specifically, the effects of P. africanum extract in restoring the contractile response to field stimulation, carbachol, and KCl were qualitatively identical when administered before or after partial outlet obstruction (28,29). Contractile dysfunction ultimately results in reduced force generation and alterations in myosin isoforms. In parallel with the improved contractile dysfunction, P. africanum extract was able to partially normalize the expression of myosin isoforms in line with improved contractility. These studies were based on the observation that alternative post-transcriptional splicing of myosin mRNA generates two isoforms of myosin, SM-A and SM-B, with lower and greater actin-activated adenosine triphosphate hydrolysis, respectively, and hence force generation. Following obstruction, detrusor smooth muscle SM-A myosin isoform expression increases threefold corresponding to reduced force generation, whereas treatment with P. africanum extract normalizes the SM-B/SM-A ratio in parallel with the improvement in field stimulation (30). Contractile dysfunctions of the obstructed bladder are directly related to ischemia (reduced blood flow) and detrusor hypoxia (31). Thus, short-term ischemia is a relevant model that recapitulates pathological aspects of contractile dysfunction inherent in partial outlet obstruction originating surgically or via prostatic enlargement. In this model, unilateral ischemia provokes direct and irrevocable ischemic insult to one side of the bladder, while partially compromising the nonischemic side. In agreement with what was observed following partial outlet obstruction, P. africanum extract pretreatment protected the nonischemic side of the bladder from the

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development of contractile dysfunction. This protective effect was further correlated with an enhanced expression of Hsp70 and c-myc. The clinical pharmacological relevance of these animal data was established by Valentini et al. (32), who demonstrated in a blinded clinical study that P. africanum extract improved detrusor contractile function after two months of treatment. The contractile dysfunctions induced by partial outlet obstruction in animal models, and by BPH-induced obstructive dysfunction in men, are secondary to denervation, mitochondrial dysfunction, and calcium storage dysfunction, which in turn is mediated partially by ischemiagenerated free radicals and calcium-activated hydrolytic enzymes. One hypothesis is that P. africanum extract acts in part by protecting neuronal and subcellular membranes from ischemia-induced damage, and by this means protects the contractile function of the bladder (7,33). Testosterone, in addition to its well-known action on stimulating prostate growth, also affects the bladder, and in the rat, administration of DHT significantly affects urodynamic parameters including frequency and volume. In DHT-stimulated rats, P. africanum extract, in addition to the aforementioned selective effect on ventral prostate growth, also significantly improves bladder frequency and volume (34). Collectively, these results clearly demonstrate that P. africanum extract directly affects bladder function. Among the limitations of the animal models employed is the lack of a concerted pathophysiology strictly representative of human BPH. The anatomic separation of prostate and bladder function in these models is, nonetheless, an advantage when attempting to demonstrate independent effects of P. africanum extract on the two organs. Furthermore, animal studies have the obvious advantage of being devoid of a placebo effect that complicates the design and interpretation of clinical trials in this indication. Treatment of LUTS is complicated by the multifactorial and multiorganelle origin, the slow evolution of the disease process, as well as the high placebo response in this patient population, which collectively limit the perceived efficacy of monotherapy in short-term clinical trials. P. africanum extract has demonstrated a reproducible efficacy in a variety of pharmacological studies addressing key aspects of lower urinary tract pathophysiology, thus altering the often encountered perception of plant extracts as poorly defined mixtures acting in an ambiguous manner to that of a reproducible molecular effector. The precise molecular component(s) responsible for the effects of P. africanum extract have not been identified. A limitation of the animal data described is the use of short-term treatment periods and the rapid evolution of the pathology in animals to investigate what in man is a chronic disease. The treatment duration of most clinical trials has also been limited, and the patient population not always ideally chosen to demonstrate beneficial effects on the bladder. The optimal patient population for showing the efficacy of an ␣-blocker, a 5-␣-reductase inhibitor, and P. africanum extract could vary, reflecting different stages of the disease process and related symptoms. The sum of the in vitro and in vivo pharmacological studies suggests a pharmacological mechanism of action of P. africanum extract affecting independently:

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r Prostate hyperplasia via a downstream target common to bFGF, EGF, as well as androgen-mediated cell proliferation at or near the level of protein kinase C. The signature of growth factor-mediated inhibition by P. africanum extract on in vitro and in vivo prostatic cell proliferation suggests that in vitro results are predictive and correlated to in vivo activity. r Bladder function with improvement in contractile dysfunction mediated via myosin isoform expression, lessened synaptic denervation and improved mitochondrial function.

ADVERSE EFFECTS Routine preclinical safety trials performed in various animal species by oral and parenteral routes, with single and repeated administrations, studied P. africanum at doses greater than 50 times the therapeutic doses. In such studies, no target organ could be identified as to potential toxic effects. P. africanum extract is devoid of any mutagenic potential. Most published open-label and placebo-controlled studies mentioned a good tolerance of the extract. Of particular note is the absence of any hormone-related adverse effects, confirming that the extract does not exert any hormonal effect. No interactions with concomitant medications such as antihypertensive agents, lipid-lowering agents, or anti-arrhythmics were reported. No significant changes were observed in biochemical safety parameters in those published studies where this is documented (10,12,13). In a recently published review, 13 of 18 randomized controlled trial studies provided information on specific adverse events. Side effects due to P. africanum were generally mild in nature and similar in frequency to placebo. The most frequently reported were gastrointestinal and occurred among seven men in five trials (15). In the most recently completed study (12), the type and overall frequency of adverse effects had similar distributions between dosage modalities during the comparative phase and were comparable for both phases of the trial. Light-to-moderate gastrointestinal effects, such as nausea, constipation, or dyspepsia, which are known to be treatment-specific, were reported most frequently (5.4% and 9.8% of patients who participated in the comparative and the extension phase, respectively). The majority of the serious adverse effects were related to the urogenital system (1.3% and 3.5% of patients who participated in the comparative and the extension phase, respectively) and appeared more related to the natural evolution of the disease than to the medication itself. It has been observed that very few severe emergent adverse effects (SEAE) appeared during the study, and the risk of presenting an SEAE during the long-term follow up was very low and constant in time. The rate of the SEAE-free patients at one year was 90% with 95% of CI 85% and 95%. Similar observations were made for the treatment-related emergent adverse events (TREAE), with the rate of the TREAEfree patients at one year equal to 92% [95% CI (88%, 96%)]. Few side effects were responsible for patients’ withdrawal from the study (15 patients during the comparative phase and 8 patients during the long-term phase). No

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significant changes were noted in blood or urine analyses in either group or during the two phases of the study. There was no statistically or clinically significant variation of the prostate-specific antigen level at 12 months compared to the baseline value. There is no report of any unwanted effect on sexual function with P. africanum. The cardiovascular effects were studied after a trial of 12 months of treatment with P. africanum (12). The treatment was not associated with any unwanted cardiovascular effects in this study.

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PRODUCTS AND DOSAGE P. africanum extracts are available worldwide under various formulations. One of them, P. africanum extract V1326, is the most common preparation Pygeum africanum Extract 573, and is marketed under the TadenanTM trade name as a prescription drug. Other preparations are available in various countries, containing P. africanum extract either as a single component (such as PronitolTM , BidrolarTM , FoudarilTM , KunzleTM , Neo UrgeninTM , NormobrostTM , NormoprostTM , ProlitrolTM , ProvolTM , etc.) or in combination with other components such as vitamins or minerals (such as ProFlowTM , PotenziaTM , Super Prostate FormulaTM ) to name a few.

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ters. A multicentre, placebo-controlled, double-blind clinical trial. Wien Klin Wochenschr 1990; 102:667–673. Dufour B, Choquenet C, Revol M, et al. Traitement symptomatique de l’ade´nome prostatique. Etude clinique controˆle´e des effets de l’extrait de Pygeum africanum. Gaz Med F 1983; 90:2238–2340. Chatelain C, Autet W, Brackman F. Therapeutic equivalence of efficacy and safety of once and twice daily dosage forms of Pygeum africanum extract in patients with symptomatic benign prostatic hyperplasia. A prospective, randomised, double-blind study. Urology 1999; 54(3):473–478. Breza J, Dziurny O, Borowka A, et al. Efficacy and acceptability of Tadenan (Pygeum africanum extract) in the treatment of benign prostatic hyperplasia (BPH): A multicentre trial in Central Europe. Curr Med Res Opin 1998; 14(3): 127–139. Wilt T, Ishani A, Mac Donald R, et al. Pygeum africanum for benign prostatic hyperplasia (Cochrane review). The Cochrane Library. Chichester, UK: John Wiley Sons, Ltd, 2004. Ishani A, Mac Donald R, Nelson D, et al. Pygeum africanum for benign prostatic hyperplasia: A systematic review and quantitative meta-analysis. Am J Med 2000; 109:654–664. Moya-Prats PP, Salva´ Verd A, Crespi Mesquida G. Valoracion estadistica de 500 pacientes con hipertrofia prostatica benigna, tratados con Pygeum africanum, y valorados estadisticamente desde el punto de vista clinico y flujometrico. Urodinamica Aplicada 1989; 1(4):150–155. Brackman F, Autet W. Once and twice daily dosage regimens of Pygeum africanum extract (PA): A double-blind study in patients with benign prostatic hyperplasia. J Urol 1999; 161(suppl 4):361. Gomella LG, Godwin BW. This month in investigative urology. Apoptosis and benign prostatic hypertrophy. J Urol 1997; 158(1):2–3. Gooren LJ, Toorians AW. Significance of oestrogens in male (patho)physiology. Ann Endocrinol (Paris) 2003; 64(2):126– 135. Paubert-Braquet M, Montboisse JC, Biochot-Lagente E, et al. Pygeum africanum extract (Tadenan) inhibits b-FGF and EGF-induced proliferation of 3T3 fibroblasts. Pharmacologist 1993; 35(3):173. Yablonsky F, Nicolas V, Riffaud JP, et al. Antiproliferative effect of Pygeum africanum extract on rat prostatic fibroblasts. J Urol 1997; 157(6):2381–2387. Levin RM, Das AK. A scientific basis for the therapeutic effects of Pygeum africanum and Serenoa repens. Urol Res 2000; 28(3):201–209. Le Brun G, Mellah I, Aubin P, et al. A rational for the use of Pygeum africanum extract during BPH course is suggested by in vitro proliferation control of human prostate and bladder fibroblasts. Eur Urol 1996; 30(suppl 2):98. Solano RM, Garcia-Fernandez MO, Clemente C, et al. Effects of Pygeum africanum extract (Tadenan) on vasoactive intestinal peptide receptors, G proteins, and adenylyl cyclase in rat ventral prostate. Prostate 2000; 45(3):245–252. Choo MS, Bellamy F, Constantinou CE. Functional evaluation of Tadenan on micturition and experimental prostate growth induced with exogenous dihydrotestosterone. Urology 2000; 55(2):292–298. Buttyan R, Jacobs BZ, Blaivas JG, et al. Early molecular response to rabbit bladder obstruction. Neurourol Urodyn 1992; 11:225–238. Buttyan R, Chen MW, Monson F, et al. Molecular control of rabbit urinary bladder hypertrophy. Biomed Pharmacother 1994; 48(suppl 1):27S–34S. Levin RM, Riffaud JP, Bellamy F, et al. Protective effect of Tadenan on bladder function secondary to partial outlet obstruction. J Urol 1996; 155(4):1466–1470.

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29. Levin RM, Hass MA, Bellamy F, et al. Effect of oral Tadenan treatment on rabbit bladder structure and function after partial outlet obstruction. J Urol 2002; 167(5):2253–2259. 30. Gomes CM, Disanto ME, Horan P, et al. Improved contractility of obstructed bladders after Tadenan treatment is associated with reversal of altered myosin isoform expression. J Urol 2000; 163(6):2008–2013. 31. Levin RM, O’Connor LJ, Leggett RE, et al. Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation. Neurourol Urodyn 2003; 22:156–163. 32. Valentini FA, Besson GR, Nelson PP. Modelised analysis of the effect of Tadenan on the bladder of patients with BPH: Blind versus open study of uroflows. 5th. Paris: International Consultation on BPH, 2000. 33. Zhao Y, Levin SS, Wein AJ, et al. Correlation of ischemia/ reperfusion and partial outlet obstruction induced spectrin proteolysis by calpain with contractile dysfunction in the rabbit bladder. Urology 1997; 49:293–300.

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34. Yoshimura Y, Yamaguchi O, Bellamy F, et al. Effect of Pygeum africanum tadenan on micturition and prostate growth of the rat secondary to coadministered treatment and post-treatment with dihydrotestosterone. Urology 2003; 61(2):474–478.

FURTHER READINGS 1. Denis LJ, et al. The 4th International Consultation on Benign Prostatic Hyperplasia (BPH), Proceedings. Paris: July 2–5, 1997; Health Publication Ltd, 1998. 2. Lowe FC, Ku JC. Phytotherapy in treatment of benign prostatic hyperplasia: A critical review. Urology 1996; 48: 12–20. 3. Lowe FC, Fagelman E. Phytotherapy in treatment of benign prostatic hyperplasia. Curr Opin Urol 1998; 8: 27–29.

Quercetin Jae B. Park

INTRODUCTION

malonyl-CoA. Briefly, 4-coumaroyl-CoA and acetyl-CoA from phenylalanine and malonyl-CoA, respectively, yield chalcone, a precursor for flavonoids including quercetin (1,5). Quercetin present in plants is mainly found conjugated to sugars as glycones (e.g., hyperin, isoquercitrin, avicularin, quercitrin, and rutin) (1,3). Besides glycosylation, it is also found modified by prenylation, acetylation, and methylation (1,3,6). Quercetin is commonly found in numerous dietary sources (onions, apples, black tea, green tea, red wine, beans, grapes, berries, vegetables, and fruits), and its dietary intake is associated with various potential health benefits (7–10).

Quercetin (3 ,4 ,5,7-tetrahydroxyflavonol, 3,3 ,4 ,5,7pentahydroxyflavone, 2-(3,4-dihydroxy-phenyl)-3,5,7trihydroxy-chromen-4-one, 2-(3,4-dihydroxyphenyl)3,5,7-trihydroxy-4H-1-benzopyran-4-one) is a flavonol belonging to a class of naturally occurring flavonoids (1– 4). It is composed of 3,5,7-trihydroxy-4H-1-benzopyran-4one (A and C) and a 3,4-dihydroxyphenyl ring (B) (Fig. 1). Synonyms for quercetin include: C.I. Natural Yellow 10; C.I. 75670; cyanidelonon 1522; flavin meletin; quercetine; quercetol; quertin; quertine; sophoretin; xanthaurine; 3,3 ,4 ,5,7-pentahydroxyflavone; 3,5,7,3 ,4 pentahydroxyflavone; 2-(3,4-dihydroxyphenyl)-3,5,7trihydroxy-4H-1-benzopyran-4-one.

ABSORPTION, METABOLISM, AND BIOAVAILABILITY There is great interest in the potential health benefits of flavonoids because of their potent antioxidant, free-radical scavenging, and other biological activities observed in vitro (9,10). Quercetin has potent antioxidant and other activities, which might contribute beneficial health effects on chronic diseases such as inflammation, cardiovascular diseases, and some types of cancers (9–11). Most beneficial health effects of quercetin would necessitate its absorption into the human body, which is interconnected with its metabolism and bioavailability. Data about these processes are to some extent available, but they are yet to be elucidated extensively.

BIOSYNTHESIS AND NATURAL SOURCES Quercetin belongs to the class of flavonoids providing flavor, color, and other functions to plants including fruits and vegetables. More than 5000 different flavonoids have been isolated, and on the basis of chemical structures, these are generally classified into several subgroups: flavanones (e.g., naringenin, hesperidin), flavones (e.g., apigenin, luteolin), flavonols (e.g., quercetin, myricetin), flavans (e.g., epicatechin, gallocatechin), anthocyanins (e.g., cyanidin, pelargonidin), and isoflavones (e.g., genistein, daidzein). In fact, quercetin belongs to the subgroup of flavonol, because of its hydroxylation at 3-position of the C ring. (1,2). Quercetin is synthesized in plants via multiple enzymatic processes from phenylalanine and

Absorption Quercetin occurs as glycones or aglycones, but in plants mainly as glycones such as rutin (quercetin rutinoside). Quercetin aglycone and glycones are likely to be quite different in their absorption and pharmacokinetics. Studies indicate that the overall kinetic behavior of quercetin changes following the ingestion of quercetin aglycones or glycones. This includes properties such as Cmax (the highest level at a given dose) and Tmax (time to reach Cmax ) (12). Currently, there are two proposed hypotheses on the absorption mechanisms of quercetin glycosides across the small intestine. One is an active uptake of quercetin glycoside by the sodium-dependent glucose transporter (e.g., SGLT1) with subsequent deglycosylation within the enterocyte by cytosolic beta-glucosidase (CBG). The other is the absorption by passive diffusion of quercetin after luminal hydrolysis of its glycones by lactase phlorizin hydrolase (LPH). Both methods seem to be utilized for the uptake of individual quercetin glycones with substrate selectivity. For instance, quercetin-4 -glucoside

OH OH B O

HO A

C OH

OH

Figure 1

O

The chemical structure of queretin.

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involves both an interaction with SGLT1, which is not able to transport aglycone quercetin, and a luminal hydrolysis by LPH, whereas quercetin-3-glucoside appears to be absorbed only following hydrolysis by LPH (13,112). The deglycosylation step seems critical for the absorption of quercetin glycosides in humans and is mediated by glucosidases such as LPH and CBG. The significant variation in their activity between individuals was even suggested as a potential cause for differences in flavonoid (quercetin) bioavailability (14). Even though the hydrolysis of the glycoside moiety from quercetin glycones is strongly believed to be a prerequisite process for quercetin absorption, there are also reports indicating that a fraction of quercetin may be absorbed as its glycones via passive diffusion and/or unidentified transporters (15,16). The discrepancy of absorption patterns between quercetin and its glycones can be accounted for by hydrolysis process of its glycoside and its diverse absorptive processes.

Metabolism Quercetin was reported to be metabolized mainly in the liver and the intestine. Absorbed quercetin is mainly metabolized in the liver, whereas the unabsorbed form can be metabolized in the gut by intestinal microorganisms. Eubacterium ramulus was reported as a microorganism involved in degrading quercetin in the intestine. This strictly anaerobic bacterium can cleave the ring structure of quercetin into 3,4-dihydroxyphenylacetic acid (17,18). Intestinal metabolism of quercetin by the microorganism may provide an alternative pathway for quercetin absorption in the gut, even though this absorption is likely to be insignificant and irrelevant to quercetin bioavailability. Primary metabolism of quercetin absorbed occurs in the liver, even though some minor metabolic processes take place in various human cells. Investigation of metabolites of quercetin in the plasma and urine revealed that the flavonol is metabolized by glucuronidation, hydroxylation, methylation, and sulfonylation. Glucuronidation occurs usually during passage across the epithelium as well as in the liver (19). The enzymes responsible for this process are UDP-glucuronosyltransferases (UGT) such as UGT1A9 (in human liver) and UGT1A1 and UGT1A8 (in intestinal epithelium) (15,20). In intestinal epithelial cell, quercetin was reported to metabolize mainly into quercetin-3- and quercetin-7-glucuronides (21). Quercetin glucuronides were also reported to metabolize further via two pathways: first, methylation of the catechol functional group of both quercetin glucuronides by methyltransferases; and second, hydrolysis of the glucuronide by endogenous beta-glucuronidase, followed by sulfation to quercetin-3 -sulfate (22,23). Quercetin is also methylated, sulfonylated, and hydroxylated in the liver (22,24). It is also found in plasma as unconjugated quercetin aglycone, even if quercetin glucuronides are considered as main circulating metabolites in humans. Occurrence of the quercetin aglycone within tissues is likely to result from the deconjugation of flavonoid glucuronides by the enzyme beta-glucuronidase. Indeed, some human tissues from the small intestine and the liver, and neutrophils, exhibit beta-glucuronidase activity against quercetin glucuronides (25). Multiple processes

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of quercetin metabolism may yield quercetin metabolites with different biochemical properties and produce some metabolites with more or less purported beneficial effects (26).

Bioavailability Bioavailability is the physiological availability of a compound in a given amount. In other words, it is the proportion of the administered amount that is absorbed into the bloodstream. Therefore, bioavailability depends mainly on the initial administered amount, absorption, metabolism, tissue distribution, and excretion. Pharmacokinetic studies are performed using several different doses and routes to determine the bioavailability of an administered compound, which can be used as a good guideline for safe human intake and a valuable criterion for verifying purported effects in in vitro studies. However, most pharmacokinetic studies of quercetin seem fragmentary and require further research to provide complete information regarding quercetin bioavailability. Originally, quercetin aglycone was thought to be able to pass through the gut wall better than its glycones. However, pharmacokinetic studies suggest that quercetin glycones are absorbed better than its aglycone (10,27,28). The studies also revealed that quercetin glycosides could show significant differences in absorption rate and bioavailability, depending on the glycosylation sites (27,28). Some pharmacokinetic studies of quercetin were performed using quercetin (aglycone) and its glycones to determine bioavailability. In the study using quercetin aglycone, it was reported that after a single 4 g dose of quercetin administered orally in humans, the flavonol was detected in the plasma. The highest peak plasma concentration (Cmax ) of quercetin was less than 100 ng/mL, and that all quercetins detected in the plasma were in the form of glucuronidated and sulfated quercetins rather than quercetin aglycone (1,29). In the same study, following single intravenous (100 mg) administration of quercetin, the highest peak plasma concentration (Cmax ) of quercetin was around 3000 ng/mL, and the time to reach Cmax (Tmax ) was 10 minutes (1,29). Human absorption of quercetin can be enhanced by quercetin conjugation with glucose (30). For instance, following ingestion of quercetin glycosides (in fried onions) equivalent to 64 mg of quercetin aglycone, Cmax was reported to be 196 ng/mL after 2.9 hours, which is higher than that following 100 mg of quercetin aglycone (31). The sugar moieties and positions of quercetin O-glycosides seem to influence their bioavailability as well (32–35). The difference in bioavailability between quercetin glycosides can also be attributed to their different solubility influencing their accessibility for absorption and to enzymes involved in the absorption (36,37). Currently, the average human intake of quercetin (glycones and aglycone) is less than 60 mg daily. On the basis of this amount, the highest concentration achieved in the plasma (Cmax ) is less than 200 ng/mL (0.6 ␮M), which includes quercetin and all its metabolites. In vivo effects of quercetin reflect its bioavailability, which can be changed on the basis of a given dose of the flavonol. Unfortunately, data on bioavailability in multiple doses are currently not available, requiring future investigation.

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CELLULAR AND MOLECULAR ACTIONS Effects of quercetin in humans are manifested in a dynamic environment. For instance, ingested quercetin (glycone and aglycone) undergoes absorption, metabolism, tissue distribution, and excretion. However, cellular and molecular actions of quercetin reported in in vitro studies are observed in a rather static environment, excluding many key physiological conditions. Also, the quercetin concentrations used in the many experiments discussed below are relatively too high to be achieved by dietary ingestion. Therefore, some biological activities reported in vitro should be regarded as having potential for future application of pharmacologic quercetin or quercetin analogs in preventing and/or treating human diseases, rather than as indicating direct use of quercetin and/or metabolites in human diseases. Flavonoids (quercetin) were once named vitamin P or vitamin C2 due to their abilities to decrease capillary permeability or spare vitamin C activities (2). Since then, quercetin has been viewed as a compound with both beneficial and harmful effects (38–40). However, it is currently recognized as a compound that is more helpful than deleterious, because epidemiological, cellular, and molecular studies of quercetin have suggested this (41,42). The antioxidant properties of quercetin are believed to explain its positive effects in a major way. The 5,7,3 ,4 -hydroxyl groups on quercetin are capable of donating electrons to quench various radical oxygen species (ROS) and other radical species (43,44), which have the potential to influence pathogenesis or treatment of chronic human diseases such as inflammation, cardiovascular diseases, and cancer.

Antioxidant Properties Oxygen radicals (superoxide, hydrogen peroxide, hydroxyl radicals, and other related radicals) have been reported to be involved in initiating and/or promoting inflammation, cardiovascular diseases, cancer, aging, and other chronic diseases (44). The radicals are quenched by endogenous antioxidant systems, including antioxidant compounds, which balance cellular redox status involved in cellular processes for cell homeostasis, such as proliferation, signaling transduction, and apoptosis (45,46). Therefore, it is proposed that improper redox balance can contribute to the progression of chronic human diseases such as inflammation, heart diseases, and cancer, and adequate intake of antioxidant chemicals from fruits and vegetables may afford significant protection against them. Generally, three criteria are considered to assess the antioxidant activity of flavonoids in vitro: first, B ring with two hydroxyl groups (adjacent); second, C ring with 2,3-double bond, 4-oxo, and 3-hydroxyl group; and third, A ring with 5,7dihydroxyl groups (2). Quercetin meets all three criteria, indicating stronger antioxidant activity than flavonoids that do not meet the criteria. Accordingly, the flavonol was reported to prevent radicals from damaging carbohydrates, proteins, nucleotides, and lipids (47). Quercetin is metabolized and found in the plasma as quercetin glucuronide conjugates, and other metabolites. What about their antioxidant properties? The glucuronide conjugates found in the plasma were also reported to have potent antioxidant activity, indicating that the activity may be

retained depending on conjugation positions (48). The antioxidant activity of quercetin is believed to contribute to its beneficial effects in a significant way.

Effects on Inflammation Inflammation is the first and necessary response of the immune system to infection and others. Inflammation is supposed to fight against on-going infection as well as initiate healing processes. However, when inflammation is not properly under control, it may attribute to developing chronic diseases (e.g., arthritis, allergy, asthma, atherosclerosis, cancer, and aging) (49–51). Inflammation processes are complex and highly orchestrated with numerous biological factors including eicosanoids, cytokines, and other immune factors. Eicosanoids are chemical mediators synthesized by cells in response to local tissue damage as well as hormonal and immunological stimuli (52,53). Eicosanoids (e.g., prostaglandins and thromboxanes) are involved in inflammatory processes via binding to their cognate receptors on a wide variety of tissues throughout the body (53). Cyclooxygenase (COX) is the key enzyme metabolizing arachidonic acid to generate prostaglandin H2, which is converted by downstream enzymes to other prostaglandins and thromboxanes (54). Therefore, COX enzyme has been a molecular target for developing antiinflammatory drugs. COX enzymes consist of two COX isoforms: COX-I and -II (55,56). COX-1 is constitutively expressed in nearly all tissues, but its activity is most strongly associated with prostaglandin production in gastric mucosa and thromboxane production in platelets. Meanwhile, COX-2 expression is upregulated in response to inflammatory stimuli. Increased prostaglandin levels are often considered as a part of inflammatory responses (56). Several in vitro studies indicated that quercetin is able to inhibit COX-I and COX-II enzymes, thereby potentially providing anti-inflammatory effects via modulating eicosanoid effects on immunological and other processes (49–51) Also, inflammation mediates various immunological effects in infected tissues. For instance, tumor necrosis factor-alpha (TNF-alpha) generated by activated macrophages induces several pathophysiological conditions during acute and chronic inflammation. Quercetin was reported to inhibit TNF-alpha overproduction and attenuate pathophysiological conditions during acute and chronic inflammation (57–60). The inhibition is not surprising at all, because quercetin is believed to inhibit a transcription factor NF-kappaB involved in the production of various immunological molecules such as TNF, IL-1beta, and iNOS. However, this seems not a specific action of only quercetin (61,62). Asthma is another wellknown inflammatory disease, characterized by constriction of lung airways. During asthma attack, the activation of mast cells and basophils by allergen releases chemical mediators and synthesizes cytokines leading to inflammatory conditions. Among these cytokines, interleukins IL-4, IL-13, and IL-5 are major ones involved in allergic inflammation. Quercetin was reported to inhibit the expression and syntheses of these cytokines in human basophils (63). In one study, a metabolite of quercetin, 3-O-methylquercetin (3-MQ), was even reported to provide beneficial effects on asthma by inhibiting cAMP- and cGMP-phosphodiesterase (PDE), counteracting harmful

Quercetin

effects of the cytokines, etc. (64). Although some reports related to beneficial effects of quercetin on inflammation are available, the reported effects of quercetin are still preliminary, necessitating future studies prior to any firm conclusions.

Effects on Cardiovascular Diseases Cardiovascular disease, also known as coronary artery disease, represents the class of diseases related to the heart and/or blood vessels such as arteries and veins where inflammation is deeply involved. One representative example of cardiovascular diseases is atherosclerosis of the coronary arteries. Atherosclerosis is a condition in which the arteries become clogged and narrowed, and the restriction of blood flow to the heart occurs. There are accumulating data indicating that quercetin is associated with beneficial effects on cardiovascular diseases with inflammatory complications. Some epidemiologic studies show that eating a diet rich in flavonoids is associated with decreased incidence of cardiovascular diseases. Quercetin has biological properties consistent with its purported effects on the cardiovascular system. For instance, quercetin has been shown to protect low-density lipoprotein (LDL) from oxidation and prevent platelet aggregation (65). It was also reported to inhibit the proliferation and migration of smooth muscle cells. These findings provide new insights and a rationale for the potential use of quercetin for preventing cardiovascular diseases. Currently, there are numerous reports supporting beneficial effects of quercetin on cardiovascular diseases. For instance, quercetin was reported to significantly lower the plasma lipid, lipoprotein, and hepatic cholesterol levels, inhibit the production of oxLDL produced by oxidative stress, and protect an enzyme that can hydrolyze specific lipid peroxides in oxidized lipoproteins and in atherosclerotic lesions (66–68). Quercetin was also reported to even induce endotheliumdependent vasorelaxation in rat aorta via increasing nitric oxide production (69). These data suggest that the protective effects of quercetin on heart diseases may result from its arterial, venous, and coronary vasodilator effects (70). The cardiovascular protective effects of quercetin may also play significant roles in attenuating hypertension. Hypertension (high blood pressure) is a condition in which the force of blood flow against artery walls is too strong. Hypertension and its related events are able to damage arteries, heart, and kidneys, leading to the progression of several types of cardiovascular diseases including atherosclerosis and stroke (71,72). Angiotensin-converting enzyme (ACE) is a well-known peptidase in the renin– angiotensin system (RAS) regulating blood pressure via controlling extracellular volume and vascular constriction through converting angiotensin I to angiotensin (Ang) II. Quercetin and its glycosides were reported to inhibit the ACE activity and also suppress Ang II–induced c-Jun Nterminal kinase activation inducing vascular smooth muscle cell (VSMC) hypertrophy (73,74). These findings suggest that the positive health effects of the flavonol on heart diseases are executed via inhibiting signal transduction pathways leading to the diseases including atherosclerosis and hypertension (75,76). Quercetin was also reported to inhibit platelet aggregation that can be beneficial for lessening conditions of cardiovascular disease (77,78). On

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the basis of all these findings, quercetin seems to have potential in the prevention and/or treatment of cardiovascular diseases. However, some effects may not be feasible or negligible in physiological conditions, because concentrations of quercetin in most studies are often too high to be achieved by dietary ingestion of quercetin. In summary, there are beneficial effects of quercetin in relation to heart disease, but many areas of uncertainty exist.

Effects on Diabetes Diabetes is a disease condition in which the body does not produce or use insulin properly, a hormone needed to convert glucose, carbohydrates, etc., into energy needed for daily life (79–83). There are two major types of diabetes, type 1 and type 2. Both types can cause blood sugar levels higher than normal. However, their mechanisms causing diabetes are different. Type 1 diabetes (insulin-dependent diabetes or juvenile diabetes) is attributed from the inability of the pancreas to produce the hormone insulin. It happens probably because the person’s own immune system jeopardizes the insulin producing cells in the pancreas. However, type 2 diabetes (non–insulin-dependent diabetes or adult onset diabetes) is different from type 1 diabetes. In type 2 diabetes, there is an inability of tissues to respond to insulin normally, even though insulin is produced (81,82). The cause of type 2 diabetes is yet to be elucidated in detail, even though there are data suggesting that both genetics and environmental factors such as obesity and lack of exercise may play roles. Patients with type 2 diabetes represent the largest portion by far of total diabetic patients compared to other types. In type 2 diabetes, the age of onset and disease progression are variable, and complications are related to control of blood glucose. There are a great number of reports indicating that flavonoids including quercetin may attenuate heath conditions and side effects derived from diabetes (84–86). In fact, potential effects of flavonoids on diabetes were highly speculated originally on the basis of an old phlorizin study (87). In the study, the reabsorption of glucose in kidney was greatly attenuated upon the intravenous administration of a relative high dose of phlorizin, even suggesting that phlorizin may be a toxic compound. Phlorizin is a glycosylated form of chalcone phloretin belonging to the subclass of flavonoids. Therefore, flavonoids have been believed to contain similar effects on glucose absorption in intestinal and other organs. In fact, quercetin-O-glycosides were proposed to interact with glucose transporters (88,89). Quercetin glycosides [quercetin-3-glucoside (isoquercitrin) and quercetin-4 -glucoside (spiraeosid)] were reported to inhibit mucosal uptake of the nonmetabolizable glucose analog methyl-alpha-D-glucopyranoside (MDG) (89). In another study, quercetin and several flavonoids were stated to prevent glucose uptake by blocking sodiumindependent glucose transporters (Glut-1 and -3) (90). Glucose and dehydroascorbic acid (oxidized vitamin C) uptakes (see chap. 92, “Vitamin C”) are interconnected in some ways, such that some sodium-independent glucose transporters are involved in both glucose and dehydroascorbic acid uptake. In HL-60, U937, and Jurkat cells, quercetin was reported to inhibit the intracellular accumulation of ascorbic acid by blocking uptake of both

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dehydroascorbic acid and ascorbic acid (vitamin C). These data may indicate new understanding of the biological effect of flavonoids on glucose and vitamin C uptake in human cells, and future application of quercetin on diseases involving abnormal glucose utilization (91,92). However, potential effects of quercetin on intestinal glucose absorption have not been investigated completely, thereby necessitating more future studies.

Effects on Cancer Human cancers are caused by numerous oncogenic and other factors, and oxidative stress is believed a key culprit implicated in the initiation and propagation of cancer. Many epidemiological studies suggest that dietary intake of quercetin may have beneficial effects on various types of human cancers, mostly via inhibiting oxidative stress (93). Lately, numerous studies have been performed for elucidating potential anticancer effects of quercetin in each type of human cancer to bolster findings from epidemiological studies. Deoxynucleotide acid (DNA) damage by oxygen radicals can be detrimental for normal cells and can change them into cancer cells. Routinely, 8hydroxy-2 -deoxyguanosine and related compounds are used as biomarkers to assess levels of DNA damage. Quercetin was shown to increase resistance of lymphocyte DNA to strand breakage, thereby decreasing the level of urinary 8-hydroxy-2 -deoxyguanosine (41). In several reports, quercetin has been described to have potent anticancer effects against various cancers by inducing programmed cell death (apoptosis). In human myelogenous leukemia cells, quercetin was reported to arrest growth of the cell by an increase in the uptake of vincristine, a chemotherapeutic agent (94). In pancreatic tumor cells, the flavonol was reported to induce cell death via inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase activity and decreasing protein phosphorylation (95). In prostate cancer cells, quercetin was stated to inhibit cell growth via suppressing protein phosphorylation (96). Quercetin was also shown to induce growth inhibition and apoptosis in MCF-7 human breast cancer cells. All these data suggest that quercetin may induce growth inhibition and apoptosis in human cancer cells by inhibiting receptor and cell cycle–related kinases and others (97). However, quercetin may have other actions against cancer cells. For instance, gastrointestinal cancer is a cancer associated with dietary and lifestyle factors. In an animal study, quercetin increased both small and large intestinal UGT enzyme activities, thereby helping detoxify compounds with carcinogenic potentials, and preventing gastrointestinal cancer. There are also several reports indicating that quercetin can have beneficial effects on colon and skin cancers (98–100). Taken together, quercetin may have capability to protect cells against mutagenic agents as well as to suppress cancerous growth via modulating numerous biological molecules via anti- and/or pro-oxidant properties. Although quercetin seems to have potential as an anticancer agent, future studies are needed. Human data come mainly from epidemiologic association studies rather than from intervention trials. Most experiments in vitro used quercetin concentrations that were too high to be achieved by dietary ingestion. In addition, beneficial

effects of quercetin and cancer are not consistently found in animal and human studies.

Effects on Bone Formation Bone formation is a balance between two major processes: bone formation by osteoblasts and bone resorption by osteoclasts. Many hormonal and biologic factors are involved, such as growth hormone, thyroid hormone, parathyroid hormone, calcitonin, estrogens, androgens, cytokines, vitamin D, and transcription factors (e.g., nuclear factor kappa B) (101–103). Several studies suggest that the receptor activator of nuclear factor kappa B (NF-␬B) ligand (RANKL) expressed on the cell surface of osteoblasts is an important factor in response to boneresorptive processes, because osteoclast precursors possess RANK (a receptor for RANKL), and the cells can differentiate into mature osteoclasts via a mechanism of RANK–RANKL recognition (103,104). During bone resorption, prostaglandin E2 (PGE2) is also produced by osteoblasts, acting as a potent stimulator of bone resorption (104). Inflammatory cytokines such as IL-1 and IL6 are known to induce PGE2 production by osteoblasts, and the produced PGE2 stimulates adenylate cyclase to accumulate cellular cAMP in osteoblasts, which induces the expression of RANKL for osteoclast differentiation (101). Quercetin and its conjugate were reported to inhibit the receptor activator of RANKL-induced osteoclast differentiation and the RANKL-stimulated expression of osteoclast-related genes. As mentioned above, quercetin and its glycosides are able to inhibit COX-I and COX-II, as well as NF-␬B, which may be involved in modulation bone-resorption processes. Although a number of reports related to potential effects of quercetin on bone homeostasis are available, the reported effects are premature, necessitating future studies before any firm decision.

Other Effects on Human Health Besides the beneficial effects mentioned above, there are also some interesting biological effects of quercetin on cognitive functions, aging, UV ray protection, and others (112–115). Nonetheless, their effects requires more data, particularly epidemiologic studies, before supporting the claimed actions.

INDICATIONS AND USAGE Quercetin is widely distributed in plant-derived dietary sources such as onions, apples, black tea, green tea, red wine, beans, and grapes (7). Humans have consumed quercetin (mostly quercetin glycosides) from dietary food sources, and the estimated average daily intake of quercetin by an individual in the United States is estimated to be less than 60 mg. Currently, no dietary recommendations regarding estimated average requirement (EAR), recommended dietary allowance (RDA), adequate intake (AI), and upper limit (UL) have been set for quercetin. Also, epidemiological studies exploring its role in human health are inconclusive (105). Future research is required because of the many biological activities

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attributed to quercetin, some of which could be beneficial or detrimental depending on the circumstances.

of quercetin supplement, because its adverse effects are yet be determined in humans (111).

Potential Uses

Compendial/Regulatory Status

Quercetin intake is associated with benefit in some chronic human diseases including cardiovascular diseases, inflammatory diseases, and some cancers. Some epidemiologic studies reveal an inverse association between quercetin intake and cardiovascular diseases, and the flavonol was shown to contain biological properties consistent with its purported effects on the cardiovascular system (106). However, the beneficial effects of quercetin on heart diseases are still inconclusive, and further studies are needed to prove these unambiguously (107). Quercetin was also reported to have anti-inflammatory potential through numerous in vitro studies. Epidemiological studies regarding effects of quercetin on inflammation are solid but still incomplete. Therefore, further research is necessary to demonstrate that inflammation can be benefited by quercetin intake. Quercetin was also stated to have anticancer potential through numerous in vitro studies. Epidemiological studies regarding effects of quercetin on cancer are less comprehensive than those on cardiovascular diseases and inflammation. In some experiments, the positive correlation between quercetin intake and risk of cancer was found, but none was found in others. Therefore, further research is necessary to demonstrate that the risk of cancer can be lowered by quercetin intake. Quercetin was also claimed as a potential compound for other human diseases such as diabetes mellitus, bone formation, and cognitive functions. However, these claims have little scientific evidence confirming its efficacy against these diseases. To note, the solubility of quercetin is comparatively lower than other well-known flavonoids (e.g., catechins, anthocyanins), which may pose a significant disadvantage in using the compound as a potential dietary supplement. In summary, the potential uses discussed herein do not suggest that quercetin supplements have therapeutic use for the prevention and treatment of the diseases. Its health effects require further study.

ADVERSE EFFECTS Although current studies emphasize beneficial effects of quercetin on cardiovascular diseases, inflammation, and cancer, its high doses are believed to have mutagenic and genotoxic activities as demonstrated in in vitro systems (108–110). Even though in vitro experiments indicate that there might be adverse effects, the concentrations in the experiments may have been too high. From animal experiments, there was also uncertainty regarding side effects of quercetin. One study was conducted using F344/N rats that were fed daily quercetin doses of 40, 400, and 1900 mg/kg, for longer than one year. No toxicity occurred at one year. However, after a longer duration, some male rats had renal tubule cell adenomas, but female rats did not. It was not clear whether quercetin induced these adenomas. Because of the uncertainties, it is suggested that quercetin should be consumed from dietary food sources, and caution should be exercised when taking high doses

Not applicable.

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20. Boersma MG, van der Woude H, Bogaards J, et al. Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol 2002; 15:662–670. 21. Gee JM, DuPont MS, Day AJ, et al. Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J Nutr 2000; 130:2765–2771. 22. O’Leary KA, Day AJ, Needs PW, et al. Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: The role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multiresistant protein 2 (MRP2) in flavonoid metabolism. Biochem Pharmacol 2003; 65:479–491. 23. Sesink AL, O’Leary KA, Hollman PC. Quercetin glucuronides but not glucosides are present in human plasma after consumption of quercetin-3-glucoside or quercetin-4 glucoside. J Nutr 2001; 131:1938–1941. 24. De Santi C, Pietrabissa A, Mosca F, et al. Methylation of quercetin and fisetin, flavonoids widely distributed in edible vegetables, fruits and wine, by human liver. Int J Clin Pharmacol Ther 2002; 40:207–212. 25. O’Leary KA, Day AJ, Needs PW, et al. Flavonoid glucuronides are substrates for human liver betaglucuronidase. FEBS Lett 2001; 503:103–106. 26. Breinholt VM, Offord EA, Brouwer C, et al. In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids. Food Chem Toxicol 2002; 40:609–616. 27. Mullen W, Graf BA, Caldwell ST, et al. Determination of flavonol metabolites in plasma and tissues of rats by HPLCradiocounting and tandem mass spectrometry following oral ingestion of [2-(14)C]quercetin-4 -glucoside. J Agric Food Chem 2002; 50:6902–6909. 28. Hollman PC, van Trijp JM, Buysman MN, et al. Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Lett 1997; 418:152–156. 29. Gugler R, Leschik M, Dengler HJ. Disposition of quercetin in man after single oral and intravenous doses. Eur J Clin Pharmacol 1975; 9:229–234. 30. Hollman PC, de Vries JH, van Leeuwen SD, et al. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr 1995; 62: 1276–1282. 31. Hollman PC, van der Gaag M, Mengelers MJ, et al. Absorption and disposition kinetics of the dietary antioxidant quercetin in man. Free Radic Biol Med 1996; 21:703– 707. 32. Graefe EU, Wittig J, Mueller S, et al. Pharmacokinetics and bioavailability of quercetin glycosides in humans. J Clin Pharmacol 2001; 41:492–499. 33. Olthof MR, Hollman PC, Vree TB, et al. Bioavailabilities of quercetin-3-glucoside and quercetin-4 -glucoside do not differ in humans. J Nutr 2000; 130:1200–1203. 34. Walle T, Otake Y, Walle UK, et al. Quercetin glucosides are completely hydrolyzed in ileostomy patients before absorption. J Nutr 2000; 130:2658–2661. 35. Morand C, Manach C, Crespy V, et al. Respective bioavailability of quercetin aglycone and its glycosides in a rat model. Biofactors 2000; 12:169–174. 36. Khaled KA, El-Sayed YM, Al-Hadiya BM. Disposition of the flavonoid quercetin in rats after single intravenous and oral doses. Drug Dev Ind Pharm 2003; 29:397–403. 37. Shimoi K, Yoshizumi K, Kido T, et al. Absorption and urinary excretion of quercetin, rutin, and alphaG-rutin, a water soluble flavonoid, in rats. J Agric Food Chem 2003; 51:2785– 2789. 38. Ross JA, Kasum CM. Dietary flavonoids: Bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002; 22:19– 34.

39. Vrijsen R, Michotte Y, Boeye A. Metabolic activation of quercetin mutagenicity. Mutat Res 1990; 232:243–248. 40. Nakayasu M, Sakamoto H, Terada M, et al. Mutagenicity of quercetin in Chinese hamster lung cells in culture. Mutat Res 1986; 174:79–83. 41. Boyle SP, Dobson VL, Duthie SJ, et al. Absorption and DNA protective effects of flavonoid glycosides from an onion meal. Eur J Nutr 2000; 39:213–223. 42. Duthie SJ, Collins AR, Duthie GG, et al. Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. Mutat Res 1997; 393:223–231. 43. Rice-Evans C. Flavonoid antioxidants. Curr Med Chem 2001; 8:797–807. 44. Uddin S, Ahmad S. Antioxidants protection against cancer and other human diseases. Compr Ther 1995; 21:41–45. 45. Jacob RA, Burri BJ. Oxidative damage and defense. Am J Clin Nutr 1996; 63:985S–990S. 46. Moran LK, Gutteridge JM, Quinlan GJ. Thiols in cellular redox signalling and control. Curr Med Chem 2001; 8:763– 772. 47. Feng Q, Kumagai T, Torii Y, et al. Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress. Free Radic Res 2001; 35:779–788. 48. Day AJ, Bao Y, Morgan MR, et al. Conjugation position of quercetin glucuronides and effect on biological activity. Free Radic Biol Med 2000; 29:1234–1243. 49. Homaidan FR, Chakroun I, Haidar HA, et al. Protein regulators of eicosanoid synthesis: Role in inflammation. Curr Protein Pept Sci 2002; 3:467–484. 50. Dogn´e JM, Hanson J, Pratico D. Thromboxane, prostacyclin and isoprostanes: Therapeutic targets in atherogenesis. Trends Pharmacol Sci 2005; 26:639–644. 51. Cl`aria J, Romano M. Pharmacological intervention of cyclooxygenase-2 and 5-lipoxygenase pathways. Impact on inflammation and cancer. Curr Pharm Des 2005; 11:3431– 3447. 52. Boyce JA. Eicosanoids in asthma, allergic inflammation, and host defense. Curr Mol Med 2008; 8:335–349. 53. Jenkins CM, Cedars A, Gross RW. Eicosanoid signalling pathways in the heart. Cardiovasc Res 2009; 82:240–249. 54. Smith WL, Meade EA, DeWitt DL. Pharmacology of prostaglandin endoperoxide synthase isozymes-1 and -2. Ann N Y Acad Sci 1994; 18:136–142. 55. Rouzer CA, Marnett LJ. Cyclooxygenases: Structural and functional insights. J Lipid Res 2009; 50:S29–S34. 56. Scher JU, Pillinger MH. The anti-inflammatory effects of prostaglandins. J Investig Med 2009; 57:703–708. 57. Manthey JA. Biological properties of flavonoids pertaining to inflammation. Microcirculation 2000; 7:S29–S34. 58. Miller AL. The etiologies, pathophysiology, and alternative/complementary treatment of asthma. Altern Med Rev 2001; 6:20–47. 59. Manjeet KR, Ghosh B. Quercetin inhibits LPS-induced nitric oxide and tumor necrosis factor-alpha production in murine macrophages. Int J Immunopharmacol 1999; 21:435– 443. 60. Bito T, Roy S, Sen CK, et al. Flavonoids differentially regulate IFN gamma-induced ICAM-1 expression in human keratinocytes: Molecular mechanisms of action. FEBS Lett 2002; 520:145–152. 61. Kim BH, Lee IJ, Lee HY, et al. Quercetin 3-O-beta-(2 galloyl)-glucopyranoside inhibits endotoxin LPS-induced IL-6 expression and NF-kappa B activation in macrophages. Cytokine 2007; 39:207–215. ¨ 62. Ruiz PA, Braune A, Holzlwimmer G, et al. Quercetin inhibits TNF-induced NF-kappaB transcription factor recruitment to proinflammatory gene promoters in murine intestinal epithelial cells. J Nutr 2007; 137:1208–1215.

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63. Higa S, Hirano T, Kotani M, et al. Fisetin, a flavonol, inhibits TH2-type cytokine production by activated human basophils. J Allergy Clin Immunol 2003; 111:1299–2306. 64. Ko WC, Chen MC, Wang SH, et al. O-Methylquercetin more selectively inhibits phosphodiesterase subtype 3. Planta Med 2003; 69:310–315. 65. Formica JV, Regelson W. Review of the biology of quercetin and related bioflavonoids. Food Chem Toxicol 1995; 33:1061–1080. 66. Bok SH, Park SY, Park YB, et al. Quercetin dihydrate and gallate supplements lower plasma and hepatic lipids and change activities of hepatic antioxidant enzymes in high cholesterol-fed rats. Int J Vitam Nutr Res 2002; 72:161– 169. 67. Pal S, Ho N, Santos C, et al. Red wine polyphenolics increase LDL receptor expression and activity and suppress the secretion of ApoB100 from human HepG2 cells. J Nutr 2003; 133:700–706. 68. Fuhrman B, Aviram M. Preservation of paraoxonase activity by wine flavonoids: Possible role in protection of LDL from lipid peroxidation. Ann N Y Acad Sci 2002; 957: 321–324. 69. Taubert D, Berkels R, Klaus W, et al. Nitric oxide formation and corresponding relaxation of porcine coronary arteries induced by plant phenols: Essential structural features. J Cardiovasc Pharmacol 2002; 40:701–713. 70. Ibarra M, Perez-Vizcaino F, Cogolludo A, et al. Cardiovascular effects of isorhamnetin and quercetin in isolated rat and porcine vascular smooth muscle and isolated rat atria. Planta Med 2002; 68:307–310. 71. Sheps SG, Kirkpatrick RA. Hypertension. Mayo Clin Proc 1975; 50:709–720. 72. Kaplan NM. Resistant hypertension. J Hypertens 2005; 23:1441–1444. 73. Hackl LP, Cuttle G, Dovichi SS, et al. Inhibition of angiotensin-converting enzyme by quercetin alters the vascular response to bradykinin and angiotensin I. Pharmacology 2002; 65:182–186. 74. Melzig MF, Escher F. Induction of neutral endopeptidase and angiotensin-converting enzyme activity of SK-N-SH cells in vitro by quercetin and resveratrol. Pharmazie 2002; 57:556–558. 75. Yoshizumi M, Tsuchiya K, Kirima K, et al. Quercetin inhibits Shc- and phosphatidylinositol 3-kinase-mediated c-Jun Nterminal kinase activation by angiotensin II in cultured rat aortic smooth muscle cells. Mol Pharmacol 2001; 60:656– 665. 76. Yoshizumi M, Tsuchiya K, Suzaki Y, et al. Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the inhibition of JNK and AP-1 signaling pathway. Biochem Biophys Res Commun 2002; 293:1458–1465. 77. Pignatelli P, Pulcinelli FM, Celestini A, et al. The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide. Am J Clin Nutr 2000; 72:1150–1155. 78. Di Santo A, Mezzetti A, Napoleone E, et al. Resveratrol and quercetin down-regulate tissue factor expression by human stimulated vascular cells. J Thromb Haemost 2003; 1:1089– 1095. 79. Mercado MM, McLenithan JC, Silver KD, et al. Genetics of insulin resistance. Curr Diab Rep 2002; 2:83–95. 80. Costacou T, Mayer-Davis EJ. Nutrition and prevention of type 2 diabetes. Annu Rev Nutr 2003; 23:147–170. 81. Sharma MD, Garber AJ, Farmer JA. Role of insulin signaling in maintaining energy homeostasis. Endocr Pract 2008; 14:373–380. 82. Chang-Chen KJ, Mullur R, Bernal-Mizrachi E. Beta-cell failure as a complication of diabetes. Rev Endocr Metab Disord 2008; 9:329–343.

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Red Clover Elizabeth C. Krause, Nancy L. Booth, Colleen E. Piersen, and Norman R. Farnsworth

INTRODUCTION

widespread around the globe. It has been cultivated since the 4th century AD and is used as livestock fodder and sometimes as green fertilizer to replenish the soil. Consumption as a food is not widespread, except occasionally as young sprouts or cooked greens. The general habit of RC is described as having “several stems arising from the same root, ascending, somewhat hairy, and varying much in its height. The leaves are ternate; the leaflets oval or obovate, entire, nearly smooth, often notched at the end, and lighter colored in the center. Stipules ovate and mucronate” (1). Flowers occur “in short, dense, ovate, sessile spikes or heads. Corollas unequal, monopetalous; lower tooth of the calyx longer than the four others, which are equal, and all shorter than the rose-red corolla” (1). Flower heads are “globose or ovoid, from 1.5 to 3 cm in length, consisting of numerous purplish red or pinkish brown papilionaceous flowers, about

Red clover (RC) is a herbaceous perennial plant that inhabits temperate and subtropical areas throughout the world. Native Americans traditionally valued RC for treatment of external skin problems and lung, nervous, and reproductive system ailments. Herbalists have employed it as a blood cleanser, expectorant, alterative, and sedative. With the recognition of its high content of mildly estrogenic isoflavones, the plant has gained popularity as a treatment for menopausal symptoms. Clinical evidence is presently lacking to support the efficacy of semipurified RC isoflavone extracts for alleviation of vasomotor and prostate cancer/benign prostatic hyperplasia (BPH) symptoms. Limited evidence suggests possible efficacy in prevention of osteoporosis and improvement of arterial compliance, a risk factor for atherosclerosis. Current RC isoflavone products do not contain a protein fraction, which precludes analogous comparison with isoflavone studies utilizing dietary soy [Glycine max (L.) Merr.] or soy protein isolates (SPIs) for various clinical outcomes. RC isoflavone extract preparations may also differ from soy isoflavone extracts in content of minor chemicals, many of which have not yet been identified, quantified, or tested for biological activity. The need for long-term studies of RC isoflavone supplements is great as placebo effects, especially for menopausal symptoms, and may persist several weeks. Also, thyroid disease and cancer patients may face a potential, yet undefined, risk from long-term exposure to isoflavones and other compounds in RC.

BOTANICAL NAME Trifolium pratense L. (Fabaceae or Leguminosae).

COMMON NAMES Bee bread, cleaver grass, clover-grass, clover rose, cow clover, creeping clover, honeysuckle, klever lugovoi, ladies’ posy, meadow clover, meadow trefoil, purple clover, red clover, rozheva konyushina, sweet clover, three-leaved grass, treboil, trefoil, trevor, wild clover, and wild red clover (Fig. 1).

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Figure 1 Drawing of RC. Source: From the USDA-NRCS PLANTS Database; Britton, N.L, Brown, A. Illustrated Flora of the Northern States and Canada; 1913; Vol. 2, 355.

BOTANICAL DESCRIPTION Trifolium pratense L. is a low-growing perennial herb that originated in the Mediterranean area and is now 665

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10 mm in length; calyx pubescent, and with subulate teeth shorter than the corolla; odor fragrant; taste somewhat sweetish and bitter” (2). Trifolium pratense should not be confused with the similarly named yellow or white “sweet clovers,” Melilotus officinalis (L.) Pall. and Melilotus alba Medikus. The flowers of M. officinalis “are in small spike-like racemes with a papilionaceous corolla and about 3 mm in length and when fresh, yellow, but on drying, a yellowish brown. The odor is fragrant, resembling coumarin, and the taste slightly bitter and pungent” (2). Melilotus alba has white flowers and is similar in appearance to M. officinalis.

FOLKLORIC, HISTORIC, AND ETHNOMEDICAL USES External Red clover blossoms were incorporated into ointments or decocted to make compresses for “ulcers,” believed by more recent authors to be cancerous lesions or growths. These preparations are also used to treat burns, bites, wounds, gout, and fungal infections. The expressed juice has been used for eye diseases.

Internal Cherokee Indians made a tea of the flowers or aboveground parts to treat fevers, “Bright’s disease” (nephritis), and leukorrhea (3). The Iroquois referred to a decoction of RC flowers as a “blood medicine.” The Ute of Nevada used a decoction as an abortifacient (4). Tea or tincture was utilized for the spasmodic coughs of whooping cough, measles, bronchitis, laryngitis, and tuberculosis in the 19th and 20th centuries (1). Red clover cigarettes were a treatment for asthma according to the National Formulary. Decoctions and infusions are still used as expectorants, alteratives, sedatives, and remedies for rheumatism, ulcers, and skin conditions. Less frequently, its utility for normalization of menses, lactogogue action, or as a fertility tonic has been reported. In 1900, a product named “Extract of Trifolium Compound,” produced by the Wm. S. Merrell Chemical Company (Cincinnati, OH), contained potassium iodide plus extracts of the following plants: T. pratense L., Stillingia sylvatica L., Lappa minor Hill, Phytolacca decandra L., Cascara amarga, Berberis aquifolium Pursh, Podophyllum peltatum L., and Xanthoxylum carolinianum (1). This preparation was recommended for treatment of syphilis, scrofula, chronic rheumatism, and glandular and various skin afflictions. The formula for the Hoxsey internal cancer remedy has likely changed over time and been customized for individual patients. However, it has probably contained, at one time or another, the following plants (plus potassium iodide): Phytolacca americana L., Arctium lappa L., B. vulgaris L., Rhamnus frangula L., S. sylvatica L., Zanthoxylum americanum Mill., C. sagrada and/or C. amarga, Glycyrrhiza glabra L., Medicago sativa L., and T. pratense L. Flor-EssenceTM , currently manufactured by Flora, Inc. (Lynden, Washington, D.C.) and Flora Manufacturing & Distributing, Ltd. (Burnaby, British Columbia, Canada), is sometimes used by cancer patients and contains the following plant extracts: A. lappa L., T. pratense L., Cnicus benedictus L., Ulmus rubra Muhl., Rumex acetosella L.,

Rheum palmatum L., Laminaria digitata Lmx., and Nasturtium officinale R. Br.

CHEMICAL CONSTITUENTS Red clover contains several general classes of compounds but is particularly rich in isoflavones, flavones, and flavonols. Both soy and RC contain the isoflavones genistein and daidzein, and soy may contain small amounts of formononetin and biochanin A. However, RC contains substantially more formononetin and biochanin A, relative to genistein and daidzein, when compared to soy. Under storage conditions of greater than 13% moisture, RC may become contaminated with the fungus Rhizoctonia legumicola, which produces the indolizidine alkaloids slaframine and swainsonine. The latter alkaloid causes lysosomal storage disease and may precipitate “locoism” (i.e., weakness, lack of coordination, trembling, and partial paralysis) in livestock. Slaframine is activated by liver metabolism to form a ketoimine that stimulates muscarinic receptors, causing excessive salivation, lacrimation, frequent urination, diarrhea, bradycardia, and bradypnea in livestock.

Compounds Used for Standardization The isoflavones genistein, daidzein, formononetin, and biochanin A are currently used to standardize chemical content of commercial RC products (Fig. 2). Standardization is based on the rationale that these four compounds exhibit significant in vitro estrogenic activity. However, RC contains various other isoflavones and compounds from distinct structural classes, some with unknown biological activity.

PHARMACOKINETICS AND METABOLISM The RC isoflavones exist in the plant primarily as inactive glucosides (genistin, daidzin, ononin, and sissotrin) or malonated glucoside forms. Microflora in the human digestive tract hydrolyzes these conjugates to their bioactive, aglycone counterparts, which are readily absorbed from the intestine. Formononetin and biochanin

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HO

O

OCH3

Formononetin

HO

O

O Daidzein

O

OH O

OCH3

Biochanin A

HO

OH

O

OH O

OH

Genistein

Figure 2 Chemical structures of the four isoflavones used in standardization of RC products.

Red Clover

O

HO

Formononetin

O OH Biochanin A

OCH3

O

HO

H3CO

O

O

O

OH

OH

OH

+ OH

+ HO

OH

OH O OH 5-Hydroxy-O-desmethylangolensin

OH

HO

O

(+/–)-Dihydrogenistein

O OH O -Desmethylangolensin

HO

OH

OH

(+/–)-Equol

HO

OH

OH

O

HO

OH

O

O Genistein

O

HO

(+/–)-Dihydrodaidzein

HO

OH

OH

Prunetin

O

OCH3

O

HO

O

OH

OH

Daidzein

HO

O

HO

O

667

OH

+

OH HO

2,4,6-Trihydroxybenzoic acid

Figure 3

O 1,3,5-Trihydroxybenzene 2-(4-Hydroxyphenyl)- p-Ethylphenol propionic acid

Structures and metabolism of the main isoflavones in RC.

A are demethylated to daidzein and genistein, respectively in the gut and the liver (Fig. 3). Prunetin, a minor component of RC, may also be converted to genistein. Only 30% to 40% of individuals can metabolize (+/−)dihydrodaidzein, a daidzein metabolite, to the potent estrogen equol. Hepatic phase II enzymes catalyze formation of isoflavone glucuronides and sulfates. Human hepatic microsomal enzymes, like gut bacteria, demethylate the 4 -O-methylated isoflavones in vitro. Isoflavones circulate in the plasma mainly as conjugates and then are excreted in urine or bile, or undergo enterohepatic circulation. Data on the tissue and fluid distribution of isoflavones in humans are limited, but isoflavones and/or isoflavone conjugates are present in prostatic fluid and secreted into breast milk. Interindi-

vidual variability is substantial, but it is estimated that isoflavone (98–99% conjugated) plasma concentrations will reach 1 ␮M in a 70-kg individual who has consumed a single 50 mg dose (5). The pharmacokinetic parameters associated with long-term administration of RC isoflavones suggest that once-daily dosing is adequate.

PHARMACOLOGICAL ACTIVITY Hormonal Effects Estrogen Receptor Research into RC mechanism of action has largely focused on interactions with estrogen receptors (ER), ER-␣ and ER␤. Crude, hydroalcoholic extracts of RC inhibit binding of

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(3) H-17␤-estradiol to endogenous and purified recombinant ER in a number of test systems. Components of RC extracts bind preferentially to ER-␤ over ER-␣, earning RC its categorization as a natural selective estrogen receptor modulator (SERM). Pure isoflavones also bind preferentially to ER-␤ but exhibit competitive binding at both receptor subtypes with the same rank order of potency: genistein > daidzein > biochanin A ∼ prunetin > formononetin. Pike et al. (6) resolved the crystal structure of genistein bound to the ER-␤ ligand-binding domain. The ER-␤:17␤-estradiol structure remains unsolved, but the ER-␣:17␤-estradiol complex is often used as a basis of comparison to deduce the mode of binding of genistein with ER-␤. Comparison of the ER-␤:genistein structure with the previously published ER-␣:genistein structure enabled identification of two conservative amino acid changes, which may contribute to isoflavones’ ER-␤ selectivity. The new structure also explains the observation that genistein is only a partial agonist. Like 17␤-estradiol, genistein is buried within the hydrophobic core of the binding cavity, but Helix 12 adopts an antagonist orientation similar to that seen for raloxifene. Figure 4 illustrates the ligand-binding modes of genistein and 17␤-estradiol coupled to the ligand-binding domains of ER-␤ and ER-␣, respectively. RC exhibits a complex array of (anti)estrogenic activities in various in vitro test systems (7). Crude extracts of RC upregulate the estrogen-inducible genes for progesterone receptor (PR) and the trefoil peptide (TFF1/pS2)

and induce alkaline phosphatase (AP) activity in various cell lines. RC downregulates ER levels in T-47D (ER+/PR+) breast cancer cells, an effect that cannot be reversed in the presence of RU486. Preparations of RC and purified isoflavones usually stimulate the proliferation of ER+ breast cancer cells in steroid-depleted media yet inhibit steroid-stimulated growth at midrange to high micromolar concentrations. Modulation of bone cell homeostasis by isoflavones is believed to occur via ERdependent mechanisms. In vivo animal studies generally support the picture of RC as a weak estrogenic agent with tissue-dependent effects. A survey of various clover and alfalfa fodders associated content of biochanin A and formononetin with uterotrophic activity in the immature rat model. Exposure of ovariectomized ewes and heifers to formononetin and RC silage, respectively, caused enlargements in teat, vulval, and uterine size and production of milky fluid by the mammae. More recently, a standardized RC extract proved weakly estrogenic by affecting uterotrophic and vaginotrophic outcomes in the ovariectomized rat model, but it did not stimulate breast cell proliferation. In a rat model of endometrial cancer, genistein upregulated estrogen-responsive genes but did not promote tumor growth. Consistent with ER-␤ selectivity, isoflavone preparations improve bone density and protect against cardiovascular disease in most ovariectomized rat and prepubertal rhesus monkey models, although bone results may vary with concentration, timing, and length of exposure.

Figure 4 Comparison of hER-␤-LBD:genistein and hER-␣LBD:17␤-estradiol ligand-binding modes. Crystal structures of genistein in the human (h) ER-␤ ligand-binding domain (LBD) (hER-␤-LBD:genistein, light grey) and 17␤-estradiol in the ER␣-LBD (hER-␣-LBD:17␤-estradiol, dark grey) are superimposed. Hydrogen bonds are drawn as dotted lines. Primary protein residue labels correspond to the hER-␤-LBD sequence. The corresponding hER-␣-LBD sequence number and residue, if different, is provided in parentheses. [This figure was kindly prepared by Barbara Calamini and Andrew Mesecar (University of Illinois at Chicago, Illinois).]

Red Clover

Isoflavone concentrations in humans routinely exceed endogenous estradiol concentrations. However, RC is not overtly estrogenizing, suggesting that alternate molecular targets must mediate the clinical actions of the isoflavones.

Nonestrogen Receptor In addition to weak estrogenic activity, RC extracts exert weak antiprogestational and antiandrogenic activities. A 50% hydroethanolic extract did not stimulate, but instead almost completely blocked, progestin-induced AP activity in T-47 cells (8). More recently, antiandrogenic action of a 5% RC extract appeared responsible for reduction in benign prostate enlargement in aromatase knockout mice. Affinity constants for the four major isoflavones at the progesterone and androgen receptors fall in the millimolar range and adhere to the same rank order of potency for both steroid receptors: biochanin A > genistein = daidzein > formononetin. A recent study of receptor binding and transactivation activities of RC isoflavones and their metabolites found that androgen and progesterone were low, but several isoflavone metabolites provided interesting ER␣ and ER␤ binding and transactivating properties: equol, and O-desmethylangolensin (both from daidzein), and the reduced metabolite of formononetin, angolensin (9).

Receptor Independent A number of receptor-independent mechanisms of action influence hormone status. Isoflavones tend to lower steroid hormone levels in part through inhibition of several enzymes involved in steroid biosynthesis (10): aromatase (biochanin A > genistein), 5-␣-reductase type 2 (genistein, daidzein, and biochanin A), and 17-␤hydroxysteroid dehydrogenase (coumestrol, biochanin A, and genistein). Daidzein sulfates inhibit the sulfatase and sulfotransferase enzymes that (i) regulate sulfation of endogenous and environmental estrogens; (ii) help determine the availability of hormones to tumors; and (iii) have been hypothesized to minimize bioactivation of xenobiotic procarcinogens. All four isoflavones—biochanin A being the most potent—expedite the elimination of inactive hormone conjugates through stimulation of UDPglucuronosyltransferase. Genistein stimulates the production of sex hormone–binding globulin (SHBG) in human hepatocarcinoma cells. Equol producers exhibit enhanced urinary ratios of 2-hydroxyestrone to 16␣-hydroxyestrone, a metabolic measure that is negatively correlated with breast cancer risk.

Cancer-Related Effects Antioxidant Crude RC extracts display antioxidant activity in several in vitro experimental systems. Known antioxidant compounds in RC include genistein, daidzein (precursor to equol), biochanin A, genistin, daidzin, formononetin, clovamide, and texasin. In addition to exerting their influence through direct chemical interactions, isoflavones lower oxidative stress via indirect mechanisms such as induction of antioxidant scavenging enzymes. In certain biological

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environments, genistein may act as a pro-oxidant with potential mutagenic and/or genotoxic consequences.

Antiproliferative At pharmacological doses, isoflavones inhibit the proliferation of both hormone-dependent and hormoneindependent cancer cells. This phenomenon has been attributed, in part or in whole, to a wide range of ERindependent effects (11): the inhibition of enzymes such as tyrosine kinase (genistein, biochanin A) and DNA topoisomerases I and II (genistein, biochanin A, equol, and orobol); regulation of growth factors and their receptors (genistein); effects on cell cycle regulatory proteins (genistein and biochanin A); regulation of stress response genes (genistein); apoptosis (genistein); and inhibition of nitric oxide synthesis (biochanin A). Biochanin A is largely responsible for the inhibition by RC of benzo(a)pyrene metabolism in hamster embryo cell culture. The isoflavones generally exert a chemoprotective effect if administered to rodents before puberty, although genistein will stimulate or suppress tumor growth depending on the cancer model employed.

Other Tumor progression and metastasis may be checked by the following actions of genistein: inhibition of angiogenesis, cell adhesion effects; tyrosine phosphorylation of membrane proteins that mediate cellular invasion; and collagenases/metalloproteases (12,13).

Inflammation and Immune Function Several lines of evidence suggest that the isoflavonoids in RC modulate inflammatory (12) and immune responses (14). In the ovariectomized mouse model, genistein in the diet or by injection induces thymic atrophy and suppresses cell-mediated immunity. Injected genistein also decreases humoral immunity. In the hairless mouse model, genistein, equol, isoequol, and dihydroequol protect against UV-induced inflammation and immunosuppression. An aqueous extract of RC strongly inhibited (82% at 0.25 mg/mL) platelet activating factor-induced exocytosis in human neutrophils. The isoflavones modulate anti-inflammatory responses in animal models of chronic ileitis, inflammation-induced corneal neovasculation, and ischemic reperfusion injury.

Thyroid Function Speculation that the isoflavones, and RC by extension, trigger thyroid disease has been based on both in vitro and in vivo findings (15). In vitro, genistein and daidzein competitively inhibit thyroid peroxidase (TPO) to form iodinated isoflavones and irreversibly inhibit the enzyme in the absence of iodide, albeit at IC50 s above typical circulating levels of free isoflavones. Rat studies have confirmed the relevance of these in vitro findings. Dietary exposure of Sprague-Dawley rats to genistein that achieved serum concentrations comparable to those seen in humans caused intrathyroidal accumulation of genistein and inactivation of TPO. Paradoxically, the rodents did not present as hypothyroid. Thyroxine, tri-iodothyronine (T3), and thyroid stimulating hormone levels remained

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usnchanged, and thyroid sections revealed no pathologies. In the ovariectomized ewe model, however, exposure to RC silage resulted in elevated free and total T3 levels, increased thyroid follicle size, and thyroid gland immunoreactivity to ER-␣.

Table 1

Glucose and Lipid Metabolism

Caryophyllene (sesquiterpene) Citrulline (amino acid) Coumestrol (coumarin)

Isoflavone supplements and RC extracts have been postulated to have beneficial effects on obesity and diabetes mellitus, and in vitro and in vivo experiments provide some support for this contention. Genistein increases glucoseinduced insulin release from either pancreatic ␤-cells or insulinoma cell lines. Also contributing to the antidiabetic effect, genistein and, to a lesser extent, daidzein inhibit insulin-stimulated glucose uptake from the intestine and other cells, and isoflavones protect against glucoseinduced oxidation of low-density lipoproteins (LDL). Anabolic effects of RC isoflavonoids have been observed in mice, rats, and cattle. At least three rabbit studies have demonstrated beneficial effects of isoflavones or RC (16) on lipid metabolism and/or progression of atherosclerosis, although reduced LDL peroxidation rather than changes in serum lipids was hypothesized to account for improvements in one study. Genistein exerts lipolytic/antilipogenic effects in ovariectomized mice.

Other Biological Activities of RC Compounds Genistein weakly antagonizes the A1, A2a, and A3 adenosine receptors at low micromolar concentrations. Genistein and daidzein inhibit GABAA receptor-mediated chloride currents. The two isoflavones, and certain structural analogues, also block calcium channels in human platelets, inhibiting thrombin-induced [Ca2+ ]i elevation. The isoflavone actions at ligand-gated ion channels occur independent of tyrosine kinase inhibition at low- to midrange micromolar concentrations. The RC isoflavones serve as effective antimicrobials (genistein, daidzein, biochanin A, and formononetin) and fungicides (genistein, biochanin A, and formononetin). See Table 1 for a noncomprehensive list of selected RC compounds with interesting biological activity.

USAGE Human Clinical Studies Caveats Inferences about RC based on soy isoflavone studies may not be valid. RC and soy not only differ in their balance of individual isoflavones, but soy foods and SPIs also contain a unique protein fraction that is not present in RC semipurified isoflavone extracts. To date, no reports have been published regarding the clinical activity of RC protein. Research of dietary soy isoflavone effects on hormone status is complicated by the relative amounts of fiber and nonstarch carbohydrates in the diet, as these influence the gut flora populations responsible for metabolism of isoflavones in the colon. In addition, it is currently assumed that the isoflavones are the only “active” constituents present in RC extracts. This may or may not eventually be shown to be true, as 20 or more minor compounds may be present in the semipu-

A Noncomprehensive Listing of Compounds Found in RC

Compound (compound class)

Selected biological activities of potential interest

Calycosin (isoflavonoid)

Antiandrogenic, cell differentiation induction, hemoglobin induction Antispasmodic, ambulatory behavior stimulation, choleretic DNA damage prevention, prolactin inhibition Prolactin stimulation, estrogenic and antiestrogenic effects in vivo, bone resorption inhibition, apoptosis induction Protein kinase C stimulation, platelet aggregation inhibition Negative chronotropism Antiestrogenic in vivo, nitric oxide synthesis inhibition, osteocalcin stimulation, prostaglandin E2 inhibition Bone resorption inhibition

Daphnoretin (coumarin) Fraxidin (coumarin) Glycitein (isoflavone)

Glycitin (isoflavone glucoside) Hyperoside (flavonol)

Medicarpin (pterocarpan) Orobol (flavonol) Pectolinarigenin (flavone) Pratensein (isoflavonoid) Prunetin (isoflavone) Scoparol (flavonol)

Scopoletin (coumarin)

Texasin (isoflavonoid) Trigonelline (alkaloid) Xanthotoxol (coumarin)

Anti-ischemic, Ca2+ uptake inhibition, antihemorrhagic, positive chronotropism, coronary vasodilator, negative inotropism Apoptosis induction, cell differentiation, hemoglobin induction 15-lipo-oxygenase inhibition, topoisomerase II induction Antiatherosclerotic, antihyperlipemic, Ca2+ -ATPase inhibition Antihypercholesterolemic Antihypercholesterolemic, aromatase inhibition, estrogenic in vivo Antispasmodic, cAMP inhibition, lipo-oxygenase inhibition, platelet aggregation inhibition, tumor necrosis factor ␣release inhibition Bronchodilator, CNS depressant, platelet aggregation inhibition, uterine relaxant, negative chronotropism/inotropism Lipid peroxidation inhibition Analgesic, antimitotic, cell cycle disruption, neuron sprouting Antiarrhythmic, antispasmodic, cell differentiation, negative chronotropism, neural transmission inhibition

Selected biological activities of potential interest are presented (17). Source: From Ref. 69.

rified extracts used in clinical trials. Studies reviewed here are limited to clinical trials of RC isoflavone supplements and/or pure isoflavones, and the above-mentioned caveats should be borne in mind when interpreting the results presented. Refer to Table 2 for details regarding specific RC isoflavone products.

Menopause Hot flashes and menopausal symptoms Initial studies administering semipurified RC preparations to women to relieve menopausal hot flashes have generally been of short duration (≤12 weeks) and effects, when present, take eight weeks to manifest. Trials broadly demonstrate a significant placebo effect during the first four weeks that may persist throughout the investigation (18,19). More recent studies have incorporated a two- or four-week run-in period to assess this placebo effect (18,20,21). Evidence

Red Clover

Table 2

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Table of RC Productsa Evaluated in Clinical Trials Isoflavone ratio (genistein + biochanin A:daidzein + formononetin)

Product name

Delivery form and dosage

Effective ingredients

Promensilb

Tablet; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A)

Dried aqueous alcoholic extract of T. pratense

1.9

Trinovinb

Tablet; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A)

Dried aqueous alcoholic extract of T. pratense

1.9

Rimostilb

Tablet; 57 mg total isoflavones (genistein + daidzein + formononetin + biochanin A) Tablet; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A) Tablet; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A) Tablet; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A) Capsule; 40 mg total isoflavones (genistein + daidzein + formononetin + biochanin A)

Dried aqueous alcoholic extract of T. pratense

0.15

Dried aqueous alcoholic extract of T. pratense; principal isoflavone is biochanin A Dried aqueous alcoholic extract of T. pratense; principal isoflavone is biochanin A Dried aqueous alcoholic extract of T. pratense; principal isoflavone is formononetin Dried aqueous alcoholic extract of T. pratense; principal isoflavones are biochanin A and formononetin Dried aqueous ethanol extract of T. pratense; principal isoflavones are biochanin A and formononetin

3.8

Relief of menopausal symptoms such as hot flushes and night sweats; maintenance of bone and cholesterol health; general well being Maintain normal prostate and urinary function. May assist in the relief of medically diagnosed benign prostatic hypertrophy Maintain bone and cholesterol health in postmenopausal women N/A

3.5

N/A

0.2

N/A

1.12

Relief of menopausal symptoms including hot flashes and vaginal dryness

1.03

Formulation developed for clinical trial—relief to menopausal symptoms

P-07c (not commercially available) P-07(b)c (not commercially available) P-083c (not commercially available) Menoflavonb (MF11RCE)

N/A Developed by UIC/NIH Botanical Centerc,d

Capsule; 116.6 mg total isoflavones (genistein + daidzein + formononetin + biochanin A)

a RC manufacturer: Novogen Ltd. b Content has been independently verified. (See Refs. 70–72.) c Content is per producer’s claim and has not been independently d From Ref. 73.

Indications

verified.

for RC efficacy in reduction of hot flashes is not compelling, and long-term trial results have varied from trials of shorter duration. One trial with a duration of 12 weeks (after a 4-week R run-in period), using 80 mg of isoflavone (Promensil 82 mg isoflavones/day, high genistein + biochanin A, Novogen Ltd., Australia) recorded a statistically significant decrease in hot flushes of 44% at week 12 among the 15 participants in the treatment group (20). Another 12-week trial, with a 2-week run-in period with a total of 246 participants in three groups testing two commerR cially available RC products (Promensil and Rimostil , 57 mg isoflavones/day, high daidzein + formononetin; Novogen Ltd., Australia) versus placebo, demonstrated no clinically important effect on hot flashes or other symptoms of menopause by either RC formulation (21). Both treatment groups showed higher response in women with body mass index (BMI) greater than 25.1. Promensil (1 mg genistein, 0.5 mg daidzein, 16 mg formononetin, 26 mg biochanin A) was evaluated in a trial, with a duration of one year, taken daily by 117 women aged between 49 and 65 years. Results demon-

strated no statistically significant changes in mean number of hot flashes or menopausal symptoms with the treatment group compared with placebo (22). Only one study has directly compared RC to a pharmaceutical proven to alleviate hot flashes. In that study RC did not show a statistically significant difference in the number of vasomotor symptoms, hot flashes, or intensity of hot flashes compared to placebo in this trial of one year that compared red clover to the active control—conjugated equine estrogens with medroxyprogesterone (CEE/MPA) (23). A subset of participants from this study participated in a separate study of objective measures of hot flashes using an ambulatory hot flash monitor. The subjects in the CEE/MPA group saw hot flashes decrease significantly; however red clover participants exhibited a 33% decline in hot flashes compared to placebo, which experienced a 0% decline in hot flashes (24). Vaginal/sexual Health In a 12-week randomized, double-blind placebocontrolled trial of 36 postmenopausal women evaluating 40 mg or 160 mg of RC (Promensil, 40 mg per

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tablet, genistein 4.0 mg, daidzein 3.5 mg, biochanin 24.5 mg, and formononetin 8.0 mg) relative to placebo, for menopausal symptoms, there were no changes in vaginal wall smear or vaginal pH relative to baseline for any group (19). There have been two reports from a six-month randomized, double-blind, placebo-controlled, crossover trial of 53 postmenopausal women, which evaluated RC R [Menoflavon (MF11RCE), 40 mg total isoflavones, Melbrosin International/Meldex International, U.K.] on vaginal health and sexuality. Results showed improvements in karyopyknotic, cornification, and basal cell maturation indices compared to placebo, which correlated with a decrease in dyspareunia, vaginal dryness, and decreased libido (25,26). Memory and Mood A six-month randomized, placebo-controlled trial in postmenopausal women found no significant short-term effects of daily RC tablets (2-tablet dose; 25 mg formononetin, 2.5 mg biochanin A, < 1 mg daidzein + genistein per tablet, Rimostil) on memory and compared to placebo showed a trend toward deterioration of digit recall. However, participants did demonstrate a trend toward improvement in the block design test (27). In contrast, postmenopausal women who participated in a oneyear study of botanicals, which included a RC arm (2capsule dose; 57.5 mg biochanin A, 56.6 mg formononetin, 1.6 mg genistein and 0.9 mg daidzein per capsule) for cognitive function compared to CEE/MPA and placebo, did not experience improvement in visuospatial abilities or decreased digit recall (24). The RC group in this study did not show a change in verbal memory, which differed from placebo. All treatment groups showed a significant decline from baseline with the CEE/MPA group showing the greatest decline. Another six-month randomized, placebocontrolled, crossover trial of 113 postmenopausal women were enrolled to examine the effect of RC (2-capsule dose; standardized to 40 mg isoflavones per capsule in a proprietary blend of biochanin A, formononetin, genistein, and daidzein, MF11RCE) on anxiety and depressive symptoms. Instruments used were the HADS (Hospital Anxiety Depressive Scale) and SDS (Zung’s Self-rating Depressive Scale). Subjects were measured at baseline, 90, and 187 days and were shown to exhibit significant decreases of 76.9% for the HADS score and 80.6% for the SDS score. In contrast, total HADS and SDS scores also decreased significantly compared to baseline after placebo, but at an average of 21.7% (28).

Breast Cancer No studies have directly evaluated the effects of RC isoflavone supplements in breast cancer patients. In a study of high-risk women, 177 subjects (49–65 years) with Wolfe P2/DY mammographic breast patterns received Promensil daily for one year and exhibited no statistically significant changes in estradiol, follicle stimulating hormone (FSH), or luteinizing hormone (LH) levels (22). Differences between densities of breast patterns were not significant between treatment and placebo groups. In a three-year study to assess the safety and tolerability of RC (40 mg tablet Promensil) taken once daily by premenopausal, perimenopausal, and postmenopausal

women with a first-degree relative with breast cancer, it was found that RC had no estrogenic effect on breast density. In this study, 18% of the 320 women who enrolled eventually withdrew; eight because they developed cancer. There was no significant difference between the treatment group and placebo for those developing cancer. On the basis of a risk calculation for each first-degree relative, 4.6 breast cancers could be expected during the study. Although there were 8 observed (5 from the placebo group), the increase (8 compared to 4.6) was not significant (P = 0.8) (29). Excretion of daidzein, genistein, and equol (from dietary sources, including soy) is reduced in women with breast cancer compared to case controls. Equol production is associated with lower concentrations of testosterone, androstenedione, dihydroxyepiandrosterone (DHEA), DHEA sulfate, and higher levels of SHBG, regardless of isoflavone consumption. Female equol producers tend to have lower midluteal phase plasma estrone, estrone sulfate, and progesterone and higher FSH levels versus nonproducers.

Cyclical Mastalgia A study for relief of cyclical mastalgia first admitted subjects to a two menstrual cycle placebo run-in period. Those with less than 30% average decrease in pain compared to baseline levels were randomized and administered 40 or 80 mg RC isoflavones (Promensil) over three menstrual cycles (30). A three-day increase in menstrual cycle length was noticed in the 80-mg group compared with the placebo group. Breast pain was significantly reduced in the 40-mg group compared with the placebo group.

Endometrial Effects A three-month study of 50 mg RC isoflavones/day (product P-07, Novogen Ltd.) in perimenopausal women found no change in the Ki-67 proliferative index of endometrial biopsies taken during the late follicular phase nor was there change in plasma estradiol, FSH, progesterone, or endometrial thickness (31). One study (32) discovered a significant inverse association between endometrial cancer risk and dietary consumption of daidzein and total isoflavones, especially at 1.2 to 1.7 mg isoflavones/day. Doses up to 85.5 mg/day of Rimostil in postmenopausal women for six months did not cause increased endometrial thickness or breakthrough bleeding. A 90-day, randomized, double-blind, placebo-controlled crossover study of 109 postmenopausal women was undertaken to examine the effects of 80 mg RC/day (2 daily capsules, MF11RCE) on the endometrium (33). Transvaginal ultrasound examinations revealed that endometrial thickness was significantly decreased with RC compared to placebo. In addition, there were no significant effects seen on hormone measurements of E2, FSH, and SHBG; however, testosterone levels were significantly increased with RC and there was a nonsignificant decrease of FSH seen with placebo. Also, a significant reduction in LH was seen for placebo, but not RC. Interestingly, in an assay of transactivational potency of MF11RCE, it was shown to be equivalent 79 ␮g E2 per gram for ER␤, and 17 ␮g for Er␣ (34). In a three-year study of women with a family history of breast cancer who took one 40-mg tablet of Promensil daily, transvaginal ultrasound examinations of

Red Clover

postmenopausal women were conducted annually and revealed no significant differences between the placebo and RC groups (29).

Prostate Cancer It is currently unclear what role, if any, serum and tissue levels of isoflavones play in prostate cancer and BPH. One study found that prostate cancer patients had higher serum levels of isoflavones compared to cancer-free controls. However, there were more equol producers in the control group versus the cancer group. Another experiment collected plasma and prostatic tissue specimens from BPH patients and bladder cancer patients with normal prostates. Prostatic genistein was lower in the BPH group, whereas equol and daidzein concentrations were similar across both groups. Plasma isoflavone concentrations were similar for both cohorts. Three clinical studies have examined the effect of RC extracts on male prostate health. The first study, unpublished but described in another report, administered 40 or 80 mg RC isoflavones/day for three months to BPH patients (exact product and methodology not provided). The International Prostate Symptom Score decreased 23.3%, urinary flow rate increased 9.8%, and quality of life improved 17% for both treatment groups. A study (35) of TrinovinTM (Novogen Ltd.) administered four 40 mg tablets daily to men with prostate cancer for 7 to 54 days before radical prostatectomy. Apoptosis of prostate cancer cells was more common in tissues from the treatment group and was especially evident in regions of low-to-moderate grade cancer. No differences were seen pre- and posttreatment for serum prostrate-specific antigen (PSA), Gleason score (grade of cancer severity), or serum testosterone. A study in healthy men using Trinovin as above showed no effects on plasma testosterone, androstenedione, dehydroepiandrosterone sulfate, androsterone, epiandrosterone sulfate, cortisol, or SHBG, but dihydrotestosterone levels increased, which is possibly a detrimental change.

Colorectal Cancer Two studies examining the effects of RC on colorectal cancer risk have been conducted; epidemiological studies have indicated that increased insulin-like growth factor IGF-I concentrations have been linked to colorectal cancer risk. In vitro and in vivo animal studies have shown that soy isoflavones may decrease IGF-I concentration. A six-month placebo-controlled, double-blind, crossover trial of 37 men with a personal history of colorectal adenomas or at least one first-degree family member with a history of colorectal cancer who were randomized to two daily tablets of RC (Promensil; 25 mg biochanin, 8 mg formononetin, 4 mg genistein, and 5 mg daidzein) exhibited no effect on serum IGF-I nor were free IGF-I, IGF-II, IGFBP-1, IGFBP-2, or IGFPB-3 concentrations significantly altered (36). A similar six-month study of 34 postmenopausal women was conducted by the same investigators and produced generally similar results: RC did not significantly affect serum total IGF-I or IGF-II, and the mean or median relative differences in IGFBP-1, IGFPB2, and IGFPB-3 between isoflavone and placebo did not deviate from zero (37).

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Osteoporosis Prevention and Treatment Results from clinical studies of RC in prevention and treatment of osteoporosis are promising but complicated by varying length of bone remodeling cycles in individuals and the biphasic effects of isoflavones. Background hormonal milieu in the body also plays a role, as does basal metabolic index, which is inversely related to rate of bone loss in postmenopausal women. Seven studies have examined the effects of RC on bone. Refer to Table 3 for a summary of these studies. Three trials in postmenopausal women observed favorable effects in terms of preservation of bone mineral density (BMD). One year of treatment with Promensil (43.5 mg total isoflavones) significantly decreased the loss of lumbar spine BMD in pre- and perimenopausal women (38). There was no effect in postmenopausal women nor was there an effect on hip BMD for any group. A six-month study documented increased BMDs of the proximal forearm (2.9%, 4.1%, 3.0% increases, respectively) but not the distal forearm, for 25, 50, or 75 mg RC isoflavones/day (Rimostil), after a one-month placebo run-in period (39). Another sixmonth study found increases in BMD of the proximal radius and ulna in postmenopausal women taking Rimostil at 57 or 85 mg RC isoflavones/day (40). The fourth study reported no measured changes in N-telopeptide and osteocalcin bone markers in perimenopausal women taking 50 mg RC isoflavones/day (product P-07, Novogen Ltd.) for thee months (31). The fifth study also reported no effect of either Promensil or Rimostil on serum osteocalcin and urinary N-telopeptide levels after daily use for 12 weeks by menopausal women (41). The sixth study also reported no statistically significant difference in N-telopeptide; bone alkaline phosphatase (BAP) rose in the placebo group at 6 and 12 months but not in the RC (Promensil, 40 mg daily) group. Osteocalcin was marginally higher in the RC group, but did not change significantly from baseline. Serum beta CTx was higher in the RC group compared to baseline. There were no significant differences in percent change in BMD versus placebo (29). The seventh trial compared the effects of four commercially available isoflavone products (two soy products, red clover, and kudzu) with estradiol + medroxyprogesterone on reduction in bone resorption (42). The soy product with the highest content of genistein produced the highest reduction and had about 5× the genistein as the RC (Rimostil, Novogen Ltd.) product. However, all of the isoflavones were variously efficacious at preventing bone loss. Early postmenopausal women taking 54 mg genistein/day showed increased bone AP, bone Gla protein levels, and increased BMD in the femur and lumbar spine.

Cardiovascular Disease Risk Vascular Effects (a) Arterial compliance: Arterial stiffness is related to the presence of atherosclerotic plaques, and this parameter has been evaluated in two studies of RC. The first (43) administered 40 mg Promensil (4 mg genistein, 3.5 mg daidzein, 8.0 mg formononetin, and 24.5 mg biochanin A; reported content differs from manufacturer specifications) daily for five weeks. The dose was then doubled to 80 mg/day for five more weeks. Treatment groups (both doses) showed increases in arterial compliance, the

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Table 3

Summary of studies evaluating effects of RC extracts on bone in women

Reference

Product and dosage

Study length (mo)

No. of subjects

Significance

Study design

Novogen Ltd. Patent: (39) WO 00/64438; PCT/AU00/00384

Not stated (15:1 to 2:1 ratio of formononetin to the sum of daidzein + genistein + biochanin A); 25, 50, or 75 mg total isoflavones given

6

50

Postmenopausal women; 1-mo placebo run-in followed by isoflavone tablet(s) daily for 6 mo

Clifton-Bligh et al. (40)

Rimostil, 28.5, 57, 85.5 mg isoflavones (daidzein + genistein + formononetin + biochanin A)

6

46

Hale et al. (31)

50 mg of Novogen Ltd.’s P-07 RC isoflavone formulation containing high amount of biochanin A 43.5 mg total isoflavones (26 mg biochanin A, 16 mg formononetin, 1 mg genistein, 0.5 mg daidzein)

3

30

50 mg group had 4.1% increase in proximal forearm BMD; 25 and 75 mg groups had 2.9%, and 3.0% increase. No significant effect seen on distal forearm BMD 57, 85.5 mg groups showed significant (4.1%, 3.0%, respectively) increase in proximal radius and ulna; no significant response in 28.5 mg group No changes in N-telopeptide or osteocalcin bone markers

12

205; 177 completed trial

3

252; 245 completed trail

36 – 12 for bone markers

401 – 77 Postmenopausal

50 days/ intervention with 50 day washout period

11

Atkinson et al. (38)

Schult et al. (41)

Powles et al. (29)

Weaver et al. (42)

Promensil, 41 mg isoflavones (24.5 mg biochanin A, 8 mg formononetin, 4 mg genistein, 5 mg daidzein) and Rimostil, 28.6 mg isoflavones (2 mg biochanin A, 25 mg formononetin, trace amount genistein + daidzein) Promensil 40 mg tablet containing defined amounts of isoflavones genistein, daidzein, formononetin and biochanin from red clover, tablet taken daily.

Four products were evaluated for their antiresorptive effects on bone: Soy Cotyledon, soy germ, kudzu, red clover (Rimostil Biochain a 6.40 mg, formononetin 31.10 mg, genistein 0.562 mg, and daidzein 1.75 mg and) estradiol + MPA

Reduced loss of lumbar spine bone mineral content and BMD in treatment group; significant increase in bone-specific AP and N-propeptide of collagen type I; no significant effect on hip BMD/ mineral content or boneresorption markers No changes in urinary N-telopeptide or serum osteocalcin bone markers

No significant difference in percentage change in BMD between RC and placebo; NTx/Cr ratio showed no significant difference from baseline at 6 or 12 mos; BAP ↑ in placebo at 6 & 12 months, but not in RC group. RC with lower level of serum beta CTx compared to placebo at 6 mos Outcome measure was 41Ca (a marker of bone resorption in urine). Serum alkaline phosphatase was lower during the RC intervention than at baseline. Serum genistein levels were highest for soy cotyledon, most effective isoflavone of the four products for suppressing bone resorption.

Perimenopausal women; 1-mo run-in period followed by 1-mo placebo period, then double-blind treatment for 6 mo Pre- and perimenopausal women; double-blind, randomized, placebocontrolled Pre-, peri- and postmenopausal women; double-blind, randomized, placebo-controlled

Peri- and postmenopausal women; double-blind, randomized, placebocontrolled

Randomized, double-blind, placebo-controlled trial

Randomized-order, crossover, blinded trial

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magnitude of which was comparable to results seen in studies of hormone replacement therapy. A six-week randomized, double-blind crossover, placebo-controlled study (44) administered two tablets of each of two different products to men and women daily: one significantly enriched in biochanin A [P-07(b)] or another in formononetin (P-083). Isoflavone treatment resulted in significant improvements in systemic arterial compliance (SAC) and pulsed wave velocity (PWV) compared to placebo. However, the formononetin-enriched product had a stronger adjusted trend toward favorable effect on SAC compared to the biochanin A–enriched product. (b) Vascular endothelial function: After six weeks in the previously mentioned study (44), plasma levels of vascular cellular adhesion molecule-1 (VCAM-1) were reduced in the group receiving 80 mg/day of the formononetinenriched RC extract (P-083). Administration of up to 85.5 mg/day isoflavones (Rimostil) to postmenopausal women for six months did not result in altered levels of serum factor V, VII, VIII, antithrombin III, or fibrinogen (45). An unpublished study observed no adverse changes in intravascular coagulation (factor VIIc), platelet activation (P-selectin), or endothelial activation (von Willebrand factor) compared to placebo after five weeks of treatment with 40 mg/day of RC isoflavones (product unspecified), although no details were provided about the patients. A small, four-month double-blind randomized, placebo-controlled, crossover study in 16 type 2 diabetic postmenopausal women showed efficacy for reducing mean ambulatory daytime systolic and diastolic blood pressure taken every 30 minutes. Study participants took two RC tablets per day, each containing 25 mg formononetin, 2.5 mg biochanin and less than 1 mg of genistein and daidzein (Rimostil, Novogen Ltd.) for four weeks. Some subjects controlled their diabetes with diet alone, and others were taking oral diabetic agents. Other physiological measures (BMI, clinic blood pressures, heart rate, glycated hemoglobin, fasting blood glucose, total-C, HDLC, and LDL-C) did not differ significantly between RC and placebo therapies. Forearm vascular response was tested and shown to be significantly greater with L-NMMA (46). Another trial in peri- and postmenopausal women taking 43.5 mg RC isoflavones daily did not demonstrate an effect on systolic or diastolic blood pressure (47). Despite the mechanisms by which RC extracts act on the vasculature having not been definitively characterized, studies on pure isoflavone compounds yield interesting clues. Orally administered genistein caused increased vasodilation in postmenopausal women, presumably via increasing basal nitric oxide (NO) levels and reducing levels of the vasoconstrictor endothelin-1 (ET-1). Two trials support this hypothesis. The first administered 54 mg genistein/day for 6 months, and plasma levels of breakdown products of NO nearly doubled compared to either the placebo group or baseline levels. Endothelin-1 levels dropped by approximately 50%. Forearm blood flow and brachial artery diameter were significantly increased during reactive hyperemia after genistein treatment. The second administered the same genistein regimen or 17␤estradiol/norethisterone acetate (1 mg/0.5 mg) for 1 year. Genistein again improved brachial artery flow-mediated dilation, and improvements in NO breakdown products and ET-1 in the genistein group were of similar magnitude

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as the results for the hormone group. It remains to be seen whether these vascular effects will also be observed for RC extracts. A recent study concludes that RC acts as an antiatherogenic and anti-inflammatory agent by reducing the expression of leukocyte adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (48). Lipid Effects Several studies have investigated the effects of RC preparations on serum lipoprotein levels. In premenopausal women, consumption of 86 mg RC isoflavones per day (product P-07, Novogen Ltd., 51.4 mg biochanin A, 18.6 mg formononetin, 8.6 mg genistein, 7.4 mg daidzein; reported content differs from the manufacturer’s claim) for two menstrual cycles had no effect on total cholesterol or triacylglycerol levels. A second study using the same product (P-07, Novogen Ltd.) and dosing regimen over three menstrual cycles found no effects on total cholesterol, LDL, high-density lipoprotein (HDL), triacylglycerol, lipoprotein(a), glucose, or insulin levels. A onemonth placebo-controlled crossover study in pre- and postmenopausal women taking two Promensil tablets (43 mg isoflavones/tablet, 25 mg biochanin A, 8 mg formononetin, 4 mg genistein, 5 mg daidzein) had a significant effect on HDL in postmenopausal women, but no effect on insulin-like growth factor (IGF) in either group (49). Postmenopausal women receiving 40 mg and then 80 mg Promensil for five wk/dose had no change in HDL, LDL, triglyceride, or total cholesterol levels. A randomized double-blind ascending dose study administered one or two tablets (26 mg biochanin A, 16 mg formononetin, 0.5 mg daidzein, and 1 mg genistein/tablet; Promensil; content differs slightly from the manufacturer’s specifications) for 4 wk/dose and also found no effect on plasma lipids (50). A three-month study in peri- and postmenopausal women taking 2 tablets Promensil (24.5 mg biochanin A, 8 mg formononetin, 4 mg genistein, 5 mg daidzein/tablet) or Rimostil (2 mg biochanin A, 25 mg formononetin, trace genistein + daidzein/tablet) had no effect on plasma lipids, but did decrease triglycerides in women with high baseline levels (41). Another study administered 28.5, 57, or 85.5 mg of RC isoflavones/day as Rimostil for 6 months. HDL for all treatment groups increased at least 15%. Apolipoprotein B declined in all groups by at least 9%. One-year treatment with 43.5 mg RC isoflavones daily decreased triglycerides and plasminogen activator inhibitor type I (PAI-1) in perimenopausal but not in postmenopausal women (47). Differences between RC extract formulations may account for some of the observed clinical variation. A randomized, placebo-controlled, parallel crossover, double-blind trial in men and postmenopausal women compared effects of a biochanin A–enriched RC product [P-07(b), Novogen Ltd.] versus a formononetinenriched one (P-083, Novogen Ltd.) (51). The former, but not the latter, lowered LDL by 9.5% in men compared to baseline levels. Neither product affected plasma lipids in the postmenopausal group. A four-month randomized, placebo-controlled, parallel study of 25 premenopausal women taking 2 tablets RC (25.7 mg biochanin A, 4.3 mg genistein, 9.3 mg

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formononetin, and 3.7 mg daidzein per tablet, Novogen, Australia) was conducted over four menstrual cycles and showed that RC had no significant impact on mean serum lipid and lipoproteins in normocholesterolemic or mildly hypercholesterolemic subjects. Lipid concentrations during the follicular and luteal phases were not affected by RC supplementation. In addition, RC had no effect on Lp(a) levels (52). Fifty-three postmenopausal women completed a six-month, randomized, double-blind, crossover study of 80 mg RC (Menoflavon 40 mg total isoflavones per capsule) to evaluate effects on serum lipids, finding that mean baseline TC, LDL-C, and TG levels were lowered by 4.4%, 11.5%, and 9.5%, respectively; however, TGs alone decreased significantly. Interestingly, serum LpA levels decreased significantly after both RC and placebo (25). A second analysis of the same trial reports results for the 35 subjects who had increased BMI values (>25 kg/m2 ) (53). For this group, RC significantly reduced baseline TC, LDLC, and LpA 4.6%, 15.6%, and 63.8%, respectively. Women with higher BMIs had higher TG levels at baseline and showed a nonsignificant decrease with supplementation. When 54 mg/day of pure genistein was administered to postmenopausal women for six months, no effects on serum lipids were observed. Homocysteine and Folate Effects Homocysteine levels in 23 premenopausal women taking RC were evaluated in a double-blind, randomized, parallel pilot study during four menstrual cycles. All subjects took placebo during the first menstrual cycle; following randomization the intervention group then received R 86 mg RC (Novogen ; formononetin 9.3 mg, biochanin A 25.7 mg, genistein 4.3 mg, and daidzein 3.7 mg) daily. Mean monthly values of folate and homocysteine averaged from weekly blood samples resulted in no significant changes from baseline in the RC or placebo groups (54).

be assayed for the presence and level of Pb, As, Cd, and Hg, and the country of origin be required to provide quality control documentation. Imported products containing the ingredients realgar (arsenic) and cinnabar (mercury) should be avoided (55,56). The first supplement to the U.S. Pharmacopoeia/National Formulary 2003 recommends a limit of not more than 10 ppm of heavy metals be present in RC products. The following pesticides have been designated by the United Nations as hazardous and are banned by some countries, including the United States: aldrin/dieldrin, chlordane, dichlorodiphenyltrichloroethane (DDT), heptachlor, lindane, malathion, and parathion. These chemicals were used on RC fields in the United States during the 1950s and 1960s. Residues persist in the soil for extended periods of time and are still present in everyday foods at low but detectable levels. It is unknown whether proprietary extraction processes may concentrate these residues in the botanical extracts that are used to make dietary supplements. Some of these pesticides remain in use in other countries and are a potential contaminant of imported plant material and extracts. Limits (mg/kg) of 34 specific organophosphorus, organochlorine, and pyrethroid pesticides for RC are given in Table 4 under method section 561 of the U.S. Pharmacopoeia/National Formulary 2004 (57).

Botanical Misidentification Trifolium pratense shares the common name “sweet clover” with the plants M. alba Medikus and M. officinalis (L.) Pall. This shared common name is unfortunate but physical misidentification is avoidable; the flowers of RC are pinkish-purple, whereas those of M. alba and M. officinalis are white and yellow and can be easily distinguished from one another. See botanical description section for more botanical characteristics of M. officinalis.

Dosage and Extract Preparation

Presence of Coumarins

Current standardized preparations of RC are based on total aglycone content of the main four isoflavones. Typical products incorporate dried aqueous alcoholic extracts of RC to deliver ≥40 mg isoflavones per dose recommended on the label. These extracts may be hydrolyzed during processing for greater isoflavone aglycone content. Standardized products are available in tablet and capsule form, and clinical doses generally range from 40 to 160 mg isoflavones per day, given in a single dose. Isoflavone doses less than 80 mg/day are considered to be higher than isoflavone exposure received by eating a diet containing soyfoods and isoflavone-containing legumes.

There are more than 3400 naturally occurring coumarins present throughout at least 160 plant families. Many do not have anticoagulant effects in vivo; many more have unknown effects. Red clover has been reported to contain some coumarins. Dicoumarol, a 4-hydroxycoumarin derivative that is known to inhibit blood coagulation, was isolated in 1941 from M. alba Medikus and/or M. officinalis (L.) Pall. (58). It is a fungal metabolite formed by Penicillium species growing in diseased M. alba and M. officinalis. Although Melilotus and Trifolium species are closely related, there are no reports of dicoumarol occurring in Trifolium species. Coumestrol, daphnoretin, fraxidin, xanthotoxol, medicagol, and scopoletin are present in trace amounts in some RC extracts (≤100 ppm) (59). A randomized, double-blind, placebo-control trial of botanicals, which included am RC arm, for the management of vasomotor symptoms examined prothrombin time of all participants at randomization and at the onemonth safety visit and found no increase in prothrombin time in women in the RC group (23). But RC has never been evaluated for long-term anticoagulant effects, or herb–drug interactions with blood-thinning drugs such as warfarin. Usual clinical doses of RC extracts are such that exposure to any particular coumarin present would

SAFETY, TOXICITY, AND ADVERSE EFFECTS Adulteration Issues Heavy Metal Contamination and Pesticide Residues Although RC does not have a particular tendency to preferentially absorb heavy metals under normal conditions, when it is grown on contaminated soil, it can accumulate high levels of Cd, Cu, Pb, As, and Zn to varying amounts, depending on the soil pH and metal solubility. It is recommended that source material and/or any resultant extracts

Red Clover

likely be below the threshold where any (hypothetical) clinical anticoagulant effects should manifest.

Inhibition of Cytochrome P450 (CYP450) Enzymes In vitro experiments with human microsomes have shown that RC extracts exhibit selective inhibition of CYP2C9, marginal inhibition of CYP1A2 and CYP3A4, and nominal inhibition of CYP2A6 and CYP2D6. Genistein and daidzein, as well as genistin and daidzin, inhibit CYP1A1 as measured by reduction of enzyme activity in a mouse hepatoma cell culture system. In other experiments, genistein and equol did not cause significant induction of xenobiotic-metabolizing enzymes in mouse (ethoxyresorufin O-deethylase, p-nitrophenol oxidase, glutathione S-transferase, CYP1A2, CYP2E1, or CYP3A1) or human hepatic cells (CYP1A1, glutathione S-transferase ␭a, or xenobiotic response elements). A recent report of the structure–activity correlation on the inhibitory effects of flavonoids on cytochromes P450 3A activity concludes that daidzein and genistein inhibited CYP3A activity in a concentration-dependent manner (60). In a study of MCF7 breast cancer cells, biochanin A inhibited the enzyme activity and suppressed the transcriptional control of CYP19 (61). There are no reports of clinically significant RC–drug interactions.

Thyroid Function Red clover products are used by menopausal women, a group that is prone to hypothyroidism and autoimmune thyroiditis and could be particularly susceptible to the antithyroid actions of the isoflavones. Individuals in this patient population on chronic RC regimens should be monitored for thyroid function. In terms of potential benefit, the San Francisco Bay Area Thyroid Cancer Study recently concluded that isoflavone intake is associated with reduced thyroid cancer risk in both pre- and postmenopausal women (62).

Safety for Cancer Patients While RC and the isoflavones exhibit anticancer activities in vitro and affect SERM-like effects in vivo, insufficient evidence exists to support their use by patients with active cancer, at elevated risk for ER+ cancer, or recovering from cancer. RC has not been rigorously tested in cancer populations, and the theoretical possibility remains that isoflavone supplementation could promote or cause progression of hormone-dependent tumors. The isoflavones also have the potential to compete with antiestrogenic chemotherapeutic agents, and their antioxidant properties may interfere with radiation and chemotherapies. Significant in vivo CYP450 interactions appear unlikely but could prove problematic in the context of chemotherapy.

Safety for Pregnant Women and Children The safety of RC or isoflavone supplements for pregnant or (the children of) lactating women has not been established, although RC is considered a class 2b herb by the American Herbal Products Association and as such is contraindicated during pregnancy (63). Red clover supplementation is also discouraged for those younger than 18 years, as the long-term consequences, if any, of high

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isoflavone consumption early in life have not yet been elucidated.

Adverse Effects Reported in Clinical Trials Because the exact chemical content of commercial RC isoflavone products is proprietary, and total isoflavone content (or a ratio of summed isoflavone content) is often reported rather than individual chemical content, it is difficult to estimate clinical doses of individual isoflavones. This vague content labeling hinders correlation of clinical effects with specific RC compounds. More trials involving chronic exposure of large patient populations to RC isoflavone extracts are needed to assess long-term risk. Novogen Ltd. provides a list of side effects, occurring at doses as low as 40 mg isoflavones/day, in their online clinical monograph for Promensil. These effects include breast tenderness, swollen neck glands, increased thyroid function, migraine/headache, dizziness, vertigo, tremor, hypertension, acne, rash, pruritus, psoriasis, bloating, constipation, diarrhea, nausea, mouth ulcer, sore throat, myalgia, osteoarthritis, bronchitis, low platelets, reflux (80 mg), epistaxis (80 mg), menstrual bleeding (80 mg), urinary tract infection (120 mg), and vaginal thrush (80 mg). Additional adverse events reported in two trials using oral administration of 54 mg/day genistein included the following: (symptomatic) hypotension, vertigo, paresthesiae, temporary return of abbreviated menses, vaginal bleeding, hot flushes, and endometrial thickness greater than 5 mm. Compounds and mechanisms responsible for triggering adverse events are currently unknown. Use of the lowest RC dose possible for treatment, with upward titration as necessary, is recommended to decrease the probability of side effects occurring.

COMPENDIAL/REGULATORY STATUS Red clover is included on the U.S. FDA generally recognized as safe (GRAS) list, and “RC isoflavones” is an approved herbal components name (HCN) designated by the Therapeutic Goods Administration of Australia (64). The flower heads are listed in the British Herbal Compendium (65), the British Herbal Pharmacopoeia (66), and in Martindale: The Extra Pharmacopoeia (67). It appears in the UK’s General Sale List, Schedule 1 of Statutory Instrument 1994 No. 2410 (68).

ACKNOWLEDGMENTS The authors thank Barbara Calamini and Andrew Mesecar for preparation of Figure 4. The NAPRALERTSM database (69) was searched to prepare Table 1. Product information for Tables 2 and 3 and some regulatory status information was provided by Novogen Ltd. Research within the UIC Botanical Center is supported by NIH grant P50 AT00155 jointly funded by the Office of Dietary Supplements, the National Center for Complementary and Alternative Medicine, the National Institute for General Medical Sciences, and the Office for Research on Women’s Health. The contents of this chapter are solely the

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responsibility of the authors and do necessarily represent the views of the funding agencies.

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symptoms compared with placebo. Maturitas 2002; 42(3): 187–193. Tice JA, Ettinger B, Ensrud K, et al. Phytoestrogen supplements for the treatment of hot flashes: the Isoflavone Clover Extract (ICE) Study: a randomized controlled trial. JAMA 2003; 290(2):207–214. Atkinson C, Warren RM, Sala E, et al. Red-cloverderived isoflavones and mammographic breast density: a double-blind, randomized, placebo-controlled trial [ISRCTN42940165]. Breast Cancer Res 2004; 6(3):R170–R179. Geller SE, Shulman LP, van Breemen RB, et al. Safety and efficacy of black cohosh and red clover for the management of vasomotor symptoms: a randomized controlled trial. Menopause 2009; 16(6):1156–1166. Maki PM, Rubin LH, Fornelli D, et al. Effects of botanicals and combined hormone therapy on cognition in postmenopausal women. Menopause 2009; 16(6):1167–1177. Hidalgo LA, Chedraui PA, Morocho N, et al. The effect of red clover isoflavones on menopausal symptoms, lipids and vaginal cytology in menopausal women: a randomized, double-blind, placebo-controlled study. Gynecol Endocrinol 2005; 21(5):257–264. Chedraui P, Hidalgo L, San Miguel G, et al. Red clover extract (MF11RCE) supplementation and postmenopausal vaginal and sexual health. Int J Gynaecol Obstet 2006; 95(3):296–297. Howes JB, Bray K, Lorenz L, et al. The effects of dietary supplementation with isoflavones from red clover on cognitive function in postmenopausal women. Climacteric 2004; 7(1):70–77. Lipovac M, Chedraui P, Gruenhut C, et al. Improvement of postmenopausal depressive and anxiety symptoms after treatment with isoflavones derived from red clover extracts. Maturitas 2009; 65:258–261. Powles TJ, Howell A, Evans DG, et al. Red clover isoflavones are safe and well tolerated in women with a family history of breast cancer. Menopause Int 2008; 14(1):6–12. Ingram DM, Hickling C, West L, et al. A double-blind randomized controlled trial of isoflavones in the treatment of cyclical mastalgia. Breast 2002; 11(2):170–174. Hale GE, Hughes CL, Robboy SJ, et al. A double-blind randomized study on the effects of red clover isoflavones on the endometrium. Menopause 2001; 8(5):338–346. Horn-Ross PL, John EM, Canchola AJ, et al. Phytoestrogen intake and endometrial cancer risk. J Natl Cancer Inst 2003; 95(15):1158–1164. Imhof M, Gocan A, Reithmayr F, et al. Effects of a red clover extract (MF11RCE) on endometrium and sex hormones in postmenopausal women. Maturitas 2006; 55(1):76–81. Dornstauder E, Jisa E, Unterrieder I, et al. Estrogenic activity of two standardized red clover extracts (Menoflavon) intended for large scale use in hormone replacement therapy. J Steroid Biochem Mol Biol 2001; 78(1):67–75. Jarred RA, Keikha M, Dowling C, et al. Induction of apoptosis in low to moderate-grade human prostate carcinoma by red clover-derived dietary isoflavones. Cancer Epidemiol Biomarkers Prev 2002; 11(12):1689–1196. Vrieling A, Rookus MA, Kampman E, et al. Isolated isoflavones do not affect the circulating insulin-like growth factor system in men at increased colorectal cancer risk. J Nutr 2007; 137(2):379–383. Vrieling A, Rookus MA, Kampman E, et al. No effect of red clover-derived isoflavone intervention on the insulinlike growth factor system in women at increased risk of colorectal cancer. Cancer Epidemiol Biomarkers Prev 2008; 17(10):2585–2593. Atkinson C, Compston JE, Day NE, et al. The effects of phytoestrogen isoflavones on bone density in women: a doubleblind, randomized, placebo-controlled trial. Am J Clin Nutr 2004; 79(2):326–333.

Red Clover

39. Kelly GE, Husband AJ. Cardovascular and bone treatment using isoflavones. PCT Int Appl: WO 00/64438, PCT/AU00/00384, 2000. 40. Clifton-Bligh PB, Baber RJ, Fulcher GR, et al. The effect of isoflavones extracted from red clover (Rimostil) on lipid and bone metabolism. Menopause 2001; 8(4):259–265. 41. Schult TM, Ensrud KE, Blackwell T, et al. Effect of isoflavones on lipids and bone turnover markers in menopausal women. Maturitas 2004; 48(3):209–218. 42. Weaver CM, Martin BR, Jackson GS, et al. Antiresorptive effects of phytoestrogen supplements compared with estradiol or risedronate in postmenopausal women using (41)Ca methodology. J Clin Endocrinol Metab 2009; 94(10):3798– 3805. 43. Nestel PJ, Pomeroy S, Kay S, et al. Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal women. J Clin Endocrinol Metab 1999; 84(3):895–898. 44. Teede HJ, McGrath BP, DeSilva L, et al. Isoflavones reduce arterial stiffness: a placebo-controlled study in men and postmenopausal women. Arterioscler Thromb Vasc Biol 2003; 23(6):1066–1071. 45. Baber RBP, Fulcher G, Liberman D, et al. The effect of an isoflavone dietary supplement (Rimostil) on serum lipids, forearm bone density an endometrial thickness in postmenopausal women. In: Annual Meeting of the North American Menopause Society; September 23–25, 1999; New York, NY. 46. Howes JB, Tran D, Brillante D, et al. Effects of dietary supplementation with isoflavones from red clover on ambulatory blood pressure and endothelial function in postmenopausal type 2 diabetes. Diabetes Obes Metab 2003; 5(5):325–332. 47. Atkinson C, Oosthuizen W, Scollen S, et al. Modest protective effects of isoflavones from a red clover-derived dietary supplement on cardiovascular disease risk factors in perimenopausal women, and evidence of an interaction with ApoE genotype in 49–65 year-old women. J Nutr 2004; 134(7):1759–1764. 48. Simoncini T, Garibaldi S, Fu XD, et al. Effects of phytoestrogens derived from red clover on atherogenic adhesion molecules in human endothelial cells. Menopause 2008; 15(3):542–550. 49. Campbell MJ, Woodside JV, Honour JW, et al. Effect of red clover-derived isoflavone supplementation on insulin-like growth factor, lipid and antioxidant status in healthy female volunteers: a pilot study. Eur J Clin Nutr 2004; 58(1):173–179. 50. Howes JB, Sullivan D, Lai N, et al. The effects of dietary supplementation with isoflavones from red clover on the lipoprotein profiles of post menopausal women with mild to moderate hypercholesterolaemia. Atherosclerosis 2000; 152(1):143–147. 51. Nestel P, Cehun M, Chronopoulos A, et al. A biochaninenriched isoflavone from red clover lowers LDL cholesterol in men. Eur J Clin Nutr 2004; 58(3):403–408. 52. Blakesmith SJ, Lyons-Wall PM, George C, et al. Effects of supplementation with purified red clover (Trifolium pratense) isoflavones on plasma lipids and insulin resistance in healthy premenopausal women. Br J Nutr 2003; 89(4):467–474. 53. Chedraui P, San Miguel G, Hidalgo L, et al. Effect of Trifolium pratense-derived isoflavones on the lipid profile of

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Reishi Solomon P. Wasser

INTRODUCTION

antlers, furniture and carpet designs, balustrades, jewelry, women’s hair combs, perfume bottles—in short, wherever an artistic urge found an outlet. The earliest mention of Ling Zhi was in the era of the first emperor of China, Shing-huang of the Ch’in Dynasty (221–207 BC). Subsequently, depictions of this fungus proliferated through Chinese literature and art. The mushroom is known by many in North America and Europe as one of the “artist’s conk” fungi (the true artist conk is Ganoderma applanatum). The mushroom is too tough to be edible. A detailed description of the Reishi mushroom and its taxonomy can be found in Refs (1,2). (Fig. 1).

Reishi or Ling Zhi (Ganoderma lucidum), a popular medicinal mushroom, has been used in China, Japan, and Korea for the promotion of longevity and health since ancient times. Over the years, fables and legends revered G. lucidum as a “heavenly herb,” which connotes auspiciousness, blissfulness, and happiness. This mushroom has become more popular all over the world in recent years. The application of modern analytical techniques has revealed the mushroom to contain numerous bioactive compounds including polysaccharides, triterpenes, and immunomodulatory proteins. This review collates the publications detailing with the activities and compounds of G. lucidum while considering the most valid claims of effectiveness; it also presents a scientific understanding of Reishi’s beneficial functions on human immune, endocrine, nervous, and cardiovascular systems. Reishi has been used in conjunction with treatment for cancer patients, chronic bronchitis, hyperlipidemia, hypertension, diabetes, hepatitis, dermal and urological dysfunctions, and viral and bacterial diseases. Reishi also has nutraceutical applications; it is a well-known dietary supplement that can help improve general health and wellness. The latest available estimates put the annual value of Reishi products worldwide at more than 3 billion USD. Further expansion of the market for Reishi products will require the introduction of more protocols for mushroom production and downstream processing to improve quality control and assure the public of medicinal mushroom benefits. It is widely grown on a commercial scale and is commonly purchased for its medicinal and spiritual properties.

Habitat This annual mushroom grows on a wide variety of dead or dying trees, for example, deciduous trees especially oak, maple, elm, willow, sweet gum, magnolia, and locust (Quercus, Acer, Alnus, Betula, Castanea, Coryolus, Fagus, Fraxinus, Populus, Pyrus, Magnolia, Tilia). G. lucidum is less frequently found on coniferous trees (e.g., Larix, Picea, Pinus) in Europe, Asia, and North and South America (in temperate rather than subtropical regions). In the

(b)

BACKGROUND Name and General Description In Latin, lucidum means shiny or brilliant and aptly describes this mushroom’s fruiting body, which has a modeled, sculptured, varnished appearance. The Chinese and Koreans know it as Ling Zhi (mushroom of herb and immortality), whereas the Japanese call this mushroom Reishi or mannentake (10,000-year mushroom). The virtues of G. lucidum extracts, handed down from generation to generation, include it as a “cancer cure” and a symbol of happy augury, good fortune, good health, longevity, and even immortality. Beginning with the Yuan Dynasty (AD 1280–1368), G. lucidum has been endlessly represented in art—in paintings, carvings of jade and deer’s

(a)

Figure 1

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Ganoderma lucidum: (A) fruit body, (B) spores.

Reishi

Orient, it grows primarily on plum trees. It is also found on stumps, generally near the soil surface, and occasionally on soils arising from buried roots.

Related Species and Artificial Cultivation Ling Zhi encompasses several Ganoderma species, which are widely used for medicinal purposes, for example, G. lucidum, G. luteum Steyaert, G. atrum Zhao, Xu and Zhang, G. tsugae Murrill, G. applanatum (Pers.: Wallr.) Pat., G. australe (Fr.) Pat., G. capense (Lloyd) Teng, G. tropicum (Jungh.) Bres., G. tenue Zhao, Xu and Zhang, and G. sinense Zhao, Xu and Zhang. According to two famous Chinese plant medical books, Shen Nong Ben Cao Jing (25–220 A.D., Eastern Han Dynasty) and Ben Cao Gang Mil, by Li Shi-Zhen (1590 A.D., Ming Dynasty), six Ling Zhi species/varieties were known in China at that time. Worldwide, more than 250 Ganoderma species have been described (1,3). However, in therapeutic practices and literature citations, Ganoderma usually refers to the species of G. lucidum. Besides being treasured for its medicinal value in China for more than 1000 years, the lack of availability of G. lucidum was also largely responsible for it being so highly cherished and expensive. During ancient times in China, any person who picked the mushroom from the natural environment and presented it to a high-ranking official was usually well rewarded. Even in the early 1950s, it was presented to Chinese leaders in Mainland China and Taiwan, following the occasional discovery in the wild. In the past, G. lucidum grew in small quantities only in the wild; therefore, it was very expensive. Artificial cultivation of this valuable mushroom was successfully achieved in the early 1970s, and since 1980, production of G. lucidum has developed rapidly, particularly in China and the United States. The process of producing G. lucidum fruiting bodies is the same as for other cultivated edible mushrooms and can be divided into two major stages. The first involves the preparation of the fruiting culture, stock culture, mother spawn, and planting spawn, while the second entails the preparation of growth substrates for mushroom cultivation. Currently, the methods most widely adopted for commercial production are the wood log, short wood segment, tree stump, sawdust bag, and bottle procedures (for cultivation details, see Refs (4,5)).

History and Traditional Uses G. lucidum has been used in folk medicine of China and Japan, especially in the treatment of hepatopathy, chronic hepatitis, nephritis, hypertension, arthritis, neurasthenia, insomnia, bronchitis, asthma, and gastric ulcers (2,6–9). In China, G. lucidum has been cherished for over 4000 years as a longevity-promoting tonic (6). According to Hikino (10), “the most important elixirs in the Orient” are ginseng (Paxax ginseng C.A. Meyer) and the fruit bodies of G. lucidum. Fascination with Ganoderma began under the name of ling chih, later transliterated to reishi in Japanese. The fungus first appeared in Chinese literature during the Han Dynasty (206 BC–AD 220). Emperor Wu associated growth of the fungus in an inner chamber of the Imperial Palace with a plant of immorality—known simply as the chih

Table 1

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The Six Types of Ling Zhi Mushroom

Color

Taste

Japanese name

Use

Blue

Sour

Aoshiba

Reda

Bitter

Akashiba

Yellow

Sweet

Kishiba

White

Hot (or pungent) Salty Sweet

Shiroshiba

Improves eyesight and liver function; calms nerves Aids internal organs; improves memory; enhances vitality Strengthens spleen function; calms the “spirit” (shen) Improves lung function; gives courage and strong will Protects kidneys Enhances function of ears, joints, muscles; helps improve complexion

Black Purple

Kuroshiba Murasakishiba

a The

red-colored variety of G. lucidum is generally regarded as the most potent and medicinal. (20).

plant or chih fungus (11). The Han Dynasty chronicler, Pan Ku, wrote a poem using the term ling chih (11). However, the association between the original chih fungus and G. lucidum had been clearly derived from legends of an earlier mysterious chih fungus or chih plant of immortality recorded in India. Indeed, versions of Indian legends concerning this mushroom are found later, in almost identical form in the Chinese literature, in reference to what would be ling chih (Reishi), while the identity of the true chih plant or fungus of immortality remains in dispute (11). In addition to its medicinal properties, Reishi has been used in the Orient as a talisman to protect a person or home against evil (6). Medicinal uses of G. lucidum in ancient Far East countries included the treatment of neurasthenia, debility from prolonged illness, insomnia, anorexia, dizziness, chronic hepatitis, hypercholesterolemia, mushroom poisoning (antidote), coronary heart disease, hypertension, prevention of altitude sickness, treatment of “deficiency fatigue,” carcinoma, and bronchial cough in the elderly (1,2,6,8,9,12). Chinese research during the past decade has focused on much the same uses, whether in the fields of antiaging/life prolongation, brain ischemia/reperfusion injury, chronic viral hepatitis, male sexual dysfunction, hypercholesterolemia, immunological function in the elderly, chemotherapy-induced toxicity, narcotic-induced immunosuppression, anticarcinogenic and antitumor activity, and immunostimulation (6,7,13–19). Different types of G. lucidum, according to Traditional Chinese Medicine, have different tastes and thus affect different organs. Based on their color, six different types of G. lucidum have been classified (20), each with different uses (Table 1).

CHEMISTRY General Nutritional Components of G. Lucidum G. lucidum contains mainly protein, fat, carbohydrate, and fiber. The artificially cultivated variety has similar contents of nutritional components compared with wild types, and the extraction significantly increases the amounts of crude protein and carbohydrates and deleted crude fiber. Mizuno (21) reported the composition of G. lucidum extract (% of dry weight), which consisted of folin-positive material (68.9%), glucose (11.1%), protein (7.3%), and metals (10.2%) (K, Mg, and Ca are the major components

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with Ge having the fifth highest metal concentration at 489 ␮g/g). These results generally agree with those reported by other authors (2,4,5). However, there are qualitative and quantitative differences in the chemical composition of G. lucidum products depending on the strain, origin, extracting process, and cultivation conditions (1,2,5,9,21).

Major Bioactive Constituents Over 300 reports have been published concerning the chemical constituents of G. lucidum and related species. The fruiting body, mycelia, and spores of G. lucidum contain approximately 400 different bioactive compounds, which mainly include triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, proteins/peptides, and trace elements (9,16,19,21,22).

Triterpenes At least 140 different triterpenes have been identified in G. lucidum (1,2,6,9,21,22). The majority are bitter tasting and largely occur as ganoderic acid (22). A new triterpenoid, named ganosporeric acid A, was recently isolated from the ether-soluble fraction of the spores (23). Min et al. (24) reported the isolation of six new lanostane-type triterpenes, and also from the spores (ganoderic acids ␥ , ␦, ε, ␨ , ␩, and ␪). Preliminary studies indicate that the spores contain considerably higher contents of ganoderic acids than other parts of the fungus and that triterpene composition of the fruit body varies according to the area in which it is grown (23) The spores also contain triterpene lactones (22), and documented triterpenoids have been divided into 10 groups based on the structural similarities and known biological and medicinal properties (Fig. 2).

Figure 2 The lanostane-type triterpenoids of Ganoderma lucidum. These triterpenoids are divided into ten groups on the basis of structural similarity.

Reishi

Polysaccharides More than 100 types of polysaccharides have been isolated from the fruiting body, spores, and mycelia, or separated from the broth of a submerged liquid culture of G. lucidum. Most have a molecular weight ranging from 4 × 105 to 1 × 106 in the primary structure. They comprise one of the major sources of G. lucidum’s pharmacologically active compounds. G. lucidum polysaccharides such as ␤-D-glucans, heteropolysaccharides, and glycoprotein have been isolated and characterized and are considered the major contributors of bioactivity of the mushroom. ␤-D-glucans consist of a linear backbone of ␤-(1→3)- linked D-glucopyranosyl groups with varying degrees of branching from the C6 position. In addition to water-soluble ␤-D-glucans, ␤-Dglucans also exist with heteropolysaccharide chains of xylose, mannose, galactose, uronic acid, and ␤-D-glucansprotein complexes that are present at 10% to 50% in dry G. lucidum (16,25–27). Some protein-bound polysaccharides and fucose-containing glycoprotein with bioactivity have been isolated (18,28,29).

Proteins Some proteins with bioactivity have also been isolated from G. lucidum. The LZ-8 is one such protein isolated from G. lucidum, which was shown, by sequencing studies, to be similar to the variable region of the immunoglobulin heavy chain in its sequence and in its predicted secondary structure. Major biological activities of LZ-8 resemble those of lectins, with mitogenic capacity toward mouse spleen cells and human peripheral lymphocytes and agglutination of sheep red blood cells in vitro. Neither was inhibited by the mono- or dimeric sugars examined, indicating that LZ-8 is not a lectin per se. It did not agglutinate human red blood cells but could function as a potent suppressor of bovine serum albumin-induced anaphylaxis in CFW mice in vitro. It appears to be related to an ancestral protein of the immunoglobulin superfamily (30).

Nucleotides and Nucleosides Nucleosides include adenosine methylsulfinylad-nosine (21).

and

5-deoxy-5

Other Constituents G. lucidum also contains sterols, amino acids, soluble proteins, oleic acid, cyclo-octasulfur, an ergosterol peroxide (5,8-epidioxy-ergosta-6,22E-dien-3-ol), and the cerebrosides (4E ,8E)-N-D-2 -hydroxystearoyl-1-O-␤-Dglucopyranosyl-9-methyl-4–8-sphingadienine, and (4E, 8E)-N-D-2 -hydroxypamitoyl-1-O-␤-D-glucopyranosyl-9methyl-4–8-sphingadienine (1,9,17,18,21). Regarding the inorganic ions, the mushroom contains Mg, Ca, Zn, Mn, Fe, Cu, and Ge. The spores themselves contain choline, betaine, tetracosanoic acid, stearic acid, palmitic acid, ergosta-7, 22-dien-3-ol, nonadecanoic acid, behenic acid, tetracosane, hentriacontane, ergosterol, and ␤-sitosterol. One of the lipids isolated from G. lucidum is pyrophosphatidic acid (13,17,21).

PRECLINICAL STUDIES G. lucidum has been reported to have a number of pharmacological effects including immunomodulating,

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antiatherosclerotic, anti-inflammatory, analgesic, chemopreventive, antitumor, radioprotective, sleep-promoting, antibacterial, antiviral (including anti-HIV), hypolipidemic, antifibrotic, hepatoprotective, diabetic, antioxidative and radical-scavenging, anti-aging, hypoglycemic, and anti-ulcer properties (1,2,6,8,9,16,19,26,31).

Antitumor Effects Polysaccharides (␤-D-glucans, heteropolysaccharides, and glycoproteins) isolated from G. lucidum demonstrated antitumor activity against Sarcoma 180 in mice (1,1,2,2,13,16,21,26,28,29,31). Triterpenoids, such as ganoderic acids T-Z isolated from G. lucidum, showed cytotoxic activity in vitro on hepatoma cells (32). A lanostanoid, 3␤-hydroxyl-26-oxo-5␣-lanosta-8,24-dien-11-one, and a steroid, ergosta-7,22-diene-3␤,3,9-triol, isolated from fruiting bodies of G. lucidum, demonstrated potent inhibitory effects on KB cells and human PLC/PRF/5 cells in vitro (33). The polysaccharide-mediated potentiation of immune function is thought to be the major mechanism of antitumor action by G. lucidum. Among the multiple polysaccharides, active ␤-D-glucans are responsible for the antitumor effect (1,2,9,13,21,29,31). This polysaccharide appears to act by binding to leukocyte surfaces or serumspecific proteins leading to activation of macrophages, Thelper, natural killer (NK), and other effector cells (34–36). All of these increase the production of cytokines such as tumor necrosis factor (TNF-␣) interleukins (IL) and interferon (IFN), nitric oxide (NO), and antibodies by the activated effector cells. Tumor regression in various animal models can be ascribed to vascular damage to tumor blood flow and necrosis caused by T cells and local TNF-␣ production. In addition to host defense potentiation, other mechanisms are also involved in the antitumor effect. A compound from G. lucidum suppressed the growth of K562 leukemic cells in a dose- and time-dependent manner and induced their differentiation into more mature erythrocytic cells (37). The conditioned medium from PSstimulated human blood mononuclear cells (PSG-MNCCM) significantly inhibited the growth of U937 cells and induced their differentiation into mature monocytes/ macrophages, which had functions of phagocytosis and of producing cytoplasmic superoxide (38). Inhibition of DNA polymerase and posttranslational modification of oncoproteins may contribute to the antitumor activity of G. lucidum (39). The organic germanium may also contribute to its antitumor activity (40). Active constituents from G. lucidum may operate through several mechanisms including enhancement of detoxification of carcinogens, increased expression and activity of Phase II enzymes, inhibition of organ exposure of carcinogens due to reduced absorption or increased excretion, decreased expression and activity of Phase I (e.g., CYPs) enzymes, decreased formation of toxic metabolites and adduct formation with macromolecules, enhanced host immune responses (e.g., activation of macrophages, T lymphocytes, and natural killers producing various cytokines such as TNF-␣, IFNs, and ILs, which improve immunosurveillance and kill preneoplastic and cancer cells), antioxidative and radicalscavenging effects, antipromotion effect, antiproliferation,

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apoptosis induction of tumor cells, induction of differentiation, direct cytotoxicity, induction of cell-cycle arrest, antiproliferation and modulation of signaling transduction molecules, antiprogression and tumor growth inhibition, antimetastasis, and antiangiogenesis (16). In summary, animal studies have demonstrated the antitumor activity of G. lucidum administered by different routes at different stages of tumor growth (1,2,16,21). Polysaccharides and triterpenoids are the major contributors to the anticancer effect of G. lucidum, but other constituents, such as proteins, also play a role (21). Possible molecular pathways that may provide an explanation for the cancer preventive and anticancer effect of G. lucidum are shown by Gao et al. (16).

Chemopreventive and Radiopreventive Effects The chemo- and radiopreventive effects of G. lucidum may result from its effects on the immune system. Ganoderma polysaccharides restored the TNF-␣ production inhibited by cyclophosphamide to normal levels in mice. Both the G. lucidum extract and krestin (protein-bound ␤-glucan isolated from Trametes versicolor) were beneficially effective in the recovery of cellular immunocompetence, measured by [3 H] thymidine incorporation with splenic cells stimulated through mitogens, such as phytohemagglutinin and concanavalin A. The extract appears more effective than krestin in repairing the damage of subset T cells in the spleens of ␥ -irradiated mice, as the relative thymus weight and CD4 and CD8 splenocytes were higher in G. lucidum extract-treated mice compared with krestin-treated mice (16). In morphine-dependent mice, a polysaccharide peptide from G. lucidum could restore several immunologic parameters depressed by morphine treatment to normal levels or even beyond (41). Both c-myb and c-myc mRNA expression in splenocytes of repetitive morphine-treated mice was significantly decreased, and the polysaccharide peptide could induce the expression of these genes indicating that the one from G. lucidum could be of a potential application in controlling abuse of opiate-induced immunodeficiency.

Enzyme-Inhibiting Activity Triterpenoids of G. lucidum have been reported to exert various enzyme inhibitory activities. Inhibitors of farnesyl protein transferase have been demonstrated to inhibit Ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. Ganoderic acids A and C were identified to be inhibitors of farnesyl protein transferase (42). Ergosterol peroxide, 5,8-epidioxy-5␣, 8␤-ergosta-6,22E-dien-3␤-ol, from G. lucidum, was reported to selectively enhance the inhibitory effect of linoleic acid on DNA polymerase-␤, but not on DNA polymerase-␣. Ergosterol peroxide itself was ineffective but completely blocked rat DNA polymerase-␤ in the presence of linoleic acid (39). Inhibitors of phospholipase A2 can be developed as potential anti-inflammatory agents for the treatment of rheumatic arthritis, asthma, and psoriasis. Ganoderic acid T was found to inhibit secreted phospholipase A2 from pig pancreas, human synovial fluid, and bee venom, but no such effect was

observed with ganoderic acids AA, O, R, S, T-OH, and T-OH-H2 (16).

Immunomodulating Effects The major immunomodulating effects of active substances derived from G. lucidum include mitogenicity and activation of immune effector cells such as T lymphocytes, macrophages, and NK cells leading to the production of cytokines including ILs, TNF-␣, and IFNs. Other effects, such as inhibition of mast cells, activation of B lymphocytes, and the complement system have also been reported (15).

Mitogenic Activity Extracts from G. lucidum (e.g., polysaccharide fractions, methanolic extracts, and LZ-8) have mitogenic effects on mouse splenocytes and human peripheral blood mononuclear cells (PBMCs) in the presence of various immunostimulating or immunosuppressive agents (e.g., phytohemagglutinin and 12-O-tetradecanoylphorbol 13acetate) (43,44). Treatment of the PBMCs with cyclosporin A (CsA) led to blockage of the cell proliferation. The methanolic fraction from G. lucidum recovered the CsAinduced inhibition of the cell proliferation, which might be due to the inhibition of the protein kinase C signal pathway and acceleration of the CsA signal pathway.

Effects on Immune Effector Cells Splenocytes In vitro and in vivo studies in mice indicated that G. lucidum water extract stimulates the production of IL-2 by splenocytes in the presence of hydrocortisone (1,2,9,12).

T Cells Extracts from G. lucidum are potent activators of T cells, inducing the production of a number of cytokines, in particular IL-2. In human PBMC (primarily T cells) in vitro, the crude G. lucidum water extract induced the expression of cytokines including IL-10 and TNF-␣, IL-1␤, IL-6, and IL-2 (44). Crude polysaccharide fractions isolated from fresh fruiting bodies potentiated the release of IFN-␥ from human T cells (38). A polysaccharide fraction (GL-B) promoted the production of IL-2 in a dosedependent manner and markedly enhanced the cytotoxicity of cytotoxic T lymphocytes, which was increased by 100% at a concentration of 200 ␮g/mL. GL-B also restored the mixed lymphocyte response to alloantigen, automatic proliferation, and IL-2 production of splenocytes in aged mice declined as compared with that in young adult mice in vitro. LZ-8 is also a potent T-cell activator mediating its effects via cytokine regulation of integrin expression. Stimulation of human peripheral blood lymphocytes with LZ-8 resulted in the production of IL-2 and a corresponding upregulation of IL-2 receptor expression (45). In addition to T-cell proliferation, microscopic examination of LZ-8stimulated peripheral blood lymphocytes revealed that LZ-8 induced cellular aggregate formation. This formation correlated with a dramatic rise in ICAM-1 expression and an increased production of IFN-␥ , TNF-␣, and IL-1␤, molecules associated with regulation of ICAM-1 expression. Both the aggregate formation and the proliferative

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effects of LZ-8 were blocked by the addition of a monoclonal antibody to either CD18 or CD11a, the counter– receptor complex components for ICAM-1. Furthermore, addition of neutralizing antibodies to both IL-2 receptor and TNF-␣ blocked aggregate formation, cellular proliferation, and ICAM-1 expression.

Natural Killer (NK) Cells A water-extracted polysaccharide fraction from G. lucidum enhanced the cytotoxicity of splenic NK cells in tumorbearing mice (1,16,38).

Macrophages Macrophages are responsible for killing pathogens in the body. Activation of macrophages by substances from G. lucidum results in the release of cytokines, NO, and other mediators (38,46). All of these responses are associated with the antitumor, antimicrobial, and anti-inflammatory effects of G. lucidum. Polysaccharides from G. lucidum, in particular ␤D-glucans, are potent stimulators of murine and human macrophages in vitro and in vivo (38,46). CR3 receptors on macrophages are bound by ␤-D-glucans and internalized, priming a series of molecular events. Crude water-extracted polysaccharides isolated from fresh fruiting bodies of G. lucidum potentiated the production of cytokines including IL-1␤, IL-6, IFN-␥ , and TNF-␣ by human macrophages, which were antiproliferative, differentiated and apoptosis inductive to the HL-60 and the U937 leukemic cells (38). IFN-␥ and TNF-␣ released from macrophages act synergistically to inhibit the growth of leukemic cells as shown by the antibody–neutralization studies. GLB7, a G. lucidum polysaccharide, decreased the production of oxygen-free radicals and antagonized the respiratory burst induced by PMA in murine peritoneal macrophages. These observations suggest that GLB7decreased production of oxygen-free radicals in murine peritoneal macrophages plays an important role in the anti-aging effect of G. lucidum polysaccharides (46). Ganoderan (GAN), a ␤-D-glucan isolated from G. lucidum, enhanced the production of NO in the RAW 264.7 macrophages (46). The ability of GANs to produce NO was based on differences in the chemical composition of GANs obtained from the mycelium on various carbon sources and mycelial fractionation. The highest NO production was observed in the polysaccharide, which was extracted from the mycelial wall. Partial removal of the protein in the extracellular GAN by TCA treatment did appreciably reduce its capacity to secrete NO. The cell proliferation of GAN-treated RAW 264.7 cell lines was inhibited compared to its control. Of the culture supernatant of macrophage activated by this glycan, the percentage of cytotoxicity against mouse leukemia L1210 cells was slightly dependent on the amount of NO in the culture supernatants of the activated macrophages. These results indicate that the ␤-glucan-related polysaccharides of the higher fungus activate macrophages and release NO, which is an important chemical messenger for the induction of many biological responses. A protein-polysaccharide fraction (GLB) from the growing tips of G. lucidum is a strong stimulator to the macrophages (47). When analyzed using a flow cytometer, GLB increased the phagocytic activity of the

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BALB/c mouse peritoneal macrophages as well as chicken macrophage BM2CL cells against FITC-labeled Candida albicans by 55.2% and 21.2%, respectively. It also enhanced the spreading and expression of MHC class II molecules of BM2CL cells as well as the mouse peritoneal macrophages.

Mast Cells Some substances from G. lucidum can act on mast cells. A water extract of the fruit body had inhibitory activity on histamine release from rat peritoneal mast cells, induced by compound 48/80 or antigen (egg white albumin)antibody reaction and on passive cutaneous anaphylaxis reaction in guinea pigs and rats. Two ganoderic acids (C and D) isolated from the fruit body by methanol inhibited the histamine release from rat mast cells, induced by compound 48/80 and concanavalin A. A chloroform extract from G. lucidum broth also significantly inhibited histamine release from rat peritoneal mast cells induced by A-23187 and compound 48/80. The mechanism for the inhibitory activity on histamine release from mast cells was further studied. Palmitic acid, stearic acid, oleic acid, and linoleic acid were isolated from the active fractions. Of these, oleic acids induced membrane stabilization in model membrane systems. Cyclo-octasulfur extracted from the culture medium of G. lucidum may decrease calcium uptake from the extracellular medium by a disulfide exchange reaction in the cell membrane leading to inhibition of histamine release from mast cells (1,2,9,14,16).

Complement System An alkali extract isolated from cultured mycelium of G. lucidum activated classical and alternative pathways of a complement system. Activated complement C3 was observed by crossed immunoelectrophoresis in mice. This fraction also activated the reticuloendothelial system of mice in the carbon clearance test and increased hemolytic plaque-forming cells of the spleen. The alkali extract consisted of 10% carbohydrate and 49% proteins.

Histamine Release Inhibition The fruiting bodies have been traditionally used as antiinflammatory agents for the treatment of asthma or allergy. In the course of a screening test for the inhibition of histamine release from rat mast cells, it was found for the first time that ganoderic acids C and D inhibited histamine release from rat mast cells (that were induced by compound 48/80 and concanavalin A). Other than the triterpenoid compounds, cyclo-octasulfur from this fungus also effectively inhibited histamine release from rat peritoneal mast cells and interacted with membrane proteins to inhibit Ca uptake causing a blockade of histamine release (12,13,19).

Hepatoprotective Activity G. lucidum has been widely used for the treatment of chronic hepatopathy of various etiologies. Data from in vitro and animal studies indicate that G. lucidum extracts (mainly polysaccharides or triterpenoids) exhibit protective activities against liver injury induced by toxic chemicals (e.g., CCl4 ) and Bacillus Calmette-Guerin plus lipopolysaccharide. Reishi also showed antihepatitis Bvirus (HBV) activity in a duckling study. The mechanisms

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of the hepatoprotective effects of G. lucidum have been largely undefined. However, accumulating evidence suggests several possible mechanisms. These include antioxidant and radical-scavenging activity, modulation of hepatic Phase I and II enzymes, inhibition of ␤-glucuronidase, antifibrotic and antiviral activity, modulation of NO production, maintenance of hepatocellular calcium homeostasis, and immunomodulating effects (17). The mushroom could represent a promising approach for the management of various chronic hepatopathies. Further studies are needed to explore the kinetics and mechanisms of action of its constituents with hepatoprotective activities.

Antidiabetic Effect Animal studies have demonstrated that the polysaccharide fractions of G. lucidum have potential hypoglycemic and hypolipidemic activities. A water extract of Reishi reduced the increase in blood glucose and blood insulin levels in rats (50 mg p.o.) following oral glucose test. Following adrenaline (IV) or oral glucose in rats, the mushroom inhibited increases in blood glucose without raising blood insulin levels. Glycans (ganoderans B and D) have shown significant hypoglycemic activity in mice.

Cardiovascular and Circulatory Functions Cholesterol and Lipid Metabolism The powdered mycelium of Reishi, at 5% of the diet of spontaneously hypertensive rats for four weeks, caused plasma total cholesterol to decrease significantly (by 18.6%) compared to controls. Total liver triglyceride and total liver cholesterol levels were also significantly lower in the Reishi-fed group (by approximately 46% and 56%, respectively) (48,49).

Hypertension A water extract of the mycelium administered to rats and rabbits produced significant hypotensive effects; an activity the researchers suggested is secondary to the primary effect that suppresses sympathetic outflow of the central nervous system (50). The powdered mycelium of G. lucidum, at 5% of the diet of spontaneously hypertensive rats for four weeks, caused systolic blood pressure to be significantly lower without causing a significant difference in the heart rate (48).

Antibacterial and Antiviral Value Antibacterial Effect of G. lucidum on Gram-Positive and Gram-Negative Bacteria Recently, more studies demonstrated that G. lucidum contained antibacterial constituents that are able to inhibit gram-positive and/or gram-negative bacteria (1,2,2,5,5,17,51). The aqueous extract from the carpophores of G. lucidum inhibited 15 types of gram-positive and gram-negative bacteria. Further studies indicate that the antimicrobial combinations of G. lucidum extract with four antibiotics (ampicillin, cefazolin, oxytetracycline, and chloramphenicol) resulted in additive effects in most instances: synergism in two instances when combined with cefazolin against Bacillus subtilis and Klebsiella oxytoca (52), and antagonism in two instances.

Helicobacter pylori Helicobacter pylori is associated with human gastroduodenal diseases such as gastritis, peptic ulcer, and gastric carcinoma. The extracts of many mushrooms inhibited the growth of this bacterium (17,53). The extract of G. lucidum and some other species of higher Basidiomycetes arrested the growth of this pathogen. When their extracts were fractionated, the ether fractions of G. lucidum and Agaricus bisporus (J. Lge) Imbach were the most effective. Among seven components separated from the ether fraction of G. lucidum extract by silica gel column chromatography, P3 was the most potent with a minimum inhibitory concentration of 200 ␮g/mL. It appears that some constituents such as ganomycin, triterpenoids, and aqueous extracts from Ganoderma species have a broad spectrum of in vitro antibacterial activity against gram-positive and gram-negative bacteria and H. pylori. Thus, it is possible that the antibacterial activity of Ganoderma species may be beneficial for those patients with chronic infection (e.g., chronic bronchitis) and those with H. pylori-positive peptic ulcer diseases, though clinical studies are required to confirm this.

Antihuman Immunodeficiency Virus (HIV) Activity HIV was isolated as an etiological agent of acquired immunodeficiency disease syndrome in 1983 (54). Acquired immunodeficiency syndrome caused by HIV infection has recently become an important social and medical problem. Anti-HIV therapy by nucleoside analogues, such as 3 -azido-thymidine, is the major effective approach for the treatment of acquired immunodeficiency syndrome (55). These agents are potent inhibitors of HIV reverse transcriptase (RT) and protease (56). However, the emergence of drug-resistant variants of HIV and toxicities severely limits the long-term effectiveness of these drugs. Recent studies have indicated that many natural products are active as anti-HIV agents. These compounds belong to a wide range of different structural classes, for example, coumarins, flavonoids, tannins, alkaloids, lignans, terpenes, naphtho- and anthraquinones, and polysaccharides (57). In vitro studies indicate that various triterpenoids from G. lucidum had potent inhibitory activity against HIV. Lucidenic acid O and lucidenic lactone, isolated from the fruiting body of G. lucidum, not only inhibited the activities of calf DNA polymerase- and rat DNA polymerase-␤, but also those of HIV-1 RT (17). Ganoderiol F and ganodermanontriol isolated from the fruiting bodies of G. lucidum are active against HIV-1 growth (2,9,17) Ganoderic acid B and ganoderiol B showed potent inhibitory effect on HIV protease. Other triterpenoids including ganoderic acid C1, 3␤-5␣-dihydroxy-6␤-methoxyergosta-7,22-diene, ganoderic acid-, ganoderic acid H, and ganoderiol A had moderate activity against HIV-1 protease (2,9,17,58). In addition, ganoderic acid-␤, lucidumol B, ganodermanondiol, ganodermanontriol, and ganolucidic acid A showed significant anti-HIV-1 protease activity (23). Ganoderic acid A, B, and C1 had minor inhibitory activity against HIV protease with IC50 values of 140–430 ␮M. It appears that there is a structure–activity relationship for triterpenoid showing anti-HIV protease activity. The C3, C24, or C25 atoms are vital for the anti-HIV activity (23).

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The aqueous low-molecular-weight fraction extracted from G. lucidum also exhibited anti-HIV activity using the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)5-[(phenylamino) carbonyl]-2 H-tetrazolium hydroxide] antiviral assay, which can quantitatively measure cytopathic effects of HIV-1 on CEM cells, a human T lymphoblastoid cell line (59,60). The IC50 and EC50 values were 125 and 11 ␮g/mL, respectively, resulting in a therapeutic index of 11.4. This aqueous low-molecular-weight extract was further fractionated to eight subfractions by methanol: GLA (methanolic extract), GLB (hexane soluble), GLC (acetic ether soluble), GLD (water soluble), GLE (neutral), GLF (acidic), GLG (alkaline), and GLH (amphoteric). All subfractions except GLD, GLF, and GLH exhibited anti-HIV activity with IC50 and EC50 values of 22–44 ␮g/mL and 14–44 ␮g/mL, respectively. GLC and GLG inhibited HIV RT. Showing consistency, incubation of GLC at 50 ␮g/mL or GLG (100 ␮g/mL) with Jurkat T cells gave a 75% and 66% inhibition of HIV growth, respectively. However, the high-molecular-weight fraction did not inhibit any HIV-induced cytopathic effect. Both low-molecular-weight and high-molecular-weight fractions from G. lucidum had negligible toxicities to CEM cells. The results indicate that the aqueous low-molecularweight fraction from the fruiting bodies of G. lucidum, and the neutral and alkaline subfractions from the methanolic extract might contain small molecular weight polysaccharides (61).

Epstein-Barr Virus Virus-induced carcinogenesis is considered a complicated process with multiple steps involving a number of cellular signaling pathways. A few polyoxygenated lanostanoid triterpenes isolated from G. applanatus inhibited the 12O-tetradecanoylphorbol-13-acetate induced Epstein-Barr virus early antigen in Raji cells. Similar effects have been observed with Zingiberaceae rhizomes, a commonly used traditional medicine in Malaysia. These results indicate that herbal medicines, such as Ganoderma species, may behave as antitumor promoters (17,62).

Other Viruses The antiviral effects of two water-soluble substances (GLhw and GLlw) and eight methanol-soluble substances (GLMe-1–8) isolated from the carpophores of G. lucidum, were investigated on influenza A virus strains and vesicular stomatitis virus Indiana and New Jersey in vitro. These activities were evaluated by the cytopathic effect inhibition assay and plaque reduction assay using Vero and HEp-2 cells. Five substances, GLhw, GLMe-1, -2, -4, and -7 significantly inhibited the cytopathic effects of vesicular stomatitis virus. GLMe-4 did not exhibit cytotoxicity up to 1000 ␮g/mL, while it displayed potent antiviral activity on the vesicular stomatitis virus New Jersey strain with a therapeutic index of more than (5,44,60,62–64).

CLINICAL STUDIES Reishi has now become recognized as an alternative adjuvant in the treatment of leukemia, carcinoma, hepatitis, and diabetes (2,8,9,14–19,26,31). Clinical studies, to date, lack the controls needed to make a scientific assessment

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of its efficacy in a given application, a situation expected to change with increasing interest from Western scientific communities. It was only since the last decade that clinical trials on the use of G. lucidum preparation used to treat cancer and other diseases have been reported in international peer-reviewed journals (62,64).

Cancer In clinical studies, G. lucidum products have been widely used as a single agent or in combination with other herbal medicines or chemotherapeutic drugs for many years, mainly in Asian countries. However, randomized, placebo-controlled and multicancer clinical studies using G. lucidum alone have rarely been reported.

G. lucidum as a Single Agent In a randomized, placebo-controlled clinical study, 143 patients with advanced previously treated cancer were given an oral G. lucidum polysaccharide extract (Ganopoly) of 1800 mg three times daily for 12 weeks (16). Twenty-seven patients were not assessable for response and toxicity, because they were lost in the follow-up or refused further therapy before the 12 weeks of treatment. Of the 100 fully assessable patients, 32.2% had progressive disease before or at the 6-week evaluation point (range: 5 day–6 wk). Sixteen subjects developed progressive disease between 6 and 12 weeks of therapy. No objective (partial or complete) responses were observed, but 26.6% had stable disease for 12 weeks or more (range: 12–50 wk). There was no significant change in the Functional Assessment of Cancer Therapy-General (FACT-G) scores in 85 assessable patients. However, palliative effects on cancer-related symptoms, such as sweating and insomnia, have been observed in many subjects. In the group with stable disease, FACT-G scores improved in 23 patients, were unchanged in five, and declined in one. Within this group, the median change from the baseline score to the 6- and 12-week score was + 7.6 and + 10.3, both statistically significant (P < 0.05). For the 38 patients with stable disease (SD), the median change from the baseline score was 28.1 ± 10.2 weeks. The prostate-specific antigen (PSA) levels in the five prostate cancer patients were reduced significantly (P < 0.05) during SD. Ganopoly was well tolerated with five moderate adverse events recorded. The results indicate that Ganopoly may have an adjunct role in the treatment of patients with advanced cancer although objective responses were not observed in this study.

G. lucidum-Containing Herbal Mixture: PC-SPES Several recently published reports have found that G. lucidum or G. lucidum-containing herbal mixtures (PC-SPES) had biological activities (e.g., cancer biomarker alteration) and beneficial effects (e.g., palliative effects in cancer patients), although striking objective responses were not observed (16,65). PC-SPES has been used as an alternative in the treatment of prostate cancer (65). Several clinical trials have been completed with patients having advanced prostate cancer (66,67). Small et al. (67) included 70 subjects with androgen-dependent (n = 33) and androgenindependent (n = 37) disease, which was refractory to surgery, radiotherapy, and hormone therapy. Treatment of PC-SPES at a dose of three capsules (320-mg cap) orally resulted in ≥80% decrease in PSA levels in all 32 patients

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with androgen-dependent cancer, while it was undetectable in 26 patients (81%). The median duration of PSA response was 57 weeks. In the 35 patients with androgenindependent cancer, 19 (54%) had a PSA decrease of ≥50% with median duration of PSA response of 18 weeks. The study by Pfeifer et al. (66), which included only 16 patients with androgen-independent disease for just a 20-week followup, showed an improvement in quality of life for the patients. PC-SPES was generally well tolerated by prostate cancer patients, but they exhibited a dose-dependent toxicity similar to that of diethylstilboestrol (67). Side effects include reduced libido, hot flashes, diarrhea, dyspepsia, leg cramps, nipple tenderness, and gynecomastia (66,67). More life-threatening adverse events are pulmonary emboli in 4% to 5% of patients and deep vein thrombosis in 2% of patients. Overall, the clinical responses to PC-SPES compare favorably with second-line hormonal therapy with agents, such as estrogens and ketoconazole (58,62). However, it must be noted that the adulteration of PCSPES products has become a serious problem. Further details may be obtained at the website of the NIH National Center for Complementary and Alternative Medicine at http://nccam.nih.gov/health/alerts/spes/.

Complement System A clinical study in elderly patients with insomnia and palpitations recently showed that taking G. lucidum essence for four to six weeks increased their serum C3 levels (1,2,21,62).

Dosage Forms G. lucidum is usually prescribed in various forms. It may be injected as a solution of powdered spore. It may be ingested as a soup, syrup, tea, tablets, capsules, tincture, or bolus (powdered medicine in honey). The dose in tincture form (20%) is 10 mL three times daily, that of tablet is one g tablet three times daily, and syrup is 4 to 6 mL/day. As an antidote for ingestion of poisonous mushrooms, dried G. lucidum (120–200 g) is decocted in water and given as a drink three to five times daily (1,2,5,9).

Safety Profile Contraindications. None known.

Drug Interactions Because Reishi potentiates the immune system, caution is advised for those receiving immunosuppressive therapies.

Side Effects In oral treatments, some patients, when initially taking a powder extract of Reishi, have experienced temporary symptoms of sleepiness, thirst, rashes, bloating, frequent urination, abnormal sweating, and loose stools (49,62) Large oral doses of vitamin C taken at the same time as Reishi powder extract reportedly counteracted loose stools (2,8,9,49,62). The inhibition of platelet aggregation by G. lucidum (1,2,9) may present an additive effect in those taking blood-thinning medications such as daily aspirin or warfarin.

Synergistic antimicrobial activity was shown with an aqueous extract of G. lucidum in combination with cefazolin against Klebsiella oxytoca ATCC 8724 and Bacillus subtilis ATCC 6603, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25933, and Salmonella typhi ATCC 6509 (56).

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39. Mizushina Y, Hanashima L, Yamaguchi T, et al. A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases. Biochem Biophys Res Commun 1998; 249:17–22. 40. Chiu SW, Wang ZM, Leung TM, et al. Nutritional value of Ganoderma extract and assessment of its genotoxicity and antigenotoxicity using comet assays of mouse lymphocytes. Food Chem Toxicol 2000; 38:173–178. 41. Lu ZW. Psychoneuroimmunological effects of morphine and the immunoprotection of Ganoderma polysaccharides peptide in morphine-dependent mice. Chin J Physiol 1995; 26:45– 49. 42. Lee S, Park S, Oh JW, et al. Natural inhibitors for protein prenyltransferase. Planta Med 1998; 54:303–308. 43. van der Hem LG, van der Vliet JA, Bocken CF, et al. Ling Zhi-8: Studies of a new immunomodulating agent. Transplantation 1995; 60:438–443. 44. Mao T, van De Water J, Keen CL, et al. Two mushrooms, Grifola frondosa and Ganoderma lucidum, can stimulate cytokine gene expression and proliferation in human T lymphocytes. Int J Immunother 1999; 15:13–22. 45. Haak-Frendscho M, Kino K, Sone T, et al. Ling Zhi-8: A novel T cell mutagen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol 1993; 150: 101–113. 46. Han MD, Lee ES, Kim YK, et al. Production of nitric oxide in RAW 264.7 macrophages treated with ganoderan, the betaglucan of Ganoderma lucidum. Korean J Mycol 1998; 26:246– 255. 47. Oh JY, Cho KJ, Chung SH, et al. Activation of macrophages by GLB, a protein-polysaccharide of the growing tips of Ganoderma lucidum. Yakhak Hoeji 1998; 42:302–306. 48. Kabir Y, Kimura S, Tamura T. Dietary effect of Ganoderma lucidum mushroom on blood pressure and lipid levels in spontaneously hypertensive rats (SHR). J Nutr Sci Vitaminol 1988; 34:433–438. 49. Soo TS. Effective dosage of the extract of Ganoderma lucidum in the treatment of various ailments. In: Royse DJ, ed. Mushroom Biology and Mushroom Products. University Park, PA: The Pennsylvania State University, 1996:177– 185. 50. Lee SY, Rhee HM. Cardiovascular effects of mycelium extract of Ganoderma lucidum: Inhibition of sympathetic outflow as a mechanism of its hypotensive action. Chem Pharm Bull (Tokyo) 1990; 38:1359–1364. 51. Suay I, Arenal F, Asensio FJ, et al. Screening of Basidiomycetes for antimicrobial activities. Antonie van Leeuwenhoek 2000; 78:129–139. 52. Yoon SY, Eo SK, Kim YS, et al. Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics. Arch Pharm Res 1994; 17:438–442. 53. Kim DH, Bae EA, Jang IS, et al. Anti-Helicobacter pylori activity of mushrooms. Arch Pharm Res 1996; 19:447–449. 54. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983; 220:868– 971. 55. Matsushita S, Kimura T. Advance in treatment strategy and immune reconstruction against HIV1 infection. Microbiol Immunol 2002; 46:231–239. 56. Menendez-Arias L. Targeting HIV: Antiretroviral therapy and development of drug resistance. Trends Pharmacol Sci 2002; 23:381–388. 57. Vermani K, Garg S. Herbal medicines for sexually transmitted diseases and AIDS. J Ethnopharmacol 2002; 80: 49–66. 58. Smith DC, Redman BG, Flaherty LE, et al. A phase II trial of oral diethylstilbestrol as a second line hormonal agent in advanced prostate cancer. Urology 1998; 52:257–260.

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59. Kim HW, Shim MJ, Choi EC, et al. Inhibition of cytopathic effect of human immunodeficiency virus-1 by water-soluble extract of Ganoderma lucidum. Arch Pharm Res 1997; 20:425– 431. 60. Mshigen KE, Mtango D, Massele A, et al. Intriguing biological treasures more precious than gold: The case of tuberous truffles, and immunomodulating Ganoderma mushrooms with potential for HIV/AIDS treatment. Discov Innov 2005; 17(3–4):105–109. 61. Lin Z-B. Lingzhi. From mystery to science. Beijing: Peking University Medical Press, 2009:162. 62. Eo SK, Kim YS, Lee CK, et al. Antiherpetic activities of various protein bound polysaccharides isolated from Ganoderma lucidum. J Ethnopharmacol 1999; 68:175– 181.

63. Eo SK, Kim YS, Lee CK, et al. Possible mode of antiviral activity of acidic protein bound polysaccharide isolated from Ganoderma lucidum on herpes simplex viruses. J Ethnopharmacol 2000; 72:475–481. 64. Zhou X, Lin J, Yin Y, et al. Ganodermataceae: Natural products and their related pharmacological functions. Am J Chin Med 2007; 35(4):559–574. 65. Pandha HS, Kirby RS. PC-SPES: Phytotherapy for prostate cancer. Lancet 2002; 359:2213–2215. 66. Pfeifer BL, Pirani JF, Hamann SR, et al. PC-SPES, a dietary supplement for the treatment of hormone-refractory prostate cancer. BJU Int 2000; 85:481–485. 67. Small EJ, Frohlich MW, Bok R, et al. Prospective trial of the herbal supplement PC-SPES in patients with progressive prostate cancer. J Clin Oncol 2000; 18:3595–3603.

Riboflavin Richard S. Rivlin

INTRODUCTION

The sequence of events in the synthesis of the flavin coenzymes from riboflavin is that the first biosynthetic enzyme, flavokinase, catalyzes the initial phosphorylation of riboflavin by ATP to FMN (Fig. 2). A fraction of FMN is directly utilized in this form as a coenzyme. The largest fraction, however, combines with a second molecule of ATP to form FAD, the predominant tissue flavin, in a reaction catalyzed by FAD synthetase, also known as FAD pyrophosphorylase. The covalent attachment of flavins to specific tissue proteins occurs after FAD has been synthesized (13,14). A sequence of phosphatases reconverts FAD to FMN and FMN, in turn, to riboflavin (15). Most flavoproteins utilize FAD rather than FMN as coenzyme for a wide variety of metabolic reactions. Microsomal nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase is highly unusual in containing both FMN and FAD as coenzymes in equimolar ratios. Riboflavin is yellow and has a high degree of natural fluorescence when excited by UV light, a property that can be utilized conveniently in its assay. There are a number of variations in structure of the naturally occurring flavins. Riboflavin and its coenzymes are sensitive to alkali and acid, particularly in the presence of UV light. Under alkaline conditions, riboflavin is photodegraded to yield lumiflavin (7,8,10-trimethylisoalloxazine), which is inactive biologically. Riboflavin is degraded by light to form lumichrome (7,8-dimethylalloxazine) under acidic conditions, a product that is also relatively inactive (10). Thus, an important physical property of riboflavin and its derivatives is their sensitivity to UV light, resulting in rapid inactivation. Therefore, prolonged phototherapy of neonatal jaundice and of certain skin disorders may promote the development of systemic riboflavin deficiency. The structure–function relationships of the various biologically active flavins have been comprehensively reviewed (14). One physical property of riboflavin needs emphasis, namely, its very limited water solubility, which greatly restricts its use as a parenteral supplement and as an oral supplement as well. The flavin coenzymes, FMN and FAD, as well as the fraction of flavins bound covalently to tissue proteins, function in a wide array of processes in intermediary metabolism, most notably in oxidation–reduction reactions. FAD is an inherent component of the respiratory chain and therefore is closely involved in the generation of energy. Flavin coenzymes participate in drug and steroid metabolism together with the cytochrome P450 enzymes. Flavins have critical roles in fat metabolism. One-electron transfers and two-electron transfers from substrate to FMN and FAD constitute the major redox functions of these flavin coenzymes (10). Other reactions

New metabolic roles for riboflavin are continually emerging since its discovery in the early part of the 20th century (1–3). We are now defining the mechanisms of action of riboflavin at the molecular, physiological, and clinical levels. In particular, we are beginning to appreciate how riboflavin may play a role in pathogenesis of chronic diseases, such as cancer, cardiovascular disease, metabolic bone disorders, inflammation, and infections.

BACKGROUND The structure of riboflavin was identified (4,5), its synthesis achieved (6,7), and its coenzyme derivatives described in 1937 (8) and 1938 (9,10). More recently, the role of this vitamin in homocysteine metabolism has become more widely appreciated; acting in concert with folic acid, vitamin B6, and vitamin B12, to lower serum levels of homocysteine (11). Riboflavin deficiency, when it occurs, has traditionally been attributed to a daily diet that contains inadequate amounts of this vitamin. In our view, insufficient attention has been paid to the many factors, both physiological and pathological, that influence the utilization of this vitamin in health and disease. Thus, a physiological state of riboflavin deficiency can result from the effects of certain drugs, hormones, and other factors in addition to a poor diet. Newer aspects of this subject are reviewed in this chapter.

BIOCHEMISTRY AND FUNCTIONS Chemically, riboflavin is 7,8-dimethyl-10-(1 -D-ribityl)isoalloxazine. The isoalloxazine ring is a planar structure that is also shared by the two major coenzyme derivatives formed from riboflavin, namely flavin mononucleotide or riboflavin-5 -phosphate (FMN) and flavin adenine dinucleotide (FAD). These structures are shown in Figure 1. A small proportion of the flavin coenzymes are linked covalently with tissue proteins (12), including some vital enzymes, such as sarcosine dehydrogenase, succinic dehydrogenase, and monoamine oxidase. Unlike humans, who cannot synthesize ascorbic acid from its precursors, some species of mammals have large amounts of the microsomal ascorbic acid synthesizing enzyme, L-gulonolactone oxidase, which contains covalently bound flavins. 691

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CH2

(CHOH)3

N

CH2OH

N

CH3

CO

CH3

NH

C O

N Riboflavin

OH CH2

N

H C O H

H C O H

H C O H

CH2 OP

O

OH

N

CH3

CO

CH3

N

NH

C O

Riboflavin phosphate (flavin mononucleotide) O CH2 N

(CHOH)3

CH2O

O

P

O

P

OH

N

CH3

CO

CH3

NH

OCH2

OH NH2

CH N

C O

HOCH

C

N

HOCH

C

O

N

CH

CH

C CH

N

N

Flavin adenine dinucleotide (FAD)

Figure 1

Structures of riboflavin, riboflavin-5 -phosphate (flavin mononucleotide, FMN), and flavin adenine dinucleotide (FAD).

catalyzed by flavoproteins include dehydrogenation, oxidative decarboxylation, dioxygenation, and reduction of oxygen to hydrogen peroxide.

RIBOFLAVIN DEFICIENCY With the onset of riboflavin deficiency, there are a number of metabolic adaptations that occur to conserve the limited

reserves. One of the adaptations is a fall in the small hepatic pool of free riboflavin to nearly undetectable levels, with a relative sparing of the pools of FMN and FAD (16). These coenzymes are needed to fulfill critical metabolic functions, whereas the vitamin itself has little biological activity. Another adaptation to deficiency in its early stages is an increased de novo synthesis of reduced glutathione (GSH) from its amino acid precursors (16). This effect

THYROID HORMONES

Flavokinase RIBOFLAVIN

FMN Phospholase

FLAVIN MONONUCLEOTIDE (FMN)

Unstable Flavoprotein Apoenzymes FAD Pyrophosphorylase Pyrophosphalase Stable Flavoprotein Holoenzymes

FLAVIN Enzyme? ADENINE DINUCLEOTIDE (FAD)

COVALENTLY BOUND FLAVINS (FAD)

Figure 2 Sequence of events in the formation of FMN, FAD, and covalently bound flavins from riboflavin and its regulation by thyroid hormones. Thyroid hormones stimulate the activity of flavokinase and FAD pyrophosphorylase as well as the formation of covalently bound flavins. The combination of unstable apoenzymes with their flavin cofactors converts them into stable flavoprotein holoenzymes.

Riboflavin

N

CH3

N

Intestinal Lumen

CO NH

CH3

N

Liver Food (FAD)

N

FMN

RIBOFLAVIN

IMIPRAMINE

CI

CH2–(CH2)2–N(CH3)2 CHLORPROMAZINE

Riboflavin

FMN FAD Mucosal Cell

S

N

Riboflavin

CH-(CH2)2-N(CH3)2 AMITRIPTYLINE

FAD

vin fla

CH2-(CH2)2-N(CH3)2

FMN bo Ri

C O

n

Ribofla vi

CH2–(CHOH)3–CH2OH

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Figure 4 Digestion of food sources of flavins to riboflavin, which is transported across the intestinal mucosa and rephosphorylated in the mucosal cell. Riboflavin bound to serum proteins is transferred to the liver, where it is rephosphorylated to FMN and FAD.

Figure 3 Structural similarities among riboflavin, chlorpromazine, imipramine, and amitriptyline.

may occur in response to the diminished reconversion of oxidized glutathione (GSSG) to its reduced form. In riboflavin deficiency, the activity of glutathione reductase, a key FAD-requiring enzyme, is greatly lessened. Reduced glutathione levels may be maintained if the increased capacity to synthesize GSH de novo is adequate to meet the mounting needs. There is increasing evidence for the emerging concept that dietary inadequacy is not the only cause of deficiency and that certain endocrine abnormalities, such as adrenal and thyroid hormone insufficiency, certain drugs, and diseases may interfere significantly with vitamin utilization (17,18). Psychotropic drugs, such as chlorpromazine, antidepressants (including imipramine and amitriptyline (19)), cancer chemotherapeutic drugs (e.g., adriamycin), and some antimalarial agents (e.g., quinacrine (20)) impair riboflavin utilization by inhibiting the conversion of this vitamin into its active coenzyme derivatives. Figure 3 shows the structural similarities among riboflavin and the psychoactive drugs imipramine, chlorpromazine, and amitriptyline. Riboflavin deficiency commonly occurs in patients who abuse alcohol chronically. Alcohol causes shortage of the vitamin by inhibiting both its digestion from dietary sources, which are largely in the form of FAD, and its intestinal absorption (Fig. 4) (21). These findings suggest that improvement of the riboflavin nutrition of alcoholics can be accomplished more rapidly and effectively by administering vitamins in pure form, as in supplements, rather than entirely from food sources. Furthermore, in riboflavin-deficient animals, decreased GSH concentrations as well as decreased activities of GSH peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase occur. These findings strongly indicate that the combination of riboflavin deficiency and alcohol administration not only lowers hepatic GSH concentrations, but also inhibits enzymes controlling GSH metabolism and therefore may intensify the hepatic injury induced by excessive alcohol consumption. The consequences of a poor diet in a patient abusing alcohol may be exacerbated by the use of certain drugs for prolonged periods.

In experimental animals, hepatic architecture is markedly disrupted in riboflavin deficiency. Mitochondria in riboflavin-deficient mice increase greatly in size, and cristae increase in both number and size (22). These structural abnormalities may disturb energy metabolism by interfering with the electron transport chain and metabolism of fatty acids. Villi decrease in number in the rat small intestine; villus length increases, as does the rate of transit of developing enterocytes along the villus (23). These findings of structural abnormalities together with accelerated rate of intestine cell turnover (24) may help to explain why dietary riboflavin deficiency leads to both decreased iron absorption and increased iron loss from the intestine. Riboflavin deficiency has many other effects on intermediary metabolism, particularly on lipid, protein, and vitamin metabolism. Of particular relevance to vitamin metabolism is the fact that the conversion of vitamin B6 to its coenzyme derivative, pyridoxal-5 -phosphate, may be impaired (25). Riboflavin deficiency has been studied in many animal species and has several consequences, foremost of which is failure of growth. Additional effects include loss of hair, skin disturbances, degenerative changes in the nervous system, and impaired reproduction. Congenital malformations occur in the offspring of female rats that are riboflavin deficient. The conjunctiva becomes inflamed, the cornea is vascularized and eventually opaque, and cataract may result (26). Changes in the skin consist of scaliness and incrustation of red-brown material consistent with changes in lipid metabolism. Alopecia may develop, lips become red and swollen, and filiform papillae on the tongue deteriorate. During late deficiency, anemia develops. Fatty degeneration of the liver occurs. Important metabolic changes occur, so that deficient rats require 15% to 20% more energy than control animals to maintain the same body weight. In all species studied to date, riboflavin deficiency causes profound structural and functional changes in an ordered sequence. Early changes are very readily reversible. Later anatomical changes, such as formation of cataract, are largely irreversible despite treatment with riboflavin (26). Clinically, riboflavin deficit is not detectable at the bedside by any unique or characteristic physical features. The classical symptoms of glossitis, angular stomatitis,

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and dermatitis are not specific to riboflavin deficiency and may be observed in other vitamin deficiencies as well. When dietary deficiency of riboflavin occurs, it is almost invariably associated with multiple nutrient deficits (27). The syndrome of dietary riboflavin deficiency in humans has many similarities to that in animals. Until recently, there was one notable exception. The spectrum of congenital malformations observed in rodents with maternal riboflavin deficiency had not been clearly identified in humans (28). It now appears from several independent reports that riboflavin deficiency in humans is indeed associated with congenital abnormalities. The National Birth Defects Prevention Study, which evaluated 324 newborn infants who had transverse limb deficiency, concluded that inadequate maternal dietary riboflavin intake is a risk factor (29). Furthermore, Smedts et al. (30) reported that reduced maternal dietary intake of riboflavin and nicotinamide increases the risk of congenital heart disease.

Antioxidant Potential As a precursor to FMN and FAD, riboflavin is a significant contributor to antioxidant activity. Riboflavin itself has little inherent antioxidant action, but the glutathione redox cycle (31) has a major protective role against lipid peroxides. Glutathione peroxidase degrades reactive lipid peroxides. This enzyme requires GSH as a substrate, which is regenerated in vivo by reduction from its oxidized form (GSSG) by glutathione reductase, a well-recognized FADcontaining enzyme, as noted above. It is for this reason that riboflavin deficiency is expected to lead to reduced antioxidant defense capabilities, as has been demonstrated in several studies (32–34). Increased lipid peroxidation has been reported in experimental riboflavin deficiency, with a return towards normal after supplementation with this vitamin (32,35). Both basal and stimulated lipid peroxidation are increased in deficiency of the vitamin (36). Furthermore, the reducing equivalents provided by nicotinamide adenine dinucleotide phosphate, the other substrate required by glutathione reductase, are primarily

B

N5 Methyl THF

A

FAD

C

generated by an enzyme of the pentose monophosphate shunt, glucose-6-phosphate dehydrogenase. Taniguchi and Hara (34), as well as our laboratory (35), have found that the activity of this enzyme is significantly diminished during riboflavin deficiency. This observation provides an additional mechanism to explain the diminished glutathione reductase activity in vivo during riboflavin deficiency and the eventual decrease in antioxidant capacity (36). By sensitizing cells to light, riboflavin may intensify the tissue damage caused by solar radiation. One possible mechanism for this effect may be that UV-A radiation together with administration of riboflavin was reported to increase gene mutations sevenfold compared to UV-A radiation without added riboflavin (37).

Homocysteine Metabolism There is much contemporary interest in the increasingly persuasive evidence that homocysteine has a role in the pathogenesis of vascular disease, including cardiovascular, cerebrovascular, and peripheral vascular disorders (11,38). A simplified sequence of homocysteine metabolism is shown in Figure 5, illustrating the sites of action of vitamins B6 and B12 , folic acid, and riboflavin. Blood levels of folic acid sensitively determine serum homocysteine concentrations (39). N-5-Methyltetrahydrofolate is a cosubstrate with homocysteine in its inactivation by conversion to methionine. Methylcobalamin is also a coenzyme in this enzymatic reaction. Vitamin B6 is widely recognized for its importance in the inactivation of homocysteine by serving as coenzyme of two degradative enzymes, cystathionine-␤-synthase and cystathioninase. However, in our view, there is insufficient appreciation of the fact that riboflavin also plays a vital role in homocysteine metabolism. The flavin coenzyme FAD is required by methylenetetrahydrofolate reductase, the enzyme responsible for converting N-5,10 -methylenetetrahydrofolate to N-5-methyltetrahydrofolate. Thus, the efficient utilization of dietary folic acid requires adequate riboflavin nutrition. As expected, therefore, riboflavin deficiency reduces the activity of

B12

N5,10 Methylene THF

Homocysteine B6

A) Methylene THF Reductase B) Homocysteine Methyltransferase C) Methionine synthase D) Cystathionine β-synthase E) Cystathioninase Figure 5

Homocystine

Methionine

D

+ Serine

Cystathionine

B6

E

Homoserine + Cysteine

Simplified representation of homocysteine metabolism to illustrate the sites of action of vitamins B6 , B12 , folic acid, and riboflavin.

Riboflavin

methylenetetrahydrofolate reductase and inhibits folic acid metabolism in rats (40). As a consequence of this effect, plasma homocysteine levels increase (41). In a large cohort of subjects in the Framingham Offspring Study, the more deficient the individual, as measured by the erythrocyte glutathione reductase activity coefficient (EGRAC), the higher the serum homocysteine concentration, particularly in those with compromised folate status (42). It is relevant to note in this context that there is a genetic variant of the methylenetetrahydrofolate reductase gene (677→T) that is common in the Caucasian population (43). Individuals homozygous (TT) for this gene have approximately half the normal activity of the enzyme and are predisposed to develop elevated serum concentrations of homocysteine (41). It is of interest that a group of investigations found an inverse correlation between plasma homocysteine and plasma riboflavin in individuals both with and without the genetic variation (44). Further research is required to determine whether the serum levels of homocysteine and the prevalence of vascular disease can be correlated directly with indices of marginal as well as overt riboflavin deficiency. Elevated serum levels of homocysteine in both hypothyroid rats (13) and hypothyroid humans are likely due to diminished conversion of dietary riboflavin to its coenzyme derivatives, flavin mononucleotide and flavin adenine dinucleotide. Treatment of hypothyroid adults with thyroid hormones without increasing dietary riboflavin intake completely corrects these defects (18).

Fat Metabolism The vital role of riboflavin in fat metabolism has been highlighted by demonstrations that in certain rare inborn errors, administration of the vitamin may be therapeutic. In acyl-CoA dehydrogenase deficiency, infants present with recurrent hypoglycemia, lipid storage myopathy, and increased urinary excretion of organic acids. Clinical improvement has occurred rapidly after riboflavin supplementation (45,46). Three varieties of the disorder occur, all of which involve flavoproteins of various types. Several patients with a mitochondrial disorder associated with NADH dehydrogenase deficiency showed improvement with riboflavin treatment (47). In patients with HIV infection, riboflavin administered together with thiamin has been noted to prevent elevation of levels of lactic acid and lactic acidosis (48). In other studies of patients with HIV, riboflavin has potential in managing lactic acidosis induced by treatment of the underlying disease with nucleoside analogs (49,50). In addition, riboflavin has been beneficial to cases of ethylmalonic acid encephalopathy (51). Therefore, this vitamin as a supplement may have a role in the management of certain rare inborn errors of fat metabolism.

Anemia Anemia is a characteristic feature in many vitamin deficiencies and it is usually multifactorial in pathogenesis. Nevertheless, there appears to be a relatively specific anemia occurring in riboflavin-deficient individuals that responds to supplementation with the vitamin (52). The anemia is associated with erythroid hypoplasia, and as a consequence, there is diminished reticulocytosis.

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A major effect of riboflavin deficiency in the pathogenesis of anemia appears to be that on iron metabolism (53). It influences tissue mobilization of iron from ferritin, particularly in the gastrointestinal mucosa (54). In addition, it now appears that the deficiency leads to a decrease in the intestinal absorption of the element (55) as well as an increase in its loss from the gastrointestinal tract (56). These mechanisms have been elucidated in rodents. Their relevance to the pathogenesis of anemia in humans remains to be firmly established. It is evident, nevertheless, that riboflavin supplementation does improve hematological function in humans, and likely has an effect on improving the metabolism of iron (53).

Carcinogenesis More recent studies (57) confirm earlier reports (58) that riboflavin deficiency may favor cancer formation by increasing the activation of certain carcinogens, particularly nitrosamines. The flavin vitamin may possibly provide protection against the damage to DNA caused by a number of carcinogens through its action as a coenzyme with a variety of cytochrome P450 enzymes. Furthermore, deficiency enhances the covalent binding of carcinogens to DNA (59). It is important to establish more firmly the potential role of riboflavin as a dietary factor capable of preventing carcinogenesis while at the same time determining the full implications of the photosensitizing actions of flavins on mutagenesis and carcinogenesis. There are reports from China (60,61) and Russia (62) raising the possibility that deficient riboflavin nutritional status, together with shortages of other vitamins, may possibly enhance development of precancerous lesions of the esophagus. Because of their photodynamic actions, flavins may have potential efficacy as adjuncts in cancer treatment. Blue light has been reported to inhibit the proliferation of B16 melanoma cells grown in culture, as well as those transplanted to rodent models (63). It has both cytostatic and cytocidal properties. Riboflavin is the only vitamin that has been observed to increase the degree of cell necrosis induced by blue light. The efficacy of riboflavin is concentration dependent and antagonized by catalase. Ohara et al. have postulated that riboflavin, by reacting with blue light to form active oxygen intermediates, may cause a greater degree of cell necrosis than blue light alone (63). Large-scale studies (64,65) have largely been disappointing in demonstrating benefits of multivitamin or multimineral supplements on cancer prevention. Nevertheless, there have been some intriguing reports that merit further study. DeSouza Quiros et al. in a preliminary report (66) observed an antiproliferative and antimetastatic effect of irradiated riboflavin in solid tumors. Other investigators have reported modifications in the riboflavin carrier protein in human prostate cancer (67) and proposed a role for vitamin-binding proteins in prostate cancer (68). This group of scientists has applied for a patent for treatment of prostate cancer with antagonists of riboflavin-binding protein (69). These considerations suggest that riboflavin deficiency may possibly play a role in carcinogenesis. Clearly, more research needs to be done before this vitamin can

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be recommended for cancer prevention for populations at risk.

ASSESSMENT AND DIETARY RECOMMENDATIONS A variety of methods are available for the analysis of riboflavin and its coenzyme derivatives. Fluorometric procedures take advantage of the inherent fluorescent properties of flavins (70). Some degree of purification of the urine or tissues may be required before analysis is undertaken. There is often significant interference by other natural substances that leads to quenching of fluorescence and methodological artifacts. Riboflavin can be measured by competitive protein binding which is applicable to studies in human urine (71). Riboflavin binds specifically to the avian egg white riboflavin-binding protein (72) and thereby provides the basis for quantitative analysis. Currently, procedures using high-performance liquid chromatography have been widely applied as they have a high degree of precision and can be utilized for the analysis of riboflavin in pure form as well as in biological fluids and tissues (73). High-performance liquid chromatography is the method most widely employed for the determination of flavins in the blood and in other tissues. In clinical studies that involve individual patients as well as population groups, the status of riboflavin nutrition is generally evaluated by determining the urinary excretion of riboflavin and EGRAC (74). Urinary riboflavin determinations may be done in the basal state, in random samples, in 24-hour collections, or after a riboflavin load test. Normal excretion in the urine is approximately 120 ␮g/g creatinine per 24 hour or higher (74). It is useful to express the value in terms of creatinine to verify the completeness of the collection and to relate excretion to this biological parameter. Expressed in terms of the total amount, riboflavin excretion in the normal adult is about 1.5 to 2.5 mg/day, which is very close to the recommended dietary allowance (RDA) of the National Academy of Sciences (75). In deficient adult individuals, outflow with urine is reduced to about 40 ␮g/g creatinine per 24 hours. Individuals deficient in riboflavin have reduced urinary excretion, reflecting diminished dietary intake and depleted body stores. Excretion is reduced with age and is stimulated by elevated body temperature as well as treatment with certain drugs, and by various stressful conditions associated with negative nitrogen balance (74). Data from urine analysis must therefore be interpreted with these factors in mind. Another potential drawback to utilizing urinary riboflavin excretion as an assessment of nutritional status of this vitamin is that the amount excreted reflects recent intake very sensitively. Thus, if an individual has been depleted for a long time but consumes food items high in riboflavin, the level of the vitamin in urine as determined a few hours later may not be in the deficient range, but is likely to be normal or even elevated. It is important, therefore, to utilize assessment techniques that more accurately reflect long-term riboflavin status. The method most widely employed and that largely meets these needs is assay of EGRAC. The principle of the method is that the degree of saturation of the apoenzyme with its coenzyme, FAD, reflects the body stores of the latter. In deficient individuals, relative un-

saturation of the apoenzyme leads to decreased basal activity of the enzyme. Therefore, the addition of FAD to the enzyme contained in a fresh erythrocyte hemolysate from deficient individuals will increase activity in vitro to a greater extent than that observed in preparations from well-nourished individuals, in whom the apoenzyme is relatively more saturated with the coenzyme. The EGRAC is the ratio of enzyme activity with, to that without, addition of FAD in vitro. In general, most studies indicate that an activity coefficient of 1.2 or less indicates adequate riboflavin status, 1.2 to 1.4 borderline-to-low status, and greater than 1.4 riboflavin deficiency (74). It must be kept in mind that a number of physiological variables influence the results of this determination as well. In the inherited disorder of glucose-6-phosphate dehydrogenase deficiency associated with hemolytic anemia, the apoenzyme has a higher affinity for FAD than that of the normal erythrocyte, which will affect the measured EGRAC. Thyroid function affects glutathione reductase activity, the coefficient being elevated in hypothyroidism (75). This disorder has many biochemical features in common with those of riboflavin deficiency, as discussed earlier (75). The RDA for riboflavin issued by the Food and Nutrition Board (76) calls for adult males aged 31 to 50 years to consume 1.3 mg/day and those aged 51 to 70 years, 1.1 mg/day. Adult females aged 31 to 50 years should consume 1.1 mg/day and the same by women aged 51 to 70 years. It is recommended that in women aged 19 to 50 years, intake be increased to 1.4 mg/day during pregnancy and 1.6 mg/day during lactation. There has been some concern as to whether these figures are applicable to other population groups around the world. The Chinese tend to excrete very little riboflavin, and their RDA may be lower than that of Americans (77). Adults in Guatemala appear to have a similar RDA in individuals older than 60 compared to those 51 years or younger (78). This finding may not necessarily be relevant to populations of other countries. The RDAs of various national groups require further study. Environmental factors, protein-calorie intake, physical activity, and other factors may have an impact on riboflavin status. More research is needed on the requirements of the extremely old, who form an increasingly large proportion of the population. They are also the group that consumes the largest number of prescribed and over-the-counter medications. A point of interest is whether riboflavin requirements are elevated in individuals who exercise compared to those who are sedentary. In women aged 50 to 67 years who exercised vigorously for 20 to 25 min/day, 6 days a week, both a decrease in riboflavin excretion and a rise in the EGRAC were noted, findings consistent with a marginal riboflavin-deficient state (79). Supplementation with riboflavin did not, however, improve exercise performance. These investigators observed compromised riboflavin status in young women exercising vigorously as well (80). Similarly reduced urinary riboflavin excretion and elevated EGRAC were observed in young Indian males who exercised actively (81). To determine whether the status of riboflavin nutrition influences metabolic responses to exercise, blood lactate levels were determined in a group of physically active college students from Finland before and after the exercise

Riboflavin

period. A number of the students were initially in a state of marginal riboflavin deficiency. Following supplementation with vitamins, including riboflavin, that produced improvement in the elevated EGRAC, the blood lactate levels were unaffected and were related only to the degree of exercise (82). Thus, to date, while it is known that exercise may lead to biochemical abnormalities in riboflavin metabolism, it has not been shown that these abnormalities lead to impaired performance; nor has it been shown that riboflavin supplementation improves exercise performance.

SAFETY AND ADVERSE EFFECTS There is general agreement that dietary riboflavin intake at many times the RDA is without demonstrable toxicity (10,83,84). Because riboflavin absorption is limited to a maximum of about 25 mg at any one time (10), the consumption of megadoses of this vitamin would not be expected to increase the total amount absorbed. Furthermore, classical animal investigations showed an apparent upper limit to the tissue storage capacity of flavins that cannot be exceeded under ordinary circumstances (85). This storage capacity is probably limited by the availability of proteins providing binding sites for flavins. These protective mechanisms prevent tissue accumulation of excessive amounts of the vitamin. Because riboflavin has very low solubility, even intravenous administration of the vitamin would not introduce large amounts into the body. FMN is more water soluble than riboflavin but is not ordinarily available for clinical use. Nevertheless, the photosensitizing properties of riboflavin raise the possibility of some potential risks. Phototherapy in vitro leads to degradation of DNA and increase in lipid peroxidation, which may have implications for carcinogenesis and other disorders. Irradiation of rat erythrocytes in the presence of FMN increases potassium loss (86) Topical administration of riboflavin to the skin may increase melanin synthesis by stimulation of freeradical formation. Riboflavin forms an adduct with tryptophan and accelerates its photo-oxidation (87). Further research is needed to explore the full implication of the photosensitizing capabilities of riboflavin and its phosphorylated derivatives.

ACKNOWLEDGMENTS Research was supported in part by Clinical Nutrition Research Unit Grant P30–29502 and R25CA 105012 from the National Institutes of Health, and by grants from the American Institute for Cancer Research, the Ronald and Susan Lynch Foundation, the Edith C. Blum Foundation, the Heisman Trophy Trust, the D’Agostino Foundation, and an industrial agreement from Wakunaga of America Co., Ltd.

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46. Bernsen PLJA, Gabreels FJM, Ruitenbeek W, et al. Treatment of complex I deficiency with riboflavin. J Neurol Sci 1993; 118:181–187. 47. Walker UA, Byrne E. The therapy of respiratory chain encephalomyopathy: a critical review of the past and present perspective. Acta Neurol Scand 1995; 92:273–280. 48. McComsey GA, Lederman MM. High doses of riboflavin and thiamine may help in secondary prevention of hyperlactatemia. AIDS Read 2002; 12:222–224. 49. Dalton DS, Rahimi AR. Emerging role of riboflavin in the treatment of nucleoside analogue-induced type B lactic acidosis. AIDS Patient Care STDS 2001; 15:611–614. 50. Posteraro AF 3rd, Mauriello M, Winter SM. Riboflavin treatment of antiretroviral induced lactic acidosis and hepatic steatosis. Conn Med 2001; 65:387–390. 51. Yoon HR, Hahn SH, Ahn YM, et al. Therapeutic trial in the first three Asian cases of ethylmalonic encephalopathy: Response to riboflavin. J Inherit Metab Dis 2001; 24:870–873. 52. Foy H, Kondi A. A case of true red cell aplastic anemia successfully treated with riboflavin. J Pathol Bacteriol 1953; 65:559–564. 53. Powers HJ. Riboflavin (vitamin B-2) and health. Am J Clin Nutr 2003; 77:1352–1360. 54. Powers HJ, Wright AJA, Fairweather-Tait SJ. The effect of riboflavin deficiency in rats on the absorption and distribution of iron. Br J Nutr 1988; 59:381–387. 55. Powers HJ, Weaver LT, Austin S, et al. Riboflavin deficiency in the rat: Effects on iron utilization and loss. Br J Nutr 1991; 65:487–496. 56. Powers HJ. Investigation into the relative effects of riboflavin on iron economy in the weanling rat and the adult. Ann Nutr Metab 1986; 29:261–266. 57. Webster RP, Gawde MD, Bhattacharya RK. Modulation of carcinogen-induced DNA damage and repair enzyme activity by dietary riboflavin. Cancer Lett 1996; 98:129–135. 58. Rivlin RS. Riboflavin and cancer: A review. Cancer Res 1973; 33:1977–1986. 59. Pangrekar J, Krishnaswamy K, Jagadeesan V. Effects of riboflavin deficiency and riboflavin administration on carcinogen-DNA binding. Food Chem Toxicol 1993; 85:1483– 1492. 60. Munoz N, Wahrendorf J, Bang LJ, et al. Vitamin intervention on precancerous lesions of the esophagus in a high-risk population in China. Ann N Y Acad Sci 1988; 534:618–619. 61. Wahrendorf J, Munoz N, Lu JB, et al. Blood retinol and zinc riboflavin status in relation to precancerous lesions of the esophagus: findings from a vitamin intervention trial in the People’s Republic of China. Cancer Res 1988; 48:2280–2283. 62. Zaridze DG, Bukin JU, Orlov YN. Relationship between esophageal mucosa pathology and vitamin deficit in population with high frequency of esophageal cancer. Vopr Onkol 1989; 35:939–945. 63. Ohara M, Fujikura T, Fujiwara H. Augmentation of the inhibitory effect of blue light on the growth of B16 melanoma cells by riboflavin. Int J Oncol 2003; 22:1291–1295. 64. Greenwald P, Anderson D, Nelson SA, et al. Clinical trials of vitamin and mineral supplements for cancer prevention. Am J Clin Nutr 2007; 85:314S–317S. 65. McGinnis JM, Birt DF, Brannon PM, et al. National Institutes of Health state-of-the-science conference statement: Multivitamin/mineral supplements and chronic disease prevention. Ann Intern Med 2006; 145:364–371. 66. DeSouza Quiroz KC, Zambuzzi WF, Santos de Souza AC, et al. A possible anti-proliferative and anti-metastatic effect of irradiated riboflavin in solid tumors. Cancer Lett 2007; 258:126–134. 67. Johnson T, Ouhtit A, Gaur R, et al. Biochemical characterization of riboflavin carrier protein (RCP) in prostate cancer. Front Biosci 2009; 14:3634–3640.

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Saw Palmetto Edward M. Croom and Michael Chan

seed inside the pulp. Flowering and fruit production are highly variable each year; most fruits mature in August and September. Saw palmetto is an important wild plant providing food and cover for many animals. The fruits are consumed by black bears, deer, raccoons, foxes, opossums, fish, and many species of birds (1–3).

INTRODUCTION Saw palmetto fruit extracts are frequently consumed for relief of the lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH). Clinical trials of saw palmetto for symptom relief of LUTS have yielded mixed results that have been attributed to product differences and clinical trial design concerns. Multiple clinical trials of extracts of saw palmetto have been shown to be superior to placebo and to have fewer side effects than ␣-blocker and 5␣-reductase inhibitor drugs for relief of symptoms in men with mild-to-moderate BPH. Because there are chemical differences in saw palmetto products and variations in the scientific substantiation including efficacy of products to improve prostate health, consumers and health care professionals must learn the scientific basis for their safety and effectiveness to make an informed product choice. When evaluating the totality of the scientific evidence including observational trials and small trials with limited information on the quality of the trial design, saw palmetto appears to have few safety concerns for any serious side effects and to be more beneficial than watchful waiting or placebo for relief of LUTS associated with BPH.

Ecological Distribution Saw palmetto is endemic to the southeastern United States. The native range is from the coastal plain of southeast South Carolina to Georgia, throughout the state of Florida, including the Florida Keys, and to the coastal plains of Alabama, Mississippi, and southeast Louisiana. Saw palmetto is a major understory plant, sometimes forming dense thickets in pinelands, dunes, sand pine scrub, mesic hammocks, and woodlands. The plant is one of the most abundant in Florida and is reported to be very well adapted to surviving fires (1–3).

Historical Uses Saw palmetto leaves, stems, roots, and fruits have had a variety of historical uses (3). In the late 1800s, saw palmetto fruits were “lauded as the ‘old man’s friend,’ giving relief from the many annoyances commonly attributed to enlarged prostate.” Felter and Lloyd, writing in the King’s American Dispensatory in 1898, commented “We would rather regard it a remedy for prostatic irritation and

BACKGROUND Serenoa repens (W. Bartram) Small (Arecaceae; also known as Palmaceae, Palmae) is commonly known as Saw palmetto. Botanical synonyms include Corypha repens W. Bartram; Brahea serrulata (Michaux) H. Wendland; Chamaerops serrulata Michaux; Corypha obliqua W. Bartram; Sabal serrulata (Michaux) Nuttall ex Schultes & Schultes f.; Serenoa serrulata (Michaux) G. Nicholson. The stems of the palm are usually prostrate, branched, and sometimes upright to a length of 3 m or more. The stiff, fan-shaped leaves range in color from yellow green to green and grayish green to silver green (Fig. 1). The saw-toothed (serrate) petioles are from 0. 5 to 1 m long and have fine to coarse teeth that account for the common name of this shrub-like, branching palm. The flower stalks are approximately the same length as the petioles. The small (4–5 mm), fragrant, spring flowers are creamy white, with three petals and six stamens. The pulpy, one-seeded fruits ripen from green to orange to black or bluish black (Fig. 2). Mature fruits are approximately 2 cm long and 1 cm in diameter, with some fruits being similar in shape and size to commercial black olives, having a large hard

Figure 1

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Saw palmetto leaves.

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inhibitors of having fewer significant side effects such as impotence, ejaculatory disorders, and dizziness.

CHEMISTRY AND PREPARATION OF PRODUCTS

Figure 2

Saw palmetto fruits. Source: Courtesy of Renato Iguera.

relaxation of tissue than for a hypertrophied prostate.” As authorities of the eclectic school of medicine, they begin their discussion of saw palmetto with “Saw palmetto appears, from clinical reports, to be a nutritive tonic.” Indeed, at that time, saw palmetto was used as a tonic to treat a variety of maladies of the male and female reproductive organs and irritations of the mucous membranes, including coughs due to different medical disorders (4). Saw palmetto fruits have been used in both traditional and homeopathic medicine for the treatment of urological symptoms associated with prostate hypertrophy. In the United States, at the beginning of the 20th century, alcohol extracts of the fruit made as tinctures and alcohol based fluidextracts were popular remedies for prostatitis and prostate hypertrophy. Although the plant was popular and was listed in the United States Pharmacopeia from 1906 to 1916 and in the National Formulary from 1926 to 1950, it is the more recent successful development of European pharmaceutical products that has stimulated more detailed studies on its pharmacology and potential mechanisms of action (5,6).

Saw Palmetto and Other Botanical Treatments Plant-derived agents may account for approximately one third of total sales of all therapeutic agents used for treating BPH in Europe (7). Saw palmetto is sold in the United States as a dietary supplement through many sales channels, including the mass market, mail order, direct sales, and multilevel marketing, as well as specialty retail and health food stores. In 2008, saw palmetto was the fourth leading botanical in total consumer sales through all types of market channels in the United States, with sales of over $17.4 million (8). In the United States and Europe, other popular phytotherapies and natural remedies include ␤sitosterol (from the tuber of the South African plant Hypoxis rooperi), a grass pollen mixture (92% rye, 5% timothy, and 3% corn), stinging nettle root, pumpkin seed oil, and Pygeum bark (see chapter “Pygeum africanum Extract”) (9). Saw palmetto is the most popular botanical used for mildto-moderate LUTS associated with BPH and has shown an advantage over standard ␣-blockers and 5␣-reductase

The dried drupe-like fruit is the plant part that has most frequently been used in traditional and allopathic medicine. The fruit consists of approximately 36% outer rind, 16% flesh, 10% seed shell, and 38% seed. The fleshy part contains large quantities of lipids, starches, polysaccharides, sugars and mannitol, and small quantities of ceramides and sphingolipids. The lipid content has been reported to consist of approximately 75% free fatty acids and 25% neutral fats. During the ripening and drying of the fruit, a lipase splits triglycerides into fatty acids. From the oil obtained by pressing fruits preserved in alcohol, C6–C18 fatty acids have been identified (5). Oleic, lauric, and myristic acids are the predominant ones, while palmitic, caproic, caprylic, and capric acids have been reported in smaller quantities in both 90% ethanol and CO2 extracts (5,10). The characteristic smell of the oil has been attributed to the secondary formation in the fruit of ethyl esters of several of the fatty acids present (5). A systematic evaluation of the total fatty acids of fruits at different stages of ripeness, the seed, fruit pulp, fruit powder, and extracts, as well as mixtures, has shown that each product has a characteristic fatty acid profile that can be used for identification and standardization of products (11). Lipophilic extracts of saw palmetto are widely used in BPH therapy. The most documented extracts, from a pharmacological and clinical point of view, are those obtained by two different extraction processes. The first process involves extraction from dried and finely ground fruits with hexane in an inert gas atmosphere and in the presence of an antioxidant such as ascorbic palmitate, and the second process involves extraction with CO2 in supercritical conditions (5). In the United States, common saw palmetto products include ethanol and CO2 extracts. In Germany, fruit extracts obtained with 90% ethanol are very popular, and a hexane extract is a registered pharmaceutical. An analysis of two commercial saw palmetto extracts produced using 90% ethanol versus under an optimized CO2 condition found that although the relative composition of free fatty acids and total fatty acids was very similar, the absolute percentage of free fatty acids was 69% in the ethanol extract versus 90% in the CO2 extract (10). A second major difference found was that the ethanol extract contained a large amount of ethyl esters that were not present in the CO2 extract (5). A CO2 extract was found to contain free fatty acids as well as their methyl- and ethyl esters, ␤-sitosterol, ␤sitosterol-3-O-␤-D-glucoside, campesterol, stigmasterol, lupeol, cycloartenol, 24-methylene-cycloartanol, longchain saturated and unsaturated alcohols, including farnesol, phytol, and alcohols with C22, C23, C24, C26, and C28 chain lengths, and polyprenolic alcohols. Carotenoids, which give a marked orange color to the oily extracts, have also been isolated (5). Flavonoids, including rutin, isoquercitrin, kaempferol-3-O-glucoside, and apigenin-7-O-rhamnoglucoside, and anthranilic acid have been reported in alcohol extracts of the fruits.

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Polysaccharide fractions from aqueous extracts have shown anti-inflammatory and immunomodulatory activities. Some of the polysaccharides found in saw palmetto are galactose, arabinose, xylose, mannose, rhamnose, glucose, and glucuronic acid (5). There exists significant chemical variation in different saw palmetto products (12). This chemical variance may be a major cause of the discrepancies observed in results from various clinical studies. For instance, a study by Scaglione et al. demonstrated that the potency of 5␣reductase inhibition, a proposed mechanism of action of the herbal, varied between brands and even batches of saw palmetto material sold in Italy (13).

PRECLINICAL STUDIES Pharmacology and Mechanisms of Action Although BPH is one of the most common diseases in aging men, its etiology is still not completely understood. The factors that cause the imbalance in prostate growth, which produce an enlarged prostate, are mostly unknown. Studies suggest that this imbalance may be related to inflammation and apoptotic mechanisms. Effective treatment of BPH is focused on the relief of the LUTS associated with the condition. The drugs currently used to relieve these symptoms are ␣-blockers and 5-␣-reductase inhibitors. Research concerning potential mechanisms of action for saw palmetto has included investigations into antiandrogenic actions, anti-inflammatory activity, and factors related to prostate cell growth as well as the mechanisms that are generally attributed to ␣-blockers and 5␣-reductase inhibitors.

Symptom Relief The use of ␣-blockers is based on the hypothesis that LUTS are partly caused by ␣1 -adrenergic-mediated contraction of prostatic smooth muscle and bladder neck (14). The ␣-blockers decrease smooth muscle tone and contraction, which reduces bladder outlet obstruction by improving urinary flow through the prostatic urethra (14). A 90% ethanol extract has been shown to reduce norepinephrine-induced contractions of rat deferential duct, and potassium chloride-induced contractions of guinea pig ileum and bladder smooth muscle tissue. Both lipid and saponifiable fractions of saw palmetto reduced the norepinephrine-induced contractions of rat aorta, in vitro as well as potassium chloride-induced contractions of rat uterus. Vanadate-induced contractions of the rat uterus have also been reduced by a lipophilic (90% ethanol) saw palmetto extract (15). Extracts of saw palmetto have potent noncompetitive inhibition of human prostatic ␣1 -adrenoceptors in vitro (16,17). Suzuki et al. reported saw palmetto inhibiting the ␣1 -adrenoceptors the prostate and spleen and also the muscarinic receptors in the bladder and submaxillary gland of rats (17). Inhibition of the muscarinic receptors in the bladder causes the detrusor muscle to relax and can reduce the frequency and intensity of contractions of the bladder. Results from a separate experiment by Suzuki et al. showed that organ selectivity was associated with the inhibitory action. Suzuki et al. reported that the muscarinic receptors in the prostate and bladder were prefer-

entially targeted for inhibition. The authors suspect that this specificity is due to saw palmetto extract preferring to accumulate in the prostate to a greater extent compared to other tissues (17,18). The other class of drug used to treat BPH symptoms is the 5-␣-reductase inhibitors. The major action of these drugs is to inhibit the enzymatic conversion of testosterone to dihydrotestosterone (DHT) by 5␣-reductase (14). Although both testosterone and DHT can activate the androgen receptors, DHT is the more potent androgen with a much greater binding affinity (19,20). The activation of the androgen receptors promotes protein synthesis and cellular growth and the overexpression of the two isoenzymes 5-␣-reductase has been associated with BPH tissue (19,20). The peripheral antiandrogenic activity of the saw palmetto extracts has been studied in vivo in mice and prepubertal rats. The animals underwent castration to remove the source of endogenous testosterone and were then given a subcutaneous (SC) injection of the hormone. Saw palmetto extract given orally for 12 days (300 mg/mouse) antagonized the stimulant effect of exogenous testosterone, reducing the weights of the ventral prostate, seminal vesicles, and the preputial glands in comparison to the control mice treated only with testosterone. Rats given saw palmetto extract (200 mg/animal) orally for six days showed similar results. Other organs were not affected (19). The antiandrogenic activity in castrated rats treated orally with a saw palmetto CO2 extract has been shown to depend on both the temperature and pressure conditions and the dose of the extract (21). Arruzazabala et al. examined the effects oral dosages of saw palmetto, coconut oil, and sunflower oil had on the prostate and body weights of rats receiving a SC injection of testosterone. The authors reported significant inhibition of testosterone-induced prostate hyperplasia in the rats receiving saw palmetto extract and coconut oil at doses of 400 mg/kg but not in rats ingesting sunflower oil (22). Unlike the synthetic steroidal 5-␣-reductase inhibitors such as finasteride, which show much greater inhibitory activity and compete with testosterone for the active androgen binding site at nanomolar concentrations, saw palmetto does not appear to bind directly to these receptors or affect their binding affinities. In vitro and in vivo studies have shown that saw palmetto does not affect serum prostate specific antigen (PSA) levels (23–27). Habid et al. demonstrated that in the presence of both DHT and Permixon, LNCaP cells demonstrated no change in accumulation of PSA protein compared to DHT alone (24). In the presence of DHT and finasteride, PSA secretion was significantly inhibited and intracellular PSA levels were reduced (24). Bayne et al. developed a BPH model that allowed for the observation of PSA secretion and the expression of functional androgen receptors. This model showed that treatment with Permixon at a dose of 10 ␮g/ml had no effect on PSA levels or on any other androgen-dependent processes that rely on binding of androgens to their receptors (25). The antiandrogenic action of saw palmetto is due primarily through the inhibition of the two isoforms of 5␣-reductase, which inhibits the production of DHT. Bayne et al.’s model showed that Permixon was a potent

Saw Palmetto

inhibitor of both isoforms of 5␣-reductase (25). In studies with cultured genital skin fibroblasts, the lipophilic extract of saw palmetto inhibits the enzymatic conversion of testosterone to DHT by 5␣-reductase and of DHT to androstanediol by 3␣-hydroxysteroid dehydrogenase. Both alcohol and CO2 extracts have also been shown to inhibit 5␣-reductase. Biopsy cores from patients receiving a saw palmetto combination product showed significant reduction in DHT levels, whereas levels in men receiving placebo exhibited no significant change (28). Wadsworth et al. reported that although feeding TRAMP mice with a dose of 50 mg/kg, a dosage considered equivalent to the recommended human dose, did not significantly change prostate DHT levels, a dose of 300 mg/kg did show reduction in prostate DHT levels (29). The free fatty acids are thought to be responsible for these inhibitory effects. In a study of the 5␣-reductase inhibiting activity of a CO2 extract and fractions made from it, inhibition was limited to the fatty acid fraction. The activity for the entire extract and the main fatty acid components was comparable (30). A study by Raynaud et al. demonstrated that lauric acid inhibited both 5␣-reductase isoforms, with IC50 of 16.7 ␮g/mL for 5␣-reductase 1 and 18.6 ␮g/mL for 5␣-reductase 2, whereas the unsaturated acids oleic and linoleic selectively inhibited 5␣-reductase 1 with IC50 of 4 ␮g/mL and 13 ␮g/mL respectively. Myristic acid was also found to strongly inhibit 5␣-reductase 2 activity with an IC50 of 4.3 ␮g/mL (31). Active 5␣-reductase enzymes are membrane-bound (30). It is suggested that saw palmetto lipids could inhibit the enzymes by changing their microenvironment through disruption of the nuclear membrane (25,30). Bayne et al. reported that Permixon appeared to disrupt the intracellular membranes, including the nuclear and mitochondrial membranes of prostatic epithelial and fibroblast cells (25). Permixon treated cells showed accumulation of lipid droplets in the cytoplasm and wide-ranging damage to the intracellular membranes. At higher magnifications polarization of the nucleus and condensation of chromatin was also observed (25).

Prostate Health and Underlying Disease Mechanisms It is suggested that inflammation and factors influencing prostate cell growth and apoptosis could play a role in the development of BPH and prostate cancer. Experimental evidence has shown that saw palmetto extract affects inflammation, cell growth, and apoptotic mechanisms. Evidence of the extract influencing cell growth and apoptosis in prostate cells has been detected through both direct observation of increased apoptotic rates versus control and through the observation of cellular apoptotic indicators. Studies on various cell lines have demonstrated that saw palmetto treated cells show increased levels of the protein p27kip1 , a cell cycle inhibitor that prevents cell cycle progression (27). Increased levels of cleaved Poly ADP ribose polymerase (PARP) fragments have also been observed in saw palmetto treated cells (27,32). Cleavage of PARP is indicative of Caspase-3 activity, part of the caspase cascade an essential sequence in the initiation of the execution-phase of cell apoptosis. Saw palmetto treatment appears to also decrease Akt phosphorylation thus inhibiting the P13 K/Akt1 signaling pathway, an im-

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portant inhibitor of apoptotic processes and inductor of protein synthesis (32–34). The herb also inhibits Signal Transducer and Activator of Transcription 3 (STAT-3) signaling by preventing the phosphorylation of STAT-3 by Interlukin 6, another pathway that has antiapoptotic and proliferative effects (27). Indications of apoptosis are also observed in in vivo settings. Wadsworth et al. reported that treatment with saw palmetto extract resulted in a significant shift in tumor pathology to less advanced grades in TRAMP mice versus control. A significant increase in detectable apoptotic cells in the TRAMP mice receiving saw palmetto was also observed (29). In a comparison between 10 patients taking 320 mg of Permixon and 15 control patients that were not prescribed Permixon, Vela-Navarrete et al. reported significantly lower levels of intact PARP in the Permixon group. The Permixon treated group had prostatic tissue with higher Bax-to-Bcl-2 ratios than those in the control group. Both Bax and Bcl-2 are proteins that belong to the Bcl-2 gene family, which encodes a number of proteins that can be either proapoptotic or antiapoptotic. Bax is thought to be a proapoptotic protein whereas Bcl-2 is considered antiapoptotic and the ratio of the two proteins is used as an indication of the susceptibility of cells to undergo apoptosis (20). Petrangeli et al. observed increased apoptosis rates and decreased levels of phosphorylated Akt in human prostate cancer PC3 cell line treated with Permixon. These effects were initially caused by rapid changes in membrane composition, notably a decrease in cholesterol content and the subsequent disruption of lipid rafts that act as organizing centers for cell signalling pathways. A second phase of membrane changes was seen 24 hours following saw palmetto treatment. In this phase the majority of the initial changes in membrane components were reversed and returned to baseline, except for a large increase in the saturated fatty acid to unsaturated fatty acid ratio. This change in ratio was predominantly due to a net decrease of omega-6 fatty acids in the membrane, most noticeably arachidonic acid. The release of these unsaturated fatty acids are thought to be responsible for the continued increased apoptosis and decreased cell proliferation rates observed in the late stage (34). It has been shown that free arachidonic acid has proapoptotic effects. The acid has been shown to cause opening of the mitochondrial permeability transition pores causing the release of cytochrome c and the activation of the caspase cascade (34,35). Baron et al. attributed the opening of the permeability transition pores in prostate cancer PC3 and LNCaP cell lines treated with Permixon to the fatty acids in the extract, noting that other studies with fatty acids have demonstrated these types of effects on mitochondria (32,34,36). Because of the high activity of cyclooxygenase (COX) and 5-lipoxygenase (LOX) in the prostate, the level of free arachidonic acid in the prostate is generally low (36). These conditions may make these cells particularly sensitive to high levels of arachidonic acid release from the membranes and could explain the apparent selectivity for prostate cell lines of saw palmetto action that have been observed. Baron et al. noted that other studies have shown that different fatty acids can induce mitochondria depolarization in other cell lines. It is possible that the

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variations in fatty acid content of different test materials could account for the reports of saw palmetto extracts inducing apoptosis in other cell lines. Typically the high level activity of COX and LOX in prostate cells would result in arachidonic acid having an antiapoptotic effect (35). These enzymes readily convert the acid into eicosanoids such as prostaglandins and leukotrienes that mediate inflammatory reactions (35). Patients with malignant prostatic disease have significant reduction in arachidonic acid and docosapentaenoic acid in their prostatic tissue (37). There is evidence that indicates saw palmetto extracts can inhibit the activity of both LOX and COX. The acid lipophilic fraction of a saw palmetto CO2 extract was found in vitro to be an inhibitor of both the COX (IC50 -value: 28.1 ␮g/ml) and LOX (IC50 value: 18.0 ␮g/ml) pathways (38). In one study, malignant prostatic tissue converted radiolabeled arachidonic acid to prostaglandin PGE2 at an almost 10-fold higher rate than BPH tissue. PGE2 production from [3 H]arachidonic acid by malignant prostatic tissue was investigated in the presence of various fatty acids, and oleic acid was found to be the most effective inhibitor (37). Oleic acid is among the most abundant of the free fatty acids in saw palmetto lipophilic extracts as it comprises approximately 30% to 35% of the free and total fatty acids in extracts obtained using 90% ethanol or supercritical CO2 (10). Saw palmetto extract, when administered orally in rodents, has shown antiedematous activity in a diversity of animal models, including centrifugation-induced tail edema in mice, histamine-and dextran-induced increase in microvascular permeability and edema in rats, IgEdependent passive cutaneous anaphylaxis in rats, and UV erythema in guinea pigs. Because the antiedematous activity was also observed in an adrenalectomized rat model, glucocorticoids cannot be the source of activity (12). The absence of estrogenic properties has been demonstrated by studying the effect of saw palmetto extract on the growth of the prepubertal female mouse uterus and on the changes in the estrus cycle of adult female mice. The absence of progestational activity has been investigated in ovariectomized female mice that were sensitized with estrone and treated daily with 100 to 400 mg saw palmetto extract orally or 100 ␮g of SC progesterone On day 10 of treatment, histamine was administered in one of the uterine horns. In contrast to progesterone, the extract of saw palmetto did not cause an increase in the weight of the uterus. Saw palmetto has both individual compounds and specific extracts with pharmacological activity that could be related to mechanisms that would provide symptomatic relief for LUTS associated with BPH. The lack of systemic hormonal and ␣-blocker activity should be important for the safe use of saw palmetto and points to the need for more detailed research on activity in prostate cells, tissues, and animals, as well as in humans, to determine whether multiple mechanisms of action are working in synergy or whether the main mechanism of action has yet to be discovered.

CLINICAL STUDIES Primary Use The current primary use of saw palmetto is for prostate health and relief of LUTS that are associated with BPH, a

condition that is common in men over 50 years. The only saw palmetto fruit extracts that have been subjected to multiple clinical trials in BPH are those produced under highly standardized conditions, by extraction with hexane or under hypercritical CO2 conditions. They have a chemical content of 85% to 95% fatty acids, and have been stability tested to assure that throughout their shelf life the chemical contents are sufficient and that the capsules will disintegrate in conditions that mimic the acidity of the stomach. Clinical trials generally evaluated the ingestion of two 160 mg soft gel capsules or one 320 mg capsule per day. Evidence from clinical trials of standardized lipophilic saw palmetto extracts has been mixed but in general it has been seen that they are a viable therapy for the relief of BPH symptoms that has been shown to be as effective and better tolerated than the use of ␣-blocker and 5␣-reductase inhibitors. Although saw palmetto may give relief as early as four to six weeks, three months or more of therapy may be required for some patients before the effects are felt.

Emerging Uses Saw palmetto has been suggested for other conditions associated with the prostate including prostatitis and prostate cancer. A trial by Kaplan et al. found no long term benefit after 12 months of saw palmetto at a dosage of 325 mg/day for category III chronic nonbacterial prostatitis/chronic pelvic pain syndrome (39). Results, however, from two clinical trials of patients with chronic bacterial prostatitis suggest that the use of a combination of saw palmetto with an ␣-blocker or other natural products and antibiotics results in a lower incidence of disease recurrence, as well as quicker and more significant relief of symptoms when compared to antibiotic therapy alone. The results of these trials cannot be definitively attributed to saw palmetto as the combination therapy of one trial included the ␣-blocker alfuzosin and the other trial included curcumin, quercetin, and stinging nettle (40,41). In vitro evidence demonstrating that saw palmetto can stimulate apoptosis and inhibit cell growth in multiple myeloma, breast, and prostate cancer cell lines has shown the possibility that the herb could be used to prevent or reduce the growth of specific types of prostate cancer cell lines (42). Results from Wadsworth et al.’s study indicated that a saw palmetto CO2 extract at doses calculated to be equivalent to and six times the recommended human dose showed no signs of toxicity, increased apoptosis, or reduced tumor grade and frank tumor incidence (29). Thus, saw palmetto supplementation could possibly be effective in slightly delaying the onset and progression of prostate cancer through its antiandrogenic action and ability to increase epithelial cell apoptosis (29). A prospective cohort study suggested saw palmetto supplement use for several years had no association with the risk of developing prostate cancer (43). Residual confounding factors and misclassifications of variables due to the reliance on self-reported data were identified as factors that would limit the interpretation of the data in this study (43).

Benign Prostatic Hyperplasia BPH is a noncancerous enlargement of the prostate that is associated with bothersome and irritative LUTS, including increased urgency and frequency, nocturia (arising from sleep at night to urinate), dribbling, hesitancy,

Saw Palmetto

straining, and incomplete bladder emptying (44). In the United States, a reported 40% of men in their 50s and nearly 90% of men in their 80s have BPH (44). Also in the United States, in 2000, nearly 8 million visits to physicians resulted in a primary or secondary diagnosis of BPH (44).

Current Medical Treatment There exist several guidelines on the diagnosis and treatment of BPH that were published by various institutions and associations (14). BPH is not always used to denote changes in the prostate but is mostly employed to describe a cluster of bothersome LUTS that increase in frequency and severity as men age. Associated mortality and even serious complications such as complete urinary retention are uncommon. Because some of the symptoms associated with BPH may also be due other medical conditions that cannot be rationally treated by saw palmetto, men should have a thorough diagnostic evaluation by a physician before and during any use of the herb to be certain that BPH and not a different disease is the source of the symptoms. All guidelines use a symptom score, the most commonly recommended is the International Prostate Symptom Score (IPSS), which has the patient answer questions concerning the severity and frequency of the main symptoms. The score on the IPSS or the identical American Urological Society (AUA) Symptom Score questionnaire is used to classify the severity of BPH as mild (0– 7), moderate (8–19), or severe (20–36). Since some individuals may chose no therapy (“watchful waiting”) whereas others with the same symptom score may be sufficiently bothered to seek relief by pharmaceuticals or surgical intervention, there exists wide latitude in the treatment of choice for a condition that mainly concerns quality of life. The main drug therapies recommended for treatment are ␣-adrenergic blockers and 5␣-reductase inhibitors. None of the guidelines recommended the use of saw palmetto or other phytotherapies citing concerns such as variations of product consistency, lack of identification of the active compound, absence of a well-documented mechanism of

action, and the lack of long-term studies on the use of these therapies (14). Phytotherapies, however, are prescribed and used to treat BPH, especially in Europe. In an observational, cross-sectional study Fourcade et al. reviewed the management of patients treated for BPH in primary care in four European countries and reported that phytotherapies did account for a significant number of prescriptions (45). An observational study by Hutchinson et al. found similar results (Table 1) (46). It should be noted that the data provided by both studies are based on prescriptions, and that the actual usage of the herb would be higher as patients can obtain phytotherapies over the counter without a prescription. Prescribing patterns are affected by a number of variables, not the least of which is drug cost. The complete lack of prescriptions for saw palmetto in the UK and the low numbers in Portugal are likely the result of the limited reimbursement policies for herbal therapies in those countries. In contrast, France, which has a more extensive reimbursement policy towards herbals, reported much higher phytotherapy prescription numbers. The impact reimbursement has on prescription is especially prominent when Hutchinson’s results are compared to numbers reported in Fourcade et al.’s study a few years later. Fourcade et al. reported prescription numbers for the period between January 2005 and June 2006, a year after the German government implemented policies that eliminated reimbursement for nonprescription medications. In Germany, saw palmetto is classified as a nonprescription medication and is not eligible for reimbursement under the new policy and the number of prescriptions for the herb fell accordingly. In contrast, in what could be an indication of the growing acceptance and increasing popularity of herbal remedies, the percentage of prescriptions for herbal remedies for France, Spain, and Portugal all increased significantly. S. repens was the most popular phytotherapy. In France phytotherapy products were the most popular class of drug and S. repens was the most popular drug.

Table 1 Prescribing Patterns by Country for Newly Presenting LUTS/BPH Patients Prescribed a Drug from January 2000 to July 2002 and from January 2005 to June 2006 January 2000–July 2002

All Countries

France

Germany

Portugal

Spain

Italy

United Kingdom

Number of Patients Drug Class Phytotherapy Alpha-blockers 5-alpha reductase Named Drug Serenoa repens Tamsulosin January 2005–June 2006 Number of Patients Drug Class Phytotherapy Alpha-blockers 5-alpha reductase Combination Named Drug Serenoa repens Tamsulosin

1516

241

106

656

248

199

66

15.6% 79.2% 5.1%

25.3% 71.8% 2.9%

36.8% 60.4% 2.8%

15.9% 77.0% 7.2%

10.5% 85.9% 3.6%

3.5% 91.0% 5.5%

0 98.5% 1.5%

5.7% 38.4%

16.5% 46.0%

16.2% 46.5%

1.1% 24.3%

5.8% 42.1%

4.0% 69.5%

39.1%

446

141

139

49

117

25.3% 67.3% 4.0% 3.4%

49.6% 46.1% 2.8% 1.4%

5.0% 87.1% 4.3% 3.6%

10.2% 63.3% 12.2% 14.3%

26.5% 70.9% 1.7% 0.9%

14.6% 35.9%

27.0% 23.4%

0 49.6%

6.1% 34.7%

20.5% 35.0%

Source: From Refs. 45 and 46.

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Efficacy in Control of Symptoms Related to BPH Using patient follow-up examinations Hutchinson et al. evaluated the efficacy of the different drug treatment regimes (46). The author’s reported that 75.3% of the 83 patients prescribed saw palmetto had IPSS improvements and an overall mean reduction in IPSS of 3.2 (46). These results are significantly higher than the results of those patients that underwent no drug therapy where only 59.2% of the 559 patients reported IPSS improvements and a mean IPSS reduction of 1.4 (46). Patients taking the herb also reported higher improvements in Quality of Life scores then the “watchful waiting” group with a mean improvement of 1.0 point versus 0.4 points (46). Although the patients were not randomized to treatments and the numbers in some categories do not allow for robust statistical analysis, these findings still correspond to the general conclusion that S. repens provides improvement of urologic symptoms and flow measures in patients suffering from BPH that were made in various clinical trial reviews (44). A recent large-scale study by Bent et al., however, has resulted in a re-assessment of the conclusions made in earlier reviews. Bent et al. investigated the effectiveness of saw palmetto, in the treatment of men suffering from moderate-to-severe BPH (23). A total of 225 men participated in the trial and were given 160 mg twice per day of either a CO2 saw palmetto extract or a placebo over a 12-month period. The study indicated that neither the saw palmetto nor the placebo showed any reduction in AUASI scores or significant improvements in any of the other clinical parameters assessed (42). In light of these results, the most recent Cochrane review concluded that S. repens was not more effective than placebo for treatment of urinary symptoms consistent with BPH (44). In their discussion the reviewer’s acknowledged the significant weight they placed on Bent et al.’s study in their analysis (44). The lack of any improvements observed for either placebo or saw palmetto obtained from Bent et al. contradict many other trials that did report data suggesting saw palmetto did relieve urinary symptoms. As data from Hutchinson et al.’s observational studies indicate, improvements were observed even when patients underwent a “watchful waiting” therapy (23). The Cochrane review did state that the evidence for the efficacy of S. repens in relief of symptoms was mixed (44). It was noted that three of the four trials that provided full data showed evidence of mean changes of −4.4, −4.4, and −6.1 in IPSS scores (44). The other trial was the one performed by Bent et al., which showed no improvements in either the saw palmetto or placebo arms. The reviewers placed greater weight on the Bent et al. trial noting that two of the trials were of smaller size and duration and the third, by Debruyne et al., lacked a placebo arm (44). Debruyne et al.’s study, which was of the same duration (12 mo) and of substantially larger size (542 patients completed the study) than Bent et al.’s trial, compared a saw palmetto hexane extract (Permixon), at a dose of 320 mg/day to Tamsulosin, the most widely prescribed therapeutic agent for BPH, at a dose of 0.4 mg/day (47). In both groups IPSS scores showed decreases by three months’ time (47). At 12 months both treatments resulted in a similar decrease of 4.4 in IPSS scores and mean changes in peak urinary flow rate of 1.89 mL/s for Tamsulosin and 1.79 mL/s for saw palmetto (47). The only

meaningful difference in side effect profiles of the agents was the 4.2% ejaculation disorders reported for Tamsulosin against 0.6% for saw palmetto (47). The results from Debruyne’s trial indicate that the effectiveness of saw palmetto is, at the least, equivalent to the effectiveness of Tamsulosin (47). A subsequent subset analysis by Debruyne et al. found that for those patients that had the most severe IPSS scores, saw palmetto was shown to be more effective than Tamsulosin in reducing the symptom score (48). A study by Carballido et al. examined the direct healthcare cost of BPH diagnosis and treatment in Spain and concluded that pharmacological therapy is responsible for 42% to 74% of the disease’s cost. Carballido et al. calculated that the annual cost of therapy, including the cost of treating any adverse events, with the Permixon was €135 less than the annual cost of therapy with Tamsulosin. Permixon was also found to be the more cost-effective option even when a generic Tamsulosin is used, costing approximately €32 less per year (49). Product-to-product variability in the chemical profiles of saw palmetto product is common in the marketplace (12). The biological and clinical activities of different extracts are dependent on the presence and amount of all the known and unknown compounds in the extract. Differences in test materials used can result in very different clinical results. Thus, direct comparison of trials utilizing different plant extracts is only valid for the entire class of a botanical extract as the different trials are effectively comparing different therapeutic agents. When trial results are homogenous then the results for the entire group of extracts are clear, but when the results are mixed then the specific extracts must also be examined individually for safety and efficacy. Although the well-designed LUTS/BPH trial by Bent et al. reported no improvement by saw palmetto, the overall clinical evidence from European observational studies and clinical trials shows that specific saw palmetto products can be a viable therapy for the relief of BPH symptoms that are more cost-effective and better tolerated than ␣-blocker and 5␣-reductase inhibitors in some patients. The important and unknown questions are whether saw palmetto can maintain prostate health for many years and prevent the worsening of LUTS symptoms or impact the initiation or slow the growth of prostate cancer in some patients.

Safety Although not available for independent scientific review, hexane/CO2 extracts of saw palmetto that have been registered as prescription drugs in Europe have had to undergo the same preclinical safety studies as drugs in the United States. The specific extracts and final pharmaceutical products registered would have been tested in vitro and in vivo for oral toxicity, teratogenicity, mutagenicity, peri- and postnatal toxicity, estrogenic activity, and effect on fertility. In the United States, saw palmetto products as dietary supplements are sold in combination with other plant products that have not undergone the safety, quality, and clinical efficacy testing required of drugs in Europe. Because saw palmetto products sold in the United States could have a different safety profile than those sold in Europe, their safety has been reviewed as a monograph by a special committee of the Food and Nutrition Board and

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the Board on Life Sciences [part of the Institute of Medicine (IOM) and National Research Council of the National Academies of Science]. Considering the weight of the current scientific evidence, the report concludes that the consumption of saw palmetto fruit (powders and extracts) does not pose a safety risk for men at the currently recommended doses (18). The report notes that the toxicity of combination products (including the 8-herb combination product PC-SPES, which was removed from commerce because of adulteration with drugs, including the anticoagulant warfarin) was evaluated only in relation to the saw palmetto component (18). The IOM report notes that no drug interactions have been documented with saw palmetto, but that more systematic drug interaction studies are needed (18). A review of the modulating effects of various herbal medicines concluded that while evidence indicated saw palmetto inhibited cytochrome activity in vitro, in vivo studies failed to show any effect on cytochrome activity leading to the conclusion that saw palmetto was unlikely to have any herb–drug interactions (50). The latest Cochrane review reported that the side effects associated with treatment with S. repens were generally mild and comparable in frequency to placebo. Their meta-analysis showed that there existed no significant difference in the relative risk of any adverse event between S. repens or placebo. In comparison to the 5␣-reductase inhibitor drug Proscar, saw palmetto had fewer adverse events, including impotence, the most frequent side effect for Proscar. Similar results were reported when comparing the herb to the ␣-blocker Tamsulosin, which had a statistically significantly higher frequency of headaches and ejaculation disorders (44). In a detailed safety assessment of saw palmetto extract using the data obtained from Bent et al.’s study, Avins et al. concluded that there was no evidence that saw palmetto extract at a dose of 320 mg/day over a year was associated with any clinically important adverse effects (51). There were few serious adverse events reported and they were more common in the placebo arm. There was no significant difference in nonserious adverse events between the extract and placebo (51). Debruyne et al. had similar results, reporting that the overall incidence of adverse events were similar between both treatment groups with a slightly higher frequency of ejaculation disorders reported in the Tamsulosin group (4.2%) versus the saw palmetto group (0.6%) (47,48). Although the results from Avins et al.’s assessment suggest that no important toxicities are associated with saw palmetto, the authors did stress that the study could not assess the association of rare serious adverse events with saw palmetto. The authors noted case reports of patients suffering from hepatitis, pancreatitis, and excessive intraoperative bleeding and prolonged bleeding time that had been associated with the herb (51). Few other adverse event reports have been submitted to the FDA. Although concerned about the prolonged bleeding time report, no clear causal relationship was found by the IOM Committee review (18). Results from a small animal study by Singh et al. did not find any evidence of hepatotoxicity due to saw palmetto (52). Singh et al. reported that their enzyme assay data actually suggested that the saw palmetto extract at doses two to five times the recommended dose appeared to have a hepatoprotective effect (52). A study

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by Tuncel et al. investigated the effects taking saw palmetto for five weeks prior to transurethral resection of the prostate would have on the intraoperative blood loss and microvessel density. The authors reported that there were no significant statistical differences in any of the variables that were measured in saw palmetto and control groups (53). A study by Beckert et al. investigated platelet function in 10 men taking saw palmetto extract (54). After two weeks of therapy, the authors could find no effect of the herb on in vivo platelet function (54). These results combined with investigations from the authors’ literature review led to their conclusion that there was no evidence to support the concern of perioperative bleeding in users of saw palmetto (54). The safe use of saw palmetto by pregnant and lactating women is more questionable, because drugs like finasteride that block the conversion of testosterone to DHT could impair the development of male genitalia in the fetus or feeding infant (18). The World Health Organization (WHO) monograph on saw palmetto states that “Owing to its effects on androgen and estrogen metabolism, the use of Fructus Serenoae Repentis during pregnancy or lactation and in children under the age of 12 years is contraindicated” (15). Because the safety and efficacy of all botanicals is based on the specific extract and final product formulation as the “active” ingredient, it is worth noting that, although rarely used today by women and children, the hexane and CO2 extracts and final products registered as prescription drugs in Europe would have been tested like all drugs for potential detrimental effects on the fetus in animal models of fertility and teratogenicity. All chemical compounds that have been reported from saw palmetto are generally nontoxic in the quantities consumed from commercial products (18). Overall, standardized lipophilic saw palmetto extracts at doses of 320 mg/day have proven risk-free in long-term clinical use in many European countries and in controlled clinical trials from 6 to 48 weeks. In the United States, saw palmetto, consumed at current levels in commercially available products, has also been very safe. For men, it should only be used to treat mild-to-moderate BPH after a complete medical examination to rule out more serious disorders. Saw palmetto products sold as dietary supplements have not undergone standard animal tests for teratogenicity and effects on fertility and therefore are contraindicated for use by pregnant or lactating women.

Pharmacokinetics Unlike pharmacodynamic studies that measure physiological changes, pharmacokinetic studies in humans with saw palmetto are difficult to conduct because the most active compounds that would logically be measured are fatty acids and sterols that are common in the normal Western diet. The pharmacokinetics has been investigated in an open, randomized, crossover study of 12 healthy males who ingested one 320-mg capsule or two 160mg capsules per day. The extract was absorbed rapidly, with a peak time (tmax ) of 1.50 to 1.58 hour and peak plasma levels (Cmax ) of 2.54 to 2.67 ␮g/ml. The area under curve value ranged from 7.99 to 8.42 ␮g hr/mL. Because the plasma concentration–time profiles of both preparations were very similar, the preparations were considered

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bioequivalent, but the validity of the methodology has been questioned (5,6). In another study, the bioavailability and pharmacokinetic profile of a rectal formulation containing 640 mg of S. repens extract were determined in 12 healthy male volunteers. The rectal was similar to the oral one but showed a slower absorption, with a tmax of 2.96 hour (5). In a study of the administration of a radioactive lipophilic sterol extract of saw palmetto in rats, tissue concentrations of lauric acid, oleic acid, and ␤-sitosterol were highest in abdominal fat tissue, the prostate, and the skin with lower concentrations in the liver and urinary bladder (18).

REGULATORY STATUS Saw palmetto products are regulated in the United States as dietary supplements since the passage of the Dietary Supplement Health and Education Act (DSHEA) in 1994. Under the regulations of the DSHEA, saw palmetto is sold without FDA premarket approval since dietary supplements are sold as a category of food. Although clinical testing is not required for inclusion in the United States Pharmacopeia or National Formulary, identity, chemical content, and quality standards for saw palmetto fruits, powders, extracts, and capsules have recently been reintroduced into the dietary supplement section of the USP32-NF27 (55). Saw palmetto products sold as dietary supplements are not allowed to make a drug claim (such as for the treatment of urological symptoms associated with BPH) but are allowed to make structure function claims (such as “to support prostate health” or “support healthy prostate function”). Saw palmetto is included in the Homeopathic Pharmacopoeia of the United States and as such may be sold as a homeopathic over-the-counter drug at strength of 1X, which is similar to a full strength alcoholic (65%) extract (56). Saw palmetto is sold in Canada as a natural health product that must be manufactured to pharmaceutical standards and meet other monograph standards, including the amount of the product to be taken and the form of the product that can be used, and is sold with labels that can state the more accurate claim, “Used in Herbal Medicine to help relieve the urologic symptoms associated with benign mild-to-moderate prostatic hyperplasia” (57). In France, it is a prescription drug reimbursable by the national health insurance. In Italy, a lipophilic hexane extract is sold as a prescription drug. The dried fruit, simple galenical preparations, and lipophilic extracts are approved as nonprescription drugs in Germany. In Belgium, saw palmetto is a prescription adjuvant for BPH, while in Sweden, it is sold as a natural remedy for selfmedication after premarketing authorization. In Switzerland, it is sold without a prescription, but sales are limited to pharmacies, and in the United Kingdom, saw palmetto is an herbal medicine on the General Sales List that requires full product licensing (6).

CONCLUSIONS Saw palmetto is a lipid-rich fruit and the fatty acids extracted from the fruit have been used to sooth irritations. In the late 1800s, Eclectic School of Medicine believed the extract was a nutritive tonic based on the high-energy

content of its lipids and the mild anti-inflammatory activity obtained from its combination of fatty acids. This still seems a rationale description of what is now widely sold as a dietary supplement. Saw palmetto fruits and extracts, on the basis of the known chemistry, pharmacology, clinical trial data, and the low number of adverse event reports, appear to be very safe for men to consume in the amounts used in clinical trials and available in dietary supplement products on the market. Mild nausea or mild gastrointestinal upset appear to be the most frequent side effects from ingesting amounts currently consumed as dietary supplements or as drugs. Saw palmetto products that have not been tested for systemic estrogen and androgen effects pose a theoretical risk to the proper development of the fetus and infants; their use is therefore contraindicated in the WHO monograph for pregnant or lactating women and children under 12 years. Based on the fact that different products vary in chemical content and amount of substantiation for efficacy, specific saw palmetto products could have different therapeutic effects. The use of saw palmetto for treating LUTS has shown mixed clinical results and it is most rationally used as a therapeutic agent on the basis of the excellent safety profile as initial therapy. If LUTS symptoms do not improve with saw palmetto, then ␣-blockers and 5-␣ reductase inhibitors or surgery are the main therapeutic options to be considered. The most interesting scientific questions that still need to be answered include: what is the long-term impact of saw palmetto to improve human health; what is the long-term impact of saw palmetto on the progression of LUTS symptoms, BPH, as well as prevention or slowing the progression of prostate cancer; and because saw palmetto is generally used as a supplement of 320 mg of a specific mixture of fatty acids and plant sterols, what is its impact on the person’s nutritional status, and even on the fatty acid content of a person’s diet, as well as on the physiological activity of supplementing the diet with saw palmetto for LUTS, cancer progression, or other improvements of health? Although saw palmetto became popular to treat LUTS associated with BPH, the impact of lipophilic components on prostate health is an important scientific question because the nutritional requirements of a healthy prostate are not well understood.

ACKNOWLEDGMENTS Thanks to Renato Iguera and Indena S.p.A., Milan, Italy, for the photographs of saw palmetto.

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Selenium Roger A. Sunde

leaching from high rainfall are generally poor in selenium. Alkaline soils yield their selenium whereas acid soils withhold it from plants. Thus, alkaline soils derived from sedimentary rock that are in arid regions tend to transmit high levels of selenium to plants. Plants grown on acidic soils tend to have low selenium content. Areas of low selenium in the United States include the Pacific Northwest and the Midwest, and grazing animals in those regions require selenium supplementation. The South Island of New Zealand and much of Scandinavia also have low selenium levels in soil. In contrast, soils in the Dakotas arising from volcanic activity are high in selenium, and wheat grown in these regions is high in selenium (4). Human selenium deficiency was firmly demonstrated in 1979, when Chinese scientists provided evidence that a cardiomyopathy occurred only in seleniumdeficient children living in regions of China where the nutrient content in plants was very low. Blood and hair selenium content of inhabitants of regions where Keshan disease was found were the lowest levels reported in human beings (5). Thus, selenium is an essential nutrient for human beings. In recent years, supplementation of selenium in pharmacological doses has been touted for a variety of health purposes. These include chemoprevention of cancer, delay of aging, and prevention of heart disease. These potential actions do not appear to relate to its nutritional effects. This brief review will focus on nutritional (physiological) functions of selenium. Other uses of the element will be addressed only briefly.

INTRODUCTION Selenium (Se) is an essential micronutrient that is incorporated into the primary structure of proteins in the form of selenocysteine. Selenoproteins facilitate redox reactions that underlie a number of biochemical processes. Among them are protection against oxidative damage, metabolism of thyroid hormones, support of DNA synthesis, and regulation of transcription factors. Selenium deficiency occurs in China and some other countries but not in the United States. The recommended dietary allowance (RDA) for selenium is 55 ␮g/day for adults, and the recommended dietary upper limit is 400 ␮g/day.

BACKGROUND Selenium was discovered by Berzelius in 1817 as a byproduct of sulfuric acid production. Its importance in biology was established in the 1930s, when it was identified as the toxic principle in plants that poisoned grazing animals in certain parts of the Great Plains of the United States. In response to this problem, the U.S. Department of Agriculture mapped the selenium content of forage from all regions of the United States and produced a selenium map to help farmers avoid grazing their animals in areas that might produce toxicity (1). In addition, the metabolism of the nutrient was studied over the next two decades with the aim of determining its mechanism of toxicity. Studies carried out during that period established that its metabolism was intertwined with that of sulfur (2). The essentiality of selenium in animals was recognized in 1957, when provision of the element to rats fed a yeast-based diet was shown to prevent the development of dietary liver necrosis (3). Vitamin E could also prevent the condition, and this fact has linked these two nutrients since that time. A number of naturally occurring animal diseases are caused by combined selenium and vitamin E deficiency. They include mulberry heart disease, nutritional muscular dystrophy, and nutritional liver necrosis of swine, exudative diathesis of chickens, gizzard myopathy of turkeys, white muscle disease of sheep, and male infertility of cattle. Reference to the selenium map produced by the U.S. Department of Agriculture shows that these conditions occur in areas where plant selenium concentrations are low (1). Geological studies led to the recognition that selenium is distributed irregularly in soil. Soils subjected to

CHEMICAL FORMS Selenium occupies a spot in the periodic table of elements just below sulfur and shares many chemical properties with that element. Its chemistry is covalent in biological systems. Most selenium in biological material is present in amino acids (Fig. 1). Plants are not known to require selenium and incorporate the element nonspecifically into selenomethionine in place of sulfur. A major source of selenium in the diet is selenomethionine. Once ingested by an animal, it enters the methionine pool and is not distinguished from methionine. Thus, much of the selenium in animal tissues is selenomethionine that is nonspecifically incorporated in proteins at methionine positions. When selenomethionine is catabolized, its selenium becomes available to the selenium metabolic pool. Thus,

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Table 1

Human Selenoproteins

Group/name

Figure 1 Common selenium supplementation forms and amino acid forms that account for most of the element in the body.

selenomethionine apparently has no biological function related to selenium other than serving as a dietary source of the element. Selenocysteine (Sec or U) is present in stoichiometric amounts in selenoproteins and is necessary for their function. It is synthesized from the selenium present in the metabolic pool. Presence of selenocysteine at active sites of enzymes such as glutathione peroxidases generally increases their activities 1000-fold above that when cysteine is present. The urinary metabolites of selenium that have been identified are trimethylselenonium ion and a methylated selenosugar (6). They are synthesized in the liver and kidney to regulate whole-body selenium. Inorganic forms of selenium such as selenite and selenate (Fig. 1) are biologically available and are often used to supplement selenium intakes. They are well tolerated and inexpensive.

BIOCHEMISTRY AND FUNCTION Selenoprotein Synthesis Animal selenoproteins contain stoichiometric amounts of selenocysteine in their primary structures. The selenocysteine that is used for this process is synthesized in the cell by modification of a serine residue that has already been ligated to tRNA[ser]sec . The resulting selenocysteinyltRNA[ser]sec recognizes a UGA in the open reading frame of the selenoprotein mRNA with the aid of at least three transacting proteins. The process of selenoprotein synthesis is complex and costly to the organism. It requires at least six gene products in addition to the ones normally used for protein synthesis. Reviews describing selenoprotein synthesis are available (7,8).

Selenoproteins and Their Functions Twenty-five genes that code for selenoproteins (Table 1) have been identified in the human genome (9). Because some genes produce more than one protein product, the number of selenoproteins in humans is probably between 25 and 50. In addition, one of the selenoproteins in humans is present in some other animals with cysteine rather than selenocysteine encoded in the gene, and a small number of selenoproteins found in other organisms are present as the cysteine-encoding genes in humans. Plants and fungi

Selenoproteins involved in thiol redox reactions Glutathione peroxidases Cellular glutathione peroxidase Gastrointestinal glutathione peroxidase Extracellular glutathione peroxidase Phospholipid hydroperoxide glutathione peroxidase Odorant metabolizing glutathione peroxidase Thioredoxin reductases Cytosolic thioredoxin reductase Thioredoxin/glutathione reductase Mitochondrial thioredoxin reductase Other U-C motif redox selenoproteins Methionine-R-sulfoxide reductase Selenoprotein 15 (ER residenta ) Selenoprotein H (may regulate glutathione metabolism) Selenoprotein M (ER resident) Selenoprotein O (largest mammalian selenoprotein) Selenoprotein T (ER resident) Selenoprotein V (related to Sepw1; expressed in testes) Membrane selenoproteins Selenoprotein I (may be phosphotransferase) Selenoprotein K (ER resident) Selenoprotein S (ER resident) Selenoproteins involved in thyroid hormone metabolism Type I iodothyronine deiodinase Type II iodothyronine deiodinase (ER resident) Type III iodothyronine deiodinase Muscle selenoproteins Selenoprotein W (binds glutathione) Selenoprotein N (ER resident) Selenocysteine synthesis selenoproteins Selenophosphate synthetase-2 Transport Selenoproteins Selenoprotein P

Abbreviation

GPX1 GPX2 GPX3 GPX4 GPX6 TXNRD1 TXNRD2 TXNRD3 SELR SEP15 SELH SELM SELO SELT SELV SELI SELK SELS DIO1 DIO2 DIO3 SEPW1 SEPN1 SEPHS2 SEPP1

a Selenoproteins

that appear to be localized in the endoplasmic reticulum (ER). Source: From Ref. 9.

lack selenoproteins, and marine organisms generally have more selenoproteins than land animals (10).

Selenoproteins Involved in Thiol Redox Reactions There are two major systems that regulate thiol redox status in cells: the glutathione and the thioredoxin systems. Both of them depend on NADPH for their reducing equivalents and contain selenoproteins as components. The glutathione peroxidases utilize reduced glutathione to catabolize hydroperoxides of various kinds. The active sites of these enzymes typically contain selenocysteine, although some of them function with cysteine in the active site. Of the seven glutathione peroxidase genes in the human genome, five code for selenoproteins. At least one of these genes produces three different protein products by the use of alternative translation start sites. Three glutathione peroxidase genes have been knocked out in mice without obvious effects on health and reproduction (11,12). Only when these knockout mice are stressed do they show increased injury compared with wild-type animals (13). A knockout of another glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, causes embryonic lethality (14). One isoform of this latter enzyme has structural functions in spermatozoa (15). The above-mentioned considerations indicate that

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these enzymes have a variety of biological functions and each will have to be evaluated separately. Thioredoxin reductases maintain thioredoxin and some other substances, including ascorbate, in a reduced state. These enzymes contain selenium in mammals, but lower life forms sometimes have cysteine homologs. Thioredoxin is responsible for providing reducing equivalents to ribonucleotide reductase and to a number of other enzymes and transcription factors. Therefore, this family is important for gene expression, signaling, and oxidant defenses (16,17). Knockout of thioredoxin in mice causes embryonic lethality (18). In addition to the glutathione peroxidases and the thioredoxin reductases, most selenoproteins contain selenocysteine-cysteine (U-C) redox motifs and thus appear to be involved in oxidation–reduction reactions. Methionine-R-sulfoxide reductase in humans contains selenium and can reduce oxidized methionine resulting from oxidative stress. Other family members have cysteine in place of selenocysteine and are specific for other forms of oxidized methionine. Selenoproteins W, V, T, H, M, and O also contain selenocysteine-cysteine motifs, although their functions are unknown (19). At least three of the selenoproteins are membrane proteins and seven of the selenoproteins appear to be associated with the endoplasmic reticulum inside cells (20). Targeted loss of all selenoproteins in mouse brain results in impaired neurological function, clearly illustrating the important roles of selenoproteins in the body’s normal processes (21,22). A full discussion of these enzyme families are beyond the scope of this chapter and can be found elsewhere (23,24).

Selenoproteins Involved in Thyroid Hormone Metabolism Thyroxine, or T4, is the hormone produced by the thyroid gland. It must be converted to triiodothyronine, or T3, to exert biological activity. T3 is inactivated by conversion to T2. All these reactions are carried out by three selenoproteins known as deiodinases (25). In animals, selenium deficiency leads to decreased activity of the deiodinases, but this is compensated for by a rise in T4, levels. When T4 production is compromised by iodine deficiency, the addition of selenium deficiency is not well tolerated (26). There have been reports of cretinism occurring in infants living in areas where combined selenium and iodine deficiency is found (27). Supplementation of selenium without iodine in these populations appears to further aggravate this disease (28).

Muscle Selenoproteins The early discoveries of the association of muscle disease in animals with selenium deficiency are being substantiated as new selenoproteins are discovered. Selenoprotein W is abundant in human and primate muscle, and levels fall in selenium deficiency (29). Selenoprotein N is highly expressed in muscle, and mutations in selenoprotein N are associated with one form of congenital muscular dystrophy (30).

Selenocysteine Synthesis Selenoproteins A selenoprotein is even required in the pathway for synthesis of selenocysteine from serine. Selenocysteinecontaining selenophosphate synthetase-2 is a major en-

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zyme in this pathway but a synthetase with threonine replacing the selenocysteine also has a role (31).

Selenium Transport Selenoproteins Selenoprotein P is essential for normal selenium distribution (32). This extracellular selenoprotein contains 10 selenocysteine residues in its full-length form. It is produced in most tissues, but selenoprotein P in plasma appears to originate largely in liver. Recently, receptors for selenoprotein P have been identified in testes, brain, and kidney, thus providing targeted transport of selenium to these tissues (33,34). Mice with selenoprotein P knocked out have been produced (35,36). Males have abnormal spermatozoa and have sharply reduced fertility. Both males and females develop neurological impairment when fed a diet containing normal amounts of selenium. They can be rescued by provision of a high-selenium diet (22,36). Knocking out the testes and brain selenoprotein P receptor results in the same defects as loss of selenoprotein P, further demonstrating the importance of targeted delivery of selenium to these organs (21,33,34).

Antioxidant Properties of Selenium The element Se is not an antioxidant and itself may be a pro-oxidant. Already discussed are the redox activities of selenoenzymes and the relationship with vitamin E, which is a free radical scavenger. When rats that are vitamin E deficient are also made selenium deficient, they undergo massive lipid peroxidation and liver necrosis (37). This indicates that selenium-dependent proteins partially compensate for the lack of vitamin E, but that loss of both nutrients leads to severe oxidative damage in the liver. A number of oxidant defense enzymes that do not contain selenium become induced in selenium deficiency. These include glutathione transferases, glutamatecysteine ligase, NAD(P)H quinone reductase, heme oxygenase-1, and other phase-II enzymes (38). These changes may occur due to the presence of response elements in genes that sense changes in cellular oxidants in selenium deficiency (39).

Cancer Chemoprevention A great deal of attention has been directed to the use of selenium to prevent cancer. The first data suggesting a correlation between low selenium intake and increased cancer incidence were presented in the 1970s. Since then, other observational studies have supported this correlation. However, they were unable to isolate selenium as the only factor responsible for the lower cancer incidence. Intervention studies have attempted to evaluate the effect of selenium administration on cancer incidence. One such study was carried out in China (40). An overall reduction in cancer mortality of 13% was achieved in a rural population by giving combined supplements of selenium, vitamin E, and carotene. Unfortunately, no subjects were given selenium alone; so the protection cannot be ascribed to it. Another study using high-selenium yeast that provided 200 ␮g of selenium per day was carried out in subjects who had a nonmelanoma skin cancer (41). The primary endpoint was development of another skin cancer.

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Secondary endpoints of other cancers were introduced after the study had been initiated. The initial analysis (10 yr of supplementation) of the primary endpoint showed no effect of the high-selenium yeast on skin cancer development, but it showed a decrease of 37% in overall cancer development. The diagnosis of prostate cancer was decreased 63% in the highselenium yeast group. Concern was raised recently by a report on the entire blinded period (12 yr) of highselenium yeast supplementation (42). It showed that the supplemented group had a higher incidence of squamous cell carcinoma of the skin than did the placebo group. Analysis of the full study also found a significantly higher diabetes incidence (hazard ratio 1.55) in Se-supplemented subjects versus placebo subjects, and a significant hazard ratio of 2.7 for Se supplementation in subjects in the highest tertile of baseline plasma Se level (43). The initial report of this study stimulated a great deal of interest in selenium as a chemoprevention agent, which contributed to the initiation of the Se and Vitamin E Cancer Prevention Trial (SELECT). The SELECT trial enrolled 35,534 participants who consumed pills containing 200 ␮g Se as L-SeMet and/or 400 mg dl-␣-tocopherol acetate or placebo pills. In 2008, however, the study was stopped because an independent monitoring committee found that Se and vitamin E, taken alone or together for an average of five years, did not prevent prostate cancer, and also there were suggestions of adverse effects due to single supplements (44). The discrepancy between these two trials is not understood, but may involve initial Se status of the two populations or the form of Se supplementation (selenized yeast vs. selenomethionine). Because of the uncertainty that exists about the effect of pharmacologic amounts of selenium on cancer development, selenium supplementation cannot be recommended at present to prevent cancer. Furthermore, analysis of 67 intervention studies looking at low-bias risk trials also found no significant effect—positive or negative—of Se supplementation on all-cause mortality (45), further indicating that selenium supplementation is not a panacea.

METABOLISM Selenium has chemical properties that account for its function in selenoproteins. Because these can also lead to catalysis of unwanted reactions, homeostatic control of selenium in the organism is necessary. The major dietary forms of selenium are selenomethionine derived from plants and selenocysteine from animal selenoproteins. Both amino acids appear to be virtually completely absorbed by intestinal amino acid transporters. The inorganic forms that are often used for supplementation, selenite and selenate, are also well absorbed. Thus, absorption of selenium is very high and not subject to regulation by selenium status. The selenium pool in the liver appears to be the site of homeostatic regulation. Absorbed selenium is removed from the portal blood by the liver, and selenomethionine is catabolized there by transsulfuration, releasing its selenium to the selenium metabolic pool. Liver selenium is used to synthesize liver selenoproteins and selenoprotein P for export. Selenium in excess of that needed for these processes appears to be converted

into excretory metabolites (trimethylselenonium ion and a methylated selenosugar) that appear in the urine. When toxic amounts of selenium are present, dimethyl selenide appears in the breath. Thus, excretion is responsible for regulating the selenium content of the body. The only transport form of selenium that has been identified is selenoprotein P (35,36). However, other form(s) must exist, because knockout of selenoprotein P does not affect selenium levels of many tissues. Selenium storage is accomplished through two mechanisms. One is unregulated and consists of selenomethionine that is present in the methionine pool. As selenomethionine is catabolized, its selenium is fed into the selenium metabolic pool. The other storage mechanism relates to the most abundant glutathione peroxidase enzyme. This protein contains a greater fraction of wholebody selenium than any other selenoprotein. When selenium is in short supply, the mRNA of this glutathione peroxidase is degraded more rapidly, reducing synthesis of this selenoenzyme. This allows selenium to be directed to other selenoproteins that are presumably more important for survival (46). In summary, selenium homeostasis is maintained by excretion of the element when it is present in amounts greater than what can be utilized for selenoprotein synthesis. When insufficient selenium is present for synthesis of all selenoproteins, hepatic cytosolic glutathione peroxidase is downregulated so that selenium can be directed to other selenoproteins.

DIETARY INTAKE AND DEFICIENCY Sources and Regional Variation of Selenium The amount of selenium in plants depends on the availability in the soil on which the plants are grown. This fact leads to a single food plant such as wheat having a selenium content that can vary by a factor of 10 or more, depending on where it is grown. This variation renders food tables for selenium in plants of reduced value. Animals, on the other hand, require selenium and have homeostatic mechanisms to maintain predictable concentrations in their tissues. Thus, foods of animal origin are more reliable sources of selenium. In areas where the soil is poor in selenium, animal foods contain more selenium than plant foods. Some marine fish such as tuna can have high levels of selenium. This selenium can diminish toxicity of high mercury levels that sometimes are found in predatory fish (47). The lowest and the highest intakes of selenium in the world have been reported in China. They vary from less than 10 ␮g/day to over 1 mg/day (48). The cause of this wide variation is the reliance of the population on plant foods and the extreme variation in available soil selenium in different regions. Intakes in other countries generally vary from around 30 ␮g/day in New Zealand and parts of Scandinavia to around 100 ␮g/day in North America. Intakes in Europe are in the range of 30 to 60 ␮g/day.

Keshan Disease and other Human Diseases The only human disease that has been clearly linked to selenium deficiency is Keshan disease. It is a childhood

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cardiomyopathy that occurs in low-selenium regions of China, where the intake of the element is approximately 10 ␮g/day. A double-blind placebo-controlled study that was carried out in the 1970s showed that selenium supplementation could prevent the development of Keshan disease (5). Because not all selenium-deficient children developed Keshan disease, a second stress was considered. Subsequent studies in mice have suggested that the second stress might be a viral infection (49). The incidence of Keshan disease has declined in the last few decades and it is now rare. This is likely due to the improvement of the Chinese economy, with increased meat intake and exchange of foodstuffs between regions. In addition to Keshan disease, muscle pain and wasting and inability to walk due to selenium deficiency occurred in TPN patients before these solutions were routinely supplemented with selenium (50), and some specific forms of male infertility are linked to selenium deficiency (51). Selenium deficiency may also contribute to a bone disorder, called Kaskin-Beck disease, that is found in certain low-selenium regions of China and Tibet (52).

Assessment of Selenium Status The endpoint usually used to set recommended intake of selenium is optimization of selenoprotein concentrations. This is based on the assumption that full expression of selenoproteins will promote optimal physiological function and health. Inadequate dietary supply of selenium limits selenoprotein synthesis and results in depressed selenoprotein concentrations. When selenoproteins are optimized, provision of additional selenium will not cause their concentrations to increase. Instead, the additional selenium is excreted. Thus, optimization of the plasma selenoproteins (as representatives of all selenoproteins) has been used to determine the selenium requirement. There are two selenoproteins in plasma, selenoprotein P and extracellular glutathione peroxidase. When optimized, these proteins contain 6.4 and 1.7 ␮g of selenium per 100 mL of plasma, respectively (53). Thus, the total selenium in the plasma selenoproteins is approximately 8 ␮g per 100 mL of plasma. A third pool of selenium is present in plasma proteins. It is selenomethionine distributed nonspecifically in the methionine pool. In the United States, the amount of selenium in this form ranges from 1 to 12 ␮g per 100 mL of plasma. This leads to the total plasma selenium concentration in the United States to vary from about 9 to 20 ␮g per 100 mL. The mean serum selenium level of 18,597 persons reported by the Third National Health and Nutrition Examination Survey (NHANES III, 1988–1994) was 12.5 ␮g per 100 mL, with 5th and 95th percentiles of 10 and 15 ␮g per 100 mL, respectively (54). On the basis of these results, plasma or serum selenium concentrations of 8 ␮g per 100 mL or higher in healthy people should indicate optimization of the plasma selenoproteins. Concentrations greater than this merely indicate that the subjects are consuming selenomethionine. People with diseases may have alterations in their selenoprotein concentrations caused by their conditions (55), so this value may not apply to them. For reference purposes, plasma concentrations of selenium in low-selenium regions of China are generally 2 ␮g per 100 mL or less. In New Zealand, they are 5–8 ␮g

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per 100 mL, and in Europe, 5–10 ␮g per 100 mL. Concentrations greater than 50 ␮g per 100 mL of plasma occur in high-selenium regions of China. Selenium status regulates the mRNA levels of glutathione peroxidase and several other selenoproteins. In rats, these mRNA levels have been used as molecular biomarkers to determine selenium status and requirements (46,56), and to investigate selenium requirements in pregnancy and lactation (57). Future research may identify panels of molecular biomarkers that can discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals who will benefit versus who will be adversely affected by selenium supplementation.

Dietary Reference Intakes Estimates of the human selenium requirement have been based on two studies. One was performed in China in the early 1980s (58). Men with a dietary intake of 11 ␮g of selenium per day were supplemented with selenium as selenomethionine for several months. The group that received a supplement of 30 ␮g of selenium per day optimized its plasma glutathione peroxidase activity. Thus, a total intake (diet plus supplement) of 41 ␮g/day optimized plasma selenoproteins. The other study was carried out in New Zealand in a group with a dietary selenium intake of 28 ␮g/day (59). Its results were more difficult to interpret because of the high basal selenium intake. However, results indicated a similar requirement for optimization to that found in China. In 2000, on the basis of these two studies, the Institute of Medicine set the RDA for selenium at 55 ␮g/day for adults of both sexes (60). Table 2 shows the corresponding values for other subjects as well. These recommendations are for healthy people and are meant to satisfy the biochemical selenium requirements of the body. Further research will be necessary to determine whether there are special populations that need a greater selenium intake. Also, this RDA does not take into consideration the possible pharmacologic use of selenium.

SUPPLEMENTATION Several forms of selenium are available for use as supplements. The two inorganic forms, selenite and selenate, are often used in animal experimental diets because they cannot be converted to selenomethionine, and therefore Table 2 Recommended Dietary Allowances (RDAs) for Selenium Group

Amount (␮g/day)

Children 1–3 yr old 4–8 yr old 9–13 yr old Adolescents (14–18 yr old) Adults ( ≥ 19 yr old) Pregnant women Lactating women

20 30 40 55 55 60 70

Source: From Ref 60.

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tissue selenium concentrations reflect only selenoproteins. These forms have similar bioavailability (50–90%), but selenite is subject to reaction with intestinal contents. Also, selenite is an oxidant and can be quite damaging when given in large quantities. Selenite is added to salt in some selenium-deficient regions of China and to fertilizer in Finland. Both these methods of supplementation have been shown to be effective in improving the selenium status of human populations. Selenomethionine makes up a large fraction of the normal dietary selenium. It is virtually completely bioavailable. It is less likely to cause acute toxicity than are the inorganic forms, although its toxicity is approximately the same as that of those forms under steady state conditions. Administration of selenium as selenomethionine can complicate interpretation of tissue selenium levels, and it is more expensive than the inorganic forms. High-selenium yeast preparations are available for selenium supplementation. These are proprietary products in which yeast is grown in a high-selenium medium. Much of the selenium in the yeast is in the form of selenomethionine, but many minor forms are also present in variable amounts, and there may be considerable variation due to differences in production. Producers and marketers of these yeast preparations make various claims, but there is no good evidence for their special efficacy. These are often expensive. All these forms of selenium are effective as supplements in delivering selenium to human beings and animals. The inorganic forms have the advantage of being inexpensive. Selenomethionine allows the person to incorporate surplus selenium into protein thus raising tissue levels of selenium. Because high-selenium yeast contains selenomethionine, it shares this property. There is, however, no evidence of beneficial effects of higher selenium levels in tissues resulting from selenomethionine supersupplementation. Scientific experiments generally use inorganic selenium or selenomethionine, depending on the design of the experiment. Research is being performed on other forms of selenium such as Se-methylselenocysteine, and on high selenium foods such as onions or broccoli, to evaluate their health-promoting activity. Such forms are not generally available at present and cannot be recommended except for research purposes.

Toxicity Selenium can be toxic. Manifestations range from severe acute multiorgan failure after ingestion of milligram-togram quantities of selenious acid (selenite) to loss of hair and nails caused by chronic ingestion of more than a milligram of selenium per day for long periods. Misformulation of selenium-containing over-thecounter products, perhaps because of addition of milligram rather than microgram quantities, is a real issue for the American public. Recent (61) and past (62) events resulted in adverse effects including significant hair loss, muscle cramps, diarrhea, joint pain, deformed fingernails, and fatigue. Studies carried out in a high-selenium region of China indicated that brittle nails and hair loss did not

occur at intakes below 1 mg/day. On the basis of this result, the Institute of Medicine set a safe upper limit of 400 ␮g/day for chronic selenium intake for adults (60). In practical terms, this is about 300 ␮g above the dietary intake of selenium in the United States. Thus, it allows room for supplements to be taken by those who believe they might be efficacious.

CONCLUSIONS Selenium is an essential element that has a variety of biochemical functions. It is lacking in the food supply of many countries, but there is no such evidence in North America. There is a need for research into the effects of marginal selenium intakes such as those in New Zealand, Scandinavia, and Europe. Also, studies are needed to identify better biomarkers of selenium status and to determine whether genetic and/or disease conditions raise the selenium requirement. Finally, further studies are needed to determine if ingestion of pharmacologic amounts of selenium, and in what form, can modulate cancer incidence.

ACKNOWLEDGMENTS The author gratefully acknowledges the author of the selenium chapter in the previous edition, Dr. Raymond F. Burk, for substantial contributions to the present chapter. The work of the author is supported by NIH grant DK74184.

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synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation. Proc Natl Acad Sci 2004; 101:16162–16167. Burk RF, Hill KE. Selenoprotein P-Expression, functions, and roles in mammals. Biochim Biophys Acta 2009; 1790:1441– 1447. Olson GE, Winfrey VP, Nagdas SK, et al. Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis. J Biol Chem 2007; 282:12290– 12297. Olson GE, Winfrey VP, Hill KE, et al. Megalin mediates selenoprotein P uptake by kidney proximal tubule epithelial cells. J Biol Chem 2008; 283:6854–6860. Schomburg L, Schweizer U, Holtmann B, et al. Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues. Biochem J 2003; 370:397–402. Hill KE, Zhou J, McMahan WJ, et al. Deletion of selenoprotein P alters distribution of selenium in the mouse. J Biol Chem 2003; 278:13640–13646. Hafeman DG, Hoekstra WG. Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J Nutr 1977; 107:666–672. Burk RF. Biological activity of selenium. Annu Rev Nutr 1983; 3:53–70. Burk RF, Hill KE, Nakayama A, et al. Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not. Free Radic Biol Med 2008; 44:1617–1623. Taylor PR, Li B, Dawsey SM, et al. Prevention of esophageal cancer: The nutrition intervention trials in Linxian, China. Linxian Nutrition Intervention Trials Study Group. Cancer Res 1994; 54:2029S–2031S. Clark LC, Combs GF, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. JAMA 1996; 276:1957–1963. Duffield-Lillico AJ, Slate EH, Reid ME, et al. Selenium supplementation and secondary prevention of nonmelanoma skin cancer in a randomized trial. J Natl Cancer Inst 2003; 95:1477–1481. Stranges S, Marshall JR, Natarajan R, et al. Effects of longterm selenium supplementation on the incidence of type 2 diabetes: A randomized trial. Ann Intern Med 2007; 147:217– 223. Lippman SM, Klein EA, Goodman PJ, et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers: The Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 2009; 301:39–51. Bjelakovic G, Nikolova D, Gluud LL, et al. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: Systematic review and meta-analysis. JAMA 2007; 297:842–857. Weiss Sachdev S, Sunde RA. Selenium regulation of transcript abundance and relative translational efficiency of glutathione peroxidase 1 and 4 in rat liver. Biochem J 2001; 357:851–858. Ganther HE, Goudie C, Sunde ML, et al. Selenium: Relation to decreased toxicity of methylmercury added to diets containing tuna. Science 1972; 175:1122–1124. Combs GF Jr, Spallholz JE, Levander OA, et al. Selenium in Biology and Medicine. New York, NY: Van Nostrand Reinhold Co. Inc, 1987. Beck MA, Kolbeck PC, Rohr LH, et al. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol 1994; 43:166–170. Johnson RA, Baker S.S, Fallon JT, et al. An occidental case of cardiomyopathy and selenium deficiency. N Engl J Med 1981; 304:1210–1212. Foresta C, Flohe L, Garolla A, et al. Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase. Biol Reprod 2002; 67:967–971.

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52. Stone R. A medical mystery in middle China. Science 2009; 234:1378–1381. 53. Burk RF, Hill KE, Motley AK. Plasma selenium in specific and non-specific forms. Biofactors 2001; 14:107–114. 54. Niskar AS, Paschal DC, Kieszak SM, et al. Serum selenium levels in the US population: Third National Health and Nutrition Examination Survey, 1988–1994. Biol Trace Elem Res 2003; 91:1–10. 55. Burk RF, Hill KE, Boeglin ME, et al. Plasma selenium in patients with cirrhosis. Hepatology 1998; 27:794–798. 56. Barnes KM, Evenson JK, Raines AM, et al. Transcript analysis of the selenoproteome indicates that dietary selenium requirements in rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity. J Nutr 2009; 139:199–206. 57. Sunde RA, Evenson JK, Thompson KM, et al. Dietary selenium requirements based on glutathione peroxidase-1 activity and mRNA levels and other selenium parameters are not increased by pregnancy and lactation in rats. J Nutr 2005; 135:2144–2150. 58. Yang GQ, Zhu LZ, Liu SJ, et al. Human selenium requirements in China. In Combs GF Jr, Spallholz JE, Levander OA, et al. eds. Selenium in Biology and Medicine. New York: AVI, 1987:589–607.

59. Duffield AJ, Thomson CD, Hill KE, et al. An estimation of selenium requirements for New Zealanders. Am J Clin Nutr 1999; 70:896–903. 60. Food and Nutrition Board. In Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids. Washington, DC: National Academy Press, 2000. 61. FDA. FDA finds hazardous levels of selenium in samples of Total Body Formula and Total Body Mega Formula. http:// www.fda.gov/bbs/topics/news/2008/new01818.html. Accessed October 30, 2009. 62. Helzlsouer K, Jacobs R, Morris S. Acute selenium intoxication in the United States [abstract]. Fed Proc 1985; 44: 1670.

FURTHER READINGS 1. Sunde RA. Selenium. In Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. 9th ed. Washington, DC: ILSI Press, 2006:480–497. 2. Hatfield DL, Berry MJ, Gladyshev VN. In Selenium. Its Molecular Biology and Role in Human Health. 2nd ed. New York, NY: Springer, 2006.

Shiitake Solomon P. Wasser

INTRODUCTION

itake, Cortinellus edodes, Lentinula edodes, whereas common names are English, Black forest mushroom, black oak mushroom, golden oak mushroom, snake butter, pasania mushroom, oakwood mushroom, Japanese forest mushroom; Japanese names include Shiitake; and in Chinese, Shiang-gu, Shing ku, Hua Gu, Xiangu, Hoang-mo.

Shiitake mushroom, the common Japanese name for Lentinus edodes (Fig. 1), derives from the mushroom associated with the shii tree (Castanopsis cuspidate Schottky) and take, the Japanese word for mushroom. Because Japan was the world leader in the production of this species of mushroom, the mushroom is now widely known by this name. These mushrooms are renowned in Far East countries (e.g., Japan, China, and Korea) as a food product and medicine for thousands of years. In the year AD 199, Kyusuyu, a native tribe of Japan, offered the Japanese Emperor Chuai a shiitake mushroom. Even older documents record its use in ancient China, where it was referred to as “ko-ko” or “hoang-mo.” Now shiitake is the second most commonly cultivated edible mushroom worldwide. Shiitake is an important ingredient in Far East cuisine and is increasingly finding its way to the dining tables of North Americans, Europeans, and people of other countries. In this chapter the artificial cultivation and the physicochemical and pharmaceutical properties of shiitake mushroom are described in detail. The application of modern analytical techniques has revealed numerous bioactive compounds including polysaccharides, -D-glucans, heterogalactans, glycoproteins, immunomodulatory proteins, organic acids, dietary fiber, and lowmolecular-weight compounds from this mushroom. The health benefits of the shiitake mushroom are rapidly increasing, demonstrating immonomodulating, antioxidant, antitumor, antidiabetic, cholesterol-regulating, antiviral, antibacterial, antifungal, and antiparasitic activities and beneficial cosmetic applications. All scientific studies have demonstrated that shiitake is one of the most bioactively safe mushrooms. Cancer is the second leading cause of death in technologically developed countries worldwide, and the proven protective effects and use of shiitake in combination with chemo- and radiation therapy in Japan may well increase its production and popularity. More randomized, double-blind, controlled studies are needed to clarify the benefits, dosages, and therapeutic regimes for the use of shiitake in the treatment of cancer and other diseases (1).

Habitat and Distribution Gregarious on fallen wood of a wide variety of deciduous trees, especially shii, oak, chestnut, beech, maple, sweet gum, poplar (aspen, cottonwood), alder, hornbeam, ironwood, chinquapin, mulberry (Castanopsis cuspidate, Quercus, Castanea, Fagus, Acer, Liquidamber, Populus, Diospyros, Alnus, Carpinus, Morus) in a warm, moist climate. Most of these are raised for artificial cultivation of shiitake mushroom. L. edodes occurs naturally throughout Southeast Asia. It has been reported from China, Japan, Korea, Vietnam, Thailand, Burma, North Borneo, the Philippines, Taiwan, and Papua New Guinea (3,4).

Cultivation The cultivation of this mushroom has been practiced for a thousand years, with its cultivation originating in China during the Sung Dynasty (AD 960–1127). Both history and legend credit Wu San Kwung as the originator of shiitake cultivation. Almost every mushroom-growing village in China has a temple in his honor (5). In 1313, Chinese author Wang Cheng recorded shiitake-growing techniques in “Book of Agriculture.” He described how to select a suitable site, choose appropriate tools, and cut down the trees on which one could cultivate the mushrooms. He outlined the basic methods, which are as follows: Cut the bark with a hatchet and cover the logs with soil. After 1year, top the soil and water frequently. Beat the logs with a wooden club to induce mushroom production. The mushrooms will appear after a rain (5,6). Shiitake mushroom cultivation techniques were probably introduced to Japanese farmers by the Chinese between AD 1500 and 1600 (2). At present, shiitake is one of the five most cultivated edible mushrooms in the world (7). Its production (3.5 million tons) is second only to button mushroom Agaricus bisporus. Grown mainly in East Asia (China is the world leader in shiitake production), shiitake is now arousing interest worldwide (3,4,7,8). Increasing markets have been spawned, partly by the exotic and well-appreciated taste of shiitake and partly by advances in research that has demonstrated its significant medicinal properties. Shiitake mushroom is becoming popular as nutritional and medicinal product throughout Asia, Europe, and North America.

Mycological Data for Shiitake Mushroom A detailed description of the shiitake mushroom (Fig. 1) can be found in literature (1–3). Formally known as Lentinus edodes (Berk.) Singer (family Pleurotaceae), its basyonym is Agaricus edodes Berk. Common synonyms are Collybia shiitake, Armillaria edodes, Agaricus russaticeps, Lentinus tonkinensis, Lepiota shiitake, Mastaleucomyces edodes, Pleurotus russaticeps, Cortinellus shiitake, Tricholoma shi719

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(e) 10 μm (a)

5 μm

5 μm

(b)

(c)

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Figure 1

Shiitake mushroom [Lentinus edodes (Berk.) Singer]. (A) Fruit body, (B) spores, (C) basidia, (D) cheilocystidia, (E) elements of pileal cuticle.

Although specialists in medicinal mushrooms and their cultivation are familiar with shiitake mushrooms as L. edodes, some alternative taxonomic classifications are discussed in literature (9–13). Although the mushroom had been grown in Asian countries for centuries, the interest there, as well as in the Western countries, has increased rapidly since World War II, especially in the last 15 to 20 years. Shiitake mushroom cultivation is now a multimillion-dollar industry worldwide. World production of shiitake mushroom reached 3 million tons per year. The process for producing shiitake mushroom fruiting bodies (Fig. 2) is the same as for other cultivated edible mushrooms and can be divided into two major stages. The first stage involves the preparation of the fruiting culture, stock culture, mother spawn, and planting spawn, and

the second stage entails the preparation of the growth substrates for cultivation. Currently, the methods most widely adopted for commercial production are wood log and synthetic sawdust bag (3,4,6,8,14). A discussion of the cultivation methods used is beyond the scope of this review. Interested readers may refer to the works already cited; growth parameters for cold- and warm-weather strains are given in Ref. 4.

Chemistry and Production of Products Shiitake mushrooms are traditionally well-known edible mushrooms of high nutritional value. Raw or dried forms, used in Chinese curative powers of shiitake mushroom, are legendary. It was stated in Ri Youg Ben Cao, Vol. 3 (1620), written by Wu-Rui of the Ming Dynasty, “shiitake accelerates vital energy, wards off hunger, cures colds, and

Figure 2 Shiitake mushroom [Lentinus edodes (Berk.) Singer]: cultivated fruiting bodies.

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defeats body fluid energy.” In later years, it was found that the mushroom contained various important nutrients. Moreover, recent scientific investigations have isolated many compounds and have found evidence of their health-promotion activities (1,3,4,15,16). Shiitake mushrooms have excellent nutritional value. Their raw fruit bodies include 88% to 92% water, protein, lipids, carbohydrates as well as vitamins and minerals. It should be noted that amounts of nutrients and biologically active compounds differ in various strains and are affected by substrate, fruiting conditions, and methods of cultivation. On a dry weight basis, they have a relatively high nutritional value when compared to commonly consumed vegetables. Dried shiitake mushrooms are rich in carbohydrates and protein. They contain 58% to 60% carbohydrates, 20% to 23% protein (digestibility of 80–87%), 9% to 10% fiber, 3% to 4% lipids, and 4% to 5% ash. The mushroom is a good source of vitamins, especially provitamin D2 (ergosterol), 325 mg%, which under ultraviolet light and heat yields calciferol (vitamin D2 ). It also contains B vitamins, including B1 (thiamine), B2 (riboflavin), B12 (niacin), and pantothenic acid (1,6,15,16). Minerals found in shiitake mushroom include Fe, Mn, K, Ca, Mg, Cd, Cu, P, and Zn. Analysis of dried cultured shiitake mycelium gives the following mineral concentrations (in mg/g of dry weight): K, 15.1; Ca, 22; Mg, 44–78; Mn, 1.2; Cd, 0.96; Fe, 2.36; Ni, 52.5; Cu, 89.1; P, 281; Zn, 282; Ge, 3; Br, 11.4; and Sr, 164. Water-soluble polysaccharides amount to 1% to 5% of the dry weight of the shiitake mushroom. In addition to glycogen-like polysaccharides, (1–4)-,(1–6)-␣-Dglucans and antitumor polysaccharides, lentinan, (1–3),(1–6)-␤-bonded heteroglucans, heterogalactans, heteromannans, xyloglucans, etc., have been identified. The mushrooms’ indigestible polysaccharides, which serve as dietary fiber, include heteroglycan, polyuronide, ␤glucan, as well as chitin. Among the free sugars present are trehalose, glycerol, mannitol, arabitol, mannose, and arabinose (1,3,4,15,16). In shiitake mushrooms, dietary fiber consists of water-soluble compounds such as ␤-glucan and protein and water-insoluble substances extractable only with salts, acids, and alkalies such as polyuronide (acidic polysaccharide), hemicellulose, ␤-glucan with heterosaccharide chains, lignin, and chitin present as cell-wall constituents (15). The aroma components include alcohols, ketones, sulfides, alkanes, fatty acids, etc. The major volatile flavor contributors are matsutakeol (octen-1-ol-3) and ethyln-amyl ketone. The characteristic aroma of shiitake mushrooms was identified as 1,2,3,5,6-pentathiepane. According to Mizuno (15), the components responsible for the delicious flavor are monosodium glutamate, 5 nucleotides, free amino acids, lower molecular weight peptides, organic acids, and sugars. Their relative ratios are responsible for the variation in flavor naturally seen in this mushroom. Organic acids contributing to the flavor of shiitake mushroom include malic acid, fumaric acid, ␣-keto-glutaric acid, oxalic acid, lactic acid, acetic acid, formic acid, and glycolic acid. The fatty acids account for 3.38% of the total lipids (14,15). Their composition is as follows: linoleic acid (18:2), 72.8%; palmitic acid (16:0), 14.7%; oleic acid (18:1), 3.0%;

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tetradecenoic acid (14:1), 1.6%; stearic acid (18:0), 0.9%; and myristic acid (14:0), 0.1%.

Dosage and Preparation of Products Shiitake mushroom concentrate can be freeze-dried or spray-dried to form a granular powder. There are many products containing powdered shiitake mushroom extract, such as a mixture of this powder with vitamin C crystals or with medicinal plants such as ginseng. In Eastern countries, the mushroom is mainly used as a concentrate when extracted with boiling water. Residues from these processes still contain substantial amounts of useful polysaccharide substances, including those effective as antitumor compounds such as ␤-glucans, nucleic acids, dietary fiber, etc. An alcohol extraction product is obtained by preserving fresh or dried shiitake mushroom in alcohol, which has been mixed with sugar or molasses. Shiitake mushroom is prescribed in various forms. It may be injected as a solution (1 mg/vial) or ingested as a sugar-coated tablet, capsule, concentrate, powdered extract, syrup, tea, wine, and/or as a medicinal dish. Tablets are usually made from a dried water extract of the mycelia or fruiting bodies because drying concentrates the lentinan and other active principles. Standardized extracts are also available, and they are preferred because the amount of lentinan present is certified and clearly stated on the bottle. Lentinan’s anticancer effect is highly dose dependent. The standard dose of the dried fruiting body in tea or in mushroom dishes is given as 6–16 g, equivalent to approximately 60–160 g of fresh fruiting bodies. The dosage, usually in the form of 2 g tablet, is 2 to 4 tablets per day. Commercial preparations can be found in many countries in health food stores and supermarkets. Although the fresh form can be a valuable dietary supplement, the quantities one would require for therapeutic doses are so great that its consumption could cause digestive upset. That is why L. edodes mycelium (LEM), which is concentrated and easily absorbed, is preferred for medicinal use (3,4,16). Fresh and dried shiitake mushrooms are used in medicinal mushroom dishes (“Yakuzen”). These dishes can be prepared in many ways, limited only by one’s ingenuity: boiled, grilled, skewered, or on aluminum foil with different types of seasoning. Concentrates, obtained by concentrating boiling water extracts of whole fruit bodies or powdered mushrooms, are used as drinks. They can also be consumed as canned “shiitake tea” or many other shiitake “healthy tea” products sold as mushroomcontaining tea bags. Some products, including “healthy shiitake wine,” are sold as a nightcap or as a tonic drink (4,15).

Preclinical Studies This section mainly discusses preclinical in vitro and in vivo (animal) studies. Shiitake is one of the best-known and bestcharacterized mushrooms used in medicine. It is the source of several well-studied preparations with proven pharmacological properties, especially polysaccharide lentinan, shiitake mushroom mycelium, and culture media extracts (LEM, glycoprotein (LAP) and KS-2) (3,14,15,17–19).

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Anticarcinogenic and Antitumor Effects By using methods of fractionation and purification of polysaccharides, Chihara et al. (20–22) isolated a watersoluble antitumor polysaccharide from fruiting bodies of shiitake, which was named “lentinan” after the genus Lentinus to which the shiitake mushroom belongs. Chihara was one of the first to report on the antitumor properties of the mushroom, stating that lentinan “was found to almost completely regress the solid type tumors of Sarcoma 180 and several kinds of tumors including methylchloranthrene-induced fibrosarcoma in synergic host-tumor system A” (21,22). The antitumor effect of lentinan was originally confirmed by using Sarcoma 180 transplanted in CD-1/ICD mice (20). Later, it showed prominent antitumor activity not only against allogenic tumors such as Sarcoma 180, but also against various synergic and autochthonous tumors, and it prevented chemical and viral oncogenesis (23). The molecular formula of ␤-D-glucan lentinan is (C6 H10 O5 )n , and the mean molecular weight is about one million (−5 ×105 Da); [␣]D + 20–22◦ (NaOH). Its structure was confirmed as ␤-(1–3)-D-glucopyranan with a branched chain of ␤(1–6)-monoglycosyl (branching degree: 2.5◦ ), showing a right-handed triple helix (3,15,17,18). It is water soluble, heat stable, and alkali labile. ␤-D-Glucan binds to lymphocyte surfaces or serum-specific proteins, which activate macrophage, T-helper cells, natural killer (NK) cells, and other effector cells. All these increase the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN-␥ ) released upon activation of effector cells (19,24). Thus, the carcinostatic effect of lentinan results from the activation of the host’s immune system. In animal testing of carcinostatic activity, IP administration is used, but oral administration (PO) is occasionally effective. The purified polysaccharide has been shown in animal studies to produce strong tumor regression and even the disappearance of sarcoma tumors in 5 weeks, ascite hepatoma 134, (18,19,25), and Ehrlich carcinoma as well as a number of other experimentally induced cancers in allogenic, syngeneic, and autologous hosts. It also exhibits preventive activity against chemical carcinogenesis. Injections of lentinan into mice produced either an 80% reduction in tumor size or complete regression in most of the animals tested. Moreover, an intact immune system and a functioning thymus gland were found to be requisite for its anticancer effect (9,10). When immunosuppressive agents such as ␤-benzylthioguanosine or X-radiation were given with lentinan, the antitumor effect decreased. The polysaccharide has also been found to restore the enzyme activity of X-prolyl-dipeptidyl-aminopeptidase, which can be depressed in cancer patients and in mice with implanted tumors (26). Laboratory tests seem to indicate a role for the adrenal–pituitary axis and central peripheral nervous system including serotonin, 5HT, histamine, and catecholamies in lentinan’s antitumor activity (1,16,17,24). The oral administration of the polysaccharide to AKR mice exerted strong antitumor activity resulting in raised levels of lymphocytokines, such as IFN-␥ , tumor necrosis factor (TNF-␣), and IL-1 ␣. Tissue cultures of murine macrophages CRL-2019, B-lymphocytes HB-284, and T-lymphocytes DRL-8179, which were treated with lentinan, showed high levels of activation by using flow

cytometry. Lentinan-activated immunocytes, particularly the T-helper cells, might render the physiological constitutions of the host highly cancer- and infection resistant. Adoptive immunotherapy of the immunodeficient mice such as the nude (athymic) mice, B-cell deficient mice, and severe combined immunodeficient mice via the transfer of the lentinan-activated immunocytes resulted in the inhibition of tumor growth. Lentinan appeared to represent a unique class of host defense potentiators (HDP), protecting the hosts from the side effects of conventional therapeutic measures and improving various kinds of immunological parameters with no toxic side effects in animal models (19,24,27,28). Lentin, a novel protein isolated from shiitake mushroom, exerted an inhibitory activity on proliferation of leukemia cells (28). Compounds that block the formation of carcinogenic N-nitroso compounds from nitrates (which occur in vegetables and meats) are produced in dried and heated mushrooms (3,4,16). The uncooked form contains no detectable amounts of the nitrite-scavenging compound thiazolidine-4-carboxylic acid, whereas the dried variety has 134 mg/100 g (dry weight basis) of this compound, and the boiled form holds 850 mg/100 g. Thus, shiitake mushrooms may have cancer-preventative properties and can be a beneficial dietary supplement.

LEM and LAP Extracts from Shiitake Mushroom Mycelium and Culture Media LEM is prepared from an extract of the powdered mycelia of the shiitake mushroom. Its yield is approximately 6 to 7 g/kg of medium. The precipitate obtained from a water solution of the mycelium by adding four volumes of ethanol was named LAP. The yield of LAP is ≈0.3 g/g of LEM. LEM and LAP are glycoproteins containing glucose, galactose, xylose, arabinose, mannose, and fructose (15). The former also contains various nucleic acid derivatives, vitamin B compounds especially B1 (thiamine), B2 (riboflavin), and ergosterol (3,4). In 1990, an immunoactive substance, EP3, was obtained by fractionation of LEM. EP3 is a lignin complex composed of approximately 80% lignin, 10% carbohydrates, and 10% protein. After removal of the last two components, biological activity was not affected, but when lignin is removed, activity was reduced. Therefore, the active substance is believed to be a water-soluble lignin containing numerous carboxyl groups (14,15). Both LEM and LAP have demonstrated strong antitumor activities orally and by injection to animals and humans. They were shown to act by activating the host’s immune system and are also useful for the treatment of hepatitis B (12–15).

KS-2-␣-Mannan Peptide Polysaccharide KS-2 was obtained by extraction of cultured mycelia of shiitake mushroom (strain KSLE 007) with hot water, followed by precipitation with ethanol (14,15,29). The product is an ␣-mannan peptide containing the amino acids serine, threonine, alanin, and proline (as well as residual amounts of the other amino acids). The polysaccharide was shown (29) to be effective on Sarcoma 180 and Ehrlich’s carcinoma, either IP or PO, and to act via interferon-inducing activity. The acute LC50

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of KS-2 was found to be extremely low in mice, more than 12,500 mg/kg when administered orally. The mechanism of action of KS-2 is not yet clear, but the results showed no direct KS-2 cytocidal effect against the tumor cells in vitro. Its antitumor activity was observed to be higher at the lower inoculum size of tumor cells, regardless of the routes of KS-2 administration (60% survival rate at 5 × 103 tumor cells/mouse, 10% survival at 1 × 106 tumor cells/mouse). The results also showed that the antitumor activity of KS-2 in mice was always accompanied by the induction of interferon in the sera. Furthermore, preliminary findings indicated that macrophages obtained from KS-2-treated mice exhibited tumoricidal activity (14,16,30), and it was reported that macrophage activation became tumoricidal when incubated in vitro with interferon. Considering these findings, the antitumor activity of KS-2 may be explained by macrophage activation with or without interferon induced by KS-2.

Immune-Modulating Effects As was stated earlier, lentinan and other polysaccharides from shiitake mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. Lentinan, for example, appears to act as HDP, which is able to restore or augment the responsiveness of host cells to lymphocytokines, hormones, and other biologically active substances by stimulating maturation, differentiation, or proliferation of cells involved in host defense mechanisms (19,24). HDPs are functionally different from biological response modifiers. Thus, lentinan is able to increase host resistance against various kinds of cancer and infectious diseases, including acquired immunodeficiency syndrome (AIDS) (3,28). The initial interactions of lentinan in the human body or animals are not presently known. However, there is a transitory but notable increase in several serum protein components in the ␣- and ␤-globulin region, namely, complement C3, hemopexin, and ceruloplasmin (3,16,19,24). Lentinan stimulates various kinds of NK-cell-, Tcell-, B-cell-, and macrophage-dependent immune reactivities. Its antitumor effect is abolished by neonatal thymectomy and decreased by the administration of antilymphocyte serum, supporting the concept that the polysaccharide requires immunocompetent T-cell compartments. The effect of lentinan was also inhibited by antimacrophage agents, for example, carrageenan. Unlike other well-known immunostimulants, lentinan is in a unique class of distal tubular-cell-oriented assistant, in which macrophages play some part (3,16,19,24). For example, lentinan can activate NK cells in vitro in the same concentrations that are achieved in the blood plasma of patients treated clinically with lentinan (14,16,24). NK-cell activity is involved in tumor suppression, and although these cells do not stimulate T-killer cell activity or do so only under certain conditions, they are strong T-helper cell stimulants both in vitro and in vivo (1,3,14,16,19,24). Using the blood of healthy donors and cancer patients, some authors have shown that the polysaccharide is able to stimulate peripheral blood lymphocytes in vitro to increase IL-2-mediated lymphokineactivated killer cell (LAK-cell) and NK cell activity at levels achievable in vivo by administration of clinical doses

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of lentinan. It has been shown to inhibit suppressor Tcell activity in vivo and to increase the ratio of activated T-cells and cytotoxic T-cells in the spleen when administered to gastric cancer patients undergoing chemotherapy (3,16,24). Many interesting biological activities of lentinan have been reported including (a) an increase in the activation of nonspecific inflammatory responses such as acute phase protein production, (b) vascular dilation and hemorrhage in vivo, (c) activation and generation of helper and cytotoxic T-cells, (d) augmentation of immune mediators like IL-1 and IL-3, colony-stimulating factor(s), and migration inhibitory factor, and (e) increasing the capacity of peripheral blood mononuclear cells of patients with gastric cancer and producing IL-1␣, IL-1␤, and a TNF-␣ (3,16,19,24,27). In an in vivo study of rats with peritonitis, combined lentinan–gentamicin treatment had a significantly better survival rate than the controls. Lentinan activated the peritoneal macrophages’ secretory activity of active oxygen and produced cytokines, thus enhancing the ability of polymorphonuclear leukocytes to produce active oxygen, which has a bactericidal effect (31). It also increases peritoneal macrophage cytotoxicity against metastic tumor cells in mice, but not against a highly metastic tumor type (32). Some patients treated with lentinan for carcinomatous pleuritis or carcinomatous peritonitis have improved with the disappearance of malignancy, whereas in another group their condition deteriorated or diminished (33). The polysaccharide can activate the normal and alternative pathways of the complement system and can split C3 into C3a and C3b enhancing macrophage activation (34). Many biological reactions are accelerated and induced by lentinan, including the very important phenomenon of infiltration of eosinophils, neutrophils, and granulocytes around target tissues. Early responses initiated by lentinan and possible pathways for inflammatory reactions are discussed by Mizuno (14). Lentinan’s immune-activating ability may be linked with its modulation of hormonal factors, which are known to play a role in tumor growth. Aoki (34) showed that the antitumor activity of lentinan is strongly reduced by administration of thyroxin or hydrocortisone. It can also restore tumor-specific antigen-directed delayed-type hypersensitivity reaction. Lentinan is not formally included among the nonspecific immunostimulants (RES stimulants), but it augments the induction of antigen-specific cytotoxic T-lymphocytes, macrophages, and other nonspecific immune responses. Possible immune system regulating actions of lentinan were summarized by Chihara et al. (23).

Cardiovascular Effects It is known that shiitake mushroom is able to lower blood serum cholesterol (BSC) via a factor known as eritadenine (also called “lentinacin” or “lentysine”). Apparently, eritadenine reduces BSC in mice, not by the inhibition of cholesterol biosynthesis, but by the acceleration of the excretion of ingested cholesterol and its metabolic decomposition. It has been shown to lower blood levels of cholesterol and lipids in animals. When added to the diet of rats, eritadenine (0.005%) caused a 25% decrease in total cholesterol in as little as 1 week. The cholesterol-lowering

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activity of this substance is more pronounced in rats fed a high-fat diet than in those on a low-fat diet. Although feeding studies with humans have indicated a similar effect, further research is needed. Hobbs (1,16) and Yang et al. (35) have shown that shiitake mushrooms lowered BSC levels. Various studies have confirmed (1,3,14,16) that the mushroom can lower blood pressure and free cholesterol in plasma, as well as accelerate the accumulation of lipids in the liver by removing them from circulation.

Hepatoprotective Effects The injection of LEM slowed the growth of cancerous liver tumors in rats (14,18,36). A polysaccharide fraction from shiitake mushrooms demonstrated liver protection in animals as well as the ability to improve liver function and enhance the production of antibodies to hepatitis B (3,37). Lentinan improved serum glutamic pyruvic transaminase and completely restored glutamic pyruvic transaminase (GPT) levels in the livers of mice with toxic hepatitis. Crude extracts of shiitake mushroom cultures have demonstrated liver-protecting actions (14,16,18,37).

Antiviral, Antibacterial, and Antiparasitic Effects Lentinan and its derivatives are effective against various kinds of bacterial, viral (including AIDS), and parasitic infections (3,16,18,28,38). An important area of this polysaccharide research deals with its ability to mobilize the body’s humoral immunity to ward off bacterial infections resistant to antibiotics (3). Many cancer and AIDS patients die of opportunistic infections due to immunodysfunction (3,27). In vitro studies show that lentinan, when used in combination with azidothymidine (AZT), suppressed the surface expression of HIV on T-cells more so than did AZT alone (39). Lentinan and the sulfated form exhibited potent in vitro anti-HIV activity resulting in inhibition of viral replication and cell fusion. Among the various therapeutic approaches used, prevention of the development of AIDS symptoms in carriers should be stressed. Based on these in vitro studies, it is possible that such prevention may be realized by the use of HDPs such as lentinan or its related substances. For example, LEM may have potential in the treatment of AIDS. It has been shown to inhibit HIV infections of cultured human T-cells, and it potentiates the effects of AZT against viral replication in vitro. The mechanism of its action is not known for certain, but the extract was found to activate macrophages and stimulate the production of IL-1 (3,16,35,39). Lentin, a novel protein isolated from shiitake mushroom, exerted an inhibitory activity on HIV-1 reverse transcriptase (28). Water-soluble lignins EP3 and EPS4 from shiitake mushroom mycelium have shown antiviral and immunomodulating effects (40). A water-soluble extract of mycelium known as JLS and JLS-18 has the ability to block the release of herpes simplex virus type 1 in animals JLS-18consisting of 65% to 75% lignin, 15% to 30% polysaccharides, and 10 to 20% protein has inhibited the herpes virus both in vitro and in vivo (41). In addition, lentinan has shown (a) antiviral activity in mice against vesicular stomatitis virus encephalitis virus, Abelson virus, and adenovirus type 12; (b) stimulated nonspecific resistance against respiratory viral infections in mice; (c) conferred complete protection against an LD75 challenge dose of virulent mouse influenza

A/SW15; (d) enhanced bronchoalveolar macrophage activity; (e) increased resistance against the parasites Schistosoma japonicum and S. mansoni; (f) exhibited activity against Mycobacterium tuberculosis bacilli resistant to antituberculosis drugs, Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Candida albicans, and Saccharomyces cerevisiae; and (h) increased host resistance to infections with the potentially lethal Listeria monocytogenes. Antibacterial polyacetylene compounds, centinamycin A and B, have also been identified in shiitake mushroom. Eritadenine, a compound that affects cholesterol metabolism, also possesses antiviral properties (3,16,37). It should be noted that a protein fraction of shiitake mushroom fruiting bodies, labeled fruiting body protein (FBP), prevented the infection of plants with tobacco mosaic virus. The binding of the virus to the plant cells was inhibited by FBP (3,14,15).

Antifungal Activity From the fruiting bodies of the shiitake mushroom, a novel protein designated lentin with potent antifungal activity was isolated in 2003 (28). It was unadsorbed on DEAEcellulose and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of the protein manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelia growth in a variety of fungal species including Physalosporia piricola, Botrytis cinerea, and Mycosphaerella arachidicola (28).

Human Clinical Studies and Medicinal Uses In the last 15 to 20 years (2), shiitake mushroom has been subject to various clinical studies in humans and is thought to be beneficial for a wide variety of disorders including different types of cancer, heart disease, hyperlipidemia (including high blood cholesterol), hypertension, infectious disease, and hepatitis. The mushroom is used medicinally for diseases involving depressed immune function (including AIDS), cancer, environmental allergies, fungal (especially Candida) infection, frequent flu and colds, bronchial inflammation, and regulating urinary incontinence. It was shown that the success of immune adjuvant in therapy depends on the type of cancer (location) being treated, the individual’s general health, immunological and hormonal status as well as the individual’s constitution.

Cancer Lentinan was demonstrated to have antitumor activity as well as to increase the survival time of patients with inoperable gastric cancer (16) and women with recurrent breast cancer following surgical therapy (for details on protocols, see Refs. 3,16,18). When the polysaccharide is administered once or twice a week with chemotherapy to a patient with progressive cancer but with no serious liver, kidney, or bone marrow dysfunction, it produced a statistically significant improvement in immune and anticancer activity when compared to chemotherapy alone (42,43). Two hundred seventy-five patients with advanced or recurrent gastric cancer were given one of two kinds of chemotherapy (mitomycin C with 5-fluorouracil or tegafur) either alone or with lentinan injections. Statistically, the best results were obtained when lentinan was administered prior to chemotherapy and in patients with a basis

Shiitake

of prolongation of life, regression of tumors or lesions, and the improvement of immune responses. Lentinan was administered into malignant peritoneal and/or pleural effusions of a group of 16 patients with advanced cancer (44). Eighty percent of the lesions showed probable clinical responses, with an improvement in performance status demonstrated in seven subjects. The survival time for those who responded immunologically to the treatment was 129 days and 45 days for those who did not respond.

Viral Diseases LEM from shiitake mushroom has been shown boost the immune response in AIDS patients (3,16,18). When it was used to treat HIV-positive patients with AIDS symptoms, the T-cell count rose from a baseline of 1250/mm3 after 30 days up to 2550/mm3 after 60 days. An improvement in clinical symptoms was also noted. Although in vitro studies have indicated that lentinan and LEM from shiitake mushroom may be more effective than AZT in the treatment of AIDS (see discussions in the section on “Preclinical Studies”), it must be stressed that more clinical trials will be necessary to assess the long-term benefit of the these products for HIV and AIDS. Lentinan has shown favorable results in treating chronic persistent hepatitis and viral hepatitis B (16). Forty patients with chronic viral hepatitis B and seropositive for Hbe antigenemia were given 6 g of LEM daily (orally) for 4 months. The study focused on the number of patients seroconverting from Hbe antigen positive to antiHbe positive, which was 25% after LEM therapy, and was higher in patients with chronic active hepatitis (36.8%). In addition, 17 patients (43%) became seronegative for Hbe antigen. Liver function tests improved even for patients who remained seropositive, and they had raised plasma albumin, and adjusted protein metabolism.

Cardiovascular Disease Dried shiitake mushroom (9 g/day) decreased 7% to 10% serum cholesterol in patients suffering with hypercholesterolemia. For many patients 60 years of age or older with hyperlipidemia, consuming fresh shiitake mushroom (90 g/day in 7 days) led to a decrease in total cholesterol blood level by 9% to 12% and triglyceride level by 6% to 7% (16,35).

Toxicity and Side Effects Shiitake mushroom is edible, but some individuals may experience minor side effects or allergic reactions. Literature describes (3,16,18,45) cases of shiitake-induced toxicodermia and shiitake dermatitis. Allergic reactions to the spores of shiitake mushrooms have been reported in workers picking mushrooms indoors, who are prone to an immune reaction to spores called “mushroom worker’s lung.” Symptoms include fever, headache, congestion, coughing, sneezing, nausea, and general malaise (46). A water extract of the fruiting body was found (47) to decrease the effectiveness of blood platelets in initiating coagulation. So people who bleed easily or who take blood thinners should be closely monitored when under long-term treatment with shiitake mushroom or its watersoluble fractions.

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LEM has shown no evidence of being acutely toxic, even in massive doses of over 50 mg/day for 1 week, though mild side effects such as diarrhea and skin rash may occur. In this respect, the author does not consider these as massive doses. As a rule, symptoms disappear after a short period, when the body has adapted to the extract. Lentinan has no known serious side effects. However, in clinical trials of patients with advanced cancer, minor side reactions occurred such as a slight increase in glutamate–oxaloacetate transminase and GPT liver enzymes and a feeling of mild pressure on the chest. But these changes disappeared after lentinan administration was stopped (34).

Drug Interactions A watery extract of the whole fruiting body of L. edodes is reported to lessen the effectiveness of the blood platelets during the process of coagulation. People who bleed easily or who take blood thinners should use caution when chronically using L. edodes extracts in therapeutic amounts or in its water-soluble fractions (LEM) (16,47). For cancer patients, smaller doses of intravenous and intramuscular lentinan are more effective than larger ones (e.g., 1 mg per injection is considered safe, whereas 10 mg may produce marked depression in the host immune response). Aoki (34) notes that what is considered an excessive dosage intravenously may be a favorable dosage when using oral administration. For treating the initial stages of AIDS or chronic hepatitis, the best oral dose of LEM is between 2 and 6 g/day in two to three divided doses. If the disease is stable, the dosage may be decreased to 0.5 to 1 g/day (3,16).

REFERENCES 1. Hobbs Ch. Medicinal Mushrooms: An Exploration of Tradition, Healing, and Culture. 2nd ed. Santa Cruz, CA, USA: Botanica Press, Inc, 1995. 2. Singer R, Harris B. Mushrooms and Truffles: Botany, Cultivation, and Utilization. 2nd ed. Koenigstein: Koeltz Sci. Books, 1987. 3. Wasser SP, Weis AL. Medicinal Mushrooms. Lentinus edodes (Berk.) Singer. Haifa, Israel: Peledfus Publ. House, 1997:95. 4. Stamets P. Growing Gourmet and Medicinal Mushrooms. 3rd ed. Berkeley, CA: Ten Speed Press, 2000. 5. Miles PG, Chang ST. Mushroom Biology: Concise Basics and Current Development. Singapore: World Scientific, 1997:193. 6. Przbylowicz P, Donoghue J. Shiitake Grower’s Handbook: The Art and Science of Mushroom Cultivation. Dubugue, Kendall: Hunt Publ. Co, 1990:199. 7. Chang ST. World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk.) Sing. in China. Int J Med Mushr 1999; 1:387–409. 8. Royse D. Specialty mushrooms and their cultivation. Horticult Rev 1997; 19:59–97. 9. Pegler D. The classification of the genus Lentinus Fr. (Basidiomycota). Kavaka 1975; 3:11–20. 10. Earle FS. The genera of the North American gill-fungi. Bull N Y Bot Gard 1909; 5:373–451. 11. Murrill WA. Additions to Florida fungi. 1. Bull Torrey Bot Club 1939; 66:29–37. 12. Singer R. The Agaricales in Modern Taxonomy. 4th ed. Koenigstein, Germany: Koeltz Sci. Books, 1986.

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13. Pegler D. The genus Lentinula (Tricholomataceae tribe Collybiaeae). Sydowia 1983; 36:227–239. 14. Mizuno T. A development of antitumor polysaccharides from mushroom fungi. Food Food Ingred J Jpn 1996; 167:69– 85. 15. Mizuno T. Shiitake, Lentinus edodes: functional properties for medicinal and food purposes. Food Rev Int 1995; 11:7–21. 16. Hobbs Ch. Medicinal value of Lentinus edodes (Berk.) Sing. A literature review. Int J Med Mushr 2000; 2:287–302. 17. Wasser SP. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol 2002; 60:258–274. 18. Smith J, Rowan N, Sullivan R. Medicinal mushrooms. Their therapeutic properties and current medical usage with special emphasis on cancer treatment. Special Report Commissioned by Cancer Research UK. Glasgow, UK: The University of Strathclyde, 2002:1–256. 19. Yap AT, Ng ML. Immunopotentiating properties of lentinan (1–3)-␤-D-glucan extracted from culinary-medicinal shiitake mushroom Lentinus edodes (Berk.) Singer (Agaricomycetideae). Int J Med Mushr 2003; 5:352–372. 20. Chihara G, Maeda YY, Hamuro J, et al. Inhibition of mouse Sarcoma 180 by polysaccharides from Lentinus edodes (Berk.). Sing. Nature 1969; 222:687–688. 21. Chihara G, Hamuro J, Maeda YY, et al. Antitumor polysaccharide derived chemically from natural glucan (pachyman). Nature (London) 1970; 225:943–944. 22. Chihara G, Hamuro J, Maeda YY, et al. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom). Cancer Res 1970; 30:2776–2781. 23. Chihara G, Hamuro J, Maeda YY, et al. Antitumor and metastasis-inhibitory activities of lentinan as an immunomodulator: an overview. Cancer Detect Rev Supp 1987; 1:423–443. 24. Yap AT, Ng ML. An improved method for the isolation of lentinan from the edible and medicinal shiitake mushroom, Lentinus edodes (Berk.) Sing. (Agaricomycetideae). Int J Med Mushr 2001; 3:9–20. 25. Moriyama M, Fukuda Y, Ishizaki M, et al. Anti-tumor effect of polysaccharide lentinan on transplanted ascites hepatoma134 in C3 H/He mice. Manipulation of Host Defence Mechanisms; International Congress Series 576. Amsterdam: Excerpta Media, 1981. 26. Mori K, Toyomasu T, Nanba H, et al. Antitumor activity of fruit bodies of edible mushrooms orally administrated to mice. Mushr J Tropics 1987; 7:121–126. 27. White RW, DeVere Hackman RM, Soares SE, et al. Effect of a mushroom mycelium extract on the treatment of prostate cancer. Urology 2002; 60:640–644. 28. Ngai PHK, Ng TB. Lentin, a novel and potent antifungal protein from shiitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells. Life Sci 2003; 73:3363–3374. 29. Fujii T, Maeda H, Suzuki K, et al. Isolation and characterization of a new antitumor polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes. J Antibiot 1978; 31:1079– 1090. 30. Suzuki C. Killing activity of experimental tumor cells given to macrophage by new antitumor immunopotentiator, KS-2. Jap J Bact 1978; 33:78–85.

31. Kurashige S, Akusawa Y, Endo F. Effects of Lentinus edodes, Grifola frondosa, and Pleurotus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butylN-butanolnitrosoamine. Immunopharmacol Immunotoxicol 1997; 19:175–183. 32. Ladanyi A, Timar J, Lapis K. Effect of lentinan on macrophage cytotoxicity against metastatic tumour cells. J Cancer Immunol Immunother 1993; 36:123–126. 33. Yoshino S, Tabata T, Hazama S, et al. Immunoregulatory effects of the anitumour polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers. Anticancer Res 2000; 20:4707–4711. 34. Aoki T. Lentinan. Immune, modulation agents and their mechanisms. Immunol Study 1984; 25:62–77. 35. Yang BK, Kim DH, Jeong S, et al. Hypoglycemic effect of a Lentinus edodes exo-polymer produced from a submerged mycelial culture. Biosci Biotechnol Biochem 2002; 66:937– 942. 36. Amagase H. Treatment of hepatitis B patients with Lentinus edodes mycelium. Proceedings of the XII International Congress of Gastroenterology, Lisbon, 1987, p. 197. 37. Wasser SP, Weis AL. Medicinal properties of substances occurring in higher Basidiomycetes mushrooms: current perspectives (review). Int J Med Mushr 1999; 1:31– 62. 38. Suay I, Arenal F, Asensio FJ, et al. Screening of Basidiomycetes for antimicrobial activities. Antonie van Leeuwenhoek 2000; 78:129–139. 39. Tochikura TS, Nakashima H, Ohashi Y, Yamamoto N. Inhibition (in vitro) of replication and of the cytopathic effect of human immunodeficiency virus by an extract of the culture medium of Lentinus edodes mycelia. Med Microbiol Immunol 1988; 177:235–244. 40. Hanafusa T, Miyamoto T, Noguchi T, et al. Intestinal absorption and tissue distribution of immunoactive and antiviral water-soluble [14 C] lignins in rats (in Japanese). Yakubutsu Dotai 1990; 5:409–436. 41. Sarkar S, Koga J, Whitley RS, et al. Antiviral effect of the extract of culture medium of Lentinus edodes mycelia on the replication of herpes simplex virus type 1. Antiviral Res 1993; 20:293–303. 42. Taguchi T, Furue H, Kimura T, et al. Clinical trials on lentinan (polysaccharide). In: Rudent A, Zalesz R, Quero AM, eds. Immunomodulation by Microbial Products and Related Synthetic Compounds. New York: Elsevier Science, 1982: 467–475. 43. Taguchi T. Clinical efficacy of lentinan on patients with stomach cancer: end point results of a four-year follow-up survey. Cancer Detect Prev 1987; 1:333–349. 44. Oka M, Hazama S, Suzuki M, et al. Immunological analysis and clinical effects of intra-abdominal and intrapleural injection of lentinan for malignant ascites and pleural effusion. Biotherapy 1992; 5:107–112. 45. Ueda A, Obama K, Aoyama K, et al. Allergic contact dermatitis in shiitake (Lentinus edodes (Berk.) Sing.) growers. Contact Dermatitis 1992; 26:228–233. 46. Van Leon PC. Mushroom worker’s lung. Detection of antibodies against Shii-take (Lentinus edodes) spore antigens in Shii-take workers. J Occup Med 1992; 34:1097–1101. 47. Yang QY, Jong SC. Medicinal mushrooms in China. Mushr. Sci. 1989; XII:631–642.

St. John’s Wort Jerry M. Cott

INTRODUCTION St. John’s wort (SJW), Hypericum perforatum L. (Hypericaceae) is a perennial plant and one of the best known and well researched of the western herbals. Though clearly a favorite medicinal among herbalists and European physicians, concerns have arisen because of reports of drug interactions and lack of efficacy. SJW (Fig. 1) has been used for millennia for its many medicinal properties, including wound healing, treatment for kidney and lung ailments, insomnia, and depression. Current uses are primarily in treating central nervous system (CNS) indications such as depression, anxiety, and insomnia, but formulations are also available for other uses as well. Although the identity of the phytochemical constituents responsible for biological activity in humans remains unknown, the chemistry of the plant has been well studied. Constituents identified to date belong to several chemical classes and include hypericin, hyperforin, and various flavonoid glycosides.

Background SFW preparations have become increasingly popular in Germany where they are approved for use in the treatment of mild-to-moderate depression and have remained a first-line treatment for many years. The plant is named for its flowering time at the end of June, around the birthday of John the Baptist. Originally brought from Europe to North America, the plant can be found growing wild along roadsides and in fields and pastures where livestock poisoning, due to photosensitivity, was not uncommon. As already noted, current uses are primarily in treating CNS indications such as depression, anxiety, and insomnia. Oilbased preparations are used for stomach upsets and are also applied topically to treat bruises, muscle aches, and first-degree burns (1). A cream-based formulation was recently shown to be more effective than the vehicle for treating atopic dermatitis (2). In 2008, SJW was the eighth best-selling herbal supplement (at just over US$8 million) in the food, drug, and mass market retailers category in the United States, up 1.5% from 2007 (3). These sales totals are generally lower than those of previous years. Causal factors for reduced sales may include published reports of poor quality control (4), herb–drug interactions (see Table 4), and concern that SJW may be ineffective due to the publication of the clinical trial data that failed to show an effect in the Journal of the American Medical Association (5,6)

Figure 1

St. John’s wort. (Compliments of Peggy Duke.)

Chemistry and Preparation of Products SJW has long been known to contain red pigments that have been postulated to be the primary active constituent(s) in this plant genus, though there is little or no evidence for this assumption, other than a weak inhibition of MAO in vitro. These compounds are the naphthodianthrones hypericin and pseudohypericin (Fig. 2), and other derivatives that make up approximately 0.1% to 0.15% by dry weight. The plant also contains flavonoid glycosides (hyperoside, quercitrin, isoquercitrin, rutin), free flavonoids (quercetin, biapigenin) (2–4%), the biflavonoid, amentoflavone, and the phloroglucinols, primarily hyperforin (2–4%) (Fig. 3). The latter constituent is the one most often invoked as the “active” principle. Other

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OH

O

OH

OH

HO

CH3 HO

HO

CH3 HO

OH

Figure 2

O

O

OH

OH CH3

OH

OH

O

OH

The naphthodianthrones hypericin and pseudohypericin.

constituents include volatile oils, tannins (6.5–15%), and caffeic acid derivatives (7). Although originally believed to be a necessary component for antidepressant activity, hypericin is now considered primarily as a marker compound for purposes of botanical identification. It is responsible for the photosensitivity side effects that have long been known (8). The early SJW clinical studies from Germany examining antidepressant activity were based on extracts standardized to hypericin only (7). More recent research has suggested that hyperforin might also be important, but that this constituent may degrade (oxidize) under normal manufacturing conditions (9). Therefore, some companies began to stabilize their formulations (such as by the addition of ascorbic acid) to prevent oxidation and standardize the hyperforin content at 3% to 5% (9). The necessity for this was called into question when the relatively hyperforin-free (30 human clinical trials precludes the selection of a NOAEL or LOAEL, so a tolerable upper level of intake cannot be established. Instead, they conducted an observed safe level risk analysis and concluded that the evidence for the absence of adverse effects is strong for taurine at supplemental intakes up to 3 g/day. Taurine is a common ingredient in energy drinks in quantities ranging from 300 to 30,000 mg taurine/L (38,54,55) but the North American mean daily exposure is probably similar to the European exposure of 500 mg taurine/day from this source (38). With the increased consumption of energy drinks in the last two decades, there has been an associated increase in reports of adverse reactions such as cardiac dysrhythmia, seizure, kidney failure, and fatalities. Because most of the cases are associated with concomitant use of alcohol and/or physical activity, a causal relationship has not been proven (38,56). In reviewing adverse reaction reports, EFSA (38) considered caffeine as the causally linked ingredient because of its known effects, whereas a causal relationship with taurine intake lacks scientific evidence. Notably, BfR (German Federal Institute for Risk Assessment) (56) recommends a maximum taurine concentration of 4000 mg/L in caffeinated energy drinks, in which the maximum caffeine concentration is 320 mg/L, with cautionary risk statements for consumers with high BP or heart disease.

INTERNATIONAL REGULATORY STATUS In the United States, taurine has been authorized by the Food and Drug Administration for addition to purified human infant formulas since 1984 (33). It is also available in prescription-only mixed amino acid injections for peR diatric parenteral nutrition, for example, TrophAmine 6% (taurine 15 mg/100 mL) and 10% (taurine 25 mg/ R 100 mL) from B. Braun Medical Inc. (57), and Aminosyn PF 7% (taurine 50 mg/100 mL) and 10% (taurine 70 mg/ 100 mL) from Hospira Inc. (58). Taurine neither has generally regarded as safe (GRAS) status (59), nor is it an ingredient of approved drug products (60). The US FDA has taken compliance actions against dietary supplement manufacturers making therapeutic claims for products containing taurine (e.g. 61). In Canada, professional-use only (but nonprescription) mixed amino acid injections for pediatric parenteral nutrition are available with taurine, for example, R Aminosyn -PF 7% and 10% from Hospira Healthcare R Corp. and Primene 10% (taurine 60 mg/100 mL) from Baxter Corp. (62). Taurine is also a medicinal ingredient of

Taurine

at least 12 authorized natural health products, including capsules for supporting cardiovascular health, liver health and healthy blood glucose levels, powders for antioxidant and nutritional support, and liquid energy drinks with health claims (63). In Australia, taurine is a constituent of pediatric R mixed amino acid IV infusions, for example, Primene 10% from Baxter Healthcare Pty Ltd. (64) and is listed as an acceptable active ingredient or excipient for use in nonprescription listed medicines (65) to support healthy liver function, gall bladder function, blood lipid levels, fat digestion, cardiovascular function, electrolyte function, eye function, and as a dietary antioxidant (66). In the United Kingdom, the Medicines and Healthcare products Regulatory Agency (MHRA) has issued marketing authorizations for several prescription-only mixed amino acid injection products for parenteral nutrition, for example, Aminoven 3.5% (taurine 350 mg/L), 5% (taurine 500 mg/L), 10% (taurine 1000 mg/L), 15% (taurine 2000 mg/L), StructoKabiven (taurine 1000 mg/L), and Smofkabiven (taurine 1000 mg/L), all from Fresenius Kabi Ltd. (67). In the European Community, taurine is authorized for use in infant formulas at a level of at least 10 ␮mol/ 100 kJ (42 ␮mol/100 kcal) (68). Regarding taurine in energy drinks and other supplements, the EFSA Panel on Dietetic Products, Nutrition and Allergies (69) has reviewed and rejected evidence for health claims associated with taurine.

CONCLUSIONS Taurine is a ␤-aminosulfonic acid obtained from dietary sources such as meat and seafood and by biosynthesis from L-methionine and L-cysteine. It can become a conditionally essential nutrient when the dietary and precursor amino acid supplies are limited, biosynthetic enzymes or their cofactors are deficient, or loss is excessive, such as in cases of long-term parenteral nutrition or digestive malabsorption. Taurine is important in bile acid synthesis for fat digestion, but more recently, physiological and potential therapeutic roles have been identified in calcium homeostasis, osmoregulation, membrane stabilization, glucose metabolism, development of the central nervous system, eyes, and reproductive system, as an antioxidant/freeradical scavenger, and in treatment or risk reduction for heart disease, hypertension, inflammation, and immune system problems. To mitigate risks to human development from taurine deficiency in cases where it may be conditionally essential, taurine is authorized as an ingredient of pediatric mixed amino acid parenteral nutrition products. With respect to its use in dietary supplements, there is a plausible mechanism for potential benefits of taurine in energy drinks with respect to improvements in mood and physical endurance. At the levels commonly present in energy drinks and other dietary supplements, that is, up to 3 g/day, taurine generally appears to be safe.

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25. Merheb M, Daher RT, Nasrallah M, et al. Taurine intestinal absorption and renal excretion test in diabetic patients: a pilot study. Diabetes Care 2007; 30:2652–2654. 26. Winiarska K, Szymanski K, Gorniak P, et al. Hypoglycaemic, antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits. Biochimie 2009; 91:261–270. 27. Chang L, Xu J, Zhao J, et al. Taurine antagonized oxidative stress injury induced by homocysteine in rat vascular smooth muscle cells. Acta Pharmacol Sin 2004; 25:341–346. 28. Zhang M, Izumi I, Kagamimori S, et al. Role of taurine supplementation to prevent exercise-induced oxidative stress in healthy young men. Amino Acids 2004; 26:203–207. 29. Hu YH, Lin CL, Huang YW, et al. Dietary amino acid taurine ameliorates liver injury in chronic hepatitis patients. Amino Acids 2008; 35:469–473. 30. Jang JS, Piao S, Cha YN, et al. Taurine chloramine activates Nrf2, increases HO-1 expression and protects cells from death caused by hydrogen peroxide. J Clin Biochem Nutr 2009; 45:37–43. 31. Chan-Palay V, Lin CT, Palay S, et al. Taurine in the mammalian cerebellum: demonstration by autoradiography with [3H]taurine and immunocytochemistry with antibodies against the taurine-synthesizing enzyme, cysteine-sulfinic acid decarboxylase. Proc. Natl Acad Sci U.S.A 1982; 79:2695– 2699. 32. Reeds PJ. Dispensable and indispensable amino acids for humans. J Nutr 2000; 130:1835S–1840S. 33. Sturman JA. Taurine in development. Physiol Rev 1993; 73:119–147. 34. Shao A, Hathcock JN. Risk assessment for the amino acids taurine, L-glutamine and L-arginine. Regul Toxicol Pharmacol 2008; 50:376–399. 35. Gaull GE. Taurine in pediatric nutrition: review and update. Pediatrics 1989, 83(3):433–442. 36. van de Poll MC, Dejong CH, Soeters PB. Adequate range for sulfur-containing amino acids and biomarkers for their excess: lessons from enteral and parenteral nutrition. J Nutr 2006; 136:1694S–1700S. 37. Hayes KC, Trautwein EA. Taurine. In: Shils ME, Olson JA, Shike M, eds. Modern Nutrition in Health and Disease. Vol. 1. 8th ed. Philadelphia, PA: Lea & Febiger, 1994; 477– 485. 38. European Food Safety Authority (EFSA) Panel on Food Additives and Nutrient Sources added to Food. Scientific opinion of the panel on food additives and nutrient sources added to food on a request from the commission on the use of taurine and D-glucurono-␥ -lactone as constituents of the socalled “energy” drinks. EFSA J 2009; 935:1–31. 39. Brosnan JT, Brosnan ME. The sulfur-containing amino acids: an overview. J Nutr 2006; 136:1636S–1640S. 40. Sturman JA, Hepner GW, Hofmann AF, et al. Metabolism of [35S]taurine in man. J Nutr 1975; 105:1206–1214. 41. Meltzer JS, Moitra VK. The nutritional and metabolic support of heart failure in the intensive care unit. Curr Opin Clin Nutr Metab Care 2008; 11:140–146. 42. Mochizuki H, Oda H, Yokogoshi H. Dietary taurine potentiates polychlorinated biphenyl-induced hypercholesterolemia in rats. J Nutr Biochem 2001, 12(2):109– 115. 43. Mochizuki H, Takido J, Oda H, et al. Improving effect of dietary taurine on marked hypercholesterolemia induced by a high-cholesterol diet in streptozotocin-induced diabetic rats. Biosci Biotechnol Biochem 1999; 63:1984–1987. 44. Fennessy FM, Moneley DS, Wang JH, et al. Taurine and vitamin C modify monocyte and endothelial dysfunction in young smokers. Circulation 2003; 107:410–415. 45. Militante JD, Lombardini JB. Treatment of hypertension with oral taurine: experimental and clinical studies. Amino Acids 2002; 23:387–393.

46. Satoh H, Kang J. Modulation by taurine of human arterial stiffness and wave reflection. Adv Exper Med Biol 2009; 643:47–55. 47. Klamt F, Zdanov S, Levine RL, et al. Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin. Nat Cell Biol 2009; 11:1241–1246. 48. Franconi F, Bennardini F, Mattana A, et al. Plasma and platelet taurine are reduced in subjects with insulindependent diabetes mellitus: effects of taurine supplementation. Am J Clin Nutr 1995; 61:1115–1119. 49. Brøns C, Spohr C, Storgaard H, et al. Effect of taurine treatment on insulin secretion and action, and on serum lipid levels in overweight men with a genetic predisposition for type II diabetes mellitus. Eur J Clin Nutr 2004; 58:1239–1247. 50. Stevens MJ. Oxidative-nitrosative stress as a contributing factor to cardiovascular disease in subjects with diabetes. Curr Vasc Pharmacol 2005; 3:253–266. 51. Seidl R, Peyrl A, Nicham R, et al. A taurine and caffeinecontaining drink stimulates cognitive performance and wellbeing. Amino Acids 2000; 19:635–642. 52. Warburton DM, Bersellini E, Sweeney E. An evaluation of a caffeinated taurine drink on mood, memory and information processing in healthy volunteers without caffeine abstinence. Psychopharmacology 2001; 158:322–328. 53. Alford C, Cox H, Wescott R. The effects of Red Bull Energy Drink on human performance and mood. Amino Acids 2001; 21:139–150. 54. Clauson KA, Shields KM, McQueen CE, et al. Safety issues associated with commercially available energy drinks. J Am Pharmacists Assoc 2008; 48:e55–e67. 55. Sawabe Y, Tagami T, Yamasaki K. Determination of taurine in energy drinks by HPLC using a pre-column derivative. J Health Sci 2008; 54:661–664. 56. BfR (German Federal Institute for Risk Assessment). New human data on the assessment of energy drinks, Information No. 016/2008, 13 March 2008. http://www.bfr .bund.de/cm/ 245/new human data on the assessment of energy drinks.pdf. Accessed December 31, 2009. R Fact Sheet. 2009. http:// 57. B. Braun Medical Inc.Trophamine www.bbraunusa.com/indexFBCE0DF865B05CD0D2CB7276494C3E4B.cfm?uuid= FBCE0DF865B05CD0D2CB7276494C3E4B. Accessed January 6, 2010. R PF 7%: An amino acid injection 58. Hospira Inc. Aminosyn  pediatric formula. 2006. http://www.hospira.com/Files/ TPN Aminosyn PF 7.pdf. Accessed January 6, 2010. 59. U.S. FDA: Food and Drug Administration. Generally Recognized as Safe (GRAS). 2010. http://www.fda.gov/Food/ FoodIngredientsPackaging/GenerallyRecognizedasSafe GRAS/default.htm. Accessed January 6, 2010. 60. U.S. FDA: Food and Drug Administration. Orange Book: Approved Drug Products with Therapeutic Equivalence Evaluations. 2009. http://www.fda.gov/Drugs/InformationOn Drugs/ucm129689.htm. Accessed January 6, 2010. 61. U.S. FDA: Food and Drug Administration. Inspections, Compliance, Enforcement, and Criminal Investigations. http://www.fda.gov/ICECI/EnforcementActions/Warning Letters/2006/ucm075961.htm. Accessed January 6, 2010. 62. Health Canada. Drug Product Database. 2010a. http:// www.hc-sc.gc.ca/dhp-mps/prodpharma/databasdon/ index-eng.php/. Accessed January 6, 2010. 63. Health Canada. Licensed Natural Health Products Database. 2010b. http://webprod.hc-sc.gc.ca/lnhpd-bdpsnh/startdebuter.do?language-langage=english. Accessed January 6, 2010. 64. Baxter Healthcare Pty Ltd. Primene 10% Amino Acids Intravenous (IV) Consumer Medicine Information. 2002. http:// www.baxterhealthcare.com.au/downloads/products/ cmi/pimene.pdf. Accessed January 6, 2010.

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65. Australian TGA: Therapeutic Goods Administration. Substances That May Be Used in Listed Medicines in Australia. 2007. http://www.tga.gov.au/cm/listsubs.htm. Accessed January 6, 2010. 66. Australian TGA: Therapeutic Goods Administration. eBS Australian Register of Therapeutic Goods – Medicines. https://www.ebs.tga.gov.au/ebs/ANZTPAR/PublicWeb. nsf/cuMedicines?OpenView. Accessed January 6, 2010. 67. U.K. MHRA: Medicines and Healthcare products Regulatory Agency. Marketing Authorizations. http://www.mhra.gov. uk/SearchHelp/Search/Searchresults/index.htm?within= Yes&keywords=taurine. Accessed January 6, 2010. 68. European Commission. Commission Directive of 14 May 1991 on infant formulae and follow-on formulae

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(91/321/EEC) consolidated with amendments to 22.06.1999. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri= CONSLEG:1991L0321:19990622:EN:PDF. Accessed January 7, 2010. 69. EFSA: European Food Safety Authority Panel on Dietetic Products, Nutrition and Allergies. Scientific Opinion on the substantiation of health claims related to taurine and protection of DNA, proteins and lipids from oxidative damage (ID 612, 1658, 1959), energy-yielding metabolism (ID 614), and delay in the onset of fatigue and enhancement of physical performance (ID 1660) pursuant to Article 13(1) of Regulation (EC) No 1924/2006 on request from the European Commission. EFSA Journal 2009; 7(9):1260. [17 pp.]. doi:10.2903/j.efsa.2009.1260. http://www.efsa.europa.eu. Accessed January 6, 2009.

Thiamin Hamid M. Said

ABBREVIATIONS NH2

RDA, recommended dietary allowances; THTR-1, thiamin transporter 1; THTR-2, thiamin transporter 2; TMP, thiamin monophosphate; TPP, thiamin pyrophosphate; TRMA, thiamin responsive megaloblastic anemia; TTP, thiamin triphosphate

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INTRODUCTION

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Thiamin (vitamin B1 ) was the first member of the watersoluble family of vitamins to be described. Reference to beriberi (a thiamin-deficiency disease) in Chinese medical literatures dates back to as early as 2700 BC. Thiamin plays an essential role in normal cellular functions, growth, and development via its involvement in critical metabolic reactions related to energy metabolism. Furthermore, this vitamin also plays a role in reducing cellular oxidative stress (1,2). Thus, low intracellular levels of thiamin lead to impairment in energy metabolism and to a propensity for oxidative stress. In addition, deficiency in intracellular thiamin level leads to apoptosis (3,4). Clinically, thiamin deficiency and suboptimal levels (which represent a significant nutritional problem) lead to a variety of abnormalities including neurological and cardiovascular disorders. On the other hand, optimization of thiamin level may be of help in the treatment of diabetic nephropathy and retinopathy (5,6). It is also effective in the treatment of many of the clinical symptoms associated with congenital disorders of thiamin metabolism and physiology. Thus sufficient intake of thiamin (from dietary or supplemental sources) is important for maintaining proper health and well-being as well as in preventing clinical abnormalities.

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Thiamin Thiamin monophosphate (TMP) Thiamin pyrophosphate (TPP) Thiamin triphosphate (TTP)

Figure 1 Structure of free thiamin (mol. wt. 337.27) and that of thiamin monophosphate. Abbreviations: TMP, thiamin diphosphate; TPP, thiamin pyrophosphate; TTP, thiamin triphosphate.

stable (with the latter derivative being less hygroscopic than the former) and are commonly used to enrich food products and in pharmaceutical preparations. Thiamin is susceptible to destruction by X-rays, gamma rays, and UV irradiation. Allithiamins are thiamin derivatives that are produced by oxidative cleavage of the thiazole ring in alkaline solutions. These compounds are biologically active and spontaneously dehydrate as a result of reductive cleavage of their disulfide bridge to regenerate thiamin. A number of allithiamins exist in plants (e.g., in members of the genus Allium), and synthetic allithiamins have also been generated (e.g., thiamin propyldisulfide, thiamin tetrahydrofurfuryldisulfide, O-benzoylthiamin disulfide, and S-benzoylthiamin-O-monophosphate). Due to their lipid solubility (which allows them to cross the intestinal epithelium easily), these derivatives are used to treat thiamin deficiency.

Structure of Thiamin and Derivatives The thiamin molecule is composed of a pyrimidine and a thiazole ring that are joined by a methylene bridge (Fig. 1). The alcohol group of the side chain of the thiamin molecule can be enzymatically phosphorylated with up to three phosphate moieties resulting in the formation of thiamin-monophosphate (TMP), -diphosphate (also called thiamin pyrophosphate; TPP), and -triphosphate (TTP) (Fig. 1). The original name used for thiamin was aneurine because of its function in preventing and curing polyneuritis in chicken that were deficient in this vitamin due to their feeding of polished rice. Following the discovery of its structure and synthesis, the name was changed to thiamin. The free base form of thiamin is unstable but its hydrochloride and mononitrate derivatives are both

THIAMIN ANTAGONISTS AND THIAMINASES Sulfites, which are widely used as preservative in food, attack thiamin at the methylene bridge, especially at acidic pH, leading to cleavage of the molecule into pyrimidine and thiazole rings. Also a number of heat-stable polyhydroxyphenolic compounds that exist in food (like ferns, 748

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THIAMIN CONTENT AND METABOLIC FORMS In adult humans, total thiamin content is estimated to be around 30 mg, and the biological half-life of the vitamin is between 10 and 20 days. Thiamin in tissues exists in the free form as well as in the form of TMP, TPP, and TTP. TPP is the predominant form of thiamin in mammalian tissues (approx. 80% of total thiamin), whereas free thiamin and TMP are the predominant forms in the plasma. TPP is synthesized from free thiamin by the action of pyrophosphokinase, whereas TMP arises mainly from sequential hydrolysis of TTP and TPP (Fig. 2). Total thiamin concentration in human whole blood is in the range of 0.1 to 0.2 ␮M (9) and it is unevenly distributed among different cell types/compartments (15% in leucocytes, 75% in erythrocytes, 10% in plasma). In the cerebrospinal fluid (CSF), thiamin exists in the free and TMP forms only (10,11). In the urine, free thiamin together with small amounts of TMP, TPP, and a number of thiamin catabolites have been found.

Metabolic Role of Thiamin Thiamin plays an essential role in a variety of cellular functions. Thiamin pyrophosphate is the predominant metabolically active form of the vitamin, although recent studies have reported additional functions for thiamin’s other derivatives. TPP acts as a coenzyme for five different enzymes involved in carbohydrate (energy) and lipid metabolism. Three of these enzymes are mitochondrial, one is cytoplasmic, and one is peroxisomal. The mitochondrial enzymes (dehydrogenases, which exist in multienzyme complexes) are involved in carbohydrate and lipid metabolism. The cytoplasmic (transketolase) enzyme is involved in the pentose phosphate cycle that supplies pentose phosphate to a variety of reactions including those

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tea leaves, blueberry, red chicory, red beetroot, black currant, red beetroot, Brussels sprouts, and red cabbage) can cleave the thiamin molecule. The latter reaction can be prevented by reducing agents such as ascorbate. Furthermore, a number of thiamin structural analogues (amprolium, oxythiamin, pyrithiamin, and chloroethylthiamin) have been chemically synthesized for experimental purposes to antagonize thiamin at the level of metabolism and/or transport. An inhibitor of thiamin synthesis has also been reported in germs (7). Thiaminolytic enzymes (i.e., enzymes that degrade thiamin) are found in a variety of microorganisms and food. Two such enzymes are known: thiaminase I and II. Thiaminase I is relatively widely spread in a variety of microorganisms (e.g., Bacillus thiaminolyticus), plants [e.g., fern, fish (e.g., carp), and insects (e.g., African silkworm Anaphe spp.). It catalyzes a base-exchange reaction between the thiazole moiety of the thiamin molecule and a variety of bases. In addition to depleting thiamin, the by-products of the latter reaction may also act as thiamin antagonists (8). Thiaminase II, which hydrolyzes thiamin into thiazole and methoxypyrimidine, is relatively rare with existence being limited to a small number of microorganisms (mainly intestinal bacteria like Bacillus thiaminolyticus and Clostridium thiaminolyticum).

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Figure 2 Diagrammatic representation of thiamin interconversion to its phosphate derivatives TMP, TPP, and TTP in mammalian cells.

involved in the synthesis of nucleotides and fatty acids as well as steroid hydroxylation. The peroxisomal enzyme (lyase) is involved in the metabolism of branched chain fatty acids. Because thiamin bridges the glycolytic and the pentose phosphate metabolic pathway, which is critical for creating chemical reducing power in cells, the vitamin is also considered to have an important role in reducing cellular oxidative stress (1,2). In the nervous system, development of neurological abnormalities in thiamin deficiency do not follow the pattern of development of impairments in the function of thiamin-dependent enzymes (pyruvate and 2-oxoglutarate dehydrogenase and transketolase). Therefore, additional functions for thiamin in the nervous system besides its role as a coenzyme have also been suggested. Other studies have shown that TTP, which can be synthesized in nerve cells, plays a role in electrical conduction in these cells. More recent investigations have reported a role for TPP in regulating the function of membrane chloride channels in nerve cells (12,13) and as a phosphate group donor to other membrane proteins (14).

Physiology of Thiamin Intestinal Absorption of Thiamin Humans and all other mammals cannot synthesize thiamin, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. The human intestine is exposed to two sources of thiamin: a dietary source, and a bacterial source (i.e., the normal microflora of the large intestine). Dietary thiamin exists mainly in the phosphorylated form, which is hydrolyzed by intestinal phosphatases to free thiamin prior to absorption (reviewed in Ref. 15). Absorption of free thiamin (which exists in the monocationic form at pH 5–7.4) then takes place predominately in the proximal half of the small intestine and involves a specialized carrier-mediated process at both the apical and basolateral membrane domains of the polarized absorptive epithelial cells (reviewed in Ref. 15). It is believed that the positively charged thiamin crosses the

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cromolar concentration) of thiamin: thiamin transporter-1 and -2 (THTR-1 and THTR-2). This was established recently in studies using THTR-1 and THTR-2 knockout mice and by studies with cultured human intestinal epithelial cells utilizing gene-silencing approach with genespecific siRNA (16,22). Absorption of high (pharmacological; >20 ␮M) concentrations of thiamin occurs mainly by simple diffusion. Studies using human and animal intestinal preparations have shown that the thiamin absorption process is adaptively regulated by the level of the vitamin in the diet, with significant up-regulation occurring in thiamin deficiency (23,24). The mechanism involved in this upregulation (at least in mice) appears to be transcriptionally mediated and affects THTR-2 only (23). Thiamin uptake by human intestinal epithelial cells also appears to be regulated by an intracellular Ca2+ -calmodulin -mediated signaling pathway (reviewed in Ref. 15).

Transport of Thiamin in Renal and Other Epithelia ?

THTR-1

Figure 3 Diagrammatic representation of our current understanding of thiamin transport across mammalian intestinal (and possibly other) epithelial cells. There is a reason to speculate the existence of transport system that can carry thiamin out of the enterocyte in the absence of functional THTR-1. This suggestion is based on the fact that intestinal thiamin uptake and plasma thiamin level are normal in THTR-1 deficient mice (16). A similar scenario may exist in TRMA patients who lack functional THTR-1, yet display normal plasma thiamin level. The compensatory induction in THTR-2 expression in the intestine of THTR-1 deficient mice may explain why transport across the brush border membrane proceeds normally.

intestinal cell membrane in exchange for H+ (Fig. 3). The diuretic amiloride was found to inhibit intestinal thiamin uptake (as well as thiamin uptake by a number of other epithelia). Some of the absorbed thiamin is converted into the phosphorylated forms of the vitamin (mainly to thiamin pyrophosphate) in intestinal epithelial cells, but only free thiamin exits the intestinal absorptive cells (reviewed in Ref. 15). As to the bacterial source of thiamin, previous studies have shown that the normal microflora of the large intestine synthesize considerable amounts of thiamin and that up to 50% of this thiamin exists in the free, that is, absorbable form (17–19). In addition, the large intestine of humans and rats is capable of absorbing thiamin from their lumens (19,20). Studies using human-derived colonic epithelial cells have shown the existence of an efficient carrier-mediated process for thiamin uptake in the large intestine (21). The latter findings suggest that bacterially synthesized thiamin in the large intestine may contribute to thiamin nutrition of the host, especially toward cellular nutrition of the local colonocytes. Further studies are needed to determine the exact level of contribution of this source toward overall host nutrition and the effect of dietary and environmental factors on this process. Two transport systems appear to be involved in intestinal absorption of physiologic levels (nano- and mi-

Normal levels of thiamin compounds in human serum averages 13, 7.1, and 3.8 nM for free thiamin, TMP, and TPP, respectively (9). Filtered thiamin in the renal glomeruli is reabsorbed by proximal renal tubular epithelial cells to prevent loss in the urine. Thus, the kidneys also play an important role in maintaining and regulating thiamin body homeostasis. Thiamin uptake by human and animal renal epithelial cells also occurs via a specialized carriermediated process that involves both THTR-1 and -2. Furthermore, studies with human renal epithelial cells and in mice have shown that the renal thiamin uptake process is adaptively upregulated in thiamin deficiency (to further minimize losses) via a transcriptionally mediated mechanism that involves both THTR-1 and THTR-2 (23,25). In the placenta, thiamin transport is again carrier mediated. The vitamin travels preferentially from the mother to the fetus, and not in the other direction. In the umbilical cord, thiamin plasma level is some 2.5-fold higher than its level in maternal plasma, and its concentration in cord vein is significantly higher than its concentration in the umbilical artery indicating significant retention of the vitamin by the fetus. Transport of thiamin across the blood–brain barrier is similarly carrier-mediated in nature.

Uptake of Thiamin by Pancreatic ␤-Cells and Islets Thiamin is important for both the exocrine and endocrine functions of the pancreas (26–28). Thiamin deficiency in rats leads to a severe reduction in pancreatic content of digestive enzymes and to a marked impairment in insulin synthesis and secretion (26–28). Of relevance to the latter is the development of diabetes mellitus in patients with thiamin-responsive megaloblastic anemia (TRMA; Rogers syndrome) a condition caused by mutation in THTR-1 (see later) (29,30). Supplementing TRMA patients with high doses of thiamin brings about a marked improvement in the clinical symptoms of the disease including a reduction or cessation in the need for exogenous insulin (29,30). The molecular mechanisms that tie thiamin to insulin synthesis and secretion await further investigations. Recent studies have delineated the mechanism of thiamin uptake by human and mouse pancreatic islets and ␤-cells and established the involvement of a carrier-mediated process (31).

Thiamin

Both THTR-1 and THTR-2 are expressed in these cells with expression of the former being significantly higher than that of the latter (31). As pancreatic ␤-cells are a major pathological target of TRMA, the pattern of expression and functionality of clinically relevant mutants of hTHTR1 were also investigated with results showing a spectrum of expression phenotypes. Certain mutants were found to be expressed at cell membrane, whereas others were either retained intracellularly or expressed at the cell membrane but with lower efficiency than wild-type hTHTR-1. However, all of the clinical mutants examined were dysfunctional in pancreatic ␤-cells (31). The thiamin uptake process of pancreatic ␤-cells was again found to be adaptively regulated by the prevailing thiamin level with higher uptake occurring by cells maintained in the presence of low compared with high thiamin levels (31). This was associated with a markedly higher level of expression of THTR-1 and THTR-2 at the protein and mRNA level, as well as higher transcription rate of their respective genes. The response of THTR-1 to changes in thiamin level, however, was markedly more pronounced when compared with THTR-2, a finding that could explain why these cells are the pathological target in TRMA. This is most likely due to the fact that THTR-1 is the predominant thiamin transporter in these cells and that dysfunction in this transporter leads to impairment in the ability of these cells to acquire sufficient amount of thiamin. With the limited capability of the cells to upregulate the other thiamin transporter, that is, THTR-2, this will lead to the development of a state of intracellular thiamin deficiency. This will in turn result in disturbances in intracellular metabolism, oxidative stress, and apoptosis.

Sources and Recommended Dietary Allowances of Thiamin Thiamin is widely distributed in foods with rice bran, dried baker’s yeast, whole grain cereal, nuts, and dried legumes being rich sources for this vitamin, whereas highly refined foods like polished rice, oils, refined sugar being poor (or very poor) sources. Thiamin of animal origin exists mostly in the phosphorylated form, whereas that of plant origin could be a mixture of free and phosphorylated forms. The recommended daily allowances (RDAs) for thiamin are 1.4, 1.1, 1.5, 1.6 mg/day for men, women, and for women during pregnancy and lactation, respectively. Because requirement for thiamin relates to the total caloric intake (especially that from carbohydrate), consumption of an unbalanced (calorie rich) diet may change the RDAs.

Assessment of Thiamin Status Three methods are available for the assessment of thiamin status in humans. The first involves determination of transketolase activity in hemolyzed erythrocytes. The second involves determination of urinary excretion before and after administration of a 5-mg dose of thiamin. The third method involves determination of thiamin concentration in whole blood and in erythrocytes. The latter method is probably the current method of choice.

Thiamin Deficiency Thiamin deficiency and suboptimal levels represent significant nutritional problems in both underdevel-

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oped/developing and developed countries. In underdeveloped and developing countries, the main cause of thiamin deficiency is poor dietary intake of the vitamin (i.e., consumption of thiamin deficient/depleted diets). In the developed countries, chronic alcoholism is probably the main cause of thiamin deficiency. However, thiamin deficiency and suboptimal levels also occur in other conditions as in patients with diabetes mellitus (32), inflammatory bowel disease, celiac disease, renal diseases, AIDS, cancer, and those with congestive heart failure, as well as in subjects on chronic diuretic therapy. In addition, thiamin deficiency and suboptimal levels have been reported in the elderly despite an average daily intake that exceeds their recommended requirement (33). Chronic thiamin deficiency leads to two distinct types of conditions: Beriberi and Wernicke’s encephalopathy. Beriberi is recognized in three different forms. The first form is dry beriberi, which is a symmetrical ascending peripheral neuritis that usually affects older individuals and is associated with wasting; also, it may or may not be associated with cardiac involvement. The second form is wet (or edematous) beriberi, which involves the heart and leads to edema of lower extremities resulting from the ensuing heart failure. The third form is the acute “fulminating” beriberi (which is also called shoshin beriberi) which occurs more frequently in infants and is associated with heart failure and metabolic abnormalities with little evidence of peripheral neuritis. Deficiency of thiamin in the human central nervous system may lead to Wernicke’s encephalopathy and Korsakoff’s psychosis. These conditions are associated with chronic alcoholism and manifest as the Wernicke– Korsakoff syndrome. Some evidence, however, exists to suggest that thiamin deficiency alone is not sufficient to cause Wernicke–Korsakoff syndrome but that alcohol is a necessary factor for the induction of this abnormality (34). Korsakoff’s psychosis is associated with confusion and loss of recent memory, although long-term memory may continue to be intact. Wernicke’s encephalopathy develops later and is associated with clear neurological abnormalities (nystagmus, extraocular palsy, ataxia, confabulation, and coma) and anatomic lesions (hemorrhagic lesions in the thalamus pontine tegmentum, and mammillary body with severe damage to astrocytes, neuronal dendrites and myelin sheaths).

Congenital Disorders Congenital defects in thiamin physiology and metabolism also occur in human. These defects include thiaminresponsive megaloblastic anemia (TRMA), maple syrup urine disease (branched-chain disease), Leigh’s disease, and lactic acidosis. TRMA (also known as Roger’s syndrome) is an autosomal recessive disorder caused by mutations in thiamin transporter-1, THTR-1 (29,30). This rare disease of infancy and childhood is characterized by megaloblastic anemia, diabetes mellitus, and sensorineural deafness; optic and cardiac abnormalities may also occur. Oral pharmacological doses of thiamin are effective in resolving the anemia, decreasing or eliminating insulin requirements, and arresting hearing loss. A recessively inherited syndrome similar to Wernicke’s encephalopathy that develops in the second

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decade of life has recently been described (35). The cause of this condition is mutations in thiamin transporter-2, THTR-2. The disease is associated with seizures, ophthalmoplegia, nystagmus, and ataxia and responds to treatment with pharmacological doses of thiamin (35). Lack of the enzyme branched-chain ␣-oxoacid dehydrogenase complex is the cause of maple syrup urine disease. Urine of the affected patients (infants) smells like maple syrup, due to the presence of high concentration of ␣-oxoacid. The disease is characterized by acidosis and seizures in the early neonatal period with some patients responding to daily high pharmacological doses of thiamin. Other diseases that develop during infancy and early childhood and respond to high pharmacological doses of thiamin are Leigh’s disease (subacute necrotizing encephalomyelopathy) and congenital lactate acidosis. Leigh’s disease is associated with weakness, anorexia, difficulties in speech and eye motion, as well as growth delay. Congenital lactic acidosis is characterized by lactic and pyruvate acidosis, neurological abnormalities, and development delay. The condition is believed to be due to a defect in the pyruvate dehydrogenase complex (35).

sources (diet and normal microflora of the large intestine). Thiamin deficiency and suboptimal levels represent significant nutritional problems and can occur due to a variety of conditions. Significant progress has been made in recent years in understanding the molecular aspects of thiamin physiology and metabolism. However, much more work is needed to fully understand the details of thiamin molecular nutrition. Studies are needed to uncover the association between thiamin level, insulin synthesis and secretion, and diabetes mellitus. Further molecular studies are needed to delineate the role of thiamin transporters in regulating cellular homeostasis in different tissues, and uncovering the function of thiamin derivatives other than TPP in the nervous system and other organs in health and disease. Finally, the contribution of the normal microflora of the large intestine toward host thiamin nutrition, and strategies to further enhance this source need to be more fully investigated.

ACKNOWLEDGMENTS We thank the NIH (DK-56061, AA-018071) and the DVA for their kind support of our work.

TOXICITY Even very high oral doses of thiamin (up to 500 mg) are well tolerated in human with no toxic effect. Also large parenterally administered thiamin (single or repeat injections) are generally well tolerated. In very rare instances, however, symptoms resembling anaphylactic shock and minor allergy have been described after parenteral administration. Despite lack of toxicity of high doses of thiamin, long-term use of such doses in normal individuals may potentially have an unintended negative effect. This relates to recent findings in mice and human epithelial cells showing downregulation of intestinal and renal thiamin uptake processes following long-term exposure to high levels of the vitamin (23,25). If the same happens in normal humans in vivo and such individuals experience serious acute illnesses that lead to abrupt cessation of food intake, this downregulation may lead to precipitous depletion of this important essential micronutrient at a time when its adequate supply is critical to meet the heightened metabolic demands. Notable among such cases are persons experiencing catastrophic accidents, stroke, acute gastrointestinal disorders (e.g., obstruction, infarction, severe gastroenteritis, acute abdominal events, etc), and fulminant infections. In such cases parenteral administration of thiamin may be considered to avoid precipitous development of a serious deficiency state. Further studies, however, are needed to examine whether such a scenario occurs and to determine the time frame required to restore normal intestinal and renal thiamin uptake in such individuals.

CONCLUSIONS The water-soluble vitamin thiamin is essential for normal cellular function, growth, and development. Humans (and other mammals) obtain the vitamin from exogenous

REFERENCES 1. Calingasan NY, Gandy SE, Baker H, et al. Noval neuritic clusters with accumulations of amyloid precursor protein and amyloid precursor-like protein 2 immunoreactivity in brain regions, damaged by thiamin deficiency. Am J Pathol 1996; 149(3):1063–1071. 2. Frederikse PH, Farnsworth P, Zigler JS Jr. Thiamin deficiency in vivo produces fiber cell degeneration in mouse lenses. Biochem Biophys Res Commun 1999; 258(3):703–707. 3. Matsushima K, MacManus JP, Hakim AM. Apoptosis is restricted to the thalamus in thiamin-deficient rats. Neuroreport 1997; 8(4):867–870. 4. Stagg AR, Fleming JC, Baker MA, et al. Defective highaffinity thiamin transporters leads to cell death in thiaminresponsive megaloblastic anemia syndrome fibroblasts. J Clin Invest 1999; 103(5):723–729. 5. Hammes HP, Du X, Edelstein D, et al. Benfotiamine blocks three major pathways of hyperglycemia damage and prevents experimental diabetic retinopathy. Nat Med 2003; 9(3):294–299. 6. Rabbani N, Alam SS, Riaz S, et al. High-dose thiamine therapy for patients with type 2 diabetes and microalbuminuria: a randomized double-blind placebo-controlled pilot study. Diabetologia 2009; 52(2):208–212. 7. Reddick JJ, Saha S, Lee J, et al. The mechanism of bacimethrin, a naturally occurring thiamin antimetabolite. Bioorg Med Chem Lett 2001; 11(17):2245–2248. 8. Edwin EE, Jackman R. Thiaminase I in the development cerebrocortical necrosis in sheep and cattle. Nature 1970; 228(1):772–774. 9. Tallaksen CM, Bohmer T, Bell H. Blood and serum thiamin and thiamin phosphate esters concentrations in patients with alcohol dependence syndrome before and after thiamin treatment. Alcohol Clin Exp Res 1992; 16(2):320–325. 10. Tallaksen CME, Bomer T, Karlsen J, et al. Determination of thiamin and its phosphate esters in human blood, plasma and urine. Methods Enzymol 1997; 279: 67–74. 11. Rindi G, Patrini C, Poloni M. Monophosphate, the only phosphoric ester of thiamin in the cerebrospinal fluid. Experientia 1981; 37(9):975–976.

Thiamin

12. Bettendorff L. A non-cofactor role of thiamin derivatives in excitable cells. Arch Physiol Biochem 1996; 104(6): 745–751. 13. Bettendorff L, Hennuy B, De Clerck A, et al. Chloride permeability of rat brain membrane vesicles correlates with thiamin triphosphate content. Brain Res 1994; 652(1):157–160. 14. Nghiem HO, Bettendorff L, Changeux JP. Specific phosphorylation of Torpedo 43 K rapsyn by endogenous kinase(s) with thiamin triphosphate as the phosphate donor. FASEB J 2000; 14(1):543–554. 15. Said HM, Seetharam B. Intestinal absorption of water-soluble vitamins. In: Johnson LR, Barrett KE, Ghishan FK, Merchant JM, Said HM, Wood JD. eds. Physiology of the Gastrointestinal Tract. Vol. 2. 4th ed. New York: Academic Press, 2006:1791–1826. 16. Said HM, Balamurugan K, Subramanian VS, et al. Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine. Am J Physiol Gastrointest Liver Physiol. 2004; 286(3):491–498. 17. Gurerrant NB, Dutcher RA. Assay of vitamins B and G as influenced by coprophagy. J Biol Chem 1932; 98(1): 225–235. 18. Gurerrant NB, Dutcher RA, Brown RA. Further studies concerning formation of B vitamins in digestive tract of rat. J Nutr 1937; 13(1):305–315. 19. Najjar VA, Holt LE. The biosynthesis of thiamin in man and its implications in human nutrition. JAMA. 1943; 123(1):683– 684. 20. Kasper H. Vitamin Absorption in the colon. Am J Proctol 1970; 21(5):341–345. 21. Said HM, Ortiz A, Subramanian VS, et al. Mechanism of thiamin uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 2001; 281(1):144–150. 22. Reidling JC, Lambrecht N, Mohammad K, et al. Impaired intestinal vitamin B1 (thiamin) uptake in thiamin transporter-2 deficient mice. Gastroenterology. In press, PMID 19879271. 23. Reidling JC, Said HM. Adaptive regulation of intestinal thiamin uptake: molecular mechanism using wild-type and transgenic mice carrying hTHTR-1 and -2 promoters. Am J Physiol Gastrointest Liver Physiol 2005; 288(6):1127–1134. 24. Laforenza U, Patrini C, Alvisi C, et al. Thiamine uptake in

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Turmeric Janet L. Funk

INTRODUCTION

Ethnobotanical Use Having a rich and long history of culinary and medicinal use, turmeric remains one of the major spices traded worldwide (1). Its culinary and medicinal uses were first developed in India, which has been its primary site of cultivation for millennia. As with many spices, turmeric’s culinary uses include food preservation, flavoring, and coloration. The average diet in India is estimated to contain 2.5 g of turmeric rhizome per day, of which 100 mg (3% by weight) are polyphenolic curcuminoids (3). In western countries, such as the United States and France, turmeric is primarily used in the food industry as a colorant (e.g., mustard), resulting in estimates of daily curcumin consumption 10-fold lower than those in India (4). Medicinal turmeric is central to many Indian systems of traditional medicine (Ayurveda, Siddha, Unani) and is part of Eastern Asian systems as well (Traditional Chinese Medicine, Japanese Kampo, Korean, Malay). Turmeric is traditionally used in India for medical disorders of (i) skin (wounds, urticaria); (ii) upper respiratory track (rhinitis, pharyngitis, and cough); (iii) joints (rheumatism); and (iv) the gastrointestinal system (digestive aid, biliary and liver disorders) (1,2). Modern scientific inquiry related to turmeric, and in particularly curcumin, has focused on its potential use in other disease states including cardiovascular disease (CVD) and cancer, which are the major causes of mortality in adults in western countries.

The various names by which turmeric is known reflect both its characteristic golden color and its cultural and historical importance as a spice and medicine [“yellow root” or “Indian saffron”; “Curcuma”, a latinization of an Arabic term for saffron (al-kurkum); “turmeric,” derived from the old French appellation, terre merite (valuable soil or clay); and “herb of the sun”], so named 4000 years ago during the Vedic period in India due to its importance as a medicinal and religious herb (1). Traditionally, turmeric has been used as an anti-inflammatory treatment for ailments such as arthritis and minor infections, and as a digestive agent. During the last two decades, as our understanding of the importance of inflammation in the etiology of other diverse disease processes such as atherosclerosis and cancer has grown, so too has interest in the use of turmeric to prevent or treat various conditions now afflicting modern societies. Drawing upon a scientific literature related to turmeric and turmeric-derived compounds that has increased exponentially in recent years, the current state of our modern understanding of the biological effects and medicinal potential of this ancient plant is summarized here.

BACKGROUND Source

Chemistry

Turmeric (Curcuma longa L.) belongs to the Zingiberaceae family, which includes other tropical medicinal plants, such as ginger. Turmeric is cultivated commercially in Southeast Asia, its area of geographic origin. India is by far the largest producer and consumer of turmeric, supplying 70% of the world’s market while only exporting 10% of its crop (1). The plant, of which there are numerous varieties, is propagated for its rhizome, which is used as a culinary spice and traditional medicine. Seed rhizomes are sown in May to August; aerial shoots and leaves form over the next 5 months, followed by a period of accelerated rhizome growth, with rhizome harvest at 8–10 months (2). Bulbs and the more highly valued fingers of this “green turmeric” cannot be stored, as they are easily damaged by insects and so are cured [boiled in water for hours until soft; dried in the sun (1–2 weeks); polished in a turning drum; and colored by application of turmeric and other colorants, such as tamarind) to obtain a dry turmeric rhizome (∼20% yield from green turmeric) (2).

Turmeric rhizomes contain two main classes of secondary metabolites: curcuminoids and essential oils (Fig. 1), each comprising approximately 3% by weight of the dried rhizome and conferring protection to the plant through insecticidal and other properties (5). Dichloromethane– methanol extraction of the dried rhizome yields an extract (9% yield), of which curcuminoids and essential oils each comprise approximately one-third by weight (6–9). Essential oils are typically discarded as a byproduct of industrial curcuminoid isolation, but can be selectively isolated (3–4% yield) from dried rhizomes by hexane extraction (9,10). While factors such as geographic origin and plant variety alter their chemical composition, turmeric essential oils are primarily composed of sesquiterpenoids, with turmerones (ar and ␣- or ␤-turmerone) and curcumenes (e.g., zingiberene) being the major classes of compounds represented (Fig. 1) (5). Alternatively, the curcuminoids can be selectively isolated, yielding products that are primarily composed of three compounds, curcumin, demethoxycurcumin, and bisdemethoxycurcumin 754

Turmeric

755

Culinary and traditional medical use

TURMERIC Dried rhizome

Curcuminoids (3%)

Essential oils (3%) Major Constituents:

Major Constituents:

turmerones CH2 curcumin (1)

Scientific evaluation

demethoxycurcumin (2)

O

OH

R2

R1

HO 1. 2. 3.

Figure 1

bis-demethoxycurcumin (3)

R1 OCH 3 H H

R2 OCH 3 OCH 3 H

H2C

o

CH2

CH2 α-turmerone

curcumenes H

OH zingiberene

Schematic indicating major secondary metabolites and classes of chemical compounds derived from dried turmeric rhizome.

(Fig. 1) (5). Again, the relative ratios of these compounds vary with geographic origin and variety of the plant from which the turmeric rhizome is harvested; curcumin usually predominates (50–70%), but the relative amounts of the other two compounds can vary significantly [e.g., bisdemethoxycurcumin content can range from 3% to 33% of total (5,8). The curcuminoids appear to be the biosynthetic product of an enzyme(s) requiring as substrates, malonyl-CoA and hydroxycinnamoyl-CoA esters that are derived from the phenylpropanoid pathway (11). The enolate form of curcumin, which is favored in basic pH (pH 7.5), is more reddish in hue, accounting for the historic use of turmeric-treated paper as a pH indicator (12).

Traditional Preparation and Dietary Supplementation During culinary use, turmeric rhizome is often heated and/or combined with oil, being responsible for the golden hue of curries (3). For topical use for wound treatment, an aqueous paste of turmeric is prepared from the whole rhizome (3). For other indications, turmeric rhizome is taken orally in various preparations (5). Regu-

latory bodies in Europe and Canada recommend the use of turmeric preparations made from the whole rhizome (rhizome itself, 1:1 aqueous infusion, or 1:5 to 1:10 tinctures utilizing 70% ethanol), with dosing usually normalized to 1–4 g equivalents of rhizome (13–15). Commercial turmeric dietary supplements sold in the United States do not contain pure curcumin but are composed of a mixture of the three major curcuminoids and are devoid of essential oils, being sold in capsules labeled to contain 400–500 g of curcumin(oids), corresponding to 13–17 g of dried rhizome (at 3% yield) (8). On average, only 50% of the curcuminoid content of randomly selected turmeric dietary supplements in the United States is composed of curcumin, the remainder being demethoxycurcumin and bisdemethoxycurcumin in varying ratios (8). The chemical composition of turmeric products used in clinical trials is often not documented (Table 1) (16–51). One exception to this is the evaluation in phase 1 and 2 trials of a commercial curcuminoid-enriched product composed of a mixture of the three major curcuminoids, whose curcumin content exceeds 70% (27–30, 32,34,35).

Published Clinical Trials

Oral lichen planus Psoriasis

CRC Liver metastases with CRC CRC Pancreatic adenocarcinoma Precancerous, non-GI Precancerous lesions Monoclonal gammopathy Skin/mucosa Oral submucous fibrosis

Gastrointestinal (GI) Dyspepsia/peptic ulcer disease Helicobacter pylori with dyspepsia Irrit bowel syndrome (IBS) Colitis (Crohn’s + UC) Ulcerative colitis (UC) Diarrhea in HIV+ Gall bladder (contraction) Gall bladder (contraction) Familial adenomatous polyps (FAP) Cancer, GI Colorectal carcinoma (CRC)

Disease process

Table 1

-/-/Yes/Yes/-/-/-/-/-

Yes/yes Yes/yes Yes/yes Yes/yes Yes/Yes/Yes/-/-

Curcumina

Turmeric extract Curcumin Curcumin Curcumin Curcumin Curcumin Curcuminb

Curcuminoids + essential oils Curcuminoids Curcuminoids Curcuminoids Curcuminoids

Curcumin Curcuminoids

Turmeric extract ± essential oil or oleoresin Curcuminoids Curcuminoids Yes/yes Yes/-

-/-

Turmeric

Material description

Chemical composition (reported/ independently verified)

Product

C C

C, NC

C C

C C C C

C

C C C Various C C C C

C

NC

Source [commercial (C) or non-C]

2 4.5

3 g TE/0.6 g EO

0.5–8 4

0.04–0.2 curc/ 0.4–2 EO 0.5–4 0.5–4 0.5–4 8

0.144 1.1–1.7 1 6–6 (20 mg X1) (20–80 mg X1) 8

0.06

3

Dose (g)

Dosing

7 16

12

r -

-

-

-

>1 1 1 8

12

-

r r r r -

-

-

Randomized, r

4

8 8 24 20%, of persons over 60 years of age (34)

MMA was found in 5% of the omnivores, 77% of the lactoovo-vegetarians, and 83% of the vegans. Mean plasma vitamin B12 concentrations of lacto-vegetarians were substantially lower than those of nonvegetarians in studies in India (36). Case reports of diet-induced vitamin B12 deficiency have been made in teenagers and incidence of severe infant deficiency associated with maternal dietary restriction has also been reported. In a study of macrobiotic children (mean age 6.4 years) who had followed a strict macrobiotic diet in early childhood but had been omnivorous since that time, MMA and Hcy were elevated and cognitive function was altered (37). In developing countries, diet-induced vitamin B12 deficiency is much more common, especially in low socioeconomic status groups where ASF intake is limited by income (38). Widespread vitamin B12 deficiency and depletion have been reported in many countries in Latin America, Africa, and Southeast Asia, where a predominantly plant-based diet is consumed (39). The reported prevalence of low plasma B12 values in various countries in Latin America was approximately 40% across the lifespan, and in both sexes. More than one-half of pregnant Nepali women had elevated Hcy and MMA (20). In a group of vegetarian and nonvegetarian adults living in Pune, India, B12 deficiency was detected in 47%, based on low plasma B12 , and in 73%, based on elevated MMA (36).

Consequences of Deficiency Clinical symptoms of deficiency in adults are often nonspecific, and include fatigue, numbness, apathy, listlessness, diarrhea, and anorexia. Some patients experience oral discomfort, such as soreness of the tongue or ulceration. Although initial clinical symptoms may be vague, nevertheless, dramatic hematological, neurological, and immunological changes may occur (Table 4).

Inadequate Ingestion Dietary vitamin B12 deficiency has been described in affluent populations, traditionally in exceptional communities that practice religious dietary restrictions, or adhere to strict dietary guidelines, such as macrobiotic or vegan diets. Hindus, for example, often restrict intake of meat, or meat and eggs, and have a higher prevalence of deficiency. More recent examination of the evidence indicates that plasma vitamin B12 concentrations are actually related to intake over a wide range but plateau at an intake >10 ug (3). Thus lacto-ovo- or lacto-vegetarians (who consume animal products, but not meat) are also at risk for developing deficiency. In a study of German vegetarians, 60% had evidence of elevated plasma Hcy and MMA (35). Elevated

Hematological Changes The hematological consequences of vitamin B12 deficiency include megaloblastic anemia due to a reduced capacity to synthesize DNA rapidly, caused by alterations in the methionine synthase pathway. Hemoglobin develops at a normal pace but mitosis lags behind. As a result, RBC production is deranged and an abnormally large nucleus is extruded, leaving behind a large cytoplasm-filled cell. A mean corpuscular volume >115 fL defines megaloblastic cells, which may be as large as 130 to 150 fL. Total erythrocyte B12 does not change as the blood cell matures because the nucleus of the red cell is extruded and B12 is largely present in this organelle. Although megaloblastic anemia

818

Allen

is recognized as a classic symptom of vitamin B12 deficiency, many individuals may not experience measurable hematological change. In addition, megaloblastic anemia is a nonspecific outcome of B12 deficiency, as folate deficiency induces the same hematological changes through the same pathway. Although the presence of megaloblastic anemia may alert clinicians to the need to assess folate and B12 status, it is important to recognize that the anemia occurs at a much later and more severe stage of B12 depletion. There is little evidence that supplementation of depleted, nonvegan populations improves hemoglobin synthesis although there are few data on this question (40). Indicators of status, such as MMA and plasma vitamin B12 , are more sensitive to improved B12 status.

Neuropathies and Cognitive Performance In the elderly, vitamin B12 deficiency produces subacute combined degeneration, a syndrome of irregular spongiform demyelination of spinal cord (SC) white matter and astrogliosis. Deficiency in the elderly primarily affects change in the SC, whereas in infants the central nervous system is damaged. In fact, vitamin B12 deficiency in this age group can produce severe brain damage that may not be completely reversible upon therapy (25). Common domains of neural symptoms in vitamin B12 -deficient elderly are (i) sensory (paresthesias, diminished proprioception, diminished vibratory sensation); (ii) motor (weakness); (iii) reflex disorder related; (iv) autonomic (incontinence, impotence); (v) gait-related (ataxia); (vi) mental (intellectual/behavioral impairment); and (vii) visual (impaired visual acuity) (41). Deficiency is often resolved, and symptoms reversed, with vitamin B12 therapy. Either megadoses of the vitamin can be injected IM or large doses (500–1000 ␮g/day) of crystalline B12 can be taken orally. Because 1% of the vitamin can be passively absorbed without the need for IF, oral treatment is effective for many patients with PA or deficiency caused by gastric atrophy, and consumption of foods fortified with B12 predicts higher plasma B12 in the elderly (42). Demyelination of nerves associated with SCD is likely responsible for the majority of symptoms experienced by the elderly. A systematic review addressed the question of whether vitamin B12 supplementation improves cognitive function in adults (43). From the relatively small number of trials conducted, only some of which were randomized and controlled, the conclusion was drawn that oral vitamin B12 is not effective for improving cognitive function, although efficacy of IM supplements could not be ruled out. However, the studies included participants over a range of ages, cognitive function, and vitamin B12 status at baseline and confounding factors such as disease duration were not often considered. In contrast, another review concluded that on the basis of more sensitive markers of status (plasma TC and MMA), cognitive function in elderly was associated with B12 status across the normal range (44). Deficiency of the vitamin could cause brain atrophy or white matter damage. Additional studies of this important topic are ongoing. There are numerous case reports of severe B12 deficiency in infants of mothers with PA or mothers practicing a vegan/lacto-vegetarian diet (25). Symptoms include regression of mental development, abnormal pigmentation, hypotonia of muscles, enlarged liver and spleen, sparse

hair, tremors, irritability, anorexia, failure to thrive, poor brain growth, refusal of solid foods, and diarrhea. Marked cerebral atrophy and ventricular enlargement may also be present. Onset of deficiency in infants occurs within a few months of dietary absence and patients are often responsive to high-dose treatment. Vitamin B12 deficiency in infancy may have long-term consequences. Although there are virtually no systematic follow-up studies, it is estimated that about half of the cases with clinical symptoms do not achieve full recovery. In the Netherlands, infants aged 4 to 18 months who were born to macrobiotic mothers developed psychomotor skills later than omnivorous controls (45).

Immune Function In developing countries, both immunomodulation associated with malnutrition and repeated exposure to infection promote high rates of morbidity and mortality. An immunomodulating role specific to vitamin B12 has been reported. Markers that may be influenced by vitamin B12 status include (i) complement component C3; (ii) CD4 and CD8 T cell counts, and CD4/CD8 ratio; (iii) natural killer (NK) cell activity; and (iv) TNF-␣ concentration. Low lymphocyte counts, elevated CD4 cells, decreased CD8 cells, elevated CD4/CD8 ratio, and suppressed NK cell activity have been observed in B12 deficiency. Therapy restored several of these abnormal values (46). Vegans in the United States have signs of compromised immune function, including lower leukocyte counts and C3, even when micronutrient levels appear normal (47). The extent to which vitamin B12 deficiency is responsible for such changes in immune function requires further exploration.

Bone Health A number of studies report an association between low plasma B12 and/or elevated tHcy concentrations with markers of bone mineral loss during aging. In the third National Health and Nutrition Examination Survey in the United States, mean age 68 years, at each higher quartile of serum MMA bone mineral density was lower and osteoporosis increased (48). Those in the highest MMA quartile had a 7.2-fold greater risk of osteoporosis compared with the lowest MMA quartile. Serum B12 was related to bone mineral density up to ≈200 pmol/L, and those with tHcy >20 umol/L had lower bone mineral than those with values 30 ng/mL or >75 nmol/L) have been proposed by some as desirable for overall health and disease prevention (21), but clinical outcome data are not available to substantiate these higher values. Serum concentrations of 25OH D3 consistently above 200 ng/mL (>500 nmol/L) are potentially toxic. Emerging evidence also suggests some increased risk of all-cause mortality and pancreatic cancer associated with 25-OH D3 levels >75 nmol/L, and evidence-based thresholds of 25-OH D3 concentrations for deficiency, sufficiency, and adverse outcomes need to be more clearly defined (1).

Standard Reference Material and Quality Control Because of variability among assays and operators conducting them, a challenge in the field remains the measurement of serum 25-OH D3 concentrations. Consequently, the National Institute of Standards and Technology recently released a 25-OH D3 standard reference material that will now permit standardization of values across laboratories (21). Four levels of 25-OH D3 are available, each with specified values for selected vitamin D metabolites. This material is intended for use as an accuracy control and quality assurances tool and should appreciably improve the accuracy and reliability of future studies focused on reporting vitamin D status.

Deficiency and Prevention Vitamin D deficiency in humans is usually the result of dietary inadequacy, impaired absorption and use, increased requirements, and/or increased excretion. Vitamin D deficiency can occur when exposure to sunlight is limited. Vitamin D–deficient diets tend to be associated with those consumed in response to milk allergy, lactose intolerance, and veganism.

Rickets and Osteomalacia Rickets and osteomalacia are the classical vitamin D deficiency diseases, although calcium deficiency can also cause both disorders. In children, vitamin D deficiency causes rickets, a disease characterized by a failure of bone tissue to properly mineralize, resulting in soft bones and skeletal deformities. The fortification of milk with vitamin D has made rickets a rare disease in the United

Vitamin D

States. However, it is still reported periodically, particularly among African-American infants and children. For instance, a 2003 report from Memphis described 21 cases of rickets among infants, 20 of whom were AfricanAmerican (22). Infancy is a period of substantial risk for vitamin D deficiency, partly because vitamin D requirements cannot always be met by human milk. Indeed, a somewhat recent review of reports of nutritional rickets found that a majority of cases occurred among young, breastfed AfricanAmericans (23). Consequently, the American Academy of Pediatrics (AAP) recommends that exclusively breastfed and partially breastfed infants should begin receiving supplements of 400 IU/day vitamin D shortly after birth and that this should continue until they are weaned and consuming ≥1000 mL/day vitamin D–fortified formula or whole milk (24). Similarly, they advise that all nonbreastfed infants ingesting 75 to

Dietary Reference Intakes for Vitamin D Through the Lifecycle ␮g/day (UI/day)

Life stage group Adequate intake (AI) levels 0–12 mo 1–18 y 19–50 y 51–70 y 70+ y Pregnancy Lactation

5 (200) 5 (200) 5 (200) 10 (400) 15 (600) 5 (200) 5 (200) Tolerable upper intake (UL) levels

0–12 mo 1–18 y 19+ y Pregnancy Lactation

25 (1000) 50 (2000) 50 (2000) 50 (2000) 50 (2000)

Source: Institute of Medicine. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Washington, DC: National Academy Press, 1997.

Vitamin D

extend far beyond those directly related to modulation of calcium economy. For example, vitamin D (whether obtained from exogenous or endogenous sources) can regulate gene expression, and this can affect both growth and differentiation of cells. This may be especially important in terms of protecting from and/or predisposing a person to unregulated cell growth that can result in cancer. However, data relating vitamin D status to cancer are conflicting. Furthermore, because of vitamin D’s potent immunomodulatory effects, some experts have suggested that perturbations in vitamin D metabolism may be involved in the etiology of a variety of autoimmune disorders, but again the available evidence is limited. Vitamin D is found in very few foods, and recent evidence suggests that several populations, such as breastfed infants and the elderly, might be especially prone to vitamin D deficiency. This has prompted a renewed interest in establishing more rigorous and effective measures of vitamin D deficiency. In addition, scientists are once again re-examining dietary recommendations (including those for supplementation) for this vitamin.

ACKNOWLEDGMENTS The authors would like to acknowledge and sincerely thank Dr. Hector DeLuca, Harry Steenbock Research Professor in the Department of Biochemistry at the University of Wisconsin-Madison and Dr. Margherita Cantorna, Associate Professor of molecular immunology at Pennsylvania State University’s Huck Institutes of the Life Sciences for their careful review and critical evaluation of this manuscript.

REFERENCES 1. Cranney C, Horsely T, O’Donnell S, et al. Effectiveness and safety of vitamin D. Evidence Report/Technology Assessment No. 158. Rockville, MD: Prepared by the University of Ottawa Evidence-Based Practice Center under Contract No. 290–02.0021, AHRQ Publication No. 07-E013, 2007. 2. Holick MF. Vitamin D: the underappreciated D-lightful hormone that is important for skeletal and cellular health. Curr Opin Endocrinol Diabetes 2002; 9:87–98. 3. Holick MF. Vitamin D. In: Shils ME, Shike M, Ross AC, et al. eds. Modern Nutrition in Health and Disease. 10th ed. Philadelphia, PA: Williams and Wilkins, 2006. 4. Wharton B, Bishop N. Rickets. Lancet 2003; 362:1389–1400. 5. Wolpowitz D, Gilchrest BA. The vitamin D questions: how much do you need and how should you get it? J Am Acad Dermatol 2006; 54:301–317. 6. American Academy of Dermatology. Position statement on vitamin D. 2009. www.aad.org/Forms/Policies/Uploads/ PS/PS-Vitamin%20D.pdf. Accessed November 1, 2009. 7. Reichel H, Koeffler H, Norman A. The role of the vitamin D endocrine system in health and disease. N Engl J Med 1989; 320:980–991. 8. Dumke CL, Nieman DC, Oley K, et al. Ibuprofen does not affect serum electrolyte concentrations after an ultradistance run. Br J Sports Med 2007; 41:492–496. 9. Konradsen S, Ag H, Lindberg F, et al. Serum 1,25-dihydroxy vitamin D is inversely associated with body mass index. Eur J Nutr 2008; 47:87–91.

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10. Cosman F, Nieves J, Dempster D, et al. Vitamin D economy in blacks. J Bone Miner Res 2007; 22:V34–V38. 11. Kutuzova GD, Sundersingh F, Vaughan J, et al. TRPV6 is not required for 1␣,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo. Proc Natl Acad Sci U S A 2008; 105:19655–19659. 12. Samuel S, Sitrin MD. Vitamin D’s role in cell proliferation and differentiation. Nutr Rev 2008; 66:S116–S124. 13. Cozzolino M, Lu Y, Sato T. A critical role for enhanced TGFalpha and EGFR expression in the initiation of parathyroid hyperplasia in experimental kidney disease. Am J Physiol Renal Physiol 2005; 289; F1096–F1102. 14. Calvo MS, Whiting SJ, Barton CN. Vitamin D fortification in the United States and Canada: current status and data needs. Am J Clin Nutr 2004; 80:1710S–1716S. 15. U.S. Department of Agriculture. USDA National Nutrient Database for Standare Reference. Release 22. www.ars.usda .gov/Services/docs.htm?docid 8964. Accessed April 17, 2010. 16. Houghton LA, Vieth, R. The case against ergocalciferol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 2006; 84:694–697. 17. Holick MF, Biancuzzo RM, Chen TC, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab 2008; 93:677–681. 18. Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes: Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press, 1997. 19. Jones G. Pharmacokinetics of vitamin D toxicity. Am J Clin Nutr 2008; 88:582S–586S. 20. Vieth R, Bischoff-Ferrari H, Boucher BJ, et al. The urgent need to recommend an intake of vitamin D that is effective. Am J Clin Nutr 2007; 85:649–650. 21. National Institute of Standards and Technology. NIST Tech Beat. [Online] July 14, 2009. www.nist.gov/public affairs/ techbeat/tb2009 0714.htm. Accessed November 2, 2009. 22. Chesney R. Rickets: an old form for a new century. Pediatr Int 2003; 45:509–511. 23. Weisberg P, Scanlon KS, Li R, et al. Nutritional rickets among children in the United States: review of cases reported between 1986 and 2003. Am J Clin Nutr 2004; 80:1697S– 1705S. 24. Wagner CL, Greer FR. American Academy of Pediatrics Section on Breastfeeding and Nutrition. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics 2008; 122:1142–1152. 25. Aloia JF. African Americans, 25-hydroxyvitamin D, and osteoporosis: a paradox. Am J Clin Nutr 2008; 88:545S–550S. 26. Davis CD, Hartmuller V, Freedman M, et al. Vitamin D and cancer: current dilemmas and future needs. Nutr Rev 2007; 65:S71–S74. 27. Stolzenberg-Solomon RZ, Vieth R, Azad A, et al. A prospective nested case-control study of vitamin D status and pancreatic cancer risk in male smokers. Cancer Res 2006; 66:10213–10219. 28. Lieberman DA, Prindiville S, Weiss DG, et al. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003; 290:2959–2967. 29. Wactawski-Wende J, Kotchen JM, Anderson GL, et al. Calcium plus vitamin D supplementation and the risk of colorectal cancer. N Engl J Med 2006; 354:684–696. 30. Parfitt AM. Metabolic bone disease and clinically related disorders. In: Krane SM, eds. Osteomalacia and related disorders. 2nd ed. Philadelphia, PA: Avioli LV. WB Saunders, 1990. 31. Freedman DM, Looker AC, Chang SC, et al. Prospective study of serum vitamin D and cancer mortality in the United States. J Natl Cancer Inst 2007; 99:1594–1602.

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32. Chung M, Balk EM, Brendel M, et al. Vitamin D and calcium: a systematic review of health outcomes. Evidence Report No. 183. Agency for Healthcare Research and Quality: Prepared by the Tufts Evidence-Based Practice Center Under Contract No. HHSA 290–2007-10055–1, August 2009. AHRQ Publication No. 09-E015. 33. Yamshchikov AV, Desai NS, Blumberg HM, et al. Vitamin D for treatment and prevention of infectious diseases: a systematic review of randomized controlled trials. Endocr Pract 2009; 15:438–449. 34. Autier P, Gandini S. Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials. Arch Intern Med 2007; 167:1730–1737.

35. Melamed ML, Michos ED, Post W, et al. 25-hydroxyvitamin D levels and the risk of mortality in the general population. Arch Intern Med 2008; 168:1629–1637. 36. Jackson RD, LaCroix AZ, Gass M, et al. Women’s Health Initiative Investigators. Calcium plus vitamin D supplementation and the risk of fractures. N Engl J Med 2006; 354:669– 683. 37. Yetley EA, Brul´e D, Cheney MC, et al. Dietary Reference Intakes for vitamin D: justification for a review of the 1997 values. Am J Clin Nutr 2009; 89:719–727. 38. Institute of Medicine. Dietary Reference Intakes for vitamin D and calcium. www.iom.edu/Activities/Nutrition/ DRIVitDCalcium.aspx. Accessed April 17, 2010.

Vitamin E Maret G. Traber

INTRODUCTION

VITAMIN E SUPPLEMENTS

Vitamin E was discovered in 1922 by Evans and Bishop (1) and was described as a dietary factor required for reproduction in rodents. Since then, great advances have been made in our understanding of the antioxidant and nonantioxidant roles of vitamin E in human nutrition. Nonetheless, no specific biochemical function, other than that of an antioxidant, has been proven as the mechanism as to why humans require it. Indeed, the nonspecific nature of the vitamin’s antioxidant role has led advocates to suggest that amounts far in excess of dietary requirements might be beneficial to promote health, delay aging, and decrease the risk of chronic diseases. This entry will address facts about vitamin E, the gaps in our knowledge, and our expectations for the future.

Most vitamin E supplements and food fortificants contain all rac-␣-tocopherol, and can also have mixtures of tocopherols or tocotrienols. Often, supplements are sold as esters, which protect ␣-tocopherol from oxidation. These can be acetates, succinates, or nicotinates of ␣-tocopherol. Either the natural stereoisomer (RRR-␣-tocopherol) or the synthetic (all rac-␣-tocopherol) form can be sold as an ester, for example, d- or dl-␣-tocopheryl acetate, respectively. However, it is important to note that only half of the vitamin E in synthetic mixtures contains the 2Rstereochemistry. Thus, only 50% of all rac-␣-tocopherol meets human requirements (2).

BIOCHEMISTRY AND FUNCTIONS Antioxidant Activity

NAME AND GENERAL DESCRIPTION

Vitamin E is the most potent lipid-soluble antioxidant in human plasma and tissues (3). Hence, it protects polyunsaturated fatty acids within membranes and plasma lipoproteins from oxidation by reactive oxygen species. For example, a peroxyl radical (ROO• ) in a membrane is 1000 times more likely to attack a vitamin E molecule than a polyunsaturated fatty acid (RH) (4). In the absence of vitamin E, a chain reaction occurs:

Vitamin E [␣-tocopherol is called RRR-␣-tocopherol; or on package labels, d-␣-tocopherol; or more formally, 2,5, 7,8-tetramethyl-2R-(4 R,8 R,12-trimethyltridecyl)-6-chromanol] is a fat-soluble vitamin (2). Positions 2, 4 , and 8 of tocopherols are chiral carbon centers that are in the R-conformation in naturally occurring tocopherols (Fig. 1), but theoretically can take on either the R- or S-conformations. The chemical synthesis of ␣-tocopherol results in an equal mixture of eight different stereoisomers (RRR, RSR, RRS, RSS, SRR, SSR, SRS, and SSS). Therefore, synthetic ␣-tocopherol is called all rac-␣-tocopherol; or on package labels, dl-␣-tocopherol; or more formally, 2,5,7,8-tetramethyl-2RS-(4 RS,8 RS,12-trimethyltridecyl)6-chromanol. Dietary components with vitamin E antioxidant activity include ␣-, ␤-, ␥ -, and ␦-tocopherols, and ␣-, ␤-, ␥ -, and ␦-tocotrienols (2). All these molecules have a chromanol ring and vary in the number of methyl groups on the chromanol ring. Tocopherols have a phytyl tail, while tocotrienols have an unsaturated tail. ␣-Tocopherol and ␣tocotrienol have three methyl groups—␤ and ␥ have two, and ␦ has one. Importantly, only ␣-tocopherol meets human vitamin E requirements because only this form has been shown to reverse human vitamin E deficiency symptoms and is recognized preferentially by the hepatic ␣tocopherol transfer protein (␣-TTP) (2). Defects in the gene for ␣-TTP result in vitamin E deficiency both in humans and in animal models, as will be discussed later. It is for this reason that vitamin E has been defined for human requirements as ␣-tocopherol (2).

R·· + O2

ROO··

ROO·· + RH

ROO·· + ROOH

However, if vitamin E (e.g., ␣-TOH) is present, the hydroxyl group on the chromanol ring reacts with the peroxyl radical (ROO• ) to form a tocopheroxyl radical (␣TO• ) and a lipid hydroperoxide (ROOH). Thus, vitamin E acts as a chain-breaking antioxidant, thereby preventing further autoxidation of lipids (5). ROO + ␣ − TOH −→ ROOH + ␣ − TO· The tocopheroxyl radical (␣-TO• ) has a number of possible fates. It can react with another radical to form nonreactive products. Alternatively, it can be further oxidized to tocopheryl quinone, a two-electron oxidation product. Another possibility is “vitamin E recycling,” where the tocopheroxyl radical is restored to its unoxidized form by other antioxidants such as vitamin C, ubiquinol, or thiols, such as glutathione (6). This “recycling” process depletes other antioxidants; hence, an adequate intake of other dietary antioxidants is important 841

842

Traber

CH3

phytyl tail

5

HO

4′

6

8′

12′ CH 3

2 H3C 7

8

O

CH3

CH3

H

CH3

H

CH3

CH3

RRR-α-tocopherol

Chromanol ring

to maintain vitamin E concentrations. In addition, the tocopheryoxyl radical, because it is relatively long lived and if there are no other coantioxidants with which it could react, can hypothetically re-initiate lipid peroxidation (7). Upston, Terentis, and Stocker (7) have called this “TMP or tocopherol-mediated peroxidation” and claim it can occur in vivo based on the detection of both oxidized lipids and unoxidized vitamin E in atherosclerotic lesions. In addition to its antioxidant activity, ␥ -tocopherol and other non-␣-vitamin E forms can also trap reactive nitrogen oxides because they have an unsubstituted position on the chromanol ring (8). Cooney et al. (9) reported that ␥ -T is more effective in detoxification of NO2 than ␣-T. Furthermore, Hoglen et al. (10) demonstrated that 5-nitro␥ -tocopherol (2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5nitro-6-chromanol; NGT) is the major reactive product between peroxynitrite and ␥ -tocopherol. NGT has been reported in the plasma of zymosan-treated rats (11), cigarette smokers (12), patients with coronary artery disease (13), as well as in brains collected postmortem from patients with Alzheimer’s disease (14). All tocopherols and tocotrienols have antioxidant activity, and in some systems many of these have been reported to have higher antioxidant activity than ␣tocopherol (15,16). Nonetheless, it must be emphasized that the relationship between biologic activity and antioxidant activity is not clear. ␣-Tocopherol has the highest biologic activity, suggesting it shows some specific molecular function.

Biologic Activity Biologic activity is a historic term indicating a disconnection between molecules having vitamin E antioxidant activity and a relative lack of in vivo biologic function. Observations in rodent experiments carried out in the 1930s formed the basis for determining the “biologic activity” of this vitamin (17). Although the various molecules with vitamin E activity had somewhat similar structures and antioxidant activities, they differed in their abilities to prevent or reverse specific vitamin E deficiency symptoms (e.g., fetal resorption, muscular dystrophy, and encephalomalacia) (18). ␣-Tocopherol, with three methyl groups and a free hydroxyl group on the chromanol ring with the phytyl tail meeting the ring in the R-orientation (Fig. 1), has the highest biological activity. This specific structural requirement for biological, but not chemical, activity is now known to be dependent upon the hepatic ␣-TTP (19).

Figure 1 Structure of RRR-␣-tocopherol showing three chiral centers with the 2-position important for biologic activity.

As will be discussed later, ␣-TTP maintains plasma, and indirectly tissue, ␣-tocopherol concentrations (20,21).

Molecular Functions In addition to antioxidant activity, there are claims for specific ␣-tocopherol-dependent functions that normalize cellular signaling and metabolism in a variety of cells (22). ␣-Tocopherol has been shown to inhibit the activity of protein kinase C (23), a central player in many signal transduction pathways. Specifically, pathways of platelet aggregation (24,25), endothelial cell nitric oxide production (26,27), monocyte/macrophage superoxide production (28), and smooth muscle cell proliferation (29) were found to be modulated by added ␣-tocopherol. Regulation of adhesion molecule expression and inflammatory cell cytokine production by ␣-tocopherol has also been reported (30). The difficulty with these studies is that animals fed vitamin E-deficient diets are genetically lacking ␣-TTP and have not been reported to have altered expression of any of these pathways. There have been reports of regulation of the expression of lipoprotein receptors by ␣-tocopherol. Both the scavenger receptor B1(SR-B1) (31), and its homolog, CD36 (32,33), are decreased by high cellular ␣-tocopherol and increased by low concentrations. ␥ -Tocopherol, as well as its metabolite (␥ CEHC; ␥ -carboxyethyl hydroxychroman), possesses antiinflammatory properties, because stimulated macrophages and epithelial cells, treated with ␥ -tocopherol, have decreased cyclo-oxygenase-2 activity and lower levels of prostaglandin E2 (PGE2 ) synthesis (34). Moreover, in rats fed a high ␥ -T diet (33 mg/kg chow) and subjected to carrageenan-induced inflammation, PGE2 and leukotriene B4 synthesis were decreased by 46% and 70%, respectively (35). Additionally, ␥ -CEHC has been shown to increase sodium excretion (36). The in vivo significance of many of these various effects and the role of vitamin E in signaling pathways remain controversial because most of the information in this area has been obtained from in vitro studies. Additionally, microarray technology has been used to show changes in gene expression in response to vitamin E (37,38), but the physiologic relevance has not yet been clearly documented. More studies in humans are needed to relate ␣-tocopherol intakes and tissue concentrations to optimal tissue responses and gene regulation. It should be recognized, however, that it is very possible that vitamin E itself has no gene regulatory function, as reviewed (39).

Vitamin E

PHYSIOLOGY Absorption and Plasma Transport Intestinal absorption of vitamin E is dependent upon normal processes of fat absorption. Specifically, both biliary and pancreatic secretions are necessary for solubilization of this vitamin in mixed micelles containing bile acids, fatty acids, and monoglycerides (Fig. 2). ␣-Tocopheryl acetates (or other esters) from vitamin E supplements are hydrolyzed by pancreatic esterases to ␣-tocopherol prior to absorption. Low-fat diets limit vitamin E absorption, especially from supplements (40). Following micellar uptake by enterocytes, it is incorporated into chylomicrons and secreted into the lymph. Once in the circulation, chylomicron triglycerides are hydrolyzed by lipoprotein lipase (LPL). During chylomicron catabolism in the circulation, vitamin E is nonspecifically transferred both to tissues and to other circulating lipoproteins (41). It is not until the vitamin E-containing chylomicrons reach the liver that discrimination between the various dietary vitamin E forms occurs. The hepatic ␣-TTP preferentially facilitates secretion of ␣-tocopherol, specifically 2R␣-tocopherols, but not other tocopherols or tocotrienols, from the liver into the plasma in very low density lipoproteins (VLDLs) (42,43) In the circulation, VLDLs are catabolized to low-density lipoproteins (LDLs). During this lipolytic process, all of the circulating lipoproteins become enriched with ␣-tocopherol. There is no evidence that vitamin E is transported in the plasma by a specific carrier protein. Instead, the vitamin is nonspecifically transported in all of the lipopro-

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tein fractions (44). An advantage of this transport is that oxidation-susceptible lipids are protected by the simultaneous transport of a lipid-soluble antioxidant. Similarly, delivery of vitamin E to tissues is dependent upon lipid and lipoprotein metabolism. Thus, as peroxidizable lipids are taken up by tissue, the tissues simultaneously acquire a lipid-soluble antioxidant. Plasma ␣-tocopherol concentrations in humans range from 11 to 37 ␮mol/L, while ␥ -tocopherol concentrations are roughly 2 to 5 ␮mol/L and tocotrienol concentrations are less than 1 ␮mol/L, even in subjects supplemented with tocotrienols (45). When plasma lipids are taken into account, the lower limits of normal level are 1.6 ␮mol ␣-tocopherol/mmol lipid (sum of cholesterol and triglycerides), or 2.5 ␮mol ␣-tocopherol/mmol cholesterol (46). The apparent half-life of RRR-␣-tocopherol in plasma of normal subjects is approximately 48 hours (47), while that of SRR-␣-tocopherol is only 15 hours (47), and that of ␥ -tocopherol is also similar to the SRR␣-tocopherol, about 15 hours (48). This relatively fast turnover of 2 S-␣-tocopherol is also accompanied by increased metabolism (49). The comparatively fast disappearance of the 2 S-␣-tocopherols indicates that by 48 hours, nearly 90% of the 2 S-forms have been removed from the plasma, while 50% of the 2R-forms remain. It is then no wonder that the plasma disappearance curves of RRR- and all rac-␣-tocopherols are parallel; they both trace the disappearance of 2R-forms (50–52).

Tissue Delivery

INTESTINE Chylomicrons α–T γ –T

α–T γ –T

Dietary Vit E

HDL α–T LDL α–T

VLDL α–T

Remnants α–T γ –T

LIVER TTP

Figure 2 Intestinal vitamin E absorption and plasma lipoprotein transport. Vitamin E absorption requires both biliary and pancreatic secretions for solubilization of vitamin E in mixed micelles. Following micellar uptake by enterocytes, vitamin E (shown as ␣- and ␥-tocopherols, ␣-T and ␥-T) is incorporated into chylomicrons and is secreted into the lymph. During chylomicron catabolism in the circulation, it is nonspecifically transferred both to tissues and to other circulating lipoproteins (not shown). It is not until the vitamin E-containing chylomicrons reach the liver that discrimination between the various dietary vitamin E forms occurs. The hepatic ␣-TTP preferentially facilitates secretion of ␣-tocopherol from the liver into the plasma in very low density lipoproteins (VLDLs). In the circulation, VLDLs are catabolized to LDLs. During this lipolytic process, all of the circulating lipoproteins (e.g., LDL and HDL) become enriched with ␣-tocopherol.

Vitamin E is delivered to tissues by three methods, none of which is specific for vitamin E. But rather its trafficking depends on mechanisms of lipid and lipoprotein metabolism. These include transfer from triglyceriderich lipoproteins during lipolysis, delivery as a result of receptor-mediated lipoprotein uptake, and exchange between lipoproteins or tissues. With respect to lipolysis, LPL facilitates the delivery of ␣-tocopherol from triglyceride-rich lipoproteins to cells, as shown in vitro (53). The importance of this pathway was demonstrated in vivo when LPL was overexpressed in muscle, resulting in increased vitamin E delivery to muscle (54). Both low-and high-density lipoproteins (LDL and HDL, respectively) have been shown to deliver vitamin E to tissues. The LDL receptor-mediated uptake of LDL delivers the lipoprotein particle via an endocytic pathway, and vitamin E is released during lipoprotein degradation (55). In contrast, HDL binds to the SR-BI allowing selective delivery of the HDL lipids, including vitamin E, to the cells (56). In SR-BI knockout mice, plasma ␣tocopherol concentrations are elevated. Some tissues (e.g., brain (57) and lung (31)) contain decreased ␣-tocopherol contents, while hepatic tocopherol concentrations are unchanged. But biliary tocopherol excretion is decreased (58). Apparently, SR-BI-mediated hepatic uptake of HDLassociated ␣-tocopherol is coupled to biliary excretion of vitamin E (58). Although vitamin E spontaneously exchanges between lipoproteins (59), the phospholipid transfer protein facilitates the exchange of phospholipids between

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lipoproteins, as well as the transfer of vitamin E from VLDL to HDL and from lipoproteins into cells (60). Phospholipid transfer protein knockout mice compared with wild types have higher vitamin E in apolipoprotein Bcontaining lipoproteins (VLDL or LDL) (61). The involvement of the plasma cholesteryl ester transfer protein in this transfer process was ruled out (61). The regulation of tissue vitamin E is not well understood, but it is seen that ␣-tocopherol is the predominant form in tissues as a result of its plasma concentrations (20). The ATP-binding cassette transporter (ABCAI) has been shown to participate in the efflux of ␣-tocopherol from cells to HDL (62). Apparently, excess vitamin E could be removed from cells via ABCAI facilitating its transfer to apolipoprotein AI, and transport via HDL to the liver where SR-BI could mediate vitamin E transfer into a liver pool destined for excretion in bile.

Metabolism and Excretion Vitamin E is excreted as intact tocopherols or tocotrienols, oxidized forms, and a metabolic product (41). ␣- and ␥ -Tocopherols as well as ␣- and ␥ -tocotrienols are metabolized to ␣- and ␥ -CEHCs [2,5,7,8-tetramethyl- and 2,7,8trimethyl-2-(2 carboxyethyl)-6-hydroxychromans], respectively, by humans (41). CEHCs were first described in rats fed high amounts of ␦-tocopherols (63). About 1% of a dose of ␣-tocopherol or tocotrienol and 5% of a dose of ␥ -tocopherol or tocotrienol are excreted in the urine as CEHCs (64). On the basis of studies in hepatocytes (65,66), it is likely that the liver synthesizes CEHCs. Studies in renal dialysis patients (67,68) suggest that in addition to urinary excretion (41), bile may be a major route for CEHC excretion. Similarly, CEHCs have been found in both rat urine and bile (69). Vitamin E metabolism appears to be a key factor in the regulation of vitamin E bioavailability (70). The various forms of vitamin E appear to be metabolized similar to xenobiotics in that they are initially oxidized by P450s, conjugated, and excreted in urine or bile. CEHCs have a shortened phytyl tail, resulting from ␻-oxidation, a cytochrome P450 (CYP)-mediated process, followed by ␤-oxidation (71–73). Hepatic CYP 4F2 is involved in ␻-oxidation of ␣- and ␥ -tocopherols (73), as is CYP 3A (65,71,72,74). It should be noted that a compound can stimulate CYPs other than those involved in its own metabolic pathway; thus, interactions with a variety of pathways are possible. CEHCs can be sulfated or glucuronidated (75–77). Both free and conjugated forms have been detected in plasma (76) urine (41), and bile (78). All of the systems involved in vitamin E metabolism could be under PXR regulation (79). However, Cho et al. (80) showed that stimulation of PXR by the mouse PXR activator, pregnenolone 16 a-carbonitrile (PCN), in wild-type compared with PXRnull mice decreased vitamin E metabolism. Thus, it appears that PXR-activation does not increase vitamin E metabolism. They also identified a new CEHC conjugate, a glycoside. Importantly, PCN treatment in wild-type mice, which stimulates PXR, decreased the urinary excretion of ␣-CEHC glucuronide and ␥ -CEHC glycoside. Thus, the regulation of vitamin E metabolism by PXR remains unclear.

Dietary vitamin E forms, such as ␥ -tocopherol (81) or ␥ -tocotrienol (82), are more actively metabolized to CEHCs than ␣-tocopherol (49,64,75). In fact, nearly all of the absorbed ␥ -tocopherol has been estimated to be metabolized to ␥ -CEHC (75). High ␣-tocopherol intakes, for example, supplements, lead to both increased ␣-CEHC (83) and ␥ -CEHC excretion (64). Thus, vitamin E metabolism may be a key factor in hepatic disposal of excess vitamin E, as well as a key determinant in vitamin E bioavailability.

HUMAN VITAMIN E DEFICIENCY Vitamin E deficiency was first described in children with fat malabsorption syndromes, principally abetalipoproteinemia, cystic fibrosis, and cholestatic liver disease (84). Subsequently, humans with severe deficit with no known defect in lipid or lipoprotein metabolism were described to have a defect in the ␣-TTP gene (85). Erythrocyte fragility, hemolysis, and anemia were described as vitamin E deficiency symptoms in various animals fed diets devoid of this antioxidant (86). However, in humans, the major symptom is a peripheral neuropathy characterized by the degeneration of large caliber axons in the sensory neurons (87).

INDICATIONS AND USAGE Food Sources Vitamin E can be readily obtained from food, but relatively few foods have high ␣-tocopherol concentrations (88). Generally, the richest sources are vegetable oils. Wheat germ oil, safflower oil, and sunflower oil contain predominantly ␣-tocopherol, while soy and corn oils have mainly ␥ -tocopherol. All of these oils are polyunsaturated. Good sources of monounsaturated oils, such as olive or canola oils, also have ␣-tocopherol to a large extent. Whole grains and nuts, especially almonds, are also good ␣-tocopherol sources. Fruits and vegetables, although rich in watersoluble antioxidants, are not good sources of vitamin E. Indeed, desserts are a major source of vitamin E in the American diet (89). In the past, it was assumed for the purpose of calculating dietary vitamin E intakes in ␣-tocopherol equivalents (␣-TEs) that ␥ -tocopherol can substitute for ␣tocopherol with an efficiency of 10% (90). However, functionally, ␥ -tocopherol is not equivalent to the latter. Caution should be exercised in applying ␣-TEs to estimates of ␣-tocopherol intakes when corn or soybean oils (hydrogenated vegetable oils) represent the major oils present in foods. These oils have high ␥ -tocopherol contents, and if food tables reporting ␣-TEs are used to estimate dietary ␣tocopherol, intakes of ␣-tocopherol may be overestimated.

Treatment of Vitamin E Deficiency Overt vitamin E deficiency occurs only rarely in humans and almost never as a result of inadequate vitamin E intakes. It does occur as a result of genetic abnormalities in ␣-TTP (87) and various fat malabsorption syndromes (91). Vitamin E supplementation halts the progression of the neurologic abnormalities caused by inadequate

Vitamin E

nerve tissue ␣-tocopherol, and in some cases, has reversed them (92). Patients with these disorders require daily pharmacologic vitamin E doses for life to overcome and prevent the deficiency symptoms. Generally, subjects with ataxia with vitamin E deficiency are advised to consume 1000 mg RRR-␣-tocopherol per day in divided doses, those with abetalipoproteinemia 100 mg/kg body weight per day, and for cystic fibrosis 400 mg/day. However, patients with fat malabsorption due to impaired biliary secretion generally do not absorb orally administered vitamin E. They are treated with special forms of vitamin E, such as ␣-tocopheryl polyethylene glycol succinate, which spontaneously form micelles, obviating the need for bile acids (93).

Chronic Disease Prevention In individuals at risk for vitamin E deficiency, it is clear that supplements should be recommended to prevent deficiency symptoms. What about vitamin E supplement in normal individuals? Dietary changes such as decreasing fat intakes (94), substituting fat-free foods for fatcontaining ones, and increasing reliance on meals away from the home have resulted in decreased consumption of ␣-tocopherol-containing foods. Therefore, intakes of the vitamin E recommended dietary allowance (RDA)— 15 mg ␣-tocopherol—may be difficult. Estimates of ␣tocopherol intakes by Americans suggest that less than 10% consume adequate amounts of the vitamin, and that women have lower intakes than men (95). Increased consumption of nuts and seeds, as well as olive and canola oils, may be useful in increasing dietary ␣-tocopherol intakes. The potential role of vitamin E in preventing or ameliorating chronic diseases has prompted many investigators to ask if supplements might be beneficial. When “excess” amounts of many vitamins are consumed, they are excreted and provide no added benefits. Antioxidant nutrients may, however, be different. Heart disease and stroke, cancer, chronic inflammation, impaired immune function, Alzheimer’s disease—a case can be made for the role of oxygen-free radicals and inflammation in the etiology of all of these disorders, and even in aging itself. Do antioxidant nutrients counteract the effects of free radicals and thereby ameliorate these disorders? And if so, do large quantities of antioxidant supplements have beneficial effects beyond “required” amounts? The 2000 DRI Report on Vitamin C, Vitamin E, Selenium, and Carotenoids stated that there was insufficient proof to warrant advocating supplementation with antioxidants (2). But it also stated that the hypothesis that antioxidant supplements might have beneficial effects was promising. Despite the lack of positive findings from various intervention studies (96,97), and some more positive findings from others (98– 101), the consequences of a long-term increased antioxidant intake in healthy people are not known. Moreover, a study examining the relationship between the genetic background of diabetic women and the benefits of antioxidant supplementation found a marked beneficial effect on coronary artery stenoses in haptoglobin 1 allele homozygotes, but not in those with the haptoglobin 2 allele (102). Moreover, haptoglobin 2–2 diabetic subjects were studied

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in a prospective, double-blind, placebo-controlled trial of vitamin E. Vitamin E supplementation reduced cardiovascular events in diabetic individuals and the Hp 2–2 genotype (ClinicalTrials.gov NCT00220831) (103). Thus, it would appear that subjects with high oxidative stress and the appropriate genetic background may benefit from antioxidant supplements, but not in those without these factors.

Dietary Reference Intakes In 2000, the Food and Nutrition Board of the Institute of Medicine, National Academy of Sciences published the Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and the Carotenoids (2). Recommendations for vitamin E intakes are shown in Table 1. The requirements for vitamin E intakes are based primarily on its long-term (5–7 yr) depletion and repletion studies in humans carried out by Horwitt et al. (104). Serum ␣-tocopherol concentrations and the corresponding hydrogen peroxide-induced erythrocyte hemolysis were determined at various intervals. Serum concentrations necessary to prevent in vitro erythrocyte hemolysis in response to known levels of vitamin E intake in subjects who had undergone experimentally induced vitamin E deficiency were used to determine estimated average requirements for vitamin E. The RDAs are levels that represent the daily ␣-tocopherol intakes required to ensure adequate nutrition in 95% to 97.5% of the population and are an overestimation of the level needed for most people in any given group.

Vitamin E Units The Food and Nutrition Board defined vitamin E for human requirements to include only ␣-tocopherol and specifically those forms with 2R-␣-tocopherol stereochemistry (2). According to the U.S. Pharmacopoeia (USP), 1 international unit (IU) of vitamin E equals 1 mg all rac-␣tocopheryl acetate, 0.67 mg RRR-␣-tocopherol, or 0.74 mg RRR-␣-tocopheryl acetate (105). These conversions were estimated on the relative “biologic activities” of the various forms when tested in the rat assay for vitamin E deficiency, the fetal resorption assay. These USP IUs are currently used in labeling vitamin E supplements and food fortificants. It should be noted that the 2000 RDA does not use vitamin E USP units; rather the recommendation is set at 15 mg 2R-␣-tocopherols. To convert IU to milligram of 2R-␣-tocopherols, the IU RRR-␣-tocopherol (or its esters) Table 1 Estimated Average Requirements (EARs), Recommended Dietary Allowances (RDAs), and Average Intakes (AIs) (mg/day) for ␣-Tocopherol in Adults and Children Lifestage 0–6 mo 7–12 mo 1–3 yr 4–8 yr 9–13 yr 14–18 yr Adult (male or female) Pregnancy Lactation Source: From Ref. 2.

EAR

RDA

AI 4 6

5 6 9 12 12 12 16

6 7 11 15 15 15 19

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is multiplied by 0.65, while the IU all rac-␣-tocopherol (or its esters) is multiplied by 0.45.

ADVERSE EFFECTS Upper Tolerable Limits High vitamin E intakes are associated with an increased tendency to bleed. It is not known if this is a result of decreased platelet aggregation caused by an inhibition of protein kinase C by ␣-tocopherol (24), some other plateletrelated mechanism (106), or decreased clotting due to a vitamin E interaction with vitamin K (107). It has also been suggested that extraordinarily high vitamin E intakes may interfere with activation of vitamin K (108). Individuals who are deficient in vitamin K or who are on anticoagulant therapy are at increased risk of uncontrolled bleeding. Thus, patients on anticoagulant therapy should be monitored when taking vitamin E supplements to ensure adequate vitamin K intakes (109). This “adverse effect” may actually be a benefit for some individuals! Glynn et al. (110) report that vitamin E supplements may decrease the risk of venous thromboembolism, especially in those with a genetic predisposition to clotting. The 2000 Food and Nutrition Board of the Institute of Medicine, National Academy of Sciences, recommended 1000 mg as an upper limit (UL) of all forms of ␣-tocopherol in supplements taken by adults 19 years and older, including pregnant and lactating women. The vitamin E UL was set for only supplements because it is impossible to consume enough ␣-tocopherol-containing foods to achieve a daily 1000 mg intake for prolonged periods of time. The UL was defined for all forms of ␣-tocopherol, not just the 2R-forms, because all eight of the stereoisometric forms in all rac-␣-tocopherol are absorbed and delivered to the liver and therefore potentially have adverse effects. The ULs for supplements containing either RRR- or all rac␣-tocopherol supplements are 1500 IU RRR-␣-tocopherol or its esters, or 1100 IU of all rac-␣-tocopherol or its esters. The UL for RRR-␣-tocopherol is apparently higher because each capsule of RRR-␣-tocopherol contains fewer milligram of ␣-tocopherol than does one containing all rac-␣-tocopherol. ULs were set for children and adolescents by adjusting the adult limit on the basis of relative body weight. No UL was set for infants because of lack of adequate data. The 2000 Food and Nutrition Board did recommend that food be the only source of vitamin E for infants. However, a UL of 21 mg/day was suggested for premature infants with birth weight of 1.5 kg, on the basis of the adult UL.

Adverse Interactions of Drugs and Vitamin E Drugs intended to promote weight loss by impairing fat absorption, such as Orlistat or olestra, can also impair vitamin E and other fat-soluble vitamin absorption. Therefore, multivitamin supplementation is recommended. Supplements should be taken with meals at times other than when these drugs are taken to allow adequate absorption of the fat-soluble vitamins. The inhibition of cholesterol absorption by Etzetimibe interferes with the function of Niemann-Pick C1-like

1 protein. This drug has also been found to decrease vitamin E absorption in rats (111). It is therefore possible that Etzetimibe also interferes with vitamin E absorption in humans. Findings from two clinical trials have suggested adverse vitamin E effects. One study was a three-year, double-blind trial of antioxidants (vitamins E and C, ␤carotene, and selenium) in 160 subjects on simvastatinniacin or placebo therapy (112,113). In subjects taking antioxidants, there was less benefit of the drugs in raising HDL cholesterol than was expected (112), while there was an increase in clinical end points [arteriographic evidence of coronary stenosis, or the occurrence of a first cardiovascular event (death, myocardial infarction, stroke, or revascularization)] (113). The other study was the Women’s Angiographic Vitamin and Estrogen (WAVE) Trial, a randomized, double-blind trial of 423 postmenopausal women with at least one coronary stenosis at baseline coronary angiography. In postmenopausal women on hormone replacement therapy, all-cause mortality was higher in women assigned to antioxidant vitamins compared with placebo group (HR, 2.8; 95% CI, 1.1–7.2; P = 0.047) (114). The reasons for these adverse effects, especially mortality, are unclear because a metaanalysis of more than 80,000 subjects taking part in vitamin E intervention trials did not find increased mortality in those taking vitamin E (115). Other meta-analyses have reported slightly increased risk of mortality, but could not identify any mechanisms of action (116,117) while other researchers have criticized the methodologies used for the meta-analyses and found no evidence of vitamin E adverse effects (118).

CONCLUSIONS One of the real difficulties in setting requirements or making recommendations for optimal vitamin E intakes is that the function of the antioxidant remains undefined. Certainly, its in vitro antioxidant function has been agreed upon for decades, but questions remain as to whether this is the only function of vitamin E, or if indeed antioxidant activity is its in vivo function (39,119,120). In addition, if the vitamin functions solely as an antioxidant, then biomarkers of oxidative stress will never be useful for setting requirements because oxidative damage certainly can be modulated by antioxidants in addition to vitamin E. Thus, one of the major thrusts is to establish the function of vitamin E. One important area that is currently under investigation is its role in inflammation (121) and immune function (122). But, here again, the role of oxidative stress confounds the findings because leukocytes release reactive oxygen species and this is attenuated by vitamin E (28). Clearly, defining vitamin E function(s) is the goal of future studies.

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116. Bjelakovic G, Nikolova D, Gluud LL, et al. Mortality in Randomized Trials of Antioxidant Supplements for Primary and Secondary Prevention: Systematic Review and Metaanalysis. JAMA 2007; 297:842–857. 117. Miller ER III, Paston-Barriuso R, Dalal D, et al. Metaanalysis: High-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005; 142: 37–46. 118. Berry D, Wathen JK, Newell M. Bayesian model averaging in meta-analysis: Vitamin E supplementation and mortality. Clin Trials 2009; 6:28–41.

119. Traber MG, Packer L. Vitamin E: Beyond antioxidant function. Am J Clin Nutr 1995; 62(suppl):1501S–1509S. 120. Zingg JM, Azzi A. Non-antioxidant activities of vitamin E. Curr Med Chem 2004; 11:1113–1133. 121. Jialal I, Devaraj S. Antioxidants and atherosclerosis: Don’t throw out the baby with the bath water. Circulation 2003; 107:926–928. 122. Meydani SN, Meydani M, Blumberg JB, et al. Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial. JAMA 1997; 277:1380–1386.

Vitamin K J. W. Suttie

INTRODUCTION

by Doisy’s group at the St. Louis University. The Doisy group also isolated a crystalline form of the vitamin from putrefied fish meal and demonstrated that this compound contained an unsaturated polyprenyl side chain at the 3position of the naphthoquinone ring. The term vitamin K is now used as a generic descriptor of 2-methyl-1,4-naphthoquinone (menadione) and in all derivatives of this compound that exhibit an antihemorrhagic activity in animals fed a vitamin K-deficient diet (Fig. 1). The major dietary source of vitamin K, the form found in green plants, is commonly called vitamin K1 , but is preferably called phylloquinone. The compound, 2-methyl-3-farnesylgeranylgeranyl-1,4naphthoquinone, first isolated from putrefied fish meal, is one of a series of vitamin K compounds with unsaturated side chains called multiprenylmenaquinones, which are produced by a limited number of anaerobic bacteria and are present in large quantities in the lower bowel. This particular menaquinone has 7 isoprenoid units in the

Vitamin K activity is exhibited by phylloquinone, found in green plants, and by a series of menaquinones, which are synthesized by a limited number of anaerobic bacteria. The metabolic role of this vitamin is as a substrate for an enzyme, the vitamin K-dependent carboxylase, which mediates a post-translational modification of a small number of proteins by converting specific glutamyl residues to ␥ -carboxyglutamyl (Gla) residues. These include a number of proteins that regulate hemostasis: prothrombin, factor VII, factor IX, factor X, and proteins C, S, and Z. The bone proteins, osteocalcin and matrix Gla protein (MGP), and several other less well-characterized proteins also require vitamin K for their synthesis. The human requirement for vitamin K is low, and the adequate intake for adult men and women is currently set at 120 and 90 ␮g/day, on the basis of the median intakes of the U.S. population. The classical symptom of a vitamin K deficiency, a hemorrhagic event, is essentially impossible to produce in adults without some underlying factor influencing absorption of the vitamin. However, newborn infants are routinely supplemented with vitamin K to prevent a condition called hemorrhagic disease of the newborn. A small amount of the protein osteocalcin circulates in plasma, and because this protein is not maximally ␥ carboxylated at normal levels of intake, there is currently a great deal of interest in a possible role of vitamin K in promoting skeletal health. Supplementation of the diet with 45 mg of menaquinone-4 is a widely used treatment for osteoporosis in Japan and other parts of Asia, but the efficacy of this treatment in North America or Europe has not yet been established. The possibility of an impact of vitamin K status on atherosclerosis outcomes through the action of MGP is also a problem of research interest.

O

O

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Phylloquinone O

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Menaquinone-9

Background, Chemistry, and Dietary Sources

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In the early 1930s, Henrik Dam observed that chicks consuming very low lipid diets developed subdural or muscular hemorrhages and that blood taken from these animals clotted slowly. This hemorrhagic disease could not be cured by supplementation with any other known dietary factor, and Dam (1) proposed the existence of a new fat-soluble factor, vitamin K. Subsequent studies by Dam and others (2) established that the antihemorrhagic factor was present both in the lipid extracts of green plants and in preparations of fish meal that had been subjected to bacterial action. The vitamin could be isolated from alfalfa as a yellow oil, and it was characterized as 2methyl-3-phytyl-1,4-naphthoquinone (3) and synthesized

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Menaquinone-4

Figure 1 Structures of vitamin K active compounds. Phylloquinone (vitamin K1 ) synthesized in plants is the main dietary form of vitamin K. Menaquinone-9 is a prominent member of a series of menaquinones (vitamin K2 ) produced by intestinal bacteria and menaquinone-4, while a minor bacterial product can also be synthesized by animal tissues from phylloquinone.

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side chain and was once called vitamin K2 . That term is currently used to describe any of the vitamers with an unsaturated side chain, and this compound is more correctly identified as menaquinone-7 (MK-7). The predominant menaquinones found in the gut are MK-7 through MK-9, but smaller amounts of others are also present. Menadione is used as a source of vitamin K activity in poultry and swine rations, and a specific compound, (MK4), is formed in animal tissues by its alkylation (4). This is the biologically active form of the vitamin present in animal tissues when menadione is used as the dietary form of vitamin K. Standardized procedures to assay the vitamin K content of foods, and sufficient values (5) to provide reasonable estimates of its daily intake are now available (Table 1). In general, foods with higher phylloquinone content are green leafy vegetables. Those providing substantial amounts of the vitamin to the majority of the population are spinach (380 ␮g/100g), broccoli (180 ␮g/100g), and iceberg lettuce (35 ␮g/100g). Fats and oils are also a major contributor to the vitamin K content of the diet. Soybean oil (190 ␮g/100g) and canola oil (130 ␮g/100g) are quite high, while corn oil (3 ␮g/100g) is a very poor source. The source of fat or oil will influence the vitamin K content of margarine and prepared foods with a highfat content. The process of hydrogenation to convert plant oils to solid margarines or shortening, converts some of the phylloquinone to 2 ,3 -dihydrophylloquinone with a completely saturated side chain. The biological activity of this form of the vitamin is not accurately known, but it has been reported that the intake of this form of the vitamin by the American population may be 20% to 25% that of phylloquinone (6).

Vitamin K-dependent Proteins The first proteins identified as requiring vitamin K for their synthesis were plasma clotting factors, and the classical sign of a vitamin K deficiency has been the development of a hemorrhagic syndrome. Many of the proteins involved in regulating blood coagulation (Fig. 2) are protease zymogens, which are sequentially activated through a series of events, many involving membrane-associated complexes with each other and with accessory proteins (7–9). Prothrombin (clotting factor II) is the circulating zymogen of the procoagulant thrombin, and was the first protein shown to be dependent on vitamin K for its synthesis. Clotting factors VII, IX, and X were all initially identified because their activity was decreased in the plasma of a patient with a hereditary bleeding disorder (10) and were subsequently shown to depend on vitamin K for their synthesis. These four “vitamin K-dependent clotting factors” were the only proteins known to require this vitamin for their synthesis until the mid-1970s. The distinguishing character of vitamin K-dependent proteins is the presence of a post-translational modified glutamic acid (Glu) residue, ␥ -carboxyglutamic acid (Gla). Proteins C and S were discovered after it had been shown that prothrombin contained Gla residues. They were subsequently shown to have an anticoagulant, rather than a procoagulant, role in hemostasis. The seventh vitamin K-dependent plasma protein, protein Z, is not a protease zymogen and also exhibits an anticoagulant role under some conditions.

Table 1 Phylloquinone Concentration of Common Foodsa Food item Vegetables Collards Spinach Salad greens Broccoli Brussels sprouts Cabbage Bib lettuce Asparagus Okra Iceberg lettuce Green beans Green peas Cucumbers Cauliflower Carrots Tomatoes Potatoes Protein sources Dry soybeans Dry lentils Liver Eggs Fresh meats Fresh fish Whole milk Fats and oils Soybean oil Canola oil Cottonseed oil Olive oil Margarine Butter Corn oil Prepared foods Salad dressings Coleslaw Mayonnaise Beef chow mein Muffins Doughnuts Potato chips Apple pie French fries Macaroni/cheese Lasagna Pizza Hamburger/bun Hot dog/bun Baked beans Bread

␮g/100g 440 380 315 180 177 145 122 60 40 35 33 24 20 20 10 6 1 47 22 5 2