Recent Advances in Polyphenol Research

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Recent Advances in Polyphenol Research

Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Recent Advances in Polyphenol Research A series for researchers and graduate students whose work is related to plant phenols and polyphenols, as well as for individuals representing governments and industries with interest in this field. Each volume in this biennial series will focus on several important research topics in plant phenols and polyphenols, including Chemistry, Biosynthesis, Metabolic Engineering, Ecology, Physiology, Food, Nutrition, and Health. Volume 1 editors: Fouad Daayf and Vincenzo Lattanzio Series Editor-in-Chief: Vincenzo Lattanzio, President of ‘Groupe Polyphénols’ (University of Foggia, Italy) Series Editorial Board: Øyvind M. Andersen (University of Bergen, Norway) Denis Barron (Nestlé Research Centre, Lausanne, Switzerland) Catherine Chèze (Université Victor Segalen, Bordeaux 2, France) Richard A. Dixon (The Samuel Roberts Noble Foundation, Ardmore, OK, USA) Ismaîl El-Hadrami (Cadi Ayyad University, Marrakech, Morocco) Tadao Kondo (Nagoya University, Japan) Paul A. Kroon (Institute of Food Research, Norwich, UK) Stéphane Quideau (IECB, Université Bordeaux 1, France) Jorge-Manuel Ricardo da Silva (University of Lisbon, Portugal) Celestino Santos-Buelga (University of Salamanca, Spain) Dieter Treutter (Technical University of Munich, Freising, Germany)

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Recent Advances in Polyphenol Research Volume 1 Edited by

Fouad Daayf Associate Professor, Molecular Plant Pathology Department of Plant Science Faculty of Agricultural and Food Sciences University of Manitoba, Winnipeg, Manitoba, Canada

Vincenzo Lattanzio Professor, Plant Biochemistry and Physiology Department of Agro-Environmental Sciences, Chemistry and Plant Protection Faculty of Agricultural Sciences University of Foggia, Foggia, Italy

A John Wiley & Sons, Ltd., Publication

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This edition first published 2008 © 2008 Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing programme has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom Editorial offices 9600 Garsington Road, Oxford, OX4 2DQ, United Kingdom 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/ wiley-blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. 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, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Recent advances in polyphenol research / edited by Fouad Daayf, Vincenzo Lattanzio. p. cm. Includes bibliographical references. ISBN-13: 978-1-4051-5837-4 (hardback : alk. paper) ISBN-10: 1-4051-5837-9 (hardback : alk. paper) 1. Polyphenols—Research. I. Daayf, Fouad. II. Lattanzio, Vincenzo, 1949– QK898.P764R43 2008 572′.2—dc22 2008006133 A catalogue record for this book is available from the British Library. Set in 10/13pt Times by Graphicraft Limited, Hong Kong Printed in Singapore by Fabulous Printers Pte Ltd 1

2008

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Dedication To Michel Bourzeix and his collaborators, Carmen Bataller and Nicolas Hérédia, whose efforts and enthusiasm have been decisive for the life of the Groupe Polyphénols.

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Acknowledgments The editors wish to thank all the members of the Groupe Polyphénols Board Committee (2004–2006) for their guidance and assistance throughout this project. Groupe Polyphénols Board 2004–2006 Professor Øyvind M. Andersen Dr Patrice André Professor Denis Barron Dr Catherine Chèze Dr Claire Dufour Professor Maria Teresa Escribano-Bailón Dr Hélène Fulcrand Dr Sylvain Guyot Dr Paul A. Kroon Dr Jean-Luc Poessel Professor Stéphane Quideau Professor Jorge-Manuel Ricardo da Silva Professor Celestino Santos-Buelga Dr Pascale Sarni-Manchado

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Contributors Preface 1

1.1 1.2

1.3 1.4

2

2.1 2.2 2.3

2.4

Plant Phenolics – Secondary Metabolites with Diverse Functions Vincenzo Lattanzio, Paul A. Kroon, Stéphane Quideau and Dieter Treutter Secondary metabolism in the interactions between plants and their environment Function and use of plant phenolics 1.2.1 UV sunscreens 1.2.2 Phenolics as signal compounds 1.2.3 Phenolics as pigments 1.2.4 Phenolics and plant growth 1.2.5 Phenolics and plant defense 1.2.5.1 Fungal pathogens 1.2.5.2 Phenolics and plant–insect interactions 1.2.6 Plant phenolics and health Note References

Lignification: are Lignins Biosynthesized via simple Combinatorial Chemistry or via Proteinaceous Control and Template Replication? John Ralph, Gösta Brunow, Philip J. Harris, Richard A. Dixon, Paul F. Schatz and Wout Boerjan Introduction The current theory Is there a need for a new theory? 2.3.1 The challenge hypothesis (proteinaceous control and template replication) 2.3.2 Has the challenge hypothesis become a theory? Are criticisms of the current theory valid? 2.4.1 Lignification as a biochemical anomaly

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1 5 6 6 8 9 11 13 17 22 24 24

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2.4.2 The β-ether frequency anomaly 2.4.3 ‘Obligatory’ linkages? 2.5 Is there anything wrong with the dirigent hypothesis? 2.5.1 Monomer substitution 2.5.1.1 Has monomer substitution been disproven? 2.5.1.2 Malleability of lignification: what makes a good monolignol substitute? 2.5.2 ‘Well-defined primary structure’ 2.5.2.1 Sequencing of lignin oligomers 2.5.2.2 Monomer-independent sequences 2.5.2.3 The ‘urgent need to sequence lignin primary structure’ 2.5.3 Lignins’ racemic nature 2.5.3.1 The ‘number of isomers’ problem 2.5.4 Comments on template replication 2.5.5 Polymer branching 2.6 Why the new hypothesis is not in contention 2.7 Is lignification a biochemical anomaly? 2.7.1 Ferulate dehydrodimerization is combinatorial 2.7.2 Polysaccharide primary structure is NOT absolutely dictated 2.8 Summary comments on the dirigent/replication hypothesis 2.9 Conclusions 2.10 Notes 2.11 References

3

3.1 3.2 3.3 3.4

3.5

3.6 3.7

Flavonoid–Protein Binding Processes and their Potential Impact on Human Health Olivier Dangles and Claire Dufour Introduction Biologically relevant chemical properties of flavonoids Binding processes prior to absorption Binding processes involved in flavonoid bioavailability 3.4.1 Absorption and conjugation 3.4.2 Transport in plasma 3.4.3 Tissue distribution and cellular metabolism Binding processes involved in the potential health effects of flavonoids 3.5.1 Inhibition of enzymes involved in ROS production 3.5.2 Modulation of the redox properties of flavonoids by binding to proteins 3.5.3 Inhibition of protein kinases 3.5.4 Inhibition of cytochrome P450 enzymes 3.5.5 Regulation of gene expression Conclusion References

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Methods for Synthesizing the Cocoa-Derived Oligomeric Epi-Catechins – Observations on the Anticancer Activity of the Cocoa Polyphenols Alan P. Kozikowski and Werner Tückmantel

4.1 4.2

Introduction Synthesis of procyanidins 4.2.1 General chemical properties of cocoa procyanidins 4.2.2 Earlier synthetic work 4.2.3 The synthesis of benzyl-protected building blocks 4.2.4 Inter-flavan bond formation using benzyl-protected building blocks 4.2.5 Establishment of inter-flavan bond stereochemistry 4.2.6 Further developments Anticancer activity Acknowledgments References

4.3 4.4 4.5

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Gene Discovery and Metabolic Engineering in the Phenylpropanoid Pathway Luzia V. Modolo, Yongzhen Pang, Li Tian and Richard A. Dixon

5.1 5.2 5.3 5.4

5.5

5.6 5.7

5.8 5.9 5.10

6

Introduction Biosynthesis and functions of isoflavones Dietary sources of isoflavones Metabolic engineering of isoflavones 5.4.1 Metabolic engineering by ectopic expression of IFS 5.4.2 Structural biology-assisted design and metabolic engineering with an artificial bifunctional IFS enzyme Gene discovery in the proanthocyanidin biosynthetic pathway 5.5.1 Structural genes for PA biosynthesis 5.5.2 Regulatory genes for PA biosynthesis Metabolic engineering of PAs in plants Glycosyltransferases for modification of phenylpropanoid compounds – in-vitro biochemistry and in-vivo function 5.7.1 UGTs active with non-flavonoid phenolic compounds 5.7.2 Glycosylation of flavonoid compounds 5.7.3 Problems for the functional annotation of UGTs Concerted strategies for metabolic engineering References Abbreviation list of the pathway genes

Recent Advances in the Molecular Biology and Metabolic Engineering of Flavonoid Biosynthesis in Ornamental Plants Kevin M. Davies, Huaibi Zhang and Kathy E. Schwinn

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88 89 89 91 92 94 97 102 105 109 109

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113 113 115 115 115 116 117 118 121 122 123 124 128 130 131 132 138

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Recent Advances in the Field of Anthocyanins – Main Focus on Structures Øyvind M. Andersen

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7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 7.10 7.11 7.12

8.1 8.2 8.3 8.4 8.5 8.6

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Introduction Metabolic engineering of flavonoid production in flowers 6.2.1 Engineering yellow flower colors 6.2.2 Engineering blue flower colors Anthocyanic vacuolar inclusions Regulation of anthocyanin biosynthesis Concluding comments References

6.3 6.4 6.5 6.6

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Introduction Anthocyanidins Anthocyanidin equilibrium forms New anthocyanin glycosides New anthocyanin acylglycosides Flavonoid complexes including at least one anthocyanidin subunit Metalloanthocyanins Biosynthesis and molecular biology Anthocyanin localization in plant cells Acknowledgments Notes References

Salicylic Acid and Induced Plant Defenses Jean-Pierre Métraux, Elisabeth Lamodière, Jérémy Catinot, Olivier Lamotte and Christophe Garcion Induced resistance and phenolics The biosynthesis of SA The network of signaling and the action of SA Conclusions Acknowledgment References

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Phenols and the Onset and Expression of Plant Disease Resistance Ray Hammerschmidt and Samantha I. Hollosy

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Introduction Biosynthetic origins of defense-associated phenolic compounds Phenolic compounds as preformed defenses

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9.3.1 9.3.2 9.3.3 9.3.4

9.4 9.5

9.6

9.7

9.8 9.9 9.10

10

10.1 10.2

10.3

Resistance of onion bulbs Fusarium wilt of carnation Mango fruit and Alternaria Regulation of preformed antifungal compounds by phenols in avocado 9.3.5 Chlorogenic acid and the infection of stone fruit by Monilinia Active defense Localized defenses 9.5.1 Phenolic phytoalexins 9.5.2 Phenolic structural defenses 9.5.3 Plant phenols and induced disease resistance Responses of the induced plant 9.6.1 Induced resistance in green bean and C. lindemunthianum 9.6.2 Acibenzolar-S-methyl mediated induced resistance 9.6.3 Plant growth-promoting rhizobacteria and induced resistance 9.6.4 Silicon as a modulator of defense and phenolic compounds Chemical induction of phenolic compounds and resistance 9.7.1 Structural phenolic compounds and induced resistance 9.7.2 Antioxidant activity of phenolic compounds and plant defense Phenols and defense: a multitude of roles Acknowledgment References

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213 214 214 214 215 215 216 216 218 219 220 220 220 220 221 221 221 222 222 223 223

Bioactivity, Absorption, and Metabolism of Anthocyanins Giuseppe (Joe) Mazza and Colin D. Kay

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Introduction 10.1.1 Structural characteristics Bioactivity 10.2.1 Antioxidant activity 10.2.2 Anti-inflammatory effects 10.2.3 Anti-atherogenic effects 10.2.4 Anticarcinogenic effects 10.2.5 Antibacterial and antiviral activity 10.2.6 Neuroprotective effects 10.2.7 Prevention of obesity 10.2.8 Gastric protective effects 10.2.9 Improvement of vision Absorption of anthocyanins 10.3.1 Variability of absorption 10.3.1.1 Variations in dosage 10.3.1.2 Chemical structure of the anthocyanins 10.3.1.3 Food matrix 10.3.1.4 Analytical methodology 10.3.2 Elimination

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10.3.3

10.4

10.5 10.6

11

11.1 11.2 11.3 11.4 11.5

11.6 11.7 11.8

12

12.1 12.2

The concentration of anthocyanins in human blood and urine is very low 10.3.4 Suggested mechanisms of anthocyanin absorption 10.3.5 Structural transformations of anthocyanins Metabolism of anthocyanins 10.4.1 Human studies 10.4.2 Animal studies 10.4.2.1 Rat studies 10.4.2.2 Pig studies 10.4.3 Potential mechanisms of anthocyanin metabolism Conclusions References

Bioavailability, Metabolism, and Bioactivity of Food Ellagic Acid and Related Polyphenols Francisco A. Tomás-Barberán, Maria Teresa García-Conesa, Mar Larrosa, Begoña Cerdá, Rocio González-Barrio, Maria José Bermúdez-Soto, Antonio González-Sarrías and Juan Carlos Espín Introduction Ellagitannins and ellagic acid as examples of bioactive polyphenols Evaluation of antioxidant activity in vitro Biological activity associated to ellagitannin-rich food intake; clinical studies Questions arising after the demonstration of the large antioxidant activity in vitro and the biological activity associated with the intake of ellagitannin-rich food 11.5.1 Bioavailability and metabolism of ellagitannins and ellagic acid and distribution of the metabolites in different tissues 11.5.2 Evaluation of the biological activity of ellagitannin metabolites produced in vivo Conclusion Acknowledgments References

Multiplicity of Phenolic Oxidation Products in Apple Juices and Ciders, from Synthetic Medium to Commercial Products Sylvain Guyot, Stéphane Bernillon, Pascal Poupard and Catherine M.G.C. Renard Introduction Preparation and characterization of the caffeoylquinic acid o-quinone solution

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263 264 265 265

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12.4 12.5 12.6 12.7

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13.3

13.4 13.5 13.6

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Incubation of caffeoylquinic acid o-quinone in model solutions 12.3.1 Incubation of caffeoylquinic acid o-quinone with caffeoylquinic acid 12.3.2 Incubation of caffeoylquinic acid o-quinone with (−)-epicatechin LC-MS analysis of oxidation products in commercial apple beverages Conclusions Acknowledgments References

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Phytoestrogens in Drug Discovery for Controlling Steroid Biosynthesis Sampo Karkola, Annamaria Lilienkampf and Kristiina Wähälä Introduction Aromatase 13.2.1 Natural phytoestrogens as aromatase inhibitors 13.2.1.1 Anti-aromatase activity of phytoestrogens in human placental microsome-based assays 13.2.1.2 Anti-aromatase activity of phytoestrogens in cell-based assays 13.2.1.3 Anti-aromatase activity of phytoestrogens in recombinant-enzyme assays 13.2.1.4 The anti-aromatase activity of various phytoestrogens 13.2.2 The structure–activity relationship of phytoestrogens 13.2.3 Phytoestrogens as lead compounds for aromatase inhibition 17β-Hydroxysteroid dehydrogenases and their inhibition by phytoestrogens 13.3.1 17β-HSD type 1 13.3.1.1 Binding of phytoestrogens to 17β-HSD1 13.3.2 17β-HSD types 2 and 4 13.3.3 Fungal 17β-HSDcl 13.3.3.1 Binding of phytoestrogens to 17β-HSDcl 13.3.4 17β-HSD type 3 13.3.5 17β-HSD type 5 13.3.6 Phytoestrogens as lead compounds for 17β-HSD inhibitors Conclusions Acknowledgments References

Recent Advances in the Chemical Synthesis and Biological Activity of Phenolic Metabolites Denis Barron Introduction The different sites of generation of phenolic metabolites 14.2.1 The saliva

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14.4

14.5

14.6

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14.2.2 The stomach 14.2.3 The small intestine 14.2.4 The colon 14.2.5 The liver 14.2.6 Metabolism at the target tissues or cells Nature of the metabolites of phenolic compounds 14.3.1 Flavone and flavonol metabolites 14.3.2 Flavanonone and flavanonol metabolites 14.3.3 Flavan 3-ol metabolites 14.3.4 Ellagic tannin metabolites 14.3.5 Lignan metabolites 14.3.6 Isoflavone metabolites 14.3.7 Hydroxycinnamic acid metabolites 14.3.8 Curcumin metabolites 14.3.9 Resveratrol metabolites The chemical synthesis of phenolic conjugates 14.4.1 The preparation of O-glucuronides 14.4.2 The preparation of O-sulfates The biological properties of phenolic conjugates 14.5.1 Antioxidant properties 14.5.1.1 Flavonol conjugates 14.5.1.2 Flavone conjugates 14.5.1.3 Flavanone conjugates 14.5.1.4 Flavan 3-ol conjugates 14.5.1.5 Isoflavone conjugates 14.5.1.6 The case of the 7-O-conjugates 14.5.2 Pro-oxidant properties 14.5.3 Interaction with signaling cascades 14.5.4 Enzyme inhibition 14.5.4.1 Aldose reductase 14.5.4.2 β-glucuronidase 14.5.4.3 Xanthine oxidase 14.5.4.4 Glycerol 3-phosphate dehydrogenase 14.5.4.5 Cyclooxygenase-2 14.5.5 Effect on vascular function and angiogenesis 14.5.6 Non-covalent binding to proteins 14.5.7 Activity of microbial and tissular metabolites The cellular transport of phenolic conjugates 14.6.1 Cellular efflux 14.6.2 Cellular uptake Conclusions References

319 319 320 320 321 321 321 322 324 324 325 325 326 327 328 328 329 332 334 334 334 340 340 340 340 341 342 343 344 344 344 344 345 345 345 345 346 346 346 347 348 350

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Polyphenols and Gene Expression Uwe Wenzel and Hannelore Daniel

15.1 15.2 15.3 15.4 15.5

Introduction The effects of polyphenols on the expression of genes underlying detoxification mechanisms Polyphenols and the expression of genes underlying cancer-relevant processes The impact of polyphenols on the expression of genes underlying atherosclerosis-relevant processes References

Index The color plate section follows page 42

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359 359 363 369 373 379

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Contributors

Øyvind M. Andersen, Department of Chemistry, University of Bergen, Allegt. 41, N5007 Bergen, Norway Denis Barron, Food Consumer Interaction Department, Nestlé Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland Maria José Bermúdez-Soto, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Stéphane Bernillon, INRA, UMR619, Centre de Bordeaux, 71, avenue Edouard Bourleaux, BP 81, F-33883 Villenave d’Ornon Cedex, France Wout Boerjan, Department of Plant Systems Biology, VIB-Ghent University, Gent 9052, Belgium Gösta Brunow, Department of Chemistry, University of Helsinki, Helsinki, Finland Jérémy Catinot, Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland Begoña Cerdá, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Fouad Daayf, Associate Professor, Molecular Plant Pathology, Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada Olivier Dangles, UMR A 408 INRA – University of Avignon, Safety and Quality of Plant Products Site Agroparc, Domaine St-Paul, 84914 Avignon Cedex 9, France Hannelore Daniel, Molecular Nutrition Unit, Department of Food and Nutrition Sciences, Technical University of Munich, Am Forum 5, D-85354 Freising, Germany

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Kevin M. Davies, New Zealand Institute for Crop & Food Research Limited, Private Bag 11-600, Palmerston North, New Zealand Richard A. Dixon, Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA Claire Dufour, UMR A 408 INRA – University of Avignon, Safety and Quality of Plant Products Site Agroparc, Domaine St-Paul, 84914 Avignon Cedex 9, France Juan Carlos Espín, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain María Teresa García-Conesa, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Christophe Garcion, Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland Rocio González-Barrio, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Antonio González-Sarrías, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Sylvain Guyot, INRA, UR117, Unité de Recherches Cidricoles, Domaine de la Motte, BP 35327, F-35653 Le Rheu Cedex, France Ray Hammerschmidt, Department of Plant Pathology, 107 CIPS Bldg, Michigan State University, East Lansing, MI 48824-1311, USA Philip J. Harris, School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand Samantha I. Hollosy, Department of Plant Pathology, 107 CIPS Bldg, Michigan State University, East Lansing, MI 48824-1311, USA Sampo Karkola, Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, AI Virtasen Aukio 1, P.O. Box 55, FIN-00014 Helsinki, Finland Colin D. Kay, Diet and Health Group, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, Norfolk, UK

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Contributors

Alan P. Kozikowski, Department of Medicinal Chemistry and Pharmacognosy, 833 South Wood Street, Room 531, Chicago, IL 60612, USA Paul A. Kroon, Institute of Food Research, Colney Lane, Norwich NR4 7UA, UK Elisabeth Lamodière, Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland Olivier Lamotte, Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland Mar Larrosa, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Vincenzo Lattanzio, Dipartimento di Scienze Agro-Ambientali, Chimica e Difesa Vegetale, Università degli Studi di Foggia, Via Napoli, 25, 71100-Foggia, Italy Annamaria Lilienkampf, Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, AI Virtasen Aukio 1, P.O. Box 55, FIN-00014 Helsinki, Finland Giuseppe (Joe) Mazza, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC, Canada V0H 1Z0 Jean-Pierre Metraux, Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland Luzia V. Modolo, Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA Stéphane Quideau, Institut Européen de Chimie et Biologie, Pôle Chimie Organique et Bioorganique, 2 rue Robert Escarpit, F-33607 Pessac cedex, France Yongzhen Pang, Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA Pascal Poupard, INRA, UR117, Unité de Recherches Cidricoles, Domaine de la Motte, BP 35327, F-35653 Le Rheu Cedex, France John Ralph, Departments of Biochemistry and Biological Systems Engineering, University of Wisconsin, Madison, WI 53706, USA; and US Dairy Forage Research Center, USDA-Agricultural Research Service, 1925 Linden Drive West, Madison, WI 53706-1108, USA Catherine M.G.C. Renard, INRA, UMR A480 ‘Sécurité et Qualité des Produits d’Origine Végétale’ Domaine St Paul, F-84914 Avignon cedex 09, France

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Paul F. Schatz, US Dairy Forage Research Center, USDA-Agricultural Research Service, 1925 Linden Drive West, Madison, WI 53706-1108, USA Kathy E. Schwinn, New Zealand Institute for Crop & Food Research Limited, Private Bag 11-600, Palmerston North, New Zealand Li Tian, Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA Francisco A. Tomás-Barberán, Research Group on Quality, Safety and Bioactivity of Plant Food, Department of Food Science & Technology, CEBAS (CSIC), P.O. Box 164, Espinardo, Murcia 30100, Spain Dieter Treutter, Center of Life and Food Science Weihenstephan, Technische Universität München, Unit Fruit Science – Fruit Tree Physiology, Alte Akademie 16, D 85350 Freising, Germany Werner Tückmantel, PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, NY 10598, USA Kristiina Wähälä, Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, AI Virtasen Aukio 1, P.O. Box 55, FIN-00014 Helsinki, Finland Uwe Wenzel, Molecular Nutrition Research, University of Giessen, Heinrich-Buff-Ring 26–32, D-35392 Giessen, Germany Huaibi Zhang, New Zealand Institute for Crop & Food Research Limited, Private Bag 11-600, Palmerston North, New Zealand

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Preface

Plant phenolics are secondary metabolites that constitute one of the most common and widespread groups of substances in plants. They represent adaptive characters that have been subjected to natural selection during evolution, when the presence of a particular secondary metabolite has conferred a selection advantage to the plant containing it. Polyphenols have a large and diverse array of beneficial effects on both plants and humans. For example, they are famous as antioxidants, hormones, constituents of essential oils, natural neurotransmitters, and as having many other biological activities. Their antioxidant ability is known to confer many health benefits such as reducing the risk of cardiovascular disease and cancer. They also provide antimicrobial activity for the plant’s own defense against invading pathogens. The diversity of structure and activity of phenolic compounds has resulted in a multiplicity of research areas such as chemistry, biotechnology, ecology, physiology, nutrition, medicine, and cosmetics. The International Conference on Polyphenols, organized under the auspices of ‘Groupe Polyphénols’, is a unique opportunity for scientists in these and other fields to get together every other year and exchange their ideas and new findings. Apart from the two-page manuscripts – Polyphenols Communications – that comprise the proceedings of this conference, a separate volume has been concurrently published, comprising full chapters by the conference guest speakers. For the first time in the history of the conference, the board of ‘Groupe Polyphénols’ decided in 2005 that, starting in Winnipeg (ICP 2006), such a volume should be published by a renowned publisher such as Wiley–Blackwell, and given the series title Recent Advances in Polyphenol Research. The present (first) volume in the series is from the 23rd conference, which was hosted by the University of Manitoba in Winnipeg, Manitoba, Canada, from August 22 to August 25 2006, and chaired by Dr Fouad Daayf. The University of Manitoba, established in 1877, is one of the oldest universities in Canada. Its education and research programs are dedicated to many areas including agriculture, art, architecture, medicine, business, and science. The interest in polyphenols involves many departments on campus including the Departments of Plant Science, Chemistry, Food Science, Nutrition, and Human Ecology, as well as other health and functional food-related centers such as the Richardson Center for Functional Foods and Nutraceuticals. In addition to the guest speakers’ chapters, the present volume also includes full chapters from other selected speakers at the 23rd International Conference on Polyphenols, and covers five topics: 1) Phenols and Polyphenols Chemistry: (a) Isolation and structural elucidation; (b) Synthesis; (c) Reactivity and physico-chemical properties; (d) Biomolecular interactions.

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Preface

xxi

2) Phenols and Polyphenols Biosynthesis and Genetic Manipulation: (a) Metabolic pathways; (b) Enzymology; (c) Biotechnology advances. 3) Ecology and Physiology of Plant Phenolics: (a) Biotic and abiotic stress; (b) Phenolic functions in plant development; (c) Role of phenolics in soil ecology. 4) Food and Nutrition: (a) Dietary intake; (b) Bioavailability; (c) Safety and toxicity; (d) Functional foods and nutraceuticals; (e) Taste. 5) Phenolics and Health: (a) Biological activities; (b) Drug discovery and development; (c) Cosmetics. These topics were presented in 59 oral communications and 222 posters, and scientists had the opportunity to debate their results, and sometimes their divergent theories, in an exciting manner. The 23rd International Conference on Polyphenols would not have been possible without the generous support of public and private donors such as the Manitoba Rural Adaptation Council and the University of Manitoba. Other sponsors include Horphag, l’Agence Universitaire de la Francophonie, Phytochemistry, Cargill, and Monsanto. Our sincere thanks go to all of our sponsors. Fouad Daayf, Conference Chair Vincenzo Lattanzio, President of ‘Groupe Polyphénols’

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DS

(a)

DS

OCH3 O OH

DS

O

DS OCH3

OCH3 OH

DS

OH

8 O

OCH3

OCH3 OH

8

O

O

8′

O 4

O 4′

OR OCH3

OCH3 OH H3CO

8

H3CO OCH3

8′

O OCH3

4 O 8′ O

OH

OR

O

OCH3 OCH3 OH

H3CO

OCH3 OH

OCH3

OH

O

H3CO

OCH3 OR

OCH3

HO

OCH3

8

H3CO

H3CO OCH3

O 8 OCH3

OH

O

OCH3

4′ O

OR

O 4′

OCH3 H3CO

8 OR

OCH3 OH

OH

OMe

(b)

O O OMe

HO

MeO

OMe

OH HO

MeO

S5

10

9

O

S4 12

11

OH

O

5

HO

MeO

S3 HO

OMe

Primary chain termination

O

O

OH

OR

OH H3CO

DS OCH3

4′ O

OH

OCH3

O 4′

OCH3 H3CO

OCH3

OCH3 OH H3CO

8′ H3CO

O 4

O 4′

DS

O

8′

DS

OR

4 O 8′

DS

Primary chain termination

OCH3 OH

O

H3CO

OR

O

O

HO

OCH3

H3CO

DS OCH3

H3CO

H3CO OCH3

O

DS

OCH3 OH

DS

OH H3CO

OCH3

MeO

OH

7

3

OMe

1

S6

S1 OMe OH 4

6

S2 8

2

OH OMe

O

O OMe

S-(b–O–4)-S-(b–O–4)-S-(b–O–4)-S-(b–b)-S-(4–O–b)-S Plate 2.2 (a) The dirigent array/replication model, redrawn from Davin & Lewis (2005). It is suggested that a protein putatively harboring an array of dirigent (monolignol radical binding) sites assembles primary lignin chains. Such chains are then hypothesized to act as templates for replication of the chain. Note that, in the scheme shown, for either the primary lignin synthesis or for template replication, mechanistic chemical principles demand that the only starting point is with the β– β-coupling in the middle. (b) An example of a hexamer, S-(β–O–4)-S-(β–O–4)-S-(β–O–4)-S-(β–β)-S-(4–O–β)-S, cf. the all-syringyl analog of the hexamer in Fig. 4 in Davin & Lewis (2005) that has been putatively sequenced (see text, section 2.5.2.1). The 12 chiral carbons are labeled.

Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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OH

(a) Coniferyl Alcohol Dehydrodimerization OH

HO

OMe

G

HO

β

HO

β

G 5

OMe

G

β

+

G

C β-β-dimer (pinoresinol)

OH

β

HO

5-5-dimer (biphenyl)

β

O

OMe

S OMe

MeO

OMe

4

OH

+

S

S

MeO

.

4

MeO

β

+ MeO

OH

OMe β

4

O

S

OMe

OH

OH

S

O

S

HO

β

+

B no β-5-dimer possible!

C β-β-dimer (syringaresinol)

(f)

(d) Monolignol cross-coupling with a G-end-unit HO

G

HO

β

O

5

OMe OH

+

G/S

G

O OH MeO

G

5

5

G

OMe

OMe OH

OH

OH

OH

.

+

G/S

OMe

OMe

4

OMe

β

4

OMe

+

G/S

G

5

HO

OH

OMe OH

4-O-5-dimer (biphenyl ether)

OMe

A β-O-4-dimer (β-ether)

5

O

OMe

HO

OH

β

OMe

OMe

HO MeO

HO

5

OMe

OH OH

5

OH

B β-5-dimer (phenylcoumaran)

(b) Sinapyl Alcohol Dehydrodimerization

OH MeO

HO

OH

A β-O-4-dimer (β-ether)

β

O

G OMe

OH

OH

OH

O

OMe

OMe

4

β

+

G

O

OMe

4

O

OMe

+

OH

OH

G

5

HO

β

G

(c)

OH

A β-O-4-unit

B β-5-unit

C no β-β-unit from cross-coupling!

D 5-5-unit

(e) Monolignol cross-coupling with an S-end-unit HO MeO

OH β

S

HO

OMe

4

O

OMe

+

G/S

β

G/S

S

MeO

OH

(g) Monolignol β-O-4-cross-coupling

B no β-5-crosscoupling possible!

a radical coupling

a

O OMe

b

H2O

OH2

B

β

HO

O 4

nucleophilic addition H2O

(β-O-4)

OMe

b HO

HO

H

B

OMe OH

C no β-β-unit from cross-coupling!

B H

O

OMe

HO

to produce lignin β-ether unit

4

G 5

O

E 4-O-5-unit

A β-O-4-unit

OH

4

OH

OH

β

G/S

.

+

OMe

OMe

4

.

+

OMe

B

β

HO

O 4

+

OMe

A

A OMe

A

OMe

OH

OMe

OH

H+

O

monolignol + oligolignol cross-coupling

β-aryl ether unit A threo-, syn-, RR/SS-isomer

quinone methide intermediate

β-aryl ether unit A erythro-, anti-, RS/SR-isomer

(h) Monolignol β-1-cross-coupling with a β-either end-unit +

to produce spirodienone unit in lignin

OMe

O

HO

A β •

HO

monolignol (radical)

O

Ar

•

+

radical coupling (β-1)

1

B

HO

A

HO β

HO nucleophilic addition 1

O Ar

B

β-O-4-end-unit (radical)

internal trapping

HO

HO β

O 1

O Ar

B OMe

OMe

OMe O

OMe

HO

A

HO

MeO O

H

O

dienone/quinone methide intermediate

O

spirodienone (β-1/β-O-4)

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Plate 2.1 The current theory of lignification is that it takes place via combinatorial radical coupling. (a)/(c) Dehydrodimerization of monolignols, the basis of lignan formation but a minor reaction in lignification. (a) Dehydrodimerization of coniferyl alcohol produces three dehydrodimers A–C, all of which involve coupling of at least one monolignol at its β-position, in comparable amounts. (b) Dehydrodimerization of sinapyl alcohol produces only two products, A and C. (c) Dehydrodimerization does not produce the 5–5- and 4–O-5-structures often shown in texts. (d)–(e) Cross-coupling of a monolignol with the growing polymer, the major reaction in lignification. (d) Cross-coupling of a hydroxycinnamyl alcohol with a guaiacyl (G) unit gives only two main products A and B, the β-ether A being major. (e) Cross-coupling of a hydroxycinnamyl alcohol with a syringyl (S) unit has only one major pathway leading to β-ether units A. When the polymer phenolic end unit is a β-ether, β–1-coupling may also occur to a relatively minor extent. (f ) Coupling of preformed oligomers is the source of the 5–5- and 4–O–5-units, D and E. (g)–(h) More detailed mechanisms for β–O–4- and β–1-cross-coupling reactions. (g) Cross-coupling of a monolignol (at its β-position) with a phenolic end-unit (G or S, at its 4–O-position) produces a new β–O–4-bond and introduces a new chiral center, the β-carbon. The intermediate quinone methide produced undergoes re-aromatization via nucleophilic addition to the α-carbon, usually by water, producing another new chiral center at the α-carbon. The product is completely racemic, indicating an independence from proteinaceous control. The unit’s two possible isomers (from the two racemic chiral centers), the RR/SS and the RS/SR isomers, also termed syn and anti or threo and erythro, are produced in ratios indicating kinetical control (see text). (h) Cross-coupling of a monolignol (at its β-position) with a phenolic β-aryl ether end-unit (G or S, at its 1-position) can produce a new β–1-bond. In this case, the intermediate quinone methide is trapped internally by the α-OH on the original β-ether unit, forming a novel spirodienone structure in the lignin (Setälä et al., 1999). Such structures, now readily found by NMR at low levels in most lignins (Zhang & Gellerstedt, 2001; Marita et al., 2003; Ralph et al., 2004b; Zhang et al., 2006), will cleave in mild acid, re-aromatizing the dienone to produce the ‘traditional β–1-unit’ with a phenolic B-ring and a glyceraldehyde-2-aryl ether. The existence of β-1-units in lignins as spirodienones explains why the B-rings in released β–1-structures are never phenol-etherified. In all products, the new bonds formed by the radical coupling reactions are drawn bolder. Red arrows indicate sites at which further radical coupling can occur during lignification; the lighter arrows to the 5-positions in (d)–(e) and (g)–(h) structures show where coupling can occur in G-units but not in S-units (where the 5-position is occupied by a methoxyl group).

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(a) HSQC (side-chain region)

(b) Mechanism: 5-hydroxyconiferyl alcohol to benzodioxane structures H in lignin

(i) Wild type

50 MeO HO β

α

60 5

HO

O

. OMe

P

5H

HO O

6

5

4

3

ppm

HO

5H

β

MeO

5H 4

O

5H

HO

OH

5

β

α

γ

. OH P5H

O

S/G/ 5H

OMe

OMe

M1.

O

O

OH

OMe QM2

H

(c) HMBC (Aα- and Hα-correlations) Aα, β-ethers

(ii) COMT-deficient

O

;

OMe OH P5H

OMe QM1

O S/ G/ 5H

90

MeO

OH

5H

MeO OH

α

γ

HO

. M15H

γ

β

OH2

OMe

4

α

4

S/G

HO

O

O

80

3

O

S/G

S/G

5H

70

MeO

OH

γ

50

Hα, Benzodioxanes (from 5-OH-CA incorp)

Bα, β-5s

60

γ

γ

γ 70

β

β

60

80

β

90

70 100

Hβ

Hα

S2

5H2

80

5H2

5H6

5H6

G2

G2

G6

G6

110

120

90

HO HO HO

α

O

5

β

β α

O

α

OMe

O

Methoxy C resinol (β-β)

B phenylcoumaran (β-5)

A β-aryl ether (β-O-4)

6.0 AcO

β

O

1

6

2

α

β

O

OMe HO OH

β

4

α

O

α

A5H

AcO H

H benzodioxane (β-O-4)

X1 (E)-cinnamyl alcohol

6

2

1

O

Ar

1

2

γ

OAc

HG

γ

5H

AcO α OAc H G5H 1

O Ar

2

6

5H

O

5H OMe

O Ar

OAc

6 1

O OAc

α

OMe β

O

H5H

H

5H

OMe O

2

OMe

(H) OAc

O

β

2

O

β

OMe

AcO O

α

OMe

α

H

G

OMe

γ β

6

Ar

5H O 6

O

O

D dibenzodioxocin (5-5/β-O-4)

Ar

1

O

OH β

O

α

OMe MeO

Not assigned here (mainly saccharides)

γ

5

AcO H

5.0

S

AcO

O O

AS

140 ppm

γ β

G

5

MeO

Ar

O

α

MeO

5′

5.5 AcO

γ β

AcO H

AG

130

4.95

ppm

5.05

3

6.09 6.04 6.00 5.95

4

G1ether G1free

5.86

5

6

5H1

5H1

G1ether G1free

γ

OAc

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Plate 2.3 COMT-deficient plants, alfalfa in this case (Marita et al., 2003), substitute 5-hydroxyconiferyl alcohol for sinapyl alcohol in the lignin polymerization. (a) Partial 2D 13C-1H correlative HMQC NMR spectra of lignins from (i) the wild-type and (ii) the COMT-deficient transgenic alfalfa. The major structural units (A-D, X1, and novel H) and color-coded chemical structures are shown. Dashed ovals in i) delineate the areas in which benzodioxane units H would correlate (if they were present). (b) The mechanism by which 5-hydroxyconiferyl alcohol incorporates into the lignin to produce novel benzodioxane structures H. (c) Gradient-selected 2D HMBC sub-spectra showing α-proton correlations to carbons within 3 bonds in β-aryl ether units A, β–5-units B (not highlighted) and benzodioxane units H. These spectra demonstrate that all types of lignin monomers (S, G, and 5H) undergo β–O–4-coupling producing β-ethers A (pink shading: AG, AS and A5H), and also glycerol units G5H and, in the yellow shading, that coniferyl alcohol and 5hydroxyconiferyl alcohol (and sinapyl alcohol at lower contour levels) all add to the new 5-hydroxyguaiacyl units formed after coupling of 5-hydroxyconiferyl alcohol, to form benzodioxanes HG, H5H (and HS). 5-Hydroxyconiferyl alcohol is clearly acting as a surrogate lignin monomer in this polymerization.

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HO

OH

HO

(a) Spruce lignin model

MeO

(b) Poplar lignin model

OMe

S

O

OH

HO HO

OMe

O

O

G

OH

MeO

HO O

HO

OMe

HO

O

MeO

OMe

S

OMe HO O

OH OH OH

MeO

O

O

OMe

OMe

O

S MeO

O

HO

OH

O

O

HO

OH

MeO

O

HO

OMe

G OH

OH OH

MeO OH

O

O

O

OMe MeO

MeO

OH O

HO

S

O

OMe

OH O

MeO

MeO

OH

O

S

OH

O HO

OH OMe O

O

OH

G

O

OMe O

OH

HO

O

G

MeO

OMe

HO HO

OMe

OMe

OH

O

HO OMe

OMe

HO

G

S O

O HO

MeO

HO OH

O

HO HO

OMe

HO

OMe

OMe O O

HO

HO

OMe OMe

O HO

O

OH

HO

O O

β-O-4, β-ether β-5, phenylcoumaran β-β, resinol 5-5/β-O-4, dibenzodioxocin 5-O-4, biphenyl ether β-1, spirodienone cinnamyl alcohol endgroup glycerol endgroup

S G

syringyl guaiacyl

OH OH

MeO

A B C D E F X1 X7

G

G O

MeO

HO

OH

G

G MeO

O OMe

HO

G

O OMe

O

OMe

S

OH OH

O HO

HO

OMe

HO

OH

S

O

MeO

MeO HO

OH

S

MeO

OH

O

OH OH

HO

O

HO

OH O OMe

O MeO

OMe

HO

OH

OH

OMe

O

MeO OMe

OH (CHO)

OMe O HO

OMe

OH OMe

S OH OMe

OH

Plate 2.4 Lignin polymer models for (a) a softwood (spruce) isolated lignin with 25 units, redrawn from Brunow (2001), and (b) a hardwood (poplar) isolated lignin with 20 units, redrawn from Boerjan et al. (2003). Color coding is uniform across the two models. Bold black bonds indicate the bonds formed during lignification, from radical coupling; lighter gray bonds are from post-coupling internal rearomatization reactions; α-OH groups (and their bonds) from nucleophilically added water assume the colors of their parent structures. The softwood lignin is more branched and contains a lower proportion of β-ether units A. The branch points (4 – O–5-units E, orange; dibenzodioxocin units D, dark blue) are differentiated by unique coloring, even though such units may also be β-ethers, for example. Note that each of these structures represents only one of billions of isomers. The softwood model (a) has 49 optical centers, as drawn. However, the relative stereochemistries of pairs of centers in ring structures (phenylcoumarans B, resinols C, and dibenzodioxocins D) are fixed; we frankly don’t know about the relationships in spirodienones. There are therefore at least 40 ‘real’ centers so we infer that there are at least 239 (over 500 billion) physically distinct isomers for the structure shown. For the poplar model (b), we calculate that there are 233 (over 8.5 billion) physically distinct isomers. Since lignins are not optically active, the optical centers must be generated randomly, i.e. under simple chemical control. Caution: these are ONLY MODELS! They do not imply any primary structure or sequencing in the lignins themselves but attempt to accommodate the main linkage types and their approximate relative frequencies.

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Chapter 1

Plant Phenolics – Secondary Metabolites with Diverse Functions Vincenzo Lattanzio, Paul A. Kroon, Stéphane Quideau and Dieter Treutter

1.1 Secondary metabolism in the interactions between plants and their environment Plant secondary metabolism constitutes a large reservoir of natural chemical diversity that encompasses an enormous range of compounds and enzymes, and a wide spectrum of mechanisms of gene regulation and of transport of metabolites and enzymes. Among the thousands of metabolites produced by plants, only a few are part of ‘primary’ metabolic pathways and the rest are termed ‘secondary’; this term is historical and was initially associated with inessentiality. Levels of secondary metabolites in plants are both environmentally induced as well as genetically controlled. In contrast with basic metabolism, which refers to the anabolic and catabolic processes required for cell maintenance and proliferation, secondary metabolism involves compounds present in specialized cells that are not directly essential for basic photosynthetic or respiratory metabolism, but are thought to be required for plants’ survival in the environment. Secondary metabolism is considered an integral part of the developmental program of plants, and the accumulation of secondary metabolites can demarcate the onset of developmental stages. The ability to synthesize secondary compounds has been selected throughout the course of evolution in different plant lineages when such compounds addressed specific needs. Secondary metabolites apparently act as defense (against herbivores, microbes, viruses or competing plants) and signal compounds (to attract pollinating or seed dispersing animals), as well as protecting the plant from ultraviolet radiation and oxidants. Therefore, they represent adaptive characters that have been subjected to natural selection during evolution (Hättenschwiler & Vitousek, 2000; Pichersky & Gang, 2000; Kutchan, 2001; Theis & Lerdau, 2003; Wink, 2003; Kliebenstein, 2004; Kutchan & Dixon, 2005; Memelink, 2005). This requirement for secondary metabolites to have highly diverse biological activities has led plants to accumulate a vast number of compounds. Plant genomes are variously estimated to contain 20,000–60,000 genes, and perhaps 15–20% of these encode enzymes for secondary metabolism, while the genetic complement of the fruit fly (Drosophila melanogaster) is substantially lower (13,601 predicted genes). One explanation for this Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Recent Advances in Polyphenol Research

discrepancy in the relationship between biological and genetic complexity may lie in the differences between the ways that plants and animals protect themselves against predators, pests, diseases, and abiotic stress. Animals have developed nervous and immune systems that enable them to detect and respond to danger, and they are capable of avoiding perilous situations. By contrast, plants cannot escape from their biotic and abiotic stressors, being linked to the ground by means of their root system, and therefore they must stay and protect themselves. Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. The production of chemicals that deter or kill pests and pathogens represents one mean of self-protection. The pattern of secondary metabolites in a given plant is complex; it changes in a tissue- and organ-specific way; regularly, differences can be seen between different developmental stages (e.g., organs important for survival and reproduction have the highest and most potent secondary metabolites), between individuals, and between populations (Wink, 1988; Pichersky & Gang, 2000; Osbourn et al., 2003; Wink, 2003; Noel et al., 2005). Plants produce a large number of secondary metabolites, which are classified into several groups according to their biosynthetic routes and structural features. Phenolic compounds are the most widely distributed secondary metabolites, ubiquitously present in the plant kingdom: it is estimated that about 2% of all carbon photosynthesized by plants, amounting to about 1 × 109 t per annum, is converted into flavonoids or closely related compounds (Robards & Antolovich, 1997). The terms ‘phenol’ and ‘polyphenol’ can be defined chemically as substances that possess an aromatic ring bearing one (phenol) or more (polyphenol) hydroxyl substituents, but in the context of plant phenolics such a definition is not satisfactory, since it would include compounds such as the phenolic carotenoid 3-hydroxyisorenieratene or the phenolic female sex hormone estrone, which are principally terpenoid in origin (Harborne, 1989). A first comprehensive definition of ‘plant polyphenols,’ based on the earlier proposal of T. White, E.C. Bate-Smith & T. Swain, was given by E. Haslam, who stated that the term ‘polyphenol’ (syn. vegetable tannin) refers to water-soluble phenolic compounds having molecular masses between 500 and 3,000–4,000 Da, possessing 12–16 phenolic groups and 5–7 aromatic rings per 1000 relative molecular mass, and expressing special properties such as the ability to precipitate proteins and alkaloids (Haslam, 1998). This original definition of polyphenols has broadened out considerably over the years to include phenolics with much simpler structures. A large number of these plant phenolics are small molecules with no tanning action. As a general rule, the terms ‘plant phenolics’ and ‘polyphenols’ should refer to secondary natural metabolites arising biogenetically from either the shikimate/ phenylpropanoid pathway or ‘polyketide’ acetate/malonate pathway, or both, producing monomeric and polymeric phenols and polyphenols, as chemically defined above, and which fulfill a very broad range of physiological roles in plants (Quideau, 2004, 2006). In fact, although the bulk of these compounds play cell wall structural roles, plant tissues synthesize a vast array of non-structural constituents that have various roles in plant growth and survival. Unless they are completely esterified, etherified or glycosylated, plant phenolics are normally soluble in polar organic solvents. Most phenolic glycosides are water-soluble but the corresponding aglycones are usually less so. With a few exceptions, water solubil-

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Plant Phenolics

3

ity increases with the number of hydroxyl groups present. Some phenolics are solubilized by sodium hydroxide and sodium carbonate, but in alkaline media their oxidation is enhanced and therefore treatment with alkaline solvents should either be performed under N2 or – preferably – be avoided. Phenolics with only a few hydroxyl groups are soluble in ether, chloroform, ethyl acetate, methanol, and ethanol. Methanol, ethanol, water, and alcohol–water mixtures are most commonly used for dissolving phenolic compounds for analytical purposes (Van Sumere, 1989). All phenolic compounds exhibit intense absorption in the UV region of the spectrum and those that are colored absorb strongly in the visible region as well. Each class of phenolic compound has distinctive absorption characteristics. For example, phenols and phenolic acids show spectral maxima in the range 250–290 nm; cinnamic acid derivatives have principal maxima in the range 290–330 nm; flavones and flavonols exhibit absorption bands of approximately the same intensity at about 250 and 350 nm; chalcones and aurones have an absorption peak of great intensity above 350 nm and a much less intense band at 250 nm; anthocyanins and betacyanins show rather similar absorption in the visible region (475–560 nm and 535–545 nm, respectively) and a subsidiary peak at about 270–275 nm (Mabry et al., 1970). Plants need phenolic compounds for pigmentation, growth, reproduction, resistance to pathogens, and many other functions. The structure of plant phenolics and polyphenols varies from simple molecules, such as phenolic acids, to highly polymerized compounds, such as proanthocyanidins, and several thousand (among them over 8,150 flavonoids) different compounds have been identified with a large range of structures. Several classes of phenolics have been categorized on the basis of their basic skeleton (Fig. 1.1): C6 (simple phenols, benzoquinones), C6–C1 (phenolic acids), C6–C2 (acetophenones, phenylacetic acids), C6–C3 (hydroxycinnamic acids, coumarins, phenylpropenes, chromones), C6–C4 (naphthoquinones), C6–C1–C6 (xanthones), C6–C2–C6 (stilbenes, anthraquinones), C6–C3–C6 (flavonoids, isoflavonoids), (C6–C1)2 (hydrolyzable tannins), (C6–C3)2 (lignans, neolignans), (C6–C3–C6)2 (biflavonoids), (C6–C3)n (lignins), (C6)n (catechol melanins), (C6–C3–C6)n (condensed tannins) (Harborne, 1980; Hättenschwiler & Vitousek, 2000; Iwashina, 2000). Phenolic compounds are found throughout the plant kingdom but the type of compound present varies considerably according to the phylum under consideration. Phenolics are uncommon in bacteria, fungi, and algae and the classes of phenols recorded are few; flavonoids are almost completely absent. Bryophytes are regular producers of polyphenols including flavonoids, but it is in the vascular plants that the full range of polyphenols is found (Swain, 1975; Harborne, 1980). Phenolic compounds have been synthesized during the course of evolution by different plant species when the presence of a particular secondary metabolite has conferred a selectionary advantage on the plant containing it. As previously stated, plants synthesize a greater array of secondary compounds than animals because they cannot rely on physical mobility to escape from their predators and have therefore evolved a chemical defense against such predators. Generally, the role of phenolic compounds in defense is related to their antibiotic, antinutritional or unpalatable properties. Besides their involvement in plant–animal and/or plant–microorganism relationships, plant phenolics also have key roles as the major red, blue, and purple pigments, as antioxidants and metal chelators, as signaling agents both above and below ground between

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CHO

COCH3

COOH

OH OCH3 OH (C6) Resorcinol

OH (C6-C1) Vanillin

OH (C6-C2) p-Hydroxyacetophenone CH3O

OCH3

O

O HO

OH

O

HO

HO

Glu

COOH (C6-C3) Sinapoyl-(S)-malate

O OH (C6-C1-C6) Mangiferin

OH O (C6-C4) Juglone

O

OH

OH HOOC

HO

HO

OH

HO (C6-C2-C6) Lunularic acid

O

OH O (C6-C3-C6) Phloretin OH

O

O

OH

(C6-C3-C6) Daidzein OH

OH GluO

O OH

CH O OH (C6-C3-C6) Aureusin HO

HO

HO

CH3

HO

CH3

OH OH HO

O OH

O O

O

O O OH

HO

O

OH

OH

O

OH

OH OH (C6-C3-C6)2 Procyanidin B-2

HO

O

O OH

OH O OH (C6-C2-C6)2 Emodin

HO OH

OO

OH

HO

OH

HO

OH

O

O

CH3O OH

OH HO OH (C6-C1)5 Tellimagrandin II

H

H

CH3O O HO

(C6-C3)2 (+)-Pinoresinol

Fig. 1.1 Some examples of phenolic structures.

plants and other organisms, and as UV light screens. This latter property has very much benefited some higher members of the Charophyceae, which are regarded as prototypes of amphibious plants that presumably preceded true land plants when they emerged from an aquatic environment onto the land; their successful adaptation to land was achieved

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largely by massive formation of ‘plant phenolic’ compounds. Finally, some studies have shown that phenolic metabolism is not only a protective mechanism against biotic and abiotic stresses but also part of the molecular programs that contribute to normal plant growth and development (Noel et al., 2005; Taylor & Grotewold, 2005). To achieve their function plant phenolics generally accumulate in specific tissues or cell types in which subcellular localization is highly regulated. Secondary metabolites are often transported from source cells to neighboring cells, or even further to other tissues or remote organs. Several studies have indicated a high degree of compartmentalization of phenolic compounds and of the enzymes involved in their biosynthesis. Phenolics usually accumulate in the central vacuoles of guard cells and epidermal cells as well as subepidermal cells of leaves and shoots. Furthermore, some phenolics are found covalently linked to plant cell wall, while others occur in waxes or on the external surfaces of plant organs. Some findings suggest also a deposition of flavonoids in the nuclei of certain tree species; it has been suggested that a flavonoid–DNA complex provides mutual protection against oxidative damage (Wink, 1997; Sarma & Sharma, 1999; Beckman, 2000; Croteau et al., 2000; Feucht et al., 2004; Yazaki, 2005).

1.2 Function and use of plant phenolics Plant phenolics have been considered for a long time to be waste products of primary metabolism. The defense hypothesis was not accepted by most botanists before the 20th century because most of them were not convinced of evolution and adaptive explanations. On the other hand, the potential value of plant secondary metabolites to taxonomy has been recognized for nearly 200 years, even if their practical application has been restricted to the 20th century, and predominantly to the last 40 years. The use of secondary compounds has clear advantages over the use of primary compounds in establishing phylogenetic relationships at species or infraspecific levels because differences in the complement of secondary compounds are qualitative differences whereas differences in the concentrations of primary compounds are quantitative differences, and these are subject to environmental as well as to genetic control. Flavonoids are particularly convenient for this purpose as they are widely distributed among plants and are chemically stable. The existence of a common pattern of secondary compounds may indeed provide much clearer evidence of common ancestry than morphological similarities attributable either to common ancestry or to convergent evolution. Moreover, the existence of a characteristic phenolic pattern, which taxonomists use to separate species, can also have enough adaptive value for survival through natural selection (Bell, 1980; Van Sumere & Vande Casteele, 1985; Lattanzio et al., 1996; Wink, 2003). In the past 30 years the view of plant phenolic metabolites as one of nature’s meaningless waste products has been replaced by the current opinion that plant phenolics play crucial roles in plant ecology and plant physiology. In addition, biomedical research has revealed that dietary phenolics, because of their antioxidant and free radical scavenging properties, play important roles in the prevention of many of the major contemporary chronic diseases (Kutchan, 2001).

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1.2.1 UV sunscreens The hypothesis of the protective role of phenolics against harmful UV rays is supported by the enhanced levels of phenolics observed in plants exposed to strong UV radiation. Plants in the field are exposed to ambient solar ultraviolet-B (UV-B) radiation (280–320 nm), which is an environmental challenge negatively affecting DNA, proteins and membranes, thus leading to altered metabolism through the generation of reactive oxygen species (ROS). Plants protect themselves from this harmful radiation by synthesizing phenolic compounds, which act as a screen inside the epidermal cell layer, and by making adjustments to the antioxidant systems at both cell and whole organism level. By means of this mechanism phenolics would prevent mutagenesis and cellular death by dimerization of thymine units in the DNA, which shows an absorption maximum at 260 nm, and possible photodestruction of coenzymes NAD or NADP, which have a maximum at 260 nm. It has been proposed that flavonoids with their high absorptivity at 250–270 and 335–360 nm act as good UV screens (Swain, 1975; Carletti et al., 2003). It is noticeable that tropical and high-altitude plants contain a higher proportion of flavonoids than temperate ones do. Several studies have demonstrated the change in flavonoid composition of plant leaves as a consequence of an excess of light or UV radiation. The activation of flavonoid biosynthetic genes by UV radiation has been shown in a number of studies (Chappell & Hahlbrock, 1984; Olsson et al., 1998; Hofmann et al., 2000; Logemann et al., 2000; Kolb et al., 2001). The importance of flavonoids in UV protection has also been proved using mutants of Arabidopsis that have a block in flavonoid production and are, therefore, UV-hypersensitive phenotypes (Ryan et al., 2001). These studies suggest that other phenolic compounds may be at least as important as flavonoids in UV protection. Speculating about the role of phenylpropanoids as sunscreens to absorb UV-B irradiation in various phenylpropanoid mutants of Arabidopsis thaliana, Kliebenstein (2004) observed that all phenylpropanoid-deficient mutants exposed to UV-B radiation were more UV-B sensitive in comparison with the wild type, but there were dramatic differences in sensitivity to UV-B between these mutants. These results have suggested that both preformed hydroxycinnamic acid sinapoyl esters and induced flavonols act as UV-B protectants and that the importance of hydroxycinnamic acids and flavonols is directly related to their relative concentrations.

1.2.2 Phenolics as signal compounds There are several reports suggesting that phenolic compounds influence the pools and fluxes of inorganic and organic soil nutrients. Polyphenols enter the soil mainly as leachates from above- and below-ground parts of plants and/or within above- and below-ground plant litter. Phenolic compounds can directly affect the composition and activity of decomposer communities thus influencing the rates of decomposition and nutrient cycling. Different types of soluble phenolics, such as ferulic acid, gallic acid or flavonoids, have been found to either stimulate or inhibit spore germination and hyphal growth of saprophytic fungi. Mycorrhizal fungi might be even more sensitive to phenolic compounds, but again different types of polyphenols can have opposite effects. Plant mycorrhizal infection, nutrient uptake and plant growth can be impaired by specific phenolics released by competitors

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(Hättenschwiler & Vitousek, 2000), a process refered to as allelopathy. Commonly this term is mainly used to describe the chemical interaction between two plants. In plants, allelochemicals can be present in leaves, bark, roots, root exudates, flowers, and fruits. The delivery of allelochemicals into the rhizosphere is often thought to occur through leaching from leaves and other aerial plant parts, through volatile emission, by root exudation, and by the breakdown of bark and leaf litter. Some identified phenolic allelochemicals are: p-hydroxy benzoic acid and p-coumaric acid (present in leaves), quercetin, juglone and 2,4-dihydroxy-1,4 (2H) benzoxazin-3-one (DIBOA) (present in leaves, bark and root exudates), and (−)catechin and sorgoleone (found in rhizosphere and root exudates) (Inderjit & Gross, 2002; Weir et al., 2004). Bais et al. (2003) present evidence that Centaurea maculosa, an invasive species in the western USA, displaces native plant species by exuding the phytotoxin (−)-catechin from its roots. This allelochemical triggers a wave of reactive oxygen species initiated at the root meristem, which leads to a Ca2+ signaling cascade triggering genomewide changes in gene expression and, ultimately, death of the root system. Resistance to allelochemicals is largely accomplished through detoxification pathways that involve the modification, followed by the secretion or the vacuolar sequestration, of xenobiotics. In addition to affecting the soil microorganisms responsible for nutrient mineralization, phenolic compounds can alter nitrogen availability by complexing proteins. Polyphenol– protein complexes originate either during senescence of plant tissues, when polyphenols stored in the vacuole come into contact with cytoplasmatic proteins, or in the soil, when polyphenols complex proteins from litter or extracellular enzymes from microorganisms. These complexes cause the brown coloring of senescent leaves and are resistant to most decomposing organisms, except basidiomycetes with the appropriate polyphenol oxidase activity, and earthworms, which can directly use a large proportion of nitrogen contained in the complexes (Haslam, 1998; Lattanzio, 2003; Papadopoulou & Frazier, 2004). Plants depend on the ability of roots to communicate with microbes. The converse is also true; many bacteria and fungi are dependent on associations with plants that are often regulated by root exudates. Biological interactions that are driven by root exudates are more complex and include signal traffic between roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil properties to the roots. Specific compounds identified in root exudates have been shown to play roles in these interactions. For example, isoflavonoids and flavonoids present in the root exudates of a variety of leguminous plants activate the Rhizobium genes responsible for the nodulation process, and might be responsible for vesicular–arbuscular mycorrhiza colonization. Flavonoid profiles in root exudates differ considerably among legumes, and this specificity enables mutualists and beneficial bacteria such as rhizobia to distinguish their hosts from other legumes (Bais et al., 2004, 2006). Although rhizobia colonize roots in a way that is reminiscent of pathogenic microorganisms, no host plant defense reactions are triggered during successful symbioses: symbiotic interactions, by definition, are beneficial to both partners. Nevertheless, the plants obviously control the invading bacteria; failure in effective nodule formation or infections with rhizobia defective in surface polysaccharides often result in pathogenic responses. Symbiosis between leguminous plants and rhizobia involves the de novo development of a specialized plant organ, the root nodule. In the nodules, rhizobia fix dinitrogen into ammonia, which is assimilated by the host plant, and, in turn, rhizobia are supplied with carbon compounds.

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The nodulation process in rhizobia–legume symbiosis requires a sequence of highly regulated and coordinated events, initiated by an exchange of specific signaling compounds between both partners. The prerequisite for the formation of a nitrogen-fixing nodule is the generation of flavonoid signal(s) that are secreted from the root exudates of the leguminous host. The flavonoid aglycone is presumed to diffuse into the rhizobial bacteria, perhaps through porins (Mithöfer, 2002). The flavonoids in root exudates induce, in conjunction with NodD protein [the product of the only nodulation (nod ) gene constitutively expressed by rizobia], the transcription of an important set of Rhizobium Nod genes. The Nod genes are responsible for the synthesis of sulfated acylated tetraglucosamine glycolipids (NodRm-1), the so-called Nod factors, that are secreted by induced rhizobia and initiate root-hair curling and cortical cell division in the infectible zone of legume roots (Long, 1989; Brewin, 1991; Aoki et al., 2000; Limpens & Bisseling, 2003; Kobayashi et al., 2004). Examples of flavonoids found to be active in the induction of Nod gene expression are eriodictyol (3′,4′,5,7-tetrahydroxyflavanone) and apigenin-7-O-glucoside isolated from pea root exudate, active at a concentration lower than 50 nM, and luteolin and chrysoeriol (3′-methoxyluteolin) released from alfalfa seeds. Other flavonoid classes released naturally from legume plants to induce nod-gene expression in their appropriate microsymbionts are flavanones, such as naringenin and hesperetin, chalcones, and isoflavonoids, such as daidzein and genistein (Hartwig et al., 1989; Mathesius et al., 1998).

1.2.3 Phenolics as pigments An important role of flavonoids is to serve as visual signals by acting as pigments in fruits and flowers, firstly to attract animals as pollinators in flowers, and later to attract animals to eat the fruits and thereby help in seed dispersal. Fruit colors are primarily determined genetically, although environmental factors such as temperature, light conditions, and availability of nutrients can have an effect on flavonoid composition and on the final hue of the fruit. Concerning anthocyanins, which are mainly responsible for the bluish to purple and reddish colors in plants, several different factors can affect the final color of the fruit or flower. Delphinidin-derived anthocyanins are known to be responsible for bluish colors, whereas cyanidin- and pelargonidin-derived anthocyanins are found from mauve and reddish tissues, respectively. Anthocyanins readily form complexes with so-called copigments that can intensify and modify the initial color given by the pigment. Apparently, almost all polyphenols, as well as other molecules such as purines, alkaloids and metallic cations, have the ability to function as co-pigments. In addition, the temperature and pH of the vacuolar solution may affect the final color (Brouillard & Dangles, 1994; Brouillard et al., 1997; Mol et al., 1998; de Freitas & Mateus, 2006). Chalcones and aurones are two classes of flavonoids that contribute to yellow flower color in a number of plants; for example, the chalcone isosalipurposide is the sole yellow coloring matter of yellow carnation, while the aurone aureusidin, occurring as 6-glucoside aureusin, is the major yellow pigment in the snapdragon (Antirrhinum majus). Variations in hydroxylation pattern of the five commonest flavones and flavonols (apigenin, luteolin, kaempferol, quercetin, and myricetin) produce structures that give white, yellow or ivory colors to the tissues in which they are located. For example, the insertion of a 2′-hydroxyl group into luteolin gives the flavone isoetin, which is a yellow flower

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pigment found in several Compositae members. The introduction of a hydroxyl group at the 6- or 8-positions of quercetin causes a significant shift in color, and the resulting compounds (e.g., quercetagetin, found in flowers of Coronilla, Lotus, Primula, and Rhododendron species, and gossypetin, the pigment of Gossypium hirsutum flowers) are yellow instead of pale cream, as is quercetin. Finally, most of the naturally occurring phenolic pigments are quinones: benzoquinones, naphthoquinones, and anthraquinones. Benzoquinones are uncommon in plants, while they occur in fungi, mainly in the Hyphomycetes and Basidiomycetes. Primin (6-methoxy-2-n-pentylbenzoquinone) is a benzoquinone, and is present in the glandular hair of leaves of Primula obconica. On the other hand, most naphthoquinone pigments are of higher plant origin such as plumbagin, an orange pigment identified in Plumbago capensis, which is also present in bound form in members of Plumbaginaceae, Droseraceae, and Ebenaceae. The largest group of natural quinones are the tricyclic anthraquinones found especially in Leguminosae, Liliaceae, Polygonaceae, Rhamnaceae, Rubiaceae, and Scrophulariaceae. Some anthraquinone pigments such as alizarin (2,3-dihydroxyanthraquinone), anthragallol (2,3,4-trihydroxyanthraquinone) and purpurin (1,2,4-trihydroxyanthraquinone) have been used in the past for dyeing textiles (Harborne, 1980).

1.2.4 Phenolics and plant growth As far as the role of plant phenolics as internal physiological regulators or chemical messengers within the intact plant is concerned, some information is available. Hydroxycinnamic acids, particularly p-coumaric acid and ferulic acid, are found in the insoluble or cell wall fraction also as esters. These pools of wall-bound acids act as a reservoir of phenylpropanoid units for lignin biosynthesis or may even represent the beginnings of lignification itself. In addition, by radical dimerization of ferulates, polysaccharide– polysaccharide cross-linking is effected: feruloylation occurs on the arabinose or galactose side-chains of pectic polysaccharides. A possible role of feruloyl pectin may be in the regulation of cell expansion, possibly through coupling reactions leading to the production of diferulate. Broadly speaking, hydroxycinnamates play an important role in plant cell walls in the maintenance of integrity, shape, and defense against the ingress of pathogens. Cross-linking among cell-wall polymers can modify its properties such as accessibility, extensibility, plasticity, digestibility, and adherence. At the completion of the expansion phase of cell growth, mechanical properties of the wall change and they are no longer capable of stress relaxation (Fry, 1983; Ralph et al., 1994; Kroon & Williamson, 1996; Hatfield et al., 1999; Ward et al., 2001; Bunzel et al., 2005; Faulds & Kroon, 2006). Another proposed role for cell-wall bound hydroxycinnamic acids, regarding phototropism in higher plants, concerns the chemical nature of the UV-A and the blue light photoreceptors and the mechanism of the transduction of light energy to physical changes in the properties of cell walls. The presence of a double bond in the lateral chain of the hydroxycinnamic acids leads to the possible existence of two isomeric forms: Z (cis) and E (trans). Native compounds are mainly of the trans form, although interconversion of the two forms in situ may lead to certain physiological responses. It has been proposed that the E (trans)/Z (cis) reversible photoisomerism, in UV-A, of cell-wall ferulate and diferulate–carbohydrate esters, concerning a large population of bound molecules, can be regarded as a mechanism for

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the transduction of light energy leading to changes in wall structure and hence water flux, turgor pressure and growth. Unilateral light would cause phototropism (Turner et al., 1993). Some intriguing effects of plant phenolics are the ones associated with the growth hormone auxin (indole-3-acetic acid, IAA). Monohydroxy B-ring flavonoids are suggested as cofactors of peroxidase functioning as an IAA oxidase that destroys the hormone, whereas dihydroxy B-ring forms act as inhibitors of the IAA degrading activity (Stafford, 1991; Mathesius, 2001). Mono- and dihydroxy-flavonoids are also implicated as inhibitors of IAA transport across the plasma membrane by binding to a plasma membrane protein known as the naphthylphthalamic acid (NPA) receptor. NPA is a synthetic compound, which is believed to bind a regulatory protein that is associated with the transmembrane efflux of IAA anions mediated by carrier. Some flavonoids, such as quercetin, apigenin, and kaempferol, do not directly compete with IAA but act through their own receptor, the NPA receptor, in the plant cell plasma membrane, thus blocking the polar auxin transport (Jacobs & Rubery, 1988; Murphy et al., 2000; Brown et al., 2001; Muday & DeLong, 2001; Cooke et al., 2002). An additional role for flavonoids in functional pollen development has been observed in perunia plants using antisense chalcone synthase (the key enzyme that catalyzes the initial step of the branch of the phenylpropanoid pathway that leads to flavonoids), and maize mutants lacking chalcone synthase. A lack of chalcone synthase activity has a pleiotropic effect in petunia and maize mutants; pollen fertility as well as flavonoid synthesis is disrupted, but fertility could be restored by adding micromolar quantities of the flavonol aglycones kaempferol or quercetin to mature pollen at pollination (Mo et al., 1992; Van der Meer et al., 1992). In general, plants are rooted and unable to move from place to place by themselves. However, some plants are known to be able to move in certain ways. Especially, the circadian rhythmic leaf movement known as nyctinasty is widely observed in all leguminous plants. Nyctinastic leaf movement is induced by the swelling and shrinking of motor cells in the pulvini, an organ located in the joint of the leaf. Nyctinastic movement are believed to be controlled by Schildknecht’s turgorins, which induce leaf-closing movement of the plants (Schildknecht, 1983). Schildknecht said that all leaf movements (nyctinastic and seismonastic movements) are controlled by turgorins, a new class of phytohormones that regulate the turgor of plants. Leaf-opening substances differ in certain aspects from IAA, which had been believed to induce the leaf-opening of nyctinastic plants: (i) the bioactivities of the leaf-opening substances (10−5 to 10−7 M) are much stronger than that of IAA (> 10−4 M), and (ii) the bioactivity of the leaf-opening substances is specific to the genus of the plant while that of IAA is non-specific (Ueda et al., 2001). Some identified phenolic turgorins are: gallic acid 4-O-(β-d-glucopyranosyl-6′-sulfate) and gentisic acid 5-O-β-d-glucopyranoside, which are localized in the pulvini in Mimosa pudica L., cis-pcoumaric acid 4-O-β-d-glucopyranoside, found in Cassia mimosoides L., and cis-pcoumaroylagmatine, identified in Albizzia julibrissin Durazz (Varin et al. 1997; Ueda et al., 2002; Ueda et al., 2003). Finally, there exist in plant material phenolics affecting seed germination and dormancy, and these substances, found in both seed coats and embryos, have been identified as phenolic acids, hydroxycinnamic acids, and coumarins. In this context, non-germinating seeds of Melilotus alba were found to possess a large amount of free coumarin, while in rapidly germinating seeds coumarinic acid β-glucoside was more prevalent. Another

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naturally occurring phenolic compound inhibiting the germination of seeds of the same or of other species is ferulic acid; a pure solution of ferulic acid gave strong inhibition of Raphanus sativus seed germination at a concentration of 10−4 M. It has been suggested that phenolics may be active as germination inhibitors by inhibiting the transport of amino acids and the formation of proteins in the seeds (Van Sumere et al., 1972). It must be stressed that the multiple cellular roles of plant phenolics can reflect their chemical diversity, or might suggest the existence of cellular targets shared between many of these seemingly disparate processes (Taylor & Grotewold, 2005).

1.2.5 Phenolics and plant defense Plants encounter numerous pests and pathogens in the natural environment. An appropriate response to attack by such organisms can lead to tolerance or resistance mechanisms that enable the plant to survive. Resistance mechanisms refer to traits that inhibit or limit attack, while tolerance strategies do not limit attack but reduce or offset the consequences to the plant’s fitness by adjusting its physiology to buffer the effects of herbivory or diseases. Resistance strategies include physical and/or chemical barriers, mechanisms that rapidly clear infection or herbivory (hypersensitive response), and processes that limit the spread of damage within the host (such as localized cell death). Tolerance often involves some degree of compensation for disease damage. Plants can tolerate infection or herbivory by increasing the chlorophyll concentration in leaves, increasing the size of new leaves or the number of new branches, advancing the timing of bud break, delaying the senescence of infected tissues, and increasing the uptake of nutrients (Paul et al., 2000; Roy & Kirchner, 2000; Taylor et al., 2004; Dietrich et al., 2005). Most plants produce a broad range of secondary metabolites that are toxic to pathogens and herbivores, either as part of their normal program of growth and development or in response to biotic stress. Preformed antibiotic compounds that occur constitutively in healthy plants are likely to represent inbuilt chemical barriers to herbivorous and fungal enemies and may protect plants against attack by a wide range of potential pests and pathogens. In contrast, induced defense compounds are synthesized in response to biotic stress as part of the plant’s defense response and are restricted to the damaged tissue. Both tolerance and resistance traits require the reallocation of host resources, therefore defensive chemicals are considered to be costly for plants, reducing the fitness of the host in the absence of disease, because resistance (R) genes might impose metabolic costs on plants (e.g., lower growth rates than their sensitive counterparts). When resistant genotypes have relatively low fitness in the absence of pests, it is often assumed that their poor performance must be explained by the energetic drain involved in building and maintaining a chemical or structural defense, metal hyperaccumulation, temporal or spatial escape. All these mechanisms include allocation costs (the costs due to diversion of limited energy and resources away from primary metabolism), storage costs, ecological (i.e. environmental) costs (negative effects of resistance on one of the myriad of interactions between a plant and its environment that affect a plant’s fitness under natural growing condition), etc. One way for a plant to reduce these costs is to synthesize defense compounds only after there has been some degree of initial damage by a pathogen or insect; this strategy is inherently risky because the initial attack may be too rapid or too severe for an effective defense response. Therefore, plants that are

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likely to suffer frequent and/or serious damage may be better off investing mainly in constitutive defenses, whereas plants that are attacked rarely may rely predominantly on induced defenses. Inducible defenses are commonly thought not to accrue fitness costs in the absence of pests and pathogens. In reality, it is more likely that inducible systems will simply be less costly than constitutive systems, because inducible resistance depends on maintaining wound-detection pathways, defense precursors, and storage vesicles, all of which require allocation of both energy and resources away from growth and reproduction. (Kombrink & Hahlbrock, 1990; Morrissey & Osbourn, 1999; Purrington, 2000; Brown, 2002; Wittstock & Gershenzon, 2002; Brown, 2003; Koricheva et al., 2004). However, the fitness costs of inducible resistance are difficult to measure because one must compare the fitness of non-induced plants that possess the above components (i.e. resistant genotypes) to the fitness of plants that lack all of the components (i.e. susceptible genotypes). In this connection, Nicholson and co-authors (Lo & Nicholson, 1998; Wharton & Nicholson, 2000; Boddu et al., 2004) presented evidence strongly suggesting that, when exposed to an inducer such as non-pathogenic fungi, sorghum (Sorghum bicolor L. Moench) ‘shuts down’ the synthesis of anthocyanin pigments, in favor of 3-deoxyanthocyanidin phytoalexin synthesis. The synthesis of these reddish-brown pigments is a site-specific response localized around the site of attempted fungal penetration. In inoculated sorghum mesocotyls 3-deoxyanthocyanidin phytoalexins accumulate between 24 and 48 h after inoculation in intracellular inclusion bodies, which migrate towards the site of fungal penetration and then release their contents, killing both the fungus and the cells that synthesize them. The authors put forward the hypothesis that the synthesis of 3-deoxyanthocyanidins and 3hydroxylated anthocyanidins in sorghum occurs by two partially overlapping but competing biosynthetic and regulatory pathways: inoculation with the non-pathogenic fungus reduced the synthesis of 3-hydroxylated anthocyanidins by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast, fungal inoculation resulted in the synthesis of 3deoxyanthocyanidin phytoalexins. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. These studies suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs of the defense response. An understanding of the regulation of the diversion of metabolism leading to resistance expression would provide insights for the development of innovative strategies to enhance disease resistance in sorghum and related crop species. A very interesting link between primary and secondary metabolism has been studied by Shetty and co-workers (Shetty, 1997; Yang & Shetty, 1998; Shetty, 2004). Phenolic metabolites in plants are efficiently produced through an alternative mode of metabolism linking proline synthesis with the oxidative pentose phosphate pathway. Proline biosynthesis coupled to the oxidative pentose phosphate pathway stimulates the synthesis of NADPH2 and sugar phosphates for anabolic pathways, including phenolic biosynthetic pathways. In this connection, the effects of nutritional stress and exogenous proline on total phenolics and rosmarinic acid (RA) content in shoot cultures of oregano (Origanum vulgare), grown on Murashige and Skoog (MS) medium, have been investigated (Table 1.1). The basal levels of total phenolics and of RA (control) in 15-day-old oregano shoots were ∼3.40 and 0.83 mg/g of fresh weight, respectively. All treatments, proline (5 mM) and half-force

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Table 1.1 Total phenolic content (as mg rosmarinic acid equivalents/g fresh weight), rosmarinic acid content (mg/g fresh weight), antioxidant activity (IC50 = μg of total phenolics to give 50% of inhibition of model reaction (Trolox), as interpolated by dose-response curves) of oregano shoot extract with different treatments, and shoot fresh weight (mg). Treatment MS MS1/2 MS + Proline5mM

Total phenolics

Rosmarinic acid

Antioxidant activity

Fresh weight/shoot

3.40 6.20 4.10

0.83 1.83 1.73

5.40 4.20 4.60

26.70 17.80 44.40

medium (MS1/2, nutritional stress), compared to the control group, showed a statistically significant increase in RA and total phenolics content (Cicco et al., 2004). It is well known that, as a result of a natural defense mechanism for dealing with a stress imposed by unfavorable conditions (both biotic stresses, such as pathogen infection, and abiotic stresses, such as nutrient deficiency), the plant is forced to accumulate a large quantity of free proline (proline accumulation, induced by stress conditions, is mediated both by increased synthesis and reduced oxidation of the amino acid). High levels of proline synthesis during stress may maintain NADP+/NADPH ratios at values compatible with metabolism under normal conditions, i.e., proline synthesis accompanied by the oxidation of NADPH. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors (ribose-5-phosphate and, in turn, erythrose-4-phosphate, which together with phosphoenolpyruvate acts as a precursor for phenolic biosynthesis via the shikimic acid pathway) to support the demand for increased phenolic metabolite production during stress. The alternating oxidation of NADPH by proline synthesis and reduction of NADP+ by the two oxidative steps of the oxidative pentose phosphate pathway serve to link both pathways and thereby facilitate the continuation of high rates of proline synthesis during stress. Although ribulose-5-phosphate and erythrose-4-phosphate are also intermediates in the reductive pentose phosphate pathway in plants, the decline in photosynthetic rates accompanying stress is likely to necessitate increased rates of their production by the oxidative pentose phosphate pathway (Hare & Cress, 1997). The increase in total phenolics and RA content in response to exogenous proline suggests that proline oxidation may have occurred in the mitochondrial matrix. If this were the case, proline oxidation could drive the oxidative pentose phosphate pathway by recycling glutamic acid into the cytosol to generate a redox cycle (Zheng et al., 2001; Shetty et al., 2002). These results suggest that perturbing proline metabolism may be a useful route for redirecting metabolites from the oxidative pentose phosphate pathway toward total phenolic and RA synthesis, thus regulating phenolic secondary metabolites at the molecular level. 1.2.5.1 Fungal pathogens It is estimated that there are about 250,000 species of higher plants, but six times as many (1.5 million) species of fungi. Fungi are, ultimately, all dependent on plants for their carbon and energy source, like most other organisms that are not able to photosynthesize.

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Fortunately for plants, the relationship between them and fungi is usually a mutually beneficial one (saprophytic fungi, mycorrhizae, and endophytes). A small minority of fungal species have developed further and broken the fine balance of mutual benefit to become plant pathogens (Grayer & Kokubun, 2001). Therefore, plants are continuously exposed to the biotic stress exerted by organisms with which plants have symbiotic or pathogenic interaction. Indeed, in interactions between plants and microbial pathogens, resistance is the rule and disease the exception. This phenomenon is known as non-host resistance or speciesspecific resistance1 and is thought to explain why a pathogen can cause disease in particular plant species but not in others. Explanations why plants may be unsuitable as hosts are: (i) plants do not support the invading pathogen’s lifestyle and therefore are not substrates for microbial growth; (ii) preformed physical or chemical barriers constitutively present on the plant surface (leaf hairs, wax layers, rigid cell walls, and antimicrobial secondary metabolites) prevent pathogen invasion and spread; (iii) the plant’s recognition of pathogens induces its endogenous multi-component defense system. There is considerable evidence that preformed defenses are a major component of non-host resistance, particularly in non-domesticated plants. Plants contain preformed peptides, proteins, and secondary metabolites (phenolics, sulfur compounds, saponins, cyanogenic glycosides, and glucosinolates). The multi-component defense response induced after the pathogen attack requires a substantial commitment of cellular resources, including extensive genetic reprogramming, because the induced expression of a large number of defense-related genes is essential for plants to counter pathogen attack. Many defense-related genes encode proteins possessing antifungal and antimicrobial activities or enzymes that catalyze defense metabolites, known as phytoalexins. Thus, defense responses are kept under tight genetic control and are activated only when the plant detects a potential invader. Other responses of invaded cells result from allosteric enzyme activation initiating tissue reinforcement by oxidative cross-linking, and apposition of callose and/or lignin. The initial perception of the signal also leads to the production of an endogenous systemically translocated signal that has the function of activating defense mechanisms in parts of the plant remotely located from the initial site of interaction, providing durable protection against challenge infection by a broad range of pathogens. Other defense genes encode these regulatory proteins, important for defense-signal transduction, and this form of induced defense response is referred to as systemic acquired resistance (SAR) (Lattanzio & Palmieri, 1994; Lattanzio et al., 1994; Heath, 2000; Huitema et al., 2003; Mysore & Ryu, 2004; Nürnberger & Lipka, 2005; Treutter, 2006). Preformed antibiotic phenolics (phytoanticipins) are stored in plant cells mainly as inactive bound forms but are readily converted into biologically active antibiotics by plant hydrolysing enzymes (glycosidases) in response to pathogen attack. These compounds are considered as preformed antibiotics since the plant enzymes that activate them are already present but are separated from their substrates by compartmentalization, enabling rapid activation without a requirement for the transcription of new gene products (Osbourn, 1996). In such cases, free phenolics are likely to be much more toxic to the invading organism than the bound forms. In addition, even if preformed antifungal phenolics are present in healthy plants at levels that are anticipated to be antimicrobial, their levels may increase further in response to challenge by pathogens. The distribution of preformed antifungal phenolics within plants is often tissue-specific and there is a tendency for many

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lipophilic compounds (e.g., flavone and flavonol methyl ethers) to be located at the plant surface (e.g., in leaf wax and bud exudates) or in the cytoplasmic fraction within the epidermal cells, suggesting that they may indeed act as deterrents to pathogens (Nicholson & Hammerschmidt, 1992; Osbourn, 1996; Wink, 1997; Morrissey & Osbourn, 1999; Beckman, 2000; Lattanzio et al., 2001; Katagiri et al., 2002). The first demonstrated example of phenolics providing disease resistance was the case of onion scales accumulating sufficient quantities of catechol and protocatechuic acid to prevent onion smudge disease, caused by Colletotrichum circinans. The colored outer onion scales of resistant onion varieties contain enough of these two phenols to reduce spore germination of C. circinans to below 2%, while susceptible varieties lack these compounds and the germination rate is over 90% (Link et al., 1929; Angell et al., 1930; Link & Walker, 1933; Walker & Stahmann, 1955). Pre-existing antifungal phenolics are simple phenols, phenolic acids, flavonols and dihydrochalcones. In addition, many flavones and flavanones have been shown to be active against fungal pathogens commonly found during the storage of fruits and vegetables, e.g., Aspergillus sp., B. cinerea and F. oxysporum (Weidenbörner et al., 1990). The fungicidal activity of a number of stilbenes and related compounds has been tested against several fungi, including some pathogens, which infect grapes during storage. Lipophilicity and/or the presence of at least one acidic hydroxyl group are, therefore, considered to be structural features essential for good antifungal activity. Lipophilicity allows active phenols to penetrate biological membranes, while hydroxyl groups may act by uncoupling oxidative phosphorylation (Wang et al., 1989; Tomás-Barberán et al., 1990; Parvez et al., 2004). The toxicity of tannins, hydrolyzable tannins and proanthocyanidins, usually estimated by measurement of the reduction in the in-vitro growth of mycelium, is well documented for several filamentous fungi, for example B. cinerea, Aspergillus niger, Colletotrichum graminicola, Gloeophyllum trabeum, Trichoderma viride, and Penicillium sp. Tannins are quite potent antibiotics. In temperate trees, tannins and related phenolic compounds preserve heartwood from fungal decay and inhibit extracellular hydrolases from invading pathogens, thus preventing their rapid deployment in the plant (Swain, 1977; Harborne, 1980; Scalbert, 1991). It is possible that inhibition of extracellular fungal enzymes (cellulase, pectinase, laccase, xylanase, etc.), nutrient deprivation of substrates (metal complexation, protein insolubilization) and action on fungal membranes (inhibition of oxidative phosphorylation) are involved in tannin toxicity. When a pathogen manages to overcome constitutive defense barriers it may become subject to recognition at the plasma membrane of plant cells. Activation of inducible plantdefense responses is probably brought about by the recognition of invariant pathogenassociated molecular patterns (PAMP) that are characteristic of whole classes of microbial organisms; PAMP perception systems trigger signaling cascades whose recognition is very likely to activate defense responses in natural plant–pathogen encounters (Nürnberger & Lipka, 2005). Plants respond to pathogens by activating broad-spectrum innate immune responses that can be expressed locally at the site of pathogen invasion as well as systemically in the uninfected tissue. Locally induced defense responses, which restrict pathogen infections to the site of attempted ingress, are characterized by a hypersensitive response (HR), a complex, early

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defense response that causes necrosis and cell death in order to restrict the growth of a pathogen. A ring of tissue around the developing lesions becomes fully refractory to subsequent infection (localized acquired resistance). Host resistance (R) genes detect the pathogen and change the membrane potential and the ion permeability of the plasma membrane. In phase 1 of the response, the R genes trigger an increase in extracellular pH and K+ concentration, while eliciting an influx of calcium and hydrogen ions into the cell. The outward K+ and the inward Ca+2 and H+ ion fluxes are interdependent and trigger the HR, resulting in cell death and formation of local lesions. In phase 2, cells undergoing the HR produce reactive oxygen species; lipid peroxidation and lipid damage may be partially responsible for some of these cell changes and probably affect membrane function. Phenolics and phytoalexins, and other compounds, are synthesized in cells surrounding the lesion. Callose and lignin are deposited and pathogenesis-related proteins (PRs), such as peroxidase, are induced. Peroxidase is of the lignin-forming type and could be involved in the strengthening of the plant cell wall. Subsequently, the hypersensitive response triggers a general resistance mechanism rendering uninfected parts of the plant less sensitive to further attack by pathogens, a phenomenon called systemic acquired resistance (SAR), suggesting that as a result of the initial infection, a signal that primes the plant to respond more effectively to subsequent infection is generated, transported and propagated (Lamb & Dixon, 1997; Sticher et al., 1997; Van Loon, 1997; Maleck & Lawton, 1998; MauchMani & Metraux, 1998; Hammerschmidt, 1999; Durrant & Dong, 2004). The potential of plants to react to an invader depends upon a signal released from the infected tissue and translocated to other parts of the plant where it induces defense reactions. Salicylic acid is considered one of the key signaling molecules that activate plant defense responses against invading pathogens (Du & Klessig, 1997; Coquoz et al., 1998; Mauch-Mani & Métraux, 1998; Métraux, 2001; Grüner et al., 2003; Shah, 2003; Mysore & Ryu, 2004). However, recent advances in understanding plant defense signaling have revealed that plants employ a network of signal transduction pathways, some of which are independent of salicylic acid. Evidence is emerging that jasmonic acid and ethylene play key roles in these salicylic acid-independent pathways. Cross-talk between the salicylic acid-dependent and the salicylic acid-independent pathways provides great regulatory potential for activating multiple resistance mechanisms in varying combinations (Costet et al., 1999; Clarke et al., 2000; van Wees et al., 2000; Traw & Bergelson, 2003). Phenolics also play a mayor role in the post-harvest resistance of fruit. The ripe fruit is the one part of the plant which is not likely to be defended chemically, since it is provided for animals in return for the widespread dispersal of the seed that lies within the fruit. By contrast, the seed and the seed coat usually possess some chemical toxins, although they are often well protected by physical structures in order to ensure that the seed is not consumed along with the fruit. The unripe fruit will, however, differ from the ripe fruit in being protected to some degree by physico-chemical barriers from herbivory and fungal pathogens, since the seed within is not yet ready for distribution. Chemical changes during ripening reduce or eliminate these barriers. In fact, it is well known that the resistance of unripe fruits to fungal decay is associated with the presence of preformed antifungal compounds in the peel. Pathogens often infect unripe fruits but then they remain quiescent, with the onset of decay coinciding with decreases in concentrations of antifungal phenolics to subtoxic levels as the fruit ripens. The levels of these chemical barriers, as well as the

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ability of the harvested fruits to synthesize phytoalexins, decline during ripening more rapidly in disease-susceptible cultivars than in resistant ones (Lattanzio & Palmieri, 1994; Lattanzio et al., 1994; Prusky, 1996; Lattanzio et al., 2001). Post-harvest treatments such as irradiation, curing (heat treatment), high CO2 concentration, superatmospheric oxygen levels and ultra low oxygen (ULO) concentration, affect the post-harvest quality of fruit and vegetables by inducing a relevant biosynthetic increase in endogenous phenolic compounds which, in turn, enhances host resistance to post-harvest pathogens (Hanotel et al., 1995; Civello et al., 1997; Cantos et al., 2000; D’hallewin et al., 2000; Kader & Ben-Yehoshua, 2000; Zhou et al., 2000; Assis et al., 2001). UV-C irradiation alters the levels of the flavanone naringin and the polymethoxyflavone tangeretin in the peel of Citrus aurantium fruits and this, in turn, reduces the growth of Penicillium digitatum on irradiated fruits. It has been suggested that the two constitutive flavonoids, naringin and tangeretin, may act as fungitoxins in the resistance mechanism against fungal attack, acting as first and second defense barriers, respectively, since tangeretin is mainly localized in the outermost tissue of the fruit while naringin is located in the albedo, which is immediately below the flavedo (Arcas et al., 2000). UV-C treatment is also used to elicit phytoalexin production in plant tissues. UV-C irradiation at 0.5 kJ m−2 reduces decay without causing damage or affecting the post-harvest quality of grapefruit; phytoalexin accumulation and decay control are dependent on cultivar, treatment dose, and harvest date. After UV-C irradiation, the coumarin phytoalexins scoparone and scopoletin accumulate in flavedo tissues. Experimental evidence indicates that the effect of UV light in reducing the citrus pathogen P. digitatum, the most important post-harvest rotting agent of citrus fruit, is an induced resistance phenomenon (production of phytoalexins) rather than a germicidal effect of the UV-C treatment as such (D’hallewin et al., 2000). In grapevine berries UV light treatment induces the production of resveratrol, which is present at concentrations 2–30-fold higher in induced berries as compared to the non-induced ones, a phenomenon that is particularly prominent in non-infected berries surrounding an infected site (Langcake & Pryce, 1976; Adrian et al., 2000). Finally, curing (post-harvest hot-air treatment) of plant commodities before cold storage has been successfully utilized in order to improve the shelf life of fruit and vegetables. Curing, for example, to cure wounds and injuries caused during post-harvest handling, can have positive effects in reducing pathogen levels and disease development by the direct effect of heat on fungal pathogens and by stimulating certain host–defense responses. These responses include an increase in levels of constitutive antifungal phenolics that inhibit fungal development in the fruit tissues, the production of a lignin-like material that serves as a mechanical barrier against pathogen invasion, and the synthesis of phytoalexins. Studies of various citrus fruit cultivars have shown that curing of fruits inoculated with Penicillium spp. prevents the development of the pathogen and promotes the biosynthesis of the phytoalexin scoparone in cells adjacent to the wound (Kim et al., 1991; Schirra et al., 2000). 1.2.5.2 Phenolics and plant–insect interactions The ecological relationship between plants and insects is a complex one with physical as well as chemical interactions. This relationship is also affected by plant factors, insect

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factors and by some insect–plant factors, including hypersensitive reactions and plant resistance to insect-borne diseases. Various environmental conditions can modify the expression of these factors by acting primarily on the insect, the plant, or the insect–plant relationship. Each of the plant or insect mechanisms indicated may be the result of one or more genetic factors (Painter, 1941). In this context, an interesting question concerns how insects select plants for both food and as sites for oviposition. Insects possess a range of chemoreceptors (gustatory and olfactory chemoreceptive systems), mainly on their antennae and mouthparts, which enable them to discriminate a wide variety of chemical compounds at often unbelievably low concentrations and encode this information. Afterwards, this information is processed by the decoding command centers localized in the central nervous system (Dethier, 1970; Schoonhoven & Derksen-Koppers, 1976; Swain, 1977). A systematic evaluation of the kinds of plants fed upon by the larvae of butterflies leads to the conclusion that secondary plant metabolites play a leading role in determining patterns of utilization. This seems true not only for butterflies but for all phytophagous insects (Thorsteinson, 1960; Ehrlich & Raven, 1964; Castellanos & EspinosaGarcía, 1997; Zagrobelny et al., 2004). Plant constituents that make hosts unpalatable are secondary metabolites present in sufficient concentration to exert an undesirable physiological effect. Therefore, plant secondary compounds have received much attention as proximate and ultimate determinants of host–plant range in phytophagous insects (Tosh et al., 2003). Phytophagous insects, however, can evolve in response to physiological obstacles. If a recombinant or mutant appears in a population of insects that enables individuals to feed on some previously protected plant group, selection could carry the line into a new adaptive zone. Changes in the choice of food plant would be especially favored in situations where the supply of the preferred plant is sufficiently limited to be an important factor in the survival of the larvae. Therefore, Ehrlich and Raven (1964) have proposed that through the process of co-evolution, insects are able to detoxify certain defensive substances that deter feeding so that, eventually, the same deterrent compounds become feeding attractants. Insects are reported to utilize secondary metabolites to increase their fitness. The ability of certain insects to sequester plant toxins from their food plants in the larval stage is well known. They then move these toxins into the adult imago, and both larva and adult generally gain protection from predation by birds. Besides clear and well-documented fitness benefits of sequestration in highly aposematic species, there are many cases in which insects deposit chemicals in the cuticle yet do not possess warning colors, and others where they gain protection from predators as a result of the gut contents alone (Bernays et al., 1991; Sword, 1999; Bernays & Chapman, 2000; Harborne, 2001). Butterflies (Polyommatus icarus) reared at larval stage on inflorescences of Vicia villosa, a plant species acceptable for larval development even if not used as a food source in nature, showed some important features of the uptake and sequestration of plant secondary metabolites by insects: (i) larvae incorporated only part of the flavonoids of their host plant (mainly quercetin and kaempferol), while other flavonoids were mostly excreted; (ii) the content of ingested flavonoids was correlated with the body mass; (iii) the flavonoids were metabolized and their conjugated forms were stored. These studies also suggested that females are better adapted for sequestering these compounds than males, and that the accumulation of flavonoids in the smaller wings of the female is used in visual communication,

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as flavonoid-rich females are more attractive to males than flavonoid-free females (Burghardt et al., 1997, 2000, 2001). Plant volatiles and visual and thigmotactic cues may be involved in an insect’s recognition of, and migration to, a host plant. When an insect has located a potential host plant, it starts to probe. The net result of individual feeding stimuli and deterrents determines whether the insect will remain and feed. Whether a plant is accepted or rejected as food by insects depends largely on its chemical composition in addition, of course, to physical factors such as toughness, thickness, hairiness, etc. In addition, chemical inhibitors play an important role in the inhibition of oviposition on the host plant and, in turn, in the growth of insect larvae and survival of the progeny (Thorsteinson, 1960; Chapman, 1974; Ferguson et al., 1983; Pereyra & Bowers, 1988; Roessingh et al., 1997; Constabel, 1999; Stotz et al., 1999; Bernays & Chapman, 2000; Harborne, 2001). Studies on the role of inhibitors in host plant selection indicate that many different chemicals may be expected to have an inhibitory effect on feeding by different insects. Among plant constituents, it is now generally accepted that plant phenolics play a role in protecting plants from both insect and mammalian herbivory (Todd et al., 1971; Elliger et al., 1980; Corcuera, 1993; Harborne & Williams, 2000; Simmonds, 2003). In addition, one of the most important advances in understanding plant–insect interactions has been the discovery of induced defense: some plants respond to insect feeding by increasing the synthesis of a particular phenolic toxin or toxins. In summary, the concentration of toxic phenolic compounds in a plant is a key factor in deterrence, and it is the accumulation of phenols in particular parts of the plant that forms a feeding barrier (Castellanos & Espinosa-García, 1997; Harborne, 2001). A well-known example of simple phenolics acting as feeding barriers to insect herbivores is represented by salicylates in Salix leaves and their role in the feeding and growth of the polyphagous larvae of Operophtera brumata. It has been observed that levels of salicylates correlated negatively with growth of the larvae; larvae exposed to leaves rich in these compounds grew slowly and consumed less material, which suggests that salicylates could be considered as feeding deterrents for O. brumata (Simmonds, 2003). Some researchers (Gronquist et al., 2001) observed that the flower of Hypericum calycinum, which appears uniformly yellow to humans, bears a UV pattern that is, presumably, visible to insects. Two categories of phenolic pigments, flavonoids (quercetin-3-O-β-D-glucoronide and the dimeric flavonoid, I3-II8-biapigenin) and dearomatized isoprenylated phloroglucinols (hypercalin A, hypercalin B, hypercalin C, chinesin I, and chinesin II), are responsible for the UV markings of this flower. The finding that isoprenylated phloroglucinols are present in the anthers and in the ovarian wall of the flower raised the possibility that these compounds also served a second function. It makes sense to presume that both pollen and developing seeds might be in need of protection, and the finding that hypercalin A is deterrent and toxic to Utetheisa ornatrix larvae suggests that the second function is defense. Most plants contain an array of flavonoids, whose fingerprints often differ among families, genera and species (Lattanzio et al., 1996). The fact that phytophagous insects usually differentiate among families suggests that flavonoids play a role in host selection. Plant flavonoids affect the behavior, development and growth of a number of insects (Simmonds & Stevenson, 2001; Simmonds, 2001, 2003). Some flavonoids are feeding

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stimulants for the boll weevil, Anthonomus grandis (Hedin et al., 1988), oviposition stimulants of a citrus-feeding swallowtail butterfly, Papilio xuthus L. (Nishida et al., 1987), or antibiotic substances efficient against phytophagous insects (Todd et al., 1971; Chan & Waiss, 1978; Chan et al., 1978; Joerdens-Roettger, 1979; Elliger et al., 1980; Hanny, 1980; Hedin et al., 1983; Peng & Miles, 1988; Ridsdill-Smith et al., 1995). In comparison with many other secondary metabolites, flavonoids are apparently not very toxic to, and have a low physiological activity in, most insects. Nevertheless, many flavonoids can act as feeding deterrents to phytophagous insects at relatively low concentrations. So, the concentrations of flavonoids in plants are normally far higher than those needed to have a deterrent effect on aphid feeding. However, aphids tend to feed from tissues such as the phloem, which are generally low in flavonoids, and thus they will normally only encounter high levels while probing the plant tissues for phloem sap, and not while feeding (Harborne & Grayer, 1993). Four isoflavonoids ( judaicin, judaicin-7-O-glucoside, 2-methoxy-judaicin, and maackiain) isolated from wild relatives of the chickpea, Cicer arietinum, were shown to deter larval feeding by Heliocoverpa armigera at 100 ppm. All four isoflavonoids showed a dosedependent effect on activity, with judaicin and maackiain retaining their deterrent activity down to 50 ppm and 10 ppm, respectively. The isoflavonoids were tested in combinations and with chlorogenic acid: the combinations containing judaicin and maackiain were most active, and chlorogenic acid enhanced the feeding-deterrent activity of all four isoflavonoids. H. armigera was the only one of four noctuids to be deterred by all four isoflavonoids. Spodoptera littoralis was deterred by judaicin alone and S. frugiperda by maackiain alone. Heliothis virescens and S. exigua were not deterred from feeding by any of the isoflavonoids. When incorporated into the diet, isoflavonoids decreased the weight gain of early-stadia larvae of H. armigera more than they did later stadia, and maackiain and judaicin were most potent. These data suggest that these isoflavonoids, especially maackiain and judaicin, could play a role in decreasing the susceptibility of Cicer to attack by H. armigera (Simmonds & Stevenson, 2001). The host plants of the native American butterfly Pieris napi oleracea include most wild mustards. However, garlic mustard, Alliaria petiolata, a highly invasive weed that was introduced from Europe, appears to be protected from this insect. Although adults will oviposit on the plant, most larvae of P. n. oleracea do not survive on garlic mustard. By using feeding bioassays with different larval stages of the insect to monitor the isolation and identification of two bioactive constituents that could explain the natural resistance of this plant, Renwick et al. (2001) found that a cyanopropenyl glycoside, alliarinoside, strongly inhibits feeding by first instars through an apparent post-ingestive feedback mechanism, while a flavone glycoside, isovitexin-6″d-β-glucopyranoside, acts as a direct feeding deterrent that is perceived by taste receptors on the mouthparts of late instars. Interestingly, the first instars are insensitive to isovitexin6″-d-β-glucopyranoside, and the late instars are little affected by alliarinoside. Most of the research concerned with insect feeding deterrents has focused on their effects upon agricultural pests, in order to determine how flavonoids can confer resistance to crops against insect attack. In this connection, flavonoid HPLC analyses of wild species of the genus Vigna supported evidence for the existence of different flavonoid chemotypes in some species of section Vigna. There are kaempferol chemotypes, kaempferol being the main aglycone detected, quercetin chemotypes, containing only quercetin glycosides, and

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two isorhamnetin chemotypes. From an ecological point of view the most interesting chemotypes are some accessions, belonging to the same species that has enabled studies into the role of endogenous flavonoids in plant resistance to aphids. Amongst V. marina accessions, two chemotypes were found: V. marina var. oblonga TVnu 1174 (isorhamnetin chemotype) contained two isorhamnetin glycosides and traces of two kaempferol glycosides, while V. marina var. marina TVnu 717 (kaempferol chemotype) contained only kaempferol glycosides. V. luteola accessions also showed two different chemotypes: the accession TVnu 475 contained only quercetin glycosides, while the other two accessions – TVnu 172 and TVnu 905, which are kaempferol chemotypes – contained robinin (kaempferol-3-robinoside7-rhamnoside). When the resistance characteristics to aphids in different chemotypes of the same species were tested, it became evident that quercetin or isorhamnetin chemotypes showed a higher level of resistance compared to the kaempferol chemotypes of the same species, thus demonstrating a direct involvement of quercetin or isorhamnetin in the resistance mechanism (Lattanzio et al., 2000). Here it must be stressed that the salivary secretions of phytophagous insects contain various enzymes that play a fundamental role in the digestion of food of sucking-piercing insects. Among these enzymes, polyphenol oxidases (PPOs) [E.C. 1.10.3.1.] and peroxidases (PODs) [E.C. 1.11.1.7] – oxidoreductases metabolizing plant phenolics – are also present. Both enzymes have been identified in the salivary secretions of several aphid species and various functions have been proposed for these enzymes. At first, it was suggested that they are involved in the chemical stabilization of the stylet sheath. Then, they were thought to enable phytophagous insects to overcome plant defenses by neutralizing phenolics and their derivatives. In fact, it has been observed that when the stylets of grain aphids penetrate plant tissues, the ruptured cells may become partially filled with aphid saliva. Thus, the salivary sheath material may absorb plant metabolites from plant tissues. As a result, the grain aphid PPO secreted in its saliva can react with the phenolics accumulated around the aphid stylet sheath, thus oxidizing and polymerizing them. Natural occurring phenolic toxins are thus converted into less toxic substances (Urbanska et al., 1998). The effectiveness of phenolics as resistance factors to insect feeding is also enhanced by oxidation to polymers, which reduce digestibility, palatability and nutritional value. Thus, high levels of PPO, the major phenolic oxidizing enzyme of plants, can be correlated with plant resistance mechanisms against insects. PPOs, presumed anti-herbivore enzymes, are anti-nutritive enzymes that decrease the nutritive value of wounded plant tissues by cross-linking proteins or catalyzing the oxidation of phenolic toxic metabolites to reactive and polymerizing quinones. The inducible expression of PPO was examined in hybrid poplar. Following mechanical wounding, simulating insect damage, PPO activity increased in wounded and unwounded leaves on wounded plants beginning at 24 h and 48 h, respectively. Feeding by forest tent caterpillars also induced PPO expression. This wound- and herbivore-induced expression of PPO in hybrid poplar supports the defensive role of this enzyme against insect pests. The results obtained showed that: (i) PPO induction is systemic, i.e. induced PPO functions in reducing additional herbivore damage rather than in wound repair; and (ii) PPO induction is mediated by increased accumulation of mRNA, which is typical of a variety of induced defense proteins (Constabel et al., 2000; Haruta et al., 2001).

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The herbaceous perennial plant Phryma leptostachya L., which is widely distributed in the Himalayas, temperate Asia, and northern East America, has been traditionally used as a natural insecticide in East Asia. From the root extracts of this plant, various lignans of the 3,7-dioxabicyclo-[3.3.0]octanes (furofuran) type, showing insecticidal activity against the larvae of some species of lepidopterous insects, have been isolated. Leptostachyol acetate, a lignan isolated from the roots of P. leptostachya var. asiatica, was lethal to third-instar larvae of Culex pipiens pallens, Aedes aegypti, and Ocheratatos togoi, three mosquito species, at 10 ppm (Park et al., 2005). In addition, tannins are generally considered to be deleterious to phytophagous insects. Tannins may affect the growth of insects in three main ways: they have an astringent taste, which affects palatability and decreases feed consumption, they form complexes of reduced digestibility with proteins, and they act as enzyme inactivators. Tannins are present at high levels in most plant seeds and grains, where they negatively impact on the use of seeds and grain in insect feed (Winkel-Shirley, 1998). In cowpea, condensed tannins (proanthocyanidins) contribute to resistance to infestation by insects such as the cowpea weevil Callosobruchus maculatus (F.) (Lattanzio et al., 2005). An increased level of proanthocyanidins during seed and grain development and maturation has been studied in a number of different plant species. A significant increase in the degree of polymerization of proanthocyanidins has been observed during the maturation of sorghum grains (Butler, 1982). In Phaseolus vulgaris a gradual increase in condensed tannins and α-amylase inhibitory activity, both considered defensive tools against phytophagous insects, was observed from anthesis to seed maturity (Coelho & Majolo, 1993). In this connection, two accessions of stored cowpea seeds, both classified as susceptible accessions and showing a different degree of bruchid damage in storage, were analyzed for their condensed tannins and α-amylase inhibitor contents as defensive compounds against cowpea weevil. IT 84E-1-108 exhibited an elevated degree of infestation (about 30%), while Vita 7 did not show any damage caused by cowpea weevil larvae. Surprisingly, no α-amylase inhibitory activity was found in the cotyledons of Vita 7 seeds, while IT 84E-1-108 exhibited a moderate level of inhibitory activity. In fact the seed-coat tannin content was found to be thirteen times higher in undamaged Vita 7 seeds than in infested IT 84E-1-108 seeds. These last results support the hypothesis that, if bruchids infest cowpea when the grain is stored after harvest, seed-coat tannins are effectively involved in the biochemical defense mechanisms, which can act to deter, poison or starve bruchid larvae that feed on these seeds (Lattanzio et al., 2005).

1.2.6 Plant phenolics and health There is considerable current interest in plant phenolics and their potential to beneficially affect human health. This interest spans from academics and health professionals to food producers and processors, and reflects the interest in dietary components that offer benefits beyond nutrition (i.e. nutrients at levels sufficient to prevent disease due to deficiency), and may prevent degenerative diseases and prolong life. In fact, there is an inverse association between the consumption of plant foods and risk for a number of age-related diseases (Hu and Willett, 2002). Plant phenolics exhibit a range of biological activities in vitro, which supports their contributing to the beneficial effects of fruit- and vegetable-rich diets. Indeed, there is

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reasonable evidence from epidemiological studies to support the notion that diets rich in phenolics (derived from fruits and vegetables) are associated with lower risks of cancer, osteoporosis, cardiovascular diseases, cataracts, and diseases associated with brain and immune dysfunction. Furthermore, phenolic compounds, which constitute the active substances found in commonly used medicinal plant extracts, modulate the activity of a wide range of enzymes and cell receptors (Middleton et al., 2000; Pietta, 2000; Manach et al., 2004; Arts & Hollman, 2005; Scalbert et al., 2005). One possible reason for this protection against diseases may be the powerful antioxidant and free radical scavenging properties of various classes of phenolic compounds, although it is worth noting that the physiological relevance of direct antioxidant action as a mechanism to explain the impact of phenolics on disease risk has been questioned (Hertog et al., 1993; Cook & Samman, 1996; Rice-Evans et al., 1997; Vinson et al., 1998a; Vinson et al., 1998b; Frankel, 1999; Hässig et al., 1999; Wang & Goodman, 1999; Prior & Cao, 2000; Kroon & Williamson, 2005; Halliwell, 2006). As more work has been carried out, a number of more specific, targeted roles for phenolics have also been identified. These include, for example, the inhibition of oxidation of target molecules such as low- and very low-density lipoproteins (crucial steps in atherosclerotic lesion formation) and DNA bases (relevant to the induction of cancer) by free radicals (Coffey et al., 1995; Peterson & Dwyer, 1998; Massaeli et al., 1999; Kaur & Kapoor, 2001). Isoflavones, stilbenes and lignans are naturally occurring phenolics that are structurally similar to the mammalian estrogen estradiol-17β, and that interact with the estrogen receptor such that the growth of hormone-dependent cancers is impaired (Cassidy et al., 2000; Parr & Bolwell, 2000). In addition, various phenolics are able to inhibit xanthine oxidase and other enzymes implicated in the production of damaging reactive oxygen species. Given that leukotriene biosynthesis from arachidonic acid is initiated by an oxygenation reaction catalyzed by 5-lipoxygenase, the inhibition of 5-lipoxygenase by various dietary phenolics such as caffeic acid, silibinin, curcumin, and various flavonoids may also be important in anti-inflammatory actions (Koshihara et al., 1984; Huang et al., 1991; de Groot & Rauen, 1998). Furthermore, Park (1999) found that some types of natural flavonoids block glucose uptake in myelocytic U937 cells as well as in lymphocytic Jurkat cells and that natural flavonoids could be used as alternative blockers of glucose uptake in vitro. Besides positive effects for controlling type 2 diabetes through the inhibition of glucose absorption and hemoglobin glycosylation, flavonoids also show benefits for stimulating insulin secretion and/or action, and improving insulin binding (Asgary et al., 1999; Jayaprakasam et al., 2005). However, much of the evidence concerning the mechanism of disease prevention by phenols and polyphenols is derived from in-vitro experiments, which are performed with doses higher than those to which human are exposed through the diet. In this connection, only partial information is available on the quantities of dietary phenolics that are consumed daily throughout the world (Manach et al., 2004). In addition, the active compounds may not be the native phenolics found in food, as most polyphenols are present in food in the form of esters, glycosides, or polymers, and these compounds must be hydrolyzed in the human body before they can be absorbed. During the course of absorption, phenolic aglycones are conjugated in the small intestine and, later, in the liver, and this process includes glucuronidation, methylation, and sulfation (Kroon et al., 2004). Therefore, much

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data concerning the putative beneficial effects of plant phenolics on human health and the mechanisms involved must be reassessed in the light of the new findings on the fate of polyphenols in the human body. Nevertheless, over the past five to seven years data from a considerable number of human intervention trials using phenolic-rich foods or extracts have been reported (Williamson and Manach, 2004), and the number of reported studies is now in excess of one hundred. These studies have been acute or short term (up to several weeks in duration) and have involved measurement of changes in surrogate disease biomarkers (as opposed to disease end-points). Overall, it is clear that for some flavonoids in relation to some diseases, there are consistent effects on disease biomarkers. In particular, food and extracts rich in flavan-3-ol, including cocoa beverages and dark chocolate, red wine, Pycnogenol (an extract of French maritime pine bark), and grape seed extracts have beneficial effects on markers of cardiovascular disease risk, especially vascular tone and platelet activity, and possibly levels of LDL-cholesterol; isoflavone-rich foods (predominantly soy products) induce weak hormonal effects and beneficial effects on markers of bone health in post-menopausal women; tea catechins influence plasma antioxidant markers and alter energy metabolism; and the flavonol quercetin alters some biomarkers related to carcinogenesis. Furthermore, a recent report indicated that improvements in vascular tone (associated with increased nitric oxide bioactivity) following consumption of a flavanol-rich cocoa beverage or pure epicatechin, were directly correlated with plasma epicatechin concentrations (Schroeter et al., 2006). Although further information is required, in particular regarding the impact of the long-term ingestion of phenolic-rich foods on disease risk in humans, it is evident that (poly)phenolics in our diets can influence short-term markers associated with certain diseases, and that they may have significant effects on the long-term risk of disease.

1.3 Note 1 Non-host resistance in plants refers to a mechanism that provides resistance against a specific parasite or pathogen throughout all the members of a plant species. It is expressed by every plant towards the majority of potentially pathogenic microbes, whereas host resistance refers to resistance expressed by plant genotypes within an otherwise susceptible host species.

1.4 References Adrian, M., Jeandet, P., Douillet-Breuil, A.C., Tesson, L. & Bessis, R. (2000). Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation. Journal of Agricultural and Food Chemistry, 48, 6103 – 6105. Angell, H.R., Walker, J.C. & Link, K.P. (1930). The relation of protocatechuic acid to disease resistance in the onion. Phytopathology, 20, 431–438. Aoki, T., Akashi, T. & Ayabe, S. (2000). Flavonoids of leguminous plants: structure, biological activity, and biosynthesis. Journal of Plant Research, 113, 475–488. Arcas, M.C., Botia, J.M., Ortuño, A.M. & Del Rio, J.A. (2000). UV irradiation alters the levels of flavonoids involved in the defence mechanism of Citrus aurantium fruits against Penicillium digitatum. European Journal of Plant Pathology, 106, 617–622. Arts, I.C.W. & Hollman, C.H. (2005). Polyphenols and disease risk in epidemiologic studies. The American Journal of Clinical Nutrition, 81 (suppl), 317–325.

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Asgary, S., Naderi, Gh., Sarrafzadegan, N. et al. (1999). Anti-oxidant effect of flavonoids on hemoglobin glycosylation. Pharmaceutica Acta Helvetiae, 73, 223–226. Assis, J.S., Maldonado, R., Muñoz, T., Escribano, M.I. & Merodio, C. (2001). Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biology and Technology, 23, 33–39. Bais, H.P., Vepachedu, R., Gilroy, S., Callaway, R.M. & Vivanco, J.M. (2003). Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science, 301, 1377–1380. Bais, H.P., Park, S.-W., Weir, T.L., Callaway, R.M. & Vivanco, J.M. (2004). How plants communicate using the underground information superhighway. Trends in Plant Science, 9, 26– 32. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. & Vivanco, J.M. (2006). The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Revue of Plant Biology, 57, 233 –266. Beckman, C.H. (2000). Phenolic-storing cells: keys to programmed cell death and periderm formation in wilt disease resistance and in general defence responses in plants? Physiological and Molecular Plant Pathology, 57, 101–110. Bell, E.A. (1980). The possible significance of secondary compounds in plants. In: Secondary Plant Products (eds E.A. Bell & B.W. Charlwood), pp. 11–21. Enciclopedia of Plant Physiology, New Series, Vol. 8. Springer-Verlag, Berlin. Bernays, E.A., Howard, J.J., Champagne, D. & Estesen, B.J. (1991). Rutin: a phagostimulant for the grasshopper Schistocerca americana. Entomologia Experimentalis et Applicata, 60, 19–28. Bernays, E.A. & Chapman, R.F. (2000). Plant secondary compounds and grasshoppers: beyond plant defenses. Journal of Chemical Ecology, 26, 1773–1794. Boddu, J., Svabek, C., Sekhon, R. et al. (2004). Expression of a putative flavonoid 3′-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins. Physiological and Molecular Plant Pathology, 65, 101–113. Brewin, N.J. (1991). Development of the legume root nodule. Annual Review of Cell Biology, 7, 191–226. Brouillard, R. & Dangles, O. (1994). Flavonoids and flower colour. In: The Flavonoids: Advances in Research since 1986 (ed. J.B. Harborne), pp. 565–588. Chapman and Hall, London. Brouillard, R., Figueiredo, P., Elhabiri, M. & Dangles, O. (1997). Molecular interactions of phenolic compounds in relation to the colour of fruit and vegetables. In: Phytochemistry of Fruits and Vegetables (eds F. Tomás-Barberán & R.J. Robins), pp. 29–49. Oxford University Press, New York. Brown, J.K.M. (2002). Yield penalties of disease resistance in crops. Current Opinion in Plant Biology, 5, 339 –344. Brown, J.K.M. (2003). A cost of disease resistance: paradigm or peculiarity? Trends in Genetics, 19, 667– 671. Brown, D.E., Rashotte, A.M., Murphy, A.S. et al. (2001). Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiology, 126, 524–535. Bunzel, M., Ralph, J., Funk, C. & Steinhart, H. (2005). Structural elucidation of new ferulic acidcontaining phenolic dimers and trimers isolated from maize bran. Tetrahedron Letters, 46, 5845 –5850. Burghardt, F., Fiedler, K. & Proksch, P. (1997). Uptake of flavonoids from Vicia villosa (Fabaceae) by the lycaenid butterfly, Polyommatus icarus (Lepidoptera: Lycaenidae). Biochemical Systematics and Ecology, 25, 527– 536. Burghardt, F., Knüttel, H., Becker, M. & Fiedler, K. (2000). Flavonoid wing pigments increase attractiveness of female common blue (Polyommatus icarus) butterflies to mate-searching males. Naturwissenschaften, 87, 304 –307. Burghardt, F., Proksch, P. & Fielder, K. (2001). Flavonoid sequestration by the common blue butterfly Polyommatus icarus: quantitative intraspecific variation in relation to larval host plant, sex and body size. Biochemical Systematics and Ecology, 29, 875–889. Butler, L.G. (1982). Relative degree of polymerization of sorghum tannin during seed development and maturation. Journal of Agricultural and Food Chemistry, 30, 1090–1094.

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Cantos, E., Garcìa-Viguera, C., de Pascual-Teresa, S. & Tomás-Barberán, F.A. (2000). Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table grapes. Journal of Agricultural and Food Chemistry, 48, 4606–4612. Carletti, P., Masi, A., Wonisch, A., Grill, D., Tausz, M. & Ferretti, M. (2003). Changes in antioxidant and pigment pool dimensions in UV-B irradiated maize seedlings. Environmental and Experimental Botany, 50, 149 –157. Cassidy, A., Hanley, B. & Lamuela-Raventos, R.M. (2000). Isoflavones, lignans and stilbenes – origins, metabolism and potential importance to human health. Journal of the Science of Food and Agriculture, 80, 1044 –1062. Castellanos, I. & Espinosa-García, F.J. (1997). Plant secondary metabolite diversity as a resistance trait against insects: a test with Sitophilus granarius (Coleoptera: Curculionidae) and seed secondary metabolites. Biochemical Systematics and Ecology, 25, 591–603. Chan, B.G. & Waiss, A.C. (1978). Condensed tannin, an antibiotic chemical from Gossypium hirsutum. Journal of Insect Physiology, 24, 113–118. Chan, B.G., Waiss, A.C., Binder, R.G. & Elliger, C.A. (1978). Inhibition of lepidopterous larval growth by cotton constituents. Entomologia Experimentalis et Applicata, 24, 94–100. Chapman, R.F. (1974). The chemical inhibition of feeding by phytophagous insects: A review. Bulletin of Entomological Research, 64, 339 – 363. Chappell, J. & Hahlbrock, K. (1984). Transcription of plant defence genes in response to UV light and fungal elicitor. Nature, 311, 76 – 78. Cicco, N., Morone Fortunato, I., Ruta, C., Tagarelli, A., Terzano, R. & Lattanzio, V. (2004). Stimulation of antioxidant phenolics in shoot cultures of oregano (Origanum vulgare L.). In: Polyphenols Communications 2004 (eds A. Hoikkala, O. Soidinsalo & K. Wähälä), pp. 503–504. Gummerus Printing, Jyväskyla, Finland. Civello, P.M., Martínez, G.A., Chaves, A.R. & Añon, M.A. (1997). Heat treatments delay ripening and postharvest decay of strawberry fruit. Journal of Agricultural and Food Chemistry, 45, 4589 – 4594. Clarke, J.D., Volko, S.M., Ledford, H., Ausubel, F.M. & Dong, X. (2000). Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. The Plant Cell, 12, 2175– 2190. Coelho, J.V. & Majolo, F.M. (1993). Evolution of phenolic compounds and condensed tannins (proanthocyanidins) during the development of bean seeds (Phaseolus vulgaris L.). Archives of Latinoamerican Nutrition, 43, 61– 65. Coffey, M.D., Cole, R.A., Colles, S.M. & Chisolm, G.M. (1995). In vitro cell injury by oxidized low-density lipoproteins involves lipid hydroperoxide-induced formation of loalkoxyl, lipid, and peroxyl radicals. Journal of Clinical Investigation, 96, 1866–1873. Constabel, C.P. (1999). A survey of herbivore-inducible defensive proteins and phytochemicals. In: Induced Plant Defenses Against Herbivores and Pathogens: Biochemistry, Ecology, and Agriculture (eds A.A. Agrawaal, S. Tuzun & E. Bent), pp. 137–166. APS Press, St Paul, MN. Constabel, C.P., Yip, L., Patton, J.J. & Christopher, M.E. (2000). Polyphenol oxidase from hybrid poplar: cloning and expression in response to wounding and herbivory. Plant Physiology, 124, 285 –295. Cook, N.C. & Samman, S. (1996). Flavonoids. Chemistry, metabolism, cardioprotective effects, and dietary sources. Journal of Nutritional Biochemistry, 7, 66–76. Cooke, T.J., Poli, D., Sztein, A.E. & Cohen, J.D. (2002). Evolutionary patterns in auxin action. Plant Molecular Biology, 49, 319 –338. Coquoz, J.-L., Buchala, A. & Métraux, J.P. (1998). The biosynthesis of salicylic acid in potato plants. Plant Physiology, 117, 1095 –1101. Corcuera, L.J. (1993). Biochemical basis for the resistance of barley to aphids. Phytochemistry, 33, 741–747. Costet, L., Cordelier, S., Dorey, S., Baillieul, F., Fritig, B. & Kauffmann, S. (1999). Relationship between localized acquired resistance (LAR) and the hypersensitive response (HR): HR is necessary for LAR to occur and salicylic acid is not sufficient to trigger LAR. MPMI, 12, 655–662.

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Advances in Phytochemistry, Vol. 4 (eds V.C. Runeckless & J.E. Watkin), pp. 165–221. AppletonCentury-Croft, New York, USA. Van Sumere, C.F. & Vande Casteele, K. (1985). Reversed phase-HPLC analysis of flavonoids and the biochemical identification of cultivars of evergreen azalea. In: Annual Proceedings of the Phytochemical Society of Europe, Vol. 25 (eds C.F. Van Sumere & P.J. Lea), pp. 17–43. Clarendon Press, Oxford, UK. Van Sumere, C.F. (1989). Phenols and phenolic acids. In: Methods in Plant Biochemistry, Vol. 1. Plant Phenolics (eds P.M. Dey & J.B. Harborne), pp. 29–72. Academic Press, London. Van Wees, S.C.M., de Swart, E.A.M., van Pelt, J.A., van Loon, L.C. & Pieterse, C.M.J. (2000). Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonatedependent defense pathways in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 97, 8711–8716. Varin, L., Chamberland, H., Lafontaine, J.G. & Richard, M. (1997). The enzyme involved in sulfation of the turgorin, gallic acid 4-O-(β-D-glucopyranosyl-6′-sulfate) is pulvini-localized in Mimosa pudica. The Plant Journal, 12, 831–837. Vinson, J.A., Hao, Y., Su, X. & Zubik, L. (1998a). Phenol antioxidant quantity and quality in foods. Journal of Agricultural and Food Chemistry, 46, 3630–3634. Vinson, J.A., Yousef, A. & Dabbagh, M.A. (1998b). Tea phenols: antioxidant effectiveness of teas, tea components, tea fractions and their binding with lipoproteins. Nutrition Research, 18, 1067–1075. Walker, J.C. & Stahmann, M.A. (1955). Chemical nature of disease resistance. Annual Review of Plant Physiology, 6, 351–366. Wang, Y., Hamburger, M., Gueho, J. & Hostettmann, K. (1989). Antimicrobial flavonoids from Psiadia trinervia and their methylated and acetylated derivatives. Phytochemistry, 28, 2323– 2327. Wang, W. & Goodman, M.T. (1999). Antioxidant property of dietary phenolic agents in a human LDL-oxidation ex vivo model: interaction of protein binding activity. Nutrition Research, 19, 191–202. Ward, G., Hadar, Y., Bilkis, I., Konstantinovsky, L. & Dosoretz, C.G. (2001). Initial steps of ferulic acid polymerization by lignin peroxidase. Journal of Biological Chemistry, 276, 18734–18741. Wharton, P.S. & Nicholson, R.L. (2000). Temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoalexins and the anthocyanin, cyanidin 3-dimalonyl glucoside. New Phytologist, 145, 457– 469. Weidenbörner, M., Hindorf, H., Jha, H.C. & Tsotsonos, P. (1990). Antifungal activity of flavonoids against storage fungi of the genus Aspergillus. Phytochemistry, 29, 1103–1105. Weir, T.L., Park, S.-W. & Vivanco, J. M. (2004). Biochemical and physiological mechanisms mediated by allelochemicals. Current Opinion in Plant Biology, 7, 472–479. Williamson, G. & Manach, C. (2004). Bioavailability and bioefficacy of polyphenols in humans. II: a review of 93 intervention studies. The American Journal of Clinical Nutrition, 81(suppl), 243 – 255. Wink, M. (1988). Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theoretical and Applied Genetics, 75, 225–233. Wink, M. (1997). Compartmentation of secondary metabolites and xenobiotics in plant vacuoles. Advances in Botanical Research, 25, 141–169. Wink, M. (2003). Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry, 64, 3–19. Winkel-Shirley, B. (1998). Flavonoids in seeds and grains: physiological function, agronomic importance and the genetics of biosynthesis. Seed Science Research, 8, 415–422. Wittstock, U. & Gershenzon, J. (2002). Constitutive plant toxins and their role in defense against herbivores and pathogens. Current Opinion in Plant Biology, 5, 1–8. Yang, R. & Shetty, K. (1998). Stimulation of rosmarinic acid in shoot cultures of oregano (Origanum vulgare) clonal line in response to proline, proline analogue, and proline precursors. Journal of Agricultural and Food Chemistry, 46, 2888–2893.

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Yazaki, K. (2005). Transporters of secondary metabolites. Current Opinion in Plant Biology, 8, 301–307. Zagrobelny, M., Bak, S., Vinther Rasmussen, A., Jørgensen, B., Naumann, C.M. & Lindberg Møller, B. (2004). Cyanogenic glucosides and plant–insect interactions. Phytochemistry, 65, 293–306. Zheng, Z., Sheth, U., Nadiga, M., Pinkham, J.L. & Shetty, K. (2001). A model for the role of the proline-linked pentose phosphate pathway in polymeric dye tolerance in oregano. Process Biochemistry, 36, 941– 946. Zhou, H.-W., Lurie, S., Lers, A., Khatchitski, A., Sonego, L. & Ben Arie, R. (2000). Delayed storage and controlled atmosphere storage of nectarines: two strategies to prevent woolliness. Postharvest Biology and Technology, 18, 133–141.

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Chapter 2

Lignification: are Lignins Biosynthesized via simple Combinatorial Chemistry or via Proteinaceous Control and Template Replication? John Ralph, Gösta Brunow, Philip J. Harris, Richard A. Dixon, Paul F. Schatz and Wout Boerjan

2.1 Introduction Lignification is the polymerization process generating lignins (complex hydroxyphenylpropanoid polymers) from phenolic monomers, notably the monolignols p-coumaryl, coniferyl and sinapyl alcohols. The current theory, based on original work by Freudenberg (Freudenberg & Neish, 1968), contends that the essential structural features of lignin could be mimicked by chemical processes involving oxidative coupling of phenols. All the structural features of lignin polymers are in accordance with the view that the actual polymerization step is solely under chemical control and that the radical coupling reactions are not, for example, directed by proteins or enzymes (Ralph et al., 2004b). An alternative but ill-defined concept, attempting to displace the current theory, has been advanced; for a recent description see (Davin & Lewis, 2005). Without offering any experimental evidence the authors propose that lignin primary structure is dictated by ‘proteins harboring arrays of dirigent (monolignol radical binding) sites,’ and that lignin macromolecules may be replicated by template replication mechanisms using the lignin chain itself as the template. The authors contended that the current free-radical coupling theory is an ‘unproven working hypothesis,’ and unilaterally raised their own hypothesis to theory status. However, the supposed replacement paradigm lacks diagnostic supporting evidence and has difficulty in explaining many of the basic facts. Despite this, the new paradigm has come to some prominence, even appearing in a textbook to the exclusion of the currently held theory (Croteau et al., 2000). This chapter is an attempt to bring balance to the current debate and to draw attention to the fact that the new contender has not earned the status of a theory. The following are a small sample of the points that should be more openly discussed. Some have already been addressed in reviews and commentary articles on the subject (Sederoff et al., 1999; Hatfield & Vermerris, 2001; Ralph et al., 2001b; Boerjan et al., 2003; Monties, 2003; Ralph et al., 2004b). Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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2.2 The current theory The current theory holds that the polymerization is a chemical process involving the radical coupling of phenols, incorporating available phenolic substrates subject to simple chemical and physical controls (Harkin, 1967; Freudenberg & Neish, 1968; Sarkanen & Ludwig, 1971; Harkin, 1973; Brunow et al., 1999; Ralph et al., 2004b). As radicals from the monolignols and from the evolving polymers are resonance-delocalized and are capable of coupling with different regiochemistries (Plate 2.1), the process is ‘combinatorial’ (Ralph et al., 2004b). The reaction of major importance in lignin polymerization is the so-called ‘end-wise’ cross-coupling of a new monomer (radical) with the free-phenolic end of the growing polymer (Sarkanen & Ludwig, 1971). Coniferyl and sinapyl alcohols invariably couple at their side-chain β-positions, in vivo or in vitro. Such reactions for adding sinapyl and coniferyl alcohol monomers onto polymers bearing syringyl and guaiacyl phenolic end-units are shown in Plate 2.1d–e. In each coupling, as shown in more detail for β–O–4-coupling, for example (Plate 2.1g), the β-carbon becomes a new chiral carbon. Since no optical activity has been observed in lignins, this coupling must yield a completely racemic mixture (i.e. 50:50 R:S). Intermediate quinone methide structures generated by the radical coupling step are re-aromatized via post-coupling nucleophilic addition reactions, by water in the case of the β–O–4-coupled product (Plate 2.1g), and internally by the phenol on the ‘B-unit’ for β–5-coupling (not shown). A less important pathway, so far authenticated only for monolignol addition to β-ether units, is via β–1-coupling (Plate 2.1h) (Setälä et al., 1999; Zhang & Gellerstedt, 2001; Ralph et al., 2004b; Zhang et al., 2006). Other important couplings, which create branch-points in the polymer, may occur via 5–5or 5–O–4-coupling of oligomers (not monomers) to produce 5–5- and 4–O–5-units (D and E, Plate 2.1f ); clearly at least one guaiacyl unit is needed in such pathways. At the start of the polymerization some dimerization of monolignols occurs (Morreel et al., 2004a), the formation of β–β-coupled structures (resinol units C) or cinnamyl endgroups being a sign of this (Plate 2.1a,b). Formation of two or three coupling products obviously cannot proceed in a statistically random manner. The proportion of each product will be governed by the relative energies of the transition states (somewhat reflected in the product energies), which will not only be different but will be differentially altered by physical reaction conditions such as pH, solvent polarity, temperature, etc. The term ‘combinatorial’ is now therefore considered more suitable than the earlier term ‘random coupling.’ And nothing more than simple chemical coupling propensity (which is not always trivial to predict) is needed to explain why dimerization of sinapyl alcohol, with two combinatorial choices (β–β and β–O–4) strongly favors β–β-coupling. It is important to stress here that the coupling propensities between monolignols when forming dimers (Plate 2.1a,b), have little to do with the coupling propensity of a monolignol with the growing oligomer (Plate 2.1d,e).

2.3 Is there a need for a new theory? Since there are no observations that are inconsistent with the existing theory, one may ask if there is a need for a new theory. The new hypothesis seems to arise from a belief

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that lack of absolute structural control over lignification would constitute a unique biochemical anomaly (see section 2.4.1). As will be described, there are certain requirements to be met before a new hypothesis can come into contention as a replacement theory.

2.3.1 The challenge hypothesis (proteinaceous control and template replication) Over several years, a challenge hypothesis has emerged (Lewis & Davin, 1998; Gang et al., 1999; Kwon et al., 1999; Davin & Lewis, 2000; Burlat et al., 2001; Davin & Lewis, 2005). It was diagrammatically illustrated in a recent paper (Davin & Lewis, 2005) and is shown in abbreviated form in Plate 2.2a. Basically, the idea is that primary lignin macromolecules are assembled via putative ‘proteins harboring arrays of dirigent (monolignol radical binding) sites’ (Davin & Lewis, 2005). It is proposed that this lignin macromolecule then acts as a replication template to create identical (or, by some unknown mechanism, mirror image) molecules. In a manner remarkably like DNA replication, the process has recently been hypothesized as being via ‘an antiparallel double-stranded lignin template’ (Chen & Sarkanen, 2006). Such a model is fraught with difficulties, as will be described below.

2.3.2 Has the challenge hypothesis become a theory? The unwary may conclude that there is a new theory for lignification. Without debate and without evidence the challenge hypothesis has been unilaterally elevated to a competing theory and beyond.1 The position seems to have arisen from seeking only supporting evidence,2 an approach distinctly at odds with Popper’s definition of a scientific method (see http://plato.stanford.edu/entries/popper/). The authors of the challenge hypothesis have chosen the risky undertaking of proclaiming it a competing theory even in the absence of supporting evidence.

2.4 Are criticisms of the current theory valid? Challenges to the current combinatorial model have been put forward. Any facts that fail to be consistent with a theory put that theory in jeopardy. But are the challenges valid? We will discuss the following challenges that purportedly falsify the current theory.

2.4.1 Lignification as a biochemical anomaly The view that uncontrolled reactions are unnatural has been widely espoused by the proponents of the dirigent/replication hypothesis.3 Firstly, it must be emphasized that the existing theory does not claim that lignification is uncontrolled. It is clear that the cell exerts exquisite control over the supply of monolignols (and other phenolics) to the lignifying zone, over the supply of H2O2 and, consequently, the radical-generation capacity, etc. Such control over monolignol supply is evident in the temporal progression from p-coumaryl through coniferyl through sinapyl alcohol incorporation (Terashima et al., 1993), in the enhanced incorporation of p-coumaryl alcohol into softwood compression wood lignins

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(Timell, 1986), and in the differences in lignin composition in various cell types (Monties & Lapierre, 1981; Chesson et al., 1997; Hatfield et al., 1999b; Chen et al., 2000; Chabannes et al., 2001; Donaldson, 2001; Li et al., 2001; Patten et al., 2003; Harris et al., 2005). ‘Evidence’ for control has often overlooked the fact that lignin composition could be controlled at the level of monolignol transport from the cytoplasm to the site of polymerization. The issue being debated, however, lies in the control over the radical coupling steps involved in the polymerization (Ralph et al., 2004b). We leave the discussion regarding the possible uniqueness of lignins’ uncontrolled reactions to the end of this chapter, section 2.7.

2.4.2 The β-ether frequency anomaly A major criticism of the current theory espoused by the proponents of the dirigent/ replication hypothesis has been that the high β-ether frequency observed in lignins is anomalous (Lewis & Davin, 1998; Lewis et al., 1998a; Davin & Lewis, 2000).4 Dehydrodimerization of sinapyl alcohol (Plate 2.1b) produces the β–β-coupling product syringaresinol, typically in over 90% yield (Tanahashi et al., 1976). As noted above (Plate 2.1a), coniferyl alcohol produces the three combinatorial dimers from coupling of one monomer at its β-position with the other at the β-, 4–O-, or 5-positions in roughly equal amounts (Tanahashi et al., 1976; Katayama & Fukuzumi, 1978). In neither case, therefore, does the β-ether frequency approach 50%. Yet lignins are reported to have 50–70% (or higher) β-ethers (Adler, 1977). How can such an apparent anomaly be explained? This assumption of an anomaly is based on a misunderstanding of the lignification process. As explained above (section 2.2), the growth of the lignin molecule occurs by coupling of a monolignol radical to a radical formed on the phenolic end group of the polymer, a process termed ‘end-wise coupling.’ It has proved difficult to reproduce this type of polymerization in vitro because the end-wise coupling is repressed when higher concentrations of monolignols are present. Monolignol radicals preferentially couple with like monolignol radicals (when available) rather than cross-couple with dimers or higher oligomers (Syrjänen & Brunow, 1998; Syrjänen & Brunow, 2000; Syrjänen & Brunow, 2001). Dehydrodimerization reactions are therefore over-represented in synthetic lignins even when attempts are made to introduce the monolignol slowly (Freudenberg, 1956; Sarkanen, 1971; Terashima et al., 1995). Consequently, the β-ether frequency is low, considerably lower than in typical lignins. Limiting the diffusion rates (and therefore monolignol radical concentrations) to favor cross-coupling reactions reveals that β-ethers are strongly favored in cross-coupling reactions. In model reactions carried out via diffusion of coniferyl alcohol through dialysis tubing where coniferyl alcohol reacted with a lignin phenolic end-unit model, apocynol, the ratio of β–O–4- to β–5-product was 10:1 (Syrjänen & Brunow, 2000), and in experiments with coupling to a guaiacyl dimer, only the β–O–4trimer was found (Syrjänen & Brunow, 1998). Selective formation of β–O–4-units in lignins can therefore be achieved by limiting the supply of monolignol radicals. In the cell, simple diffusion control may suffice. No other agents are necessary to ‘explain the mystery of specificity of radical precursor coupling in lignin biosynthesis’ (Davin & Lewis, 2000). This feature was recognized early on as evidenced by eloquent passages in various treatises (Sarkanen & Ludwig, 1971; Adler, 1977), and has been emphasized repeatedly, by, for example, Lundquist (1973); Syrjänen & Brunow

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(1998); Boerjan et al. (2003); and Ralph et al. (2004b). The notion that the β-ether frequency is an anomaly that the current theory cannot accommodate must therefore be dismissed.

2.4.3 ‘Obligatory’ linkages? Softwood resinols (β–β-linked structural units) have been claimed to be ‘obligatorily 5-linked’ (Chen & Sarkanen, 2003; Sarkanen, 2005). The claim arises from extrapolating various published observations.5 If pinoresinol units in softwood lignins were obligatorily 5-linked, an explanation under the combinatorial theory would be needed. But again, what are the facts? First, the level of resinol units in softwood lignins is very low, typically about 2% (Adler, 1977; Zhang et al., 2003). Second, such units obviously have two phenolic ends, each of which is capable of radical coupling at its 4–O- or 5-position. Third, and rather importantly, pinoresinol is produced by dehydrodimerization of the monolignol coniferyl alcohol. At the time it is formed, early in the process of lignification, it is likely that there will be other dehydrodimers and small oligomers present. The 5-coupling of pinoresinol with another pinoresinol (Freudenberg & Sakakibara, 1959), or its cross-coupling with a β-ether dimer, has been shown (Kim, H., 2006, unpublished) to be efficient reactions. The idea therefore that at least one end of a pinoresinol unit becomes 5-coupled is hardly surprising. But are such units obligatorily 5-linked? The answer appears to be no. Although early reports are that the β–β-dimer derived from pinoresinol is not released by acidolysis or thioacidolysis, it is released by another milder ether cleaving reaction, derivatization followed by reductive cleavage (the DFRC method). As illustrated in a recent review (Ralph et al., 2004b, Fig. 8), the level of the DFRC dimer from pinoresinol in spruce or pine lignin is about a quarter the level of the secoisolariciresinol product. Secoisolariciresinol units are present in lignins at about half the level of pinoresinol units (Zhang et al., 2003) and are also capable of coupling at both phenolic ends. Regrettably, neither we nor others have yet quantified the release of such units but they are not difficult to detect. Quantification is now required to determine just what fraction of the pinoresinol can be released. There may also be a need to determine why thioacidolysis and DFRC appear to differ here. In summary, the evidence is that pinoresinol units are not obligatorily 5-linked and it is not difficult to come up with reasonable explanations under the current theory (different coupling propensities, coupling with other oligomers present in the lignifying zone at the same time) to rationalize why only a fraction of the particularly minor (∼2%) pinoresinol components in softwood lignins can be released by ether-cleaving reactions. The claim that resinols are obligatorily 5-linked, and that proteinaceous control over lignification is therefore demanded, is not supported.

2.5 Is there anything wrong with the dirigent hypothesis? 2.5.1 Monomer substitution The remarkable statement that ‘There is, however, no known precedent for the free interchange of monomeric units in any biopolymer assembly, then or now . . .’ (Lewis, 1999), was made in response to suggestions that plants finding themselves with reduced capacity to supply traditional monolignols appeared to respond by (partial) substitution with other

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available phenolics, for example, monolignol pathway intermediates such as the hydroxycinnamaldehydes, or 5-hydroxyconiferyl alcohol (Ralph et al., 1997; Ralph et al., 2001b). Although this evidence continues to be denied (see Section 2.5.1.1), monomer substitution has now been compellingly established, as reviewed by Ralph et al. (2001b, 2004b). Monomer substitution in more highly structurally controlled polysaccharide biosynthesis had already been unequivocally illustrated. Fucose-deficient Arabidopsis mur1 mutants substitute l-galactose for l-fucose in their xyloglucans (Zablackis et al., 1996; Sederoff et al., 1999). Since the dirigent/replication hypothesis had already been declared to be incompatible with the concept of monomer substitution (Lewis, 1999),6 establishing monomer substitution should be sufficient to dispel the challenge hypothesis. 2.5.1.1 Has monomer substitution been disproven? It has been claimed that ‘Furthermore, most – if not all – claims of non-lignin monomers acting as lignin monomer surrogates have now been unequivocally disproven (Anterola & Lewis, 2002)’ (Patten et al., 2005). 5-Hydroxyconiferyl alcohol’s unmistakable substitution for sinapyl alcohol in COMT-deficient angiosperms (see section 2.5.1.2) was carefully avoided. In each case addressed, the proof offered was simply inaccurate. Three of the most criticized examples will suffice here to make the point that monomer substitution has not been disproven and is in fact continuing to gain validation. Hydroxycinnamaldehyde incorporation into lignins The hydroxycinnamaldehydes coniferaldehyde and sinapaldehyde incorporate integrally into angiosperm lignins, as reviewed by Boerjan et al. (2003) and Ralph et al. (2004b). The evidence comes from several sources. First, NMR reveals additional aldehyde peaks at different chemical shifts from simple cinnamaldehyde endgroups (Ralph et al., 1999b; Kim et al., 2000; Ralph et al., 2001b; Kim et al., 2003). Diagnostic 2D NMR experiments reveal the nature of such aldehydes and, in particular, demonstrate that coniferaldehyde monomers β–O–4-cross-couple with syringyl phenolic end-units on the growing polymer, whereas sinapaldehyde β–O–4-cross-couples with both guaiacyl and syringyl units. Incidentally, such cross-coupling of coniferaldehyde with guaiacyl units cannot be attained in vitro either. Second, the occurrence of hydroxycinnamaldehyde β–O–4-coupled units in lignins is validated by diagnostic marker compounds released from such units by analytical thioacidolysis (Kim et al., 2002; Lapierre et al., 2004). The marker levels increase in conjunction with the degree of CAD-deficiency in transgenic poplar. How can these data be reconciled with attempts to impugn the notion of hydroxycinnamaldehyde incorporation? For example, the ability to extract the red coloration from CAD-deficient fiber using methanolic-HCl was recently presented (Laskar et al., 2004). The implication was that such extraction removed aldehyde components (‘polyaldehydes’), which could, therefore, not have been integrated into lignin polymers. A simple test would have revealed that such color removal had nothing to do with the aldehyde components in the lignin.7 As noted above, thioacidolysis produces diagnostic marker compounds from hydroxycinnamaldehydes that are incorporated end-wise into lignins by β–O–4-coupling. Methanolic-HCl extraction, even after multiple such extractions, does not reduce the release of such markers by thioacidolysis. In fact, on a weight basis the marker yield is slightly

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higher, logically due to the loss of mass from extracted components (including some hemicellulosic components) from the material; the data for a CAD-deficient poplar with 10% residual CAD-activity are: 7.0 μM/g of cell wall for unextracted material and 8.7 μM/g for MeOH/HCl-extracted cell walls vs. 213 and 247 μM/g of normal syringyl and guaiacyl monomers (Ralph, Kim et al., 2008). It is therefore obvious that the hydroxycinnamaldehydes that are incorporated into cell wall lignins in CAD-deficient plants are integral components of the polymer. They have been established to be coupled via end-wise β–O–4-coupling with the phenolic end of the growing polymer, by pathways completely analogous with the coupling of the monolignols, qualifying them as authentic lignin monomers in these plants. Since they replace monolignols (to an extent depending on the degree of CAD-deficiency), it is hard to refute the notion of monomer substitution during lignification. Clearly, the monolignol substitution by hydroxycinnamaldehydes has not been unequivocally disproven. DHCA incorporation into lignins Dihydroconiferyl alcohol (DHCA) is a well-established minor component (of unknown origin) of gymnosperm lignins (Ralph et al., 1999a; Ralph et al., 1999b; Sederoff et al., 1999). It is far from an ideal lignin monomer – without the cinnamyl alcohol double bond, the only coupling possible is to 4–O- and 5-positions on the aromatic ring, so DHCA units are always end-units. A CAD-deficient pine lignin contained enormously enhanced levels of this component (Ralph et al., 1997; Sederoff et al., 1999; Lapierre et al., 2000a; Dimmel et al., 2001; MacKay et al., 2001). As was clearly indicated in the initial report (Ralph et al., 1997), it was found cross-coupled with coniferyl alcohol, the normal monolignol whose availability for lignification was severely curtailed in the mutant, in products such as the β–5-coupled dihydro-dehydrodiconiferyl alcohol. But about half of the DHCA units were 5-coupled, including in dibenzodioxocin structures (Sederoff et al., 1999). Coniferyl alcohol monomers have never been found to 5–5-couple, so the idea that 5–5-coupled DHCA units could arise from post-coupling benzylic reduction reactions of coniferyl alcohol is illogical. Ignoring all such evidence, DHCA units continue to be ascribed to post-coupling transformations (Lewis et al., 1998a;8 Lewis, 1999). Nevertheless, additional evidence continued to accumulate to support the original premise that DHCA is an authentic monomer. This includes data from thioacidolysis and pyrolysis (Lapierre et al., 2000a; Dimmel et al., 2001; MacKay et al., 2001), along with the significant observation that the derived guaiacylpropane-1,3-diol is also incorporated into gymnosperm lignins (Ralph et al., 1999a,b).9 These diols are found incorporated into normal gymnosperm lignins and at significantly elevated levels in the CAD-deficient pine lignin. Logically guaiacylpropane1,3-diol is a monomer derived from DHCA under the oxidative conditions. When formed and present during lignification, it is incorporated into the polymer like any other phenolic component present in the lignifying zone, subject to simple chemical principles. All of the above provide unequivocal proof that DHCA is an authentic monomer that is incorporated into the lignification process. Tyramine ferulate incorporation into lignins The levels of tyramine ferulate appear to be enhanced in tobacco down-regulated in cinnamoyl-CoA reductase (CCR) (Piquemal et al., 1998; Ralph et al., 1998b). Substrates

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at the oxidation level of an acid, ester, or amide might logically build up when reduction to the aldehyde is compromised. However, the knowledge that such amides were also well known wound-response products prompted caution about whether tyramine ferulate was really a component of the lignin in CCR-deficient or wild-type plants where the component is also readily observed (Ralph et al., 1998b). Ralph et al., it was however, carefully established that tyramine ferulates were integral to the phenolic polymer component isolated in typical fashion (Ralph et al., 1998b). More recently it has been claimed that such components are not part of the polymer fraction (Anterola & Lewis, 2002). The argument presented was that the tyramine peaks in 1D 13C-NMR spectra were much sharper than those from the bulk lignin polymer and therefore were relegated to simple low-molecular-mass impurities. The ‘sharp-peak’ argument is flawed, and reflects unfamiliarity with NMR relaxation phenomena. While it is true that solution-state NMR peaks broaden with increasing molecular mass owing to the slower molecular tumbling rate and consequent faster relaxation rates, other factors determine line-widths. Terminal units that are especially rotationally mobile also have lower relaxation rates and hence sharper peaks. This is exactly the situation with the terminal free-phenolic tyramine units attached to ferulates incorporated into the backbone of these isolated polymers (Ralph et al., 1999b). Free-phenolic p-coumarate units are found acylating the γ-methylol of side-chain units in grass lignins (Ralph et al., 1994), and p-hydroxybenzoates are similarly found acylating lignin units in palms, poplars and willows (Smith, 1955; Nakano et al., 1961; Landucci et al., 1992; Sun et al., 1999; Meyermans et al., 2000; Li & Lundquist, 2001; Lu & Ralph, 2003). All display analogously sharp peaks in their 13C NMR spectra. A simple argument, beyond noting the fact that the lignins were molecular-mass-fractionated, as stated in the original CCR paper (Ralph et al., 1998b), would have attenuated the criticism – if the tyramine ferulate units were from low molecular mass components, the ferulate moieties would also display observably sharp peaks! However, finding the ferulate moieties, and proving that they were attached to the tyramines, required more sophisticated NMR experiments. The simple reason is that the ferulate moiety cross-couples integrally into the polymer, combinatorially coupling at its 4–O-, 5-, and 8-positions like the monolignols themselves, as had been well established in a number of studies (Ralph et al., 1995; Ralph et al., 1998a; Hatfield et al., 1999a; Grabber et al., 2002; Ralph et al., 2004a). Ferulates are consequently involved in such a variety of structures that they become difficult to detect in simple 1D spectra. Whether tyramine ferulates are truly part of lignin can certainly be debated, but the contention that they are have been ‘unequivocally disproven’ as being incorporated into the polymer by combinatorial radical coupling reactions is not supported by the facts. 2.5.1.2 Malleability of lignification: what makes a good monolignol substitute? Lignins are complex enough when considered to be derived from the three monolignols. However, it is abundantly clear that, in normal and transgenic plants, there may be many other lignin precursors (Boerjan et al., 2003; Lu et al., 2004; Morreel et al., 2004b; Ralph et al., 2004b). Combinatorial coupling reactions and the malleability of a process that is limited only by its chemistry (and the availability of phenolic monomers), under the current theory, allow researchers to contemplate substantial engineering of the polymer. Basically,

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any phenolic transported to the lignifying zone of the cell wall can, subject to simple chemical concerns, be incorporated into the polymer. There remain obvious limits, but we suggest that the perturbations that have been achieved to date by down- and up-regulating monolignol biosynthetic pathway genes merely indicate what is possible. From what is now known about ‘non-traditional monomers,’ some insights into what makes a good monolignol substitute emerge. They are not particularly surprising. Those that function the best, allowing the plant to grow without obvious phenotypic alteration, are those that function like the monolignols themselves (Ralph, 2006). The most important reaction is the linear end-wise β–O–4-coupling of the monomer with the phenolic end of the growing polymer. Ideal candidates are the acylated monolignols, such as sinapyl acetate (in kenaf, palms), sinapyl and coniferyl p-coumarate (grasses) and sinapyl p-hydroxybenzoate (palms, poplars and willows). The acylation of the γ-hydroxyl does not significantly interfere with coupling reactions at the β-position. Post-coupling reactions may be significantly altered, however. For example, after β–β-coupling (or cross-coupling with a monolignol), the normal internal trapping of the quinone methide moieties by the γ-OH cannot occur when the γ-OH is acylated (Lu & Ralph, 2002). The coupling still occurs but the products are altered; re-aromatization of the quinone methide takes place via water addition and then may involve the newly formed α-OH in trapping of the other quinone methide moiety. More subtly, the presence of the acyl group alters the isomer-ratio of β-ethers arising from water addition to those quinone methides. Once again, the emerging observations of the same isomer distributions in vitro and in vivo in these diverse products strongly suggests that post-coupling reactions are not under proteinaceous control. Partial substitution of sinapyl alcohol with 5-hydroxyconiferyl alcohol, in plants deficient in caffeate/5-hydroxyferulate O-methyltransferase (COMT), does not appear to adversely affect plant growth and development (Van Doorsselaere et al., 1995; Vignols et al., 1995; Morrow et al., 1997; Baucher et al., 1998; Lapierre et al., 1999; Osakabe et al., 1999; Boudet, 2000; Jouanin et al., 2000; Lapierre et al., 2000b; Li et al., 2000; Akim et al., 2001; Bajrovic et al., 2001; Guo et al., 2001a; Guo et al., 2001b; Marita et al., 2001; Parvathi et al., 2001; Pincon et al., 2001; Ralph et al., 2001a; Ralph et al., 2001b; Guo et al., 2002; Pilate et al., 2002; Piquemal et al., 2002; Barrière et al., 2003; Boerjan et al., 2003; Bout & Vermerris, 2003; Fontaine & Barrière, 2003; Higuchi, 2003; Jouanin et al., 2004; Morreel et al., 2004b; Ralph et al., 2004b). Again, 5-hydroxyconiferyl alcohol beautifully undergoes the end-wise β–O–4-coupling reaction (Plate 2.3b). β–O–4-Addition of another monomer to the new dihydroxylated end-unit P5H produces a quinone methide intermediate QM2, the re-aromatization of which now results in novel benzodioxane units H in the lignin (Plate 2.3a,b). Such units presumably confer enhanced rigidity on the polymer. It has been clearly established that any of the monolignols (coniferyl alcohol, sinapyl alcohol, or further 5-hydroxyconiferyl alcohol) can and do add to the new o-diphenol end-unit produced after 5-hydroxyconiferyl alcohol addition to the polymer (Plate 2.3c) (Marita et al., 2003). 5-Hydroxyconiferyl alcohol is therefore readily incorporated into the lignification process, making it an excellent monolignol substitute. Hydroxycinnamaldehydes present a more interesting case. As noted above (see section 2.5.1.1) both coniferaldehyde and sinapaldehyde will, like the monolignols, β–O–4-couple with the phenolic end of a growing lignin polymer (Ralph et al., 2001b). Coniferaldehyde, however, will only undergo such coupling with syringyl units. Thus both monomers can

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extend the chain in angiosperm lignification. It is not yet fully understood how the subsequent coupling reactions are affected by the more electron-deficient phenolic ends produced from the coupling of hydroxycinnamaldehydes, i.e. whether further extension of the polymer is attenuated, as has been suggested (Lapierre et al., 2004). The inability of coniferaldehyde to β–O–4-cross-couple with guaiacyl end-units explains why the CADdeficient pine did not have a lot of new aldehyde structures, despite other evidence that the level of the monomer was significantly elevated during lignification (MacKay et al., 1997; Ralph et al., 1997; Ralph et al., 1999b; Sederoff et al., 1999; Lapierre et al., 2000a; Kim et al., 2003). Without being able to couple end-wise into the polymer, the coniferaldehyde in gymnosperms is relegated to end-units arising from coupling of coniferyl alcohol with the coniferaldehyde, and a few other reactions. One interesting but still minor reaction is coniferaldehyde’s coupling with ‘syringyl-like’ units in softwoods, namely the 5-substituted guaiacyl units in 5–5- and/or 5–O–4-units. β–O–4-cross-coupling of coniferaldehyde with such structures appear to be evidenced in NMR spectra (Kim et al., 2003).

2.5.2 ‘Well-defined primary structure’ Notions of well-defined and repeating structures for lignin polymers have emerged from time to time. The most infamous is perhaps the Forss model, in which repeating 18-mer units were postulated (Forss, 1966; Forss & Fremer 2003). Structurally, the cyclic lignin structure proposed does not fit the known structural features in any way. For example, a common rendition (see http://www.lignin.fi/lignin.html) had some 12 of its 18 units as non-cyclic α-aryl ethers – there are few if any such structures in lignins (Ede & Kilpeläinen, 1995; Ralph et al., 2004b). With the emergence of the dirigent hypothesis, and putative proteinaceous control of lignification, repeating units are again being entertained.10 The ‘evidence’ comes from alleged sequencing of small oligomers. As will be shown (see section 2.5.2.1), such observations not only fall short of diagnostic evidence for well-defined primary structures in lignin, they are fully consistent with the current theory. More recent claims that the apparent linearity of monomer yield (on a cell wall basis) vs. lignin content demand proteinaceous control are also preliminarily addressed in section 2.5.2.2. 2.5.2.1 Sequencing of lignin oligomers A recent contention was that putatively ‘sequenced’ fragments, such as the hexamer S-(β–O–4)-S-(β–O–4)-S-(β–O–4)-S-(β–β)-S-(4–O–β)-G, the all-syringyl analog of which is shown in Plate 2.2b (cf. Fig. 4 in Davin & Lewis, 2005), require programmed assembly by protein templates (Davin & Lewis, 2005). It should, however, be pointed out that the structure of the hexamer is based on mass spectral evidence and no sequencing has been carried out; the position of the β−β-unit is not known. The reader is encouraged to read the paper by Evtuguin and Amado describing some beautiful mass-spectrometric methods (Evtuguin & Amado, 2003), bearing in mind the following observations that relate to the programmed assembly notion. First, it is evident from Fig. 1 in this paper that the putative hexamer masses were chosen from a virtual continuum of low-abundance peaks. Second, note that the eucalyptus lignin sample chosen has a particularly high syringyl content.

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The constitution of such a syringyl-rich hexamer is entirely in agreement with the combinatorial coupling theory. For example, since the only coupling mode of a monomer to a syringyl unit is β–O–4 (Plate 2.1e), the syringyl oligomers must be linear and can contain at most a single β–β-unit (resinol).11 Thus, small oligomers containing all β-ethers or one resinol and a remainder of β-ethers are patently unremarkable – they are exactly what would be expected in combinatorial coupling.12 There are, quite simply, few other choices in a high-syringyl polymer. The structures ‘sequenced’ therefore in no way repudiate the theory of combinatorial coupling and cannot be used as evidence for other levels of control during lignification. Concluding that the putative sequencing of such a hexamer rules out combinatorial coupling13 overlooks the constraints placed on polymer formation by simple mechanistic chemical principles. Moreover, the mass spectral evidence does not reveal anything about the possible occurrence of stereoisomers. When postulating that lignification is mediated by proteins the authors have evaded addressing the problem of explaining how a racemic polymer like lignin can be formed with protein templates when no racemic proteins are known. Instead of admitting this serious gap in the challenge hypothesis the authors question simple calculations of the possible numbers of isomers of lignin molecules. In polymeric lignin most linkages contain two chiral carbon atoms. And since a structure with n asymmetric carbons has 2n possible stereoisomers (March, 1985), the hexamer in the above example (Plate 2.2b) has 212 = 4096 total stereoisomers but, taking into account that the β–β-unit has fixed relative stereochemistry, there are 29 = 512 actual possible stereoisomers, 28 = 256 of which are not enantiomers and are therefore physically distinct, and all of which have the same m/z and are indistinguishable by mass spectrometry. Ignoring the simple fact that there are many possible isomers to be considered for any of these oligomers or the polymer (see section 2.5.3.1) does not solve the problem. The fact is that there is no scientific evidence that the isolated oligomer is a single isomer. Finally, the notion of repeating units should be re-addressed. If the putative protein encodes a chain, say an 18-mer, containing a syringaresinol (β–β-coupled) unit, then this can never repeat!11 The reason again is that the radical coupling mechanism simply does not allow for more than one syringaresinol unit in any linear chain, regardless of its length. The whole idea of repeating macro-units therefore also needs to be dismissed. 2.5.2.2 Monomer-independent sequences Among the more intriguing recent claims14 (based largely on unpublished data) is the following: evidence for control over lignin assembly is provided by the fact claim that putatively invariant and predetermined distributions of identical linkages within the primary lignin chains are independent of the degree of monomer methoxyl group substitution patterns and/or lignin contents (Davin & Lewis, 2005; Lewis & Davin, 2005). Evidence for evidence for control over lignin assembly is provided by the claim that putatively invariant and predetermined distributions of identical linkages within the primary lignin chains are independent the designation of well-defined primary structures was claimed from monitoring lignin deposition by thioacidolysis and nitrobenzene oxidation in Arabidopsis stems (Davin & Lewis, 2005). It is suggested that the linearity of plots of monomer yield (μM/g of cell wall) vs. lignin level (acetyl bromide lignin as a percent of the cell wall) supports

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the notion of a monomer-independent sequence (Davin & Lewis, 2005). This conclusion does not have any logical connection with the experimental results, which are, in themselves, open to different interpretations: for instance, the degree of syringyl or guaiacyl enrichment in the lignins is not stated.15 The linearity of the monomer yield versus lignin content (the reality of which is open to doubt – the curve does not go through the origin!16) is perfectly explicable with a constant ratio of end-wise to bulk polymerization in the time frame studied. The only physically relevant measure is the monomer yield to lignin level on a strictly molar basis; the moles of lignin units have not been calculated from the phydroxyphenyl: guaiacyl:syringyl (H:G:S) ratio, also confounding the analysis. The structure of lignins is complex and there are many competing factors that contribute to thioacidolysis and nitrobenzene oxidation monomer yields, but the data refute any notion that there is mysterious monomer-independent control over the lignification. High-syringyl lignins are obviously depleted in β–5-units, but are elevated in β–β-units, even in the same transgenic lines. For example, down-regulation of F5H produces guaiacyl-rich lignins, and an F5H-deficient Arabidopsis mutant is, like softwoods, essentially guaiacyl-only (Meyer et al., 1998), whereas F5H-up-regulation produces lignins with high-syringyl and only very low guaiacyl contents (Marita et al., 1999; Huntley et al., 2003; Li et al., 2003). Furthermore, p-hydroxyphenyl-rich alfalfa lignins (deriving largely from the typically minor monomer p-coumaryl alcohol) appear to be totally depleted in β–1-coupled products that are present in the control (Ralph et al., 2006a). In all cases, the distribution of lignin linkages is sufficiently disparate that the notion of sequence invariance is irreconcilable. In summary, the purported linearity probably lacks a physical basis. But even if it is real, more diagnostic experiments and data are required if they are to provide evidence for the existence of well-defined primary (macro-)structure. 2.5.2.3 The ‘urgent need to sequence lignin primary structure’ The espoused need to sequence lignin primary structure (Davin & Lewis, 2005) would only be valid if the dirigent/replication model were correct. Facts regarding lignin structure suggest, however, that lignins have no regularly repeating macro-structures. Attempts at sequencing extended structures are likely therefore Quixotian endeavors, for reasons that were covered in section 2.5.2.1.

2.5.3 Lignins’ racemic nature In a variety of studies over decades, using a variety of methods, fragments released from the lignin structure (under conditions that have been proven to retain optical activity) have never been found to be optically active (Ralph et al., 1999c; Akiyama et al., 2000). And entire isolated polymer fractions have no detectable optical activity (Ralph et al., 1999c). Until these papers were published, reservations continued to be expressed about the lack of optical activity in lignins.17 The lack of optical activity is entirely expected from the current combinatorial chemical coupling theory, but is a major problem for the dirigent/replication hypothesis. To address the lack of optical activity, two arguments have been advanced. First, it was suggested that there could be two sets of dirigent protein arrays, equally represented, encoding opposite

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antipodes (Lewis & Davin, 1998; Gang et al., 1999). It should not be necessary to point out that encoding the opposite enantiomer of a product requires a different protein. For example, a dirigent protein that produces (+)-pinoresinol has a primary structure that must obviously be different from a dirigent protein involved in producing (−)-pinoresinol. A different protein will have different kinetics. But even if the kinetics were the same, the two proteins would have to be available at the same time and space to produce racemic products. This is why, even when both enantiomers of a lignan are produced in planta, the lignans are not racemic – they have a net optical activity. The putative protein harboring arrays of dirigent sites would have to have an antipode in which all those dirigent sites were re-engineered to produce the opposite enantiomer. Applying Occam’s razor that it is the aim of science to present the facts of nature in the simplest and most economical conceptual formulations (Thorburn, 1918), this proposal must be dismissed. The second proposed solution was to have template replication produce the opposite isomers, the implications of which will be addressed in section 2.5.4. 2.5.3.1 The ‘number of isomers’ problem A consequence of racemic polymerization is extraordinary isomeric complexity that results in lignins and the implication that a lignin molecule has a low probability of being identical to other lignin molecules. Lignin monomers do not have any optical activity – unlike the protein and polysaccharide monomers, they possess no chiral centers. Chiral centers are however created in each coupling reaction involving the monolignol side-chain β-position, and another is created when the intermediate quinone methide is re-aromatized (Plate 2.1). The result is that the number of isomers of any ‘randomly’ formed lignin structure increases with its degree of polymerization, quickly becoming astronomical. This topic was fully addressed in a recent review (Ralph et al., 2004b) and only the following will be noted here. The models illustrated in Plate 2.4 are not lignins, nor are they assumed ‘sequences’ of any kind, but are useful models conforming as closely as possible to available unit frequency data. The pine model, as drawn with 25 units has over 500 billion possible isomers, all of which are physically distinct. The poplar model, with 20 linkages, has over 8.5 billion possible isomers. The implications of the fact that lignins are racemic polymers needs to be more widely appreciated. This challenge has not been met by proponents of the dirigent/replication hypothesis, who continue to question this unequivocal chemical fact18 (Davin & Lewis 2005; Lewis & Davin 2005) instead of providing experimental evidence. A priority before ‘sequencing lignin primary structure’ should be to determine whether released lignin oligomers comprise only a single enantiomeric pair, as predicted by the dirigent/replication hypothesis, or many isomers, as predicted by the combinatorial theory. If the number of isomers is found to be consistent with combinatorial coupling, the dirigent/replication hypothesis can be unequivocally dismissed.

2.5.4 Comments on template replication The hypothesis that lignin primary chains might replicate by template polymerization (Guan et al., 1997; Sarkanen, 1998), where one chain dictates the exact mirror opposite

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chain in the next assembled molecule (Lewis & Davin, 1998), is problematic. The coupling mechanism proposed is not compatible with the known mechanism of oxidative phenolic coupling. Identification of the forces that govern the replication has been attempted, but there is no discernible hierarchy among the dimer–dimer attractions that are proposed in the article (Chen & Sarkanen, 2003). To have real replication, attractions that are clearly stronger than the alternatives need to be identified, and what is being replicated must be clearly defined. Replication means replication, and that means that identical structures are being formed. This leads to a regular structure with identical units, which must be chiral (see section 2.5.3). The template hypothesis of lignin biosynthesis has no precedent. Of the ‘several thousand papers’ proclaimed as describing template effects (Davin & Lewis, 2005), not one was cited that pertains to lignin biosynthesis, nor was a documented example from nature of a protein template producing racemic polymers provided. Additionally, the problems facing the concept of template replication as a mechanism of lignin formation were evaded. One concept that has not been appreciated again has to do with the presence of resinol units in a lignin chain. The polymerization of the chain shown in the dirigent/replication model (Plate 2.2a), must begin with β–β-coupling to first form the resinol unit. After that, coupling can occur sequentially from either end of this dimer. There is no alternative method that can produce the same polymer. In the same way, template replication of the chain shown in Plate 2.2a cannot begin at either of the ends of a lignin molecule – the only place it can begin is again with the resinol somewhere within the chain. It is therefore difficult to understand the concept of ‘double-stranded template replication’ – certainly it cannot operate in anything like the manner of DNA replication, which proceeds from one end to the other. To be taken seriously, the template replication hypothesis has to be brought into accordance with the mechanism of oxidative phenolic coupling, and it must be demonstrated that lignin indeed consists of two enantiomers in equal amounts.

2.5.5 Polymer branching Only two structures, containing 5–5- and 4–O–5-linkages, are known to result in lignin branching. Neither are formed during end-wise monolignol coupling reactions; they require the coupling of the phenolic ends of two pre-formed oligomers or polymers. And neither can be formed without the participation of at least one guaiacyl unit. For that reason, high-syringyl lignins are logically more linear. The oldest estimates are that 4–O–5- and 5–5-structures in softwood guaiacyl lignins comprise a total of about 13–15% of the linkages (Adler, 1977). Some doubt is cast on the levels of 4–O–5-linkages as they cannot be authenticated in NMR spectra of softwood lignins, either isolated lignins or from dissolution of the entire cell-wall fraction (Ralph, 2005). And there are suggestions that dibenzodioxocin structures, formed following addition of a monolignol to a 5–5-linked unit, may terminate the chain; for reasons that need to be examined, the chemical propensity for monolignols to couple to the phenolic unit in a dibenzodioxocin may be low. As a result of these factors, the structural picture is evolving to recognize lignins as more linear polymers, as reflected by recent models that have been proposed, e.g. the two shown in Plate 2.4. The criticism recently leveled (Davin & Lewis, 2005), that ‘none of these data are in agreement with the unproven random coupling assembly hypothesis depicted as leading

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to highly cross-linked three-dimensional polymers’ is therefore outdated. Nevertheless, branching is a characteristic feature of lignin polymers, particularly in guaiacyl lignins, and must be accommodated by any lignification model. Branching of the lignin chains is not envisaged in the replication theory. In a discussion of this topic, two explanations appear to be offered (Chen & Sarkanen, 2003). First, that there is in fact no branching in the native lignin macromolecule, although in this work the authors used kraft lignin, derived from cooking wood at 170°C in caustic soda and sodium sulfide, instead of native lignin. The second is that, if there is branching, it is ‘incidental.’ Neglecting for now the problems in validating 4–O–5-structures in softwood lignins, there is no debate over the existence of 5 –5-coupling between guaiacyl units nor the dibenzodioxocin structures that result in the polymer. Biphenyls (5–5-linkages) are generally accepted as accounting for ∼9.5–11% of the C9-units in softwood lignins (Adler, 1977). Dibenzodioxocins, readily detected in all but the most syringyl-rich lignins by NMR, have been estimated to involve ∼11% of the C9-units in softwood lignins (Argyropoulos et al., 2002). They can hardly therefore be dismissed as being incidental. There is also mention of ‘errant’ monolignol radicals by Chen & Sarkanen (2003). These explanations of uncontrolled oxidative coupling in lignin biosynthesis sound dangerously close to the Freudenberg concept!

2.6 Why the new hypothesis is not in contention Promoting ambiguous observations and ideas without direct experimental support is not what is required to bring a hypothesis toward theory status. If lignification is in fact under proteinaceous control, as suggested many years ago now, by putative proteins harboring arrays of dirigent sites, the evidence required is clear. Where is this protein or its gene? It should not be hard to find the gene for a protein harboring perhaps 18 or more dirigent binding sites, assuming that dirigent sites that bind monolignols would share some sequence identity with dirigent proteins, and also have features of externally exported proteins. Do such proteins appear in analysis of the cell wall proteome? An in-depth informatic analysis of the Arabidopsis genome sequence, coupled with analysis of the large amount of publicly available microarray data for co-expression of candidates with other lignin biosynthetic genes, might also reveal candidates. Has such an analysis been carried out? Furthermore, forward genetic analysis of Arabidopsis T-DNA insertion- and/or activation-tagged lines might be expected to reveal mutants in the presumed protein template with severe lignin phenotypes. Have such screens been performed? The standard tests then are the traditional loss-of-function/gain-of-function for the gene of interest. Such tests may be lacking even for the dirigent protein/genes themselves.19 Prolonging the case for the competing hypothesis by continuing to publish non-diagnostic data that may or may not support the hypothesis does not convince the scientific community. It is time to call for delivery of the protein and its gene. And, if replication on a lignin template is to be advanced, real replication of lignin structure needs to be demonstrated. We suggest that replicating a β-ether homopolymer, generated by recently published methods (Kishimoto et al., 2006), would be a good start.

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2.7 Is lignification a biochemical anomaly? Is lignification, the polymerization step, under absolute biochemical control? Or is it, as the current theory maintains, simply a chemical process? And is it true that ‘Such claims (that the monolignols are combinatorially coupled according to chemical principles), if ever shown to be correct, would place lignin formation – Nature’s second most abundant polymer, no less – with no biochemical equivalent of any kind’ (Lewis et al., 1998b)? We suggest that there is useful elegance in chaotic processes, and in combinatorial chemistry. Among the many natural processes that could be considered here, we choose just two to illustrate the concepts. The first, cell-wall cross-linking by ferulate dehydrodimerization, is a process related to lignification in that it involves combinatorial radical coupling processes. The second, polysaccharide biosynthesis, is used to illustrate that even highly enzymatically controlled processes can have random elements that result in enormously variable primary structure.

2.7.1 Ferulate dehydrodimerization is combinatorial Ferulates acylating polysaccharides, particularly arabinoxylans in grasses, are now highly implicated in vital cell-wall cross-linking mechanisms (Ralph et al., 1998a; Hatfield et al., 1999a; Ralph et al., 2004a). Polysaccharide–polysaccharide cross-linking is accomplished by ferulate dehydrodimerization as well as dehydrotrimerization (Bunzel et al., 2003; Rouau et al., 2003; Bunzel et al., 2004; Bunzel et al., 2005; Funk et al., 2005; Bunzel et al., 2006) and perhaps even higher oligomerization (via radical coupling mechanisms). And polysaccharide–lignin cross-linking occurs when ferulates and their dehydrodimers incorporate into lignins via radical reactions with lignin monomers and oligomers. The simple point to be made here is that the range of combinatorial dehydrodimers (1A–E, Fig. 2.1) found in plant materials is the same range as produced by chemical radical coupling in vitro. Notably, all three 8–8-coupling products 1C are found in vivo, implicating several post-coupling re-aromatization pathways. Bearing Occam’s razor (‘Pluralitas non est ponenda sine neccesitate’) in mind again, is it more logical to conclude that ferulate dehydrodimerization takes place via a simple combinatorial chemical process or via a carefully controlled biochemical process with enzymes or proteins specific for all of the coupling modes, to give the same array of products as an uncontrolled reaction?

2.7.2 Polysaccharide primary structure is NOT absolutely dictated The (1→3),(1→4)-β-glucans from the cell walls of barley grains are a good example of polysaccharides in which there is tight control over subunit structure, but a random distribution of the subunits within the molecule. These linear polysaccharides contain approximately 30% (1→3)- and 70% (1→4)-linkages. Approximately 90% of the polysaccharides consist of cellotriosyl and cellotetraosyl subunits composed of three and four glucosyl residues, respectively, joined by (1→4)-linkages; these subunits are joined by single (1→3)-linkages. Blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions (Woodward et al., 1983). However, the distribution of the cellotriosyl and cellotetraosyl

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Fig. 2.1 Ferulate dehydrodimerization is an important mechanism for polysaccharide–polysaccharide cross-linking in grasses. All of the dimers 1A–E have been authenticated, indicating that combinatorial coupling (8–O–4, 8–5, 8–8, 5–5, and 4–O–5) all occur in planta, and that post-coupling re-aromatization reactions follow at least three courses, in vitro and in planta, giving distinct 8–8-coupling products 1C1, 1C2 and 1C3.

subunits within the molecule is random (Staudte et al., 1983). Mechanisms for the biosynthesis of (1→3),(1→4)-β-glucans must account for these features in the molecule. Interestingly, in-vitro studies showed that substrate concentration influenced the structure of the polysaccharides (Henry & Stone, 1982). The concentration of UDP-glucose significantly affected the relative proportions of cellotriosyl and cellotetraosyl subunits. With the recent demonstration that proteins encoded by CSLF genes are involved in the biosynthesis of (1→3),(1→4)-β-glucans, further understanding of the biosynthesis of these molecules may soon be available (Burton et al., 2006).

2.8 Summary comments on the dirigent/replication hypothesis The dirigent/replication hypothesis as it has been put forward (Chen & Sarkanen, 2003; Davin & Lewis, 2005) falls short of being a ‘constructive challenge’ for several reasons. A challenge hypothesis must offer a solution to a problem, to something not explained by the current theory, and offer a stimulus for further research. What is the problem for which the dirigent/replication hypothesis is a solution? There is no clear statement of

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such a valid problem in these papers. The hypothesis is instead offered as a ‘conceivable mechanism whereby particular sequences of inter-unit linkages in lignin macromolecules can be replicated’ (Chen & Sarkanen, 2003). A constructive challenge should suggest new experiments that allow the hypothesized mechanism to be tested, and falsified if necessary. This is where the dirigent/replication hypothesis fails completely. The Freudenberg concept of oxidative coupling in lignin biosynthesis has enabled researchers to explain many features of the structure of lignin. Any alternative hypothesis has to be better than the accepted one to be taken seriously. Synthesis of lignin on a protein array of dirigent sites that direct the coupling was initially an intriguing idea to consider. But there is a problem: all proteins are chiral and exist as single enantiomers, i.e. as only one of two mirror images. This fact constitutes a serious impediment to the acceptability of the dirigent hypothesis. If lignin is racemic, as researchers all agree, surely the simplest explanation is that the coupling occurs without the intervention of chiral dirigents. Regarding the replication aspect itself, the idea of molecular attraction between the growing lignin polymer and the monolignol radical is quite plausible and undoubtedly of some importance. But the replication hypothesis as it is stated poses problems of its own. Replication means that identical structures will be formed. This leads to lignin macromolecules with a regular structure and identical units in identical sequences. In order to explain the lack of optical activity, the authors claim that replication inexplicably produces enantiomeric structures. What regularities of the replicating process have the authors observed that necessitate this complicated hypothesis? In addition to the structural aspects of the polymers that have been revealed over many decades of research, more recent studies with transgenic plants indicate that lignification is a particularly malleable process. The dirigent/replication hypothesis does not tolerate monomer substitution, a process that is now well authenticated with several compelling examples in plants with reduced capacities to biosynthesize the traditional lignin monomers. By contrast, the existing theory readily accommodates monomer substitution along with the plethora of non-monolignol monomers that are becoming increasingly well authenticated in various lignins. Bonding patterns in lignins have been successfully replicated in laboratory cross-coupling experiments, demonstrating that the cross-coupling propensities of a number of monomers observed in vitro can explain the couplings observed in vivo. Overall the oxidative coupling concept as originally formulated by Freudenberg still offers the best basis for understanding the lignification process.

2.9 Conclusions The examples documented here, and countless more, illustrate how ostensible support for the dirigent array/template replication hypothesis hinges on carefully selected data. For a new hypothesis to be considered viable, it has to stand the scrutiny of experiment. An idea or a hypothesis is not a theory, as science editor Alan L. Leshner eloquently reminded researchers recently (Leshner, 2005).20 To date, presentations such as that of Davin & Lewis (2005) have failed to specify (in terms of experimental data) the shortcomings of the Freudenberg mechanism of lignification,

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and failed to show how the dirigent/replication hypothesis resolves these shortcomings. Presenting ambiguous or irrelevant observations as evidence cannot supplant diagnostic experiments that validate or invalidate hypotheses. And declaring that ‘recent progress has provided crucial evidence to support the theory that lignin primary structure is controlled at the proteinaceous level’ and that ‘evidence for control over lignin assembly has been demonstrated . . .’ (Davin & Lewis, 2005) without producing the evidence, simply does not make it so. Regarding the controversy, it has been said so well previously that another quote is warranted. Gary Taubes wrote the following in an article on ‘The soft science of dietary fat’ (Taubes, 2001): ‘To the outside observer, the challenge in making sense of any such long-running scientific controversy is to establish whether the skeptics are simply on the wrong side of the new paradigm, or whether their skepticism is well founded. In other words, is the science at issue based on sound scientific thinking and unambiguous data, or is it what Sir Francis Bacon, for instance, would have called “wishful science,” based on fancies, opinions, and the exclusion of contrary evidence? Bacon offered one viable suggestion for differentiating the two: the test of time. Good science is rooted in reality, so it grows and develops and the evidence gets increasingly more compelling, whereas wishful science flourishes most under its first authors before going downhill.’ Defending a 50-year-old theory is hardly a highlight in a scientist’s career. Our aim in this chapter has been to avoid the potential tragedy whereby a perfectly good theory that remains consistent with all the current facts is overthrown, without challenge, by an unsupported runaway hypothesis. The danger to unwary researchers is that adherence to a principle that may not be correct alters experimental design approach and conclusions.21 The logical conclusion from examining the facts is that the dirigent/replication hypothesis fails to challenge the current theory and should cease to be entertained. Further research into determining the number of lignin oligomer isomers, for example, may be worthwhile to confirm that lignins are truly combinatorial polymers. Heeding recommendations that lignin primary structure needs urgent sequencing would then be futile. Finally, lignin should not be labeled an enigma because its exact primary structure is not absolutely stipulated. Lignins are certainly not unique in this regard. It is logical that their irregular structure has biological significance (Sederoff et al., 1999). An ordered structure would be more sensitive to destructive influences. As succinctly stated by Denton: ‘Biological defense is well served by loosely ordered chemistry. Where many randomly linked products are needed, sloppy pathways are economical’ (Denton, 1998). A role of lignin is to fill available space in the cell wall, making it strong and hydrophobic. If the cell wall is already largely established by cellulose microfibrils and various complex hemicelluloses, it is difficult to imagine filling up the available space with rigid and ordered molecules in which the primary structure is defined. Indeed, the process is most facile either with small molecules, or with large molecules if the structure of the latter is flexible and able to respond to local needs. The combinatorial coupling of monolignols is a superb solution of Nature for accommodating this task. Perhaps it is opportune to re-evaluate the elegance in chaotic systems, particularly for plant defense. Plants have evolved a route to the crucial cell-wall component lignin that allows them tremendous flexibility to respond to environmental factors, and even to the tinkerings of genetic engineers bent on impeding monolignol production.

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2.10 Notes 1 ‘Thus more and more evidence is gathering that reveals that lignin formation is under full biochemical control, in harmony with formation of all other known biopolymers’ (Kwon et al., 1999). ‘In summary, there is now little doubt that the biochemical control of lignification is implemented at the levels of both monomer deposition and linkage specification’ (Gang et al., 1999). ‘Taken together, this evidence suggests that lignin assembly in vivo is subject to biochemical regulation, whereby the appropriate monomers are linked in a specific manner to yield a limited number of coupling modes in characteristic proportions’ (Croteau et al., 2000). ‘This is further evidence for full biochemical control of lignin assembly, i.e. via monomer transport, oxidation, and directed polymerization’ (Patten et al., 2003). ‘. . . which serve to underscore the fact that complexity of assembly need not be confused any further with random formation’ (Anterola & Lewis, 2002). ‘Recent progress has provided crucial evidence to support the theory that lignin primary structure is controlled at the proteinaceous level’ (Davin & Lewis, 2005). 2 Lewis is quoted as stating: [In hypothesis-driven research, the way to move forward is] ‘not to look for evidence that doesn’t support it. You look for evidence that will support your hypothesis, and you do the controls’ (Rouhi, 2001). 3 ‘It is inconceivable that lignin formation would be left to the vagaries of such a wide range of enzymes or be realized in a haphazard manner’ (Davin et al., 1997). ‘This supposition represented a departure from all other known biochemical processes, since no explicit control of the final configuration of the product was envisaged’ and ‘which has no counterpart elsewhere in biochemistry’ and ‘the prevailing dogma has insisted that lignin biosynthesis occurs in a manner whereby . . . no direct control of macromolecular assembly is exercised at the enzymatic level’ (Lewis et al., 1998a). ‘Such claims, if ever shown to be correct, would place lignin formation – Nature’s second most abundant polymer, no less – with no biochemical equivalent of any kind’ (Lewis et al., 1998b). ‘The one favored by the author is that of full biochemical control over the outcome of phenoxy radical coupling in vivo, in harmony with that of all other biological systems’ (Lewis, 1999). ‘For decades, the perceived formation of lignins in vivo has been biochemically incongruous’ (Croteau et al., 2000). ‘If this (random coupling) were correct, then the formation of approximately 20 –30% of all plant organic matter would have been left essentially to chance’ (Davin & Lewis, 2000). 4 ‘This early working hypothesis did not, however, explain the preponderance of 8–O–4′ linkages in lignin biopolymers’ (Lewis et al., 1998a). ‘The perspective could not, however, explain many biological aspects of lignification, including . . . the observed regiospecificity in coupling resulting in approximately 50% to 70% of all interunit linkages being 8–O–4′ bonded’ (Davin & Lewis, 2000). [Note: 8–O–4′ is equivalent to the β –O–4-terminology used throughout this chapter]. 5 Pinoresinol is not significantly released from β-ether cleaving reactions. Thus acidolysis of spruce did not release detectable pinoresinol (Lundquist, 1970); thioacidolysis released only traces of the same dimeric products as were produced from pinoresinol (Lapierre et al., 1991; Jacquet et al., 1997; Önnerud & Gellerstedt, 2003). When thioacidolysis trimers (following Raney Ni desulfurization) were examined, most of the β–β-products appeared to be 5–O–4-linked (Önnerud & Gellerstedt, 2003), explaining why β–β-dimers were released in such low quantities. This group suggested, as Lundquist had earlier (Lundquist, 1992), that pinoresinol units might derive not from monolignol–monolignol coupling reactions but from the 5-coupling of a monolignol to the growing oligomer, leading to an in-chain resinol formation (Zhang et al., 2003). At this point, evidence for 5-coupling of monolignols is not compelling and it is not currently possible to distinguish this route from one in which preformed pinoresinol dimers couple at their 5-positions with other lignin oligomers (Ralph et al., 2004b). 6 ‘. . . this concept of the free-interchange of lignin monomeric units is diametrically opposite to the working hypotheses that guide our own research undertakings’ (Lewis, 1999). 7 Higuchi was the first to note that the red coloration of CAD-deficient plants could be attributed to products derived from the hydroxycinnamaldehydes (Higuchi et al., 1994). Indeed, synthetic lignins incorporating hydroxycinnamaldehydes are distinctly reddish in color. The basis for the

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color was not however conjectured. Dehydrodimers of hydroxycinnamaldehydes are at most only pale yellow colored (Kim et al., 2003). The products from β–O–4-cross-coupling with guaiacyl or syringyl units are also not highly colored (Kim et al., 2003). Interestingly, the latter structures do not stain with phloroglucinol-HCl either – this stain has long been used as a test for lignins owing to the diagnostic staining coloration with hydroxycinnamaldehyde end-groups. Since hydroxycinnamaldehydes incorporated into lignins by β–O–4-coupling do not stain, however, this stain can not reliably be used to infer anything about the incorporation of hydroxycinnamaldehydes into angiosperm lignins (Kim et al., 2003). This paper also utilized cyclic arguments to ascribe DHCA components to minor impurities. ‘Careful evaluation of the reported data revealed that only about 17% of this “lignin” had been extracted from the tissues for analysis, and that the total contribution to the plant lignin from the dihydroconiferyl alcohol unit was < 5–6%’ (Lewis et al., 1998a). The DHCA component was erroneously assumed to be a fully-extractable impurity (and this assumption was then used in the proof that it was an impurity); subsequent analyses showed that significant levels of DHCA remained in the lignin that was not extracted and in the total lignin fraction (Ralph et al., 1999b; Lapierre et al., 2000a; Dimmel et al., 2001; MacKay et al., 2001). The accusation ‘The researchers had failed to recognize that formation of dihydrodehydrodiconiferyl alcohol results from dehydrogenative dimerization of at least one, and more probably two, coniferyl alcohol moieties’ (Lewis, 1999) is verifiably false. It was clearly stated in the original reference (Ralph et al., 1997) that coniferyl alcohol was involved in the formation of this product. The claim that dihydroconiferyl alcohol units arose from the reduction of pre-formed coniferyl alcohol dimers apparently ignored evidence that DHCA was undeniably a monomer. If DHCA monomer is present under peroxidase-H2O2 conditions, not only will it undergo radical coupling reactions, but it can be converted to guaiacylpropane-1,3-diol (Ralph et al., 1999a, 2001b). As has been seen previously with benzyl monomers (Zanarotti, 1982, 1983), singleelectron oxidations produce quinone methides by apparent double H·-abstraction; the mechanism is more likely to be disproportionation of two phenolic radicals to yield the one molecule of the original DHCA and one quinone methide. Under the aqueous conditions, the quinone methide derived from DHCA adds water, producing guaicylpropane-1,3-diol, which will then incorporate into lignin. ‘Both [Sarkanen and Lewis] say they wouldn’t be surprised to find some repeating unit, such as an 18-mer’ (Rouhi, 2001). ‘Current evidence would indicate that there are only a few native lignin primary structures’ (Davin & Lewis, 2005). The restriction to only a single syringaresinol unit in a chain derives from the chemistry of lignification. Although β–β-coupling can and does occur between sinapyl alcohol lignin monomers (Plate 2.1b), the resulting syringaresinol dimer, like any other syringyl phenolic end-unit on the polymer, can only couple 4 –O- (Plate 2.1e), (and, to a minor extent, 1-). And, since there is never single electron density that can be delocalized from a phenolic radical to the β-carbon of any lignin oligomer, it is simply not possible to produce another syringaresinol unit in the growing chain. Contemplation of simple distributions is even possible here for the all-syringyl analog. For example, if it is assumed that a chain initiates with β–β-coupling to form a resinol R, a hexamer will have 5 inter-unit linkages, 4 of which will be β-ethers B, i.e. structures B4R. If adding sinapyl alcohol to a phenolic end was random then, in free solution, the distribution of the three possible hexamers B4R:B3RB:B2RB2 will be 1:4:3. Of course, the addition propensities are not equal; adding to a resinol phenol is electronically different than adding to the phenol of a β-ether unit. In the wall, one end of the resinol may also be less accessible. Finally, the hexamers available for MS study were not from interrupted biosynthesis but followed ball-milling, which breaks β-ether linkages, so rationalizing the distribution of hexamers, were that data available, would remain complex. ‘Thus recent claims . . . that lignins have no primary sequences have no sound scientific basis. Even the most cursory consideration that a lignin-derived fragment has already been sequenced might temper such claims’ (Davin & Lewis, 2005).

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14 ‘Evidence for control over lignin assembly has been demonstrated with the discovery of monomerinvariant aryl-O-ether linkages in lignins that upon alkaline cleavage release the corresponding monomers in equimolar amounts, regardless of monolignol composition’ (Davin & Lewis, 2005); ‘. . . the existence of invariant and predetermined distributions of identical linkages with the primary lignin chains that are independent of the degree of monomer methoxyl group substitution patterns and/or lignin contents. Accordingly, the main primary chains of the biopolymers are considered to be of a predetermined regularity’ (Lewis & Davin, 2005). ‘We have identified severe restrictions imposed in vivo on lignin macromolecular configuration indicative on nonrandom assembly. Specifically, there is a predesignated template periodicity in placement of dominant (alkali-cleavable) inter-unit linkages in lignins invariant of methoxyl group substitution pattern’ (Jourdes et al., 2006). 15 Existing data clearly and predictably show that (syringyl-rich) hardwood lignins give significantly higher yields of thioacidolysis monomers than (guaiacyl-rich) softwood lignins (Lapierre, 1993). Guaiacyl lignins with non-cleavable β–5- and 5–5-linkages, comprising some 15–20% of the linkages and being irregularly distributed, typically yield monomers at ∼1200 μM per g of lignin (e.g. 1230 μM/g-lignin in spruce). High-syringyl lignins, which are essentially linear β-ether polymers with typically 10% non-cleavable β–β-units (resinols), and only one resinol per chain (see above, section 2.5.2.1), of course release significantly higher levels of monomers, typically twice those of softwoods (e.g. 2460 μM/g-lignin for a birch lignin that is ∼76%-syringyl). These data, predictable from what has been long known about the structure of guaiacyl vs. syringyl lignins, simply cannot be reconciled with a model in which the sequence of units is independent of lignin composition, i.e. independent of the extent to which the polymers were derived from monomers p-coumaryl, coniferyl, or sinapyl alcohols. 16 If such plots are to have any basis in reality, obviously they must be forced through the origin – one can hardly debate that a plant with no lignin will not release any thioacidolysis monomers, or the corollary that zero release of monomers implies that there is no (true polymeric) lignin. Yet the projected line in the published plot (Davin & Lewis, 2005) implies that no monomers at all are released if the lignin content is below 6.5%. Constraining such fitting-curves to logically go through the origin suggests that they are not linear. 17 Davin and Lewis observed: ‘Yet, although the questions of lack of optical activity in lignin formation had already been carefully addressed* by ourselves and others, several proponents of random coupling re-analyzed the enantiomeric composition of various lignin-derived dimeric products; as expected they were racemic’ (Davin & Lewis, 2005). Response: to that point in time, these authors expressed doubt that lignins were racemic, as noted by Rouhi (2001) and as these examples show: ‘This contrasts with the racemic linkages which are believed to be incorporated into lignin biopolymers’ (Lewis et al., 1998a); ‘even the apparent lack of optical activity in lignins . . .’ (Lewis, 1999); ‘perhaps two of the most intriguing aspects about lignification is the apparent lack of any optical activity’ and ‘such a process could also explain the perceived lack of optical activity of lignins . . .’ (Lewis & Davin, 1998); ‘Indeed, such a process could also explain the reputed lack of optical activity in native lignins . . .’ (Gang et al., 1999); ‘. . . account for the reported lack of optical activity of polymeric lignins’ (Davin & Lewis, 2000); ‘In contrast to the lignans, lignins are thought to be racemic (optically inactive)’ and ‘. . . the presumed lack of optical activity in lignins might result . . .’ (Croteau et al., 2000). *‘Carefully addressed’ apparently refers to the following distinctly anti-Occam suggestions: ‘it could be that the primary chains, for example, form complementary “mirror images” via template replication, or that there are two distinct types of proteins each encoding formation of complementary chains that effectively cancel out any measurable optical activity’ (Lewis & Davin, 1998). 18 The following quotes illustrate how this basic chemical principle is not understood: ‘For example, unproven assertions were made that a lignin molecule of relative molecular weight 21,500 could have as many as 1066 possible isomers, this being noted to approximate the number of atoms in the galaxy, whereas a lignin chain composed of 20 monomers has over 17 billion possible isomers. Such unproven assertions beg the question as to how much longer such extravagant claims will continue to be made’ (Davin & Lewis, 2005) [Response: as long as they continue

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to be true!]. ‘Reinterpretation of previous (often error prone) claims of putative lignin random assembly/combinatorial biochemistry, including that of 20-mer lignin structures allegedly harboring 17 billion plus isomers’ and ‘these researchers take the unusual approach of developing a model with the scientifically incomprehensible suggestion of 17 billion plus isomers being possible in a short chain lignin’ and ‘it is timely to unequivocally refute the unparalleled notion that a 20-mer unit poplar lignin contains 17 billion plus isomeric forms. This scientifically untenable notion, being espoused in the 21st century, envisages nearly 1,000,000 times more isomers in a short chain lignin (i.e. 17,000,000,000 isomers!) than that, for example, of the total number of genes (28,000) present in the Arabidopsis genome. Such notions cannot be seriously entertained, not only because they fully lack scientific credibility, but because such pronouncements starkly run counter to the detailed and painstaking enzyme/protein work of ourselves and others as described above’ (Lewis & Davin, 2005). [Note: It was never stated that this was lignin, just a model, and not that it contains this many isomers, just that there are 17 billion possible isomers. Such calculations are factual and based on the principle, found in any elementary chemistry text, that a non-symmetrical molecule with n optical centers will have 2n optical isomers, 2n−1 of which will be physically distinct compounds (March, 1985). A random 20-mer β -ether linear polymer has 38 optical centers leading to 238 optical isomers and 237 (over 137 billion) physically distinct isomers: The lesser number, 17 billion, arises because the model being considered was not a homo-β-ether polymer but incorporated structures in which certain of the centers were constrained relative to others (see Plate 2.4). The idea that there are not enough genes to account for such an astronomical number of isomers is absolutely correct, reinforcing the idea that biosynthesis of this racemic polymer cannot be under proteinaceous control!] 19 What evidence has been obtained to rule out a role for dirigent proteins in lignin, as opposed to lignan, biosynthesis? Gene-array experiments by an independent group (Ralph et al., 2006b) [no relation to the joint-author of this Chapter!] suggest that dirigent proteins themselves are not involved in cell-wall biosynthesis. What phenotypes are observed in Arabidopsis knockouts of the various dirigent protein genes? Is there a lignin phenotype? Have any experiments been done to test dirigent function by loss- or gain-of-function experiments in transgenics? This would not be difficult in Arabidopsis. The one issue that might arise would be genetic redundancy, such that there is no phenotype. Have the experiments been done, or do the comparisons of sequences among the dirigent protein family suggest that this will be a major problem? It should be possible to see if some do not have overlapping expression and should therefore give a phenotype if down-regulated. 20 ‘There is an important distinction between a belief and a theory. . . . In our business, a theory is not an educated guess nor, emphatically, is it a belief. Scientific theories attempt to explain what can be observed, and it is essential that they be testable by repeatable observations and experimentation. In fact, “belief ” is a word you almost never hear in science. We do not believe theories. We accept or reject them based on their ability to explain natural phenomena, and they must be testable with scientific methodologies’ (Leshner, 2005). 21 For example, a study into wheat straw lignins recently concluded that the lignin was linear and regular, despite the data presented demonstrating just the opposite (Benjelloun-Mlayah et al., 2005). The carbon NMR spectra are far more complex than would result from a lignin with a well-defined primary structure. Indeed, the extraordinary broadness and complexity of both proton and carbon spectra, compared to those from other natural polymers with well-defined primary structures, is attributable to the combinatorial nature by which the polymer was generated and the incredible stereochemical complexity resulting from racemic coupling reactions.

2.11 References Adler, E. (1977). Lignin chemistry – past, present and future. Wood Science and Technology, 11, 169 – 218.

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Akim, L.G., Argyropoulos, D.S., Jouanin, L. et al. (2001). Quantitative P-31 NMR spectroscopy of lignins from transgenic poplars. Holzforschung, 55, 386–390. Akiyama, T., Magara, K., Matsumoto, Y., Meshitsuka, G., Ishizu, A. & Lundquist, K. (2000). Proof of the presence of racemic forms of arylglycerol-β-aryl ether structure in lignin: studies on the stereo structure of lignin by ozonation. Journal of Wood Science, 46, 414–415. Anterola, A.M. & Lewis, N.G. (2002). Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry, 61, 221– 294. Argyropoulos, D.S., Jurasek, L., Kristofova, L., Xia, Z.C., Sun, Y.J. & Palus, E. (2002). Abundance and reactivity of dibenzodioxocins in softwood lignin. Journal of Agricultural and Food Chemistry, 50, 658 – 666. Bajrovic, K., Ipekci, Z., Ogras, T., Altinkut, A., Ozturk, N. & Gozukirmizi, N. (2001). Investigations on biotechnologically developed poplar clones as raw material for pulp and paper industry. Biotechnology & Biotechnological Equipment, 15, 39–43. Barrière, Y., Guillet, C., Goffner, D. & Pichon, M. (2003). Genetic variation and breeding strategies for improved cell wall digestibility in annual forage crops. A review. Animal Research, 52, 193–228. Baucher, M., Christensen, J.H., Meyermans, H. et al. (1998). Applications of molecular genetics for biosynthesis of novel lignins. Polymer Degradation and Stability, 59, 47–52. Benjelloun-Mlayah, B., Le Bigot, Y., Banoub, J.H. & Delmas, M. (2005). The linear and regular character of straw lignin. In: Thirteenth International Symposium on Wood, Fiber, and Pulping Chemistry, Auckland, New Zealand, pp. 359–364, APPITA, Australia. Boerjan, W., Ralph, J. & Baucher, M. (2003). Lignin biosynthesis. Annual Reviews in Plant Biology, 54, 519 –549. Boudet, A.M. (2000). Lignins and lignification: Selected issues. Plant Physiology and Biochemistry, 38, 81– 96. Bout, S. & Vermerris, W. (2003). A candidate-gene approach to clone the sorghum Brown midrib gene encoding caffeic acid O-methyltransferase. Molecular Genetics and Genomics, 269, 205–214. Brunow, G. (2001). Methods to Reveal the Structure of Lignin. In: Lignin, Humic Substances and Coal (eds M. Hofrichter & A. Steinbüchel), pp. 89–116. Wiley-VHC, Weinheim, Germany. Brunow, G., Lundquist, K. & Gellerstedt, G. (1999). Lignin. In: Analytical Methods in Wood Chemistry, Pulping, and Papermaking (eds E. Sjöström & R. Alén), pp. 77–124. Springer-Verlag, Berlin, Germany. Bunzel, M., Funk, C. & Steinhart, H. (2004). Semipreparative isolation of dehydrodiferulic and dehydrotriferulic acids as standard substances from maize bran. Journal of Separation Science, 27, 1080 –1086. Bunzel, M., Ralph, J., Brüning, P. & Steinhart, H. (2006). Structural identification of dehydrotriferulic and dehydrotetraferulic acids isolated from insoluble maize fiber. Journal of Agricultural and Food Chemistry, 54, 6409 – 6418. Bunzel, M., Ralph, J., Funk, C. & Steinhart, H. (2003). Isolation and identification of a ferulic acid dehydrotrimer from saponified maize bran insoluble fiber. European Food Research and Technology, 217, 128 –133. Bunzel, M., Ralph, J., Funk, C. & Steinhart, H. (2005). Structural elucidation of new ferulic acid-containing phenolic dimers and trimers isolated from maize bran. Tetrahedron Letters, 46, 5845 –5850. Burlat, V., Kwon, M., Davin, L.B. & Lewis, N.G. (2001). Dirigent proteins and dirigent sites in lignifying tissues. Phytochemistry, 57, 883–897. Burton, R.A., Wilson, S.M., Hrmova, M. et al. (2006). Cellulose synthase-like CslF genes mediate the synthesis of cell wall (1,3;1,4)-β-d-glucans. Science, 311, 1940–1942. Chabannes, M., Ruel, K., Yoshinaga, A. et al. (2001). In situ analysis of specifically engineered tobacco lignins reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and sub-cellular levels. The Plant Journal, 28, 271–282. Chen, C.Y., Meyermans, H., Burggraeve, B. et al. (2000). Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar. Plant Physiology, 123, 853–867.

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Terashima, N., Atalla, R.H., Ralph, S.A., Landucci, L.L., Lapierre, C. & Monties, B. (1995). New preparations of lignin polymer models under conditions that approximate cell well lignification. I: Synthesis of novel lignin polymer models and their structural characterization by 13C NMR. Holzforschung, 49, 521–527. Terashima, N., Fukushima, K., He, L.-F. & Takabe, K. (1993). Comprehensive model of the lignified plant cell wall. In: Forage Cell Wall Structure and Digestibility (eds H.G. Jung, D.R. Buxton, R.D. Hatfield & J. Ralph), pp. 247–270. American Society of Agronomy (ASA)-Crop Science Society of America (CSSA)-Soil Science Society of America (SSSA), Madison, USA. Thorburn, W.M. (1918). The myth of Occam’s razor. Mind, 27, 345–353. Timell, T.E. (1986). Compression wood in gymnosperms. Springer-Verlag, Berlin, Germany. Van Doorsselaere, J., Baucher, M., Chognot, E. et al. (1995). A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity. The Plant Journal, 8, 855 – 864. Vignols, F., Rigau, J., Torres, M.A., Capellades, M. & Puigdomenech, P. (1995). The brown midrib 3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. The Plant Cell, 7, 407– 416. Woodward, J.R., Fincher, G.B. & Stone, B.A. (1983). Water soluble (1→3),(1→4)- β-d-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure. Carbohydrate Polymers, 3, 207–225. Zablackis, E., York, W.S., Pauly, M. et al. (1996). Substitution of l-fucose by l-galactose in cell walls of Arabidopsis mur1. Science, 272, 1808–1810. Zanarotti, A. (1982). Preparation and reactivity of 2,6-dimethoxy-4-allylidene-2,5-cyclohexadien1-one (vinyl quinone methide). A novel synthesis of sinapyl alcohol. Tetrahedron Letters, 23, 3815 –3818. Zanarotti, A. (1983). Synthesis and reactivity of lignin model quinone methides. Biomimetic synthesis of 8.0.4′ neolignans. Journal of Chemical Research, Synopses, 306–307. Zhang, L. & Gellerstedt, G. (2001). NMR observation of a new lignin structure, a spiro-dienone. Journal of the Chemical Society, Chemical Communications, 2744 – 2745. Zhang, L., Gellerstedt, G., Ralph, J. & Lu, F. (2006). NMR studies on the occurrence of spirodienone structures in lignins. Journal of Wood Chemistry and Technology, 26, 65–79. Zhang, L., Henriksson, G. & Gellerstedt, G. (2003). The formation of β–β structures in lignin biosynthesis – are there two different pathways? Organic and Biomolecular Chemistry, 1, 3621–3624.

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Chapter 3

Flavonoid–Protein Binding Processes and their Potential Impact on Human Health Olivier Dangles and Claire Dufour

3.1 Introduction Dietary flavonoids are currently the subject of intense research for their possible role in preventive nutrition (Hollman & Katan, 1999; Harborne & Williams, 2000; Scalbert & Williamson, 2000; Clifford & Brown, 2006). At stake is the demonstration that flavonoids and other polyphenols that are abundant in a diet rich in plant products are bioactive food components whose regular intake helps to fight the development of chronic diseases such as cardiovascular diseases, cancers and age-related disorders. Flavonoid–protein binding processes (Havsteen, 2002; Dangles & Dufour, 2006) and their possible role in the development of the potential health effects of flavonoids are the special focus of this chapter. It is important to note that most of the information available about the redox and binding properties of flavonoids deals with in-vitro studies. In particular, their affinity for specific proteins is typically assessed from purified proteins or cell extracts. The biological significance of these binding processes is highly related to bioavailability data and validation on whole cells. The main questions to address in this perspective are: l

l

l

What are the structures and concentrations of the flavonoid metabolites circulating in the plasma? Are these metabolites delivered to specific tissues and further metabolized in cells (e.g., deconjugation, oxidative metabolism)? What is the cellular distribution of flavonoids (membranes, cytosol, organites)? What are the main signaling pathways affected by flavonoids and their metabolites in cells? Are these biochemical signals affected by specific binding to protein targets (if so, which ones?) or by the redox changes involving the electron- or H-atom-donating properties of flavonoids? Can we relate in-vivo investigations in animals (using, for instance, transgenic reporter animals) or in humans (using the nutrigenomic approach of microarray analysis) to the data generated by in-vitro cell studies?

In spite of the remarkable progress achieved in the fields of flavonoid bioavailability and flavonoid-mediated cellular effects (Walle, 2004; Williams et al., 2004; Clifford & Brown, 2006), the whole task remains quite complex. Part of this complexity certainly arises Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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from the fact that dietary flavonoids are not drugs with well-defined pharmacokinetics and pharmacological targets. They are only moderately bioavailable, heavily metabolized in intestinal, hepatic and bacterial (microflora) cells, and fairly reactive since their phenolic nuclei confer on them a reducing character and an affinity for proteins that was once considered indiscriminate. In the perspective of preventive nutrition, dietary flavonoids are expected to act at low concentrations (except, possibly, in the gastrointestinal tract) following daily intake and in the long term, since the chronic pathologies they are supposed to help fight typically develop over decades. Hence, the low concentrations of flavonoid metabolites that bioavailability studies have disclosed are not expected to trigger spectacular changes in cell functioning. To circumvent this difficulty, cell studies typically make use of much higher concentrations of flavonoids, often in the biologically irrelevant form of aglycons or native glycosides, so that the relevance of the conclusions as to their nutritive effects remains speculative.

3.2 Biologically relevant chemical properties of flavonoids From a chemical viewpoint, flavonoids can be considered as fairly reactive compounds. They typically display π-electron-rich aromatic nuclei and labile phenolic -OH groups that confer on them a reducing (electron- and hydrogen-donating) character. This property largely underlies the well-known in-vitro antioxidant properties of flavonoids (van Acker et al., 1996; Pietta, 2000), i.e. their ability to rapidly reduce reactive oxygen or nitrogen species (ROS/RNS) overproduced in the inflammatory conditions that frequently accompany the development of chronic pathologies (oxidative stress), or regenerate endogenous amphiphilic antioxidants (bound to membranes or lipoproteins) such as α-tocopherol. In addition, through their catechol (1,2-dihydroxybenzene) or hydroxyketo groups, flavonoids bind iron and copper ions (El Hajji et al., 2006), whose redox cycling is typically involved in ROS production. The formation of inert metal complexes sustains part of the flavonoids’ antioxidant activity (van Acker et al., 1998). A third property of flavonoids especially critical to their biological effects from plants to humans is their affinity for a wide variety of proteins, including enzymes and receptors (Havsteen, 2002; Dangles & Dufour, 2006). Among the common chemical groups, the phenolic nucleus is probably the one that is most prone to developing molecular interactions with proteins. Indeed, the phenolic -OH group is both a hydrogen bond donor and acceptor and the aromatic ring can develop strong dispersion (van der Waals) interactions with nonpolar amino-acid residues or polarizable cofactors. Additionally, the keto group frequently found on the C-ring – as well as the glycosyl residues – can also be involved in hydrogen bonding. The fundamental chemical properties of flavonoids in relation to their biological role are schematically represented in Fig. 3.1. A recent example of the diverse molecular interactions that flavonoids can establish with a protein is provided by the ternary complex between prostaglandin F synthase, NADPH (cofactor) and rutin, a common quercetin glycoside (Komoto et al., 2004). In the complex, rutin adopts a U-shaped conformation with most of the H-bond network to the protein being established by the B-ring and the terminal sugar residue (l-rhamnose). In addition, the rutin B-ring is stacked onto the NADPH nicotinamide moiety through dispersion interactions (Fig. 3.2).

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O FeII/III R

Antioxidant activity

O •

+

X

+FeII/III/–2H

XH

O

OH

•

Flavonoid OH

R

R

Protein α-Toc-O

D1

•

O Stabilized aryloxyl H radical

α-Toc-OH

H A1 O

H D2

H R

O H

Flavonoid–protein noncovalent binding A2

Fig. 3.1 The biologically significant chemical properties of flavonoids. Notes: α-Toc, α-tocopherol.

The redox and binding properties of flavonoids are combined in the process of flavonoid– protein covalent coupling (Fig. 3.3). In a first step, flavonoids are enzymatically or chemically oxidized into highly electrophilic ortho-quinones and/or para-quinonemethides. In a second step, nucleophilic thiol or amino groups of the protein side-chains add to these electrophiles and form the covalent bonds (Kaldas et al., 2005). Currently, a lot of research about flavonoids is devoted to the in-vivo validation of their health effects and the elucidation of the corresponding mechanisms. In the debate over health effects dominated by antioxidant or cell-specific effects (Williams et al., 2004; Halliwell et al., 2005), the following considerations must be kept in mind: l

l

After ingestion, flavonoids are typically detected in the plasma as conjugates (glucuronides and/or sulfates of aglycons and their methylethers) in low concentrations (Cmax < 10 μM) in comparison with those of the endogenous antioxidants such as ascorbate, tyrosine and α-tocopherol. Conjugation in intestinal or hepatic cells generally lowers the antioxidant potential. This is especially true for methylation by catechol-O-methyltransferase (COMT), which affects

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Tyr55 His117 N

N

+ NH2

H O Rutin

O

H

H

H

B

O H

O

Lys84

H

H O

O

C2(Rha) H

H2N NADPH

N

– O

O P

O

HO

O

O HO OH B HO

O A

OH C O-β-D-Glc-1,6-α-L-Rha OH

O

Rutin

Fig. 3.2 Schematic representation of the prostaglandin F synthase–NADPH–rutin complex (adapted from Komoto et al., 2004).

l

flavonoids having a 3′,4′-dihydroxy substitution in their B-ring, a critical determinant of their reducing and metal chelating properties. More than 90% of the ingested flavonoids reach the colon, where they undergo extensive catabolism by the microflora enzymes before eventual absorption. Except for glycoside hydrolysis, these transformations (e.g., C-ring cleavage, deoxygenation of phenolic nuclei) are also expected to lower the reducing and metal chelating properties.

Hence, it currently appears more likely that flavonoids and their metabolites exert their biological activity by modulating intracellular signaling cascades via interactions with specific target proteins, rather than by their antioxidant activity. Finally, a major site for the antioxidant action of flavonoids independently of their bioavailability could be the gastrointestinal tract, which can be exposed to pro-oxidant species of dietary origin (e.g., hydrogen peroxide, nitrite, lipid oxidation products) and ROS-generating processes involving dietary iron (Kanner & Lapidot, 2001; Halliwell et al., 2000, 2005). Hence, a protective action

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71

R

OH

S

OH

Protein

Protein

O

OH –2H

R

OH

R

O –H2O

Oxidizable flavonoid

Protein

Protein

N NH2

R

O

Fig. 3.3 Oxidative flavonoid–protein covalent coupling.

of dietary antioxidants accumulating in this site under their native form after ingestion of plant products is an interesting perspective. In this respect, it is significant that quercetin and other flavonoids (but not α-tocopherol and β-carotene) quickly reduce the iron-oxo center of ferrylmyoglobin, an activated form of metmyoglobin (a common form of dietary heme iron) that is a potent initiator of lipid peroxidation (Vulcain et al., 2005). In the following sections, some important examples of binding processes involving flavonoids and human proteins will be discussed in connection to the role they play in flavonoid bioavailability and possibly in disease prevention.

3.3 Binding processes prior to absorption Upstream of gastrointestinal absorption, a variety of binding processes can take place. Some are listed below: l

l

Interactions with food proteins: some examples reported in the literature concern proteins from beverages (Siebert, 1999), plant lectins (Ohno et al., 2006), lipoxygenases (Richard-Forget et al., 1995) and milk caseins (Jobstl et al., 2006). Interactions with salivary proteins (Baxter et al., 1997; Wroblewski et al., 2001; Charlton et al., 2002): this process is especially significant with oligomeric procyanidins, whose chains can wrap around the weakly structured proline-rich salivary proteins. At large

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polyphenol:protein molar ratios, precipitation occurs. Further aggregation to form colloidal particles is then governed by surface charges. The whole process is the early event triggering the sensorial perception of astringency that is experienced upon consumption of tannin-rich foods. Interactions with human digestive enzymes (Maliar et al., 2004).

Most of these binding processes are probably too weak and superficial to significantly transform the bulk of the ingested flavonoids or mask them to the gastrointestinal barrier where absorption takes place. A possible exception could be the salivary glucosidases that have been demonstrated to rapidly hydrolyze some flavonoid glucosides in the oral cavity (Walle et al., 2005). On the other hand, investigations using the Caco-2 cell model of intestinal barrier have shown that the interaction of dietary isoflavone glucosides with soybean lectin favors their transport across the cell barrier, whereas the isoflavone aglycons are unaffected (Ohno et al., 2006). It is thus likely that soybean lectin acts by selective binding to the glucosyl moiety of the isoflavone glucosides.

3.4 Binding processes involved in flavonoid bioavailability 3.4.1 Absorption and conjugation Whereas absorption of flavonoid aglycons takes place by passive diffusion, some dietary flavonoids cross the gastrointestinal barrier via transporters. For instance, anthocyanins can be absorbed from the gastric compartment via the organic anion carrier bilitranslocase (Passamonti et al., 2002; Talavera et al., 2003). Moreover, some flavonoid glucosides can be taken up from the small intestine upon deglucosylation by the enzyme lactase phlorizin hydrolase and subsequent passive diffusion of the aglycons through the enterocyte layer. Another (minor) route consists in the active transport of the glucosides through the sodiumdependent intestinal d-glucose carrier SGLT1 and subsequent deglucosylation by cytosolic β-glucosidase (Day et al., 2003). However, the bulk of the ingested flavonoids have to reach the colon and undergo degradation by microflora enzymes (depolymerization, hydrolysis of glycosidic bonds, aglycon fragmentation) before absorption (Deprez et al., 2000; Aura et al., 2002). Flavonoid aglycons are extensively conjugated in intestinal and hepatic cells (O’Leary et al., 2003; Walle, 2004). Quercetin glucuronidation in the liver has been studied in detail using Michaelis–Menten kinetic analysis (Day et al., 2000). The UDP-glucuronyltransferase isoforms having the highest affinity for quercetin (lowest Km) perform glucuronidation on the B-ring while the isoform for glucuronidation at 7-OH shows the highest catalytic efficiency (highest Vmax). Interestingly, the quercetin 3- and 7-glucuronides formed in intestinal cells can be absorbed by hepatic cells and either methylated (COMT activity) or hydrolyzed (β-glucuronidase activity) with transient formation of the aglycon, which is finally sulfated at the 3′-OH position. On the other hand, incubation of 14C-labeled quercetin in cultures of intestinal or hepatic cells has shown that after cell internalization up to 10% of total quercetin is oxidized (probably by cell peroxidases) and become bound to specific proteins (Walle et al., 2003). Hence, flavonoid–protein covalent coupling in cells involved

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in absorption and conjugation could significantly decrease the bioavailability of the most oxidizable flavonoids.

3.4.2 Transport in plasma Human serum albumin (HSA), the most abundant plasma protein, displays three homologous domains, each divided into two sub-domains and delineated by α-helices. The different sub-domains act as binding sites for a large variety of ligands including free fatty acids (asymmetrically distributed over up to seven binding sites), vitamins and drugs. In addition, flavonol conjugates in plasma have been shown to bind HSA (Manach et al., 1998). Since then, several investigations, essentially involving flavonoid aglycons, have allowed the estimation of the corresponding binding constants and shed some light about the possible binding sites (Boulton et al., 1998; Zsila et al., 2003; Dufour & Dangles, 2005). The main conclusions from investigations by fluorescence spectroscopy are as follows: l

l

l

Flavones and flavonols bind to sub-domain IIA (strong quenching of the fluorescence of the single Trp residue, which is located in this site) with binding constants in the range 1–30 × 104 M−1 depending on the substitution pattern. Methylation or sulfation only moderately affects the binding strength, whereas glycosyl groups are typically more destabilizing (one exception being baicalein 7-O-β-glucuronide, which is as good a ligand as its aglycon). Given the high HSA concentration in the blood (ca. 0.6 mM), even the weakest flavone and flavonol ligands must be mostly bound to HSA. In competition with quercetin used as a fluorescent marker, isoflavones and flavanones having similar hydroxylation patterns appear as poorer ligands. No evidence can be obtained for the binding of flavanols. Addition of up to four equivalents of linoleic acid to a solution of quercetin and HSA in a 1:1 molar ratio only weakly affects the fluorescence of the quercetin–HSA complex (Dufour & Loonis, 2007), thereby suggesting that linoleic acid and quercetin bindings are largely independent.

3.4.3 Tissue distribution and cellular metabolism The possibility of direct uptake by cells of the highly polar flavonoid conjugates circulating in the plasma (sulfates, glucuronides) and subsequent metabolism inside tissues (e.g., deconjugation) is still poorly documented. A possible route could also involve prior deconjugation by β-glucuronidases secreted by cells under inflammatory conditions. Some flavonoid aglycons and methylethers have been incubated in cultures of skin cells, neurons and cancer cells and shown to undergo cell metabolism after absorption (Spencer et al., 2004). For instance, the citrus flavanones hesperetin and naringenin are taken up by fibroblasts, glucuronidated and excreted. By constrast, the redox-active flavonol quercetin undergoes oxidative metabolism involving formation of o-quinone/p-quinonemethide and subsequent addition of glutathione (GSH) at the 2′ position. The oxidative metabolism of flavonoids has been studied in detail in noncellular models using horseradish peroxidase (Galati et al., 2001, 2002). Whereas flavonoid–glutathione conjugation is the dominant pathway with 3′,4′dihydroxyflavonoids such as quercetin and catechin, 4′-hydroxyflavonoids have been shown

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to form aryloxyl radicals that abstract the labile H-atom from the SH group of GSH. The thiyl radical thus formed reacts with a second GSH molecule, with concomitant reduction of dioxygen and ROS formation. This process provides a possible metal-independent pro-oxidant mechanism. At low GSH concentration, the oxidative metabolism of flavonoids may lead to covalent modifications of proteins at reactive Cys residues. For instance, quercetin time-dependently inhibits human GSH S-transferase P1-1, an enzyme involved in the cellular defense against electrophiles, whereas mutants lacking the reactive Cys47 residue remain unaltered (van Zanden et al., 2003). It is thus quite likely that quercetin undergoes a slow autoxidation with concomitant o-quinone/p-quinonemethide formation and subsequent coupling to Cys47. Tyrosinase enhances the inhibition, whereas ascorbate and GSH abolish it.

3.5 Binding processes involved in the potential health effects of flavonoids Flavonoids partially owe their affinity for enzymes and receptors to their formal resemblance to the corresponding natural substrates and ligands. Here are some examples: l

l

l

Flavones and flavonols display some structural similarities with the adenine nucleus. As such, they frequently inhibit adenosine receptors (Moro et al., 1998) as well as enzymes and receptors using ATP as a cofactor by binding to the ATP site in competition with the nucleotide. An especially important case is the inhibition of protein kinases (Bridges, 2001). Isoflavones more obviously mimick estrogen hormones. For instance, 4′,5,7trihydroxyisoflavone (genistein) strongly interacts with the estrogen receptors α and β and triggers gene activation as a full agonist (Kuiper et al., 1998; Kostelac et al., 2003). Flavones and flavonols display flat unsaturated polycyclic nuclei that make them similar to the typical substrates of cytochromes P450 (phase I enzymes), such as polyaromatic hydrocarbons and dibenzo-p-dioxins (Hodek et al., 2002).

It is important to note that the flavonoid–protein binding processes reported in the literature for their possible health implications have been demonstrated in vitro using purified proteins or cell extracts and mostly with flavonoid aglycons, not the circulating conjugated forms. Hence, their in-vivo biological significance remains unclear. Furthermore, given the complexity and interdependence of the intracellular signaling cascades that appear to be modulated by flavonoids in studies on whole cells (Schroeter et al., 2002; Williams et al., 2004), it is no easy task to relate these studies to those involving purified proteins or cell extracts and disclose the true cellular protein targets that bind flavonoids. In the following sections a selection of important examples is discussed.

3.5.1 Inhibition of enzymes involved in ROS production The inhibition of ROS production – a possible antioxidant mechanism for flavonoids – can proceed by direct binding to the enzyme (formation of inhibitor–enzyme complexes)

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O

O N

HN

75

H N

HN

O N H

O

N H

O

Xanthine

MoVI-Pterin

•– 2 O2

N H

N H Uric acid

MoIV-Pterin

2 O2

Fig. 3.4 Schematic representation of the catalytic cycle of xanthine oxidase.

and/or by ROS scavenging. This dual behaviour is well-illustrated by the case of xanthine oxidase, the enzyme of purine catabolism that catalyzes the conversion of xanthine into uric acid (Fig. 3.4) (Cos et al., 1998). The enzyme displays a molybdenum (VI)-pterin cofactor whose regeneration from the Mo (IV) state by dioxygen is accompanied by superoxide production. Flavonoids having a flat C-ring (flavones, flavonols) form enzyme–inhibitor complexes in competition with the xanthine substrate. On the other hand, flavanols having a 3′,4′-dihydroxylated B-ring (the crucial structural determinant for a strong reducing activity), such as catechin, efficiently scavenge superoxide (IC50 = 0.48 μM) but do not bind to the enzyme. Finally, antioxidant flavones and flavonols such as quercetin combine the two mechanisms, i.e. they form enzyme–inhibitor complexes (IC50 = 2.38 μM) and scavenge the residual superoxide leaking from the enzyme cavity (IC50 = 0.33 μM). An investigation by molecular modeling of the apigenin (4′,5,7-trihydroxyflavone)– xanthine oxidase complex suggests a binding dominated by dispersion interactions with hydrophobic residues (e.g., the stacking of the apigenin A- and C-rings onto the phenyl group of Phe 914) (Lin et al., 2002). Interestingly, the main quercetin glucuronides formed by conjugation of quercetin in the liver have been tested for their ability to inhibit xanthine oxidase (Day et al., 2000). Glucuronidation at the B-ring essentially maintains the affinity for the enzyme. Thus, with a Kd value of 0.25 μM, quercetin 4′-O-β-D-glucuronide is as good a ligand as quercetin, although its superoxide scavenging activity (unchecked in this study) is probably much lower, since a free 4′-OH is typically critical to the antioxidant activity. On the other hand, quercetin 3- and 7-O-β-D-glucuronides, which must retain a large superoxide scavenging activity, do not bind to xanthine oxidase. Other metalloenzymes involved in oxidative stress such as lipoxygenase (Sadik et al., 2003) are also inhibited by flavonoids.

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3.5.2 Modulation of the redox properties of flavonoids by binding to proteins It sounds common sense that flavonoid–protein binding builds a steric barrier around the flavonoid that must lead to its being masked to ROS/RNS delivered outside the complex (Arts et al., 2002). However, much to our surprise, we have repeatedly observed that serum albumin-bound quercetin is as prone as, if not more prone than, free quercetin to react with one- or two-electron oxidants such as nitrosodisulfonate (C. Dufour et al., unpublished observations) and periodate (Dangles et al., 1999). From Fig. 3.5, for instance, it is quite clear that oxidation of quercetin by potassium nitrosodisulfonate is actually much faster in the presence of HSA, which in addition allows the detection of a transient oxidation product of quercetin displaying a remarkable low-energy absorption band (around 700 nm).

0.5 0.4 0.3

0.3

Absorbance

Absorbance

0.4

0.2

0.1

0.1 0.0 300 (a)

0.0 350

400

450

500

Wavelength (nm)

0 (b)

Absorbance

Absorbance

200

300

Time (s)

0.4

0.4 0.3 0.2

0.3 0.2 0.1

0.1

(c)

100

0.5

0.5

0.0 300

0.2

0.0 500

700

Wavelength (nm)

900

0 (d)

20

40

Time (s)

Fig. 3.5 UV-vis monitoring of the oxidation of quercetin (25.6 μM) by potassium nitrosodisulfonate (256.7 μM) in 50 mM phosphate–100 mM NaCl buffer at pH 7.4 and 25°C without and with human serum albumin (HSA) (37.3 μM). (a) UV-vis spectra, time interval between two consecutive spectra = 70 s, without HSA. (b) Absorbance vs. time curves at 330 nm (䊏), 400 nm (䉱) and 460 nm (䊉), without HSA. (c) UV-vis spectra, time interval between two consecutive spectra = 10 s, in the presence of HSA. (d) Absorbance vs. time curves at 330 nm (䊏), 400 nm (䉱) and 690 nm (䊉) in the presence of HSA.

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This transient intermediate could be the p-quinonemethide derived from quercetin upon H-atom abstraction at O3-H and O4′-H (possibly stabilized by charge transfer interactions with an HSA residue acting as a donor) or a quercetin radical. These highly reactive species are expected to rapidly evolve by addition of water and disproportionation, respectively, and both pathways could actually be strongly impeded by the protein matrix. The oxidizability of HSA-bound quercetin was confirmed in the investigation of the inhibition by quercetin of the peroxidation of HSA-bound linoleic acid (Dufour & Loonis, 2007 and Dufour et al., 2007). The main results of this study are: l

l

l

l

Under conditions where both linoleic acid (LH) and quercetin (AH) are tightly bound to HSA, quercetin retains a high ability to inhibit the accumulation of lipid hydroperoxides, whereas other common antioxidants are either inactive (ascorbate) or poorly active (α-tocopherol). The antioxidant activity is prolonged much beyond quercetin consumption. This observation suggests that some of the quercetin oxidation products are also potent inhibitors, which was independently confirmed with two of them (3,4-dihydroxybenzoic acid and the benzofuranone derivative formed upon water addition on the p-quinonemethide derived from quercetin). The rate of quercetin consumption under the flow of initiating peroxyl radicals (produced by the thermal decomposition of diazo compound AAPH) is as high as in the absence of HSA. In this model, the kinetics of LH consumption and LOOH formation show no evidence for a propagation step (LOO• + LH → LOOH + L•) taking place and quercetin is expected to basically react by inhibiting initiation (reduction of the AAPH-derived peroxyl radicals: ROO• + AH → ROOH + A•) rather than reducing the lipid-derived peroxyl radicals LOO•.

However, it remains surprising that inhibition of initiation proceeds as effectively in the absence or presence of HSA. It can thus be concluded that quercetin in its binding site (sub-domain IIA) remains fully available to the hydrophilic AAPH-derived peroxyl radicals produced in the aqueous phase. Two interpretations can be proposed: 1) The AAPH-derived peroxyl radicals have full access to the quercetin binding site. 2) The AAPH-derived peroxyl radicals react at the periphery of HSA and oxidation efficiently propagates along a set of hydrogen-bonded residues to reach quercetin. Since HSA can even accelerate quercetin oxidation (as shown in Fig. 3.5 with nitrosodisulfonate as the oxidant), we favor the second hypothesis. In summary, the binding of flavonoids to redox-inactive enzymes such as HSA does not necessarily depress their antioxidant activity. In fact, the influence of proteins on the redox properties of flavonoids could well be specific to each flavonoid–protein couple under consideration. Ultimately, the fate of quercetin undergoing oxidation in the presence of HSA could be covalent coupling to HSA as evidenced in the case of oxidation by H2O2 catalyzed by horseradish peroxidase (Kaldas et al., 2005).

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3.5.3 Inhibition of protein kinases Flavones and flavonols display a general affinity to ATP-binding proteins as a consequence of their formal structural analogy with ATP. Within this protein class, protein kinases are especially important targets. Protein kinases catalyze the transfer of a phosphate group from the ATP co-factor to a protein or peptide substrate. Serine/threonine kinases and tyrosine kinases are distinguished according to the ligand residue accepting the phosphate group. Kinase-catalyzed phosphorylation is a very important cell signaling mechanism, especially involved in cell growth, proliferation, survival and apoptosis. In-vitro studies on purified enzymes or cell extracts have investigated direct flavonoid–protein kinase binding. For example, 3′,4′-dihydroxylated flavones and flavonols have been reported to efficiently inhibit protein kinase C (PKC), a serine/threonine kinase, by binding to the ATP site in competition with the cofactor (EC50 values in the range 1–10 μM) (Gamet-Payrastre et al., 1999). By contrast, 3′,4′,5,7-tetrahydroxychalcone has been shown to act as a selective tyrosine kinase inhibitor (Yang et al., 1998). However, these data are often difficult to correlate with investigations on whole cells showing PKC inhibition upon treatment by flavonoids. In addition, there is increasing evidence that flavonoids selectively modulate intracellular signaling pathways involving mitogen-activated protein kinases (MAPK) in a variety of cells (Schroeter et al., 2002; Yoshizumi et al., 2002; Ahn et al., 2004; Williams et al., 2004). MAPKs include extracellular signal-related kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 kinase, which display several isoforms and distinct regulation pathways. In response to extracellular or intracellular stimuli, MAPKs catalyze the phosphorylation of cytosolic or nuclear proteins with subsequent activation of transcription factors (TFs) or downstream kinases (Schroeter et al., 2002). For example, the transcription factor AP-1 is activated by JNK-catalyzed phosphorylation of its c-Jun component. Each MAPK requires activation by upstream kinases that catalyze the double phosphorylation of a MAPK Thr-X-Tyr motif. On the other hand, MAPKs are inactivated by specific phosphatases. It is also noteworthy that MAPKs are especially sensitive to oxidative stress stimuli, i.e. the increase in intracellular concentrations of oxidants (e.g., H2O2, lipid oxidation products, NO, peroxynitrite), or the decrease in intracellular concentrations of reductants (e.g., glutathione). For instance, JNK displays a redox-sensitive Cys residue that can be nitrosated by NO. Among this formidable complexity, it is not clear whether flavonoid-induced MAPK regulation operates via direct kinase–flavonoid binding, by interfering with cytosolic MAPK-regulated TF components, by inhibition of oxidative stress (intracellular antioxidant action), or by promoting any biochemical process liable to affect MAPK activity, such as changes in intracellular Ca2+ concentrations. As an example, neuronal cell death induced by oxidized LDL was inhibited as efficiently by epicatechin and its metabolite 3′-O-methylether, which, however, is a much less potent antioxidant (Schroeter et al., 2001). Consistently, the protective mechanism does not involve inhibition of the intracellular oxidative stress but is selectively correlated to JNK inactivation. As a possible mechanism for the prevention of cardiovascular diseases, quercetin-3O-glucuronide inhibits the growth of rat vascular smooth muscle cells by preventing JNK activation by angiotensin II (Yoshizumi et al., 2002). This is one of the rare examples

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demonstrating that an important circulating flavonoid metabolite (although quite polar) is incorporated in whole cells and triggers a specific response. However, since JNK can be activated by superoxide released by the NAD(P)H oxidase enzyme, the protective mechanism of quercetin-3-O-glucuronide could involve binding to specific proteins (JNK and other upstream or downstream kinases, NAD(P)H oxidase) as well as an antioxidant action (superoxide scavenging).

3.5.4 Inhibition of cytochrome P450 enzymes Cytochrome P450 enzymes (CYPs) are membrane-bound heme monoxygenases of liver microsomes that catalyze oxygen transfer reactions from dioxygen to a variety of substrates (e.g., hydrocarbons, alkenes, aromatic compounds, sulfur- or nitrogen-containing compounds) (Fig. 3.6). CYPs exist as several isoforms (1A1, 1A2, 1B1, 3A4 . . .) that play an important role in detoxification by converting lipophilic xenobiotics into more hydrophilic products (phase I conjugation) for further conjugation by phase II enzymes (e.g., glucuronyl- and sulfotransferases, glutathione S-transferase) and final excretion in urine. However, a typical CYP-mediated adverse effect consists in their activation of procarcinogens such as polyaromatic hydrocarbons (PAHs) into potent electrophiles. Flavonoids can regulate CYP activities by two distinct mechanisms (Hodek et al., 2002): 1) The up- or down-regulation of CYP 1A1 expression depending on the flavonoid and cell type considered: this mechanism occurs via the agonist or antagonist binding of

O2 source of electrons

H2O

•+ P-FeIV=O

P-FeIII P = porphyrin nucleus

C

H

H

S C

OH

O

OH

O S

Fig. 3.6 Schematic representation of the catalytic cycle of cytochromes P450.

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flavonoids to the arylhydrocarbon receptor (AhR) in the cytosol. Upon binding to an agonist ligand, AhR translocates to the nucleus and associates to a protein partner (the AhR nuclear translocator), thus forming a transcription factor that binds to the xenobiotic-responsive elements of CYP 1A1 and triggers transcription. Among flavonoids, those having a planar C-ring (flavones, flavonols) mimicking PAHs are the best AhR ligands. The balance between agonist and antagonist behaviors may be fairly sensitive to flavonoid substitution. For instance, quercetin acts as an agonist for CYP 1A1 transcription in MCF-7 human breast cancer cells whereas its 3′-deoxy analog kaempferol acts as an antagonist (Ciolino et al., 1999). Both bind to AhR in competition with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an environmental contaminant and typical CYP 1A1 gene inducer. On the other hand, in human hepatic G2 cells, quercetin binds to AhR as an antagonist and inhibits benzo[a]pyrene-induced expression of CYP 1A1 (Kang et al., 1999). 2) Direct flavonoid-CYP binding: the binding of flavones and flavonols is strong (typical Ki < 100 nM). Depending on the flavonoid, different isoform specificities are observed (Silva et al., 1997; Zhai et al., 1998; Doostdar et al., 2000). During the inhibition, the flavonoids can be hydroxylated or demethylated. For instance, the flavonol kaempferol is hydroxylated to quercetin by rat CYP 1A1 (Silva et al., 1997) whereas the flavanone hesperetin is demethylated to eriodictyol by human CYPs 1A1 and 1B1 (Doostdar et al., 2000). Hence, CYPs could cause partial flavonoid deconjugation in cells with subsequent increase in the redox activity.

3.5.5 Regulation of gene expression The case of CYP enzymes clearly shows that flavonoids can regulate protein functions via direct binding to proteins and/or at the gene level. The development of the new tools of nutrigenomics (microarray analysis, transgenic reporter animal models; Afman & Muller, 2006) has allowed researchers to gain evidence that dietary flavonoids can actually up- or down-regulate many genes in vivo (Moskaug et al., 2004). Besides the genes displaying xenobiotic responsive elements involved in detoxification (e.g., CYP genes, see section 3.5.4), the genes typically responsive to flavonoids are: l

l

Genes regulated by transcription factors NF-κB (immune and inflammatory responses, protection against apoptosis), AP-1 (cell growth, cellular stress) and PPARγ (antiinflammatory responses) (Liang et al., 2001; Yoshizumi et al., 2002; Kim et al., 2003; Ahn et al., 2004; Chen et al., 2005). Genes with antioxidant/electrophile responsive elements, e.g. genes regulated by transcription factors Nrf1/2 and involved in detoxification and antioxidant defense (e.g., enzymes for glutathione synthesis and conjugation, NAD(P)H-quinone reductase) (Myhrstad et al., 2002; Talalay et al., 2003).

The regulation of these genes provides a strong basis for interpreting some health effects attributed to flavonoids such as their anti-inflammatory and anti-atherosclerotic properties. The question of the biochemical mechanisms underlying flavonoid-mediated gene regulation is still open but could involve a combination of redox and specific binding processes.

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Repressor TFC

TFC Activator

Kinase

Kinase

TFC

Phosphate group

TFC

Phosphate group

Transcription factor assembly and translocation into the nucleus

Fig. 3.7 Flavonoid–protein interactions and the regulation of gene expression by flavonoids’ inhibition of kinases, direct binding to transcription factor components (TFC) or to repressor/activator proteins.

Binding processes could take place via direct association in the cytosol between the flavonoid and a transcription factor component or activator/repressor protein. In addition to flavonoidAhR binding (see section 3.5.4), a weak association to peroxisome proliferator-activated receptor-γ (PPAR-γ) has also been reported in vitro (Liang et al., 2001). Transcription factor assembly in the cytosol can involve phosphorylation steps catalyzed by protein kinases, which could themselves be the targets of flavonoids (Fig. 3.7). For example, NF-κB, AP-1 and STAT1 are all important TFs for the expression of NO synthase (iNOS), an enzyme involved in the inflammatory response. Activation of NF-κB requires the phosphorylation of the repressor protein IκBα, to which it is bound in the cytosol. It has been shown that quercetin consistently inhibits the activity of the corresponding kinase (IKK) in mouse neurons (Chen et al., 2005). On the other hand, quercetin induces heme oxygenase-1 expression, possibly via MAPK inhibition. Hence, flavonoids could modulate the expression of pro-inflammatory enzymes (down-regulation of iNOS) or anti-inflammatory enzymes (up-regulation of heme oxygenase-1) via inhibition of specific kinases. In another work, quercetin has been shown to increase the intracellular glutathione level by activating the promoter of the catalytic subunit of γ-glutamylcysteine synthetase (Myhrstad et al., 2002). This effect is fairly specific since myricetin (5′-hydroxyquercetin), quercetin 3-glucoside and rutin are inactive. As a possible molecular mechanism, quercetin could help release specific nuclear factors (from inert cytosolic complexes), thereby facilitating their translocation into the nucleus. However, some flavones, isoflavones and flavonols are also known to activate PPAR-γ, thus leading to suppression of inducible cycloxygenase-2 and NO synthase in mouse macrophages. A likely mechanism involves allosteric binding of the flavonoids to PPAR-γ and subsequent modification of the receptor conformation (Liang et al., 2001). However, it must be pointed out that some TFs involved (especially NF-κB) are also redox-sensitive, so their activation or inactivation could also involve the flavonoids’

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GS

OH

R

OH

Flavonoid–glutathione conjugate

GSH

O

OH Peroxidase, H2O2 R

R

OH Flavonoid

S

O o-Quinone

S

SH

SH

Repressor protein and its bound transcription factor Free transcription factor

Translocation to nucleus, gene expression Fig. 3.8 Possible redox regulation of gene expression by flavonoids.

reducing properties. In particular, genes with antioxidant/electrophile-responsive elements (A-EpRE) are known to be regulated by a large variety of structurally unrelated redox-active compounds susceptible to alter di-Cys/cystine ratios in transcription factor components or possibly involved in covalent binding to these proteins (Talalay et al., 2003). Hence, flavonoids could induce A-EpRE genes through their ability to form electrophilic o-quinones and/or p-quinonemethides (Fig. 3.8). Recently, quercetin was shown to be more prone than dihydroquercetin (taxifolin) at inducing A-EpRE genes in reporter cells (Boerboom et al., 2006). In addition, although the selected flavonols were potentially all able to undergo two-electron oxidation to o-quinones and/or p-quinonemethides, the most potent inducers were not necessarily the most redox active. Hence, flavonol– protein interactions could also play a role in A-EpRE gene expression. A possible target

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for the flavonols could be the repressor protein Keap1, which binds the main TF involved in A-EpRE gene expression (Nrf2) as an inert complex. Flavonol–Keap 1 binding could help release Nrf2 in the cytosol, thereby triggering its translocation to the nucleus and subsequent binding to DNA.

3.6 Conclusion Over the last two decades, spectacular advances have been made in the understanding of the biochemical mechanisms underlying flavonoid bioavailability and flavonoid-mediated cellular effects. However, some links are still missing. In particular, the way the circulating flavonoid metabolites are delivered to tissues and eventually further processed to form the cellular active metabolites is still poorly understood. Anyway, it is now clear that the antioxidant activity by ROS scavenging (itself modulated by flavonoid–protein interactions) is only one of the possible mechanisms involved in the flavonoid-mediated health effects, its contribution being possibly most important in the gastrointestinal tract. However, some intracellular redox processes of flavonoids (pro-oxidant activity involved in cell death, redox regulation of cysteine-rich transcription factors) may be quite important. Besides their redox activity, the interactions of flavonoids with specific protein kinases and transcription factors (or proteins involved in their activity) emerge as their main mode of action. It is, however, clear that, depending on the flavonoid structure, a combination of intracellular binding and antioxidant processes can operate (Ishige et al., 2001; Chow et al., 2005). The most difficult task ahead is now to understand how this complex in-vitro cell biochemistry correlates with the even more complex in-vivo data that suggest that flavonoids can regulate specific metabolic pathways, such as lipid metabolism (Jung et al., 2006).

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van Zanden, J.J., Ben Amman, O., van Iersel, M.L.P.S. et al. (2003). Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin. Chemico-Biological Interactions, 145, 139–148. Vulcain, E., Goupy, P., Caris-Veyrat, C. & Dangles, O. (2005). Inhibition of the metmyoglobininduced peroxidation of linoleic acid by dietary antioxydants: action in the aqueous phase vs. lipid phase. Free Radical Research, 35, 547–563. Walle, T. (2004). Absorption and metabolism of flavonoids. Free Radical Biology & Medicine, 36, 829 – 837. Walle, T., Browning, A.M., Steed, L.L., Reed, S.G. & Walle, U.K. (2005). Flavonoid glucosides are hydrolyzed and thus activated in the oral cavity in humans. Journal of Nutrition, 135, 48–52. Walle, T., Vincent, T.S. & Walle, U.K. (2003). Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells. Biochemical Pharmacology, 65, 1603 –1610. Williams, R.J., Spencer, J.P.E. & Rice-Evans, C. (2004). Flavonoids: antioxidants or signalling molecules. Free Radical Biology & Medicine, 36, 838–849. Wroblewski, K., Muhandiram, R., Chakrabartty, A. & Bennick, A. (2001). The molecular interactions of human salivary histatins with polyphenolic compounds. European Journal of Biochemistry, 268, 4384 – 4397. Yang, E.B., Zhang, K., Cheng, L.Y. & Mack, P. (1998). Butein, a specific protein tyrosine kinase inhibitor. Biochemical & Biophysical Research Communications, 245, 435–438. Yoshizumi, M., Tsuchiya, K., Suzaki, Y. et al. (2002). Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the inhibition of JNK and AP-1 signaling pathway. Biochemical & Biophysical Research Communications, 293, 1458–1465. Zhai, S., Dai, R., Friedman, F.K. & Vestal, R.E. (1998). Comparative inhibition of human cytochromes P450 1A1 and 1A2 by flavonoids. Drug Metabolism & Disposition, 26, 989–992. Zsila, F., Bikádi, Z. & Simonyi, M. (2003). Probing the binding of the flavonoid, quercetin to human serum albumin by circular dichroism, electronic absorption spectroscopy and molecular modelling methods. Biochemical Pharmacology, 65, 447–456.

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Methods for Synthesizing the Cocoa-Derived Oligomeric Epi-Catechins – Observations on the Anticancer Activity of the Cocoa Polyphenols Alan P. Kozikowski and Werner Tückmantel

4.1 Introduction Our research into the chemistry and biology of the cocoa-derived polyphenols got started in a rather serendipitous way, after we relocated to the Georgetown University Medical Center. There the senior author met with the director of the Lombardi Cancer Center, Dr Marc Lippman, who told him about the interesting results they were seeing in some breast tumor xenograft studies using agents derived from cocoa that had possible anticancer activity. In particular, he related the fact that the Mars candy company was supporting this work, and that they would like to have someone carry out the total synthesis of these materials in order to authenticate the structure of the natural cocoa-derived materials, as well as to provide pure material for the biology studies. Of course, today much ado is made about the virtues of chocolate, and in particular dark chocolate. It is no longer simply a good tasting treat, but it is also supposed to be good for you. Chocolate is rich in antioxidant flavonoids, which include the procyanidins and their monomeric building blocks, epicatechin and catechin. If one takes a moment to look into the history of chocolate, and, in particular, its use in medicine, it is interesting to find that it was used for therapeutic purposes as long ago as the 4th century. Not unexpectedly, warriors consumed cacao beverages to boost their energy and used cacao butter to dress their wounds. The Aztecs mixed cacao with the bones of their ancestors in order to create a cure for diarrhea, while the Spanish conquistadors use it to make a paste to calm their stomach (http://groups.msn.com/FoodiesCorner/ chocolateandmedicine.msnw). Reports dating from the 1600s in Europe exist referring to cocoa’s good taste as well as its medicinal values for angina and heart pain. Healers were also reported to grind cocoa beans into a paste and to use the powder to make a drink that they gave to individuals suffering from fevers, liver disease, and kidney disorders. A cocoa drink was reputed to foster needed weight gain – especially if augmented with ground maize. Hot chocolate was even prescribed as a laxative and an aid to digestion. Of course it has long been known Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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to serve as a stimulant for lovemaking and even to help women to conceive and to boost milk production (http://scicom.ucsc.edu/scinotes/0001/chocolate.htm). In the 1800s cocoa was mixed with amber dust to treat the effects of drinking too much alcohol. In more recent times, because of the belief that chocolate is just another way of taking something sweet and caloric into the body, it has been viewed as something that is likely to be unhealthy, and certainly to lead to obesity if consumed in large quantities. However, after determining that cocoa contains substantial concentrations of certain polyphenols (Osakabe et al., 2002), like those found in wines, fruits and vegetables, it was considered possible that chocolate could, in fact, have a positive effect on the heart by reducing cardiovascular disease. In recent studies, the consumption of cocoa has been shown to reduce platelet reactivity, as well as to increase prostacyclin levels and to reduce level of leukotrienes (Keen, 2001). Such changes in the prostacyclin and leukotriene levels will in turn reduce platelet aggregation and reduce the risk of thrombosis as well as the entry of low-density lipoproteins into the arterial walls. Another provocative finding relates to a study showing that the pentameric procyanidins isolated from chocolate are able to inhibit expression of the tyrosine kinase Erb2 gene. In turn, this leads to a reduction in the growth of cultured human aortic endothelial cells. Using human dermal microvascular endothelial cells, investigators have also shown that this pentamer is able to modulate the activity of several crucial proteins involved in angiogenic signaling through alteration of tyrosine phosphorylation; down-regulation of Erb2 tyrosine kinase is thus observed in these endothelial cells (Kenny et al., 2004). It would thus appear that these polyphenols are able to effect endothelial growth signaling, and thereby exert an effect in vitro that may be useful in the treatment of certain neoplasias. Below, we shall first hightlight the efforts of our synthetic undertakings, which now serve to provide any of the oligomeric epicatechin-derived procyanidines in pure form. These efforts required that we discover an efficient means for generating a carbo-cation at the 4-position of the epicatechin ring system. After providing the chemistry details, we then summarize the cancer biology studies that have been carried out using these compounds by us and our colleagues at the Lombardi Cancer Center of the Georgetown University Medical Center.

4.2 Synthesis of procyanidins 4.2.1 General chemical properties of cocoa procyanidins Cocoa procyanidins are largely based on (−)-epicatechin building blocks, which form predominantly linear oligomers with inter-flavan bonds extending from the 4-position of one building block to the 8-position of another (Porter et al., 1991) (Fig. 4.1). As the separation of the oligomers from each other and from minor constituents becomes progressively more difficult with increasing molecular size, only the dimer 3 through hexamer 7 had been characterized in the literature at the onset of this work (Morimoto et al., 1986; Foo & Karchesy, 1989; Foo & Karchesy, 1991; Porter et al., 1991). We depict the inter-flavan bond with β stereochemistry, as had been widely assumed earlier and was confirmed by us employing a degradative method (see below).

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Fig. 4.1 Structures of epicatechin, catechin, and epicatechin oligomers.

The most important aspect in the chemistry of polyphenols is their sensitivity to common chemical agents. Firstly, like other phenols, they are susceptible to complex freeradical oxidation reactions by air and reactive oxygen species, especially in the presence of bases. This is exactly the reason why this class of compounds has been so thoroughly investigated with regard to their antioxidant capabilities in biological systems. Polyphenol isolates from cocoa provided to us by Mars, which had gone through chromatographic separation processes entailing lengthy exposure to adsorbents and air, had a dark brown to reddish-brown color, indicating partial oxidation. Secondly, they are, at elevated temperatures or in the presence of bases, subject to a reversible opening of their C ring by way of a transient quinone methide, which leads to stereochemical scrambling at C-2 (Foo & Porter, 1983) (Fig. 4.2) or, after ring closure involving different hydroxyl groups or aromatic carbon atoms, to rearrangement products (Laks et al., 1987; Burger et al., 1990;

Fig. 4.2 Epimerization of epicatechin and catechin in acidic and basic reaction media.

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Steynberg et al., 1990). Thirdly, the Friedel–Crafts alkylation reaction through which interflavan bonds are formed naturally and synthetically is reversible, and both degradation and re-condensation reactions occur upon acid (Hemingway et al., 1982; Foo & Porter, 1983) as well as base (Burger et al., 1990) treatment of oligomeric procyanidins. In our hands, epicatechin oligomers were stable to the brief exposure to very dilute acid that occurs during analytical reverse-phase HPLC (typically performed with addition of 0.1% of trifluoroacetic acid to the eluent to suppress peak broadening), but the evaporation of eluates after preparative HPLC resulted in partial decomposition, as evidenced by the appearance of additional peaks. With this information in mind, most chemists would probably agree that it is advisable to perform all requisite synthetic steps and separations on protected precursors and to de-protect pure precursors at the very end of the synthesis under the mildest possible conditions.

4.2.2 Earlier synthetic work The original biomimetic protocol for procyanidin formation involved the acid-catalyzed condensation of unprotected monomers with building blocks that bear a leaving group in the 4-position, such as 4-hydroxylated flavanols like dihydroquercetin – obtained by reduction of the expensive natural product taxifolin (Kolodziej, 1986) – or oligomer fractions that had first to be isolated from plant materials (Hemingway et al., 1982; Foo & Porter, 1983). In the latter case, use was made of the reversibility of the inter-flavan bond-forming reaction, which is basically a Friedel–Crafts alkylation of the A-ring of the nucleophilic component (the ‘flavanyl acceptor,’ by analogy with the terminology of oligosaccharide synthesis) with a carbenium ion generated from the 4-substituted building block (the ‘flavanyl donor’). Inherent characteristics of this synthetic approach are the formation of regioisomers in which inter-flavan bonds issue from either the 6- or the 8-position of the flavanyl acceptor, as well as the re-entry of already formed oligomers into the process to generate higher oligomers, even if an excess of the flavanyl acceptor is employed. The difficult separation of the resulting mixtures of sensitive, unprotected procyanidins was as much a deterrent for us to follow this route as was the absence of a commercially available, reasonably priced, flavanyl donor. Methylation of the phenolic hydroxyls has commonly been employed for characterization purposes, and in a few cases methylated building blocks have been entered into oligomer-forming condensation reactions. The combination of difficult cleavage of methyl ethers with the high sensitivity of the target compounds, however, renders methyl unsuitable as a protecting group for procyanidins. Benzyl appeared more promising, and some fundamental results in this area had already been published at the onset of our own work. Thus, (+)-catechin had been benzylated at all four of its phenolic hydroxyls, leaving the 3-OH group unreacted, with benzyl bromide and sodium hydride in DMF. The alcohol function had been oxidized with DMSO/acetic anhydride to the ketone, and a mixture of benzylated catechin and epicatechin obtained by reduction of this mixture with NaBH4. Furthermore, the protecting groups had been removed by hydrogenolysis over Pd/C without affecting the O-C2 bond, which is also benzylic but sterically hindered (Miura et al., 1983). And finally, a few examples of inter-flavan bond formation or related reactions had already been reported using benzylated flavanyl donors with catechin (2,3-trans)

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stereochemistry obtained by total synthesis (Kawamoto et al., 1989, 1991a, 1991b, 1993). Encouraged by this information, we decided to pursue the partial synthesis of cocoa procyanidins from natural monomers by way of benzylated intermediates.

4.2.3 The synthesis of benzyl-protected building blocks (Tückmantel et al., 1999) Since (+)-catechin is several times less expensive than (−)-epicatechin, we decided to start from this natural product and invert its C-3 stereocenter (Fig. 4.3). The O-benzylation of catechin has been reported to proceed in 45% yield (Miura et al., 1983); we typically obtained yields of the tetra-O-benzyl derivative 10 in the low 20% to low 30% range. C-Benzylated and incompletely benzylated by-products account for the balance. CBenzylation has been reported for other polyhydroxylated aromatics as well (Anthoni et al. 2001). After column chromatography and crystallization, our compound 10 was at least 97% pure by HPLC. A typical batch contained 1.2% of the epi-isomer. This material could have originated from a small amount of epicatechin in the starting material or by base-induced epimerization, in which case it would be of the unnatural absolute configuration. When benzyl chloride was substituted for benzyl bromide in the hope that this harder electrophile would reduce the extent of C-benzylation, an improved product yield was indeed realized. Unfortunately, increased formation of tetra-O-benzylepicatechin (12) was also observed. As the same batch of catechin was used throughout these studies, we concluded that substantial epimerization had occurred, presumably owing to the slower reaction involving the chloride as compared to the bromide, and discontinued this direction of work (unpublished results). A near-quantitative yield has, however, recently been obtained by others under optimized conditions (Mustafa et al., 2004). A Mitsunobu reaction on the alcohol 10 with p-nitrobenzoic acid resulted in the elimination of water to form the 3,4-olefin rather than the inverted ester. The reported oxidation (Miura et al., 1983) of intermediate 10 to the ketone 11 with activated DMSO failed in our hands. However, a clean and high-yielding oxidation was observed with the Dess–Martin periodinane in moist CH2Cl2 (Meyer & Schreiber, 1994). It was deemed essential to obtain a high degree of stereoselectivity in the subsequent reduction step to avoid a potentially difficult separation of large amounts of material. Lithium tri-sec-butylborohydride (LSelectride®) fulfilled this requirement. A stereoselectivity of at least 200:1 was obtained,

Fig. 4.3 Synthesis of the flavanyl donor 13 from (+)-catechin.

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but enolization occurred to some extent as indicated by the presence of unreacted starting ketone, despite the use of an excess of the reducing agent. The enolate did not survive the alkaline hydrogen peroxide work-up, and the yield of inverted alcohol was considered inadequate. In addition reactions of Grignard or organolithium reagents to enolizable ketones, recourse is often made to transmetalation to a less basic organolanthanoid reagent by adding anhydrous CeCl3. This measure was found effective only if a large excess of the expensive hydride reagent was employed. From visual inspection, it appeared that little or none of the additive had gone into solution. Since some transition metal salts that are insoluble in THF alone can be solubilized in the form of -ate complexes by addition of lithium halides, we made another attempt using a 1:4 mixture of CeCl3 and LiBr as the additive. This time, enolization was almost completely suppressed; however, it still appeared that the CeCl3 had not dissolved. Logically, we continued by testing LiBr alone as the additive, and obtained an almost equally good result. On a 77-mmolar scale, reduction with 1.3 equivalents of L-Selectride® and 5.2 equivalents of LiBr finally furnished an 81% yield of recrystallized, inverted alcohol 12. A similar yield was recently obtained by others without addition of LiBr in the analogous reduction of a gallocatechin-type ketone (Li & Chan, 2001; Zaveri, 2001). The origin of the discrepancy between these and our findings is not clear at present. The oxidation of C-3 to a ketone carbonyl offers in principle another opportunity for racemization through enolization towards C-2, in addition to the potential formation of the ent-epicatechin derivative in the initial O-benzylation step, which would be expected to pass unchanged through the above sequence. There is a good reason to be concerned about the presence of even small amounts of the incorrect enantiomer in the intermediate 12, and that is the oligomeric nature of our target compounds. For the pentamer, even 2% of ent-12 translates to only 0.985 = 90% purity, and this deficiency is in addition to other impurities that arise from side-reactions. It was therefore mandatory to ascertain a reasonable enantiomeric purity of the intermediate, and this was achieved by preparation of its diastereoisomeric Mosher esters with both enantiomers of the Mosher acid chloride. Both esters exhibited sharp and distinct signals in their 19F and 1H NMR spectra, thereby allowing their diastereoisomeric purity to be estimated to at minimum 98% (there is a 99% limit because of the imperfect enantiomeric purity of the agents used). A good precedent existed for the functionalization of the 4-position of compound 12. Thus, the reported oxidation of both 5,7,3′,4′-tetra-O-methylcatechin and –epicatechin with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in chloroform/methanol gave moderate yields of the respective 4-methoxy derivatives (Steenkamp et al., 1991). In our experience, the alcohol 12 on DDQ oxidation with either methanol or isopropanol as the nucleophile gave mixtures of 4-alkoxy and dialkoxy derivatives. In the case of methanol, these were readily separable; the isopropoxy derivatives, however, eluted closely together. Hypothesizing that the mono- and dialkoxy derivatives might be better separable if a polar alkoxy group was introduced, we oxidized 12 with DDQ in CH2Cl2 in the presence of ethylene glycol and found that the resulting 4-alkoxy derivative 13 was indeed easily purified by column chromatography. The pre-purified material crystallized and was in this form storable at −20°C; the yield was around 50%. It should be noted that others have subsequently improved the yield of an analogous reaction in the catechin series to above 70% by extending the reaction period, a somewhat surprising result since dialkoxylated byproducts are already formed under our conditions, and obtained furthermore a near-quantitative yield

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of the corresponding 4-(2-ethoxyethoxy) derivative when employing ethyl cellosolve as the alcohol reactant (Saito et al., 2002, 2003). Again counterintuitively, the same derivative was formed in only 64% yield from benzylated epicatechin (Saito et al., 2003).

4.2.4 Inter-flavan bond formation using benzyl-protected building blocks (Tückmantel et al., 1999; Kozikowski et al., 2003) With both the protected inter-flavan donor and acceptor readily accessible, their TiCl4mediated union as described by Kawamoto et al. (Kawamoto et al., 1989, 1991a, 1991b, 1993) was investigated next (Fig. 4.4). It was found necessary to moderate the Lewis acidity of the condensing agent by addition of a Lewis-basic co-solvent (THF) to achieve reasonable yields. Employing a four-fold excess of flavanyl acceptor, the 4,8-linked oligomers 14, 15, and 16 as well as an impure 4,6:4,8-linked trimer – 17 – were isolated by preparative HPLC in 53, 22, 1.5, and 4.5% yields, respectively. Product characterization proved rather difficult. The 1H and 13C NMR spectra of all benzylated oligomers, complex enough to begin with because of the great number of similar protons, were further complicated by the occurrence of hindered rotation around the inter-flavan bonds, which – as a consequence of the steric influence of the multiple benzyl groups – is frozen on the NMR timescale at room temperature, thus giving rise to at minimum two sets of sharp signals. It was not a priori certain that minor sets of signals did not stem from impurities rather than additional rotamers; indeed, this scenario proved to be the case for compound 17. In this early work, separations were achieved by normal-phase HPLC on silica gel with an isocratic ethyl acetate/hexane mixture. This protocol was adequate for the isolation of the lower oligomers, but the product peaks were broad. For larger oligomers, 4,6-linked or branched by-products would almost certainly not have been separable from the all-4,8 major isomers. A better separation was achieved with an ethyl acetate/isooctane gradient on a β-cyclodextrin column (Fig. 4.5), and sharp, narrow peaks were finally obtained by reverse-phase HPLC on C8 or C18 silica gel with an acetonitrile–water gradient. It is owing to this last analytical method, in conjunction with the obtention of materials by different condensation techniques that exhibited the same ratio of major and minor peaks in their 1H NMR spectra, that we are convinced of the homogeneity of our protected all-4,8-oligomers. This separation protocol proved amenable to translation to a preparative scale and subsequently enabled the isolation of larger protected oligomers. We have

Fig. 4.4 Inter-flavan bond formation with TiCl4 as the condensing agent.

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15 14 12

16 17

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Fig. 4.5 Chromatogram of a mixture of benzylated epicatechin oligomers on β-cyclodextrin (Astec Cyclobond I), 4.6 × 250 mm, with an ethyl acetate/isooctane gradient: 0–30 min, 35–60%; 30–60 min, 60–70% ethyl acetate, at 1 mL/min. UV detection at 280 nm.

depicted the regio- and stereochemistry of our major oligomers as all-4β,8 but defer the discussion of this matter to the following section. The benzylated dimer 14, trimer 15, and tetramer 16 were deprotected by hydrogenolysis over Pearlman’s catalyst, and the free oligomers 3–5 were obtained by lyophilization from water in the form of amorphous, off-white solids, which by combustion analysis were found to contain up to two molecules of water per epicatechin moiety. Purities ranging from 94 to 96% (this includes the pentamer 6 discussed below) were determined by normal-phase HPLC. The NMR spectra are even less characteristic than those of their precursors, as they are, at room temperature, near the coalescence point owing to faster but still hindered rotation about the inter-flavan bond. On the other hand, the free polyphenols are best suited for molecular weight determination by LC-MS. For NMR characterization, which is essential for comparison with isolates from plant material, the free oligomers were acetylated with acetic anhydride and pyridine, a reaction that gave only moderate yields of the peracetates because of the formation of multiple minor by-products, which sometimes included lower oligomer fragments (as evidenced by HPLC). The peracetates exhibit characteristic 1H signal patterns for their acetate methyl groups, which at a given spectrometer frequency are suitable as fingerprints. Since part of the dimer formed in the above condensation reaction had already entered into further condensation steps, it appeared that it would be straightforward to extend the protocol to chain elongation from the dimer to the trimer, and so on. The expectation was that by purifying each oligomer carefully before subjecting it to further chain elongation, the total level of impurities resulting from alternative regiochemical outcomes could be kept in check, at least for the next few members of the series. The experiment showed otherwise. When the protected trimer 15 was reacted with the electrophile 13 (in a 2:1 ratio because of the relative scarcity of the trimer), the tetramer, pentamer, and small amounts of higher

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oligomers were indeed formed, but acid-catalyzed degradation also took place. This sidereaction led to the formation of monomer 12 and dimer 14, which then became substrates for chain-extension steps leading not only to all-4,8-oligomers but also to their regioisomers such as 17. Without much other rationale than curiosity, we examined the condensation of the building blocks 12 and 13 using a solid acid, the clay mineral Bentonite K10. There actually is an advantage to the use of a solid acid, namely the convenience of its removal by simple filtration. What we could not expect was the resulting product distribution. With the previously employed flavanyl acceptor to donor ratio of 4:1, dimer 14 was isolated in an impressive 88–92% yield, while 2–5% of trimer 15 was also formed. No trace of the regioisomeric trimer 17 was detected. While the reason for this astounding selectivity remains unknown, a simple consequence is that the method is not well suited for further chain extension. Thus, a 3:1 mixture of dimer 14 and flavanyl donor 13 produced only 40% of trimer 15 (again as a single isomer) as well as 13% of tetramer 16. At this point a decision had to be made as to whether to optimize the reaction conditions of the acid-catalyzed chain extension reaction so as to reduce the extent of concomitant degradation, or to find an alternative process that is inherently free of this problem. Fortunately, a literature report existed that pointed a path towards attaining the latter objective (Steynberg et al., 1998). 4-(Benzylthio)catechin and –epicatechin are commonly formed in the course of the analytical characterization of procyanidins by acid-catalyzed degradation with benzyl mercaptan. It was reported that these compounds could conversely be utilized as flavanyl donors in the synthesis of procyanidins if sulfur-specific activating agents were employed. As such, dimethyl(methylthio)sulfonium tetrafluoroborate and especially silver tetrafluoroborate were found suitable. The reaction conditions were neutral, thus issues of the degradation of already formed oligomers were not expected to surface, and a high selectivity for 4,8-regiochemistry was observed even in the absence of protecting groups. However, we regarded the noxious nature of benzyl mercarptan as a deterrent and were gratified to find that for our purpose this reagent could be replaced with odorless 2-mercaptobenzothiazole. The displacement of the 4-(2-hydroxyethoxy) substituent in the flavanyl donor 13 was brought about in 67% yield by treatment with an organoaluminum reagent prepared in situ from 2-mercaptobenzothiazole and trimethylaluminum (Fig. 4.6). Reaction of the resulting thioether 18 with an excess of protected epicatechin 12 and AgBF4 resulted in the formation of dimer 14 (56%) and trimer 15 (14%). In addition, a small amount of a by-product was isolated from the recovered starting material (12), which was tentatively assigned (by 1H NMR) the structure of a 3-O-4 dimer. The formation of such a compound, which would hardly have survived in the usual acidic reaction medium, was an unwanted effect of operating under neutral conditions. To avoid this side-reaction, the 3-hydroxyl groups of both reactants were blocked by acetylation in near-quantitative yields. In these experiments, dimer 14 rather than monomer 12 was employed as the flavanyl acceptor. Exposure of these reactants to AgBF4 did generate the hoped-for trimer 21 and tetramer 22, albeit in low yields. The major product isolated was the 4-hydroxy derivative of 12, that is, the reaction product of the flavanyl donor with adventitious moisture, most likely introduced by AgBF4, which is very hygroscopic. An attempt to suppress this unwanted hydrolysis by drying the solution of flavanyl acceptor and AgBF4 over 4-Å molecular sieves prior to adding the flavanyl donor 19 inexplicably

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Fig. 4.6 Silver-induced, regioselective inter-flavan bond formation employing 2-mercaptobenzothiazole as the leaving group in the flavanyl donor.

failed; only starting materials were recovered. Vacuum drying of the silver salt, on the other hand, produced the desired result. An exceptionally clean reaction (Fig. 4.7) resulted in the isolation of a series of linear oligomers ranging from trimer 21 to octamer 26. By starting from trimer 21 or tetramer 22, small amounts of larger oligomers, up to undecamer 29, were also obtained. The main purpose of these reactions, however, was the procurement of adequate quantities of the pentamer, on which the biological work was focused. Fully protected oligomers up to nonamer 27 were deacetylated with tetrabutylammonium hydroxide, a base chosen for its solubility in the relatively nonpolar reaction medium required by the lipohilicity of the reactants. The benzylated oligomers up to octamer 33 were then hydrogenolyzed in the same manner as their lower homologs, and portions of the free epicatechin oligomers 4–9 were peracetylated for additional characterization.

4.2.5 Establishment of inter-flavan bond stereochemistry (Kozikowski et al., 2000) Historically, the elucidation of the structures of oligomeric procyanidins followed a rocky path. Two general formulae of the dimers proposed early on (Forsyth & Roberts, 1960) were incorrect. The subsequently accepted gross structure was arrived at by chemical intuition (Freudenberg, 1960) and confirmed by 1H NMR data (Geissman & Dittmar, 1965) and synthetic studies (Creasy & Swain, 1965; Weinges et al., 1970). Only after our own work on the B2 isomer established its structure, were the regio- and stereochemistry of procyanidin B1 (epicatechin-4β,8-catechin) elucidated by X-ray crystallographic analysis of

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OBn O

BnO

OBn

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Fig. 4.7 AgBF4-mediated reaction between compounds 19 and 20 – chromatogram of the reaction mixture after filtration over silica gel. Column: Hewlett-Packard RP-8, 200 × 4.6 mL. Acetonitrile/water gradient: 0–30 min, 80 –100% acetonitrile, then 100%, at 1 mL/min. UV detection at 280 nm. Reprinted with permission from Kozikowski et al. (2003). Journal of Organic Chemistry, 68, 1641. Copyright 2003, American Chemical Society.

its peracetate (Weinges et al., 2001). No crystallographic information has been reported to date for procyanidin B2, of which no crystalline derivatives have, as yet, been prepared. The regiochemistry (6- vs. 8-substitution) of catechin and epicatechin derivatives was originally inferred from small chemical shift differences in their NMR spectra between the remaining aromatic protons, an effect that was of insufficient magnitude to begin with,

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not understood with regard to its origin, and eventually shown to be erratic (Kiehlmann & Tracey, 1986). More recently developed NMR methods were, on the other hand, highly successful. The two-dimensional, heteronuclear HMQC and HMBC (Bax & Summers, 1986) NMR experiments permit the establishment of single- and two- or three-bond C-H connectivity, respectively. This set of methods finally settled the regiochemistry issue first for the pair catechin-4α,6-catechin and catechin-4α,8-catechin (Balas & Vercauteren, 1994) and subsequently for epicatechin-4β,8-epicatechin (procyanidin B2) (Khan et al., 1997). 4,8-Regiochemistry follows from the following steps of reasoning (Fig. 4.8): (1) The two protons H-4F were readily identified by their unique chemical shifts (δ 2.74, 2.91) and their multiplicity (AB part of an ABX multiplet). These signals exhibit a one-bond correlation with that of C-4F at δ 28.6. The only other high-field 13C signal, that of C-4C, appears at δ 36.5. (2) The resonances of H-2C, H-3C, H-2F, and H-3F were located by a COSY experiment. (3) An aromatic 13C signal at δ 155.6 exhibits an HMBC (two- or three-bond) correlation with both H-2F and H-4F and was therefore assigned to C-8aD/F. (4) The same signal also correlates with H4-C. This is only possible if the inter-flavan bond is in position 4,8; if it were in position 4,6, the distance between H-4C and C-8aD/F would be five bonds, and therefore no C-H correlation should be observed. The relative inter-flavan bond configuration is quite readily established for dimers with a top catechin unit, as a trans relationship between H-3 and H-4 typically results in the observation of a large trans-diaxial coupling constant. The other dimer having the same monomer sequence and regiochemistry is then automatically the 3,4-cis isomer. With epicatechin at the top, the situation is ambiguous as either relative configuration around the 3,4-bond entails ideally (in cyclohexane) a dihedral angle of 60°, thus not resulting in a characteristic coupling constant. In the actual flexible molecules, deviations from this ideal dihedral angle lead to both small and medium coupling constants, but there is no unequivocal way of knowing in which sense a given 3,4-stereochemistry should influence the outcome. Our goal was a rigorous proof of structure that would not rely on any assumption, no matter whether based on intuition or computation, about a preferred conformation of a flexible molecule. This same reason militates against another frequently used analytical technique, circular dichroism. With the X-ray crystal structure of procyanidin B1 peracetate still a year ahead when our own work went into print, the less laborious approach

Fig. 4.8 Structure elucidation of procyanidin B2 (3) by means of an HMBC NMR experiment; reported three-bond correlations in bold print.

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Fig. 4.9 Synthesis of a mixed methyl- and benzyl-protected derivative of procyanidin B2.

of chemically correlating suitably protected procyanidin B1 and B2 by alcohol inversion was not available. Instead, we pursued the isolation of the stereocenter under scrutiny by an oxidative degradation approach, and comparison of the resulting product with a reference compound to be synthesized independently. Our strategy called for exhaustive oxidation of the bottom epicatechin unit to carboxyl while preserving the aromatic rings of the top unit. To this end, since electron-rich aromatics are more readily oxidized than others, O-methylation was implemented in the bottom unit to ‘lock in’ its electron-rich character. In the top unit, benzyl protection was chosen for the phenolic oxygens, permitting deoxygenation after de-protection to lead to a less electron-rich, more oxidation-resistant aromatic system. The alcoholic hydroxyls give rise to unnecessary stereocenters and were viewed as a source of complications arising from their reactivity and were therefore also slated for deoxygenation. Figure 4.9 shows the early stages of the execution of this plan. O-Methylation of catechin according to an established procedure (Mehta & Whalley, 1963) was followed by alcohol inversion employing the same protocol as in the case of the O-benzylated intermediates. Tetra-Omethylepicatechin (37) was then condensed with the O-benzylated flavanyl donor 13 using the TiCl4 method. In order to gain proof of the structure of procyanidin B2, we had first to establish that the resulting dimer 38 belonged to the same regio- and stereochemical series. This was effected by transforming 38 to the octamethyl ether 39 by hydrogenolytic debenzylation and further O-methylation. As hoped for, the same compound 39 was obtained from procyanidin B2 by methylation under the same conditions. Moving forward, the thionocarbonate 40 was prepared and deoxygenated under modified Barton conditions (Fig. 4.10) (Barton et al., 1993). Debenzylation was followed by formation of the tetratriflate 41 by reaction with N-phenyltrifluoromethanesulfonimide and a base. The hydrogenolysis of compound 41 surprisingly did not stop at the dimer devoid of its sulfonyloxy substituents but additionally cleaved the benzylic C-O bond of the now defunctionalized top flavan moiety. This outcome was viewed favorably as it stripped the top moiety of the last additional stereocenter and simplified the structure of the reference compound that needed to be subsequently synthesized. The phenolic hydroxyl formed in this process was removed by a repetition of the preceding steps. Compound 43 was then ready to be used as a degradation substrate. Sharpless’ ruthenium(III) chloride/NaIO4 system (Carlsen et al., 1981) typically takes 1–2 days to oxidize a phenyl group to carboxyl at room temperature. To preserve the phenyl

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Fig. 4.10 Degradation of a procyanidin B2 derivative to (R)-(−)-2,4-diphenylbutyric acid.

groups present in compound 43, its oxidation was terminated after two hours. The mixture of products was esterified with diphenyldiazomethane and the benzhydryl ester of the degradation product 44 (2,4-diphenylbutyric acid) isolated by preparative HPLC in a meager 10% yield. This material was levorotatory. The racemate resolution of 2,4-diphenylbutyric acid with α-methylbenzylamine was known in the literature, but the absolute configuration of the individual enantiomers had not previously been established. We synthesized the racemate by alkylation of phenylacetonitrile with phenethyl bromide and acidic nitrile hydrolysis and crystallized one of the diastereoisomeric salts with (R)-(+)-α-methylbenzylamine in the form of long needles that turned out to be of sufficient quality for X-ray crystallography. It was found that the enantiomer of 44 contained therein is R-configured. Both the free acid obtained from this salt, and its derived benzhydryl ester were to be found levorotatory. The sample of 44 obtained by degradation thus has R-configuration as well, which translates into β stereochemistry for the inter-flavan bond in procyanidin B2, as hitherto assumed. The larger oligomers require an additional comment. For all the 4,8-linked epicatechin oligomers isolated to date from natural sources, thiolytic degradation has been performed to establish their building-block sequence. The treatment of oligomers with benzyl mercaptan in the presence of acid brings about a reversal of their formation in the sense of a retro-Friedel–Crafts reaction. Cleavage of the chain of flavanyl moieties can occur at any position and therefore produces segments that can be compared with known smaller oligomers. In the case of the 4-(benzylthio)oligomers issuing from the top portion of a larger oligomer, desulfurization leads to the sulfur-free oligomers. In this way, all interflavan linkages in the known 4,8-linked epicatechin oligomers have been correlated with that in procyanidin B2 and are thus proven to be 4,8-linkages and furthermore to be βconfigured. NMR data on derivatives that exhibit sharp signals, specifically the peracetates, have regrettably been published only for oligomers up to the tetramer. These are in good agreement with ours, and it is therefore certain that our oligomers up to the tetramer are the all-4β,8 isomers. Beyond the tetramer, we can at present only say that it stands to reason that further inter-flavan bonds, after the first three of them have formed in the 4β,8 fashion, would form in the same manner. It is noteworthy that under the harsh conditions of exhaustive thiolysis side-reactions have been reported (McGraw et al., 1992) that led the authors to caution about the reliability of quantitative interpretations of thiolysis results.

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Others posited subsequently that these findings were ‘calling into question all of the structures established through such a degradative method’ (Balas & Vercauteren, 1994). This would appear as an overreaction to us, as such side-reactions have not been observed under the mild, controlled conditions employed for establishing flavan sequences.

4.2.6 Further developments Before the question of the relative inter-flavan bond configuration in procyanidin B2 was settled for good, we became interested in obtaining the isomer of opposite stereochemistry for comparison purposes. The stereochemical outcome of the inter-flavan bond-forming process is subjected to the influence of both pre-existing stereocenters at C-2 and C-3. In the case of epicatechin, the flavan nucleophile enters anti to the bulky 2-aryl group and also anti to the 3-hydroxy or 3-acetoxy group, which presumably shields the α-face of the 4-carbenium ion through a neighboring-group effect. This situation had been suspected all along before a firm proof of the dimer’s stereochemistry was at hand, and formed the basis of a working hypothesis for generating the desired stereoisomer. For, if the inter-flavan bond was already in place and the 4-carbenium ion reacted with a hydride nucleophile, then it should by the same token be the hydrogen that ends up in the β-position, and thus the flavanyl group in the α-position. The experiment confirmed the validity of the concept. The requisite tertiary alcohol, with methyl rather than benzyl derivatization of the phenolic hydroxyls (and thus of course unsuitable for obtention of the free dimer), had been prepared by organolithium addition to the 4-ketone (Weinges et al., 1970), an approach that we readily modified towards our standard O-benzyl phenolic protection (Fig. 4.11) (Kozikowski et al., 2001). TBDMS or (not shown) benzyl were both suitable for protection of the 3-hydroxyls. The 4-ketone 47 was obtained by Ley oxidation (Griffith et al., 1987) of the alcohol 46, which in turn resulted from a variant of the usual DDQ oxidation protocol in which water rather than ethylene glycol was employed as nucleophile and the solvent replaced with THF. In parallel, tetra-O-benzylepicatechin (12) was brominated with NBS, a reaction that yielded the 8-bromide 48 in excellent yield and regioselectivity. After protection of the 3-hydroxyl in this compound as well, halogen-metal exchange was

Fig. 4.11 Stereoselective synthesis of the unnatural procyanidin epicatechin-4α,8-epicatechin.

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effected with tert-butyllithium and the 4-ketone 47 added. This resulted in a fair yield of the tertiary alcohol 50 with unknown configuration at C-4 of the top epicatechin moiety. This compound underwent reduction to the protected dimer 51 with tri-n-butyltin hydride and trifluoroacetic acid, which furnished the free dimer 52 after two consecutive deprotection steps. This compound, as had been expected, is different from procyanidin B2, and after proof of the latter’s inter-flavan linkage β-stereochemistry is thus recognized by default as the 4α,8-isomer. We were rather unlucky in our efforts to extend the described methodology to the formation of dimers containing a catechin top unit, in that reduced yields were obtained compared to procyanidin B2, and mixtures of the α- and β-stereoisomers were formed. These isomers had to be laboriously separated by preparative HPLC, an undesirable situation if larger oligomers were subsequently to be prepared. The likely reason for the observed lack of stereoselectivity is the opposite directing effects of the 2-aryl and 3hydroxyl groups, which had been working in concert in the epicatechin series. However, others have in the meantime succeded in optimizing α-selectivity (Fig. 4.12) (Saito et al., 2002). The flavanyl donor 53 carrying an acetyl group at O-3 and a 2-ethoxyethoxy group at C-4, apparently under almost exclusive steric control by the 3-substituent, gave on reaction with tetra-O-benzylcatechin (10) in the presence of trimethylsilyl triflate at −78°C a quantitative yield of the 4α,8-dimer 54 with a stereoselectivity of >48:1. Other 4-alkoxy groups and Lewis acids investigated by the authors led to lower selectivities and/or yields. Noteworthy, even though the excess of the acceptor 10 is higher (4.5 equivalents) than typically employed by us, is the total absence of further condensation of the resulting dimer to form higher oligomers. In an even more remarkable publication, Saito et al. reported that the imposition of a steric constraint by tethering the acceptor and donor together through a dicarboxylic acid linker produced a near-quantitative yield of the 4β,8-linked macrodiolide 56 as a single stereoisomer (Saito et al., 2003). The analogous cyclization substrate having a bottom epicatechin unit reacted much less efficiently and then produced only the 4α,8-isomer, and an inter-flavan bond between two epicatechin units could not be formed in this manner. It would be worthwhile to pursue the synthesis of catechin-4β,8-epicatechin by hydroxyl inversion of the monoester 57 that resulted from 56 through transesterification

Fig. 4.12 Stereoselective synthesis of catechin-4β-catechin by intramolecular condensation of a flavanyl donor tethered to a flavanyl acceptor.

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Fig. 4.13 Orthogonal strategy for the condensation of two flavanyl donors resulting in a dimeric flavanyl donor, and its further chain elongation.

involving only the less hindered of its two ester linkages. Cleavage of the remaining ester required a reductive approach using diisobutylaluminum hydride. Another beautiful piece of work by Japanese researchers is concerned with the generation of activated dimers, which would enable a convergent synthesis of well-defined mixed oligomers (Ohmori et al., 2004). In our own research, low yields of benzylated epicatechin oligomers bearing a 4-(2-benzothiazolyl)thio group on their bottom unit were isolated as products of self-condensation of the building block 19, and the dimer could be used as a biflavanyl donor to extend the chain of the tetramer 22 by two members. However, in order to synthesize oligomeric flavanyl donors efficiently, two requirements have to be met: (1) The 8-position of the top unit has to be reversibly blocked so as to prevent selfcondensation; and (2) the leaving group on the bottom unit has to survive the inter-flavan bond formation protocol used in the connection of the top and bottom units, which requires the availability of at least two different chemistries. The authors demonstrated, on examples involving catechin building blocks, that variants of the existing acid-catalyzed condensation reaction applied to 4-oxygenated units, and of the silver(I)-induced condensation protocol applied to 4-thiolated units are well suited for this purpose (Fig. 4.13). A bromo substituent was employed to block the donor’s 8-position and was later removed by a rather unusual method, namely reduction with LiAlH4. The difficulty of effecting this transformation had discouraged us from pursuing this avenue of work (catalytic hydrogenation, as we also found, being poorly suitable owing to the generation of HBr as a by-product, and halogen-metal exchange with tert-BuLi giving a rather low yield). What remains to be done? To date no procedure is known that would permit the formation of 4,6-inter-flavan linkages using benzyl-protected intermediates, and reactions involving unprotected building blocks typically deliver mixtures of regioisomers. The only exception is a report involving the 8-methylated donor 64 and acceptor 65 (Kawamoto et al., 1991b) (Fig. 4.14), in which the 8-substituents block reaction in this position but cannot afterwards be conveniently removed. Even though a bromine atom and a methyl group are

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Fig. 4.14 Formation of a 4,6-inter-flavan bond by condensation of building blocks with blocked 8-positions.

comparable in size (and should thus have a similar tendency to induce the 7-benzyloxy group to point its phenyl ring towards the 6-position, which could result in diminished reactivity for steric reasons), 8-bromination in our hands completely suppressed inter-flavan bond formation. This result, which is the basis for the above-discussed strategy to prevent self-condensation of the electrophile, would therefore appear to be based on the substituent’s electronic effect.

4.3 Anticancer activity As noted above, the proanthocyanidins have attracted a great deal of recent attention in the fields of nutrition, medicine, and health owing to their wide range of potentially significant biological activities. In particular, there is a growing body of evidence suggesting that these compounds act as potent antioxidants in vitro, ex vivo, and in vivo and may thus alter the pathophysiology of imbalances or perturbations of free radical and/or oxidatively driven processes in many diseases or directly interfere with many cellular processes. Our interest has been focused in the cancer area, where initial observations have shown that procyanidin-rich fractions from cocoa cause growth inhibition in several human cancer cell lines (Kozikowski et al., 2003). The development of improved analytical and preparative HPLC methods has allowed us to obtain quantities of the purified oligomers, which were screened against certain human breast cancer cell lines together with our synthetic products (up to the pentamer). Cytotoxic effects were clearly associated with high molecular weight oligomers (pentamer and higher) at the 100 μg/mL dose level (Fig. 4.15). This effect was also observed in MCF-7, MDA MB 231, and MDA 435 human breast cancer cell lines treated in a similar fashion (data not shown). Curiously, no activity was observed for the dimer, trimer, and tetramer, which others have reported to show activity on small cell lung and colorectal cancer cell lines. Cytotoxicity thus increases sharply with the degree of oligomerization. Several studies have demonstrated an association between cell cycle regulation and cancer. The cell cycle machinery regulates cell proliferation, and dysregulated cellular proliferation is a hallmark of cancer. Many synthetic cell cycle inhibitors such as flavopiridol, olomoucine, roscovitine, and puvalanol B are viewed as a new generation of anticancer

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Fig. 4.15 Cytotoxicity of procyanidin fractions purified from cocoa on the human breast cancer cell line SKBR-3 at 100 and 25 μg/mL. Reprinted with permission from Kozikowski et al. (2003). Journal of Organic Chemistry, 68, 1641. Copyright 2003, American Chemical Society.

drugs, and some are being investigated in clinical trials (Buolamwini, 2000). Cell cycle analysis of procyanidin-treated MDA MB 231 cells clearly showed a G0/G1 arrest by the pentamer, no effect by the dimer or trimer, and only a slight effect by the tetramer (Table 4.1). The increase in G0/G1 was accompanied by a decrease of cell numbers in the S phase and in G2/M. The pentamer-caused G0/G1 arrest was reversible in cells treated for up to 8 h and irreversible after a 24 h treatment. No difference in activity was observed between natural and synthetic trimer 4; an approximately 15% increase in G0/G1 Table 4.1 Cell cycle analysis of MDA MB 231 human breast cancer cells treated with oligomeric procyanidins purified from cocoa. Reprinted with permission from Kozikowski et al. (2003). Journal of Organic Chemistry, 68, 1641. Copyright 2003, American Chemical Society. Treatment

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36.69 38.26 38.13 42.28 43.60 43.22 40.33 43.71 51.46 38.33 37.84 66.03 88.31

23.39 22.43 22.43 35.61 34.10 35.98 36.25 34.42 28.25 21.05 21.39 17.23 6.07

39.92 39.30 39.45 22.12 22.30 20.80 23.42 21.87 20.30 40.61 40.77 16.67 5.62

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Table 4.2 Comparison cell cycle analysis of MDA MB 231 human breast cancer cells treated with natural versus synthetic oligomeric procyanidins. Reprinted with permission from Kozikowski et al. (2003). Journal of Organic Chemistry, 68, 1641. Copyright 2003, American Chemical Society. Treatment

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Control Vehicle Natural trimer (4) (200 μg/mL; 24 h) Synthetic trimer (4) (200 μg /mL; 24 h) Natural tetramer (5) (200 μg/mL; 24 h) Synthetic tetramer (5) (200 μg/mL; 24 h) Natural pentamer (6) (200 μg/mL; 24 h) Synthetic pentamer (6) (200 μg/mL; 24 h)

28.65 27.19 28.46 26.98 36.82 43.49 45.99 64.15

49.28 49.61 48.49 49.57 43.37 39.39 38.25 23.36

22.06 23.2 23.05 23.45 19.02 17.03 15.76 12.49

arrest was seen for synthetic tetramer 5 compared to natural material, and an approximately 30% increase for synthetic vs. natural pentamer 6 (Table 4.2). These differences can be attributed to the higher purities of the synthetic procyanidins. Cell cycle arrest induced by monomeric catechins, especially epigallocatechin gallate (EGCG), has previously been observed by others (Ahmad et al., 1997). Cell cycle arrest may result in cell death, which was indeed observed in the present experiments. The manner of cell death (apoptosis or necrosis) was investigated by the annexin V-fluorescein isothiocyanate (FITC) assay using Trevigen’s TACS Annexin V-FITC kit. Cell cycle analysis of MDA MB 231 cells treated with natural and synthetic oligomers 4, 5, and 6 is shown in Fig. 4.16. We also checked for the possibility that these effects may be due to polyphenol-induced hydrogen peroxide formation, which has been observed previously, and found that this was not the case. Collectively, these results confirm the cytotoxicity to human breast cancer cell lines by an epicatechin pentamer purified from cocoa and assigned the structure 6. This procyanidin causes a G0 /G1 arrest in MDA MB 231 cells that is independent of any effects caused by H2O2. An increase in annexin V and propidium iodide positive cells suggests that pentamer-treated cells quickly enter into a necrotic phase of cell death. However, additional efforts are required to elucidate the molecular mechanisms by which 6 exerts its effects in vitro and to conclusively determine whether apoptosis or necrosis is the pathway of cell death. In continuation of this earlier work, the Dickson group at the Lombardi Cancer Center investigated the effects of the pentamer on the proliferation of human breast cancer cells (MDA MB-231, MDA MB-436, MDA MB-468, SKBR-3, and MCF-7) and benzo[a]pyreneimmortalized 184A1N4 and 184B5 cells (Ramljak et al., 2005). While the pentamer was shown to inhibit the proliferation of these malignant cells, it was found that both normal human mammary epithelial cells in primary culture and spontaneously immortalized MCF10A cells were largely unaffected. In studies using estrogen receptor-positive MCF-7 breast cancer cells bearing wild-type p53, it was concluded that the growth inhibitory effects of the pentamer are not dependent on the status of estrogen receptor or p53 in the cells. Additionally, other work by these investigators has established that the effects of the pentamer are unlikely to be due to changes in mitochondrial membrane potential. In order to learn something about the proteins that are affected by the pentamer, a proteomic multilayered dot blot analysis was undertaken. This proteomics approach provides information on both the expression and the activity of proteins. A total of 46 different

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Fig. 4.16 Flow cytometry of procyanidin-treated MDA MD 231 human breast cancer cells using Annexin V-FITC and propidium iodide (control versus 24 h treatment with 200 μg/mL of oligomer): row (a) trimer (4); row (b) tetramer (5); row (c) pentamer (6). Lower left quadrants: viable cells. Lower right quadrants: early apoptotic events. Upper right quadrants: late apoptotic events. Upper left quadrants: non-viable cells. Reprinted with permission from Kozikowski et al. (2003). Journal of Organic Chemistry, 68, 1641. Copyright 2003, American Chemical Society.

antibodies were used in this screening study, with proteins being selected based upon their role in cell growth, proliferation, survival and apoptosis. The pentamer was found to affect the phosphorylation status of eight proteins, and of these, the phosphorylation status of five proteins (Cdc2, PKCy, pRb, stress-activated protein kinase/c-Jun-NH2-kinase, Stat5) was decreased by the cocoa pentamer within 48 hours. As increased phosphorylation of p53 at Ser392 correlates with increased aggressiveness of tumors (Furihata et al., 2002), the ability of the pentamer to cause a reduction in phosphorylation at Ser392 may be of considerable importance to its anti-tumor effects. While undoubtedly more work will be required to obtain a detailed understanding of the in-vitro mechanism of anticancer action of these oligomeric epicatechins, the situation

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may be very different in vivo. In particular, as these chemically sensitive cocoa oligomers can be degraded by acidic conditions in the gut as well as by metabolizing enzymes in the body, the actual utility of such compounds may be compromised. Thus, we call attention to a study in which procyanidin dimer B3 was administered in purified form to rats; this dimer could not be found in the plasma (Williamson & Manach, 2005). Thus, the effects that have been measured in humans upon ingestion of procyanidin-rich foods may well be a consequence of the formation of some unknown metabolites, or, more likely, may be due to the effects of the monomeric species. In summary, the highly efficient methods we have developed in order to prepare the oligomeric epicatechins provide a means of obtaining pure material for biological studies. As this methodology can be used to assemble a host of ‘designer catechins’ that can be modified in ways that may improve their stability, it may well be possible to generate compounds that have more drug-like properties, and thereby show greater efficacy in vivo.

4.4 Acknowledgments The authors are indebted to Mars Inc. for their support of these research studies, and to Leo Romancyzk for providing information on the natural products isolated from chocolate.

4.5 References http://groups.msn.com/FoodiesCorner/chocolateandmedicine.msnw. http://scicom.ucsc.edu/scinotes/0001/chocolate.htm. Ahmad, N., Feyes, D.K., Nieminen, A.L., Agarwal, R. & Mukhtar, H. (1997). Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. Journal of the National Cancer Institute, 89, 1881–1886. Anthoni, U., Christophersen, C. & Nielsen, P.H. (2001). O-Benzylation of polyphenolics. Preparation of 1,2,4-tribenzyloxybenzene. Synthetic Communications, 31, 2223–2229. Balas, L. & Vercauteren, J. (1994). Extensive high-resolution reverse 2D NMR analysis for the structural elucidation of procyanidin oligomers. Magnetic Resonance in Chemistry, 32, 386– 393. Barton, D.H.R., Jang, D.O. & Jaszberenyi, J.C. (1993). The invention of radical reactions. 32. Radical deoxygenations, dehalogenations, and deaminations with dialkyl phosphites and hypophosphorous acid as hydrogen sources. Journal of Organic Chemistry, 58, 6838–6842. Bax, A. & Summers, M.F. (1986). 1H and 13C Assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2d multiple quantum NMR. Journal of the American Chemical Society, 108, 2093 –2094. Buolamwini, J.K. (2000). Cell cycle molecular targets in novel anticancer drug discovery. Current Pharmaceutical Design, 6, 379 –392. Burger, J.F.W., Kolodziej, H., Hemingway, R.W., Steynberg, J.P., Young, D.A. & Ferreira, D. (1990). Oligomeric flavanoids. Part 15. Base-catalyzed pyran rearrangements of procyanidin B-2, and evidence for the oxidative transformation of B- to A-type procyanidins. Tetrahedron, 46, 5733 – 5740. Carlsen, P.H.J., Katsuki, T., Martin, V.S. & Sharpless, K.B. (1981). A greatly improved procedure for ruthenium tetroxide catalyzed oxidations of organic compounds. Journal of Organic Chemistry, 46, 3936 –3938. Creasy, L.L. & Swain, T. (1965). Structure of condensed tannins. Nature, 208, 151–153.

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Kozikowski, A.P., Tückmantel, W. & Hu, Y. (2001). Studies in polyphenol chemistry and bioactivity. 3. Stereocontrolled synthesis of epicatechin 4α,8-epicatechin, an unnatural isomer of the B-type procyanidins. Journal of Organic Chemistry, 66, 1287–1296. Laks, P.E., Hemingway, R.W. & Conner, A.H. (1987). Condensed tannins. Base-catalysed reactions of polymeric procyanidins with phloroglucinol: Intramolecular rearrangements. Journal of the Chemical Society, Perkin Transactions 1, 1875–1881. Li, L. & Chan, T.H. (2001). Enantioselective synthesis of epigallocatechin 3-gallate (EGCG), the active polyphenol component from green tea. Organic Letters, 3, 739–741. McGraw, G.W., Steynberg, J.P. & Hemingway, R.W. (1992). Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage. Tetrahedron Letters, 34, 987– 990. Mehta, P.P. & Whalley, W.B. (1963). The stereochemistry of some catechin derivatives. Journal of the Chemical Society, 5327–5332. Meyer, S.D. & Schreiber, S.L. (1994). Acceleration of the Dess–Martin oxidation by water. Journal of Organic Chemistry, 59, 7549 –7552. Miura, S., Midorikawa, T. & Awata, N. (1983). Synthesis of [3-2H]- and [3-3H]-cianidanol. Radioisotopes, 32, 225 –230. Morimoto, S., Nonaka, G. & Nishioka, I. (1986). Tannins and related compounds. XXXVIII. Isolation and characterization of flavan-3-ol-glucosides and procyanidin oligomers from cassia bark (Cinnamomum cassia BLUME). Chemical and Pharmaceutical Bulletin, 34, 633–642. Mustafa, J., Khan, R., Khan, I.A. & Ferreira, D. (2004). Benzylation of flavan-3-ols (catechins). Organic Preparations and Procedures International, 36, 61–67. Ohmori, K., Ushimaru, N. & Suzuki, K. (2004). Oligomeric catechins: An enabling synthetic strategy by orthogonal activation and C(8) protection. Proceedings of the National Academy of Sciences of the USA, 101, 12002 –12007. Osakabe, N., Yasuda, A., Natsume, M., Takizawa, T., Terao, J. & Kondo, K. (2002). Catechins and their oligomers linked by C4 → C8 bonds are major cacao polyphenols and protect low-density lipoprotein from oxidation in vitro. Experimental Biology and Medicine, 227, 51–56. Porter, L.J., Ma, Z. & Chan, B.G. (1991). Cacao procyanidins: Major flavanoids and identification of some minor metabolites. Phytochemistry, 30, 1657–1663. Ramljak, D., Romanczyk, L.J., Metheny-Barlow, L.J., Thompson, N., Knezevic, V., Galperin, M., Ramesh, A. & Dickson, R.B. (2005). Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells. Molecular Cancer Therapeutics, 4, 537–546. Saito, A., Nakajima, N., Tanaka, A. & Ubukata, M. (2002). Synthetic studies of proanthocyanidins. Part 2: Stereoselective gram-scale synthesis of procyanidin-B3. Tetrahedron, 58, 7829–7837. Saito, A., Nakajima, N., Tanaka, A. & Ubukata, M. (2003). Synthetic studies of proanthocyanidins. Part 4. The synthesis of procyanidin B1 and B4: TMSOTf-catalyzed cyclization of catechin and epicatechin condensation. Heterocycles, 61, 287–298. Steenkamp, J.A., Mouton, C.H.L. & Ferreira, D. (1991). Regio- and stereoselective oxidation of flavan-3-ol-, 4-arylflavan-3-ol-, and biflavanoid derivatives with 2,3-dichloro-5,6-dicyano-1,4benzoquinone (DDQ). Tetrahedron, 47, 6705–6716. Steynberg, J.P., Bezuidenhoudt, B.C.B., Burger, J.F.W., Young, D.A. & Ferreira, D. (1990). Oligomeric flavanoids. Part 7. Novel base-catalysed pyran rearrangements of procyanidins. Journal of the Chemical Society, Perkin Transactions 1, 203–208. Steynberg, P.J., Nel, R.J.J., van Rensburg, H., Bezuidenhoudt, B.C.B. & Ferreira, D. (1998). Oligomeric flavanoids. Part 27. Interflavanyl bond formation in procyanidins under neutral conditions. Tetrahedron, 54, 8153–8158. Tückmantel, W., Kozikowski, A.P. & Romanczyk, L.J., Jr. (1999). Studies in polyphenol chemistry and bioactivity. 1. Preparation of building blocks from (+)-catechin. Procyanidin formation. Synthesis of the cancer cell growth inhibitor, 3-O-galloyl-(2R,3R)-epicatechin-4β,8-[3-O-galloyl(2R,3R)-epicatechin]. Journal of the American Chemical Society, 121, 12073–12081. Weinges, K., Perner, J. & Marx, H.-D. (1970). Synthese des octamethyl-diacetyl-procyanidins B 3. Chemische Berichte, 103, 2344 – 2349.

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Weinges, K., Schick, H. & Rominger, F. (2001). X-Ray structure analysis of procyanidin B1. Tetrahedron, 57, 2327–2330. Williamson, G. & Manach, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. American Journal of Clinical Nutrition, 81, 243S–255S. Zaveri, N.T. (2001). Synthesis of a 3,4,5-trimethoxybenzoyl ester analogue of epigallocatechin-3gallate (EGCG): A potential route to the natural product green tea catechin, EGCG. Organic Letters, 3, 843 – 846.

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Chapter 5

Gene Discovery and Metabolic Engineering in the Phenylpropanoid Pathway Luzia V. Modolo, Yongzhen Pang, Li Tian and Richard A. Dixon

5.1 Introduction Plant polyphenols impact on many traits of plants including quality, yield, resistance and stress tolerance, in addition to being important dietary components and phytomedicines. Much of the rich chemical diversity of the plant kingdom arises from a limited number of chemical scaffolds. In the case of polyphenols, these scaffolds range from simple hydroxycinnamic acids to more complex polyketide-derived flavonoid compounds, modified by specific chemical substitutions (hydroxylation, glycosylation, acylation, prenylation, O-methylation, etc.) brought about by substrate- and/or regio-specific substitution enzymes, and by additional oxidation or reduction reactions affecting the scaffold itself. The molecular genetic basis underlying these chemistries is becoming apparent through concerted genomic and genetic approaches, and this is facilitating metabolic engineering of polyphenol biosynthetic pathways through transgenic technology. In this article, we illustrate how the recent discovery of key biosynthetic genes is enabling the engineering of isoflavones and proanthocyanidins, two classes of polyphenols with important properties benefiting human health, ruminant nutrition and plant defense. We also review progress in understanding the biochemistry and molecular biology of plant polyphenol glycosyltransferases, enzymes that will be critical for enabling stable accumulation of engineered polyphenols in plant tissues.

5.2 Biosynthesis and functions of isoflavones Isoflavones are a group of polyphenolic compounds derived from a branch of the flavonoid biosynthetic pathway. Genistein and daidzein represent basic isoflavone scaffold structures (Fig. 5.1) and are commonly further modified by hydroxylation, methylation, glycosylation or prenylation reactions (Dixon & Ferreira, 2002). Isoflavone aglycones may be conjugated to glucose, galactose or other sugar moieties, and this alters the polarity and bioactivity of the core molecules (Bowles et al., 2006). Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Fig. 5.1 Biosynthetic pathways to isoflavonoids and flavonoids in plants. Enzymes, selected structures of intermediates, and the steps blocked by the tt3, tt5 and tt6 mutations in Arabidopsis, are shown. CHI, chalcone isomerase; IFS, isoflavone synthase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase.

The first step in isoflavone biosynthesis is the aryl migration reaction that converts flavanone via 2-hydroxyisoflavanone to isoflavone (Fig. 5.1). Genes encoding 2-hydroxy isoflavanone synthase (commonly, but incorrectly, called isoflavone synthase, IFS) have been cloned and characterized from various leguminous plants and the non-legume sugarbeet (Steele et al., 1999; Jung et al., 2000). IFS belongs to the cytochrome P450 family and requires both NADPH and O2 as co-factors for the formation of 2-hydroxyisoflavanone. This compound is then dehydrated to form isoflavone (genistein or daidzein), either spontaneously or catalyzed by 2-hydroxyisoflavanone dehydratase (Akashi et al., 2005). Isoflavones are well known for their phytoestrogenic activities, as a result of the common structural features they share with estrogens (Dixon & Ferreira, 2002; Dixon, 2004). Isoflavones can bind to estrogen receptors and exhibit estrogenic or anti-estrogenic activities. There have been numerous reports correlating isoflavone intake and protection against hormone-related illnesses, such as easing post-menopausal symptoms, and reducing the risk of breast cancer, prostate cancer and cardiovascular diseases (Mazur, 1998; Setchell & Cassidy, 1999; Alekel et al., 2000; Mentor et al., 2001; Singletary et al., 2002). Isoflavones also have potential antioxidant activities (Wiseman et al., 2000; Rufer & Kulling, 2006).

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In plants, isoflavones and their derivatives have been classified as ‘phytoalexins’ that accumulate in response to microbial pathogen attack (Dixon, 1986). In contrast to this defensive role, leguminous plants also use isoflavones as signals for the recognition of symbiotic rhizobium bacteria to form nitrogen-fixing root nodules (Phillips & Kapulnik, 1995). In the host-specific symbiotic relationship between legumes and soil rhizobia, the bacteria fix nitrogen into ammonia, and plants assimilate the ammonia into amino acids and in return supply rhizobia with carbohydrates. The expression of a set of nodulation (nod) genes is required for the rhizobia to synthesize and release chitooligosaccharide ‘nod factors’ and induce plants to form root nodules. Flavonoids and isoflavonoids act as signals to induce the expression of nod genes in symbiotic rhizobia (Rolfe, 1988).

5.3 Dietary sources of isoflavones Isoflavones are present in most members of the Leguminosae family (Mazur, 1998). Soybean and red clover are the main sources of isoflavone-rich food, food supplements and pharmaceutical products. Other dietary sources of isoflavones include chickpea and alfalfa (Reinli & Block, 1996; Kaufman et al., 1997). Currants and raisins were also reported to be rich in daidzein and genistein (Liggins et al., 2000). In soybeans, isoflavones are glycosylated and the aglycone form is released after fermentation or digestion. Isoflavones are relatively stable under most cooking conditions and will not be destroyed during food processing (Stintzing et al., 2006).

5.4 Metabolic engineering of isoflavones 5.4.1 Metabolic engineering by ectopic expression of IFS IFS catalyzes the conversion of flavanone to isoflavanone and therefore manipulation of IFS expression has become the focus of isoflavonoid engineering strategies. In recent years several efforts have been made to produce/over-produce isoflavones in plants by expressing IFS ectopically (Jung et al., 2000; Yu et al., 2000; Liu et al., 2002; Yu et al., 2003; Deavours & Dixon, 2005; Sreevidya et al., 2006). In general, genistein aglycones did not accumulate in these IFS transgenic plants. Instead, the introduced isoflavones accumulated as various sugar conjugates, depending on the plant species that were transformed and the tissue where IFS was ectopically expressed (Table 5.1). Expression of soybean IFS in wild-type Arabidopsis led to the production of genistein glycoside (Jung et al., 2000; Yu et al., 2000; Liu et al., 2002). When IFS was introduced into the Arabidopsis tt6/tt3 double-mutant background, where expression of flavanone 3hydroxylase (F3H) and dihydroflavonol reductase (DFR) was eliminated by gene knockout, genistein accumulation was 10–50-fold higher compared to that in plants with IFS expressed in the wild-type Arabidopsis background, indicating that the limiting factor for enhanced isoflavone production was competition between endogenous flavonol biosynthesis and the introduced isoflavonoid pathway (Liu et al., 2002). When a Medicago truncatula IFS gene was over-expressed in alfalfa (M. sativa), genistein and the genistein derivatives biochanin A and pratensein were produced. However,

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Table 5.1 Accumulation of isoflavones in various plant species transformed by the manipulation of IFS expression. Genistein level quantified after acid or β-glucosidase hydrolysis

Reference

Plant

Tissue

Transgene

Promoter

Product

Arabidopsis

Leaf and stem

Soybean IFS1

35S

Genistein conjugates

∼2 ng mg−1 fresh weight

Jung et al. (2000)

Tobacco

Flowers

Soybean IFS1

35S

Genistein glucoside, genistein malonylglucoside

∼2 ng mg−1 fresh weight

Yu et al. (2000)

Arabidopsis

Leaf

Soybean IFS

35S

Glucose-rhamnose genistein, rhamnose genistein, unidentified genistein glucoside

31–169 nmol g−1 fresh weight (in tt6/tt3 double mutant background)

Liu et al. (2002)

Alfalfa

Leaf

Medicago truncatula IFS

Double 35S

Genistein glucoside, biochanin A glucoside, pratensein glucoside

Up to 50 nmol g−1 fresh weight

Deavours & Dixon (2005)

Rice

Root

Soybean IFS

35S

Genistein conjugates

Not quantified

Sreevidya et al. (2006)

all these compounds were found as glucose conjugates, whereas the endogenous flavones in alfalfa are glucuronidated (Deavours & Dixon, 2005). In an attempt to increase the overall isoflavone content in soybean seeds, the maize transcription factors C1 and R were co-expressed in conjunction with the suppression of F3H, and the total isoflavone levels in the seeds from the transgenic plants reached up to 4-fold higher than in the seeds from control plants (Yu et al., 2003). To test the possibility of producing isoflavones in monocot cells, maize BMS cell lines were transformed with a 35S/IFS construct together with a chimeric transcription factor consisting of the maize C1 and R transcription factor functions; genistein was detected in its conjugated form in the transgenic BMS cells (Yu et al., 2000). The soybean IFS gene was expressed under the control of the 35S promoter and transformed into rice through Agrobacterium-mediated transformation in an attempt to produce isoflavones as nodulation signals in a non-nodule-forming plant. Production of genistein glycoside rather than genistein aglycone was detected in root tissue of the transgenic rice plants. In addition, both leaf and root extracts were able to stimulate nod gene expression in some of the rhizobia tested (Sreevidya et al., 2006). However, no root nodule formation was observed in the 35S–IFS transgenic rice plants.

5.4.2 Structural biology-assisted design and metabolic engineering with an artificial bifunctional IFS enzyme To more efficiently manipulate isoflavonoid biosynthesis, a bifunctional IFS/chalcone isomerase (CHI) enzyme was designed with the assistance of structural biology tools. In this

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approach, the open reading frames of genes encoding the two consecutive biosynthetic enzymes, CHI and IFS (Fig. 5.1), were directly fused to form two independent domains of a single enzyme (Tian & Dixon, 2006). Molecular modeling of the predicted fusion enzyme showed flexible linkage between the two domains and predicted the formation of a functional enzyme in vivo without steric constraint from the individual domains. The order of IFS and CHI in the hybrid polypeptide chain (IFS at the N-terminus) was selected to take advantage of the 5′ transit peptide of IFS, which directed the fusion enzyme to localize to the outer surface of the endoplasmic reticulum, with the catalytic domains of both enzymes in the cytosol. The recombinant protein was functionally tested in yeast (Saccharomyces cerevisiae). When microsomes from yeast transformed with the IFS/CHI fusion were incubated with isoliquiritigenin or naringenin chalcone (substrates for the CHI reaction), daidzein or genistein (products of the IFS reaction) were formed respectively, indicating that the IFS/CHI fusion has both IFS and CHI activities in vitro. To establish the isoflavonoid pathway in non-legume tobacco plants, the IFS/CHI fusion was expressed constitutively under control of the 35S promoter, and this led to the accumulation of genistein and genistein glycoside in both young leaves and petals of the transgenic plants (Tian & Dixon, 2006). Tobacco flowers contain chalcone and flavonoid substrates for isoflavone production (Winkel-Shirley, 2001). Genistein production was two-fold higher in the flowers of IFS/ CHI fusion plants compared to plants transformed with IFS alone. The higher yield of genistein glucosides in the IFS/CHI transgenic plants could be due to the physical proximity of the two sequentially operating enzymes, which might create high local concentrations of the intermediates around the enzyme complex, or could simply be due to the over-expression of the CHI gene. However, total flavonol levels were not significantly different between IFS/CHI and IFS transformed tobacco plants, indicating that over-expressing CHI alone did not increase flux through the flavonoid pathway, and thereby suggesting that chalcone is converted to genistein more efficiently in IFS/CHI transgenic tobacco plants. In general, a fusion protein approach can simplify the metabolic engineering of complex pathways. In addition to potentially increased catalytic efficiency and the ability to create or ‘engineer around’ metabolic channels, fusion proteins require a single promoter to drive the expression of more than one enzyme, thereby reducing the potential for gene silencing resulting from the expression of multiple copies of the same promoter within a plant cell. Furthermore, the signaling element of one protein/enzyme for a particular subcellular organelle or compartment can alter the subcellular localization of the other biosynthetic enzyme in the complex, possibly resulting in the production of novel metabolites.

5.5 Gene discovery in the proanthocyanidin biosynthetic pathway Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric or polymeric end-products of the flavonoid biosynthetic pathway. They are widespread in fruits, bark, leaves and seeds throughout the plant kingdom, where they provide protection against predation. They are also determinants of astringency or flavor in beverages such as wines, teas and fruit juices, and their presence in forage crops is a major quality factor (Dixon

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et al., 2005). Much attention has recently been paid to this class of secondary metabolite since there is increasing evidence to show that PAs and related flavonoids possess beneficial effects on human health (Scalbert et al., 2005).

5.5.1 Structural genes for PA biosynthesis During the last few years, the late, specific stages of the PA biosynthetic pathway have become better understood, primarily through the results of molecular genetic experiments. PA monomer biosynthesis occurs via two distinct branches of the flavonoid pathway, with which it shares the same upstream biosynthetic pathway to 2R,3S,4S-leucoanthocyanidin (Fig. 5.2). Leucoanthocyanidins can be converted either to 2R,3S-trans-flavan-3-ols, one precursor class for PAs, by the action of leucoanthocyanidin reductase (LAR) (Tanner et al., 2003), or to anthocyanidins by anthocyanidin synthase (ANS, also known as leucoanthocyanidin dioxygenase, LDOX) (Saito et al., 1999; Wilmouth et al., 2002). Subsequently, anthocyanidins can be converted to 2S,3S-cis-flavan-3-ols, the other precursor class for PAs, by the action of anthocyanidin reductase (ANR) (Xie et al., 2003) (Fig. 5.2). The only difference between the 2R,3S-trans-flavan-3-ols and the 2S,3S-cis-flavan-3-ols is the stereochemical configuration at the C2 and C3 positions, this being introduced by totally different biosynthetic mechanisms for the cis- and trans-isomers. The three structural genes LAR, ANS and ANR play crucial roles at the branch points of the PA biosynthetic pathway. LARs from the tannin-rich legume Desmodium uncinatum, and the grapevine Vitis vinifera, have been characterized via recombinant protein assay in vitro (Tanner et al., 2003; Bogs et al., 2005). Both LARs were found to convert 3Hleucocyanidin to (+)-3H-catechin. However, product yields were small, and this precluded identification of chemically authenticated (+)-catechin; rather, the assay measured incorporation of radioactivity into a compound that co-eluted with a (+)-catechin standard. Furthermore, neither catechin nor PA accumulation was found in transgenic tobacco or Arabidopsis plants constitutively expressing the Desmodium LAR gene, although LAR activity could be detected in transgenic tobacco extracts (Tanner et al., 2003). It is possible that the 2R, 3S-trans-flavan-3-ols produced via LAR contribute primarily as starter units of PAs in planta; LAR activity may be lower in vivo than ANR activity, which produces a large amount of 2S, 3S-cis-flavan-3-ols as the predominant extension units in most plants (Porter, 1993). LAR activity in vivo could also be inhibited by the substrate of ANR, anthocyanidin, or other flavonoids as part of a feedback mechanism controlling the flux into either anthocyanidins or 2R, 3S-trans-flavan-3-ols. Additional biochemical and genetic experiments are needed to better define the role of LAR (and ANR) in PA biosynthesis. ANS, a member of the 2-oxoglutarate iron-dependent dioxygenase (2-ODD) family, shares the same substrate as LAR. ANS was first cloned and characterized from Perilla frutescens (Saito et al., 1999). However, recent in-vitro enzyme studies have questioned the exact physiological roles of this enzyme, as it was shown that ANS is capable of catalyzing the conversion of 2R, 3R-trans-dihydroquercetin to quercetin, a reaction previously believed to be catalyzed only by flavonol synthase (FLS) (Turnbull et al., 2000). The mechanism of action of ANS, together with those of FLS and flavanone 3-hydroxylase (F3H), two other members of the 2-ODD family having overlapping substrate and product specificity with ANS, was studied with respect to oxygen exchange with a variety of substrates

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Fig. 5.2 Biosynthesis of anthocyanins and proanthocyanidins. Enzymes are: FLS, flavonol synthase; DFR, dihydroflavonol reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase. Other symbols next to reaction arrows indicate regulatory genes or transporters (see text for details). Mutations in Arabidopsis genes designated by TT cause a transparent testa phenotype. AHA10 encodes a proton pump.

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(Turnbull et al., 2004), and the oxidation mechanism of ANS was further calculated by quantum mechanical methods. Surprisingly, the results suggested that the oxidation of leucoanthocyanidin at the C3 position to yield anthocyanin can be regarded as a ‘side-reaction’ from the viewpoint of the quantum mechanics of the enzymatic reactions (Nakajima et al., 2006). Recombinant ANS from Gerbera hybrida showed the same FLS activity as ANS from Arabidopsis thaliana. Gerbera ANS protein is also capable of converting the product of the LAR enzyme, (+)-catechin, to a dimeric oxidized form, suggesting a role for ANS upstream of the oxidation of leucocyanidins. However, the dimerization function of Gerbera ANS was not seen with other PA monomers such as (−)-epicatechin, ent (−)-catechin or ent (+)-epicatechin, and the product from catechin was not linked in the normal manner seen in PAs (Wellmann et al., 2006). It is not clear whether the FLS and dimerization activities of ANS have functional significance in vivo, whether the proposed dimerization function of ANS toward (+)-catechin is species-specific, or whether other ANS enzymes also possess a dimerization function toward other PA monomers. Clearly, the enzymatic activity of ANS is complex and requires further study. The function of the ANR protein encoded by the BANYULS gene, a DFR-like enzyme that was previously believed to function as LAR (Devic et al., 1999), was determined by Xie et al. (2003) in a study that answered the long-standing question of the biosynthetic origin of cis-epicatechin. Ectopic expression of the Arabidopsis ANR protein in transgenic tobacco plants resulted in conversion of the colored anthocyanidin pigments found in the wild type, pink tobacco flowers into colorless epicatechins, leading to white flowers on the transgenic plants. Further kinetic studies showed that the ANRs from Medicago truncatula and Arabidopsis can convert the three anthocyanidins (delphinidin, cyanidin and pelargonidin) to the corresponding 2,3-cis-flavan-3-ols with different relative substrate preferences (Xie et al., 2004) (Fig. 5.2). Although the suggested roles of LAR, ANS and ANR in PA biosynthesis are supported by in-vitro data and, in the case of ANS and ANR, they are also supported by genetic evidence, questions still remain as to how flux is controlled at the interface between the three reactions, and whether control is exerted through metabolic channeling between the three enzymes (Winkel, 2004). PA monomers are believed to be sequestered into vacuoles by a member of the protoncoupled multi-drug and toxic compound extrusion (MATE) family of secondary transporterlike proteins, encoded in Arabidopsis by TT12, the mutation in which results in a transparent testa phenotype caused by reduction of PA levels in the seed coat (Debeaujon et al., 2001). In addition to TT12, a member of the glutathione S-transferase (GST) multigene family, encoded by TT19, is also involved in accumulation of PAs. An ortholog of GST from petunia, AN9, can complement the tt19 mutation and allow the vacuolar uptake of anthocyanidin, but not the restoration of PA accumulation (Kitamura et al., 2004). Recently, a proton pumping ATPase (isoform 10, AHA10) was reported to be required for the accumulation of PAs in Arabidopsis, and is possibly involved in vesicular trafficking, although it is predicted to be localized to the plasma membrane (Baxter et al., 2005). Although the discovery of TT12, TT19 and AHA10 has shed some new light on our understanding of PA monomer transport, the exact biochemical functions and possible functional relationships of these proteins are still unclear.

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In the early stage of seed development, PA monomers first accumulate in vacuoles, and then migrate to the cell wall in the senescing testa, until they are no longer visible in the vacuole (Kitamura et al., 2004). The PAs then become oxidized through the action of the protein encoded by TT10, a laccase that can oxidize epicatechin oligomer units into the corresponding quinones, leading to the formation of the brown material characteristic of the mature Arabidopsis seed coat (Pourcel et al., 2005). The actual mechanism of initial oligomerization of PA monomers remains unclear. We have speculated that epicatechin or catechin may be converted to the corresponding o-quinones by a polyphenol oxidase enzyme. The quinones would then be converted to carbocations through coupled non-enzymatic oxidation. Nucleophilic attack on the carbocations by epicatechin or catechin would produce dimers and then oligomeric PAs linked through C4-C8 or C4-C6 (Dixon et al., 2005). This is only one of many potential mechanisms that have been proposed, none of which has direct experimental support. Hopefully these outstanding questions can be resolved by combined biochemical and genetic approaches.

5.5.2 Regulatory genes for PA biosynthesis Expression of the individual structural genes of the PA pathway in Arabidopsis is controlled by several regulatory genes functioning in seed-coat development after fertilization. To date, more than 25 Myb, Myc and bHLH type transcription factors have been characterized as involved in the anthocyanidin/proanthocyanidin biosynthetic pathway in different plant species (Broun, 2004, 2005), and those from Arabidopsis can be grouped into three classes based on the gene(s) they regulate and their developmental functions. The sites of actions of these genes are indicated in Fig. 5.2. The first group of regulatory genes controls the general flavonoid pathway to anthocyanidin; this group includes the maize R and C1 genes and their orthologs from other plant species. R gene orthologs from Arabidopsis, GLABRA3 (GL3) and MYC-146/ENHANCER OF GLABRA3 (EGL3), encode basic helix-loop-helix (bHLH) proteins that can complement the biosynthesis of anthocyanins in petals of a white-flowered mutant of Matthiola incana following transient expression (Ramsay et al., 2003). The over-expression of the C1 Myb gene orthologs PAP1 and PAP2 from Arabidopsis results in constitutive anthocyanin production in Arabidopsis and tobacco, but PA production was not reported (Borevitz et al., 2000). Recently, expression of a chimeric PAP1 repressor in Arabidopsis was shown to suppress the expression of ANS, ANR and two early flavonoid pathway genes, CHS and DFR, indicating that PAP1 has the potential ability to regulate the biosynthesis of PAs in addition to anthocyanidin (Matsui et al., 2004). In another study, over-expression of PAP1 in Arabidopsis activated most of the genes in the anthocyanidin pathway, including ANS, but there was no reported induction of ANR (Tohge et al., 2005). In addition to R (MYC/bHLH) and C1 (MYB) orthologs, TTG1, a WD domain protein known to interact physically with MYB (PAP1 and PAP2) and MYC (GL3 and EGL3) in a yeast two-hybrid system, is also required for anthocyanin biosynthesis (Payne et al., 2000; Zhang et al., 2003). The second class of regulatory genes includes TT2, TT8 and TTG1, which modulate the late flavonoid biosynthetic genes DFR and ANS as well as ANR and TT12. TT2 encodes an R2–R3 MYB protein; its mutation affects the expression of DFR, ANS and ANR in developing siliques, and its ectopic expression can induce ANR and TT8 expression and

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also up-regulate DFR and ANS (Nesi et al., 2001). TT2 over-expression in Arabidopsis under the control of the double 35S promoter induces expression of AHA10 as well as TT12 and ANR (Sharma & Dixon, 2006). TT2 therefore appears to be a critical regulator in the PA biosynthetic pathway. The TT8 gene encodes a basic helix-loop-helix domain protein, which is required for expression of DFR and ANR in Arabidopsis siliques (Nesi et al., 2000). Yeast two-hybrid and three-hybrid assays indicated that TT8 interacts with TT2 as a Myb/HLH pair, which then physically interacts with the WD repeat domain protein TTG1 in a ternary complex to activate ANR expression (Walker et al., 1999; Baudry et al., 2004). The third group of transcription factors regulate organ and cell development for PA deposition, in addition to regulating the transcription of PA pathway-specific genes; this group includes TT1, TT16 and TTG2. TT1 is a WIP domain-containing zinc finger protein that is necessary for PA biosynthesis as it is involved in endothelial cell development in the seed coat (Sagasser et al., 2002). TT16 encodes an ARABIDOPSISBSISTER (ABS) MADS domain protein, which is also necessary for ANR expression and PA accumulation in a manner similar to that controlled by TT1 in the endothelial cells of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggest that TT16 is also involved in the specification of endothelial cells (Nesi et al., 2002). TTG2 modulates trichome differentiation, mucilage production and PA biosynthesis; it is a zinc finger-like protein of the plant-specific WRKY family, and requires TTG1 function for regulating PA biosynthesis (Johnson et al., 2002). Although the genetic regulation of the anthocyanidin and PA biosynthetic pathways in Arabidopsis now seems clear, the network in other plants may differ from, or be even more complex than, this model. For example, the Rc gene, encoding an HLH family transcription factor, was recently reported to be required for the red pericarp phenotype in rice and proposed to be a positive regulator of PAs. However, Rc is orthologous with INTENSIFIER1 from maize, which is a negative regulator of anthocyanidin biosynthesis (Sweeney et al., 2006).

5.6 Metabolic engineering of PAs in plants The metabolic engineering of PAs has been largely focused on forage plants, with the aim of developing high-quality, bloat-safe cultivars with increased ruminal bypass protein. By binding to protein and reducing its rate of degradation in the rumen, PAs can protect ruminant animals from potentially lethal pasture bloat. The problem is that the leaves and stems of protein-rich forages such as alfalfa are essentially devoid of PAs and anthocyanins (alfalfa leaves accumulate flavone glycosides), so the whole pathway has to be introduced. One strategy is to transform plants with the transcription factors that together control the whole pathway to anthocyanins and on to PAs. Introduction of the maize Lc anthocyanin regulatory gene into alfalfa led to a visible phenotype of red-purple aerial tissue, but accumulation of polymeric anthocyanidin or PA material was only obtained under conditions of high light intensity and low temperature stress (Ray et al., 2003). Transgenic alfalfa expressing the maize anthocyanin pathway transcription factors B-Peru or C1 did not produce a red-purple phenotype or PA-like materials even under stress condition (Ray

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et al., 2003). In another study, three transcription factors, TT2, PAP1 and Lc, were introduced into Arabidopsis alone or in combination. Expression of TT2 alone resulted in the ectopic expression of ANR, AHA10 and TT12, and when coupled with the expression of PAP1, accumulation of PAs was found, but only in a subset of cells. However, the simultaneous high-level expression of the three transcription factors led to the accumulation of PA in young leaves and cotyledons, but this was accompanied by necrosis and death of the transgenic lines, possibly as a result of the biosynthesis of cytotoxic compounds or cellular dysregulation (Sharma & Dixon, 2006). Another approach is to express PA-specific structural genes in transgenic plants. Expression of the M. truncatula ANR gene led to a low level of PA accumulation in tobacco flowers, but not in the vegetative tissue (Xie et al., 2003). However, when MtANR was over-expressed in M. truncatula itself, anthocyanidin pigmentation in the red spots that occur naturally on the leaves was decreased by up to 50%, and PA oligomers of mainly two specific size classes were detected (Xie et al., 2006). The third strategy is to introduce both regulatory genes of the PA/anthocyanidin pathway and additional PA-specific genes into plants. In a successful example of this approach, Arabidopsis PAP1 and an ANR gene were co-expressed in transgenic tobacco. The transgenic plants accumulated epicatechin and gallocatechin monomers and a series of dimers and oligomers as detected by TLC analysis using dimethylaminocinnamaldehyde (DMACA) as stain (Xie et al., 2006).

5.7 Glycosyltransferases for modification of phenylpropanoid compounds – in-vitro biochemistry and in-vivo function The structural diversity of plant secondary metabolites is obtained from relatively simple reactions that modify a set of scaffold molecules. Among the modification reactions, glycosylation plays a particularly important role in plants, contributing to the biosynthesis and storage of secondary metabolites (Bowles et al., 2005), to the regulation of hormone homeostasis (Lim & Bowles, 2004), to the detoxification of xenobiotics (Hefner et al., 2002; Lim et al., 2003; Messner et al., 2003), to the enhancement of molecules’ solubility (Kaminaga et al., 2003; Kaminaga et al., 2004), and to plant self-defense (Price et al., 1987; Matros & Mock, 2004; Langlois-Meurinne et al., 2005). Small molecule uridine diphosphate sugar glycosyltransferases (UGTs) catalyze the transfer of activated sugar residues to an acceptor. These proteins comprise a superfamily of enzymes ubiquitous in living organisms (http://afmb.cnrs-mrs.fr/CAZY/fam/acc_GT. html), the magnitude of which in the plant kingdom is likely comparable to that of the O-methyltransferase and cytochrome P450 enzymes (Ibrahim, 1997; Chapple, 1998). The number of UGTs and glycosyl hydrolases in Arabidopsis thaliana is much higher than in humans (Coutinho et al., 2003). Phenylpropanoid compounds, particularly flavonoids, are among the most studied plant glycosides (Harborne & Williams, 2000). The activities of flavonoids affecting plant and human health can be modified (enhanced or reduced) by glycosylation. Thus, understanding the molecular basis for the specificity of plant UGTs is important for developing metabolic engineering strategies for the production and cellular storage of bioactive polyphenols.

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5.7.1 UGTs active with non-flavonoid phenolic compounds Figure 5.3 presents the structures of some cinnamyl alcohols, aldehydes, and acids that serve as reported substrates for plant UGTs. The first report of a coniferyl alcohol glycosyltransferase (CAGT) described an enzyme from cell cultures of Paul’s scarlet rose (Ibrahim & Grisebach, 1976). This CAGT catalyzed the transfer of glucose from UDP-glucose to coniferyl alcohol and exhibited the highest substrate specificity yet reported for a CAGT (Km 3.3 nM for coniferyl alcohol and 20 μM for UDP-glucose). A CAGT from Picea abies was shown to be feedback inhibited when the levels of coniferyl aldehyde 4-O-glucoside exceeded 10 mmol/g tissue (Schmid & Grisebach, 1982). A UDP-glucose: CAGT capable of 4-O-glycosylation of coniferyl and sinapyl alcohols and their corresponding aldehydes was isolated from Pinus strobus (Steeves et al., 2001). Km values for coniferyl alcohol and sinapyl alcohol were 120 and 154 μM, respectively, with higher values for coniferyl aldehyde (317 μM) and sinapyl aldehyde (242 μM). P. strobus CAGT also catalyzed the 4-O-glycosylation of dihydroconiferyl alcohol. Production of dihydroconiferyl alcohol 4-O-glucoside has been demonstrated in other Pinus species (Higuchi et al., 1977), and dihydroconiferyl alcohol was suggested to be a constituent of lignin in a mutant loblolly pine (Ralph et al., 1997). UGTs from Arabidopsis thaliana that conjugate lignin monomers and related metabolites were recently reported (Lim et al., 2005). UGT72E1 is specific for coniferyl and sinapyl aldehydes, while UGT72E2 also recognizes the corresponding alcohol derivatives and other phenylpropanoid compounds such as ferulic and sinapic acids. Although UGT72E1 only glycosylates the aldehyde derivatives, UGT72E2 exhibits much higher specificity toward these compounds. The Kcat/Km ratio for UGT72E2 toward coniferyl and sinapyl aldehydes was 69 and 53 mM−1 s−1, respectively, whereas the corresponding values for UGT72E1 were 3.1 and 4.5. The Km values for UGT72E2 with coniferyl and sinapyl aldehydes (20 μM)

Fig. 5.3 Structures of some phenylpropanoid alcohol, aldehyde, and acid derivatives that are substrates for plant UGTs.

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were much lower than those for UGT72E1, which were in the range of 270–460 μM. However, both enzymes showed similar turnover with the aldehyde derivatives (Kcat values < 1.5 s−1) (Lim et al., 2005). Low Km values (6–15 μM) were observed for coniferyl and sinapyl alcohols in reactions catalyzed by UGT72E2, with a turnover around 4-fold slower than that for the corresponding aldehydes. In planta, monolignols are mainly used for lignin biosynthesis, and their corresponding aldehydes may also be precursors for the synthesis of ferulic and sinapic acids (Nair et al., 2004). Thus, glycosylation of coniferyl and sinapyl aldehydes may cause an impact on lignin and cell-wall phenolic biosynthesis. The generation and analysis of transgenic plants down- or up-regulated for the genes encoding CAGTs will provide a better understanding of the roles of these enzymes in lignin biosynthesis and/or production of other phenylpropanoid compounds. Salicylic acid (SA) is an important component of plant signaling pathways during interactions with microbes. Inoculation of tobacco leaves with incompatible pathogens triggers the synthesis of SA that is subsequently converted to the 2-O-glucoside and other glucosyl SA derivatives (Lee & Raskin, 1999). A UDP-glucose: salicylate glucosyltransferase was isolated from inoculated tobacco tissues (Lee & Raskin, 1999). The gene encoding this enzyme was also cloned and expressed in E. coli. Both plant-purified and recombinant enzymes displayed activity toward several phenolic acids (Fig. 5.4, right), but not toward the plant hormones indole-3-acetic acid (IAA), gibberellic acid (GA3) or zeatin. Km values for SA and UDP-glucose were 270 and more than 1,000 μM, respectively. A high Km value for SA was seen as acceptable since this UGT is inducible during plant–pathogen interactions when the levels of SA increase dramatically (Yalpani et al., 1993). The enzyme was also active with 3-hydroxybenzoic and 4-hydroxybenzoic acids, catalyzing glucosylation of hydroxyl, but not the carboxyl group (Lee & Raskin, 1999). Activity for phenolic acids was up to 7-fold higher than that exhibited for SA. However, the Km values for these compounds were not determined. SA rapidly induced UDP-glucose: salicylate glucosyltransferase mRNA, and the correlation between the increase in the levels of mRNA and enzyme activity suggests that induction of UGT activity occurs as a direct consequence of de novo enzyme synthesis.

Fig. 5.4 Structures of some coumarins and substituted benzoic acids that are substrates for pathogeninduced UGTs.

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A coumarin UGT exhibiting 7-O-glycosylation activity toward o-dihydroxy coumarins was partially purified from non-inoculated tobacco cells (Ibrahim & Boulay, 1980) (Fig. 5.4). Daphnetin and esculetin were the best substrates, with Km values of 95 and 111 μM, respectively, while the Km for UDP-glucose was 50 μM. Other tobacco coumarin UGTs identified in cell cultures or plants are associated with pathogen defense (Taguchi et al., 2001a). Salicylate-induced NtGT1a and NtGT1b glycosylated 3-hydroxycoumarin, esculetin, 4methylumbelliferone, umbelliferone, and scopoletin in vitro. Scopoletin 7-O-glucoside was the most abundant phenolic compound in tobacco cell cultures (Taguchi et al., 2001b). NtGT1a exhibited Km values of 1,400 and 77 μM for scopoletin and 4-methylumbelliferone respectively, although it was argued that scopoletin might nevertheless be an in-vitro substrate in view of its high concentration in tobacco cell protoplasts. The Km for UDPglucose was low (37 μM). TOGT is another tobacco UGT induced by SA during pathogen interactions, and transgenic plants over-expressing TOGT showed enhanced resistance against potato virus Y. The activity of TOGT in the leaves and roots of those plants was several-fold increased, with subsequent accumulation of large amounts of coumarin glucosides (Matros & Mock, 2004). TOGT exhibited high activity toward esculetin and scopoletin (Fraissinet-Tachet et al., 1998). Reduction of the capacity to generate scopoletin 7-O-glucoside by altering TOGT activity through antisense transgene expression reduced the resistance of tobacco against viruses (Chong et al., 2002). p-Coumaric acid, an intermediate of the central phenylpropanoid pathway, is usually produced by plants as the trans-isomer (Fig. 5.5). However, cell cultures and axenicallycultured Sphagnum fallax plants synthesize cis-p-coumaric acid (Rasmussen et al., 1996; Rasmussen & Rudolph, 1997). A UGT with pronounced specificity for cis-p-coumaric acid was purified from S. fallax (Rasmussen & Rudolph, 1997). Km values for UDP-glucose and cis-p-coumaric acid were 110 and 44 μM, respectively. Glycosylation occurred solely on the hydroxyl group, and trans-p-coumaric acid was not a substrate. In fact, the only

Fig. 5.5 Structures of some cinnamic acids and a quinone that are substrates for plant UGTs.

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other phenolic substrate glycosylated by this enzyme was cis-caffeic acid (Fig. 5.5), although the activity was 10% of that exhibited for cis-p-coumaric acid. Caffeic acid (Fig. 5.5), a phenylpropanoid precursor of lignin (Whetten et al., 1998), also contributes to the synthesis of a large number of other secondary metabolites. The first UGT (UGT71C1) specific for generating caffeoyl 3-O-glucoside was recently reported from Arabidopsis thaliana (Lim et al., 2003). Among the polyphenols tested, caffeic acid gave the lowest Km value (320 μM), followed by m-coumaric acid (690 μM), and o-coumaric acid (800 μM). UGT71C1 displayed high specificity for caffeic acid as attested by the highest Kcat/Km ratio (0.91 mM−1s−1). The enzyme was inactive with ferulic and sinapic acids, in which the -OH group(s) at C3 (former) and C3 and C5 (latter) are methylated, and with p-coumaric acid. The regioselectivity of UGT71C1 was maintained in A. thaliana overexpressing UGT71C1 (Lim et al., 2003). It was suggested that UGT71C1 may participate in the detoxification of excess levels of caffeic acid (Lim et al., 2003). Arbutin synthase, a multifunctional UGT capable of transforming many natural products and xenobiotics, is widely distributed among plants of the families Ericaceae, Rosaceae, and Saxifragaceae. Dihydroquinone (Fig. 5.5) is its natural substrate and is converted to a monoglucoside derivative (Hefner et al., 2002). Recombinant arbutin synthase from Rauvolfia serpentine was expressed in E. coli and shown to glycosylate 45 out of 74 natural and synthetic phenols tested (Hefner et al., 2002). However, none of the recognized substrates exhibited conversion rates comparable to that of dihydroquinone (Km < 1 μM). UDP-glucose was the best sugar donor for the R. serpentine arbutin synthase (Km 77 μM), while the Km for TDP-glucose was 1.1 mM. The broad spectrum of arbutin synthase activity may have physiological relevance for the detoxification of harmful phenolics produced in high amounts. CaUGT2 from Catharanthus roseus cell culture glycosylates both curcumin aglycone and curcumin monoglucoside (Fig. 5.6). CaUGT2 gene expression was up-regulated when a C. roseus cell culture was treated with methyl jasmonate (Kaminaga et al., 2004). The reactions catalyzed by this enzyme were strictly dependent on UDP-glucose. Diglucoside derivative production occurred in two steps, by which a monoglucoside was first produced followed by the glycosylation of the second hydroxyl group. [6]-Gingerol and capsaicin

Fig. 5.6 Structures of curcumin and structurally-related phenolics tested as substrates for CaUGT2.

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(Fig. 5.6), two compounds structurally related to curcumin, were not substrates for CaUGT2, suggesting that the conjugated double bonds in the curcumin structure are crucial for the enzyme activity. CaUGT2 can use both UDP-glucose and UDP-galactose, but the enzyme is 24-fold more specific for the former compound (Kaminaga et al., 2004). Curcumin was the best substrate for CaUGT2 (Km 19 μM) with higher specificity (1.7-fold) when compared with curcumin monoglucoside (Km 63 μM). Other phenolic compounds were also recognized by CaUGT2. However, the Km values were greater than 660 μM. It is likely that CaUGT2 is defense-related, acting in the biosynthesis of antimicrobial compounds.

5.7.2 Glycosylation of flavonoid compounds Apple skins accumulate high levels of flavonoid 3-O-glycosides, particularly flavonol and anthocyanin galactosides (Lister et al., 1994, 1997). Consistent with this observation, UDP-galactose was the preferred sugar donor for apple skin glycosyltransferase (UFGT), although some activity was observed with UDP-glucose and UDP-xylose (Lister et al., 1997). UFGT accepted anthocyanins and flavonols, but not flavanones, flavones or dihydroflavonols (Lister et al., 1997), consistent with the lack of these latter compounds in apple. The anthocyanidin cyanidin was the best substrate (217% of the activity with quercetin) followed by the flavonols myricetin (124%), kaempferol (114%), quercetin (100%), and 3′-methoxy quercetin (93%), and the anthocyanidins delphinidin (92%) and pelargonidin (69%) (Fig. 5.7). Malvidin (3′,5′-dimethoxy delphinidin) was, however, a poor substrate. UFGT was highly regiospecific, and produced only 3-O-glycoderivatives. A UDP-glucuronic acid: anthocyanin glycosyltransferase (BpUGAT) was recently identified in flowers of the red daisy (Bellis perennis) (Sawada et al., 2005). BpUGAT was regioselective, catalyzing the transfer of a glucuronosyl unit to the 2″-hydroxyl group of glucosides of cyanidin and delphinidin, but not of pelargonidin. The enzyme recognized both cyanidin 3-O-glucoside and cyanidin 3-O-6″-O-malonylglucoside (Sawada et al., 2005). Km values of 19 and 476 μM were recorded for cyanidin 3-O-6″-O-malonylglucoside and UDP-glucuronic acid, respectively. Native BpUGAT was highly specific for UDPglucuronic acid as sugar donor. Real time quantitative RT-PCR demonstrated that BpUGAT transcripts accumulated in red petals consistent with the temporal and spatial distribution of enzyme activity and a role for BpUGAT in pigment biosynthesis (Sawada et al., 2005). The roots of chickpea (Cicer arietinum) contain a UGT that catalyzes the transfer of glucose to 4′-methoxy isoflavones (Koster & Barz, 1981), the best substrates being biochanin A and formononetin (Fig. 5.7). The corresponding 4′-hydroxy isoflavones genistein and daidzein (Fig. 5.7) were poor substrates, indicating that an –OCH3 group at C4′ is a requirement for enzyme activity. Biochanin A and formononetin were converted to the corresponding 7-O-glucoderivatives with Km values of 12 and 24 μM, respectively. Isoflavanones, flavones, flavanones, flavonols, coumarins, cinnamic acids and benzoic acids were not substrates for the enzyme. Another isoflavone-specific glycosyltransferase (UGT73F1) was found in cell culture of Glycyrrhiza echinata (Nagashima et al., 2004). UGT73F1 mRNA was transiently induced in the cultures in response to yeast extract. The enzyme was highly specific for formononetin and daidzein, leading to the production of the corresponding 7-O-glucosides (Nagashima et al., 2004).

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Fig. 5.7 Structures of some flavonoid compounds that act as substrates for plant UGTs.

UGT71G1, a triterpene glycosyltransferase from Medicago truncatula, efficiently glycosylated genistein and biochanin A at the 7-OH position (Achnine et al., 2005), with Km values in the range of 30–45 μM. UGT73J1 was identified in the epidermal layer of yellow onion (Allium cepa) and displayed high activity against genistein and weak activity against quercetin 3-O-glucoside (Kramer et al., 2003). Each substrate was converted into the respective 7-O-glucoside. UGTs from several different plant species have been found with activity toward flavonols. NtGT1s and NtGT3 from cell cultures of tobacco exhibit high specificity for kaempferol (Taguchi et al., 2001a, 2003). NtGT3 and NtGT1a were induced by treatment of cells with naphthol, and the latter enzyme was also induced by salicylic acid. The Km for quercetin was in the range of 3.5–13 μM for both enzymes. However, NtGT3 exhibited much higher

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specificity for quercetin than did NtGT1a (Taguchi et al., 2003). At least two UGTs from Arabidopsis thaliana were characterized as specific for flavonoids. AtGT2 is a 7-Oglycosyltransferase with high specificity for kaempferol (Km 26.3 μM, Kcat/Km 1.3 μM−1 s−1) and the flavanone eriodictyol (Km 18 μM, Kcat/Km 2.7 μM−1 s−1), although some activity was also observed for the flavones apigenin and luteolin (Kim et al., 2006b). UGT73B2 was previously classified as a UDP glucose: flavonoid 7-O-glycosyltransferase (http://afmb. cnrs-mrs.fr/CAZY/fam/GT1.html). A recent report describes the activity of UGT73B2 toward flavanones, flavones, and flavonols and, based on the relative conversion rates, this enzyme prefers substrates that yield 3-O-glucoderivatives rather than 7-O-glucoderivatives (Kim et al., 2006a). 7-O-glycosylation occurred only in cases where the substrate lacked a hydroxyl group at C3 (Kim et al., 2006a). Flavonols were the best substrates, and flavanones (eriodictyol and naringenin) were better than flavones. Levels of UGT73B2 mRNA were highest in flowers and leaves (Kim et al., 2006a). However, A. thaliana leaves produce kaempferol 3-O-rhamnoside-7-O-rhamnoside as the major flavonol, whereas flowers synthesize not only kaempferol glycoderivatives, but also quercetin 7-O-glucoside (Jones et al., 2003). These results, together with the controversial classification for UGT73B2, indicate that further studies are required to evaluate the substrate specificity of UGT73B2 in leaves and flowers of A. thaliana. The most efficient substrate for recombinant rice glycosyltransferase RUGT-5 was kaempferol, followed by apigenin, genistein and luteolin (Ko et al., 2006). Kaempferol was converted to three products: the 3-O-glucoside (major product), 7-O-glucoside, and 3′-O-glucoside. Quercetin was converted to the 7-O-glucoside as the main product, but some 4′-O-glucoside was also produced (Ko et al., 2006). Both apigenin and luteolin were converted to the corresponding 7-O-(major for apigenin) and 4′-O-(major for luteolin) glucoderivatives. Kaempferol was the best substrate of those tested (Km 239.5 μM, and highest Kcat/Km ratio). The specificity of RUGT-5 for apigenin and genistein was very similar, with genistein exhibiting the lowest Km value (121 μM). Considering the flavonoid composition in rice plants, it is reasonable to assume that kaempferol and apigenin are the in-vivo substrates for RUGT-5.

5.7.3 Problems for the functional annotation of UGTs The above examples indicate that UGTs can glycosylate a wide variety of metabolites with different regioselectivities. At one end of the specificity spectrum, Rauvolfia serpentine arbutin synthase glycosylates many compounds with different basic scaffold structures, whereas A. thaliana UGT72E1 appears specific for coniferyl and sinapyl aldehydes (Hefner et al., 2002; Lim et al., 2005). Amino acid sequence identity by itself may be misleading for the prediction of UGT function. For example, the amino acid sequence of M. truncatula UGT71G1 shares 44% similarity, and clusters phylogenetically, with UGT71C1 or UGT71C4 from A. thaliana, enzymes known to glycosylate benzoic acid derivatives, but does not display activity toward these compounds. Also, in-vitro observations may not reflect true in-vivo function. For example, quercetin was found to be the best substrate for UGT71G1 in vitro (Km 25 μM, Km/Kcat 4,800 M−1 s−1) and the triterpene hederagenin exhibited poor specificity and a Km value 6-fold higher (Achnine et al., 2005). However, no quercetin glucoderivatives were

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detected in M. truncatula cell cultures, and induction of UGT71G1 by methyl jasmonate was followed by an accumulation of triterpene glycosides (Achnine et al., 2005). Determination of the three-dimensional structures of plant UGTs is crucial for comprehending structure–activity relationships. As of the time of writing, structures are only available for UGT71G1 from M. truncatula and VvGT1 from Vitis vinifera (Shao et al., 2005; Offen et al., 2006). Small changes in amino acid sequence may have profound effects on regio-selectivity of UGTs. For example, in in-vitro assays with the flavonol quercetin as acceptor, UGT71G1 catalyzes the formation of all five potential monoglucosides (Shao et al., 2005), with the 3′-O-glucoside accounting for approximately 70% of the total activity. Reducing the size of large aromatic side-chains at one end of the substrate binding pocket altered regioselectivity for quercetin from predominantly B-ring 3′-O-glucosylation to predominantly C-ring 3-O-glucosylation. Mutation of tyrosine 202 to alanine resulted in retention of 96% of wild-type activity, with the 3-O-glucoside now accounting for 95% of the conversion and production of the 3′-O-glucoside reduced to just 1% of the wild-type value (He et al., 2006). Although the Phe148Val mutant lost 80% of the wild-type activity, it produced a single product identified as quercetin 3-O-glucoside. Residues Phe148 and Tyr202 are close to each other in the three-dimensional structure of UGT71G1, located at one end of the acceptor binding pocket (Shao et al., 2005). Reducing the sizes of the side-chains at either of these positions would increase the volume of the binding pocket around where the C-ring of quercetin is located. This binding-pocket volume change allows the 3-hydroxyl to move closer to the C1 reaction center on the UDP-glucose and be more favorably placed for glucosylation (He et al., 2006). Owing to the large number of UGTs present in plants, and their often overlapping substrate specificities, it is important to understand how UGTs manage their activities toward the same substrate, and the implications of this for plant metabolism. Studies integrating spatially and temporally resolved metabolome and transcriptome analysis will be crucial for elucidating the individual roles of UGTs in vivo, and such approaches are beginning to yield results (Rosati et al., 2003; Gachon et al., 2004; Achnine et al., 2005; Lee et al., 2005; Tohge et al., 2005). From the point of view of human health, plant UGTs are promising biocatalysts for regioselective generation of natural product glycosides that are already finding uses in medicine as glycodrugs or new lead compounds (Lim et al., 2004; Weis et al., 2006; Modolo et al., 2007).

5.8 Concerted strategies for metabolic engineering As shown in the example of engineering isoflavone (genistein) accumulation in Arabidopsis, the level of production was largely controlled by the endogenous competing pathways (Liu et al., 2002). In addition to competing pathways, substrate availability, subcellular localization of substrates and biosynthetic enzymes, the true substrate for in-vivo biosynthesis and product stability are all factors that will affect the outcome of metabolic engineering. A thorough understanding of plant metabolism and a concerted strategy are therefore essential for manipulation of polyphenol biosynthesis/accumulation.

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One outstanding example of such a strategy, involving both a scaffold-modifying enzyme and a UGT, is the generation of yellow flowers that produce aurone 6-O-glucoside as the predominant pigment (Ono et al., 2006). Aurones are a family of flavonoid pigments that account for the yellow color of some ornamental flowers, such as snapdragon (Antirrhinum majus). The enzyme aureusidin synthase (AS) converts chalcones to aureusidin (Fig. 5.7). AS had previously been identified from snapdragon, and belongs to the plant polyphenol oxidase family (Nakayama et al., 2000). However, expressing snapdragon AS in a non-aurone accumulating blue-flowered plant, Torenia hybrida, did not result in aurone production in the petals (Ono et al., 2006). Co-expression of chalcone 4′-O-glucosyltransferase (4′CGT ) and AS led to the accumulation of aureusidin 6-O-glucoside in petals, indicating that the chalcone substrate was first glycosylated at the 4′-hydroxyl group in the cytoplasm followed by transport to the vacuole, where aureusidin 6-O-glucoside was formed. Torenia hybrida naturally has blue colored flowers owing to the presence of a malvidin class of anthocyanin (Fig. 5.7). Although aureusidin 6-O-glucoside was produced in Torenia hybrida petals by co-expression of 4′CGT and AS, the yellow color was still largely masked by the blue anthocyanin. Anthocyanin biosynthesis, and hence the competition for chalcone substrates, was depleted by reducing either endogenous DFR or F3H expression through RNA interference. The production of yellow flowers was eventually achieved by co-expressing 4′CGT and AS, together with depletion of the competing anthocyanin biosynthesis (Ono et al., 2006). As has been seen from examples given elsewhere in this article, this type of concerted strategy will also be necessary for the efficient engineering of isoflavones and proanthocyanidins in plants.

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Sreevidya, V., Srinivasa Rao, C., Sullia, S., Ladha, J.K. & Reddy, P.M. (2006). Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia. Journal of Experimental Botany, 57, 1957–1969. Steele, C.L., Gijzen, M., Qutob, D. & Dixon, R.A. (1999). Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Archives of Biochemistry and Biophysics, 367, 147–150. Steeves, V., Forster, H., Pommer, U. & Savidge, R. (2001). Coniferyl alcohol metabolism in conifers – I. Glucosidic turnover of cinnamyl aldehydes by UDPG: coniferyl alcohol glucosyltransferase from pine cambium. Phytochemistry, 57, 1085–1093. Stintzing, F.C., Hoffmann, M. & Carle, R. (2006). Thermal degradation kinetics of isoflavone aglycones from soy and red clover. Molecular and Nutritional Food Research, 50, 373–377. Sweeney, M.T., Thomson, M.J., Pfeil, B.E. & McCouch, S. (2006). Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice. Plant Cell, 18, 283–294. Taguchi, G., Yazawa, T., Hayashida, N. & Okazaki, M. (2001a). Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin. European Journal of Biochemistry, 268, 4086 – 4094. Taguchi, G., Yoshizawa, K., Kodaira, R., Hayashida, N. & Okazaki, M. (2001b). Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells. Plant Science, 160, 905 – 911. Taguchi, G., Ubukata, T., Zhao, P., Yamamoto, H., Hayashida, N. & Okazaki, M. (2003). Exogenously added naphthols are accumulated as malonylglucosides in tobacco cells. Plant and Cell Physiology, 44, S192. Tanner, G.J., Francki, K.T., Abrahams, S., Watson, J.M., Larkin, P.J. & Ashton, A.R. (2003). Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. Journal of Biological Chemistry, 278, 31647–31656. Tian, L. & Dixon, R.A. (2006). Engineering isoflavone metabolism with an artificial bifunctional enzyme. Planta, 224, 496 – 507. Tohge, T., Nishiyama, Y., Hirai, M.Y. et al. (2005). Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant Journal, 42, 218 –235. Turnbull, J.J., Nakajima, J., Welford, R.W.D., Yamazaki, M., Saito, K. & Schofield, C.J. (2004). Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis. Journal of Biological Chemistry, 279, 1206 –1216. Turnbull, J.J., Sobey, W.J., Aplin, R.T. et al. (2000). Are anthocyanidins the immediate products of anthocyanidin synthase? Chemical Communciations, 24, 2473–2474. Walker, A.R., Davison, P.A., Bolognesi-Winfield, A.C. et al. (1999). The TRANSPARENT TESTA GLABRA 1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell, 11, 1337–1350. Weis, M., Lim, E.K., Bruce, N. & Bowles, D. (2006). Regioselective glucosylation of aromatic compounds: Screening of a recombinant glycosyltransferase library to identify biocatalysts. Angewandte Chemie International Edition (English), 45, 3534–3538. Wellmann, F., Griesser, M., Schwab, W. et al. (2006). Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin. FEBS Letters, 580, 1642–1648. Whetten, R.W., MacKay, J.J. & Sederoff, R.R. (1998). Recent advances in understanding lignin biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology, 49, 585–609. Wilmouth, R.C., Turnbull, J.J., Welford, R.W., Clifton, I.J., Prescott, A.G. & Schofield, C.J. (2002). Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure, 10, 93 –103. Winkel, B.S.J. (2004). Metabolic channeling in plants. Annual Review of Plant Biology, 55, 85–107. Winkel-Shirley, B. (2001). Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 126, 485–493.

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5.10 Abbreviation list of the pathway genes ANR, anthocyanidin reductase or BANYULS ANS, anthocyanidin synthase (LDOX leucoanthocyanidin dioxygenase) AS, aureusidin synthase 4′CGT, chalcone 4′-O-glucosyltransferase CHI, chalcone isomerase CHS, chalcone synthase DFR, dihydroflavonol reductase F3H, flavonoid-3-hydroxylase FLS, flavonol synthase GST, glutathione-S-transferase LAR, leucoanthocyanidin reductase tt, transparent testa UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase.

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Chapter 6

Recent Advances in the Molecular Biology and Metabolic Engineering of Flavonoid Biosynthesis in Ornamental Plants Kevin M. Davies, Huaibi Zhang and Kathy E. Schwinn

6.1 Introduction The flavonoid biosynthesis pathway is one of the most extensively studied of all plant metabolite pathways. From some of the earliest scientific studies onwards, angiosperms have been important model systems for understanding the pathway, principally owing to the availability of flower-color mutants. Flavonoids are key flower pigments and flower color provides an excellent visual reporter system, allowing changes in pigmentation to be linked to changes in enzyme and gene activity. The control of flavonoid synthesis, particularly that of anthocyanins, has provided one of the main models for understanding the combinatorial transcriptional control of gene activity in plants. Furthermore, the range of functions that flavonoids have in plants, along with their aesthetic value and an emerging awareness of their beneficial activities in humans and animals, is driving efforts to modify their production in commercially important plants. As a result there is an abundance of scientific literature addressing the molecular biology of the flavonoid pathway. In this chapter we have chosen to focus on aspects of flavonoid biosynthesis for which there has been significant progress in recent years. These are the genetic modification of the pathway in ornamentals (with an emphasis on blue and yellow flower colors); the formation of ‘Anthocyanic Vacuolar Inclusions’; and the regulation of anthocyanin production in flowers. We include a brief summary of the flavonoid biosynthetic pathway, introducing the key enzymes, but more extensive recent reviews can be found in Davies & Schwinn (2006) and Grotewold (2006). There are also reviews focused on specific enzyme types involved in the pathway such as acyltransferases (Nakayama et al., 2003), cytochrome P450-dependent enzymes (Tanaka, 2006), glycosyltransferases (Vogt, 2000; Vogt & Jones, 2000), flavone synthases (Martens & Mithofer, 2005), flavonoid dioxygenases (Gebhardt et al., 2005) and methyltransferases (Ibrahim & Muzac, 2000). Moreover, interesting reviews that illustrate the importance of ornamentals as model species are those on antirrhinum (Antirrhinum majus; Schwarz-Sommer et al., 2003), gerbera (Gerbera hybrida; Teemu et al., 2006) and petunia (Petunia hybrida; Gerats & Vandenbussche, 2005). Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Anthocyanins are the most important pigments formed in the flavonoid pathway, being responsible for the great majority of flower colors in the range from red to blue. Flavonoids of the flavone and flavonol type are widespread ‘colorless’ floral compounds. However, when they are produced in conjunction with anthocyanins in a flower, they help in the development and stability of the flower color. The yellow flavonoids, chalcones and aurones are important pigments in a limited range of species. The biosynthetic pathway to all these flavonoid types is well defined at the genetic and enzymatic level. DNA sequences are available for all the enzymes catalyzing the biosynthetic steps to the base compounds and also for many of the secondary modification activities. A diagrammatic representation of the core pathway is presented in Fig. 6.1. Starting from the hydroxycinnamic acid-CoA ester and malonyl-CoA precursors, three biosynthetic steps are common to the flavonol and anthocyanin pathways. These are catalyzed by chalcone synthase (CHS), chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H), which act sequentially and lead to the formation of the dihydroflavonols (DHFs). The flavonols are formed from DHFs by the action of flavonol synthase (FLS), and are glycosylated by flavonoid glycosyltransferases (GTs), frequently at the 3-hydroxyl. The formation of the simplest common anthocyanin is via the actions of dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and anthocyanidin 3-O-glycosyltransferase (A3GT). The biosynthetic scheme commonly shows the conversion proceeding through an anthocyanidin intermediate. However, in vivo a more complex reaction pathway may be followed involving a glycosylated pseudobase intermediate (Nakajima et al., 2006). It is also worth noting that assays of recombinant enzymes have revealed that many of the flavonoid enzymes carry out a variety of different reactions in addition to the in-vivo ones defined to date (e.g. Turnbull et al., 2004; Wellmann et al., 2006). Flavones are formed from flavanones by the action of flavone synthase (FNS). The formation of chalcones and aurones is discussed in detail later in this review. As can be seen in Fig. 6.1, the common flavonoids associated with pigmentation in ornamentals have a 15-carbon (C15) structure comprised of two aromatic rings (the A and B rings) joined by a third ring (the C-ring). The degree of oxidation of the C-ring defines the various flavonoid types. The degree of B-ring hydroxylation in anthocyanins is of prime importance in flower color. It has a major effect on the color of anthocyanin pigment, with increasing hydroxylation causing a shift towards the blue end of the spectrum. The 4′-hydroxyl group is incorporated in the flavonoid precursor biosynthetic pathway, by cinnamate 4-hydroxylase (C4H). Hydroxyl groups present at the C-3′ and C-5′ positions of the B-ring are most commonly added to flavonoid precursors of the anthocyanins, through the action of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′,5′H). Over 500 different anthocyanins have been identified to date, and about 90% are 3-hydroxyanthocyanins with 5,7-hydroxylation of the A-ring (Andersen & Jordheim, 2006). This hydroxylation pattern is common to the six anthocyanidins that are the basis of most anthocyanins. These anthocyanidins are pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin, which vary in the hydroxylation and the subsequent methylation of the B-ring (Fig. 6.1). A few species primarily have anthocyanins that lack the hydroxyl group at the C-3 position (3-deoxyanthocyanins) or have an additional hydroxyl group at the C-6 position of the A-ring (6-hydroxyanthocyanins).

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Fig. 6.1 A section of the general phenylpropanoid and flavonoid biosynthetic pathways leading to the anthocyanins and other flavonoids found in flowers. For ease of presentation, generally only the route for flavonoids with 4′-hydroxylation of the B-ring is shown and the simplified biosynthetic scheme from leucoanthocyanidins to anthocyanins is used. Enzyme abbreviations are defined in the text except for PAL (phenylalanine ammonia lyase), 4CL (4-coumaroyl CoA:ligase), ACC (acetyl-CoA carboxylase) and FNR (flavanone 4-reductase). The box shows the structures of the other common anthocyanidin types besides pelargonidin. The structure for malvidin includes the numbering of some of the carbons that is used for the anthocyanidins and anthocyanins (the numbering for chalcones and aurones differs from this).

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6.2 Metabolic engineering of flavonoid production in flowers As has been demonstrated in a range of species, several approaches for engineering flavonoid biosynthesis in ornamentals are now well established. The most successful have been: the inhibition of flavonoid biosynthesis, the introduction of anthocyanin regulatory genes, and the introduction of biosynthetic activities that are novel to the target species. We discuss examples of these briefly followed by discussions in greater detail of some of the recent advances in gene technology for engineering yellow and blue flower colors. Reducing flavonoid or anthocyanin biosynthesis by blocking the activity of a single biosynthetic gene has now been shown to work in several species, including carnation (Dianthus caryophyllus; Zuker et al., 2002), chrysanthemum (Courtney-Gutterson et al., 1994), cyclamen (Cyclamen persicum; Tanaka et al., 2005), gerbera (Elomaa et al., 1993), lisianthus (Eustoma grandiflorum; Deroles et al., 1998), petunia (van der Krol et al., 1988, 1990; Napoli et al., 1990), rose (Rosa hybrida; Gutterson, 1995), Nicotiana (Nishihara et al., 2005) and torenia (Torenia fournieri or T. hybrida; Aida et al., 2000a, 2000b; Suzuki et al., 2000; Fukusaki et al., 2004). A comprehensive list can be found in Davies & Schwinn (2006) and To & Wang (2006). Reducing anthocyanin biosynthesis could be desirable in order to develop cultivars with white or pale-colored flowers or flowers in which the carotenoid pigments are seen more clearly. A side-effect of this approach in some species has been novel floral pigmentation patterns (from ordered to chaotic) in transgenic lines over-expressing sense or antisense RNA of an endogenous anthocyanin biosynthetic gene. For a spectacular example of a chaotic pattern achieved in a transgenic ornamental (lisianthus) see Deroles et al. (1998). This effect appears to be limited to species that naturally display pigmentation patterns. In this regard, it is interesting to note that the ‘Red Star’ flower color pattern of non-transgenic petunia has been shown to be due to sequence-specific degradation of CHS RNA through short interfering RNAs (Koseki et al., 2005). Another application of gene inhibition technology is to redirect substrate within the flavonoid pathway to increase production of the desired compounds. There are surprisingly few examples of this approach for ornamental species, but those published include inhibiting production of colorless flavonols to increase anthocyanin production (Holton et al., 1993; Davies et al., 2003), and the inhibition of the F3′H and/or F3′,5′H to generate transgenic lines with increased levels of cyanidin- or pelargonidin-based anthocyanins (Shimada et al., 2001; Tsuda et al., 2004; Ueyama et al., 2006). There may be additional biosynthetic blocks that need to be overcome when using this approach for conferring the capacity for pelargonidin biosynthesis. For example, Ueyama et al. (2006) used antisense RNA to inhibit F3′,5′H in a purple-flowered cultivar of Nierembergia in an attempt to promote pelargonidin biosynthesis. Although the transgenic plants had reduced levels of delphinidin-based anthocyanins, kaempferol-based flavonols rather than cyanidin- or pelargonidin-based anthocyanins accumulated, suggesting that the endogenous DFR has low activity with DHK. Tsuda et al. (2004) showed for petunia that a combination of transgenes was effective for developing cultivars with novel flower colors, one to inhibit F3′H activity and one to introduce a DFR that efficiently uses DHK. Anthocyanin regulatory gene technology has proved effective in increasing pigmentation in the foliage of several species, and for increasing petal anthocyanin levels in species

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with very pale or white flowers. A variety of different regulatory genes have been used, although the majority of experiments have used cDNAs for transcription factors from maize (Zea mays). Only a few of the studies have used ornamental species, specifically, Caladium bicolor (Li et al., 2005), chrysanthemum (Boase et al., 1998), petunia (Bradley et al., 1998) and lisianthus and Pelargonium (Bradley et al., 1999). Novel phenotypes were observed only in C. bicolor and petunia, in both cases due to over-expressing a transgene for the LC transcription factor from maize. The transgenic petunia showed deep purple foliage and an associated up-regulation of at least eight anthocyanin biosynthetic genes, however the phenotype was unstable in varying glasshouse and field conditions (Boase et al., 2006). Transgenic C. bicolor plants had enhanced anthocyanin accumulation in the roots, leaves and stems, including the vascular bundles. The reviews of Schijlen et al., (2004) and Davies & Schwinn (2006) contain more extensive listings of the biotechnological applications of anthocyanin regulatory genes. Also, the role of transcription factor genes in regulating anthocyanin biosynthesis in flowers is discussed in more detail in Section 6.4 of this review. There are surprisingly few examples of generating novel flower colors using biosynthetic genes to introduce enzyme activities that are lacking in the target species. The first example published was for the promotion of pelargonidin biosynthesis in petunia by expressing a DFR that efficiently uses DHK in a line lacking F3′H and F3′,5′H activities (Meyer et al., 1987), and this has since been replicated using RNAi against the F3′H (as discussed previously). Another approach has been the introduction of delphinidin synthesis, which has led to the first (and, as yet, only) commercial success of an engineered ornamental. The approach is based around introducing a F3′,5′H transgene, but can also involve the use of mutant backgrounds or additional transgenes to promote the accumulation of delphinidin-based anthocyanins over those based on cyanidin or pelargonidin (Tanaka et al., 2005; Tanaka, 2006). A range of carnation cultivars with mauve to deep purple flower colors are in the market place, and rose cultivars with mauve flowers are in the commercial pipeline (Tanaka et al., 2005; Tanaka, 2006). The prospects of using F3′,5′H gene technology to produce cultivars with blue flowers are discussed in more detail in Section 6.2.2.

6.2.1 Engineering yellow flower colors Many commercial ornamental species such as cyclamen, gentian, impatiens, lisianthus and pelargonium lack good yellow flower colors. Most yellow colors are based on carotenoid pigments, but there are many obstacles to the successful manipulation of this pathway (Fraser & Bramley, 2004), not least of which is the need for plastids/chromoplasts in the petal cells. The yellow flavonoid pigments, chalcones and aurones, provide an attractive alternative for engineering strategies. Chalcone is the first flavonoid produced in the pathway, and as such it is ubiquitous in flowers. However it rarely accumulates, being converted to colorless flavanone and ultimately other flavonoid end-products. Chalcone accumulation has been achieved in target species through either the use of the polyketide reductase to direct biosynthesis of the stable 6′-deoxychalcones (Davies et al., 1998) or RNAi of chalcone isomerase (CHI) gene activity (Nishihara et al., 2005). Unfortunately, only pale yellow flower colors resulted, most likely owing to either the level of chalcones

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being insufficient or the cellular conditions being unsuitable for them to generate yellow colors. Recent developments indicate that engineering aurone production is more promising for introducing yellow pigmentation. Aurones (characteristic pigments of a few families, e.g. Scrophulariaceae) are produced from chalcone glycoside precursors. A cDNA for aureusidin synthase (AUS), a key biosynthetic enzyme for the formation of aurones in antirrhinum, has been isolated (Nakayama et al., 2000; Davies et al., 2006), but initial attempts to use it to introduce aurone biosynthesis into other species proved unsuccessful (Ono et al., 2006a). However, the understanding of aurone biosynthesis has recently advanced, and gene technology is now available for engineering aurone production. AUS, which converts chalcone to aurone, is a vacuolar-located bifunctional polyphenol oxidase variant, catalyzing both hydroxylation and oxidation of its substrate (Ono et al., 2006b). For the AUS to function for aurone production it requires a second enzyme to be active, the uridine diphosphate-glucose (UDP-Glc): chalcone 4′-O-glucosyltransferase (C4′GT). C4′GT generates a glycosylated chalcone intermediate that is transported into the vacuole, where it is converted to aureusidin 6-O-glucoside by AUS. The introduction of transgenes for both AUS and C4′GT is sufficient for enabling the production of aureusidin 6-Oglucoside in transgenic plants of the ornamental species torenia, another member of the Scrophulariaceae (Ono et al., 2006a). When combined with down-regulation of anthocyanin biosynthesis, using RNAi against F3H, transgenic torenia with yellow flowers were obtained. Theoretically, this approach is likely to be successful in other, distantly related species.

6.2.2 Engineering blue flower colors Blue flower colors are lacking from commercial crops such as carnation, chrysanthemum, cyclamen, daffodil, gerbera, impatiens, lisianthus, pelargonium, rose and tulip. While blue flower colors are most commonly associated with the presence of delphinidin-based anthocyanins (Harborne & Williams, 2000), the introduction of delphinidin biosynthesis to those species that lack it is unlikely in itself to generate blue flower colors. This is illustrated by the engineering experiments described previously and by the flower colors naturally seen in cyclamen, impatiens, lisianthus, pelargonium and tulip. Cultivars of these exist that already produce delphinidin-based anthocyanins, but purple flower colors are generated rather than blue. In addition to the presence of delphinidin-derived anthocyanins, suitable vacuolar pH (> pH 5.5 is preferable) and intra- or inter-molecular (co-pigmentation) interactions are required to enable blue colors to form (Andersen & Jordheim, 2006). In particular, extensive chemical studies have found that many of the brilliant blue flower colors in nature are based on anthocyanins with complex structures, such as the presence of multiple monosaccharide and acyl units, the covalent attachment of co-pigments or the formation of anthocyanin–metal ion complexes (e.g. Shiono et al., 2005; Andersen & Jordheim, 2006; Mori et al., 2006; Yoshida et al., 2006). Indeed, there are examples where the vacuolar conditions and anthocyanin secondary structure enable true blue color to be formed from cyanidin-based anthocyanins (Yoshida et al., 2006). In the case of the blue cornflower (Centaurea cyanus) a ‘superpigment’ is formed consisting of six molecules of cyanidin 3-O-(6-O-succinylglucoside)-5-O-glucoside, six molecules of apigenin 7-Oglucuronide-4-O-(6-O-malonylglucoside), one ferric ion, one magnesium ion and two

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calcium ions. X-ray crystallography studies suggest that the pigment self-associates in response to molecular crowding (Shiono et al., 2005; Ellestad, 2006). Acylation does not in itself substantially alter the absorption spectrum of the anthocyanin, but has a marked effect on the stability of the pigment in the vacuole and the likelihood that the ‘stacking’ of pigments into a complex will occur. The influence on stability and color arises from the chemical changes that can occur with anthocyanins in aqueous solutions, and the reduction in hydration that acylation and stacking of anthocyanins can bring about. Acylated glycosyl groups at the C-3, C-5 and C-3′ positions have been suggested as being particularly effective in generating stable (and bluer) anthocyanins. It is clear that, although the gene technology for introducing delphinidin biosynthesis into new species has been established, altering vacuolar pH or the introduction of enzymatic activities for extensive secondary modifications of the base anthocyanidin to trigger formation of in-vivo anthocyanin complexes may also be required if blue colors are to be obtained in transgenic ornamental crops. We will now discuss recent advances in the molecular biology of anthocyanin modification activities, and the prospects of using these for engineering blue flower colors. In most plants the first anthocyanin formed is an anthocyanidin 3-O-glycoside, and the most common glycosyltransferase involved in this is A3GT, which adds a glucose residue. The anthocyanin may subsequently be extensively modified by the addition of other sugar residues, aromatic or aliphatic acylation, and methylation at hydroxyl groups. The additional sugar moieties may be added at hydroxyl groups (e.g. frequently at C-5) and/or to previously added sugars, and the acyl groups are attached to the sugars. There has been much interest in obtaining DNA sequences for the enzymes that carry out anthocyanin secondary modifications. Sequences were identified for A3GT as far back as 1984 (Fedoroff et al., 1984), however, it was ten years before reports of the cloning of anthocyaninmodification genes were published, these being for the UDP-Rha:anthocyanidin 3-Oglucoside 6″-O-rhamnosyltransferase (Brugliera et al., 1994; Kroon et al., 1994). In the last decade there has been substantial progress made on isolating cDNAs for other modification enzymes, resulting from research on a wide variety of plant species. There are now cDNA clones available for at least nine distinct anthocyanin Oglycosyltransferases (AGTs) (Table 6.1). Of particular note recently are the anthocyanidin 3-O-galactosyltransferase (Kubo et al., 2004), anthocyanin O-glucuronosyltransferase (Sawada et al., 2005), anthocyanin 3′-O-glucosyltransferase (A3′GT; Fukuchi-Mizutani et al., 2003), anthocyanin 3′5′-O-glucosyltransferase (Noda et al., 2004), and an enzyme from rose that catalyzes the sequential addition of glucose at the hydroxyl groups at the C-5 then C-3 positions (Ogata et al., 2005). Analyses of the modification activities using recombinant proteins have shown that there is much variation in the enzyme substrate specificity, dependent on the source species of the cDNA. For example, recombinant gentian (Gentiana trifolia) A3′GT will accept only anthocyanins with 3′4′5′-OH substitution of the B-ring, having no activity with pelargonidin or cyanidin-based anthocyanins (Fukuchi-Mizutani et al., 2003); and recombinant petunia A5GT accepts only delphinidin 3-O-(p-coumaroyl)-rutinoside (Yamazaki et al., 2002). In contrast to the petunia enzyme, the recombinant A5GTs from perilla (Perilla frutescens) and Verbena hybrida accept a range of 3-O-glycosides and acyl-glycosides (Yamazaki et al., 1999). There is consistently high specificity with regard to the sugar donor accepted. All the anthocyanin-related GT

UGT1 UGT1 UGT1 UGT1 UGT1 UGT1

A3′GT A3′5′GT A5,3GT (RhGT1) A3RT A3G2″GT (BpUGAT)

UDP-Glc:anthocyanin 3′,5′-O-glucosyltransferase

UDP-Glc:anthocyanidin 5,3-O-glycosyltransferase

UDP-Rha:anthocyanidin 3-O-glucoside 6″-Orhamnosyltransferase

UDP-Glucuronic acid:anthocyanidin 3-O-glucoside 2″-Oglucuronosyltransferase

A3AT (Pf3AT) A5MaT (Ss5MaT1) A3MaT (Sc3MaT, Dm3MaT1, Dv3MaT) A5MaT (Ss5MaT2) A3diMaT (Dm3MaT2)

Hydroxycinnamoyl-CoA:anthocyanidin 3-O-glucoside-6″-Ohydroxycinnamoyltransferase

Malonyl-CoA:anthocyanin 5-O-glucoside-6′″-O-malonyltransferase

Malonyl-CoA:anthocyanidin 3-O-glucoside-6″-O-malonyltransferase

Malonyl-CoA:anthocyanin 5-O-glucoside-4′″-O-malonyltransferase

Malonyl-CoA:anthocyanidin 3-O-glucoside-3″,6″-O-dimalonyltransferase

VAT

VAT

VAT

VAT

Yoshihara et al., 2006

Suzuki et al., 2004a

Suzuki et al., 2004b

Suzuki et al., 2002, 2003b, 2004a

Suzuki et al., 2001

Yonekura-Sakakibara et al., 2000

Fujiwara et al., 1998

2

Reference is given only to the first publication on the isolation and characterization of the corresponding cDNA/gene. Some A3GTs analyzed will accept other flavonoids in addition to anthocyanidins as substrates, with acceptance of flavonols most commonly demonstrated. 3 The recombinant A5GT proteins studied have shown varying degrees of anthocyanin substrate specificity. 4 The AAT nomenclature follows Nakayama et al. (2003). The positional number of the hydroxyl that is modified on the sugar moiety is given followed by prime symbols to indicate which sugar is affected. The double and triple primes indicate the 3-O-glycosyl and 5-O-glycosyl, respectively. Recombinant AATs show varying degrees of substrate specificity, and only a general indication of substrate acceptance is given in this table.

1

VAT

A5AT (Gt5AT)

Hydroxycinnamoyl-CoA:anthocyanin 5-O-glucoside-6′″-Ohydroxycinnamoyltransferase4 VAT

Versatile acyltransferase (VAT)

A3AT (Ih3AT1 and Ih3AT2)

Coumaroyl-CoA:anthocyanin 3-O-glycoside-O-coumaroyltransferase

Patent application WO03/062428

SAM O-Methyltransferase (OMT) Patent application WO03/062428 OMT

A3′OMT A3′5′OMT

SAM:anthocyanin 3′,5′-O-methyltransferase

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Brugliera et al., 1994; Kroon et al., 1994

Ogata et al., 2005

Noda et al., 2004

Fukuchi-Mizutani et al., 2003

Yamazaki et al., 1999, 2002

Morita et al., 2005

Kubo et al., 2004

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SAM:anthocyanin 3′-O-methyltransferase

UDP-Glc:anthocyanin

A5GT

UGT1

A3G2″GT

UDP-Glc:anthocyanin 3-O-glucoside-2″-O-glucosyltransferase

UDP-Glc:anthocyanin 3′-O-glucosyltransferase

UGT1

A3GalT

UDP-Gal:anthocyanidin 3-O-galactosyltransferase

Fedoroff et al., 1984

Reference1

146

5-O-glucosyltransferase3

UDP-Glycosyltransferase, sub-family 1 (UGT1)

A3GT

UDP-Glc:anthocyanidin 3-O-glucosyltransferase2

Protein family

Abbreviation

Enzyme

Table 6.1 Anthocyanidin/anthocyanin modification enzymes for which DNA sequences have been characterized.

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sequences identified to date contain the ‘plant secondary product glycosyltransferase’ (PSPG) motif, thought to be the binding site of the nucleotide–diphosphate sugar (Gachon et al., 2005). The recent solving of the crystal structure of a flavonoid GT (Offen et al., 2006) should greatly aid in identifying the protein motifs important for substrate and regiospecificity. Anthocyanin acylation is common, with over two-thirds of known anthocyanin structures having this modification (Andersen & Jordheim, 2006). Anthocyanin acyltransferases (AATs) catalyze the transfer of either aromatic or aliphatic acyl groups from CoA-donor molecules to hydroxyl residues of sugars attached to the anthocyanin (frequently at the 6-position of glucose groups). A wide range of AAT activities has been characterized (Table 6.1), and examples of the products of aromatic and aliphatic AATs are shown in Fig. 6.2. Biochemical studies have identified activities that use acetyl-CoA, caffeoyl-CoA, 4-coumaroyl-CoA, malonyl-CoA or succinyl-CoA donors (Nakayama et al., 2003; Davies & Schwinn, 2006). The published DNA sequences cover three aromatic and six aliphatic AATs. Recombinant enzyme studies show that these AATs do indeed use a range of donor

(a)

(b) Fig. 6.2 Examples of the products formed by aromatic or aliphatic anthocyanin acyltransferases. Recombinant A5AT of gentian (Gt5AT) will use either caffeoyl-CoA or 4-coumaroyl-CoA donors to transfer a hydroxycinnamic acid moiety to a glucose residue at the C-5 hydroxyl of pelargonidin, cyanidin or delphinidin derivatives (Fujiwara et al., 1998). The example shows a delphinidin 3,5-di-O-glucoside with coumaroyl added ((a) circled). Recombinant A3diMaT of chrysanthemum (Dm3MaT2) uses malonyl-CoA to perform sequential transfers of the aliphatic malonyl group to cyanidin 3-O-glucoside (Suzuki et al., 2004), forming cyanidin 3-O-(6″-O-malonylglucoside) and cyanidin 3-O-(3″,6″-di-O-malonylglucoside) ((b) structure shown with the malonyl additions circled).

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groups but, as with the AGTs, they can have narrow specificity with regard to the target acceptor. The specificity is generally towards the secondary modification pattern of the anthocyanin rather than the hydroxylation status of the B-ring. For example, recombinant cineraria (Senecio cruentus) malonyl-CoA:anthocyanidin 3-O-glucoside-6″-O-malonyltransferase accepts pelargonidin-, cyanidin- or delphinidin 3-O-glucosides but does not use the anthocyanin diglycosides (Suzuki et al., 2003b). Likewise, the recombinant gentian A5AT can use either caffeoyl-CoA or 4-coumaroyl-CoA as the donor and will accept pelargonidin, cyanidin or delphinidin derivatives, but specificity is shown with regard to the anthocyanin glycosylation and acylation pattern (Fujiwara et al., 1998). Almost all of the molecular studies on AATs to date have been with dicot plants. Recently the first AAT sequence from a monocot plant was published, for an anthocyanin 3-O-coumaroyltransferase of Iris hollandica that catalyzes the transfer of a coumaroyl moiety to anthocyanidin 3-O-rhamnosylglucoside5-O-glucoside or anthocyanidin 3-O-rhamnosylglucoside (Yoshihara et al., 2006). Anthocyanin methylation most commonly occurs at the 3′- and 5′-hydroxyl groups, but methylation at the C-5 and C-7 hydroxyl groups also occurs in some species (Andersen & Jordheim, 2006). Enzymes of the S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MT) family, typically class II O-MTs (OMTs), catalyze this type of modification. The OMTs of petunia that act at the 3′ and 5′-hydroxyls of anthocyanins have been well-characterized biochemically, but to date DNA sequences for these (and other) anthocyanin OMTs have been reported only in the patent literature (International Patent Application WO03/062428). Additional information on the molecular biology of anthocyanin modification enzymes and the enzyme families to which they belong can be found in reviews by Ibrahim & Muzac (2000), Vogt & Jones (2000), Nakayama et al. (2003) and Davies & Schwinn (2006). Also, recent molecular phylogenies are presented in Gachon et al. (2005) and Yoshihara et al. (2005) for the GTs and Yoshihara et al. (2006) for the AATs. One of the drives for investigating anthocyanin modification activities is to advance knowledge and technology with regard to the formation of blue flower pigments. So do the range of cDNAs isolated for the anthocyanin modification activities enable the engineering of the complex anthocyanin structures often associated with blue flower colors? There are cDNAs for anthocyanin modification enzymes that do indeed catalyze some of the structural changes thought to be associated with blue colors. However, although there are few transgenic plant data to date, it is likely that it would still be difficult to engineer blue flower colors in most target species. One reason is the specificity shown by some of the enzymes, so the required substrate may not be present in the target species. Furthermore, although there has been much progress in identifying DNA sequences for modification enzymes, there is still a lack of understanding of other key factors such as the incorporation of metal ions into the pigment structures. Also, for some species several transgenes may be required to direct production of the target anthocyanin type. The initial introduction of delphinidin biosynthesis may require a combination of two or three other transgenes (Tanaka et al., 2005) in addition to those required for different modification activities. While there are examples of using several transgenes to achieve a desired outcome (e.g. Wu et al., 2005), this requires a range of suitable gene promoters and a plant species amenable to the introduction of multiple transgenes. There are few published examples of attempts to engineer complex anthocyanin structures. In one case, a line of petunia that

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accumulates delphinidin 3-O-glucoside in the petals was transformed with transgenes for the gentian A3′GT and torenia A5GT (Fukuchi-Mizutani et al., 2003). The transgenic plants did indeed produce the new anthocyanin delphinidin 3,5,3′-tri-O-glycoside, but only at 2–6% of the total anthocyanins, illustrating some of the potential hurdles to be overcome. Finally, although some genes have been identified that can alter vacuolar pH (Yamaguchi et al., 2001; Yoshida et al., 2005; Quattrocchio et al., 2006), no data on reliable gene technology for creating a significant shift in vacuolar pH have been published.

6.3 Anthocyanic vacuolar inclusions Unlike for most plant secondary metabolite pathways, there are data on both the nature of the anthocyanin biosynthetic enzyme complex and the subsequent transport of the compounds to their sites of accumulation. Cytosolic multi-enzyme complexes that are anchored on endoplasmic reticulum (ER) via the component cytochrome P450 enzymes, such as C4H or F3′H, are likely to be involved (Grotewold, 2006). Such complexes could allow for metabolic channeling of substrates between enzymes, or between diverse complexes that contain different mixes of biosynthetic enzymes or isoenzymes (Winkel, 2004). The anthocyanins’ final destination is the vacuole, an acidic environment in which they form the familiar brightly colored structures (Kitamura, 2006). Although there has been progress from molecular studies in deciphering aspects of the transport process to the vacuole, this is one of the least understood stages of the biosynthetic pathway. Diverse transport models have been proposed for different flavonoids, only some of which may apply to anthocyanins. The availability of mutant lines of arabidopsis, carnation, maize and petunia has demonstrated the requirement for members of the glutathione S-transferase (GST) family for successful anthocyanin sequestration into the vacuole (Marrs et al., 1995; Alfenito et al., 1998; Larsen et al., 2003; Kitamura et al., 2004). This would suggest a pathway similar to that for detoxification, in which the addition of glutathione residues by GST forms stable water-soluble conjugates that are sequestered by ATP-binding cassette (ABC) transmembrane transporters (Marrs et al., 1995). However, no anthocyanin–glutathione conjugates have been observed in vivo, and it is possible that the GST works as an anthocyanin-binding protein to escort anthocyanins to the tonoplast from the site of synthesis (Mueller et al., 2000). A second possible transport mechanism identified through mutant analysis in tomato (Solanum lycopersicum; Mathews et al., 2003) and maize (Goodman et al., 2004) is via multi-drug and toxic compound extrusion (MATE) transporters located in the tonoplast membrane. The tt12 mutant line of arabidopsis supports a role for MATE proteins in proanthocyanidin transport also (Debeaujon et al., 2001). Further information on possible components of the anthocyanin/proanthocyanidin transport system has come from ultrastructural studies on plant tissues or cell lines. Data from maize cell cultures, arabidopsis mutants and flower petals of lisianthus suggest the involvement of membrane vesicles and a coordination of the transport process with vacuole biogenesis. The observations of apparent vesicle transport in lisianthus petals have come from studies on the formation of anthocyanic vacuolar inclusions (AVIs), and we discuss both the nature of AVIs and the association of pre-vacuolar vesicles with anthocyanin transport in the following section.

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AVI is a term suggested in 2000 (Markham et al., 2000) that has become established for referring to the dense concentrations of anthocyanins that occur in the vacuoles of some cells. Anthocyanin-concentrated bodies were reported some time ago for a range of species, and were suggested as ‘anthocyanoplasts’ that were possible sites of anthocyanin biosynthesis (Pecket & Small, 1980; Small & Pecket, 1982). Subsequently, more detailed studies of the anthocyanin bodies identified different forms, specifically, spherical vesicle-like bodies and irregularly shaped dense bodies. The vesicle-like bodies were first characterized in detail in suspension cell cultures of sweet potato (Ipomoea batatas) and maize (Nozue & Yasuda, 1985; Grotewold et al., 1998), and in both cases were associated with light-induction of anthocyanin biosynthesis. In sweet potato, upon exposure to light a large number of smaller vesicles form that gradually fuse into a small number of larger bodies (Nozue & Yasuda, 1985). The term AVI was first coined for the non-vesicle, dense and compact bodies found in petals of lisianthus and carnation, which can be extracted as particles from the cell (Markham et al., 2000). However, the term is now often used for any anthocyanin bodies observed. AVIs occur in a range of angiosperm species, without any obvious phylogenetic association. In addition to those already mentioned, vesicle-like AVIs have been observed in grape (Vitis vinifera) cell cultures (Conn et al., 2003), in epidermal cells of grape skins (Mizuno et al., 2006) and some lisianthus petal cells (Zhang et al., 2006). Also, more compact forms, either regular or irregular in shape, have been found in cells of red-cabbage leaves (Small & Pecket, 1982), carnation petals (Markham et al., 2000), rose petals (Gonnet, 2003), Sinningia cardinalis petals (Winefield et al., 2005), apple (Malus domestica) skins (Bae et al., 2006) and some lisianthus petal cells (Zhang et al., 2006). No boundary membrane has been observed for the sweet potato, lisianthus or carnation AVIs, but it has been suggested that specific proteins are associated with them (Markham et al., 2000). The large irregularly shaped AVIs are probably insoluble, as they can be isolated as stable particles, and they also impart their irregular shape to protoplasts prepared from AVI-containing cells (Zhang et al., 2006). Detailed analysis of the formation of AVIs in lisianthus has revealed heterogeneity of structure, and the possible role of pre-vacuolar vesicles in transport of anthocyanins from the site of formation in the cytoplasm to the growing AVI in the vacuole (Zhang et al., 2006). The petal may contain both vesicle-like and particle-like AVIs, with the occurrence of the different types varying between the adaxial and abaxial epidermis (Fig. 6.3). It is possible that the various AVI forms reflect differences in the associated vacuolar contents of the cells, for example the anthocyanin concentration and structures, but how this would cause such distinct characters of anthocyanin accumulation is not clear. Furthermore, as with the petals of many species, the occurrence of anthocyanins across the epidermis is sporadic, so that one cell may have an evenly colored vacuole while neighboring cells have either AVI bodies or no apparent anthocyanins (Fig. 6.3). An interesting finding from ultrastructural studies on AVI formation is the presence of colored, electron-dense bodies in the cytoplasm of lisianthus petals (Zhang et al., 2006) and transgenic Black Mexican Sweet suspension cell lines of maize (Grotewold et al., 1998; Lin et al., 2003). It has been suggested that they are pre-vacuolar vesicles that transport the newly synthesized phytochemicals into the central vacuole. Figure 6.4 shows a possible sequence in which vesicles within a lisianthus petal cell migrate to the central vacuole, where they rupture and release their contents onto the growing AVI. This indicates

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a

b

c

Fig. 6.3 Anthocyanic vacuolar inclusions (AVIs) of lisianthus flowers. (a) An abaxial epidermal peel of the basal petal region observed under bright light, showing vesicle-like AVIs in the vacuoles of some cells, and an even anthocyanin distribution or no anthocyanins in the vacuoles of neighboring cells. (b) Bright field image showing the large irregular AVIs in the deeply-pigmented adaxial epidermal cells of the basal petal region. (c) Bright field image of isolated AVIs mounted on glass slide in buffer solution (pH 7.0).

Fig. 6.4 Transmission electron micrograph of an adaxial epidermal cell of the strongly pigmented inner petal region of a lisianthus flower. The edge of a large, irregularly shaped anthocyanic vacuolar inclusion (AVI) is shown. Material, probably originating from pre-vacuolar compartments (PVC), is being deposited onto the forming AVI. CW, cell wall; DEP, material being deposited.

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that the cytoplasm is not only the site of anthocyanin biosynthesis but also a site where anthocyanins can accumulate to a certain level prior to mass transport to the central vacuole. In this case, GSTs and other transporters would probably be associated with transfer of the anthocyanins into these pre-vacuolar vesicles. How common this process is in different species is not known. However, ER-derived vesicles are involved in the mass transport of other cellular components, for example proteins into protein storage vacuoles (Mori et al., 2004; Takahashi et al., 2005). Furthermore, the ANS mutation of arabidopsis (tds4) not only inhibits proanthocyanidin production, but also prevents normal vacuole development and causes the accumulation of small vesicles (Abrahams et al., 2003). The function of AVIs, if any, is not known. It has been suggested that they may facilitate the storage of high concentrations of anthocyanins (Markham et al., 2000), and that they may have a role in generating flower color variation in lisianthus and rose (Markham et al., 2000; Gonnet, 2003). Furthermore, it is also not known whether AVIs effect the self-association and intra- or inter-molecular copigmentation processes that are thought to assist in generating colored anthocyanin forms in the vacuole (Andersen & Jordheim, 2006; Ellestad, 2006). Perhaps such structures can form under conditions of high anthocyanin concentration in the pre-vacuolar vesicles prior to formation of AVIs in the vacuole.

6.4 Regulation of anthocyanin biosynthesis Flower pigmentation is under tight developmental and spatial control, presumably to maximize pollination success. Typically, bright pigmentation is mainly in the petals. Within the petal, pigments may have a distribution that assists in further directing pollinators to the appropriate region of the flower. For example, in antirrhinum, the yellow face (aurone pigments) on the background of red anthocyanins is thought to provide a visual target for foraging bumblebees and may guide them to the mouth of the corolla (Lunau et al., 2006). Furthermore, flower color may help the timing of pollinator visits to coincide with the period that a flower is fertile. Thus, pigmentation may immediately precede fertility, and change following pollination as the result of alteration of the pH of petal cells, degradation of pigments, or de novo pigment biosynthesis (Bohm, 1998; Farzad et al., 2002, 2003). For anthocyanin pigmentation, there is now information available on the range of signals that contribute to the spatial and temporal regulation of pigment production in the flower and the molecular mechanisms by which these signals result in changes in anthocyanin biosynthesis. Transcription of the biosynthetic genes is the key control point for anthocyanin production. This has been determined from studies in maize and subsequently in many other species including ornamentals such as antirrhinum, gerbera, Japanese morning glory (Ipomoea nil) and petunia. Since the isolation of the first transcription factor gene from plants, which was the anthocyanin regulator C1 from maize (Paz-Ares et al., 1986, 1987), the regulation of anthocyanin production has become the best-characterized example of combinatorial control of gene transcription in plants. Transcriptional activation of the biosynthetic genes is mediated by transcription factors (TFs) of the R2R3-MYB and basic Helix-Loop-Helix (bHLH) (or MYC) type, along with a co-regulator of the WD40/WDrepeat type (WDR) (reviewed in Vom Endt et al., 2002; Davies & Schwinn, 2003; Koes

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et al., 2005). The current model of the transcriptional complex has the WDR protein as a component. It may have a role in stabilizing the complex and/or be involved in nuclear localization of the bHLH/MYB factors or modulating chromatin conformation (reviewed in Broun, 2005). Table 6.2 provides details of some of the MYB, bHLH and WDR sequences identified as regulating anthocyanin production in flowers, along with examples from arabidopsis, maize and rice. Sequence analysis of the anthocyanin-related MYB and bHLH factors shows two distinct phylogenetic clades for each type of TF (Table 6.2; authors’ own data; reviewed in Koes et al., 2005). What this means within the context of anthocyanin regulation remains to be determined. In antirrhinum flowers, three structurally very similar MYB factors (from the same clade) and two bHLH factors (from separate clades) control pigmentation (see Table 6.2). Interestingly, one of the MYB factors (ROSEA2) shows a specificity in its interactions with the bHLH factors (DELILA and MUTABILIS) in that it interacts effectively only with DELILA (Schwinn et al., 2006). This accounts for at least some of the differences that have also been determined between the three MYB factors in their anthocyanin target gene specificities (Schwinn et al., 2006). Further information on functional aspects of the transcription complex are coming from studies of the maize TFs C1 (MYB) and R (bHLH) (Grotewold et al., 2000; Hernandez et al., 2004; Feller et al., 2006). For example, key protein domains and amino acid residues behind the interaction of C1 and R have been identified, and dimerization of R through an ACT-like domain has been shown to be important to its function. It has not been determined yet whether in vivo R is functioning as a homodimer or a heterodimer, and indeed, much remains to be elucidated about the transcriptional complexes, including the exact components and the stoichiometry. Spatial regulation of anthocyanin biosynthesis involves both the association of anthocyanins with the flower, or other organs for pollinator attraction, and pigmentation patterning within the flower. Patterning within the flower may involve color variation between petals, or between petals and sepals, or differential coloration between individual cells within the petal, to generate spots, stripes, irregular blotches, or combinations of all of these. Features common to many species include a dark throat region (bi-colors) or stripes associated with the veins (venation). Some species have evolved complex pigment patterning in association with morphological features and scent production to attract specific pollinators. A good example of this is found in orchids that mimic female insects to achieve pollination through pseudocopulation by the male (Paxton & Tengo, 2001). Pigmentation patterning may be programmed by the flower’s development genes, may be induced by viral infection, or may be due to the presence of genetic instability and heterogeneity, for example transposons and chimeral dispositions (Olbricht et al., 2006). The programmed, stable pigmentation patterns are of particular interest. Antirrhinum is a model for these studies. The three Myb genes controlling anthocyanin pigment produce very different patterns and intensities of pigmentation (Schwinn et al., 2006). Rosea1 gives strong, ‘wild-type’ pigmentation, with dark red pigment in both the inner and outer epidermis of the corolla. Rosea2, which with Rosea1 forms a complex locus, only colors the inner epidermis weakly. Venosa causes the production of anthocyanins in epidermal cells above the veins of the petal (venation), a phenotype only visible when Rosea1 is inactive. Furthermore, differences in the activity of these Myb genes underpin the observed

DELILA

GL3, EGL3

JAF13 MYC-RP

–

TT23

– –

OsC1

– MYB-C055 C1 PL

ROSEA1 ROSEA2 VENOSA

AtMYB75 (PAP1) AtMYB90 (PAP2) AtMYB113 AtMYB114

GMYB10

INMYB1 INMYB2 INMYB3

–

AN2 AN4

–

–

Antirrhinum majus

Arabidopsis thaliana

Gerbera hybrida

Ipomoea nil

Oryza sativa

Petunia hybrida

Perilla frutescens

Zea mays

PFWD MP1 PAC1

MYC-F3G1 IN16

AN11

–

Rc4

AN1

INWDR1 INWDR2

INBHLH2

–

Paz–Ares et al., 1986, 1987; Chandler et al., 1989; Ludwig & Wessler, 1990; Cone et al., 1993; Carey et al., 2004

Gong et al., 1999a, 1999b; Sompornpailin et al., 2002; Yamazaki et al., 2003

de Vetten et al., 1997; Quattrocchio et al., 1999; Spelt et al., 2002; Kroon, 2004

Hu et al., 1996; Reddy et al., 1998; Sakamoto et al., 2001; Nagabhushana & Reddy, 2004; Sweeney et al., 2006

Park et al., 2004; Morita et al., 2006

Elomaa et al., 1998

Walker et al., 1999; Borevitz et al., 2000; Payne et al., 2000; Ramsay et al., 2003; Zimmermann et al., 2004

Goodrich et al., 1992; Schwinn et al., 2006

AMWDR12

TTG1

References1

WDR

2

Reference is given only to the first publication on the isolation and characterization of each corresponding cDNA/gene. K. Schwinn, unpublished data. 3 TT2 is involved in regulating the production of proanthocyanidins. 4 Rc regulates proanthocyanidin production for the red pericarp trait of rice. 5 MYB-C05 (Yamazaki et al., 2003) is a putative anthocyanin-related regulator of perilla that is closer in sequence similarity to the confirmed anthocyanin-related MYBs of other species than is another putative anthocyanin-related regulator MYB-P1 (Gong et al., 1999b). 6 Intensifier activity inhibits anthocyanin production in maize.

B-PERU LC R SN

PLw-OSB1 (Ra) PLw-OSB2

INBHLH1 INBHLH3

–

TT8

MUTABILIS

Group 2

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1

Group 1

Group 2

Group 1

Species

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GMYC1

BHLH (TF)

R2R3-MYB (TF)

Table 6.2 Anthocyanin-related regulatory proteins of some ornamental and/or model plant species, showing groupings within transcription factor (TF) types based on phylogenetic similarity.

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variation in the floral anthocyanin pigmentation intensity and patterning in the Antirrhinum genus, indicating that it is activity of the Myb genes that is likely to be the major cause of the natural variation in anthocyanin pigmentation that we see in plants (Schwinn et al., 2006). This key role of the Myb genes is supported by studies of petunia flowers, grape berry skin, apple flesh and potato (Solanum tuberosum) tubers (Quattrocchio et al., 1999; De Jong et al., 2004; Kobayashi et al., 2005; Takos et al., 2006). Consistent with the key role of MYB factors as determinants of pigmentation variation is evidence that they provide specificity in regulatory networks, determining which developmental pathway is activated. The complex WDR/MYB/bHLH network in arabidopsis that controls anthocyanin production, trichome and root hair initiation and seed mucilage illustrates this point (reviewed in Broun, 2005; the reader is referred in particular to Fig. 2 in that review). The WDR protein (TTG1) is involved in regulating all the processes, and the individual bHLH factors (GL3, EGL1 and TT8) function in more than one of the regulatory pathways. It therefore appears to be down to the MYB factor to specify the process that is regulated. For example, TT8 may interact with TT2 for the regulation of flavonoid production in the seed coat but with AtMYB61 for the production of seed-coat mucilage. Whether the arabidopsis results are a relevant model for other species remains to be determined. Environmental factors such as temperature, water stress and light quantity and quality have been shown to influence flower coloration, with light the principal environmental signal (reviewed in Weiss, 2000). Preventing light exposure of the flowers of some species will inhibit anthocyanin formation. For gerbera this is probably owing to a direct action of light as a positive regulatory signal for the anthocyanin biosynthetic genes, rather than indirectly through promoting photosynthetic activity in the flower, and blue light in particular has been shown to promote CHS and DFR gene expression (Meng & Wang, 2004). However, in contrast to the wealth of data on the regulation by light of pigment production in vegetative tissues (reviewed in Weiss, 2000 and Davies & Schwinn, 2003), there are few data on the molecular mechanisms that may be involved in mediating the light signals in flowers. Interestingly, a diurnal rhythm of abundance of DFR transcript has been observed in the colored spathe of anthurium (Anthurium andraeanum; Collette et al., 2004). It is not known if this is related to the fact that the spathe, the major colored organ in the Araceae, is a modified leaf (which subtends the inflorescence), or whether diurnal regulation may occur in petals also. The developmental signals that control floral pigmentation have been examined in a few species, but are generally not well understood. It is probable that a range of signals are required, for example pigmentation in Viola cornuta flowers requires at least light, gibberellic acid (GA) and pollination (Farzad et al., 2002, 2003). One of the best characterized systems is petunia. In this species, anther-produced GA is thought to be a developmental signal translocated to the corolla to induce petal growth and anthocyanin pigmentation (reviewed in Weiss, 2000). In contrast, GA is not required for floral pigmentation in antirrhinum, at least for the venation phenotype caused by the Myb gene Venosa (Yongjin Shang, Crop & Food Research, personal communication). In petunia, other hormones, specifically abscisic acid, may also be involved; and exogenously supplied carbohydrate increases anthocyanin production and up-regulates CHS expression (Weiss, 2000). In species that show a color change in the open flowers, for example following pollination, ethylene may act as a

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signal (Farzad et al., 2002). Which of the various signals known to influence anthocyanin biosynthesis are key developmental regulators is not known. However, the constitutive signals, such as light and carbohydrate, are likely to be general influences on flower development, rather than specific signals controlling the timing of anthocyanin formation. Developmental regulation of petal pigmentation is linked to the action of the genes that determine petal identity. Loss of function of the MADS box B-function protein DEFICIENS in antirrhinum results in reduced expression of CHS and F3H and lower anthocyanin levels (Bey et al., 2004), and in petunia, inhibition of the activity of the E-function floral binding protein2 gene results in plants with small green corollas that fail to produce anthocyanins or express CHS (Angenent et al., 1994; Ferrario et al., 2003). However it remains to be determined whether these genes directly regulate the MYB (or bHLH) factors controlling anthocyanin biosynthetic genes. The flowers of gerbera, a member of the Asteraceae, have been shown to regulate pigmentation in association with various anatomical domains during inflorescence development. Typical of the species of this genus, it bears different types of flowers in its inflorescence. There are inconspicuous central hermaphrodite disc flowers, and more showy marginal female ray and trans flowers. In different cultivars only some of the flowers, or parts of flowers, within the complex inflorescence become pigmented, generating patterns. The variation in pigmentation is probably due to variation in expression of the anthocyaninrelated TFs, with DFR one of the key target biosynthetic genes (Teemu et al., 2006). Despite the success in elucidating aspects of the transcriptional regulation of anthocyanin biosynthesis, much is still not known, including how activities of the TFs are modulated or the role of repressor TFs in regulating pigmentation. Although there are MYB and bHLH proteins that are known to have a repressive effect on anthocyanin biosynthetic gene transcription (Paz-Ares et al., 1990; Burr et al., 1996; Aharoni et al., 2001; Chen et al., 2004), none have been reported that are active in flower coloration. It has recently been proposed that one of the roles of the bHLH in the activation of the target genes is to relieve the MYB from the effect of an inhibitory factor (Hernandez et al., 2004). A single repeat MYB (PhMYBX) has been identified in petunia, which is structurally similar to two MYBs (CPC and TRY) that are part of the network controlling trichome and root-hair initiation in arabidopsis (Kroon, 2004). In yeast two-hybrid assays, PhMYBX bound the petunia bHLH factors AN1 and JAF13, and its over-expression in petunia downregulated the production of anthocyanin. Not surprisingly, the production of other flavonoid end-products in the flower, such as flavonols or flavones, is under independent control, as this enables metabolic flexibility. For example, as flavonols and anthocyanins differ in their UV-absorbing properties, it allows for the formation of floral nectar guides, given the ability of some pollinators to detect both UV- and visible-light wavelengths. Similar MYB factors, ATMYB12 and P1/P2, regulate flavonol production in arabidopsis and flavone C-glycoside (and phlobaphene) production in maize, respectively (Zhang et al., 2003; Mehrtens et al., 2005). However, there are few data on the regulation of flavonol or flavone O-glycoside production in flowers. The MYB factors MYB305 and MYB340 of antirrhinum can up-regulate some of the early genes in the flavonoid pathway (Moyano et al., 1996), but their role in antirrhinum flowers has not been determined. In vegetative tissues of some species light is a key signal, with the potential involvement of the photoreceptors phytochrome B and cyptochrome (Jenkins

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et al., 2001; Hemm et al., 2004). Based on analysis of the promoters of the biosynthetic genes, and the isolation of candidate TFs, the light signal may be mediated by MYBs and basic region/leucine zipper (bZIP) TFs (Hartmann et al., 2005; and reviewed in Jakoby et al., 2002 and Davies & Schwinn, 2003).

6.5 Concluding comments Knowledge of flavonoid biosynthesis in ornamentals is well advanced. Gene sequences are available for all of the enzymes of the ‘core’ pathway to anthocyanins, as well as for a range of anthocyanin modification activities. There is also some information on the final step of the biosynthesis of anthocyanins, their transport to the vacuole. However, there are many unanswered questions on the nature of the anthocyanin bodies observed in some species. The regulation of anthocyanin biosynthesis is one of the best-characterized examples of transcriptional regulation in plants, with much knowledge of the TFs involved. Points of current interest include the nature of the regulatory signals acting upstream of the direct regulators of the anthocyanin biosynthetic genes, and the subtleties of combinational control by families of similar anthocyanin-related TFs. Notably, although the first published account of metabolic engineering for new flower colors was 20 years ago (Meyer et al., 1987), and there have been many field trials of transgenic cultivars with novel phenotypes (Kuehnle & Mudalige-Jayawickrama, 2007), molecular breeding within the ornamental industry is still in its infancy. This is not through a lack of success with the scientific approaches. For example, the experiments of Meyer et al. led to cultivars with striking colors novel to petunia (Oud et al., 1995), but these have not become commercially available. Clearly the requirements that must be met before market release of transgenic crops, principally regulatory approval costs and freedom to operate for intellectual property, form a major economic barrier to the commercialization of relatively minor crops such as ornamentals. Gene technology that would allow the introduction of high-impact, novel colors (e.g. yellow, scarlet or true blue) into leading commercial species might help justify the costs involved in bringing transgenic cultivars to market. In this respect, the recent identification of the aurone biosynthetic genes could prove significant for engineering yellow coloration. Effective gene technology to deliver true blue colors (from complex anthocyanin structures) or bright orange-red colors (e.g. from 3-deoxyanthocyanins) may still be some time away.

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Schwarz-Sommer, Z., Davies, B. & Hudson, A. (2003). An everlasting pioneer: the story of Antirrhinum research. Nature Reviews Genetics, 4, 657–666. Schwinn, K., Venail, J., Shang, Y.-J. et al. (2006). A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. The Plant Cell, 18, 831– 851. Shimada, Y., Ohbayashi, M., Nakano-Shimada, R. et al. (2001). Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-3′,5′-hydroxylase: specific switching of the pathway in petunia. Plant Cell Reports, 20, 456 – 462. Shiono, M., Matsugaki, N. & Takeda, K. (2005). Structure of the blue cornflower pigment – packaging red-rose anthocyanin as part of a ‘superpigment’ in another flower turns it brilliant blue. Nature, 436, 791. Small, C.J. & Pecket, R.C. (1982). The ultrastructure of anthocyanoplasts in red-cabbage. Planta, 154, 97– 99. Sompornpailin, K., Makita, Y., Yamazaki, M. & Saito, K. (2002). A WD-repeat-containing putative regulatory protein in anthocyanin biosynthesis in Perilla frutescens. Plant Molecular Biology, 50, 485 – 495. Spelt, C., Quattrocchio, F., Mol, J. & Koes, R. (2002). ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms. The Plant Cell, 14, 2121–2135. Suzuki, H., Nakayama, T., Yonekura-Sakakibara, K. et al. (2001). Malonyl-CoA:anthocyanin 5-Oglucoside-6′″-O-malonyltransferase from scarlet sage (Salvia splendens) flowers: enzyme purification, gene cloning, expression, and characterization. Journal of Biological Chemistry, 276, 49013 – 49019. Suzuki, H., Nakayama, T., Yonekura-Sakakibara, K. et al. (2002). cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6″O-malonyltransferase from dahlia flowers. Plant Physiology, 130, 2142–2151. Suzuki, H., Nakayama, T. & Nishino, T. (2003a). Proposed mechanism and functional amino acid residues of malonyl-CoA:anthocyanin 5-O-glucoside-6′″-O-malonyltransferase from flowers of Salvia splendens, a member of the versatile plant acyltransferase family. Biochemistry, 42, 1764 –1771. Suzuki, H., Sawada, K., Yonekura-Sakakibara, K. et al. (2003b). Identification of a cDNA encoding malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6″-O-malonyltransferase from cineraria (Senecio cruentus) flowers. Plant Biotechnology, 20, 229–234. Suzuki, H., Nakayama, T., Yamaguchi, M.A. & Nishino, T. (2004a). cDNA cloning and characterization of two Dendranthema × morifolium anthocyanin malonyltransferases with different functional activities. Plant Science, 166, 89 – 96. Suzuki, H., Sawada, S., Watanabe, K. et al. (2004b). Identification and characterization of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers: an enzyme that is phylogenetically separated from other anthocyanin acyltransferases. The Plant Journal, 38, 994 –1003. Suzuki, K., Xue, H.M., Tanaka, Y. et al. (2000). Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes. Molecular Breeding, 6, 239–246. Sweeney, M.T., Thomson, M.J., Pfeil, B.E. & McCouch, S. (2006). Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice. The Plant Cell, 18, 283–294. Takahashi, H., Saito, Y., Kitagawa, T. et al. (2005). A novel vesicle derived directly from endoplasmic reticulum is involved in the transport of vacuolar storage proteins in rice endosperm. Plant and Cell Physiology, 46, 245 –249. Takos, A.M., Robinson, S.P. & Walker, A.R. (2006). Transcriptional regulation of the flavonoid pathway in the skin of dark-grown ‘Cripps’ Red’ apples in response to sunlight. Journal of Horticultural Science and Biotechnology, 81, 735–744. Tanaka, Y. (2006). Flower colour and cytochromes P450. Phytochemistry Reviews, 5, 283–291. Tanaka, Y., Katsumoto, Y., Brugliera, F. & Mason, J. (2005). Genetic engineering in floriculture. Plant Cell Tissue and Organ Culture, 80, 1–24.

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Yoshida, K., Kawachi, M., Mori, M. et al. (2005). The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of morning glory, Ipomoea tricolor cv. Heavenly Blue. Plant and Cell Physiology, 46, 407–415. Yoshida, K., Kitahara, S., Ito, D. & Kondo, T. (2006). Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis. Phytochemistry, 67, 992–998. Yoshihara, N., Imayama, T., Fukuchi-Mizutani, M. et al. (2005). cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Iris hollandica. Plant Science, 169, 496 – 501. Yoshihara, N., Imayama, T., Matsuo, Y. et al. (2006). Characterization of cDNA clones encoding anthocyanin 3-p-coumaroyltransferase from Iris hollandica. Plant Science, 171, 632–639. Zhang, H., Wang, L., Deroles, S.C., Bennett, R. & Davies, K. (2006). New insight into the structures and formation of anthocyanic vacuolar inclusions in flower petals. BMC Plant Biology, 6, 29. Zhang, P., Wang, Y., Zhang, J., Maddock, S., Snook, M. & Peterson, T. (2003). A maize QTL for silk maysin levels contains duplicated Myb-homologous genes which jointly regulate flavone biosynthesis. Plant Molecular Biology, 52, 1–15. Zimmermann, I.M., Heim, M.A., Weisshaar, B. & Uhrig, J.F. (2004). Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. The Plant Journal, 40, 22 – 34. Zuker, A., Tzfira, T., Ben-Meir, H. et al. (2002). Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Molecular Breeding, 9, 33–41.

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Chapter 7

Recent Advances in the Field of Anthocyanins – Main Focus on Structures Øyvind M. Andersen

This chapter is based on the lecture Recent Advances in the Field of Anthocyanins, which was presented by the author after he had been given the Groupe Polyphenol award for 2006 at the XXIIIrd International Conference on Polyphenols in Winnipeg, Manitoba, Canada (August, 2006).

7.1 Introduction The anthocyanins constitute a major flavonoid group, which is responsible for colors ranging from salmon-pink through red and violet to dark blue in plant tissues. These pigments are particularly brilliant in flowers, fruits and leaves of angiosperms. Although the desire to understand and identify the source of natural colors has fascinated the practicing chemist and his predecessors since before the Renaissance, the last two decades have experienced increased interest in anthocyanins mainly because of their potential health-promoting properties and their use as natural food colorants, as well as their appearance in cultivars and plant mutants with new colors and shapes (Fig. 7.1). The main aim of this chapter is to give an overview of anthocyanin structures in plants. A detailed account on anthocyanins reported from various sources during the last two years (Tables 7.1 & 7.2) includes 36 new structures (sections 7.4 & 7.5). Together with the various anthocyanins reported until 2005 (Andersen & Jordheim, 2006) the total number of anthocyanins isolated from plants is, at the time of writing, 575. Improvements in the methods and instrumentation used for separation and structure elucidation of anthocyanins (Rivas-Gonzalo, 2003; Andersen & Francis, 2004; Mazza et al., 2004; Andersen & Fossen, 2005; Rodriguez-Saona & Wrolstad, 2005; Giusti & Wrolstad, 2005; Durst & Wrolstad, 2005; Marston & Hostettmann, 2006; Fossen & Andersen, 2006) have made it easier to use smaller quantities of material for analysis, and to achieve results at increasing levels of precision. Thus, during the last decade a variety of new anthocyanidin structures have been reported (section 7.2), and important information about flavonoid complexes including anthocyanidins as subunits has been obtained (sections 7.6 & 7.7). The topics ‘anthocyanidin equilibrium forms’, ‘biosynthesis and molecular biology’ and ‘anthocyanin localization Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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1000 900 800 700 600 500 400 300 200 100 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Fig. 7.1 Records in the literature search within the database SciFinder giving the numbers of articles containing the word ‘anthocyanins’ in the period 1990–2006.

in plant cells’ have seen important progress in recent years, and are treated in sections 7.3, 7.8 and 7.9, respectively.

7.2 Anthocyanidins Thirty-two naturally occurring monomeric anthocyanidins have been properly identified (Table 7.3), however, most anthocyanins are based on cyanidin (31%), delphinidin (22%) and pelargonidin (18%), respectively. The other three common anthocyanidins (peonidin, malvidin and petunidin), which contain methoxy group(s) on their B-rings, constitute together 21% of the reported anthocyanins. During the last decade one new methylated anthocyanidin, 7-O-methylcyanidin, five new desoxyanthocyanidins, and a novel type of anthocyanidin called pyranoanthocyanidin have been reported (Table 7.3). The 3-O-β-d-galactopyranoside of 7-O-methylcyanidin, 1 (Fig. 7.2), was isolated from red-colored mango peels of the cultivar ‘Tommy Atkins’ and completely identified by extensive use of 2D nuclear magnetic resonance (NMR) studies, mass spectrometry (MS), and chemical transformations (Berardini et al., 2005). This pigment has previously most probably been wrongly reported to occur in mango as peonidin 3-O-galactoside (Proctor & Creasy, 1969). The same aglycone, which has been reported to occur in 7-O-methylcyanidin 3-O-glucoside isolated from the flowers of Bombax malabaricum, was tentatively identified by paper-chromatography, IR- and UV-vis spectroscopy and chemical reactions (Niranjan & Gupta, 1973). The 3-desoxyanthocyanidin, 7-O-methylapigeninidin, has been isolated in low amounts from grains and leaf sheaths of Sorghum caudatum (Pale et al., 1997). Its UV/visible spectrum recorded in methanol with 0.1% HCl showed absorption maxima at 278.6 and 476.4 nm. A similar 3-desoxyanthocyanidin has been detected in grains of Sorghum bicolor after incubation with the fungus Colletotrichum sublineolum (Lo et al., 1996). In addition

Flowers

Leaves

Berries

Oxalidaceae Oxalis triangularis

Ruscaceae Ruscus aculeatus

Flowers

Flowers

Flowers

Orchidaceae Dendrobium spp. section Phalaenanthe Dendrobium × superbiens

Liliaceae Tricyrtis formosana ‘Fujimusume’ (toad lily) Tulipa (17 species and 25 cultivars) (tulips)

Kernel

Pg3-[6-(rha)glc], Pg3-glc, (Pg3-[6-(E-cum)glc], Pg3-[6-(Z-cum)glc])†

Mv3-[6-(4-(mal)(rha)glc] (10), Mv3-[6-(4-(mal)(rha)glc]-5-glc (11), Mv3-[6-(rha)glc]-5-[6-(mal)glc] (12), Mv3-[6-(4-(mal)(rha)glc]-5-[6-(mal)glc] (13), Mv3-[6-(Z-cum)glc]-5-glc (14), Mv3-[6-(rha)glc]5-glc, Mv3-[6-(E-cum)glc]-5-glc

(Cy3-glc-7,3′-di[X-(X-(glc)hba)glc], (Cy3-[X-(mal)glc]-7,3′-di[X-(X-(glc)hba)glc], Cy3-glc-7,3′di[X-(sin)glc], Cy3-[X-(mal)glc]-7,3′-di[X-(sin)glc])† Cy3-[6-(mal)glc]-7-[6-(hba)glc]-3′-[6-(4-(glc)hba)glc](9), Cy3-[X-(mal)glc]-7-[X-(hba)glc]-3′-[X(hba)glc]† + four anthocyanins†

Cy3-[6-(mal)glc]-8C-[6-(E-sin)glc] (2), Cy3-[6-(mal)glc]-8C-glc, Cy3-glc, Cy3-[6-(rha)glc], Cy3-[6-(mal)glc] Dp3-[6-(rha)glc], Cy3-[6-(rha)glc], Pg3-[6-(rha)glc], Cy3-[2-(ace)-6-(rha)glc], Pg3-[2-(ace)-6-(rha)glc]

Cy3-glc, Pg3-glc, Pn3-glc, Cy3-[6-(mal)glc], Cy3-[3-(mal)-6-(mal)glc], Cy3-glc, Cy3-[2-(xyl)glc], Pg3-[6-(rha)glc], Pn3-glc

Cy3-[6-(rha)glc], Cy3-glc, Cy3-[2-(xyl)glc], Pg3-[6-(rha)glc], Pn3-glc

Longo & Vasapollo, 2005b

Fossen et al., 2005

Tatsuzawa et al., 2006b

Tatsuzawa et al., 2005b

Torskangerpoll et al., 2005

Tatsuzawa et al., 2004

Moreno et al., 2005

Schauss et al., 2006

Byamukama et al., 2006

References

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Gramineae Zea mays ‘Arrocillo’, ‘Conico’, ‘Peruano’, ‘Purepecha’ (maize)

Fruits

Cy3-[6-(rha)glc], Pg3-[6-(rha)glc]

Pigment

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Arecaceae Euterpe oleracea (acai)

Flowers

Organ

Occurrence of anthocyanins reported after 2004§.

MONOCOTYLEDONEAE Amaryllidaceae Hippeastrum hybridum hort. (‘Red Lion’, ‘Royal Velvet’, ‘Liberty’, ‘Jungle Star’, ‘Magic Green’, ‘La Paz’)

Taxon

Table 7.1

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Flowers

Flowers

Evolvulus pilosus

Fruits

Flowers

Fruits & leaves

Convolvulaceae Ipomoea nil (duskish mutant)

Caprifoliaceae Sambucus spp, Lonicera spp, Viburnum spp

Campanulaceae Campanula medium

Berberidaceae Berberis spp

Leaves Peels

Pn3-[2-(6-(caf)glc)glc] (16), Pn3-[2-(6-(caf)glc)glc]-5-glc (I7), Pn3-[2-(6-(caf)glc)-6-(4-(6-(3(glc)caf)glc)caf)glc] (18), Pn3-[2-(6-(3-(glc)caf)glc)-6-(4-(6-(3-(glc)caf)glc)caf)glc] (19), Pn3-[2(6-(caf)glc)-6-(4-(6-(3-(glc)caf)glc)caf)glc]-5-gl (20) Dp3-[6-(4-(6-(4-(glc)caf)glc)caf)glc]-5-[6-(mal)glc] (22), Dp3-[6-(4-(6-(4-(glc)caf)glc)caf)glc]-5-glc (21)

Cy3-glc, Cy3-gal, Cy3-[6-(ara)glc] (6), Cy3-[6-(rha)glc], Cy3-[2-(xyl)-6-(rha)glc], Cy3-[2-(xyl)gal], Cy3-[2-(xyl)glc], Cy3-[2-(xyl)glc]-5-glc, Cy3-[2-(xyl)-6-(Z-cum)-glc]-5-glc, Cy3-[2-(xyl)-6-(E-cum)glc]-5-glc, Pg3-glc, Dp3-glc

Cy3-[6-(rha)glc]-7-[6-(4-(6-(4-(6-(hba)glc)hba)glc)hba)glc] (15), Pg3-[6-(rha)glc]-7-[6-(4-(6-(4-(6(hba)glc)hba)glc)hba)glc]

Cy3-glc, Pg3-glc, Dp3-glc, Pn3-glc, Pt3-glc, Mv3-glc

Cy3-glc, Cy3-[6-(mal)glc], Dp3-[6-(mal)glc], Cy3-glc, Cy3-glc-5-glc, Pn3-glc, (Cy3-sop, Cy3-[3-(mal)glc], Cy3,5-(mal)diglc, Cy3-[6-(mal)glc], Pn3-[3-(mal)glc], Dp-gly)†

7-O-MeCy3-gal (1), Cy3-gal

Mori et al., 2006

Saito et al., 2005

Jordheim et al., 2007

Toki et al., 2006

Sorokopudov et al., 2005

Innocenti et al., 2005 Schuetz et al., 2005

Berardini et al., 2005

Montefiori et al., 2005 Montefiori et al., 2005

Longo & Vasapollo, 2005

References

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Asteraceae Cichorium intybus (chicory) Cynara scolymus (artichoke)

Peels

(Cy3-[2-(xyl)gal], Cy3-gal)† (Cy3-gal, Cy3-glc)†

Pg3-[6-(rha)glc], Cy3-[6-(rha)glc]

Pigment

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Anacardiaceae Mangifera indica ‘Tommy Atkins’ (mango)

Fruits Fruits

DICOTYLEDONEAE Actinidiaceae Actinidia chinensis (Kiwifruit) Actinidia deliciosa (Kiwifruit)

Organ

Berries

(Cont’d )

Smilacaceae Smilax aspera

Taxon

Table 7.1

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Berries

Fruits

Elaeocarpaceae Aristotelia chilensis (maqui)

Ericaceae Arbutus unedo (strawberry tree)

Hydrophyllaceae Phacelia campanularia Flowers

Flowers

Flowers

Orychophragonus violaceus

Dp3-[6-(4-(6-(4-(glc)caf)glc)caf)glc]-5-[6-(mal)glc] (22), Dp3-[6-(4-(6-(4-(glc)caf)glc)caf)glc]-5-glc (21)

Cy3-glc, Cy3-[2-(xyl)glc], Cy3-[6-(rha)glc], Cy3-[2-(xyl)-6-(rha)glc], Cy3-[2-(xyl)-6-(rha)glc]-7-glc (8)

Cy3-gal, Cy3-ara, Dp3-glc

Dp3-glc, Dp3-glc-5-glc, Dp3-sam, Dp3-sam-5-glc, Cy3-glc, Cy3-glc-5-glc, Cy3-sam, Cy3-sam-5-glc

Mori et al., 2006

Tatsuzawa et al., 2005a

Pawlowska et al., 2006

Escribano-Bailon et al., 2006

Honda et al., 2005

Tatsuzawa et al., 2006a

Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a

Bjorøy et al., 2007

Bjorøy et al., 2007

Vareed et al., 2006

Del Caro et al., 2006

6:10 PM

Fumariaceae Corydalis elata, C. flexuosa, C. ambigua, C. lineariloba, C. decumbens, C. curvicalcarata, C. speciosa

Flowers

Lunaria annua

Cy3-gal, Dp3-gal, Pg3-gal

Fruits

Cy3-[2-(xyl)-6-(E-cum)glc]-5-glc, Cy3-[2-(xyl)-6-(Z-cum)glc]-5-glc Cy3-[2-(xyl)-6-(4-(glc)E-cum)glc]-5-glc (23), Cy3-[2-(2-(E-caf)xyl)-6-(E-cum)glc]-5-glc (24), Cy3-[2-(2-(E-fer)xyl)-6-(E-cum)glc]-5-glc (25), Cy3-[2-(2-(E-caf)xyl)-6-(4-(glc)E-cum)glc]-5-glc (26) Cy3-[2-(xyl)-6-(E-cum)glc]-5-[6-(mal)glc] (27), Cy3-[2-(xyl)-6-(Z-cum)glc]-5-[6-(mal)glc] (28), Cy3-[2-(xyl)-6-(E-fer)glc]-5-[6-(mal)glc] (29) Cy3-[2-(2-(4-(6-(4-(glc)caf)glc)caf)xyl)-6-(4-(glc)fer)glc]-5-glc (30), Cy3-[2-(2-(4-(6-(4(glc)caf)glc)caf)xyl)-6-(4-(glc)cum)glc]-5-[6-(mal)glc] (31), Cy3-[2-(2-(4-(6-(4-(glc)caf )glc)caf )xyl)-6(4-(glc)sin)glc]-5-[6-(mal)glc] (32)

Dp3-gal-3′-glc-5′-glc (3), Dp3-gal-3′-glc (4), Cy3-gal-3′-glc (5), Cy3-gal, Dp3-gal

Fruits

Flowers Flowers

Dp3-glc, Dp3-rut, Cy3-gal

Cy3-glc, Cy3-rut

Fruits

Fruits

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Cruciferae (Brassicaceae) Cheiranthus cheiri Lobularia maritima

Cornaceae Cornus alternifolia, C. controversa, C. kousa, C. florida Cornus alba ‘Sibirica’ (Siberian dogwood) Cornus mas (cornelian cherry dogwood)

Combretaceae Bucida buceras ‘Bosana’ (black olive)

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(Cont’d )

Fruits Fruits Berries

Moraceae Ficus carica (fig)

Myrtaceae Myrciaria dubia (camu-camu) Myrtus communis (common myrtle)

Rosaceae Crataegus opaca (mayhaw) Prunus armeniaca (apricot) Prunus cerasus, four cvs. (sour cherries)

Rhamnaceae Rhamnus alaternus Fruits Fruits Flowers

Berries

Fruits

Fruits

Polygonaceae Eskemukerjea megacarpum (bhote khair)

Cy3-[2-(glc)ara] (7)

Seeds

Malpighiaceae Malpighia emarginata

Cy3-glc, Cy3-gal Cy3-[6-(rha)glc], Cy3-glc Cy3-[6-(rha)glc], Cy3-[2-(glc)-6-(rha)glc]

Dp3-rut, Cy3-rut, Pt3-rut, Pg3-rut, Pn3-rut, Mv3-rut,

Cy3-gal

Cy3-glc, Dp3-glc Cy3-glc, Dp3-glc, Pn3-glc, Pt3-glc, Mv3-glc, Cy3-ara, Dp3-ara, Pt3-ara, Mv3-ara

Cy3-rut

Trappey et al., 2005 Ruiz et al., 2005 Simunic et al., 2005

Longo et al., 2005

Miyaichi et al., 2006

Zanatta et al., 2005 Montoro et al., 2006

Solomon et al., 2006

Hanamura et al., 2005

Takeoka et al., 2005

Kazuma et al., 2004 Kim et al., 2005

Longo & Vasapollo, 2005a Ashton et al., 2006

References

6:10 PM

Cy3-rh, Pg3-rh

Dp3-[6-(mal)glc]-3′-glc (33) Dp3-glc, Cy3-glc, Pt3-glc,

Cy3-glc, Cy3-rut, Pn3-glc, Pn3-rut Cy3-glc

Pigment

Flowers Seeds

Berries Fruits

Organ

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Leguminosae Clitoria ternatea (butterfly pea) Glycine max (59 Korean varieties) (black soybeans) Lens culinaris (lentils)

Lauraceae Laurus nobilis (bay) Persea americana (avocado)

Taxon

Table 7.1

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Seeds

Cy3-gal, Cy3-ara

Niemenak et al., 2006

Ueyama et al., 2006 Tatsuzawa & Ando, 2005 Ichiyanagi et al., 2005 Eichhorn & Winterhalter, 2005

ace, acetyl; mal, malonyl; hba, p-OH-benzoyl; gao, galloyl (tri-OH-benzoyl); cum, p-coumaroyl; caf, caffeoyl; fer, feruloyl; sin, sinapoyl; gly, glycosyl; ara, arabinosyl; xyl, xylosyl; rha, rhamnosyl; gal, galactosyl; glc, glucosyl; glu, glucuronosyl; 2-(xyl)glc, sambubiosyl; 2-(xyl)gal, lathyrosyl; 6-(rha)glc, rutinosyl; 2-(glc)glc, sophorosyl; 3-(glc)glc, laminariobiosyl. See Table 7.3 for anthocyanidin abbreviations and linkage positions. § Numbers in bold represent new anthocyanins not previously listed by Andersen & Jordheim, 2006. See Figs 7.2, 7.7 and 7.8 for structures of new anthocyanins. † Incompletely defined structures. * Implausible structure.

Sterculiaceae Theobroma cacao L. (cacao)

Cy3-glc, Cy3-[2-(xyl)glc], Cy3-[X-(xyl)-6-(rha)glc], Cy3-[6-(rha)glc], Pg3-[6-(rha)glc] Cy3-[6-(rha)glc], Cy3-glc Cy3-gal, Cy3-glc, Cy3-ara

Berries Berries Berries

Dp3-[6-(4-(4-(6-(caf)glc)cum)rha)glc]-5-glc Mv3-glc, Pt3-glc Mv3-[6-(4-(E-p-cum)(rha)glc]-5-glc, Mv3-[6-(4-(Z-p-cum)(rha)glc]-5-glc Pt3-[6-(X-(p-cum)(rha)glc]-5-glc, Pl3-[6-(X-(p-cum)(rha)glc]-5-glc, Pn3-[6-(X-(p-cum)(rha)glc]-5-glc, Mv3-[6-(X-(p-cum)(rha)glc]-5-glc, Pt3-[6-(rha)glc]-5-glc, Pl3-[6-(rha)glc]-5-glc, Pn3-[6-(rha)glc]-5-glc, Mv3-[6-(rha)glc]-5-glc

Cy3-[2-(glc)glc], Cy3-[X-(glc)-6-(rha)glc]

Berries

6:10 PM

Flowers Flowers Fruit Tubers

Fan-Chiang & Wrolstad, 2005 Mcghie et al., 2006

Cy3-glc, Cy3-[6-(rha)glc], Cy3-xyl, Cy3-[6-(mal)glc] + one anthocyanins†

Berries

Tian et al., 2006 Byamukama et al., 2005 Hukkanen et al., 2006

Zhao et al., 2006 Novruzov, 2005

Cy3-[6-(rha)glc], Cy3-[3-(6-(gao)glc)glc] (34)* Cy3-glc, Cy3-glc-5-glc, Pn3-glc-5-glc

Flowers Flowers

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Solanaceae Nierembergia sp. Petunia sp. (petunia) Solanum melongena (eggplant) Solanum tuberosum (‘Hermanns Blaue’, ‘Highland’, ‘Burgundy Red’, ‘Shetland Black’, ‘Vitelotte’ (potato))

Rubus loganbaccus x R. baileyanus (boysenberry) Rubus occidentalis (black raspberry) Rubus pinnatus, R. rigidus Sorbus aucuparia (various cvs.) (rowanberries)

Prunus mume ‘Nanjing Hongxu’ Rosa foetida, R. spinosissima, R. canina, R. corymbifera, R. azerbaidzhanica, R. iberica, R. komarovii, R. nisami, R. prilipkoana, R. cuspidata, R. tomentosa, R. pulverulenta Rubus sp. (blackberry)

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7-O-methyl-Cy 3-galactoside Cy 3-[6-(malonyl)glucoside]-8C-[6-(E-sinapoyl)glucoside] Cy 3-galactoside-3′-glucoside Dp 3-galactoside-3′-glucoside Dp 3-galactoside-3′-glucoside-5′-glucoside Cy 3-[6-(arabinosyl)glucoside] Cy 3-[2-(glucosyl)arabinoside] Cy 3-[2-(xylosyl)-6-(rhamnosyl)glucoside]-7-glucoside Cy 3-[6-(malonyl)glucoside]-7-[6-(hydroxybenzoyl)glucoside]-3′-[6-(4-(glucosyl)hydroxybenzoyl) glucoside] Mv 3-[6-(4-(malonyl)(rhamnosyl)glucoside] Mv 3-[6-(4-(malonyl)(rhamnosyl)glucoside]-5-glucoside Mv 3-[6-(rhamnosyl)glucoside]-5-[6-(malonyl)glucoside] Mv 3-[6-(4-(malonyl)(rhamnosyl)glucoside]-5-[6-(malonyl)glucoside] Mv 3-[6-(Z-p-coumaryl)glucoside]-5-glucoside Cy 3-[6-(rhamnosyl)glucoside]-7-[6-(4-(6-(4-(6-(hydroxybenzoyl)glucosyl)hydroxybenzoyl)glucosyl) hydroxybenzoyl)glucoside] Pn 3-[2-(6-(caffeoyl)glucosyl)glucoside] Pn 3-[2-(6-(caffeoyl)glucosyl)glucoside]-5-glucoside Pn 3-[2-(6-(caffeoyl)glucosyl)-6-(4-(6-(3-(glucosyl)caffeoyl)glucosyl)caffeoyl)glucoside] Pn 3-[2-(6-(3-(glucosyl)caffeoyl)glucosyl)-6-(4-(6-(3-(glucosyl)caffeoyl)glucosyl)caffeoyl)glucoside] Pn 3-[2-(6-(caffeoyl)glucosyl)-6-(4-(6-(3-(glucosyl)caffeoyl)glucosyl)caffeoyl)glucoside]-5-glucoside Dp 3-[6-(4-(6-(4-(glucosyl)caffeoyl)glucosyl)caffeoyl)glucoside]-5-glucoside Dp 3-[6-(4-(6-(4-(glucosyl)caffeoyl)glucosyl)caffeoyl)glucoside]-5-[6-(malonyl)glucoside] Cy 3-[2-(xylosyl)-6-(4-(glucosyl)E-p-coumaryl)glucoside]-5-glucoside Cy 3-[2-(2-(E-caffeoyl)xylosyl)-6-(E-p-coumaryl)glucoside]-5-glucoside Cy 3-[2-(2-(E-feruloyl)xylosyl)-6-(E-p-coumaryl)glucoside]-5-glucoside] Cy 3-[2-(2-(E-caffeoyl)xylosyl)-6-(4-(glucosyl)E-p-coumaryl)glucoside]-5-glucoside Cy 3-[2-(xylosyl)-6-(E-p-coumaryl)glucoside]-5-[6-(malonyl)glucoside] Cy 3-[2-(xylosyl)-6-(Z-p-coumaryl)glucoside]-5-[6-(malonyl)glucoside] Cy 3-[2-(xylosyl)-6-(E-feruloyl)glucoside]-5-[6-(malonyl)glucoside] Cy 3-[2-(2-(4-(6-(4-(glucosyl)caffeoyl)glucosyl)caffeoyl)xylosyl)-6-(4-(glucosyl)feruloyl)glucoside]5-glucoside Cy 3-[2-(2-(4-(6-(4-(glucosyl)caffeoyl)glucosyl)caffeoyl)xylosyl)-6-(4-(glucosyl)pcoumaryl)glucoside]-5-[6-(malonyl)glucoside] Cy 3-[2-(2-(4-(6-(4-(glucosyl)caffeoyl)glucosyl)caffeoyl)xylosyl)-6-(4-(glucosyl)sinapoyl)glucoside]5-[6-(malonyl)glucoside] Dp 3-[6-(malonyl)glucoside]-3′-glucoside Cy 3-[3-(6-(galloyl)glucosyl)glucoside]*

1 2 3 4 5 6 7 8 9

Saito et al., 2005 Saito et al., 2005 Saito et al., 2005 Saito et al., 2005 Saito et al., 2005 Mori et al., 2006 Mori et al., 2006 Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Tatsuzawa et al., 2006a Honda et al., 2005 Honda et al., 2005 Honda et al., 2005 Kazuma et al., 2004 Zhao et al., 2006

Ipomoea nil Ipomoea nil Ipomoea nil Ipomoea nil Ipomoea nil Phacelia campanularia Phacelia campanularia Lobularia maritima Lobularia maritima Lobularia maritima Lobularia maritima Lunaria annua Lunaria annua Lunaria annua Orychophragonus violaceus Orychophragonus violaceus Orychophragonus violaceus Clitoria ternatea Prunus mume

C37H39O19 C43H49O24 C67H71O35 C73H81O40 C73H81O40 C57H63O33 C60H65O36 C47H55O27 C50H51O25 C51H53O25 C56H61O30 C44H47O25 C44H47O25 C45H49O26 C78H89O44 C80H89O46 C82H93O48 C30H33O20 C34H35O20

See Table 7.3 for anthocyanidin abbreviations. † Not listed previously by Andersen and Jordheim, 2006. * Implausible structure. See Figs 7.2, 7.7 and 7.8 for structures of new anthocyanins. Rosacyanins A1 and A2 with rosacyanin B linked covalently to ellagitannins are not included in this table.

33 34

32

31

Bjorøy et al., 2007 Bjorøy et al., 2007 Bjorøy et al., 2007 Bjorøy et al., 2007 Bjorøy et al., 2007 Toki et al., 2006

Oxalis triangularis Oxalis triangularis Oxalis triangularis Oxalis triangularis Oxalis triangularis Campanula medium

C32H37O19 C38H47O24 C38H47O24 C41H49O27 C38H41O19 C66H73O36

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16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Berardini et al., 2005 Tatsuzawa et al., 2004 Vareed et al., 2006 Vareed et al., 2006 Vareed et al., 2006 Jordheim et al., 2007 Takeoka et al., 2005 Tatsuzawa et al., 2005a Tatsuzawa et al., 2006b

Mangifera indica Tricyrtis formosana Cornus alba ‘Sibirica’ Cornus alba ‘Sibirica’ Cornus alba ‘Sibirica’ Viburnum opulus Lens culinaris Corydalis spp Dendrobium × superbiens

C22H23O11 C41H42O24 C27H31O16 C27H31O17 C33H41O22 C26H29O15 C26H29O15 C40H53O26 C56H61O33

Reference

Source

Molecular formula

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10 11 12 13 14 15

Compound

New anthocyanins reported after 2004†.

No.

Table 7.2

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Structures of naturally occurring anthocyanidinsa.

Table 7.3

3′ 4′ 9 8

3′ 4′ 8 7

9 O

+

6 5

10

1′

+

9b

7 6a 6O

5′

2 3

9a O

4

O

1′

5′

2 3

3a 4 5 OH

Substitution pattern Anthocyanidinsb

3

5 (6a)

6 (7)

7 (8)

3′

4′

5′

Common anthocyanidins Pelargonidin (Pg) Cyanidin (Cy) Delphinidin (Dp) Peonidin (Pn) Petunidin (Pt) Malvidin (Mv)

OH OH OH OH OH OH

OH OH OH OH OH OH

H H H H H H

OH OH OH OH OH OH

H H OH OMe OMe OMe

OH OH OH OH OH OH

H H OH H OH OMe

Rare methylated anthocyanidins 5-MethylCy 7-MethylCyc 7-MethylPn (Rosinidin) 5-MethylDp (Pulchellidin) 5-MethylPt (Europinidin) 5-MethylMv (Capensinidin) 7-MethylMv (Hirsutidin)

OH OH OH OH OH OH OH

OMe OH OH OMe OMe OMe OH

H H H H H H H

OH OMe OMe OH OH OH OMe

OH OH OMe OH OMe OMe OMe

OH OH OH OH OH OH OH

H H H OH OH OMe OMe

6-hydroxylated anthocyanidins 6-HydroxyPg 6-HydroxyCy 6-HydroxyDp

OH OH OH

OH OH OH

OH OH OH

OH OH OH

H OH OH

OH OH OH

H H OH

H H H H H H

OH OH OH OH OMe OMe

H H H H H OH

OH OH OH OMe OH OH

H OH OH H OH H

OH OH OH OH OH OH

H H OH H H H

H

OMe

OH

OH

H

OMe

H

H H

OMe OMe

OH OH

OH OH

OH OH

OH OMe

H H

OH OH

OO-

H H

OH OH

H OH

OH OH

H H

3-Desoxyanthocyanidins Apigeninidin (Ap) Luteolinidin (Lt) Tricetinidin (Tr) 7-MethylApc 5-MethylLtc 5-Methyl-6-hydroxyAp (Carajurone)c 5,4′-Dimethyl-6-hydroxyAp (Carajurin) 5-Methyl-6-hydroxyLtc 5,4′-Dimethyl-6-hydroxyLtc Pyranoanthocyanidins 5-CarboxypyranoPgc 5-CarboxypyranoCyc,d a

The numbering of the structures on the left and right is used for anthocyanins and pyranoanthocyanins, respectively. b Riccionidins A and B and Sphagnorubins A–C have not been included. c New anthocyanidins (reported 1996 –2006). d See Fig. 7.3 for rosacyanin B.

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Fig. 7.2 Structures of new anthocyanins reported in the recent literature. See Table 7.2 for references. glc, glucosyl.

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to plasma desorption MS data, bathochromic shift analyses indicated that the structure of the compound was consistent with that of 5-O-methylluteolinidin. The spectrum of this phytoalexin, which showed greater fungitoxicity than luteolinidin, revealed an absorption maximum at 495 nm. Although the synthesis of the desoxyanthocyanidin carajurin, 6,7-dihydroxy-5,4′dimethoxy-flavylium, (isolated from leaves of Arrabidaea chica) was published in 1953 (Ponniah & Seshadri), the structure of this pigment was considered to be only partially described (Timberlake & Bridle, 1975; Mazza & Miniati, 1993). Later two groups (Zorn et al., 2001; Devia et al., 2002) nearly simultaneously confirmed the structure of carajurin – even by presenting a crystal structure (Devia et al., 2002). The structure of carajurone was revised to be 6,7,4′-trihydroxy-5-methoxy-flavylium (Zorn et al., 2001). Additionally, two new 3-desoxyanthocyanidins, 6,7,3′-trihydroxy-5,4′-dimethoxy-flavylium (Zorn et al., 2001) and 6,7,3′,4′-tetrahydroxy-5-methoxy-flavylium (Zorn et al., 2001; Devia et al., 2002) were isolated from theses leaves, which are traditionally used by some indigenous populations of South America for body painting and for dyeing fibres. There have been many reports on the isolation and identification of pyranoanthocyanins from wines, juices and other processed foodstuff (Vivar-Quintana et al., 2002; Schwarz et al., 2003, 2004; Hillebrand et al., 2004; Rein et al., 2005; Cheynier, 2006; Cheynier et al., 2006; De Freitas & Mateus, 2006), but only a few reports related to fresh material. The first pyranoanthocyanidin isolated from intact plants, rosacyanin B (35) (Fig. 7.3) was isolated in small amounts from the petals of Rosa hybrida cv. ‘M’me Violet’ (Fukui et al., 2002). Rosacyanin B is very stable in acidic alcoholic solutions, however, under neutral or weakly acidic aqueous conditions it is precipitated before forming the colorless pseudobase. The structure of this violet pigment, which was revealed mainly by highresolution fast atom bombardment mass spectrometry (FAB MS) and NMR, contains no sugar units. However, in a recent paper the same group showed that rosacyanin B was

Fig. 7.3 Rosacyanin B (35) has been isolated from the violet petals of Rosa hybrida cv. ‘M’me Violet’ (Fukui et al., 2002). The 3-glucoside (37) and 3-[6-(malonyl)glucoside] (38) of 5-carboxypyranocyanidin were isolated from extracts of red onion (Fossen & Andersen, 2003), while 5-carboxypyranopelargonidin 3-glucoside (36) has been isolated from extracts of strawberries (Andersen et al., 2004). mal, malonyl.

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conected to tannins in two novel pigments, rosacyanins A1 and A2 (Fukui et al., 2006). These blue pigments had a common chromophore, which contained cyanidin with a galloyl group link between positions 4 and 5 of the hydroxy group of the flavylium nucleus, and tellimagrandin 1 and 2, respectively, at C-3 of the cyanidin nucleus. These are the first known natural pigments in which the flavylium nucleus binds to ellagitannins. Four anthocyanins with the same aglycone, 5-carboxypyranocyanidin, have been isolated from acidified, methanolic extracts of the edible scales as well as from the dry outer scales of red onion (Allium cepa) (Fossen & Andersen, 2003). Two of the structures were elucidated by 2D NMR spectroscopy and liquid chromatography–MS as the 3-glucoside (37) and 3-[6-(malonyl)glucoside] (38) of this 4-substituted aglycone (Fig. 7.3). The two analog pigments methylated at either the terminal carboxyl group of the acyl moiety, or at the aglycone carboxyl, were most probably formed by esterification of 38 with the solvent (acidified methanol) during the isolation process. Another 3-glucoside (36) with the new 4-substituted aglycone, 5-carboxypyranopelargonidin, has been isolated in small amounts from an acidified methanolic extract of strawberries (Fragaria × ananassa) (Andersen et al., 2004) (Fig. 7.3). By comparison of UV-Vis absorption spectra, 36 showed in contrast to ordinary pelargonidin 3-glucoside a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 36 in various acidic and neutral buffer solutions implied restricted formation of the unstable colorless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity of 36 varied little with pH contrary to the behavior of, for example, pelargonidin 3-glucoside. Four reported methylpyranoanthocyanins isolated from blackcurrant seeds (Lu et al., 2000) were later shown to be the oxidative cycloaddition products of the extraction solvent (acetone) and the natural anthocyanins (Lu & Foo, 2001). Even though anthocyanidins, but not desoxyanthocyanidins, now and then have been reported to occur in vivo as free aglycones, these anthocyanidins have normally been treated as artifacts formed during the extraction and/or isolation stages. Recently, the natural presence of cyanidin, peonidin and pelargonidin in extracts of beans has been suggested after careful consideration of the processes of extraction and purification followed by application of LC–MS for identification purposes (Macz-Pop et al., 2006b).

7.3 Anthocyanidin equilibrium forms For simple anthocyanins it is well known that each anthocyanidin is involved in a series of equilibria giving rise to different forms, which exhibit their own properties including color. Thermodynamic and kinetic studies have led to a generally accepted scheme with respect to the different transformations of the flavylium cation of simple anthocyanins under various pH conditions (Brouillard & Dangles, 1994; Pina, 1998). The flavylium cation is the predominant equilibrium form in strongly acidic solutions. In addition, the existence of tautomeric quinonoidal bases derived from the flavylium cation by deprotonation, and hemiacetal and chalcone forms related to the flavylium form by nucleophilic reaction with water, under various pH conditions, have been described. The various forms – including the colorless hydroxy-adducts known as carbinol bases, pseudobases, or hemiacetals – have

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been examined using pH-jump methods, UV–visible and fluorescence spectroscopy, and NMR spectroscopy (Cheminat & Brouillard, 1986; Mistry et al., 1991; Santos et al., 1993; Terahara et al., 1993; Brouillard & Dangles, 1994; Bakker et al., 1997). However, experimental proof for accurate determination of the forms other than the flavylium cation has been limited. Recently, the hemiacetal forms of the 3-glucosides of delphinidin, petunidin and malvidin, and cyanidin 3-galactoside, dissolved in deuterated methanolic solutions were characterized as two epimeric 2-hydroxy-hemiacetals on the basis of assignments of both proton and carbon NMR signals together with chemical shift considerations (Jordheim et al., 2006). No 4-hydroxy-hemiacetal form was detected for any of the pigments. For each anthocyanin dissolved in deuterated methanol, the equilibrium between each of the two epimeric hemiacetals and the corresponding flavylium cation was confirmed by the observed positive exchange cross-peaks in the 2D 1H NOESY1 spectra. The molar proportions of the flavylium cation and the two hemiacetals forms of the four pigments in deuterated methanol were very similar for all pigments, even after storage for weeks. However, when the 1D 1H NMR spectrum of a covalently linked anthocyanin–flavone C-glycoside, (43) (Fig. 7.4), dissolved in CD3OD–CF3CO2D (95:5) was recorded 45 min after sample preparation, this pigment occurred mainly as flavylium cation (38%) and two equilibrium forms assigned to be quinonoidal bases (54%) (Fossen et al., 2007). Only minor amounts of the two hemiacetal forms were present. After five days storage at 300 K, the hemiacetals (39%) and flavylium cation (38%) constituted the main forms of 43. Most simple anthocyanins are normally considered to be in the flavylium cation form in this acidified deuterated methanolic solvent (Jordheim et al., 2006). The cross-peaks observed in the NOESY NMR spectra of 39 (Fig. 7.5) indicated the presence of vertical ‘π–π’ stacking between the B-ring of the flavone unit and the A-ring of each of the two forms assigned to be quinonoidal bases (Fossen et al., 2007). It was not possible to discriminate between inter- or intramolecular association mechanisms. The equilibria between the various forms of 43 were studied by two-dimensional NOESY and ROESY2 NMR spectroscopy. 2D HSQC-TOCSY3 NMR spectroscopy was among the methods used for characterization of the various forms. Structural transformations of cyanidin 3-glucoside, from the flavylium cation through the colorless hemiacetal and chalcone forms to the mixture of quinoidal-bases, have also been characterized on the basis of thermochemical parameters calculated by the functional theory B3LYP4 method (Borkowski et al., 2005). The results showed that the stabilization of the flavylium cation by the net of intramolecular H-bonds within the sugar moiety and between the sugar and the anthocyanidin molecule affected its structural transformations. In a re-examination, the intricate pH-dependent set of chemical reactions involving synthetic flavylium compounds (e.g. 4′-hydroxyflavylium) have been confirmed to be basically identical to those of natural anthocyanins (e.g. malvidin 3,5-diglucoside) in acidic and neutral media (Pina et al., 2003). For each process a kinetic expression has been deduced allowing calculation of all the equilibrium constants and most of the rate constants in the system. By a systematic investigation of photochemical and thermal reactions it was shown that 4′,7-dihydroxyflavylium (AH+) in water–ionic liquid biphasic systems can be used as a write-read-erase system (Pina et al., 2003). In acid media, the trans-chalcone form

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Fig. 7.4 (4″″-(malvidin 3-[6″-(rhamnosyl)glucoside]-5-glucoside)) (6′″-(apigenin 6-C-[2″-(glucosyl) glucoside]))malonate (43) from purple leaves of Oxalis triangularis, (6′″-(delphinidin 3-[6″-(glucosyl) glucoside])) (6″-(apigenin 7-glucoside))malonate (44) and (6′″-(delphinidin 3-[6″-(glucosyl)glucoside])) (6″-(luteolin 7-glucoside))malonate (45) from blue-violet flowers of Eichhornia crassipes, (6″-(cyanidin 3-glucoside)) (2″″-(kaempferol 3-[2″-(glucosyl)(glucoside)]-7-glucuronoside))malonate (46) and (6″-(cyanidin 3-[3″-(acetyl)glucoside])) (2″″-(kaempferol 3-[2″-(glucosyl)(glucoside)]-7-glucuronoside))malonate (47) from pale-purple flowers of chive (Allium schoenoprasum), and (6′″-(delphinidin 3-[6″-(p-coumaryl)glucoside]7-glucoside)) (6″″-(kaempferol 3-glucoside-7-xyloside-4′-glucoside))succinate (48) and (6′″-(delphinidin 3-[6″-(p-coumaryl)glucoside]-7-glucoside)) (6″″-(kaempferol 3-glucoside-7-glucoside-4′-glucoside))succinate (49) from blue Agapanthus flowers.

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Fig. 7.5 Afzelechin(4α→8)Pg3glc (39), epiafzelechin (4α→8)Pg3glc (40), catechin(4α→8)Pg3glc (41), epicatechin(4α→8)Pg3glc (42) isolated from extracts of strawberries. Pg3glc, pelargonidin 3-glucoside.

was soluble in ionic liquids, thermally metastable, but photochemically reactive (write) to give the yellow flavylium salt, which may be read optically without being erased. The system was prepared for a new cycle by two consecutive ‘pH jumps’ (Pina et al., 2004). These results are very interesting since the flavylium compounds represent examples of multistate/multifunctional chemical systems that may be used for information processing at the molecular level according to principles similar to those that govern information transfer in living organisms. In particular, flavylium compounds can behave as optical memories and logic gate systems: a write-read-erase molecular switch. Another interesting anthocyanin application involves the blue-violet anthocyanin extracts of Jaboticaba (Myrtus cauliflora) and Calafate (Berberis buxifolia) as TiO2 dyesensitizers (Polo et al., 2006). The results show a successful conversion of visible light into electricity by using natural dyes as wide band-gap semiconductor sensitizers in dye-sensitized solar cells. The results indicate an environmentally friendly alternative for dye-sensitized solar cells with low-cost production and an excellent system for educational purposes.

7.4 New anthocyanin glycosides Although most new anthocyanins discovered during the last decade have been based on the six common anthocyanidins (Table 7.3), some exceptions with limited distribution have been reported. The major anthocyanin in peel of the red-colored mango cultivar ‘Tommy Atkins’ has been identified as 7-O-methylcyanidin 3-galactopyranoside, 1 (Fig. 7.2), (Berardini et al., 2005). The new 3-[6-(p-coumaryl)glucoside] and 3-glucoside of hirsutidin (Table 7.3) together with the known corresponding petunidin and malvidin derivatives have been identified in extracts of both cell suspensions and fresh flowers of Catharanthus roseus (Piovan et al., 1998). The extracts were analyzed by electrospray MS, and collision experiments were performed on molecular ions by means of ion trap facilities. In flowers of

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various Alstroemeria cultivars the 3-[6-(malonyl)glucoside] of 6-hydroxycyanidin and 6hydroxydelphinidin, the 3-glucoside of 6-hydroxydelphinidin and 6-hydroxypelargonidin, in addition to 6-hydroxydelphinidin 3-glucoside have been identified (Nørbæk et al., 1996; Tatsuzawa et al., 2001, 2002, 2003). The positions of the 6-hydroxyl group of these anthocyanidins were unambiguously assigned by homo- and heteronuclear NMR techniques (Nygård et al., 1997). The major 3-deoxyanthocyanin isolated from the fern Blechnum novae-zelandiae has been determined to be luteolinidin 5-[3-(glucosyl)-2-(acetyl)glucoside] by HPLC, NMR (1D, 2D) and electrospray MS (Swinny, 2001). Anthocyanins have glycosidic moieties in the anthocyanidin 3-, 5-, 7-, 3′- or 5′-position. With the exception of the desoxyanthocyanins, nearly all anthocyanins have a sugar located at the 3-position. The only exceptions are the 3′-[2-(galloyl)galactoside] and 3′-[2-(galloyl)6-(acetyl)galactoside] of delphinidin isolated from blue flowers of the African water lily Nymphaéa caerulea (Fossen & Andersen, 1999), and the 4′-glucoside and 7-[3-(glucosyl)6-(malonyl)glucoside]-4′-glucoside of cyanidin from red onion (Allium cepa) (Fossen et al., 2003). Several anthocyanidin 5-glycosides and anthocyanidin 7-glycosides without sugar in their 3-positions have been reported (Harborne & Baxter, 1999). However, all of these may be classified as tentative structures owing to there being limited data for complete identification of the linkage positions of the sugar group. The sugar moieties of anthocyanins are normally connected to the anthocyanidins through O-linkages. However, both cyanidin 3-O-[6-O-(malonyl)-β-glucopyranoside]-8-C-βglucopyranoside and cyanidin 3-O-[6-O-(malonyl)-β-glucopyranoside]-8-C-[6-O-(transsinapoyl)-β-glucopyranoside] (2, Fig. 7.2) have been isolated from the purple flowers of Tricyrtis formosana cultivar Fujimusume (Liliaceae) together with four known common cyanidin derivatives (Saito et al., 2003; Tatsuzawa et al., 2004). The anthocyanin monosaccharides are represented by glucose, galactose, rhamnose, arabinose, xylose and glucuronic acid; no new monosaccharide attached to anthocyanidins has been reported during the last decade. Three new anthocyanins, cyanidin 3-O-βgalactopyranoside-3′-O-β-glucopyranoside (3), delphinidin 3-O-β-galactopyranoside-3′-O-βglucopyranoside (4) and delphinidin 3-O-β-galactopyranoside-3′,5′-di-O-β-glucopyranoside (5) (Fig. 7.2), together with the 3-galactosides of delphinidin and cyanidin, have recently been isolated from the bluish-white berries and compound umbel of Siberian dogwood, Cornus alba ‘Sibirica’ (Bjorøy et al., 2007). Anthocyanins with sugars moieties in both the 3′- and 5′-positions have previously been isolated only from Leguminosae, Lobeliaceae and Liliaceae (Andersen & Jordheim, 2006). Most anthocyanins contain two, three or just one monosaccharide unit. No tetrasaccharide has yet been found in an anthocyanin, however, as many as seven monosaccharide units have been found in ternatin A1 (Clitoria ternatea) (Terahara et al., 1990) and cyanodelphin (Delphinium hybridum) (Kondo et al., 1991). Altogether 260 anthocyanins have been reported to contain a disaccharide. In recent years Yoshida et al. (2002) have isolated delphinidin 3-[6-(E-p-coumaryl)glucoside]-5[6-(malonyl)-4-(rhamnosyl)-glucoside] (muscarinin A) from purplish-blue spicate flower petals of Muscari armeniacum (Liliaceae), which contained an interesting 1→4 linkage between the rhamnose and one of the glucose units. Another new disaccharide moiety, 2glucosylgalactose, has been found in pelargonidin 3-[2-(2-E-caffeyl)glucosyl)-galactoside] isolated from the sepals of Pulsatilla cernua (Ranunculaceae) (Yoshitama et al., 1997), in the major anthocyanin cyanidin 3-[2-(glucosyl)galactoside], isolated from the scarlet

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fruits of Cornus suecica (Cornaceae) (Slimestad & Andersen, 1998), and in seven exciting acylated cyanidin and delphinidin derivatives from flowers of Anemone coronaria (Saito et al., 2002; Toki et al., 2003). The identification of cyanidin 3-O-[6-O-(α-arabinopyranosyl)β-glucopyranoside], 6 (Fig. 7.2) – isolated from Viburnum opulus – is the first complete identification of the disaccharide vicianose linked to an anthocyanidin (Jordheim et al., 2007), while the report of delphinidin 3-O-[2-O-(β-d-glucopyranosyl)-α-l-arabinopyranoside] 7 (Fig. 7.2), isolated from Beluga black lentils, is the first identification of this disaccharide connected to an anthocyanidin (Takeoka et al., 2005). Altogether 25 anthocyanins based on the seven trisaccharide moieties 2-glucosyl6-rhamnosylglucose, 2-xylosyl-6-rhamnosylglucose, 2-xylosyl-6-glucosylgalactoside, 2xylosyl-6-glucosylglucose, 6-(6-glucosylglucosyl)glucose, 3-(3-glucosylglucosyl)glucose and 3-glucosyl-6-glucosylglucose, have been identified. Among the new anthocyanins containing a trisaccharide reported during the last decade, cyanidin 3-[2-(xylosyl)-6(rhamnosyl)glucoside]-7-glucoside has been found in the flowers of Corydalis elata and C. flexuosa cultivars (Tatsuzawa et al., 2005a), 8 (Fig. 7.2), while the 3-[6-(rhamnosyl)2-(xylosyl)glucosides] of petunidin and peonidin has been isolated in minor amounts from fruits of Vaccinium padifolium (Cabrita et al., 2000).

7.5 New anthocyanin acylglycosides Anthocyanin diversity is highly associated with the nature, number and linkage positions of the acyl groups, and around 66% of the reported anthocyanins with properly identified structures have one or more acyl moieties linked to their sugar unit(s). As many as 301 anthocyanins have aromatic acylation, which includes various hydroxycinnamic acids (pcoumaric, caffeic, ferulic, sinapic and 3,5-dihydroxycinnamic acids) and two hydroxybenzoic acids (p-hydroxybenzoic and gallic acids). Malonic acid, which is identified in 25% of the anthocyanins, is the most frequent acyl moiety of anthocyanins. This acyl unit plus acetic, malic, oxalic, succinic, and tartaric acids comprise the various aliphatic acyl moieties, of which one or more have been identified in 191 anthocyanins. The only anthocyanins found conjugated with sulfate, malvidin 3-glucoside-5-[2-(sulfato)glucoside] and malvidin 3-glucoside-5-[2-(sulfato)-6-(malonyl)glucoside], have been isolated from violet flowers of Babiana stricta (Iridaceae) (Toki et al., 1994c). As many as four different acyl groups located at four different glycosyl moieties have been identified in Lobelinin B isolated from the flowers of Lobelia erinus (Kondo et al., 1989). The numbers of anthocyanins containing the various acyl moieties are presented in Fig. 7.6. Anthocyanin diversity is highly associated with the nature, number and linkage positions of the acyl groups. More details regarding the acyl groups of anthocyanins, including some chemotaxonomic considerations, are treated elsewhere (Honda & Saito, 2002; Andersen & Jordheim, 2006). Here follow references to new acylated anthocyanins that have not been reviewed previously (Andersen & Jordheim, 2006). Within monocotyledonea four new acylated malvidin 3-rutinosides (10–13) (Fig. 7.7), malvidin 3-[6-(Z-p-coumaryl)glucoside]-5-glucoside (14) two known malvidin derivatives have been isolated from the purple leaves of Oxalis triangularis (Oxalidaceae) (Fossen et al., 2005). In the pigments 10–13 a malonyl unit is linked to the rhamnose 4-position,

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160 140 120 100 80 60 40 20 0 cum

caf

fer

sin

hca

hba

gao

mal

ace

mly

suc

tar

oxa

sul

Fig. 7.6 Numbers of anthocyanins containing the various acyl moieties identified in anthocyanins. cum, p-coumaryl; caf, caffeoyl; fer, feruloyl; sin, sinapoyl; hca, 3,5-dihydroxycinnamoyl; hba, p-hydroxybenzoyl; gao, galloyl; mal, malonyl; ace, acetyl; mly, malyl; suc, succinyl; tar, tartaryl; oxa, oxalyl; sul, sulfate.

which has not been reported previously for any anthocyanin. The identifications were mainly based on 2D NMR spectroscopy and electrospray MS. In the Orchidaceae the new anthocyanins Cy 3-[6-(malonyl)glucoside]-7-[6-(hydroxybenzoyl)glucoside]-3′-[6(4-(glucosyl)hydroxybenzoyl)glucoside] (9, Fig. 7.2) and cyanidin 3-malonylglucoside-7glucosyl-p-hydroxybenzoylglucoside-3′-p-hydroxybenzoylglucoside (tentatively identified) have been isolated from the purple flowers of Dendrobium × superbiens, along with four known pigments (Tatsuzawa et al., 2006b), while cyanidin 3-[6-(malonyl)glucoside]-8C-[6-(E-sinapoyl)-glucoside] (2, Fig. 7.2) has been isolated from the purple flowers of Tricyrtis formosana cultivar Fujimusume (Liliaceae), together with five known pigments (Saito et al., 2003; Tatsuzawa et al., 2004). Within the dicotyledoneae the new major anthocyanin cyanidin 3-[6-(rhamnosyl)glucoside]7-[6-(4-(6-(4-(6-(hydroxybenzoyl)glucosyl)hydroxybenzoyl)glucosyl)hydroxybenzoyl) glucoside], 15 (Fig. 7.7), has together with its known pelargonidin analog rubrocampanin, been isolated from the red-purple flowers of Campanula medium (Campanulaceae) (Toki et al., 2006), while a postulated intermediate in the biosynthesis of ternatins, delphinidin 3-[6-(malonyl)glucoside]-3′-glucoside, 33 (Fig. 7.8), has been found to occur in the blue petals of Clitoria ternatea (Leguminosae) (Kazuma et al., 2004). In the Convolvulaceae five new acylated peonidin glycosides (16–20) (Fig. 7.7) have been isolated as major pigments from the pale gray-purple flowers of Japanese morning glory (Ipomoea nil, duskish mutant), along with the known anthocyanin Heavenly Blue Anthocyanin (Saito et al., 2005). Mainly based on HR-FABMS and 1D- and 2D 1H NMR it was found that 16, 18 and 19 were based on peonidin 3-sophoroside, while 17 and 20 contained peonidin 3-sophoroside-5-glucoside as their deacylanthocyanins. All the anthocyanins were acylated with caffeic acid and/or

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Fig. 7.7 Structures of some new anthocyanins reported in the recent literature. See Table 7.2 for references. caf, caffeoyl; glc, glucosyl; mal, malonyl.

glucosylcaffeic acid. Several anthocyanins from species within each of the families Compositae, Orchidaceae, Solanaceae and Convolvulaceae have a glucosyl in the phenolic 4-OH group of caffeic acid, making a chain of alternating glucosyl-caffeoyl-glucosyl-units (Andersen & Jordheim, 2006). In fifteen previously reported anthocyanins from species in Convolvulaceae, the terminal caffeoyl unit with a glucosyl linked to its phenolic end, has the sugar linked to the caffeoyl 3-OH position. In a recent paper by Mori et al. (2006) the structure of the major anthocyanin from the blue petals of Evolvulus pilosus (22) (Fig. 7.7) were found to be identical to phacelianin isolated from Phacelia campanularia (Hydrophyllaceae). Phacelianin has its terminal glucosyl linked to the 4-hydroxyl of the

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Fig. 7.8 Structures of some new anthocyanins reported in the recent literature. See Table 7.2 for references. caf, caffeoyl; fer, feruloyl; cum, p-coumaroyl; sin, sinapoyl; glc, glucosyl.

caffeic acid, which also revises the caffeoyl 3-OH linkage reported for the pigment(s) of Evolvulus pilosus (Toki et al., 1994b; Figueiredo et al., 1996). In the Cruciferae, the anthocyanins are based on two typical glycoside patterns; anthocyanidin 3-sophoroside-5-glucoside or anthocyanidin 3-sambubioside-5-glucoside (Harborne, 1967; Andersen & Jordheim, 2006). Recently, the latter anthocyanin pattern has been reported to occur in four species of this family: three new acylated pigments from the violet-blue flowers of Orychophragmus violaceus (Honda et al., 2005) (30 –32), four new acylated pigments from the purple-violet flowers of Lobularia maritima ‘Easter Bonnet’ (23–26), together with a known pigment, three new acylated pigments from the red-purple flowers of Lunaria annua (27–29), and two known pigments from the rose-red flowers of Cheiranthus cheiri L. (Tatsuzawa et al., 2006a) (Fig. 7.8, Table 7.1). The distribution and biosynthesis of these pigments have been discussed according to the variations of acylation and glucosylation at their 3-sambubiose residues by Tatsuzawa et al. (2006a). The major anthocyanins in flower extracts of Prunus mume ‘Nanjing Hongxu’ have, based on chromatography data and 1H-NMR, 13C-NMR and FAB MS spectra been indicated to be cyanidin 3-rutinoside and cyanidin 3-[6-(galloyl)-3-(glucosyl)glucoside], 34 (Fig. 7.8) (Zhao et al., 2006). If this latter structure is correct, this is the first report of the disaccharide laminariobiose linked to an anthocyanidin found outside the genus Allium (Terahara et al., 1994; Fossen & Andersen, 2003) and the fern Blechnum novae-zelandiae (Swinny, 2001). The distribution of galloylated anthocyanins has previously been thought to be restricted to species of the Nymphaéaceae and Aceraceae and in Acalypha hispida (Euphorbiaceae) (Andersen & Jordheim, 2006).

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7.6 Flavonoid complexes including at least one anthocyanidin subunit Two classes of flavonoid complexes including at least one anthocyanidin subunit have been identified in plants in recent years. One class includes pigments formed by direct condensation between an anthocyanidin and a flavanol (Fig. 7.7), and the other class includes pigments where an anthocyanin and a flavone or flavonol are linked to each end of a dicarboxylic acyl unit (Fig. 7.4). Metalloanthocyanins are treated in section 7.7. Pigments resulting from direct condensation between anthocyanins and flavanols have been assumed to be formed exclusively during storage and processing in plant-derived foods, including wines (Remy-Tanneau et al., 2003). However, this type of pigment also seems to occur naturally, although in small quantities, in extracts of unprocessed plants. In extracts of fresh strawberries four purple pigments were characterized by spectroscopic methods to be afzelechin(4α→8)pelargonidin 3-O-β-glucopyranoside (39), epiafzelechin(4α→8)pelargonidin 3-O-β-glucopyranoside (40), catechin(4α→8)pelargonidin 3-O-β-glucopyranoside (41) and epicatechin(4α→8)pelargonidin 3-O-β-glucopyranoside (42) (Fig. 7.5) (Fossen et al., 2004). The stereochemistry at the 3- and 4-positions of the flavan-3-ols was elucidated after assumption of the R-configuration at C-2. Each of the four pigments occurred in the NMR solvent as a pair of rotamers. Later the same four dimers and afzelechin-(4→8)-pelargonidin 3-rutinoside were tentatively identified by HPLCDAD/ESI-MS5 in extracts of the strawberry cultivar ‘Camarosa’ (Gonzalez-Paramas et al., 2006). Similarly, (epi)catechin-cyanidin 3,5-diglucoside has been tentatively identified in extract of purple corn, (epi)catechin-peonidin 3-glucoside and (epi)catechin-malvidin 3glucoside in extract of grape skin, and (epi)catechincyanidin-3-glucoside, (epi)gallocatechindelphinidin and (epi)catechin linked to cyanidin, petunidin, and peonidin in extracts of various beans (Gonzalez-Paramas et al., 2006; Macz-Pop et al., 2006a). Putative flavanol– anthocyanin condensation products have been detected in a concentrate from black currant (Ribes nigrum L.) (McDougall et al., 2005). These latter compounds were partly purified by semipreparative HPLC and gave MS and MS2 spectra consistent with delphinidin-3rutinoside and cyanidin-3-rutinoside covalently linked to epigallocatechin or gallocatechin. Further studies are necessary to confirm the in-vivo formation of flavanol–anthocyanin condensed pigments; however, the results given above suggest that these pigments may occur naturally in plants and not only as products formed during the storage and ageing of processed food, wine and other beverages. The structure of catechin-(4α→8)-malvidin 3-O-glucoside obtained by the in-vitro reaction of taxifolin and malvidin 3-O-glucoside (Mv3-glc), following a protocol adapted from proanthocyanidin dimer synthesis, has been elucidated by NMR spectrometry (Salas et al., 2004). Incorporation of the anthocyanin moiety into a covalently-linked flavanol– anthocyanin dimer did not modify the color properties of the anthocyanin moiety (i.e. hydration equilibrium constants and copigmentation). The mechanism of direct reactions between Mv3-glc and (−)-epicatechin has been studied in detail in model solutions (Duenas et al., 2006). The direct reactions were strongly related to pH, however, nucleophilic addition of epicatechin onto the flavylium form of Mv3-glc took place at all pH values between 2.0 and 6.0. Recently a covalently-linked anthocyanin–flavone C-glycoside (43) (Fig. 7.4), has been isolated from the purple leaves of Oxalis triangularis (Fossen et al., 2007). This pigment

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has an apigenin 6-C-sophoroside molecule attached with an ester bond to one end of malonic acid, and malvidin 3-O-rutinoside-5-O-glucoside linked to the other end (Section 7.3). Two covalently-linked anthocyanin–flavone pigments (44, 45) (Fig. 7.4) have been isolated from the blue-violet flowers of Eichhornia crassipes (Pontederiaceae) by Toki et al. (1994a, 2004). The major Eichhornia anthocyanin A (44) has an apigenin 7-glucoside molecule attached with an ester bond to one end of malonic acid, and delphinidin 3gentiobioside linked with a similar bond to the other end. The minor Eichhornia anthocyanin B (45) has a similar structure with apigenin 7-glucoside replaced with luteolin 7-glucoside. The chromophore (delphinidin) and the co-pigment (flavone) occupy a folding conformation as a binary complex (Toki et al., 1994a, 2004). The existence of intramolecular hydrophobic interactions between the chromophoric skeleton and the flavone group was indicated by a reduction in the hydration constant when compared with the parent delphinidin 3-glycoside (Figueiredo et al., 1996). Eichhornia anthocyanin A exhibited remarkable color stability in aqueous solution at mildly acidic pH values. The existence of other anthocyanin–flavone complexes has been indicated in orchids (Uphoff, 1982; Strack et al., 1989), lupins (Takeda et al., 1993) and Salvia patens (Takeda et al., 1994). Two covalently-linked anthocyanin–flavonol pigments (46, 47) (Fig. 7.4) have been isolated from the pale purple flowers of chive (Allium schoenoprasum) (Fossen et al., 2000). These pigments, which have been supplied with complete NMR assignments, were based on either cyanidin 3-glucoside or cyanidin 3-[3-(acetyl)glucoside] esterified to one end of malonic acid, and kaempferol 3-[2-(glucosyl)glucoside]-7-glucosiduronic acid connected to the other end. Compared to similar spectra of the same monomeric anthocyanins, the bathochromic shifts (9 nm) in the UV-vis spectra of the complexes revealed intramolecular association between the anthocyanin and flavonol units, which influenced the color of the pigment. Two similar anthocyanin–flavonol pigments (48, 49) (Fig. 7.4) have been isolated from the blue Agapanthus flowers (Agapanthaceae) (Bloor & Falshaw, 2000). In these structures succinate is involved instead of malonate to connect delphinidin 3-[6-(pcoumaloyl)glucoside]-7-glucoside to either kaempferol 3,4′-di-glucoside-7-xyloside or kaempferol 3,7,4′-tri-glucoside.

7.7 Metalloanthocyanins Commelinin from the blue flowers of Commelina communis has been found to consist of six molecules of delphinidin 3-[6-(p-coumaryl)glucoside]-5-[6-(malonyl)glucoside] (malonylawobanin) copigmented with six flavone (flavocommelin) molecules complexed with two Mg2+ ions (Kondo et al., 1992). Self-association was shown to exist between the anthocyanin moieties. The blue flower-color development and the stability of the color were explained by metal complexation of the anthocyanin and intermolecular hydrophobic association. Another metalloanthocyanin, protodelphin, which is similar to commelinin, has been isolated from flowers of Salvia patens (Takeda et al., 1994; Kondo et al., 2001). Like commelinin, protodelphin includes six molecules of the anthocyanin malonylawobanin and two Mg2+ -ions, but complexed with six molecules of another flavone, apigenin 7,4′diglucoside. Based on experiments with synthetic apigenin 7,4′-diglucosides derived from

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d- and l-glucose, Kondo et al. (2001) concluded that restricted chiral and structural recognition controlled the entire self-assembly of the metalloanthocyanin, and was responsible for the blue flower color. In 1998 Kondo et al. proposed a new molecular mechanism for blue color expression with protocyanin from the cornflower, Centaurea cyanus. The protocyanin structure was also suggested to be similar to that of commelinin, composed of six molecules each of apigenin 7-glucuronide-4′-[6-(malonyl)glucoside] and succinylcyanin, complexed with Mg2+ and Fe3+ ions (Kondo et al., 1994). However, recently it has been shown that the presence of two Ca2+ ions is essential for the formation of protocyanin (Shiono et al., 2005; Takeda et al., 2005). Metal complexes involving anthocyanins have also been proposed in the blue flowers of Hydrangea macrophylla (Brouillard & Dangles, 1994). However, it has been suggested that the color-change in hydrangea is caused by free Al3+ complexation of those components of the complex responding to slight vacuolar pH change (Yoshida et al., 2004). It has been suggested that the blue petal color of the Himalayan blue poppy, Meconopsis grandis, is based on a new type of anthocyanin complex containing a cyanidin derivative, two or more equivalents of kaempferol derivatives, 1/6 equivalent of Fe3+ and excess of Mg2+ ions (Yoshida et al., 2006). The ferric ions supposedly chelate the ortho-dihydroxy group of the B-ring of the anthocyanidin and are essential for the development of the blue color. The flavonols may stack on both sides of cyanidin and are stabilized by a co-pigmentation effect. The blue pigment might be composed of a very weak molecular association, and is only realized in an aqueous solution similar to that of the hydrangea pigment. Previous investigations of commelinin, protocyanin, protodelphin and hydrangea blue pigment have recently been reviewed by Takeda (2006), while Ellestad (2006) similarly has reviewed the experimental results obtained over the past 30 years for elucidating the self-assembly of the same metalloanthocyanins. This latter review also focuses on the role of the pendant sugars in directing the observed stacking chirality, and ends with speculation on the biological significance of the stacking chirality of the pigments in flower petals and its importance as to the possibility that insects might be sensitive to reflected circularly polarized light from flowers.

7.8 Biosynthesis and molecular biology Domestication of wild species in conjunction with classical breeding has long been the principal route to the generation of novelty in the flower industry. More recently genetic engineering has expanded the floriculture gene pool, thus promoting the generation of new commercial varieties. However, commercialization of genetically engineered flowers is currently confined to novel-colored carnations (http://www.florigene.com/). Several excellent reviews of the general biosynthetic pathway leading to flavonoids (Forkmann & Heller, 1999), and recent advances in the molecular biology and biotechnology of flavonoid biosynthesis, including targeted manipulation of flavonoid production in transgenic plants (Tanaka et al., 2005; Davies & Schwinn, 2006), have been published. Springob et al. (2003) covered the biochemistry, molecular biology and regulation of anthocyanin biosynthesis, with particular emphasis on mechanistic features and the late steps of anthocyanin biosynthesis, including glycosylation and vacuolar sequestration. Irani et al. (2003) focused

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on molecular mechanisms of the regulation of anthocyanin biosynthesis, and the factors that influence the pigmentation properties of anthocyanins, while Ben-Meir and collaborators (2002) have outlined the biochemistry and genetics of the pathway leading to anthocyanin production, and provided an overview on the application of the generated knowledge toward the molecular breeding of ornamentals. Griesbach (2005) has discussed anthocyanin biosynthesis, Mendelian inheritance and transgene technology of flower color, while Grotewold (2006) has reviewed the biosynthesis, regulation, and contribution to flower coloration of betalains, the carotenoids, and anthocyanins. More details have been presented by Kevin Davies elsewhere in this book. Here are given some references to important findings in the field. It has been proposed that anthocyanidins are biosynthetically derived from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leucocyanidin(s), which are eventually oxidized by anthocyanidin synthase (ANS) (Nakajima et al., 2001). The role of ANS has, however, been put into question, because the recombinant enzyme from Arabidopsis was shown to exhibit primarily flavonol synthase (FLS) activity with negligible ANS activity (Welford et al., 2001). This and other studies led to the proposal that ANS as well as FLS may select for dihydroflavonoid substrates carrying a β-face C-3 hydroxyl group and initially form the 3-geminal diol by α-face hydroxylation. Studies on assays with recombinant ANS from Gerbera hybrida have recently supported this proposal, and were extended with catechin and epicatechin isomers as potential substrates to delineate the enzyme’s specificity (Wellmann et al., 2006). The results of Winefield et al. (2005), in studies of the biosynthesis of 3-deoxyanthocyanins in Sinningia cardinalis, suggested a mechanism similar to that reported for Zea mays, in which lack of flavanone 3-hydroxylase activity allowed action of the dihydroflavonol 4-reductase/flavanone 4-reductase on flavanone substrates and production of flavan-4-ols. These intermediates are then probably converted to 3-deoxyanthocyanins through the action of ANS and subsequent glucosylation. Based on the finding that a small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum, Schwinn et al. (2006) have more generally proposed that the activity of MYB-related genes is a primary cause of the natural variation in anthocyanin pigmentation in plants. The genetic basis of color variation is, however, reckoned to be species specific. In this context the findings of major genetic determinants of berry color variation in a large collection of table grape cultivars and somatic variants is relevant (Lijavetzky et al., 2006). By providing optimized protocols for the determination of the activities of the key flavonoid enzymes in developing strawberry, Halbwirth et al. (2006) have been able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes, with the exception of flavonol synthase. The first activity peak corresponded to the formation of flavanols, while the second peak was clearly related to anthocyanin and flavonol accumulation. The results indicated that flavonoid 3-O-glucosyltransferase activity was not essential for redirection from flavanol to anthocyanin formation in the strawberry. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway has been constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, ANS also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida

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(Yan et al., 2005). Using two rounds of polymerase chain reaction each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosomebinding site, and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-glucoside or cyanidin 3-glucoside. The anthocyanins produced were, however, present at low concentrations. This is the first time plant-specific anthocyanins have been produced from a microorganism.

7.9 Anthocyanin localization in plant cells Although the biosynthetic pathways for anthocyanins and their regulation have been regularly studied, the mechanisms of anthocyanin accumulation in the cell are less clear. These pigments are considered to be synthesized in the cytoplasm by a multi-enzyme complex anchored on endoplasmic reticulum via the cytochrome P450 enzymes, which are part of the complex (Grotewold, 2006; Kitamura, 2006), and are normally found dissolved uniformly in vacuolar solutions of epidermal cells. However, in species from more than thirtythree families, anthocyanins have been found located in pigmented bodies in vacuoles described as anthocyanoplasts (ACPs) (Pecket & Small, 1981; Nozzolillo & Ishikura, 1988). Not much had been documented about the chemical nature and functional significance of these inclusions in petal cells before Markham et al. (2000) reported intensely-colored intravascular bodies in the petals of carnations (Dianthus caryophyllus) and lisianthus (Eustoma grandiflorum), which they named anthocyanic vacuolar inclusions (AVIs). The AVIs occurred predominantly in the adaxial epidermal cells, and their presence was shown to have a major influence on flower color by enhancing both intensity and blueness. Conn et al. (2003) later reported that AVIs in two lines of grapevine (Vitis vinifera) cell suspension culture appeared as dark red-to-purple spheres of various sizes in vacuoles owing to their interaction with anthocyanins. Electron microscopy studies on lisianthus epidermal tissue failed to detect a membrane boundary in AVI bodies, and the isolated AVIs were shown to have a protein matrix (Markham et al., 2000). Recently, Zhang et al. (2006) observed that AVIs in the epidermal cells of different regions of the lisianthus petal occurred in three different forms: vesicle-like, rod-like and irregularly-shaped. Again no membrane encompassing the AVIs was observed, however, the AVI itself consisted of membranous and thread structures throughout. The results strongly suggest the existence of mass transport for anthocyanins from biosynthetic sites in the cytoplasm to the central vacuole. The anthocyanins accumulate first as vesicle-like bodies in the cytoplasm, which themselves are contained in prevacuolar compartments (PVCs). The vesicle-like bodies seem then to be transported into the central vacuole through the merging of the PVCs and the central vacuole in the epidermal cells (Zhang et al., 2006). Anatomical observations of anthocyanin-rich cells in apple skins carried out by light microscopy and electron microscopy showed that skins with fully developed red color had more layers of anthocyanin-containing epidermal cells than those of green skins (Bae et al., 2006). The anthocyanins were frequently found in clusters or in round agglomerations in the epidermal cells of the red skins. There was no distinct envelope membrane on the anthocyanin granules in the vacuoles. The anthocyanin

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seemed to be synthesized around the tonoplast and condensed on the inward side of the vacuole. Though anthocyanins are widely found as flower and fruit pigments in higher plants, betacyanins have largely replaced anthocyanins in the Caryophyllales, except in the families Caryophyllaceae and Molluginaceae. With one exception (Mammillaria vivapara var. vivapara; Chapman et al., 2006), there has been no report of anthocyanins and betacyanins existing together in the same plant. Grotewold (2006) has thus expressed the notion that a molecular explanation for the observed mutual exclusion of the anthocyanins and betalains is an unresolved issue. However, Shimada et al. (2005) have found that spinach (Spinacia oleracea) and pokeweed (Phytolacca americana) – belonging to Caryophyllales – have functional anthocyanidin synthases (ANSs). The ability of ANSs of the Caryophyllales to oxidize trans-leucocyanidin to cyanidin was comparable to that of ANSs in anthocyanin-producing plants. It was thus suggested that the suppression or limited expression of dihydroflavonol 4-reductase and ANS was one possible explanation for the lack of anthocyanins in the Caryophyllales.

7.10 Acknowledgments The author is very grateful for being awarded the Groupe Polyphenols prize for 2006. Collaboration with other colleagues and former and present students in the field of anthocyanins during more than two decades has made his life very colorful. For scientific contributions to this presentation he particularly thanks Dr Torgils Fossen and Monica Jordheim, MSc and Ørjan Bjorøy, MSc.

7.11 Notes 1 NOESY, nuclear overhauser enhancement spectroscopy 2 ROESY, rotational nuclear overhauser effect spectroscopy 3 HSQC, heteronuclear single quantum correlation [spectroscopy]; TOCSY, total correlation spectroscopy 4 B3LYP, Becke 3-Parameter (Exchange), Lee, Yang and Parr 5 HPLC-DAD, High performance liquid chromatography with diode array detection ESI-MS, Electrospray ionization mass spectrometry

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Montoro, P., Tuberoso, C.I.G., Perrone, A., Piacente, S., Cabras, P. & Pizza, C. (2006). Characterisation by liquid chromatography-electrospray tandem mass spectrometry of anthocyanins in extracts of Myrtus communis L. berries used for the preparation of myrtle liqueur. Journal of Chromatography A, 1112, 232–240. Moreno, Y.S., Sanchez, G.S., Hernandez, D.R. & Lobato, N.R. (2005). Characterization of anthocyanin extracts from maize kernels. Journal of Chromatographic Science, 3, 483–487. Mori, M., Kondo, T., Toki, K. & Yoshida, K. (2006). Structure of anthocyanin from the blue petals of Phacelia campanularia and its blue flower color development. Phytochemistry, 67, 622–629. Nakajima, J., Tanaka, Y., Yamazaki, M. & Saito, K. (2001). Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. Journal of Biological Chemistry, 276, 25797–25803. Niemenak, N., Rohsius, C., Elwers, S., Omokolo Ndoumou, D. & Lieberei, R. (2006). Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phenolics and anthocyanins contents. Journal of Food Composition and Analysis, 19, 612–619. Niranjan, G.S. & Gupta, P.C. (1973). Anthocyanins from the flowers of Bombax malabaricum. Planta Medica, 24, 196 –199. Novruzov, E.N. (2005). Pigments of species in the genus Rosa and their chemotaxonomic value. Acta Horticulturae, 690, 225 –230. Nozzolillo, C., & Ishikura, N. (1988). An investigation of the intracellular site of anthocyanoplasts using isolated protoplasts and vacuoles. Plant Cell Reports, 7, 389–392. Nygård, A.M., Aksnes, D.W., Andersen, Ø.M. & Bakken, A.K. (1997). Structure determination of 6-hydroxycyanidin- and 6-hydroxydelphinidin-3-(6″-O-α-l-rhamnopyranosyl-β-d-glucopyranosides) and other anthocyanins from Alstroemeria cultivars. Acta Chemica Scandinavica, 51, 108–112. Nørbæk, R., Christensen, L.P., Bojesen, G. & Brandt, K. (1996). Anthocyanins in chilean species of Alstroemeria. Phytochemistry, 42, 97–100. Pale, E., Kouda-Bonafos, M., Nacro, M., Vanhaelen, M., Vanhaelen-Fastré, R. & Ottinger, R. (1997). 7-O-methylapigenidin, an anthocyanidin from Sorghum caudatum. Phytochemistry, 45, 1091– 1092. Pawlowska, A.M., De Leo, M. & Braca, A. (2006). Phenolics of Arbutus unedo L. (Ericaceae) fruits: identification of anthocyanins and gallic acid derivatives. Journal of Agricultural and Food Chemistry, 54, 10234 –10238. Pecket, R.C. & Small, C.J. (1981). Occurrence, location and development of anthocyanoplasts. Phytochemistry, 19, 2571–2576. Pina, F. (1998). Thermodynamics and kinetics of flavylium salts. Journal of the Chemical Society – Faraday Transactions, 94, 2109 –2116. Pina, F., Maestri, M. & Balzani, V. (2003). Photochromic systems based on synthetic flavylium compounds and their potential use as molecular-level memory elements. Handbook of Photochemistry and Photobiology, 3, 411– 450. Pina, F., Lima, J.C., Parola, A.J. & Afonso, C.A.M. (2004). Thermal and photochemical properties of 4′,7-dihydroxyflavylium in water-ionic liquid biphasic systems: A write-read-erase molecular switch. Angewandte Chemie (International edition), 43, 1525–1527. Piovan, A., Filippini, R. & Favretto, D. (1998). Characterization of the anthocyanins of Catharanthus roseus (L.) G. Don in vivo and in vitro by electrospray ionization in trap mass spectrometry. Rapid Communications in Mass Spectrometry, 12, 361–367. Polo, A.S., Murakami, I. & Neyde, Y. (2006). Blue sensitizers for solar cells: Natural dyes from Calafate and Jaboticaba. Solar Energy Materials & Solar Cells, 90, 1936–1944. Ponniah, L. & Seshadri, T.R. (1953). Nuclear oxidation in flavones and related compounds, Part XLV: A synthesis of carajuridin chloride and carajurin. Proceedings of the Indian Academy of Science, A38, 77– 83. Proctor, J.T., & Creasy L.L. (1969). The anthocyanin of the mango fruit. Phytochemistry, 8, 2108. Rein, M.J., Ollilainen, V., Vahermo, M., Yli-Kauhaluoma, J. & Heinonen, M. (2005). Identification of novel pyranoanthocyanins in berry juices. European Food Research and Technology, 202, 239 – 244.

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Tian, Q., Giusti, M.M., Stoner, G.D. & Schwartz, S.J. (2006). Characterization of a new anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry. Food Chemistry, 94, 465–468. Timberlake, C. F. & Bridle, P. (1975). Anthocyanins. In: The Flavonoids (eds J.B. Harborne & T.J. Mabry), pp. 214 – 266. Chapman and Hall, London, UK. Toki, K., Saito, N., Iimura, K., Suzuki, T. & Honda, T. (1994a). (Delphinidin 3-gentiobiosyl) (Apigenin 7-glucosyl)malonate from the flowers of Eichhornia crassipes. Phytochemistry, 36, 1181– 1183. Toki, K., Saito, N., Kawano, K., Lu, T.S., Shigihara, A. & Honda, T. (1994b). An acylated delphinidin glycoside in the blue flowers of Evolvulus pilosus. Phytochemistry, 36, 609–612. Toki, K., Saito, N., Ueda, T., Chibana, T., Shighihara, A. & Honda, T. (1994c). Malvidin 3-glucoside5-glucoside sulphates from Babiana stricta. Phytochemistry, 37, 885–887. Toki, K., Saito, N., Shigihara, A. & Honda, T. (2003). Acylated cyanidin glycosides from the purple-red flowers of Anemone coronaria. Heterocycles, 60, 345–350. Toki, K., Saito, N., Tsutsumi, S., Tamura, C., Shigihara, A. & Honda, T. (2004). (Delphinidin 3gentiobiosyl) (luteolin 7-glucosyl) malonate from the flowers of Eichhornia crassipes. Heterocycles, 63, 899 – 902. Toki, K., Saito, N., Ito, M., Shigihara, A. & Honda, T. (2006). An acylated cyanidin 3-rutinoside7-glucoside with p-hydroxybenzoic acid from the red-purple flowers of Campanula medium. Heterocycles, 68, 1699 –1703. Torskangerpoll, K., Nørbæk, R., Nodland, E., Øvstedal, D.O. & Andersen, Ø.M. (2005). Anthocyanin content of Tulipa species and cultivars and its impact on tepal colours. Biochemical Systematics and Ecology, 33, 499 – 510. Trappey, A., Bawadi, H.A., Bansode, R.R. & Losso, J.N. (2005). Anthocyanin profile of mayhaw (Crataegus opaca). Food Chemistry, 91, 665–671. Ueyama, Y., Katsumoto, Y., Fukui, Y. et al. (2006). Molecular characterization of the flavonoid biosynthetic pathway and flower color modification of Nierembergia sp. Plant Biotechnology, 23, 19 –24. Uphoff, W. (1982). Identification of European orchids by determination of the anthocyanin concentration during development of the blossoms. Experientia, 38, 778–780. Vareed, S.K., Reddy, M.K., Schutzki, R.E. & Nair, M.G. (2006). Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida fruits with health benefits. Life Sciences, 78, 777–784. Vivar-Quintana, A.M., Santos-Buelga, C. & Rivas-Gonzalo, J.C. (2002). Anthocyanin-derived pigments and colour of red wines. Analytica Chimica Acta, 458, 147–155. Welford, R.W.D., Turnbull, J.J., Claridge, T.D.W., Schofield, C.J. & Prescott, A.G. (2001). Evidence for oxidation at C-3 of the flavonoid C-ring during anthocyanin biosynthesis. Chemical Communication, 1828 –1829. Wellmann, F., Griesser, M., Schwab, W. et al. (2006). Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)catechin to cyanidin and a novel procyanidin. FEBS Letters, 580, 1642–1648. Winefield, C.S., Lewis, D.H., Swinny, E.E. et al. (2005). Investigation of the biosynthesis of 3deoxyanthocyanins in Sinningia cardinalis. Physiologia Plantarum, 124, 419–430. Yan, Y., Chemler, J., Huang, L., Martens, S. & Koffas, M.A.G. (2005). Metabolic engineering of anthocyanin biosynthesis in Escherichia coli. Applied and Environmental Microbiology, 71, 3617–3623. Yoshida, K., Aoki, H., Kameda, K. & Kondo, T. (2002). Structure of muscarinin A, an acylated anthocyanin, from purplish-blue spicate flower petals of Muscari armeniacum. ITE Letters on Batteries, New Technologies & Medicine, 3, 35–38. Yoshida, K., Oyama, K. & Kondo, T. (2004). Flower color development and nano-science. Yuki Gosei Kagaku Kyokaishi, 62, 490 – 499. Yoshida, K., Kitahara, S., Ito, D. & Kondo, T. (2006). Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis. Phytochemistry, 67, 992–998.

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Chapter 8

Salicylic Acid and Induced Plant Defenses Jean-Pierre Métraux, Elisabeth Lamodière, Jérémy Catinot, Olivier Lamotte and Christophe Garcion

8.1 Induced resistance and phenolics Plants, like other organisms, defend themselves against a wide variety of pathogenic microorganisms. The earliest reports – dating back to the 19th century – describe the surprising observations made by attentive plant pathologists (for a review see Gäumann, 1946). In recent years, the study of plant resistance mechanisms has received increased attention, given its potential for practical applications to improve crop protection. Interestingly, phenolics are closely associated with the power of plants to ward off pathogens. This article is based on a conference given on this topic at the XIII International Conference on Polyphenols held in Winnipeg, August 22–25, 2006. Interested readers might refer to various recent reviews for more complete information on this general topic (Friend, 1985; Nicholson & Hammerschmidt, 1992; Harborne, 1999; Garcion & Métraux, 2006). Plants exhibit both constitutive defenses, characterized by histological structures or the accumulation of a variety of antibiotic substances. These substances are termed ‘phytoanticipins’ (van Etten et al., 1994) and include various preformed secondary metabolites such as terpenoids or phenolics. Often these compounds are stored as inactive glycosyl precursors that will be activated upon membrane leakage inflicted by the attack of a pathogen. Plants are also able to recognize pathogens and to induce resistance mechanisms. This induced resistance (also referred to as basal resistance or basal induced resistance) involves the build-up of histological barriers to the ingress and progression of pathogens, mainly by various cell-wall reinforcements. In addition, chemical barriers in the form of antimicrobial molecules are synthesized de novo, including pathogenesis-related (PR) proteins (van Loon & van Strien, 1999) and phytoalexins. The latter group includes a range of secondary metabolites, some of which are phenolic derivatives. Basal induced resistance might be further enhanced by an encounter with a pathogen or exposure to an inducing chemical or stress. This additional resistance can then be expressed locally or systemically in uninfected tissues of the plant and is termed induced systemic resistance (ISR) or, alternatively, systemic acquired resistance (SAR). It is based on priming or conditioning whereby the intensity of a physiological response to a stimulus is enhanced by a previous exposure to the stimulating agent. The molecular basis of priming for induced Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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resistance has not yet been deduced. The somewhat misleading use of the terms ISR or SAR has historical reasons. As will be described below, SAR was initially described in many plants as taking place after a local infection with a necrotizing pathogen, and depends on the simple phenolic salicylic acid (SA) for its activation in the plant (for a recent account of SA see Sticher et al., 1997; Garcion & Métraux, 2006). Later, systemic resistance was also observed to take place upon localized exposure of plants to non-pathogenic microorganisms such as soil-borne rhizobacteria. This form of resistance was found to be independent of SA and termed ISR to distinguish it from SAR. The correct use of the terms SAR and ISR was discussed during a plenary discussion of the First International Symposium on Induced Resistance, which took place in 2000. It was proposed that ISR should be used as a generic term, while SAR should be used in those cases limited to pathogen-induced SA-dependent systemic resistance (Hammerschmidt et al., 2001). An interesting comparison has been drawn between basal induced resistance in plants and innate immune responses in animals. Both processes have been shown to display surprising similarities. Both plant and animal responses rely on the perception by receptorlike-kinases of molecules released during the interaction (elicitors) or on pathogenassociated molecular patterns (Gomez-Gomez & Boller, 2002). After perception, both types of organism present downstream events that include ion fluxes and activation of protein kinase cascades. This culminates in the transcription of genes associated with the defense response. A paradigm of such a signal transduction sequence in plants is the activation of defense mechanisms by flagellin, a bacterial molecular pattern derived from a protein present in the flagellum of many phytopathogenic bacteria (Asai et al., 2002). SA has also attracted considerable interest. It was initially discovered – in this context – in tobacco and cucumber during a search for endogenous signals involved in the regulation of systemic acquired resistance, and was also reported to function in Arabidopsis. This observation made it possible to study the action of SA using a genetically tractable system and opened the door to a vigorous research effort. As intimated above, phenolic substances participate in various important steps in the defense of plants to pathogens. The sections below will mainly focus on our current knowledge of SA, in particular its biosynthesis and mode of action.

8.2 The biosynthesis of SA The following section represents a summary of some of the major features of the biosynthesis of SA. Further information on the metabolism and conjugation of SA and SA-derived compounds can be found in a recent book by Garcion & Métraux (2006). SA was shown in the 1960s to be a product of the conversion of l-phenylalanine to trans-cinnamate (CA) – catalyzed by the phenylalanine–ammonia lyase (PAL) enzyme (see review by Lee et al., 1995) – which is converted to SA either by an o-hydroxylation of CA to o-coumarate before decarboxylation to SA, or by conversion to benzoic acid (BA) followed by hydroxylation. There are only a few reports supporting the o-coumarate pathway (Lee et al., 1995), most studies having documented the BA route. In cucumber and in potato 14C-phenylalanine can be converted to 14C-CA (Meuwly et al., 1995; Coquoz et al., 1998), and feeding 14C-CA to cucumber, rice, potato and tobacco

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results in the formation of 14C-SA (Meuwly et al., 1995; Silverman et al., 1995; Coquoz et al., 1998; Chong et al., 2001). The biosynthesis of SA via a PAL-catalyzed step was also supported by experiments using PAL inhibitor 2-aminoindan-2-phosphonic acid (AIP; Zon & Amrhein, 1992). In cucumber and in potato, inactivation of PAL by AIP inhibits the transformation of 14C-SA into SA (Meuwly et al., 1995; Coquoz et al., 1998). AIP has been reported to prevent the accumulation of SA in Arabidopsis in response to an infection (Mauch-Mani & Slusarenko, 1996). In cultured tobacco cells, 14C-CA is converted to SA via BA (Yalpani et al., 1993). The conversion of CA into BA has also been observed in potato (Coquoz et al., 1998) but not in cucumber (Meuwly et al., 1995). A later study in tobacco proposed conjugated rather than free forms of BA to be the precursors of SA. Support for this was obtained using the inhibitor piperonylic acid to block the cinnamate-4-hydroxylase, thus redirecting CA to the formation of SA (Chong et al., 2001). In summary, results from radiolabeling experiments provide support for the synthesis of SA from free BA or conjugated forms of BA. It is possible that some of these results are the consequence of metabolic channeling effects in the phenylpropanoid pathway (Jorgensen et al., 2005). The side-chain shortening of CA to BA might occur via an oxidative CoA-dependent, reaction, involving 3-hydroxy-3-phenylpropanoate-coA, much like fatty acid β-oxidation. Alternatively, CA might be converted by a CoA-independent route through benzaldehyde (reviewed in Lee et al., 1995). In cucumber and tobacco, 14C-SA is formed after labeling with 14C-3-hydroxy-3-phenylpropanoic acid (Jarvis et al., 2000), whereas benzaldehyde is unlikely to be an intermediate in SA biosynthesis in tobacco (Ribnicky et al., 1998). The enzymes catalyzing the steps from CA to BA have not yet been characterized. The final step in the conversion of BA to SA is a 2-hydroxylation. A benzoic acid 2-hydroxylase is induced in tobacco mosaic virus (TMV)-infected tobacco (Leon et al., 1995) and in rice (Silverman et al., 1995). The tobacco enzyme was reported to be a cytochrome P450 oxygenase (Leon et al., 1995). A genetic approach has recently yielded an interesting breakthrough in this phytochemical problem. Screening of Arabidopsis for inability to accumulate SA after pathogen infection (Nawrath & Métraux, 1999) or for enhanced disease susceptibility (Dewdney et al., 2000) made it possible to identify two mutants of isochorismate synthase (ICS; Wildermuth et al., 2001). The Arabidopsis ICS gene shows 57% identity at the amino acid level with an ICS gene of Catharanthus roseus. The product of the Catharanthus gene had previously been characterized and confirmed biochemically (van Tegelen et al., 1999). Given its homology with the Catharanthus gene, the Arabidopsis ICS gene was assumed to have a similar biochemical activity. We have confirmed recently that the Arabidopsis ICS gene can rescue E. coli deficient in the EntC gene coding for an ICS. The sequence of the Arabidopis ICS gene includes a predicted plastid-targeting signal (Wildermuth et al., 2001). Using an ICS-GFP construct, we have demonstrated the localization of ICS-GFP at the plastids (Lamodière et al., in preparation) This is in agreement with the localization of much of the shikimic acid pathway in the plastidic compartment (Schmid & Amrhein, 1999; Viitanen et al., 2004). The localization of ICS on the chloroplast hints to a plastidic localization of the full SA synthesis and opens interesting and unanswered questions on the possible transit of SA to the cytoplasm, where it is likely to act.

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Interestingly, the biosynthesis of SA seems also to proceed from isochorismate in Nicotiana benthamiana, a species closely related to N. tabacum. The sequence of the chorismate-binding domain of the N. benthamiana isochorismate gene is homologous with those of other plants (Catharanthus, tomato, Arabidopsis) and has been used for virusinduced gene suppression (VIGS). VIGS of the isochorismate synthase in N. benthamiana led to a strong decrease in the accumulation of SA after infection with Pseudomonas syringae or treatment with UV (Catinot & Métraux, 2008). Taken together these recent results from Arabidopsis and N. benthamiana support a pathway for SA that derives from isochorismate similar to that described for the bacteria. For instance, P. aeruginosa and P. fluorescens produce SA in two steps: isomerization of chorismate to isochorismate by an ICS and conversion of isochorismate into SA by an isochorismate pyruvate-lyase (IPL) (MercadoBlanco et al., 2001; Gaille et al., 2002, 2003). The enzyme for the conversion of isochorismate to SA, presumably an IPL, has not been described in plants so far. We are now left with two major pathways for the synthesis of SA in plants, one deriving from phenylalanine and the other from chorismate. The genetic data obtained from Arabidopsis and N. benthamiana indicate that most pathogen-induced SA derives in fact from chorismate. Possibly, this pathway has escaped attention in most radiolabeling studies, in which phenylalanine or metabolites downstream of it (CA, BA) were used as precursors for SA (see discussion in Garcion & Métraux, 2006). A dilution of the specific activity of SA would indicate a source of SA derived from an unlabeled precursor. In most of the labeling studies cited above this has not been observed and might provide an explanation for our ignorance of this branch of the SA pathway. This poses the interesting question of how to elucidate the biological relevance of phenylalanine-derived SA.

8.3 The network of signaling and the action of SA The importance of SA for induced resistance was determined with studies with mutants and transgenic plants that exhibit altered levels of SA. A number of Arabidopsis mutants displaying low endogenous levels of SA are impaired in induced resistance. Transgenic plants expressing a gene from Pseudomonas putida coding for naphthalene hydroxylase (NahG), which catalyzes the conversion of SA to catechol, cannot accumulate wild-type levels of SA after an infection (Delaney et al., 1994). Such plants are strongly impaired in induced resistance. Conversely, mutants or transgenic plants with constitutively high levels of SA exhibit increased tolerance to pathogens (reviewed in Métraux & Durner, 2004). Thus, SA seemed to emerge as an endogenous regulator of induced resistance directly linked to an effective concentration of SA in the tissue. This somewhat simplified and thus appealing picture turned out to be far from complete. Other endogenous signal molecules have been associated with the SA-independent activation of resistance responses. These compounds include octadecanoic acid derivatives such as jasmonic acid (JA), methyl jasmonate (MeJA) and 12-oxo-phytodienoic acid (OPDA), and ethylene (ET). Interestingly, in Arabidopsis thaliana, SA-dependent responses can only provide resistance to a defined spectrum of pathogens such as f. ex. Hyaloperonospora parasitica or Pseudomonas syringae, while JA- and ET-dependent resistance responses seem to operate against another group (f. ex. Alternaria brassicicola or Botrytis cinerea)

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(Thomma et al., 1998). In addition, defense-signaling pathways independent of SA, JA or ET have also been observed in Arabidopsis. For example, PAD2-mediated resistance to Phytophthora porri (Roetschi et al., 2001), the RPP7-, RPP8- and RPP13-mediated resistance to Peronospora parasitica (Bittner-Eddy & Beynon, 2001) and resistance to Botrytis in cuticle-depleted or wounded plants function independently of these signals (Chassot & Métraux, 2005; Chassot et al., in press; Chassot et al., 2007). These responses do not appear simply to be parallel and independent pathways. In fact, they are interconnected as a network, since both positive and negative cross-talk or interference (Penninckx et al., 1998; Thomma et al., 1998; Dewdney et al., 2000; Gupta et al., 2000; Norman-Setterblad et al., 2000; Berrocal-Lobo et al., 2002) has been observed between different pathways. This network can also cross-talk with signaling pathways activated by environmental stimuli such as light (Genoud et al., 2002). This defense network presumably increases the flexibility of the plant to optimize its defensive reactions to a given situation. A digital approach based on Boolean logic has been proposed for the representation of the plant defense network (Genoud et al., 2003). Positive and negative cross-talk can be described by combinations of logical steps performed by Boolean operators, making it possible to simulate the network on a computer. A general view of the defensive network was recently published for Arabidopsis responding to pathogenic fungi or bacteria or to various insects. The changes in the transcripts were followed using Affymetrix genome GeneChips. Specific combinations of genes associated with changes in the tissue levels of SA, JA and ET were recorded for each type of invader. These signatures showed some degree of overlap among certain pathogens, but also displayed remarkable degree of specificity with regard, for example, to JA-induced genes. The study highlights the relative importance of SA, JA and ET-induced gene expression, but also hints at other, as yet unidentified, signals and cross-talk as regulatory mechanisms taking place after attack by pathogens (De Vos et al., 2005). Investigation of the mode of action of SA has often been approached in the same way as classical plant and animal hormone research, by ligand-receptor studies. A first study identified catalase as an SA-binding protein (SABP). It was thought that catalase activity is inhibited upon binding with SA (Kd,14 μM), and the resulting increase in intracellular H2O2 was supposed to activate the expression of defense genes or to inhibit pathogen growth by virtue of its antibiotic properties (Chen et al., 1993). The biological relevance of catalase binding by SA was later questioned for several reasons, and H2O2 was placed upstream rather than downstream from SA (reviewed in Garcion & Métraux, 2006). Another SABP (SABP2) was isolated some years later (Du & Klessig, 1997). SABP2 exhibits high affinity for SA (Kd, 90 nM) and has an esterase and a SA-inducible lipase activity (Kumar & Klessig, 2003). Local and systemic induced resistance to TMV in tobacco is lost if SABP2 is silenced using RNA interference (Kumar & Klessig, 2003). SABP2 belongs to the α/β hydrolases superfamily, based on an analysis of its crystalline structure, and a methylated form of SA (MeSA) can bind to the Ser-His-Asp triad of the active site of SABP2. MeSA might be the translocated and/or a stored form of SA (Seskar et al., 1998), and it has been proposed that binding to SABP2 might control intracellular SA levels (Forouhar et al., 2005). Carbonic anhydrase (CAN) is the third identified SABP (SABP3), with a moderate affinity (Kd, 3.7 μM) for SA. It localizes to the soluble fraction of tobacco

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leaf chloroplasts (Slaymaker et al., 2002). Although SA does not modify the activity of CAN, it was proposed that it modifies its antioxidant properties, as a result of which there is a putative increase in the amount of active oxygen species that might activate SA production and induce defense responses to pathogens (Slaymaker et al., 2002). The formation of free radicals upon binding of SA to heme-containing enzymes such as peroxidase or catalase has also been suggested as a possible mode of action of SA. Such free radicals might activate defense reactions upon formation of lipid peroxides and products deriving from them (Durner & Klessig, 1995, 1996).

8.4 Conclusions Phenolics play an essential role in various aspects of the defense of plants against pathogens. The discovery of the role of SA as a regulatory molecule in induced resistance provided us with an interesting tool for understanding the molecular events taking place in infected plants. It was soon followed by the identification of other regulators such as JA and ET, and the list is probably incomplete. Clearly, the regulation of induced resistance defense is a complex network integrated with other physiological processes. There are still many open ends in our understanding of the action of SA and other signals. Given the worldwide activity in this research field, we should soon learn more about their biosynthetic regulation and mode of action, as well as their integration into the defense network. From a practical point of view, this knowledge will fuel the development of new approaches for crop protection, an area that – much like research on antibiotics in medicine – is a never-ending struggle against ever-evolving pest varieties. From a methodological point of view, the largest advances have been achieved through the use of combinations of approaches, a good example of which is the deciphering of the biosynthesis of SA, which was made possible by a combination of genetic studies and classic biochemistry. The complexity of the network of plant defenses might therefore require the use of an appropriate combination of approaches including genome-wide expression analysis, proteomics, metabolomics, signalomics, forward and reverse genetics, bioinformatics and biomathematics.

8.5 Acknowledgment The Swiss National Science Foundation (grant 3100A0-104224/1 to J.P.M.) is gratefully acknowledged for its generous financial support.

8.6 References Asai, T., Tena, G., Plotnikova, J. et al. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature, 415, 977– 983. Berrocal-Lobo, M., Molina, A. & Solano, R. (2002). Constitutive expression of ETHYLENERESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. The Plant Journal, 29, 23 – 32.

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Bittner-Eddy, P.D. & Beynon, J.L. (2001). The Arabidopsis downy mildew resistance gene, RPP13Nd, functions independently of NDR1 and EDS1 and does not require the accumulation of salicylic acid. Molecular Plant–Microbe Interactions, 14, 416–421. Catinot, J., Buchala, A., Aboo-Mansoor, E., Métraux, J.P. (2008). Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiane benthamina. FEBS Letters, 582, 473 – 478. Catinot, J. & Métraux, J.P. SA is made via isochorismate synthase in tobacco. In preparation. Chassot, C. & Métraux, J.P. (2005). The cuticle as source of signals for plant defense. Plant Biosystems, 139, 28 – 31. Chassot, C., Nawrath, C. & Métraux, J.P. (2007). Cuticular defects lead to full immunity to a major pathogen. Plant Journal, 49, 972 – 980. Chassot, C., Buchala, A., Métraux, J.P. & Lamotte, O. Wound-induced resistance to Botrytis cinerea. In preparation. Chen, Z.X., Silva, H. & Klessig, D.F. (1993). Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science, 262, 1883–1886. Chong, J., Pierrel, M.A., Atanassova, R., Werck-Reithhart, D., Fritig, B. & Saindrenan, P. (2001). Free and conjugated benzoic acid in tobacco plants and cell cultures. Induced accumulation upon elicitation of defense responses and role as salicylic acid precursors. Plant Physiology, 125, 318 –328. Coquoz, J.L., Buchala, A. & Métraux, J.P. (1998). The biosynthesis of salicylic acid in potato plants. Plant Physiology, 117, 1095 –1101. Delaney, T.P., Uknes, S., Vernooij, B. et al. (1994). A central role of salicylic acid in plant disease resistance. Science, 266, 1247–1249. De Vos, M., van Oosten, V.R., van Poecke, R.M.P. et al. (2005). Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Molecular Plant–Microbe Interactions, 18, 923 – 937. Dewdney, J., Reuber, T.L., Wildermuth, M.C. et al. (2000). Three unique mutants of Arabidopsis identify eds loci required for limiting growth of a biotrophic fungal pathogen. The Plant Journal, 24, 205 –218. Du, H. & Klessig, D.F. (1997). Identification of a soluble, high-affinity salicylic acid-binding protein in tobacco. Plant Physiology, 113, 1319–1327. Durner, J. & Klessig, D.F. (1995). Inhibition of ascorbate peroxidase by salicylic acid and 2,6dichloroisonicotinic acid, two inducers of plant defense responses. Proceedings of the National Academy of Sciences of the USA, 92, 11312–11316. Durner, J. & Klessig, D.F. (1996). Salicylic acid is a modulator of tobacco and mammalian catalases. Journal of Biological Chemistry, 271, 28492–28501. Forouhar, F., Yang, Y., Kumar, D. et al. (2005). Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proceedings of the National Academy of Sciences of the USA, 102, 1773–1778. Frien, J. (1985). Phenolic substances and plant disease. In: The biochemistry of plant phenolics (eds C.F. van Sumere & P.J. Lea), pp. 367–392. Clarendon Press, Oxford, UK. Gaille, C., Kast, P. & Haas, D. (2002). Salicylate biosynthesis in Pseudomonas aeruginosa. Purification and characterization of pchb, a novel bifunctional enzyme displaying isochorismate pyruvate-lyase and chorismate mutase activities. Journal of Biological Chemistry, 277, 21768– 21775. Gaille, C., Reimmann, C. & Haas, D. (2003). Isochorismate synthase (PchA), the first and ratelimiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa. Journal of Biological Chemistry, 278, 16893 –16898. Garcion, C. & Métraux, J.P. (2006). Salicylic acid. In: Plant Hormone Signaling (eds P. Hedden & S.G. Thomas), pp. 229 –255. Annual Plant Reviews, Vol. 24. Blackwell Press, Oxford, UK. Gäumann, E. (1946). Pflanzliche Infektionslehre. Birkhäuser, Basel, CH. Genoud, T., Buchala, A.J., Chua, N.H. & Métraux, J.P. (2002). Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis. The Plant Journal, 31, 87–95.

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Genoud, T., Trevino Santa Cruz, M.B. & Métraux, J.P. (2003). Networks of cellular information processing: digital description and simulation. Current Genomics, 4, 27–36. Gomez-Gomez, L. & Boller, T. (2002). Flagellin perception: a paradigm for innate immunity. Trends in Plant Science, 7, 251–256. Gupta, V., Willits, M.G. & Glazebrook, J. (2000). Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)- dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA. Molecular Plant–Microbe Interactions, 13, 503–511. Hammerschmidt, R., Métraux, J.P. & van Loon, L.C. (2001). Inducing resistance: a summary of papers presented at the First International Symposium on Induced Resistance to Plant Diseases, Corfu, May 2000. European Journal of Plant Pathology, 107, 1–6. Harborne, J.B. (1999). The comparative biochemistry of phytoalexin induction in plants. Biochemical Systematics and Ecology, 27, 335–367. Jorgensen, K., Rasmussen, A.V., Morant M. et al. (2005). Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Current Opinion in Plant Biology, 8, 280–291. Kumar, D. & Klessig, D.F. (2003). High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity. Proceedings of the National Academy of Sciences of the USA, 100, 16101–16106. Lamodière, E., Métraux, J.P. & Garcion, C. Activity and localization of the isochorismate ICS1 gene from Arabidopsis thaliana. In preparation. Lee, H.I., Leon, J. & Raskin, I. (1995). Biosynthesis and metabolism of salicylic acid. Proceedings of the National Academy of Sciences of the USA, 92, 4076–4079. Leon, J., Shulaev, V., Yalpani, N., Lawton, M.A. & Raskin, I. (1995). Benzoic-acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic-acid biosynthesis. Proceedings of the National Academy of Sciences of the USA, 92, 10413–10417. Mauch-Mani, B. & Slusarenko, A.J. (1996). Production of salicylic acid precursors is a major function of phenylalanine ammonia-lyase in the resistance of Arabidopsis to Peronospora parasitica. The Plant Cell, 8, 203 –212. Mercado-Blanco, J., van der Drift, K., Olsson, P.E., Thomas-Oates, J.E., van Loon, L.C. & Bakker, P. (2001). Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374. Journal of Bacteriology, 183, 1909 –1920. Métraux, J.P. & Durner, J. (2004). The role of salicylic acid and nitric oxide in programmed cell death and induced resistance. In Ecological studies (ed. H. Sandermann), pp. 111–150. Springer, Berlin, DE. Meuwly, P., Molders, W., Buchala, A. & Métraux, J.P. (1995). Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiology, 109, 1107–1114. Nawrath, C. & Métraux, J.P. (1999). Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. The Plant Cell, 11, 1393 –1404. Nicholson, R.L. & Hammerschmid, R. (1992). Phenolic compounds and their role in disease resistance. Annual Review of Phytopathology, 30, 369–389. Norman-Setterblad, C., Vidal, S. & Palva, E.T. (2000). Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora. Molecular Plant–Microbe Interactions, 13, 430–438. Penninckx, I., Thomma, B., Buchala, A., Métraux, J.P. & Broekaert, W.F. (1998). Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. The Plant Cell, 10, 2103–2113. Ribnicky, D.M., Shulaev, V. & Raskin, I. (1998). Intermediates of salicylic acid biosynthesis in tobacco. Plant Physiology, 118, 565 –572. Roetschi, A., Si-Ammour, A., Belbahri, L., Mauch, F. & Mauch-Mani, B. (2001). Characterization of an Arabidopsis–Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling. The Plant Journal, 28, 293 – 305.

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Schmid, J. & Amrhein, N. (1999). The Shikimate Pathway. In: Plant Amino Acids – Biochemistry and Biotechnology (ed. B. Singh), pp. 147–169. Marcel Dekker, New York, USA. Seskar, M., Shulaev, V. & Raskin, I. (1998). Endogenous methyl salicylate in pathogen-inoculated tobacco plants. Plant Physiology, 116, 387–392. Silverman, P., Seskar, M., Kanter, D., Schweizer, P., Métraux, J.P. & Raskin, I. (1995). Salicylic acid in rice – biosynthesis, conjugation, and possible role. Plant Physiology, 108, 633–639. Slaymaker, D.H., Navarre, D.A., Clark, D., del Pozo, O., Martin, G.B. & Klessig, D.F. (2002). The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proceedings of the National Academy of Sciences of the USA, 99, 11640–11645. Sticher, L., Mauch-Mani, B. & Métraux, J.P. (1997). Systemic acquired resistance. Annual Review of Phytopathology, 35, 235 –270. Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A. et al. (1998). Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proceedings of the National Academy of Sciences of the USA, 95, 15107–15111. van Etten, H.D., Mansfield, J.W., Bailey, J.A. & Farmer, E.E. (1994). Two classes of plant antibiotics–phytoalexins versus phytoanticipins. The Plant Cell, 6, 1191–1192. van Loon, L.C. & van Strien, E.A. (1999). The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 55, 85 – 97. van Tegelen, L.J.P., Moreno, P.R.H., Croes, A.F., Verpoorte, R. & Wullems, G.J. (1999). Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus. Plant Physiology, 119, 705 –712. Viitanen, P.V., Devine, A.L., Khan, M.S., Deuel, D.L., van Dyk, D.E. & Daniell, H. (2004). Metabolic engineering of the chloroplast genome using the Echerichia coli ubic gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiology, 136, 4048 – 4060. Wildermuth, M.C., Dewdney, J., Wu, G. & Ausubel, F.M. (2001). Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 417, 562–565. Yalpani, N., Leon, J., Lawton, M.A. & Raskin, I. (1993). Pathway of salicylic-acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiology, 103, 315–321. Zon, J. & Amrhein, N. (1992). Inhibitors of phenylalanine ammonia-lyase: 2-aminoindan-2-phosphonic acid and related compounds. Liebigs Annalen der Chemie, 6, 625–628.

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Chapter 9

Phenols and the Onset and Expression of Plant Disease Resistance Ray Hammerschmidt and Samantha I. Hollosy

9.1 Introduction Although there are thousands of plant pathogens, individual plant species are infected by only a small percentage of the pathogens that exist in nature. This suggests that plants are well equipped with the means to defend against most attacks, and that successful infection may only occur where these defenses are breached or nullified in some way. Plant defenses against pathogens are those processes, structures or metabolites that contribute to stopping the ingress and growth of pathogens in host tissues. Because of the myriad pathogens and the many different mechanisms of pathogenesis that have been identified, it is not surprising that a variety of approaches to defense have evolved. Defense mechanisms consist of a combination of preformed or constitutive defenses along with those defenses that are produced only after infection (Hammerschmidt & Nicholson, 1999). The preformed defenses provide a passive form of defense, and must be produced in tissues that come into contact with the potential pathogen. Defenses induced as a result of infection are referred to as active defenses and can be localized at the site of the attempted infection or may be produced throughout the plant. The many different types of pathogen that a plant encounters require not only different types of defense strategies (preformed vs. activated after infection) to protect against pathogens but also different biochemical mechanisms of defense. Many different types of natural product with antimicrobial activity have been associated with both passive and active defense (e.g. Grayer & Harborne, 1994; Treutter, 2005, 2006). Almost all classes of secondary metabolites (e.g. alkaloids, terpenoids, polyketides, and a wide variety of phenolic compounds) have been associated with host defense as illustrated by their invitro antimicrobial activity (Grayer & Harborne, 1994; Hammerschmidt, 1999b). Induced defenses also come in a wide range of chemical forms and include a variety of antifungal and hydrolytic enzymes, low molecular weight antimicrobial compounds produced at the site of infection (i.e. phytoalexins), cell-wall strengthening materials such as callose, lignins and hydroxyproline-rich glycoproteins, and several species of active oxygen (Hammerschmidt & Nicholson, 1999). However, as pointed out in other publications (e.g. Nicholson & Hammerschmidt, 1992; Hammerschmidt, 1999b), demonstration of antimicrobial activity in an in-vitro assay or

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presumed ability to strengthen structural barriers is insufficient to show a role in plant defense against a pathogen. For antimicrobial compounds to be a functional defense, it must be demonstrated that the compounds are present in tissues that come into contact with the pathogen and be present in concentrations that are high enough to inhibit microbial growth. Finally, if a host plant fails to produce a specific compound implicated in defense, this lack of production must be shown to result in an increased ability of the pathogen to invade the host (Hammerschmidt, 1999b). Defenses against pathogens have also been associated with structural barriers such as suberized periderms and lignified cell walls (Nicholson & Hammerschmidt, 1992; Hammerschmidt, 1999a). Once again, the presence of these in response to a pathogen may or may not provide the evidence needed to demonstrate a role in defense. In later parts of this review, some of the key approaches needed to demonstrate if a putative defense is truly a defense will be discussed along with examples of defenses in several host–pathogen systems.

9.2 Biosynthetic origins of defense-associated phenolic compounds Phenolic compounds have been shown to play a role in both active and passive forms of defense. Most classes of phenolic compounds have been shown to be involved in defense, and these include simple hydroxybenzoic acid, free and conjugated hydroxycinnamic acids, coumarins, flavonoids and stilbenes (Grayer & Harborne, 1994). While most of the phenolic compounds implicated in defense are derived in part or fully from the phenylpropanoid pathway, others are produced via the acetate–malonate (polyketide) pathway or acetate– mevalonate (terpenoid) pathway. Other plant-defense-associated phenolic compounds, such as the hydrolysable tannins, are derived from intermediates of the shikimic acid pathway (Strack, 1997). Thus, depending on the plant species, the types of phenol that are implicated in defense may differ greatly.

9.3 Phenolic compounds as preformed defenses Most plants contain preformed compounds that are antimicrobial, and there is good evidence to support the role of pre-formed compounds in resistance (Nicholson and Hammerschmidt, 1992; Grayer & Harborne, 1994; Treutter, 2005, 2006). Pre-existing chemicals that may be involved in plant defense represent a broad spectrum of secondary plant metabolites that includes phenolic compounds. An example of a phenolic compound that has been implicated as a preformed defense is the flavanone pinocembrin from Populus deltoides (Shain & Miller, 1982). This compound is found on leaf surfaces and it is proposed that it functions in defense by inhibiting fungal spore germination. Many other preformed, antimicrobial compounds have been documented (e.g. Grayer & Harborne, 1994; Prusky & Keen, 1993). However, more evaluation is needed to determine the role of these and other antimicrobial compounds in passive defense. In the following sections, some examples of the various roles that preformed phenolic compounds play in defense are described.

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R=H, Catechol R=COOH, Protocatechuic acid (onion)

Chlorogenic acid (widely distributed)

3-Hydroxyacetophenone (carnation)

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Epicatechin (avocado)

Caffeic acid (widely distributed)

5-Pentadecylresorcinol (mango)

Pinocembrin (cotton wood)

Fig. 9.1 Structures of phenolic compounds associated with preformed defenses (the plant names refer to examples discussed in the text).

9.3.1 Resistance of onion bulbs Onion smudge is caused by the soil borne pathogen Colletotrichum circinans (Walker, 1925). Early work by Walker and colleagues (Link et al., 1929; Walker et al., 1929; Angell et al., 1930) showed that resistance to this pathogen correlated with pigmentation of the bulb. If spores of C. circinans were incubated in aqueous extracts of dry outer scales of pigmented bulbs, hyphal growth was inhibited and germ tubes appeared abnormal. Aqueous extracts from dry scales of white onions were not inhibitory. Analysis of the extracts revealed that the antimicrobial activity was not due to the anthocyanin and flavonoid pigments, but rather to two simple phenolic compounds: catechol and protocatechuic acid (Fig. 9.1). Because these two phenols are water soluble, they are easily extracted from the dead scales. Since the spores need free water to germinate, the very water needed to promote infection also provides the delivery vehicle for these defense compounds. Thus, diffusion of protocatechuic acid and catechol from dead onion scales into the spore-containing water droplets results in inhibition of fungal development and thus provides resistance. However, if the outer scales of pigmented onions are removed, the underlying living scale tissues are just as susceptible as the white bulbs. Thus, these compounds provide protection against infection as long as the tissues containing them are intact (Walker et al., 1929).

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9.3.2 Fusarium wilt of carnation One of the most important diseases of carnation is fusarium wilt cause by Fusarium oxysporum f. sp. dianthi. Curir et al. (1996) found that varieties of carnation that were resistant to infection by this pathogen had high concentrations of 3-hydroxyacetophenone (Fig. 9.1). This compound was found to be antifungal in in-vitro assays, and evidence for an in-planta role in defense has also been provided by the authors. Another phenolic compound, a triglycoside of the flavonol kaempferide, has also been associated with resistance to F. oxysporum f. sp. dianthi (Curir et al., 2001). A recent report indicates that F. oxysporum f. sp. dianthi can detoxify 3-hydroxyacetophenone to a less toxic metabolite (Curir et al., 2000). Two NADH-dependent oxidoreductases are produced by the fungus in the presence of 3-hydroxyacetophenone. It will be interesting to learn if the ability to detoxify this compound is associated with virulence of the pathogen.

9.3.3 Mango fruit and Alternaria Fruits are often resistant to disease development while immature, yet they quickly express symptoms of disease as the ripening process begins (Prusky & Keen, 1993). This expression of symptoms is not related to when infections of the fruits occur, but rather to how immature fruit respond to infection as compared to ripe fruit. The fungus can begin to infect immature fruit but is held in check by fungistatic compounds that decrease in concentration upon ripening of the fruit. One example of this is found in mango fruit. Unripe mango fruit is thought to be resistant to Alternaria alternata because of the presence of two preformed antifungal compounds in the peel of the fruit: the resorcinol derivatives 5-12-cis-heptadecenylresorcinol and 5-pentadecylresorcinol (Fig. 9.1) (Cojocaru et al., 1986; Prusky & Keen, 1993). As the fruit ripens, the concentrations of these compounds decrease and the fruit becomes susceptible to Alternaria infection (Droby et al., 1986; Prusky & Keen, 1993).

9.3.4 Regulation of preformed antifungal compounds by phenols in avocado Another well-studied example of the role of preformed compounds in defense is the resistance of avocado fruit to fungal pathogens. This is similar to the case of mango, in which preformed antimicrobial compounds in the peel inhibit the growth of pathogens until the fruit matures. In this case, however, the phenolic compounds appear to play a role in maintaining the presence of antifungal compounds and not being directly involved in inhibiting the pathogens. Prusky and co-workers demonstrated a correlation between the concentration of antifungal fatty-acid derivatives in the peel of unripe avocado fruits with resistance to Colletotrichum gloeosporioides (Prusky et al., 1982; Prusky et al., 1991; Prusky & Keen, 1993). As the avocado fruit matures, the concentrations of the antifungal fatty acid derivatives decrease and disease development by previously quiescent C. gloeosporioides begins (Prusky et al., 1982; Prusky & Keen, 1993). Prusky et al. reported that the loss of the diene and monoene appears to be associated with a near doubling of lipoxygenase activity in the

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avocado tissue. This provides a reasonable explanation for how the fruits combat infection, but what role do phenolic compounds play in this defense? The increase in lipoxygenase activity and decrease in diene concentration was correlated with levels of epicatechin (Fig. 9.1), an inhibitor of lipoxygenase (Karni et al., 1989). As the fruit ripens, the concentration of epicatechin declines, and thus it is likely that this decrease in epicatechin allows endogenous lipoxygenase to degrade (and thus detoxify) the antifungal diene, and this permits the colonization of the fruit tissue by the pathogen. Further support for the important role of epicatechin comes from studies on the ability of C. gloeosporioides to degrade epicatechin with a laccase (Guetsky et al., 2005). The laccase is produced in vitro in the presence of epicatechin, and the fungal enzyme can also be detected in infected tissue. Treatment of fruits with compounds that inhibit laccase activity resulted in a decrease in disease development, thus further supporting the role that epicatechin plays in resistance modulation. Similarly, isolates of the pathogen that are deficient in laccase production are less virulent (Guetsky et al., 2005). Finally, Wattad et al. (1994) found that epicatechin can also inhibit the activity of a pectin lyase produced by C. gloeosporioides. This type of enzyme is thought to be important in pathogen virulence, and thus the inhibition of this enzyme may further contribute to the resistance of immature fruits to the pathogen.

9.3.5 Chlorogenic acid and the infection of stone fruit by Monilinia Monilinia fructicola causes a serious disease of stone fruit known as brown rot. In peach, resistance to this disease has been correlated with the amount of cholorogenic and caffeic acids (Fig. 9.1) in the outer peel. As the fruit ripens the amount of these phenols decreases and susceptibility increases. Bostock et al. (1999) examined the role of chlorogenic and caffeic acids in resistance to brown rot and found that the concentrations of these phenolic acids found in the peel were not inhibitory to Monilinia growth or spore germination. Thus, the role of these compounds in defense is probably not through fungistatic or fungitoxic properties. However, they did find that these phenolic compounds may contribute to defense by blocking the production of cutinase, an esterase involved in penetration of the plant cuticle. Growing cultures of Monilinia on cutin in the presence of caffeic acid prevented the production of cutinase and other esterases. Caffeic and chlorogenic acids had no effect on the activity of cutinase, thus these compounds are probably not involved in defense by inhibiting enzyme activity. Bostock et al. (1999) proposed that chlorogenic and caffeic acids may function in defense by blocking the production of enzymes needed in the infection of fruit.

9.4 Active defense In most plant–pathogen interactions, defense responses are induced after the infection process has been initiated (Hammerschmidt & Nicholson, 1999). This interaction, which is initiated by recognition of the pathogen by the host plant, results in a series of events that lead to the expression of a wide variety of responses, of which accumulation of phenolic compounds is a common feature. These responses are often highly localized and may involve

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only a few host cells. In other interactions, the initial infection results in the systemic expression of defenses and a change in the physiology of the host plant that allows rapid deployment of defenses upon infection. This phenomenon is referred to as ‘induced resistance’.

9.5 Localized defenses In the early 20th century Noel Bernard found that infection of orchid tubers with a non-pathogenic strain of Rhizoctonia would protect the tissue against infection with a pathogenic strain. Bernard and later workers reported that a diffusible antimicrobial substance was associated with the resistance and that production of the antimicrobial compounds was enhanced by the infection (reviewed in Allen, 1959; Gäumann, 1950). Perhaps the first clear experimental evidence for induced, localized defenses was present by Müller & Börger in 1940 (Reviewed in Gaumann, 1950; Muller, 1959), who found that preinoculation of potato tuber tissue with an incompatible race of Phytophthora infestans resulted in a defense response that not only stopped the incompatible race but also induced resistance to subsequent inoculations with compatible races of the pathogen that should have caused disease. They hypothesized that the induced resistance was due to the production of what they called phytoalexins (reviewed in Allen, 1959). Within twenty years, the first phytoalexins were identified and many of these compounds were found to be phenolic. In the 1950s the first biochemical studies of active defense responses began in earnest. For example, Kuc´ and co-workers reported that inoculation of potato tuber tissue slices with Cochliobolus (Helminthosporium) carbonum, a pathogen of maize but not of potato, resulted in the production of extractable, fungistatic compounds (Kuc´ et al., 1955, 1956). A subsequent study found that the inoculated tubers produced both caffeic and chlorogenic acids (Fig. 9.1) as part of the response to C. carbonum. Although these phenolic acids constitute part of the defense response, Kuc´ later reported that it was unlikely that they would be able to account for all of the antifungal activity in the extracts (Kuc´, 1957), and this was proven to be true by the subsequent identification of other antimicrobial compounds induced in potato tubers (Kuc´, 1995; Hammerschmidt, 1999b). By the end of the 1950s, the first phytoalexin had been characterized by Cruickshank and Perrin (reviewed in Cruicskshank, 1963). This was the isoflavonoid derivative pisatin (Fig. 9.2), which was isolated from immature pea pods that had been inoculated with spores of a Monilinia (a non-pathogen of peas). In subsequent years many more phytoalexins were discovered from many plant families (Cruickshank, 1963; Kuc´, 1995; Hammerschmidt, 1999b). Some of these turned out to be phenolic compounds, and this will be the focus of the rest of this section.

9.5.1 Phenolic phytoalexins An important consideration to take into account in studying induced antimicrobial compounds is determining whether they play a direct role in defense or are only part of a general metabolic response to infection. Several criteria have been proposed to help show that these compounds play an important role (Hammerschmidt, 1999b), and one of these

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Pisatin (pea)

Glyceollin I (soybean)

217

Luteolinidin (sorghum)

Lacinilene c (cotton)

Cucumerin B (cucumber) Avenanthramide A (oats) Fig. 9.2 Structures of phenolic compounds associated with induced or active defenses (th plant names refer to examples discussed in the text).

involves demonstrating that the compounds accumulate at the right time and in the right location. One of the very best examples of localizing and in-vivo quantification of phytoalexins in a host pathogen interaction comes from the work of Nicholson and co-workers on the resistance of sorghum to fungal pathogens. They found that sorghum produces a variety of flavan-3-ol phytoalexins such as luteolinidin (Fig. 9.2), apigeninidin and derivatives of these flavonoids (Nicholson et al., 1987; Hipskind et al., 1990; Lo et al., 1999). These compounds are also pigments and this allows direct visualization in the tissue. Using light microscopy and microspectrophotometry, Nicholson and co-workers were able to show that the phytoalexins did accumulate at the right time and place to be effective defenses, and they were also able to demonstrate that the concentration of the phytoalexins in the infected cells was more than sufficient to inhibit growth of the pathogen (Snyder & Nicholson, 1990; Snyder et al., 1991). There are other examples of the localization of phenolic phytoalexins that help demonstrate their roles in defense, and these include studies with cotton phytoalexins such as lacinilene c (Fig. 9.2) (Essenberg et al., 1992; Pierce et al., 1996) and the avenanthramides (Fig. 9.2) from oats (Mayama & Tani, 1982). In a more recent study, McNally et al. (2003) were able to take advantage of the autofluorescent properties of cucumber C-glycosyl flavonoid phytoalexins to localize these phytoalexins in host cells. Using the Podosphaera xanthii–cucumber interaction, they were able to localize the production of autofluorescent C-glycosyl flavonoid phytoalexins such as cucumerin B (Fig. 9.2) in relation to pathogen development using fluorescence microscopy. The authors demonstrated the accumulation of the autofluorescent phytoalexins in

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the epidermis of resistant plants (the cell type colonized by this pathogen). Fluorescence and laser scanning confocal microscopy showed the localization of the phytoalexins in relation to fungal structure development. The eventual collapse of the pathogen could be demonstrated and closely correlated with the production of phytoalexins at the infection site. After the pathogen had collapsed, the amount of phytoalexins began to decline, as is typical for phytoalexins in resistant plant–pathogen interactions (Hammerschmidt, 1999b; Kuc´, 1995). Additional information on the role and nature of phenolic and non-phenolic phytoalexins can be found in several recent reviews (Kuc´ 1995; Hammerschmidt, 1999b).

9.5.2 Phenolic structural defenses In the early 1960s, Hijwegen (1963) reported that treatment of cucumber seedlings with the amino acid phenylserine promoted the formation of lignin and increased resistance to the pathogen Cladosporium cucumerinum. From this study, Hijwegen concluded that lignification could be a part of host defense against pathogens. Since that report, there have been numerous studies showing a correlation between active host resistance and the deposition of lignin or some type of insoluble phenolic material (often the determination of the deposited materials was based only on histochemical reagents, which are useful in localizing the deposition of the polymers in relation to the pathogen, but provide limited chemical data). Several reviews have detailed much of the work on lignin in host defense, and this section will provide only a few representative examples. Ride and colleagues along with Vance and Sherwood demonstrated that the resistance of several graminaceous monocots was associated with the deposition of a lignin-like material at the site of attempted penetration by fungal pathogens (Vance & Sherwood, 1976, 1977; also reviewed in: Ride, 1978; Ride & Pearce, 1979; Vance et al., 1980; Bird & Ride, 1981). Ride (1978) proposed that lignification could play several roles in host defense. These include increasing the mechanical strength of plant cell walls, chemical modification of host cell walls to reduce the effect of pathogen-produced cell-wall degrading enzymes, and forming barriers that would restrict the flow of nutrients from host to pathogen as well as preventing the movement of pathogen toxins into living host cells. Ride also suggested that the process of lignification may be an important part of defense. Lignification is associated with the production of activated oxygen species such as hydrogen peroxide, and these anti-microbial oxygen species have been detected at sites of attempted fungal penetration and phenolic compound deposition (Lu & Higgins, 1998; Huckelhoven et al., 1999). In addition, the monolignol lignin precursors also have antifungal activity and it has been proposed that they function as phytoalexins in flax (Keen & Littlefield, 1979). A variation on how lignification may function in defense was proposed by Hammerschmidt & Kuc´ (1982), who suggested that fungal hyphae might themselves serve as a matrix upon which lignin could be deposited (thus stopping hyphal extension or branching). Using transmission electron microscopy and staining with manganese as a detection reagent for lignin, Stein et al. (1993) provided evidence that hyphae could become coated with a phenolic polymer in plant tissues actively defending against infection. Using a variety of metabolic inhibitors (e.g. of phenylalanine ammonia-lyase (PAL) or cinnamyl alcohol dehydrogenase (CAD)), further evidence for lignification as an

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active defense have been reported. Treatment of potato tuber tissue with aminooxyacetic acid blocked the production of lignin in tissue that was inoculated with the cucumber pathogen Cladosporium cucumerinum, and resulted in the colonization and sporulation of this fungus on tuber tissue (Hammerschmidt, 1984). Treatment of barley and wheat with the CAD inhibitor decreased the resistance to powdery mildew and rust pathogens, respectively, and also resulted in a decrease in the amount of structural phenolics (based on cytological analysis) (Moerschbacher et al., 1990; Zeyen et al., 1995).

9.5.3 Plant phenols and induced disease resistance Another form of actively expressed disease resistance is known as induced resistance. This is a form of resistance that develops in susceptible plants following some type of biotic or abiotic treatment that increases the ability of the host plant to defend itself following attempted infection (Hammerschmidt, 1999a). There are many reviews on this topic (e.g. Hammerschmidt & Kuc´, 1995; Sticher et al., 1997; van Loon et al., 1998), and further details on this form of resistance will focus on the role of plant phenols in it. Plant phenolic compounds have been shown to function both in the regulation of this type of resistance and as defense factors that are believed to restrict pathogen development. In 1979 R.F. White reported that treatment of tobacco plants carrying the N gene for resistance to tobacco mosaic virus (TMV) with acetyl salicylic and salicylic acids resulted in increased resistance to the virus. Although salicylic acid was a well-known natural plant product, it was not until 1990 that a relationship between induced disease resistance and salicylic acid was first reported. Both cucumber and tobacco plants that were treated to induce a resistant state systemically accumulated salicylic acid just prior to the expression of resistance (Malamy et al., 1990; Métraux et al., 1990). Although the initial thought was that salicylic acid might be the mobile signal that was involved in the expression of systemic induced resistance, further experiments suggested that it may not have that function (Rasmussen et al., 1991; Métraux, 2001; Métraux et al., 2002). However, it is clear that salicylic acid is a critical factor needed for the expression of certain types of host resistance (e.g. Gaffney et al., 1993; Mauch-Mani & Métraux, 1998) and the regulation of genes involved in defense mechanisms (see reviews by Dempsey et al., 1999; Dong, 2001; Glazebrook, 2001; Kunkel & Brooks, 2002). How salicylic acid functions in the induced-resistance phenomenon has also been intensively studied. There have been numerous attempts to find proteins that may bind to and thus be a receptor for salicylic acid. For example, it has been shown that salicylic acid will bind to catalase, thus inhibiting its activity and allowing hydrogen peroxide to accumulate (Chen & Klessig, 1991). The hydrogen peroxide that accumulates may then be involved in defense signaling (Chen et al., 1993; Lamb & Dixon, 1997). Another protein (SABP2) shown to bind salicylic acid has lipase activity, and silencing the gene coding for this protein inhibits the induction of induced resistance (Kumar & Klessig, 2003). One possible function of SABP2 may lie in its ability to release fatty-acid signals. Further work on SABP2 has shown that it may be a methyl salicylate esterase capable of releasing biologically active salicylic acid from its methyl ester (Forouhar et al., 2005). Salicylic acid itself is a strong inhibitor of the conversion of methyl salicylate to free salicylic acid. More recently, another salicylic acid binding protein, SABP3 has been characterized. SABP3

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is the chloroplast carbonic anhydrase (Slaymaker et al., 2002). This enzyme has antioxidant activity and it also appears that it is important in the expression of the hypersensitive resistance response. More recently, it has been proposed that salicylic acid functions in the defense of plant cells by affecting their redox state through the following series of events (Despres et al., 2003; Fobert & Despres, 2005). Systemic induced resistance responses and other types of resistance require the action of the NPR1 protein. This protein interacts with transcription factors required for the induction of salicylic acid-mediated resistance. The redox state of the cell controlled by salicylic acid appears to be involved in the oxidation state of NPR1.

9.6 Responses of the induced plant Once defense has been induced in a plant, the expression of resistance to a subsequent infection has been shown to be related to a rapid deployment of such defense. This heightened state of defensive readiness is known as ‘priming’ (Conrath et al., 2002). Although this phase of induced resistance has not been studied as intensively as other parts of this phenomenon, in many cases a role of phenolic compounds as putative defenses has been demonstrated. This section will describe a few cases where phenols may be important factors in stopping pathogens in induced plants.

9.6.1 Induced resistance in green bean and C. lindemunthianum One of the first host–pathogen systems in which induced resistance was studied was the green bean (Phaseolus vulgaris)–Colletotrichum lindemuthianum interaction. Elliston et al. (1971) were able to demonstrate that bean tissue at a short distance from a resistanceinducing inoculation became resistant to pathogenic races of C. lindemuthianum. The induced tissues showed an enhanced ability to produce isoflavonoid phytoalexins when challenged with C. lindemuthianum (Elliston et al., 1977).

9.6.2 Acibenzolar-S-methyl mediated induced resistance Acibenzolar-S-methyl (ASM) is a synthetic plant resistance activator that functions as an analog of salicylic acid. Latunde-Dada & Lucas (2001) demonstrated that this material induced resistance in cowpea plants to Colletotrichum destructivum. When challenged by C. destructivum, the induced cowpea plants produced isoflavonoid phytoalexins as part of the defense response.

9.6.3 Plant growth-promoting rhizobacteria and induced resistance Select rhizosphere bacteria, collectively known as plant growth promoting rhizobacteria or PGPR, have been shown to induce a form of systemic resistance. This resistance requires ethylene and jasmonic acid signaling for its expression. However, like resistance that depends on salicylic acid, this form of resistance also primes tissue for enhanced defense (van Loon et al., 1998). Resistance to Fusarium wilt can be induced in carnation by prior treatment with PGPR. Challenge of the PGPR-treated plants with Fusarium oxysporum f. sp. dianthi

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resulted in the formation of the dianthramide phytoalexins (van Peer et al., 1991). Inoculations with only the PGPR or F. oxysporum did not result in phytoalexin production, thus suggestion that the prior root colonization by the PGPR induced or primed the plant to rapidly respond when inoculated with the pathogen. In some cases, the PGPR may directly induce the production of phenolic phytoalexins. Ongena et al. (2000) reported that treatment of cucumber plants with a resistance-inducing Pseudomonad resulted in a systemic accumulation of phytoalexins in both the treated and untreated tissues. This suggests that some forms of induced resistance may be due to a pre-challenge accumulation of fungistatic or fungitoxic phenolic compounds.

9.6.4 Silicon as a modulator of defense and phenolic compounds Silicon treatments have been shown to be effective in managing certain diseases (Fauteux et al., 2005). Fawe et al. (1998) demonstrated an increase in glycosides of the antifungal flavonol rhamnetin and increased resistance to powdery mildew in cucumber plants pretreated with silicon.

9.7 Chemical induction of phenolic compounds and resistance Treatment of soybean plants with herbicides that inhibit protoporphyrinogen oxidase increases the resistance of the plants to white mold caused by Sclerotinia sclerotiorum (Dann et al., 1999; Nelson et al., 2002a, 2002b). Treatment of foliage with these herbicides results in the accumulation of the isoflavonoid phytoalexin glyceollin and increased resistance to infection by the pathogen. Over time, the amount of glyceollin decreases in the tissue and the level of resistance declines. As such, there is some correlation between the production of the phytoalexin and resistance. Mechanistically, these herbicides result in the accumulation of protoporphyrin IX that, in the presence of light, generates singlet oxygen (Scalla & Matringe, 1994). It is hypothesized that the damage caused by the singlet oxygen is involved in the induction and accumulation of the phytoalexins. The growth regulator prohexadione (Roemmelt et al., 2003) has been reported to increase the accumulation of novel flavonoids in apple including luteoliflavan, luteoliflavan 5glucoside, eriodictyol 7-glucoside and 6″O-trans-p-coumaroyleriodictyol 3′-glucoside. This increase is associated with increased resistance of prohexadione-treated trees to the fire blight bacterium Erwinia amylovora, although further work is needed to establish a role for these compounds in defense.

9.7.1 Structural phenolic compounds and induced resistance Induced resistance in cucumber plants to Colletotrichum orbiculare is due, in part, to the inability of the pathogen to penetrate into induced tissues (reviewed in Hammerschmidt & Yang-Cashman, 1995). Histochemical observations of the penetration of C. orbiculare into induced leaf tissue revealed the deposition of a lignin-like material based on reactions with phlorglucinol-HCl and toluidine blue (Hammerschmidt & Kuc´, 1982). Challenge of induced tissues with another cucumber pathogen, Cladosporium cucumerinum, also resulted in the deposition of lignins at the point of pathogen ingress into the tissues

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(Hammerschmidt & Kuc´, 1982). Deposition of insoluble phenolic compounds at the site of penetration may only be part of the induced structural defense. Stein et al. (1993) showed that silicon was also being deposited at the same place where lignin was being deposited. This suggests that phenolics are probably functioning in combination with other wallstrengthening materials to block infection.

9.7.2 Antioxidant activity of phenolic compounds and plant defense The role of salicylic acid as a redox agent in the modulation of plant defense expression has already been discussed in this review, but it has been known for some time that the antioxidant activity of phenols may play an important role in disease resistance responses. The reactions that occur in cells that are actively mounting a defense to pathogen ingress are often associated with the localized production of active oxygen species such as hydrogen peroxide and superoxide (Lu & Higgins, 1998). In addition, the formation of structural polymers such as lignin results in the concomitant formation of phenolic free radicals as part of polymer assembly. All of these products of the defense response have the ability to damage cells that surround the site of infection but are not directly attacked by the pathogen. As these cells may be needed to produce new metabolites for defense, it is important that they are not damaged by these by-products of defense. In maize lines resistant to fungal pathogens an accumulation of anthocyanin pigments occurs in the cells surrounding the infection site (Heim et al., 1983). Hipskind et al. (1996) characterized the anthocyanins that accumulated around lesions and suggested that these compounds may function as antioxidants that protect the living cells surrounding the infection site. Kangatharalingam et al. (2002) reported that anthocyanins accumulated in cotton leaf epidermal cells near the sites of active defense against Xanthomonas infection. They also suggested that the formation of anthocyanins may be involved in protection against the oxidative reactions that are part of host defense. Along these lines, Baker et al. (2002, 2005a,b) have shown that phenolic antioxidants are produced by cultured tobacco cells when challenged with a strain of Pseudomonas syringae pv. syringae that elicits a hypersensitive response. In a subsequent study, Baker et al. demonstrated that inoculation with hypersensitive response-inducing or non-inducing strains of P. syringae induced the release of hydroxycinnamic acid amides and acetophenones into the cell-culture medium. In those cultures inoculated with a hypersensitive responseinducing strain of Pseudomonas, the quantity of these extracellular phenols in the culture medium began to decline by about 7 hours after inoculation. At this time the plant cells were producing hydrogen peroxide, and it is thought that the decline in phenols was the result of their oxidation. It is thus very possible that the production of extracellular phenols plays an important role in modulating the accumulation or production of extracellular active oxygen species.

9.8 Phenols and defense: a multitude of roles Phenolic compounds have long been studied as possible factors in disease resistance. The obvious antimicrobial activity of many phenolic compounds and the ability of simple

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phenols to form structural barriers are two obvious ways that phenols contribute to defense. However, it is becoming increasingly clear that the role of these ubiquitous compounds is far broader than as part of a direct defense response. Defense signaling, regulation of cellular redox states and functioning as protective antioxidants are additional roles that phenolic compounds are being shown to play in plant–pathogen interactions. The challenge ahead lies in determining the contribution or contributions that phenols play in defense and conclusively demonstrating how phenols function in plant defense.

9.9 Acknowledgment Support from the Michigan Agricultural Experiment Station is gratefully acknowledged.

9.10 References Allen, P.J. (1959). Physiology and biochemistry of defense. In: Plant Pathology an Advanced Treatise, Vol. 1 (eds J.G. Horsfall & A.E. Dimond), pp. 435–467. Academic Press, New York, USA. Angell, H.R., Walker, J.C. & Link, K.P. (1930). The relation of protocatechuic acid to disease resistance in the onion. Phytopathology, 20, 431–438. Baker, C.J., Mock, N.M., Whitaker, et al. (2005a). Involvement of acetosyringone in plant-pathogen recognition. Biochemical and Biophysical Research Communications, 328, 130–136. Baker, C.J., O’Neill, N.R., Deahl, K. & Lydon, J. (2002). Continuous production of extracellular antioxidants in suspension cells attenuates the oxidative burst detected in plant microbe interactions. Plant Physiology and Biochemistry, 40, 641–644. Baker, C.J., Whitaker, B.D., Roberts, D.P., Mock, N.M., Rice, C.P., Deahl, K.L. & Aver’yanov, A.A. (2005b). Induction of redox sensitive extracellular phenolics during plant-bacterial interactions. Physiological and Molecular Plant Pathology, 66, 90–98. Bird, P.M. & Ride, J.P. (1981). The resistance of wheat to Septoria nordorum fungal development in relation to host lignification. Physiological Plant Pathology, 19, 289–299. Bostock, R.M., Wilcox, S.M., Wang, G. & Adaskaveg, J.E. (1999). Suppression of Monilinia fructicola cutinase production by peach fruit surface phenolic acids. Physiological and Molecular Plant Pathology, 54, 37 – 50. Chen, Z. & Klessig, D.F. (1991). Identification of a soluble salicylic acid-binding protein that may function in signal transduction in the plant disease resistance response. Proceedings of the National Academy of Sciences of the USA, 88, 8179 –8183. Chen, Z., Silva, H. & Klessig, D.F. (1993). Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science, 262, 1883–1886. Cojocaru, M., Droby, S., Glotter, E. et al. (1986). 5-(12-heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit. Phytochemistry, 25, 1093–1095. Conrath, U., Pieterse, C.M.J. & Mauch-Mani, B. (2002). Priming in plant–pathogen interactions. Trends in Plant Science, 7, 210 – 216. Cruickshank, I.A.M. (1963). Phytoalexins. Annual Review of Phytopathology, 1, 351–374. Curir, P., Danieli, B., Dolci, M., Pasini, C., Guglieri, L. & Sacco, M. (2000). Reductive detoxification of the acetophenone skeleton of the carnation phytoanticipin by Fusarium oxysporum f. sp. dianthi. Plant Pathology, 49, 742 –747. Curir, P., Dolci, M., Lanzotti, V. & Taglialatela-Scafati, O. (2001). Kaempferide triglycoside, a possible factor of resistance of carnation (Dianthus caryophyllus) to Fusarium oxysporum f. sp. dianthi. Phytochemistry, 56, 717–721. Curir, P., Marchesini, A., Danieli, B. & Mariani, F. (1996). 3-Hydroxyacetophenone in carnations is a phytoanticipin active against Fusarium oxysporum f. sp. dianthi. Phytochemistry, 41, 447–450.

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Roemmelt, S., Zimmermann, N., Rademacher, W. & Treutter, D. (2003). Formation of novel flavonoids in apple (Malus × domestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca. Phytochemistry, 64, 709 –716. Scalla, R. & Matringe, M. (1994). Inhibitors of protoporphyrinogen oxidase as herbicides: diphenyl ethers and other photobleaching molecules. Reviews in Weed Science, 6, 103–132. Shain, L. & Miller, J.B. (1982). Pinocembrin – an antifungal compound secreted by leaf glands of eastern cottonwood. Phytopathology, 72, 877–880. Slaymaker, D.H., Navarre, D.A., Clark, D., del Pozo, O., Martin, G.B. & Klessig, D.F. (2002). The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proceedings of the National Academy of Sciences of the USA, 99, 11640–11645. Snyder, B.A., Leite, B., Hipskind, J., Butler, L.G. & Nicholson, R.L. (1991). Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site. Physiological and Molecular Plant Pathology, 39, 463 – 470. Snyder, B.A. & Nicholson, R.L. (1990). Synthesis of phytoalexins in sorghum as a site specific response to fungal ingress. Science, 248, 1637–1639. Stein, B.D., Klomparens, K.L. & Hammerschmidt, R. (1993). Histochemistry and ultrastructure of the induced resistance response of cucumber plants to Colletotrichum lagenarium. Journal of Phytopathology, 137, 177–188. Sticher, L.B., Mauch-Mani, B. & Métraux, J.P. (1997). Systemic acquired resistance. Annual Review of Phytopathology, 35, 235 –270. Strack, D. (1997). Phenolic metabolism. In: Plant Biochemistry (eds P.M. Dey & J.B. Harborne), pp. 397– 416. Academic Press, New York, USA. Treutter, D. (2005). Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biology, 7, 581–591. Treutter, D. (2006). Significance of flavonoids in plant resistance, a review. Environmental Chemistry Letters, 4, 147–157. van Loon, L.C., Bakker, P.A.H.M. & Pieterse, C.M.J. (1998). Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology, 36, 453–483. van Peer, R., Niemann, G.J. & Schippers, B. (1991). Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology, 81, 728 –734. Vance, C.P., Kirk, T.K. & Sherwood, R.T. (1980). Lignification as a mechanism of disease resistance. Annual Review of Phytopathology, 18, 259 –288. Vance, C.P. & Sherwood, R.T. (1976). Regulation of lignin formation in reed canary grass in relation to disease resistance. Plant Physiology, 57, 915–919. Vance, C.P. & Sherwood, R.T. (1977). Lignified papilla formation as a mechanism for protection in reed canary grass. Physiological Plant Pathology, 10, 247–256. Walker, J.C. (1925). Studies on disease resistance in the onion. Proceedings of the National Academy of Sciences of the USA, 11, 183 –189. Walker, J.C., Link, P.K. & Angell, H.R. (1929). Chemical aspects of disease resistance in the onion. Proceedings of the National Academy of Sciences of the USA, 15, 845–850. Wattad, C., Dinoor, A. & Prusky, D. (1994). Purification of pectate lyase produced by Colletotrichum gloeosporioides and its inhibition by epicatechin – a possible factor involved in the reistance of unripe avocado fruits to anthracnose. Molecular Plant–Microbe Interactions, 7, 293–297. White, R.F. (1979). Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology, 99, 410 – 412. Zeyen, R.J., Bushnell, W.R., Carver, T.L.W. et al. (1995). Inhibiting phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase suppresses Mla1 (HR) but not Mlo5 (non-HR) barley powdery mildew resistances. Physiological and Molecular Plant Pathology, 47, 119–140.

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Chapter 9

Phenols and the Onset and Expression of Plant Disease Resistance Ray Hammerschmidt and Samantha I. Hollosy

9.1 Introduction Although there are thousands of plant pathogens, individual plant species are infected by only a small percentage of the pathogens that exist in nature. This suggests that plants are well equipped with the means to defend against most attacks, and that successful infection may only occur where these defenses are breached or nullified in some way. Plant defenses against pathogens are those processes, structures or metabolites that contribute to stopping the ingress and growth of pathogens in host tissues. Because of the myriad pathogens and the many different mechanisms of pathogenesis that have been identified, it is not surprising that a variety of approaches to defense have evolved. Defense mechanisms consist of a combination of preformed or constitutive defenses along with those defenses that are produced only after infection (Hammerschmidt & Nicholson, 1999). The preformed defenses provide a passive form of defense, and must be produced in tissues that come into contact with the potential pathogen. Defenses induced as a result of infection are referred to as active defenses and can be localized at the site of the attempted infection or may be produced throughout the plant. The many different types of pathogen that a plant encounters require not only different types of defense strategies (preformed vs. activated after infection) to protect against pathogens but also different biochemical mechanisms of defense. Many different types of natural product with antimicrobial activity have been associated with both passive and active defense (e.g. Grayer & Harborne, 1994; Treutter, 2005, 2006). Almost all classes of secondary metabolites (e.g. alkaloids, terpenoids, polyketides, and a wide variety of phenolic compounds) have been associated with host defense as illustrated by their invitro antimicrobial activity (Grayer & Harborne, 1994; Hammerschmidt, 1999b). Induced defenses also come in a wide range of chemical forms and include a variety of antifungal and hydrolytic enzymes, low molecular weight antimicrobial compounds produced at the site of infection (i.e. phytoalexins), cell-wall strengthening materials such as callose, lignins and hydroxyproline-rich glycoproteins, and several species of active oxygen (Hammerschmidt & Nicholson, 1999). However, as pointed out in other publications (e.g. Nicholson & Hammerschmidt, 1992; Hammerschmidt, 1999b), demonstration of antimicrobial activity in an in-vitro assay or Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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presumed ability to strengthen structural barriers is insufficient to show a role in plant defense against a pathogen. For antimicrobial compounds to be a functional defense, it must be demonstrated that the compounds are present in tissues that come into contact with the pathogen and be present in concentrations that are high enough to inhibit microbial growth. Finally, if a host plant fails to produce a specific compound implicated in defense, this lack of production must be shown to result in an increased ability of the pathogen to invade the host (Hammerschmidt, 1999b). Defenses against pathogens have also been associated with structural barriers such as suberized periderms and lignified cell walls (Nicholson & Hammerschmidt, 1992; Hammerschmidt, 1999a). Once again, the presence of these in response to a pathogen may or may not provide the evidence needed to demonstrate a role in defense. In later parts of this review, some of the key approaches needed to demonstrate if a putative defense is truly a defense will be discussed along with examples of defenses in several host–pathogen systems.

9.2 Biosynthetic origins of defense-associated phenolic compounds Phenolic compounds have been shown to play a role in both active and passive forms of defense. Most classes of phenolic compounds have been shown to be involved in defense, and these include simple hydroxybenzoic acid, free and conjugated hydroxycinnamic acids, coumarins, flavonoids and stilbenes (Grayer & Harborne, 1994). While most of the phenolic compounds implicated in defense are derived in part or fully from the phenylpropanoid pathway, others are produced via the acetate–malonate (polyketide) pathway or acetate– mevalonate (terpenoid) pathway. Other plant-defense-associated phenolic compounds, such as the hydrolysable tannins, are derived from intermediates of the shikimic acid pathway (Strack, 1997). Thus, depending on the plant species, the types of phenol that are implicated in defense may differ greatly.

9.3 Phenolic compounds as preformed defenses Most plants contain preformed compounds that are antimicrobial, and there is good evidence to support the role of pre-formed compounds in resistance (Nicholson and Hammerschmidt, 1992; Grayer & Harborne, 1994; Treutter, 2005, 2006). Pre-existing chemicals that may be involved in plant defense represent a broad spectrum of secondary plant metabolites that includes phenolic compounds. An example of a phenolic compound that has been implicated as a preformed defense is the flavanone pinocembrin from Populus deltoides (Shain & Miller, 1982). This compound is found on leaf surfaces and it is proposed that it functions in defense by inhibiting fungal spore germination. Many other preformed, antimicrobial compounds have been documented (e.g. Grayer & Harborne, 1994; Prusky & Keen, 1993). However, more evaluation is needed to determine the role of these and other antimicrobial compounds in passive defense. In the following sections, some examples of the various roles that preformed phenolic compounds play in defense are described.

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R=H, Catechol R=COOH, Protocatechuic acid (onion)

Chlorogenic acid (widely distributed)

3-Hydroxyacetophenone (carnation)

213

Epicatechin (avocado)

Caffeic acid (widely distributed)

5-Pentadecylresorcinol (mango)

Pinocembrin (cotton wood)

Fig. 9.1 Structures of phenolic compounds associated with preformed defenses (the plant names refer to examples discussed in the text).

9.3.1 Resistance of onion bulbs Onion smudge is caused by the soil borne pathogen Colletotrichum circinans (Walker, 1925). Early work by Walker and colleagues (Link et al., 1929; Walker et al., 1929; Angell et al., 1930) showed that resistance to this pathogen correlated with pigmentation of the bulb. If spores of C. circinans were incubated in aqueous extracts of dry outer scales of pigmented bulbs, hyphal growth was inhibited and germ tubes appeared abnormal. Aqueous extracts from dry scales of white onions were not inhibitory. Analysis of the extracts revealed that the antimicrobial activity was not due to the anthocyanin and flavonoid pigments, but rather to two simple phenolic compounds: catechol and protocatechuic acid (Fig. 9.1). Because these two phenols are water soluble, they are easily extracted from the dead scales. Since the spores need free water to germinate, the very water needed to promote infection also provides the delivery vehicle for these defense compounds. Thus, diffusion of protocatechuic acid and catechol from dead onion scales into the spore-containing water droplets results in inhibition of fungal development and thus provides resistance. However, if the outer scales of pigmented onions are removed, the underlying living scale tissues are just as susceptible as the white bulbs. Thus, these compounds provide protection against infection as long as the tissues containing them are intact (Walker et al., 1929).

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9.3.2 Fusarium wilt of carnation One of the most important diseases of carnation is fusarium wilt cause by Fusarium oxysporum f. sp. dianthi. Curir et al. (1996) found that varieties of carnation that were resistant to infection by this pathogen had high concentrations of 3-hydroxyacetophenone (Fig. 9.1). This compound was found to be antifungal in in-vitro assays, and evidence for an in-planta role in defense has also been provided by the authors. Another phenolic compound, a triglycoside of the flavonol kaempferide, has also been associated with resistance to F. oxysporum f. sp. dianthi (Curir et al., 2001). A recent report indicates that F. oxysporum f. sp. dianthi can detoxify 3-hydroxyacetophenone to a less toxic metabolite (Curir et al., 2000). Two NADH-dependent oxidoreductases are produced by the fungus in the presence of 3-hydroxyacetophenone. It will be interesting to learn if the ability to detoxify this compound is associated with virulence of the pathogen.

9.3.3 Mango fruit and Alternaria Fruits are often resistant to disease development while immature, yet they quickly express symptoms of disease as the ripening process begins (Prusky & Keen, 1993). This expression of symptoms is not related to when infections of the fruits occur, but rather to how immature fruit respond to infection as compared to ripe fruit. The fungus can begin to infect immature fruit but is held in check by fungistatic compounds that decrease in concentration upon ripening of the fruit. One example of this is found in mango fruit. Unripe mango fruit is thought to be resistant to Alternaria alternata because of the presence of two preformed antifungal compounds in the peel of the fruit: the resorcinol derivatives 5-12-cis-heptadecenylresorcinol and 5-pentadecylresorcinol (Fig. 9.1) (Cojocaru et al., 1986; Prusky & Keen, 1993). As the fruit ripens, the concentrations of these compounds decrease and the fruit becomes susceptible to Alternaria infection (Droby et al., 1986; Prusky & Keen, 1993).

9.3.4 Regulation of preformed antifungal compounds by phenols in avocado Another well-studied example of the role of preformed compounds in defense is the resistance of avocado fruit to fungal pathogens. This is similar to the case of mango, in which preformed antimicrobial compounds in the peel inhibit the growth of pathogens until the fruit matures. In this case, however, the phenolic compounds appear to play a role in maintaining the presence of antifungal compounds and not being directly involved in inhibiting the pathogens. Prusky and co-workers demonstrated a correlation between the concentration of antifungal fatty-acid derivatives in the peel of unripe avocado fruits with resistance to Colletotrichum gloeosporioides (Prusky et al., 1982; Prusky et al., 1991; Prusky & Keen, 1993). As the avocado fruit matures, the concentrations of the antifungal fatty acid derivatives decrease and disease development by previously quiescent C. gloeosporioides begins (Prusky et al., 1982; Prusky & Keen, 1993). Prusky et al. reported that the loss of the diene and monoene appears to be associated with a near doubling of lipoxygenase activity in the

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avocado tissue. This provides a reasonable explanation for how the fruits combat infection, but what role do phenolic compounds play in this defense? The increase in lipoxygenase activity and decrease in diene concentration was correlated with levels of epicatechin (Fig. 9.1), an inhibitor of lipoxygenase (Karni et al., 1989). As the fruit ripens, the concentration of epicatechin declines, and thus it is likely that this decrease in epicatechin allows endogenous lipoxygenase to degrade (and thus detoxify) the antifungal diene, and this permits the colonization of the fruit tissue by the pathogen. Further support for the important role of epicatechin comes from studies on the ability of C. gloeosporioides to degrade epicatechin with a laccase (Guetsky et al., 2005). The laccase is produced in vitro in the presence of epicatechin, and the fungal enzyme can also be detected in infected tissue. Treatment of fruits with compounds that inhibit laccase activity resulted in a decrease in disease development, thus further supporting the role that epicatechin plays in resistance modulation. Similarly, isolates of the pathogen that are deficient in laccase production are less virulent (Guetsky et al., 2005). Finally, Wattad et al. (1994) found that epicatechin can also inhibit the activity of a pectin lyase produced by C. gloeosporioides. This type of enzyme is thought to be important in pathogen virulence, and thus the inhibition of this enzyme may further contribute to the resistance of immature fruits to the pathogen.

9.3.5 Chlorogenic acid and the infection of stone fruit by Monilinia Monilinia fructicola causes a serious disease of stone fruit known as brown rot. In peach, resistance to this disease has been correlated with the amount of cholorogenic and caffeic acids (Fig. 9.1) in the outer peel. As the fruit ripens the amount of these phenols decreases and susceptibility increases. Bostock et al. (1999) examined the role of chlorogenic and caffeic acids in resistance to brown rot and found that the concentrations of these phenolic acids found in the peel were not inhibitory to Monilinia growth or spore germination. Thus, the role of these compounds in defense is probably not through fungistatic or fungitoxic properties. However, they did find that these phenolic compounds may contribute to defense by blocking the production of cutinase, an esterase involved in penetration of the plant cuticle. Growing cultures of Monilinia on cutin in the presence of caffeic acid prevented the production of cutinase and other esterases. Caffeic and chlorogenic acids had no effect on the activity of cutinase, thus these compounds are probably not involved in defense by inhibiting enzyme activity. Bostock et al. (1999) proposed that chlorogenic and caffeic acids may function in defense by blocking the production of enzymes needed in the infection of fruit.

9.4 Active defense In most plant–pathogen interactions, defense responses are induced after the infection process has been initiated (Hammerschmidt & Nicholson, 1999). This interaction, which is initiated by recognition of the pathogen by the host plant, results in a series of events that lead to the expression of a wide variety of responses, of which accumulation of phenolic compounds is a common feature. These responses are often highly localized and may involve

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only a few host cells. In other interactions, the initial infection results in the systemic expression of defenses and a change in the physiology of the host plant that allows rapid deployment of defenses upon infection. This phenomenon is referred to as ‘induced resistance’.

9.5 Localized defenses In the early 20th century Noel Bernard found that infection of orchid tubers with a non-pathogenic strain of Rhizoctonia would protect the tissue against infection with a pathogenic strain. Bernard and later workers reported that a diffusible antimicrobial substance was associated with the resistance and that production of the antimicrobial compounds was enhanced by the infection (reviewed in Allen, 1959; Gäumann, 1950). Perhaps the first clear experimental evidence for induced, localized defenses was present by Müller & Börger in 1940 (Reviewed in Gaumann, 1950; Muller, 1959), who found that preinoculation of potato tuber tissue with an incompatible race of Phytophthora infestans resulted in a defense response that not only stopped the incompatible race but also induced resistance to subsequent inoculations with compatible races of the pathogen that should have caused disease. They hypothesized that the induced resistance was due to the production of what they called phytoalexins (reviewed in Allen, 1959). Within twenty years, the first phytoalexins were identified and many of these compounds were found to be phenolic. In the 1950s the first biochemical studies of active defense responses began in earnest. For example, Kuc´ and co-workers reported that inoculation of potato tuber tissue slices with Cochliobolus (Helminthosporium) carbonum, a pathogen of maize but not of potato, resulted in the production of extractable, fungistatic compounds (Kuc´ et al., 1955, 1956). A subsequent study found that the inoculated tubers produced both caffeic and chlorogenic acids (Fig. 9.1) as part of the response to C. carbonum. Although these phenolic acids constitute part of the defense response, Kuc´ later reported that it was unlikely that they would be able to account for all of the antifungal activity in the extracts (Kuc´, 1957), and this was proven to be true by the subsequent identification of other antimicrobial compounds induced in potato tubers (Kuc´, 1995; Hammerschmidt, 1999b). By the end of the 1950s, the first phytoalexin had been characterized by Cruickshank and Perrin (reviewed in Cruicskshank, 1963). This was the isoflavonoid derivative pisatin (Fig. 9.2), which was isolated from immature pea pods that had been inoculated with spores of a Monilinia (a non-pathogen of peas). In subsequent years many more phytoalexins were discovered from many plant families (Cruickshank, 1963; Kuc´, 1995; Hammerschmidt, 1999b). Some of these turned out to be phenolic compounds, and this will be the focus of the rest of this section.

9.5.1 Phenolic phytoalexins An important consideration to take into account in studying induced antimicrobial compounds is determining whether they play a direct role in defense or are only part of a general metabolic response to infection. Several criteria have been proposed to help show that these compounds play an important role (Hammerschmidt, 1999b), and one of these

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Pisatin (pea)

Glyceollin I (soybean)

217

Luteolinidin (sorghum)

Lacinilene c (cotton)

Cucumerin B (cucumber) Avenanthramide A (oats) Fig. 9.2 Structures of phenolic compounds associated with induced or active defenses (th plant names refer to examples discussed in the text).

involves demonstrating that the compounds accumulate at the right time and in the right location. One of the very best examples of localizing and in-vivo quantification of phytoalexins in a host pathogen interaction comes from the work of Nicholson and co-workers on the resistance of sorghum to fungal pathogens. They found that sorghum produces a variety of flavan-3-ol phytoalexins such as luteolinidin (Fig. 9.2), apigeninidin and derivatives of these flavonoids (Nicholson et al., 1987; Hipskind et al., 1990; Lo et al., 1999). These compounds are also pigments and this allows direct visualization in the tissue. Using light microscopy and microspectrophotometry, Nicholson and co-workers were able to show that the phytoalexins did accumulate at the right time and place to be effective defenses, and they were also able to demonstrate that the concentration of the phytoalexins in the infected cells was more than sufficient to inhibit growth of the pathogen (Snyder & Nicholson, 1990; Snyder et al., 1991). There are other examples of the localization of phenolic phytoalexins that help demonstrate their roles in defense, and these include studies with cotton phytoalexins such as lacinilene c (Fig. 9.2) (Essenberg et al., 1992; Pierce et al., 1996) and the avenanthramides (Fig. 9.2) from oats (Mayama & Tani, 1982). In a more recent study, McNally et al. (2003) were able to take advantage of the autofluorescent properties of cucumber C-glycosyl flavonoid phytoalexins to localize these phytoalexins in host cells. Using the Podosphaera xanthii–cucumber interaction, they were able to localize the production of autofluorescent C-glycosyl flavonoid phytoalexins such as cucumerin B (Fig. 9.2) in relation to pathogen development using fluorescence microscopy. The authors demonstrated the accumulation of the autofluorescent phytoalexins in

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the epidermis of resistant plants (the cell type colonized by this pathogen). Fluorescence and laser scanning confocal microscopy showed the localization of the phytoalexins in relation to fungal structure development. The eventual collapse of the pathogen could be demonstrated and closely correlated with the production of phytoalexins at the infection site. After the pathogen had collapsed, the amount of phytoalexins began to decline, as is typical for phytoalexins in resistant plant–pathogen interactions (Hammerschmidt, 1999b; Kuc´, 1995). Additional information on the role and nature of phenolic and non-phenolic phytoalexins can be found in several recent reviews (Kuc´ 1995; Hammerschmidt, 1999b).

9.5.2 Phenolic structural defenses In the early 1960s, Hijwegen (1963) reported that treatment of cucumber seedlings with the amino acid phenylserine promoted the formation of lignin and increased resistance to the pathogen Cladosporium cucumerinum. From this study, Hijwegen concluded that lignification could be a part of host defense against pathogens. Since that report, there have been numerous studies showing a correlation between active host resistance and the deposition of lignin or some type of insoluble phenolic material (often the determination of the deposited materials was based only on histochemical reagents, which are useful in localizing the deposition of the polymers in relation to the pathogen, but provide limited chemical data). Several reviews have detailed much of the work on lignin in host defense, and this section will provide only a few representative examples. Ride and colleagues along with Vance and Sherwood demonstrated that the resistance of several graminaceous monocots was associated with the deposition of a lignin-like material at the site of attempted penetration by fungal pathogens (Vance & Sherwood, 1976, 1977; also reviewed in: Ride, 1978; Ride & Pearce, 1979; Vance et al., 1980; Bird & Ride, 1981). Ride (1978) proposed that lignification could play several roles in host defense. These include increasing the mechanical strength of plant cell walls, chemical modification of host cell walls to reduce the effect of pathogen-produced cell-wall degrading enzymes, and forming barriers that would restrict the flow of nutrients from host to pathogen as well as preventing the movement of pathogen toxins into living host cells. Ride also suggested that the process of lignification may be an important part of defense. Lignification is associated with the production of activated oxygen species such as hydrogen peroxide, and these anti-microbial oxygen species have been detected at sites of attempted fungal penetration and phenolic compound deposition (Lu & Higgins, 1998; Huckelhoven et al., 1999). In addition, the monolignol lignin precursors also have antifungal activity and it has been proposed that they function as phytoalexins in flax (Keen & Littlefield, 1979). A variation on how lignification may function in defense was proposed by Hammerschmidt & Kuc´ (1982), who suggested that fungal hyphae might themselves serve as a matrix upon which lignin could be deposited (thus stopping hyphal extension or branching). Using transmission electron microscopy and staining with manganese as a detection reagent for lignin, Stein et al. (1993) provided evidence that hyphae could become coated with a phenolic polymer in plant tissues actively defending against infection. Using a variety of metabolic inhibitors (e.g. of phenylalanine ammonia-lyase (PAL) or cinnamyl alcohol dehydrogenase (CAD)), further evidence for lignification as an

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active defense have been reported. Treatment of potato tuber tissue with aminooxyacetic acid blocked the production of lignin in tissue that was inoculated with the cucumber pathogen Cladosporium cucumerinum, and resulted in the colonization and sporulation of this fungus on tuber tissue (Hammerschmidt, 1984). Treatment of barley and wheat with the CAD inhibitor decreased the resistance to powdery mildew and rust pathogens, respectively, and also resulted in a decrease in the amount of structural phenolics (based on cytological analysis) (Moerschbacher et al., 1990; Zeyen et al., 1995).

9.5.3 Plant phenols and induced disease resistance Another form of actively expressed disease resistance is known as induced resistance. This is a form of resistance that develops in susceptible plants following some type of biotic or abiotic treatment that increases the ability of the host plant to defend itself following attempted infection (Hammerschmidt, 1999a). There are many reviews on this topic (e.g. Hammerschmidt & Kuc´, 1995; Sticher et al., 1997; van Loon et al., 1998), and further details on this form of resistance will focus on the role of plant phenols in it. Plant phenolic compounds have been shown to function both in the regulation of this type of resistance and as defense factors that are believed to restrict pathogen development. In 1979 R.F. White reported that treatment of tobacco plants carrying the N gene for resistance to tobacco mosaic virus (TMV) with acetyl salicylic and salicylic acids resulted in increased resistance to the virus. Although salicylic acid was a well-known natural plant product, it was not until 1990 that a relationship between induced disease resistance and salicylic acid was first reported. Both cucumber and tobacco plants that were treated to induce a resistant state systemically accumulated salicylic acid just prior to the expression of resistance (Malamy et al., 1990; Métraux et al., 1990). Although the initial thought was that salicylic acid might be the mobile signal that was involved in the expression of systemic induced resistance, further experiments suggested that it may not have that function (Rasmussen et al., 1991; Métraux, 2001; Métraux et al., 2002). However, it is clear that salicylic acid is a critical factor needed for the expression of certain types of host resistance (e.g. Gaffney et al., 1993; Mauch-Mani & Métraux, 1998) and the regulation of genes involved in defense mechanisms (see reviews by Dempsey et al., 1999; Dong, 2001; Glazebrook, 2001; Kunkel & Brooks, 2002). How salicylic acid functions in the induced-resistance phenomenon has also been intensively studied. There have been numerous attempts to find proteins that may bind to and thus be a receptor for salicylic acid. For example, it has been shown that salicylic acid will bind to catalase, thus inhibiting its activity and allowing hydrogen peroxide to accumulate (Chen & Klessig, 1991). The hydrogen peroxide that accumulates may then be involved in defense signaling (Chen et al., 1993; Lamb & Dixon, 1997). Another protein (SABP2) shown to bind salicylic acid has lipase activity, and silencing the gene coding for this protein inhibits the induction of induced resistance (Kumar & Klessig, 2003). One possible function of SABP2 may lie in its ability to release fatty-acid signals. Further work on SABP2 has shown that it may be a methyl salicylate esterase capable of releasing biologically active salicylic acid from its methyl ester (Forouhar et al., 2005). Salicylic acid itself is a strong inhibitor of the conversion of methyl salicylate to free salicylic acid. More recently, another salicylic acid binding protein, SABP3 has been characterized. SABP3

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is the chloroplast carbonic anhydrase (Slaymaker et al., 2002). This enzyme has antioxidant activity and it also appears that it is important in the expression of the hypersensitive resistance response. More recently, it has been proposed that salicylic acid functions in the defense of plant cells by affecting their redox state through the following series of events (Despres et al., 2003; Fobert & Despres, 2005). Systemic induced resistance responses and other types of resistance require the action of the NPR1 protein. This protein interacts with transcription factors required for the induction of salicylic acid-mediated resistance. The redox state of the cell controlled by salicylic acid appears to be involved in the oxidation state of NPR1.

9.6 Responses of the induced plant Once defense has been induced in a plant, the expression of resistance to a subsequent infection has been shown to be related to a rapid deployment of such defense. This heightened state of defensive readiness is known as ‘priming’ (Conrath et al., 2002). Although this phase of induced resistance has not been studied as intensively as other parts of this phenomenon, in many cases a role of phenolic compounds as putative defenses has been demonstrated. This section will describe a few cases where phenols may be important factors in stopping pathogens in induced plants.

9.6.1 Induced resistance in green bean and C. lindemunthianum One of the first host–pathogen systems in which induced resistance was studied was the green bean (Phaseolus vulgaris)–Colletotrichum lindemuthianum interaction. Elliston et al. (1971) were able to demonstrate that bean tissue at a short distance from a resistanceinducing inoculation became resistant to pathogenic races of C. lindemuthianum. The induced tissues showed an enhanced ability to produce isoflavonoid phytoalexins when challenged with C. lindemuthianum (Elliston et al., 1977).

9.6.2 Acibenzolar-S-methyl mediated induced resistance Acibenzolar-S-methyl (ASM) is a synthetic plant resistance activator that functions as an analog of salicylic acid. Latunde-Dada & Lucas (2001) demonstrated that this material induced resistance in cowpea plants to Colletotrichum destructivum. When challenged by C. destructivum, the induced cowpea plants produced isoflavonoid phytoalexins as part of the defense response.

9.6.3 Plant growth-promoting rhizobacteria and induced resistance Select rhizosphere bacteria, collectively known as plant growth promoting rhizobacteria or PGPR, have been shown to induce a form of systemic resistance. This resistance requires ethylene and jasmonic acid signaling for its expression. However, like resistance that depends on salicylic acid, this form of resistance also primes tissue for enhanced defense (van Loon et al., 1998). Resistance to Fusarium wilt can be induced in carnation by prior treatment with PGPR. Challenge of the PGPR-treated plants with Fusarium oxysporum f. sp. dianthi

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resulted in the formation of the dianthramide phytoalexins (van Peer et al., 1991). Inoculations with only the PGPR or F. oxysporum did not result in phytoalexin production, thus suggestion that the prior root colonization by the PGPR induced or primed the plant to rapidly respond when inoculated with the pathogen. In some cases, the PGPR may directly induce the production of phenolic phytoalexins. Ongena et al. (2000) reported that treatment of cucumber plants with a resistance-inducing Pseudomonad resulted in a systemic accumulation of phytoalexins in both the treated and untreated tissues. This suggests that some forms of induced resistance may be due to a pre-challenge accumulation of fungistatic or fungitoxic phenolic compounds.

9.6.4 Silicon as a modulator of defense and phenolic compounds Silicon treatments have been shown to be effective in managing certain diseases (Fauteux et al., 2005). Fawe et al. (1998) demonstrated an increase in glycosides of the antifungal flavonol rhamnetin and increased resistance to powdery mildew in cucumber plants pretreated with silicon.

9.7 Chemical induction of phenolic compounds and resistance Treatment of soybean plants with herbicides that inhibit protoporphyrinogen oxidase increases the resistance of the plants to white mold caused by Sclerotinia sclerotiorum (Dann et al., 1999; Nelson et al., 2002a, 2002b). Treatment of foliage with these herbicides results in the accumulation of the isoflavonoid phytoalexin glyceollin and increased resistance to infection by the pathogen. Over time, the amount of glyceollin decreases in the tissue and the level of resistance declines. As such, there is some correlation between the production of the phytoalexin and resistance. Mechanistically, these herbicides result in the accumulation of protoporphyrin IX that, in the presence of light, generates singlet oxygen (Scalla & Matringe, 1994). It is hypothesized that the damage caused by the singlet oxygen is involved in the induction and accumulation of the phytoalexins. The growth regulator prohexadione (Roemmelt et al., 2003) has been reported to increase the accumulation of novel flavonoids in apple including luteoliflavan, luteoliflavan 5glucoside, eriodictyol 7-glucoside and 6″O-trans-p-coumaroyleriodictyol 3′-glucoside. This increase is associated with increased resistance of prohexadione-treated trees to the fire blight bacterium Erwinia amylovora, although further work is needed to establish a role for these compounds in defense.

9.7.1 Structural phenolic compounds and induced resistance Induced resistance in cucumber plants to Colletotrichum orbiculare is due, in part, to the inability of the pathogen to penetrate into induced tissues (reviewed in Hammerschmidt & Yang-Cashman, 1995). Histochemical observations of the penetration of C. orbiculare into induced leaf tissue revealed the deposition of a lignin-like material based on reactions with phlorglucinol-HCl and toluidine blue (Hammerschmidt & Kuc´, 1982). Challenge of induced tissues with another cucumber pathogen, Cladosporium cucumerinum, also resulted in the deposition of lignins at the point of pathogen ingress into the tissues

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(Hammerschmidt & Kuc´, 1982). Deposition of insoluble phenolic compounds at the site of penetration may only be part of the induced structural defense. Stein et al. (1993) showed that silicon was also being deposited at the same place where lignin was being deposited. This suggests that phenolics are probably functioning in combination with other wallstrengthening materials to block infection.

9.7.2 Antioxidant activity of phenolic compounds and plant defense The role of salicylic acid as a redox agent in the modulation of plant defense expression has already been discussed in this review, but it has been known for some time that the antioxidant activity of phenols may play an important role in disease resistance responses. The reactions that occur in cells that are actively mounting a defense to pathogen ingress are often associated with the localized production of active oxygen species such as hydrogen peroxide and superoxide (Lu & Higgins, 1998). In addition, the formation of structural polymers such as lignin results in the concomitant formation of phenolic free radicals as part of polymer assembly. All of these products of the defense response have the ability to damage cells that surround the site of infection but are not directly attacked by the pathogen. As these cells may be needed to produce new metabolites for defense, it is important that they are not damaged by these by-products of defense. In maize lines resistant to fungal pathogens an accumulation of anthocyanin pigments occurs in the cells surrounding the infection site (Heim et al., 1983). Hipskind et al. (1996) characterized the anthocyanins that accumulated around lesions and suggested that these compounds may function as antioxidants that protect the living cells surrounding the infection site. Kangatharalingam et al. (2002) reported that anthocyanins accumulated in cotton leaf epidermal cells near the sites of active defense against Xanthomonas infection. They also suggested that the formation of anthocyanins may be involved in protection against the oxidative reactions that are part of host defense. Along these lines, Baker et al. (2002, 2005a,b) have shown that phenolic antioxidants are produced by cultured tobacco cells when challenged with a strain of Pseudomonas syringae pv. syringae that elicits a hypersensitive response. In a subsequent study, Baker et al. demonstrated that inoculation with hypersensitive response-inducing or non-inducing strains of P. syringae induced the release of hydroxycinnamic acid amides and acetophenones into the cell-culture medium. In those cultures inoculated with a hypersensitive responseinducing strain of Pseudomonas, the quantity of these extracellular phenols in the culture medium began to decline by about 7 hours after inoculation. At this time the plant cells were producing hydrogen peroxide, and it is thought that the decline in phenols was the result of their oxidation. It is thus very possible that the production of extracellular phenols plays an important role in modulating the accumulation or production of extracellular active oxygen species.

9.8 Phenols and defense: a multitude of roles Phenolic compounds have long been studied as possible factors in disease resistance. The obvious antimicrobial activity of many phenolic compounds and the ability of simple

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phenols to form structural barriers are two obvious ways that phenols contribute to defense. However, it is becoming increasingly clear that the role of these ubiquitous compounds is far broader than as part of a direct defense response. Defense signaling, regulation of cellular redox states and functioning as protective antioxidants are additional roles that phenolic compounds are being shown to play in plant–pathogen interactions. The challenge ahead lies in determining the contribution or contributions that phenols play in defense and conclusively demonstrating how phenols function in plant defense.

9.9 Acknowledgment Support from the Michigan Agricultural Experiment Station is gratefully acknowledged.

9.10 References Allen, P.J. (1959). Physiology and biochemistry of defense. In: Plant Pathology an Advanced Treatise, Vol. 1 (eds J.G. Horsfall & A.E. Dimond), pp. 435–467. Academic Press, New York, USA. Angell, H.R., Walker, J.C. & Link, K.P. (1930). The relation of protocatechuic acid to disease resistance in the onion. Phytopathology, 20, 431–438. Baker, C.J., Mock, N.M., Whitaker, et al. (2005a). Involvement of acetosyringone in plant-pathogen recognition. Biochemical and Biophysical Research Communications, 328, 130–136. Baker, C.J., O’Neill, N.R., Deahl, K. & Lydon, J. (2002). Continuous production of extracellular antioxidants in suspension cells attenuates the oxidative burst detected in plant microbe interactions. Plant Physiology and Biochemistry, 40, 641–644. Baker, C.J., Whitaker, B.D., Roberts, D.P., Mock, N.M., Rice, C.P., Deahl, K.L. & Aver’yanov, A.A. (2005b). Induction of redox sensitive extracellular phenolics during plant-bacterial interactions. Physiological and Molecular Plant Pathology, 66, 90–98. Bird, P.M. & Ride, J.P. (1981). The resistance of wheat to Septoria nordorum fungal development in relation to host lignification. Physiological Plant Pathology, 19, 289–299. Bostock, R.M., Wilcox, S.M., Wang, G. & Adaskaveg, J.E. (1999). Suppression of Monilinia fructicola cutinase production by peach fruit surface phenolic acids. Physiological and Molecular Plant Pathology, 54, 37 – 50. Chen, Z. & Klessig, D.F. (1991). Identification of a soluble salicylic acid-binding protein that may function in signal transduction in the plant disease resistance response. Proceedings of the National Academy of Sciences of the USA, 88, 8179 –8183. Chen, Z., Silva, H. & Klessig, D.F. (1993). Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science, 262, 1883–1886. Cojocaru, M., Droby, S., Glotter, E. et al. (1986). 5-(12-heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit. Phytochemistry, 25, 1093–1095. Conrath, U., Pieterse, C.M.J. & Mauch-Mani, B. (2002). Priming in plant–pathogen interactions. Trends in Plant Science, 7, 210 – 216. Cruickshank, I.A.M. (1963). Phytoalexins. Annual Review of Phytopathology, 1, 351–374. Curir, P., Danieli, B., Dolci, M., Pasini, C., Guglieri, L. & Sacco, M. (2000). Reductive detoxification of the acetophenone skeleton of the carnation phytoanticipin by Fusarium oxysporum f. sp. dianthi. Plant Pathology, 49, 742 –747. Curir, P., Dolci, M., Lanzotti, V. & Taglialatela-Scafati, O. (2001). Kaempferide triglycoside, a possible factor of resistance of carnation (Dianthus caryophyllus) to Fusarium oxysporum f. sp. dianthi. Phytochemistry, 56, 717–721. Curir, P., Marchesini, A., Danieli, B. & Mariani, F. (1996). 3-Hydroxyacetophenone in carnations is a phytoanticipin active against Fusarium oxysporum f. sp. dianthi. Phytochemistry, 41, 447–450.

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Dann, E.K., Diers, B.W. & Hammerschmidt, R. (1999). Suppression of Sclerotinia stem rot of soybean by lactofen herbicide treatment. Phytopathology, 89, 598–602. Dempsey, D., Shah, J. & Klessig, D.F. (1999). Salicylic acid and disease resistance in plants. Critical Reviews in Plant Science, 18, 547–575. Despres, C., Chubak, C., Rochon, A. et al. (2003). The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. The Plant Cell, 15, 2181–2191. Dong, X. (2001). Genetic dissection of systemic acquired resistance. Current Opinion in Plant Biology, 4, 309 – 314. Droby, S., Prusky, D., Jacoby, B. & Goldman, A. (1986). Presence of antifungal compounds in the peel of mango fruits and their relation to latent infections of Alternaria alternata. Physiological and Molecular Plant Pathology, 29, 173 –183. Elliston, J., Kuc´, J. & Williams, E.B. (1971). Induced resistance to bean anthracnose at a distance from the site of the inducing interaction. Phytopathology, 61, 1110–1112. Elliston, J., Kuc´, J., Williams, E.B. & Rahe, J.E. (1977). Relationship of phytoalexin accumulation to local and systemic protection of bean against anthracnose. Journal of Phytopathology, 88, 114– 130. Essenberg, M., Pierce, M.L., Cover, E.C., Hamilton, B., Richardson, P.E. & Scholes, V.E. (1992). A method for determining phytoalexin concentrations in fluorescent hypersensitively necrotic cells in cotton leaves. Physiological and Molecular Plant Pathology, 41, 101–109. Fauteux, F., Remus-Borel, W., Menzies, J.G. & Belanger, R.R. (2005). Silicon and plant disease resistance against pathogenic fungi. FEMS Microbiology Letters, 249, 1–6. Fawe, A., Abou-Zaid, M., Menzies, J.G. & Bélanger, R.R. (1998). Silicon-mediated accumulation of flavonoid phytoalexins in cucumber. Phytopathology, 88, 396–401. Fobert, P.R. & Despres, C. (2005). Redox control of systemic acquired resistance. Current Opinion in Plant Biology, 8, 378 – 382. Forouhar, F., Yang, Y., Kumar, D. et al. (2005). Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proceedings of the National Academy of Sciences of the USA, 102, 1773–1778. Gaffney, T.P., Friedrich, L., Vernooij, B. et al. (1993). Requirement of salicylic acid for the induction of systemic acquired resistance. Science, 261, 754–756. Gäumann, E. (1950). Principles of plant infection: a text-book of general plant pathology for biologists, agriculturists, foresters and plant breeders. Hafner, New York, USA. Glazebrook, J. (2001). Genes controlling expression of defense responses in Arabidopsis – 2001 status. Current Opinion in Plant Biology, 4, 301–308. Grayer, R.J. & Harborne, J.B. (1994). A survey of antifungal compounds from higher plants, 1982–1993. Phytochemistry, 37, 19 – 42. Guetsky, R., Kobiler, I., Wang, X. et al. (2005). Metabolism of the flavonoid epicatechin by laccase of Colletotrichum gloeosporioides and its effect on pathogenicity on avocado fruits. Phytopathology, 95, 1341–1348. Hammerschmidt, R. (1984). Rapid deposition of lignin in potato tuber tissue as a response to fungi non-pathogenic on potato. Physiological and Molecular Plant Pathology, 24, 33–42. Hammerschmidt, R. (1999a). Induced disease resistance. How do induced plants stop pathogens? Physiological and Molecular Plant Pathology, 55, 77–84. Hammerschmidt, R. (1999b). Phytoalexins. What have we learned after 60 years? Annual Review of Phytopathology, 37, 285 –306. Hammerschmidt, R. & Kuc´, J. (1982). Lignification as a mechanism for induced systemic resistance in cucumber. Physiological Plant Pathology, 20, 61–71. Hammerschmidt, R. & Kuc´, J. (1995). Induced resistance to disease in plants. Kluwer Academic Publishers, Doredrecht, NL. Hammerschmidt, R. & Nicholson, R.L. (1999). A survey of plant defense responses to pathogens. In: Induced Plant Defenses Against Pathogens and Herbivores (eds A.A. Agrawal & S. Tuzun), pp. 55 –71, APS Press, St. Paul, USA.

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Roemmelt, S., Zimmermann, N., Rademacher, W. & Treutter, D. (2003). Formation of novel flavonoids in apple (Malus × domestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca. Phytochemistry, 64, 709 –716. Scalla, R. & Matringe, M. (1994). Inhibitors of protoporphyrinogen oxidase as herbicides: diphenyl ethers and other photobleaching molecules. Reviews in Weed Science, 6, 103–132. Shain, L. & Miller, J.B. (1982). Pinocembrin – an antifungal compound secreted by leaf glands of eastern cottonwood. Phytopathology, 72, 877–880. Slaymaker, D.H., Navarre, D.A., Clark, D., del Pozo, O., Martin, G.B. & Klessig, D.F. (2002). The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proceedings of the National Academy of Sciences of the USA, 99, 11640–11645. Snyder, B.A., Leite, B., Hipskind, J., Butler, L.G. & Nicholson, R.L. (1991). Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site. Physiological and Molecular Plant Pathology, 39, 463 – 470. Snyder, B.A. & Nicholson, R.L. (1990). Synthesis of phytoalexins in sorghum as a site specific response to fungal ingress. Science, 248, 1637–1639. Stein, B.D., Klomparens, K.L. & Hammerschmidt, R. (1993). Histochemistry and ultrastructure of the induced resistance response of cucumber plants to Colletotrichum lagenarium. Journal of Phytopathology, 137, 177–188. Sticher, L.B., Mauch-Mani, B. & Métraux, J.P. (1997). Systemic acquired resistance. Annual Review of Phytopathology, 35, 235 –270. Strack, D. (1997). Phenolic metabolism. In: Plant Biochemistry (eds P.M. Dey & J.B. Harborne), pp. 397– 416. Academic Press, New York, USA. Treutter, D. (2005). Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biology, 7, 581–591. Treutter, D. (2006). Significance of flavonoids in plant resistance, a review. Environmental Chemistry Letters, 4, 147–157. van Loon, L.C., Bakker, P.A.H.M. & Pieterse, C.M.J. (1998). Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology, 36, 453–483. van Peer, R., Niemann, G.J. & Schippers, B. (1991). Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology, 81, 728 –734. Vance, C.P., Kirk, T.K. & Sherwood, R.T. (1980). Lignification as a mechanism of disease resistance. Annual Review of Phytopathology, 18, 259 –288. Vance, C.P. & Sherwood, R.T. (1976). Regulation of lignin formation in reed canary grass in relation to disease resistance. Plant Physiology, 57, 915–919. Vance, C.P. & Sherwood, R.T. (1977). Lignified papilla formation as a mechanism for protection in reed canary grass. Physiological Plant Pathology, 10, 247–256. Walker, J.C. (1925). Studies on disease resistance in the onion. Proceedings of the National Academy of Sciences of the USA, 11, 183 –189. Walker, J.C., Link, P.K. & Angell, H.R. (1929). Chemical aspects of disease resistance in the onion. Proceedings of the National Academy of Sciences of the USA, 15, 845–850. Wattad, C., Dinoor, A. & Prusky, D. (1994). Purification of pectate lyase produced by Colletotrichum gloeosporioides and its inhibition by epicatechin – a possible factor involved in the reistance of unripe avocado fruits to anthracnose. Molecular Plant–Microbe Interactions, 7, 293–297. White, R.F. (1979). Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology, 99, 410 – 412. Zeyen, R.J., Bushnell, W.R., Carver, T.L.W. et al. (1995). Inhibiting phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase suppresses Mla1 (HR) but not Mlo5 (non-HR) barley powdery mildew resistances. Physiological and Molecular Plant Pathology, 47, 119–140.

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Chapter 10

Bioactivity, Absorption, and Metabolism of Anthocyanins Giuseppe (Joe) Mazza and Colin D. Kay

10.1 Introduction Anthocyanins are flavonoids widely distributed in the human diet through fruit, vegetable, and grain products (Mazza & Miniati, 1993). They are distinguished from other flavonoids as a separate class by virtue of their ability to form positively charged flavylium cations (2-phenylbenzopyrylium) (Brouillard, 1982; Mazza & Miniati, 1993). Anthocyanins are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. They are particularly abundant in berries and other fruits with red, blue, or purple color, and in red wines (Mazza & Miniati, 1993). Approximately 550 individual anthocyanins have been identified to date (Wrolstad, 2000; Mateus et al., 2001, 2003; Andersen & Jordheim, 2006). Anthocyanins are of considerable nutritional interest as it has been suggested that they may play an important role in the promotion of health and disease prevention. Anthocyanins have been shown to improve risk factors for cardiovascular health, to aid in preventing obesity, to have antioxidant, anti-inflammatory and anti-cancer properties, and to improve vision, skin and possibly other bioactivities. Their daily intake has been estimated at between 180 and 215 mg/d in the United States (Kühnau, 1976) and 82 mg/d in Finland. These values are considerably higher than the intake estimated for many other flavonoids, including quercetin, kaempferol, myricetin, apigenin, and luteolin (Kühnau, 1976; Hertog et al., 1993; Middleton et al., 2000). Major sources of anthocyanins in the ‘Western’ diet are purple grapes, grape juice and red wine, blueberries, cherries, raspberries, strawberries, blackcurrants, and red oranges. Servings of 100 g of some berries can provide up to 500 mg of anthocyanins (Mazza & Miniati, 1993). Interest in anthocyanins and their biological effects has increased significantly during the past decade, and in the last few years much has been learned about their absorption and metabolism. In this chapter, we will present and discuss the results of recent studies on bioactivity, absorption and metabolism of anthocyanins, with a focus on human studies.

10.1.1 Structural characteristics The six anthocyanidins commonly found in plants are classified according to the number and position of hydroxyl and methoxyl groups on the flavan nucleus or B-ring, and are Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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R1 3′ 2′ 1+

8

HO 7

O

A

C

6 5

4

1′

OH 4′

B

5′

2 6′

R2

3

R3

OH Delphinidin Cyanidin Petunidin Peonidin Malvidin Pelargonidin

Substitution pattern R1

R2

R3

OH OH OCH3 OCH3 OCH3 H

OH H OH H OCH3 H

OH OH OH OH OH OH

Fig. 10.1 Structural classification of common anthocyanidin species.

named pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin (Fig. 10.1). The most commonly occurring anthocyanidin in nature is cyanidin, present in 90% of fruits. Anthocyanins are polyhydroxylated or polymethoxylated glycosides or acylglycosides of anthocyanidins, which are oxygenated derivatives of 2-phenylbenzopyrylium or flavylium salts (Mazza & Miniati, 1993). Color differences between anthocyanins are largely determined by the substitution pattern of the B-ring of the aglycone, the pattern of glycosylation, and the degree and nature of esterification of the sugars with aliphatic or aromatic acids and the presence of copigments (Brouillard, 1982; Brouillard et al., 1989; Mazza & Brouillard, 1990; Mazza & Miniati, 1993; Figueiredo et al., 1996; Mazza et al., 2004). One unique characteristic of anthocyanins is that they undergo reversible molecular transformations within varying pH ranges. The molecule is colorless at pH 4.5 while it forms an intensely red colored flavylium at lower pH, and a blue and relatively unstable quinoidal form is generated at a more alkaline pH. Acylation and position of the glycosidic groups also affect the exact pH at which these molecular changes occur (Brouillard, 1982; Mazza & Miniati, 1993). The importance of understanding the molecular characteristics of anthocyanins becomes apparent when we discuss the implications of identifying these compounds in biological fluids and tissues.

10.2 Bioactivity In recent years numerous studies have shown that anthocyanins display a wide range of biological activities (Mazza, 2000; Kong et al., 2003) including antioxidant (Wang et al., 1997; Tsuda et al., 1998; Fukumoto & Mazza, 2000; Mazza et al., 2002), anti-inflammatory (Wang & Mazza, 2002; Youdim et al., 2002), antimicrobial (Pisha & Pezzuto, 1994) and anti-carcinogenic activities (Kamei et al., 1995; Kang et al., 2003; Katsube et al., 2003); improvement of vision (Mercier et al., 1965; Matsumoto et al., 2003); induction of apoptosis (Katsube et al., 2003); and neuroprotective effects (Youdim et al., 2000; Galli et al., 2002). In addition, anthocyanins have exhibited a variety of effects on cells, blood vessels (Andriambeloson et al., 1998; Martin et al., 2003) and platelets (Morazzoni &

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Magistretti, 1990; Demrow et al., 1995) that may reduce the risk of coronary heart disease (Renaud & Lorgeril, 1992). One important point to note is that much of the mechanistic evidence for the prevention of diseases by anthocyanins and other polyphenols is derived from in-vitro or animal experiments, which are often performed with doses much higher than those observed in the human diet.

10.2.1 Antioxidant activity Oxidation is one of the most important mechanisms responsible for the major degenerative diseases of aging, including cancer, heart disease, cataracts, and cognitive dysfunction. Evidence from intervention studies indicates that antioxidants may prevent many of the oxidative processes that contribute to the causation of these chronic diseases. In the past decade, several authors have reported that anthocyanins are potent antioxidants and in certain environments superior to ‘classical’ or ‘traditional’ antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoulene (BHT), and α-tocopherol (Wang et al., 1997; Narayan et al., 1999; Fukumoto & Mazza, 2000). Tsuda et al. (1996) investigated the reaction products of cyanidin 3-glucoside with the radical generator 2,2′-azobis (2,4-dimethylvaleronitrile) and concluded that the antioxidative mechanism of cyanidin 3-glucoside appeared different than that of ‘traditional’ antioxidant nutrients such as α-tocopherol. Additionally, in a clinical trial with elderly women, Cao et al. (1998) reported increased serum antioxidant capacity utilizing various in-vitro methods including the oxygen radical absorbance capacity (ORAC), the trolox equivalent antioxidant capacity (TEAC), and the total antioxidant potential (TRAP) assays, after the consumption of strawberries or red wine. This was corroborated by a more recent study by Mazza et al. (2002) that showed that the concentration of anthocyanins in the serum of male subjects who had consumed 1.2 g anthocyanins from freeze-dried blueberries was positively correlated with the serum antioxidant capacity (Fig. 10.2). Anthocyanins have demonstrated antioxidant activity in a number of different ex-vivo and in-vitro systems including preventing low-density lipoprotein (LDL) (Teissedre et al., 1996; Satué-Gracia et al., 1997; Aviram & Fuhrman, 2002), lipid (Narayan et al., 1999;

Antioxidant capacity

7 6 5 4 3

R 2 = 0.4667 P < 0.05

2 1 0 5 10 15 Total serum anthocyanins (ng/mL)

20

Fig. 10.2 Correlation between serum antioxidant capacity as measured by ORAC-total and concentration of total serum anthocyanins. Antioxidant value expressed as 1000 × μm Trolox equivalents/L, and anthocyanins expressed as cyanidin-3-glucoside chloride equivalents (Adapted from Mazza et al., 2002).

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Wang et al., 1999; Pilaczynska-Szczesniak et al., 2005) and DNA oxidation (RamirezTortosa et al., 2001) and inhibiting immune cell activation (Wang & Mazza, 2002). For example, we found that pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvidin 3-glucoside, and malvidin 3,5-diglucosides have strong inhibitory effects on NO production in LPS/IFN-γ-activated RAW 264.7 macrophages (Wang & Mazza, 2002). Additionally, Narayan et al. (1999) investigated the antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation. Their results showed that in-vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dosedependent manner by a purified anthocyanin pigment from carrot cell culture. The kinetics showed that anthocyanins were non-competitive inhibitors of lipid peroxidation in this model. Concurrently, this was corroborated by Wang et al. (1999), who additionally concluded that the antioxidant activities of the anthocyanins and cyanidin were comparable to the antioxidant activities of tert-butylhydroquinone and butylated hydroxytoluene, and superior to vitamin E at equivalent concentrations. Beneficial antioxidant effects have also been observed in models of exercise- and antioxidant-deficiency induced oxidative stress. For example, in rats fed a diet deficient in vitamin E for 12 weeks and then repeated with a diet containing a highly purified anthocyanin-rich extract of Abies coreana (1 g/kg diet), a significant improvement in plasma antioxidant capacity and a decrease in the vitamin E deficiency-enhanced hydroperoxide and 8-oxo-deoxyguanosine (a marker of DNA damage) concentrations in the liver were observed. The authors (Ramirez-Tortosa et al. (2001)) suggested that a dietary intake of anthocyanin-rich foods may contribute to overall antioxidant status, particularly in individuals habitually consuming a diet deficient in vitamin E. More recently, PilaczynskaSzczesniak et al. (2005) reported that an intake of approximately 34.5 mg/d of anthocyanin (from chokeberry juice) could lower the blood parameters of redox status in rowers performing physical exercise during a 1-month training camp. After the supplementation period, the concentrations of thiobarbituric acid reagent substances (TBARS) in blood samples collected 1 min after an exercise test and following a 24-h recovery period were significantly lower in the subjects receiving chokeberry juice than in the control group. The investigators suggested that the anthocyanins may have enhanced the endogenous antioxidant defense system. The antioxidant activity of anthocyanins in vitro has been attributed to many structural characteristics, and involves direct radical scavenging, metal chelation and protein binding. Factors that will affect the in-vivo antioxidant effects of anthocyanins and other polyphenols include quantities consumed, absorbed and/or metabolized, and plasma and/ or tissue concentrations. Anthocyanins as parent structures and metabolized derivatives probably have varying antioxidant activities. Glycosylation of an anthocyanin has been shown to decrease radical-scavenging activity compared with the aglycone, as it reduces the ability of the anthocyanin radical to delocalize electrons. The same is probably true for glucuronidated derivatives in human blood. In accordance with this, Fukumoto & Mazza (2000) reported increased antioxidant activity with increase in the number of hydroxyl groups in the molecule, and decreased antioxidant activity with glycosylation of anthocyanidins (Table 10.1). This said, it is important to note that aglycones/anthocyanidins have not been identified in any appreciable amount in human fluids or tissues, which is probably a result of their relative instability.

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Table 10.1 Antioxidant and Prooxidant Activity of Selected Anthocyanins Selected results from literature

Current study

HPLC method

Compound Cyanidin Cyanidin 3-glucoside Cyanidin 3,5-diglucoside Delphinidin Malvidin Malvidin 3-glucoside Malvidin 3,5-diglucoside Pelargonidin Pelargonidin 3-glucoside Pelargonidin 3,5-diglucoside Peonidin Peonidin 3-glucoside STANDARDS Ascorbic acid α-Tocopherol BHA BHT 1

DPPH assay1 antiradical power7 prooxidant [antiradical activity6 activity]

ORAC assay2 ORAC slope8

β-carotene method initial slope3 (× 10−6)

DPPH method antiradical activity4

836 ± 69 278 ± 32

−7.40 ± 0.15 200 – 300 −6.81 ± 0.30 300 – 400

+ +

2.239 ± 0.029 3.491 ± 0.011

220 ± 39

−3.32 ± 0.07 500 –1000

+

1.689 ± 0.052

897 ± 147 288 ± 34 448 ± 40

−8.86 ± 0.28 500 –1000 −4.49 ± 0.28 1500 –2000 −4.29 ± 0.42 500 –1000

+ + +

1.809 ± 0.068 2.009 ± 0.167 1.404 ± 0.052

266 ± 27

−2.56 ± 0.10 2000 – 2500

+

1.550 ± 0.062

nc 444 ± 94

−4.63 ± 0.25 1500 – 2000 −3.95 ± 0.22 2000 – 2500

+ +

1.540 ± 0.033 1.560 ± 0.145

nc

−2.04 ± 0.10 2000 – 2500

+

1.067 ± 0.043

169 ± 22 251 ± 4

−4.05 ± 0.17 1500 – 2000 −3.38 ± 0.15 2500 – 3000

+ +

1.693 ± 0.035 1.805 ± 0.014

nc 870 ± 21 835 ± 50 864 ± 76

−1.83 ± 0.07 −1.95 ± 0.07 −2.61 ± 0.01 −3.17 ± 0.07

antioxidant activity5 (μM of compound added)

> 4000 + 2000–2500 (50%) nd 1000 –1500 nd 200 – 300 nd

3.7 [−1.85] 4.17 [−2.09] 4.2 [−2.1]

The DPPH assay used by Brand-Williams et al. (1995). The oxygen radical absorbing capacity (ORAC) assay measuring reaction with peroxyl radicals expressed as μM of Trolox equivalent per μM of compound. Results for flavonols were taken from Cao et al. (1997) and results for anthocyanidins/anthocyanins were taken from Wang et al. (1997). 3 Values are means of slope coefficients calculated by linear regression ± standard deviations (n = 3) in A450 nm after 90 minutes of incubation in the dark/μM of compound added. 4 Values are means of slope coefficients calculated by linear regression ± standard deviations (n = 3) in μM of DPPH/μM of compound. 5 Antioxidant activity was defined by the concentration range of compound added needed to reach 0% malonaldehyde of the control. 6 Prooxidant activity was positive (+) if the % malonaldehyde of the control was > 100% in the concentration range tested. 7 Antiradical power was defined as the reciprocal of the amount of antioxidant needed to decrease the initial DPPH concentration by 50%. The antiradical activity was equivalent to negative half of the antiradical power. 8 Values are slope coefficients calculated by linear regression ± standard error. a Not calculated since linear regression r2 < 0.800. b Values were obtained after reaction for 48 hours. c Not detected. (Adapted from Fukumoto & Mazza, 2000.) 2

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10.2.2 Anti-inflammatory effects Reports of anthocyanins possessing anti-inflammatory activity were first published in the 1970s (Lietti et al., 1976). More recent studies have reported anti-inflammatory mechanisms such as inhibition of arachidonic acid metabolism (Ferrandiz & Alcaraz, 1991) and prostaglandin synthase cyclooxygenase activity (Hoult et al., 1994; Hou et al., 2005). Recently, a study by Hou et al. (2005) utilizing a mouse macrophage cell line stimulated with LPS showed that anthocyanidins inhibited lipopolysaccharide (LPS)-induced cyclooxygenase isozyme (COX-2) expression. The inhibitory effects depended on the orthodihydroxyphenyl structure on the B-ring, and delphinidin with this structure inhibited LPS-induced COX-2 expression by blocking the signaling cascades of MAPK with the attendant activations of NF-κB, C/EBPd and AP-1. Anthocyanins have been reported to affect the permeability of blood capillaries and inflammatory conditions of the joints. The property of anthocyanins to decrease the fragility and permeability of blood capillaries is also common to other flavonoids and was originally identified by Albert Szent-Györgyi, who coined the term Vitamin P (Rusznyàk & Szent-Györgyi, 1936) for those compounds that reduce capillary permeability. Several prescription and non-prescription pharmaceutical products containing anthocyanins from bilberry (Vaccinium myrtillus) as the principal active component have been used to control capillary permeability and fragility (Wagner, 1979; Boniface et al., 1986; Pizzorno & Murray, 1987; Beretz & Cazahave, 1988; Morazzoni & Bombardelli, 1996). The antiinflammatory activity of these anthocyanin extracts accounts for their significant anti-edema properties and their reported action on diabetic microangiopathy (Boniface et al., 1986; Morazzoni & Bombardelli, 1996). Bilberry fruit anthocyanin extract is reported to be useful in the treatment of inflammatory conditions of the joints, as well as in the treatment of glaucoma and retinopathies, including diabetic retinopathy, owing to its positive effects on collagen structure. Additionally, small clinical trials have demonstrated the efficacy of bilberry extract in the treatment of peripheral vascular disease in the lower limbs and in the pre- and post-operative treatment of varicose veins and hemorrhoids (Morazzoni & Bombardelli, 1996). To our knowledge, there are no apparent scientific reasons for the use of bilberry anthocyanin exclusively over other anthocyanin-rich plant foods such as elderberries, chokeberries, blueberries, raspberries, grapes, etc. However, the in-vitro antioxidant activity of anthocyanins is higher in delphinidin and cyanidin than other anthocyanins; thus, the high concentration of glycosides of delphinidin and cyanidin in bilberries (Baj et al., 1983; Mazza & Miniati, 1993) may account for their apparently higher anti-inflammatory activity in certain models of inflammation or oxidation.

10.2.3 Anti-atherogenic effects Several epidemiological studies have shown that coronary heart disease (CHD) mortality can be decreased by moderate consumption of red wine (Rimon et al., 1991; Graziano et al., 1993; Klatsky, 1994). Although one mechanism responsible for this reduction in risk level is the effect of ethanol on platelet coagulability (Elwood et al., 1991; Renaud et al., 1992) and high-density lipoprotein cholesterol (HDL) synthesis (Graziano et al.,

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1993; Klatsky, 1994), there is a great deal of evidence for the added benefit of wine phenolics and polyphenolics on CHD risk (Frankel et al., 1993; Kinsella et al., 1993; Kanner et al., 1994). For example, various studies have shown that phenolics and polyphenolics such as anthocyanins in wine can improve vasodilation capacity or flow-mediated dilatation (Kay et al., 2006). Furthermore, a study of the relationships between vasodilatation capacity, antioxidant activity and phenolic content of 16 red wines reported that the total phenol content correlated well with vasodilatation capacity and antioxidant activity of the wines, but only anthocyanins were correlated with vasodilatation capacity (Burns et al., 2000). Additionally, Andriambeloson et al. (1998) found that only the anthocyanin and oligomeric condensed tannin-containing fractions of red wine showed vasorelaxant activity comparable to the original polyphenol fraction of the red wine. The phenolic acid derivatives, hydroxycinnamic acids and flavanol classes tested failed to induce this type of response. Various in-vitro and clinical studies have shown that grape and red-wine polyphenols can also affect various other CHD risk factors such as the inhibition of lipid and lipoprotein oxidation and the reduction of inflammation and modulation of eicosanoid metabolism (Bors & Saran, 1987; Afanas’ev et al., 1989; Steinberg et al., 1989; Esterbauer et al., 1992). Although a few animal studies have suggested that anthocyanins are capable of reducing cholesterol, triglyceride and apolipoprotein B plasma concentrations as well as decreasing aortic fatty streak area (Auger et al., 2001; Xia et al., 2006) evidence for this in humans has not been established.

10.2.4 Anticarcinogenic effects The consumption of phenolics has been correlated with the inhibition of colon, esophagus, lung, liver, mammary, and skin cancers (Decker, 1995). The anti-tumor promoting activity of flavonoids appears to involve many mechanisms, including the inhibition of various growth factors, receptors (Kay & Holub, 2005) and enzymes such as polymerases and decarboxylases (Ohno & Nakane, 1990). Anthocyanins have been found to significantly suppress the growth of cultured tumor cells and have a greater inhibitory effect than many other flavonoids (Kamei et al., 1995). The anticancer mechanisms of anthocyanins are often attributed directly to their antioxidant capacities, which include free radical scavenging and direct inhibition of DNA oxidation; however, many of the reported anticancer activities of anthocyanins appear to occur through mechanisms other than radical scavenging. Recently Meiers et al. (2001) showed that cyanidin and delphinidin have the ability to inhibit the epidermal growth-factor receptor by shutting off downstream signaling cascades using the human carcinoma cell line A431. Additionally, in our laboratory, we demonstrated for the first time that anthocyanin and anthocyanin-rich extracts induced the production of tumor necrosis factor and acted as modulators of the immune response in activated macrophages (Wang & Mazza, 2002). Many further studies in the literature have reported anticancer properties unrelated to antioxidant effects, including interactions with mammalian enzymes, growth factors and receptors, direct antimutagenic activity, and the inhibition of the metabolic activation of carcinogens (Tsuda et al., 1996; Middleton et al., 2000; Kay & Holub, 2005).

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10.2.5 Antibacterial and antiviral activity Most phenolic compounds possess antibacterial or antiviral activities at certain concentrations (Pisha & Pezzuto, 1994). For example, the phenolic acids hydroxybenzoic, salicylic, gallic, and protocatechuic acid have all been reported to have antibacterial effects (Balansard et al., 1980; Dumenil et al., 1980). Most of the phenolic compounds tested for antimicrobial properties, have however, been shown to display considerably lower activity than products such as antibiotics (Wagner, 1985). The antiviral properties of epicatechin have been observed in vitro and are suggested to be the result of the inhibition of reverse transcriptase, as determined by utilizing the Moloney murine leukemia virus (Pisha & Pezzuto, 1994). Additionally, chlorogenic acid has been found to be active against the Epstein– Barr and the HIV viruses (Pisha & Pezzuto, 1994), and recently Knox et al. (2001) reported that cyanidin 3-α-l-rhamnopyranosyl-β-d-glucopyranoside and cyanidin 3-glucoside extracted from Ribes nigrum L. fruits display potent antiviral activity against influenza viruses A and B and herpes simplex virus 1.

10.2.6 Neuroprotective effects Joseph et al. (1999) associated the ingestion of some antioxidant-rich foods, including blueberry and strawberry (both containing relevant amounts of cyanidin-glycosides), with a marked decrease in age-related declines of neuronal signal transduction and cognitive and motor behavioral deficits in rats. Additional studies by this group showed that blueberry supplementation enhances signaling and prevents behavioral deficits in an Alzheimer disease model (Joseph et al., 2003). More recently, Ramirez et al. (2005) demonstrated that, in rats fed lyophilized anthocyanins from the blueberry or bilberry (at 3.2 mg/kg/d), there was significantly enhanced short-term memory. The feeding of these berries to rats also improved working memory in the radial maze, but diet did not alter reference memory in this task. Finally, Kang et al. (2006), using a mouse-brain-injury model, showed that a mulberry fruit extract containing cyanidin 3-glucoside had neuroprotective effects. In vitro, a 1% HCl-MeOH mulberry fruit extract was shown to have a cytoprotective effect on PC12 cells that had been exposed to hydrogen peroxide, and the extract also inhibited cerebral ischemic damage caused by oxygen glucose deprivation in PC12 cells.

10.2.7 Prevention of obesity Recently a study by Tsuda et al. (2003) reported that an anthocyanin extract from purple corn reduced adipose weight and ameliorated hyperglycemia, hyperinsulinemia and hyperleptinemia in mice following a high-fat diet. In this study, mice were fed a control diet, a diet high in cyanidin 3-glucoside-rich purple corn color extract and a high-fat diet with or without anthocyanin-rich purple corn extract for 12 weeks. Dietary purple corn anthocyanins significantly suppressed the high-fat diet-induced increase in body weight and white and brown adipose tissue weights. The high-fat diet induced hyperglycemia, hyperinsulinemia and hyperleptinemia, which were normalized in mice fed a high-fat diet with the anthocyanin-rich purple corn extract. The cyanidin 3-glucoside-rich diet appeared to function by suppressing the mRNA levels of key enzymes in fatty acid synthesis. Additional

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work by Tsuda et al. (2005) showed that the gene expression profile of isolated rat adipocytes treated with cyanidin or cyanidin-3-glucoside indicated up-regulation of hormone sensitive lipase and enhanced lipolytic activity. These results suggest that dietary anthocyanins may ameliorate high-fat diet-induced insulin resistance and adiposity in mice and rats.

10.2.8 Gastric protective effects Three gastric protective effects of a cyanidin-rich bilberry extract (IdB 1027) have been reported by independent studies. The protective effect of IdB 1027 against aspirin-induced mucosa damage in man was reported by Barzaghi et al. (1991), who found that the drop in gastric transmucosal potential difference induced by aspirin was significantly reduced by dietary administration of IdB 1027. These results corroborate results reported by Cristoni et al. (1989), who showed that the gastroprotective activity of IdB 1027 is mediated by enhancement of the efficiency of the gastric mucosal barrier, as evidenced by the observed increase in gastric bicarbonate secretion. These data are additionally supported by those of Mertz-Nielsen et al. (1990), who demonstrated increases in the rate of the luminal release of prostaglandin E2 in healthy subjects following the consumption of cyanidin chloride.

10.2.9 Improvement of vision There is some evidence to suggest that bilberry anthocyanin extracts may improve visual effects related to night-time visual acuity and low-light adaptation, and to decrease recovery time after exposure to glare. In an Italian human study from the 1960s, patients given a combination of bilberry (400 mg/day) and beta-carotene (20 mg/day) had improved night vision and adaptation to changes in light intensity (Fiorni et al., 1965). Even though it is difficult to determine from this study how much of the reported effect is attributed to the consumption of beta-carotene, the protective effect on human vision has been reported by others. The protective effect exerted by the anthocyanins in Vaccinium myrtillus has been reviewed by Morazzoni & Bombardelli (1996), who also discussed the beneficial clinical effects obtained in human patients with different ocular disorders such as myopia, simple glaucoma, retinitis pigmentosa, and other ocular diseases. In a recent study on the effect of cyanidin 3-glycosides on the regeneration of rhodopsin, Matsumoto et al. (2003) found that the oral intake of a blackcurrant anthocyanin concentrate containing cyanidin 3-glucoside and cyanidin 3-rutinoside improved darkness adaptation and the transient alteration of vision induced by long-term exposure to video display terminals in healthy human subjects. However, a study by Levy & Glovinsky (1998) on the effect of anthocyanins on night vision concluded that a single oral administration of 12–36 mg of anthocyanins had no significant effect on militarily relevant night-vision tests. It is clear that more evidence is required to establish the effects of anthocyanins on visual conditions.

10.3 Absorption of anthocyanins Prior to 1995, it was widely believed that anthocyanins and other flavonoids could not be absorbed intact after oral ingestion, but were hydrolyzed to their aglycones by bacterial enzymes in the lower gastrointestinal tract (GIT). It was further suggested that the agly-

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cones might then be partially absorbed or may undergo further biotransformation by bacteria (Griffiths & Barrow, 1972; Bokkenheuser et al., 1987). In 1995 Hollman et al. showed that quercetin glucoside as present in onions, could be absorbed intact in the small intestine, and suggested that the sodium-dependent glucose transporter 1 (SGLT1) could be the possible transport mechanism. In 1997 Paganga & Rice-Evans described evidence for the absorption of anthocyanins, rutin and phloridzin, and their presence in human plasma in the glycosylated form by high-pressure liquid chromatography (HPLC) analysis with photodiode array detection. The polyphenols were detected in plasma from nonsupplemented humans at levels in the range 0.5–1.6 μM. Other studies on anthocyanin absorption soon followed. A recent study by Vitaglione et al. (2007) supports Tsuda’s work in rats (Tsuda et al., 1999) and suggests that protocatechuic acid is the major human metabolite of cyanidin-3-glucoside. Lapidot et al. (1998) fed six healthy human subjects anthocyanins from red wine. In this investigation the subjects, on a polyphenols-free diet, underwent three treatments: either 300 mL of water, white wine or red wine. In the 300 mL dose of red wine the subjects received 218 mg of anthocyanins, which were subsequently detected in their urine by HPLC analysis with a photodiode array detector. Two of the wine anthocyanins were found unchanged in the urine. Other anthocyanin compounds, which seemed to have undergone molecular modifications, were detected in the urine after incubation. The anthocyanin level in the urine reached a peak within 6 h of drinking the wine. Subsequently, Miyazawa et al. (1999) reported the results of a human study involving seven male and five female adults. The subjects orally ingested 2.7 mg cyanidin 3-glucosides and 0.25 mg cyanidin 3,5-diglycosides/kg body weight. Thirty min after intake, the level of cyanidin 3,5-diglycosides in plasma was 11 μg/L. At 60 min after intake, the plasma cyanidin 3-glucoside level was 13 μg/L. Only traces of cyanidin 3,5-diglycosides were identified. These general findings were corroborated by Cao & Prior (1999), who fed 1.5 g of total anthocyanins – consisting of cyanidin 3-glucoside and cyanidin 3-sambubioside – from elderberries to humans. In the above-mentioned studies, evidence for the absorption of the cyanidin glycosides in human plasma was based on octadecylsilane solid-phase extraction for sample preparation and an HPLC analysis with diode array. No pharmacokinetic data or evidence of metabolites in the blood or urine were reported at the time. Currently there is little direct evidence of anthocyanin aglycones existing in the blood circulation or urine of humans (Felgines et al., 2003). Most recent animal and human studies have reported that either anthocyanins are absorbed intact as glycosides (Milbury et al., 2002; Mulleder et al., 2002) or that anthocyanins appear in the circulation as both intact parent glycosides and as metabolized conjugates (Wu et al., 2002; Felgines et al., 2003, 2005; Kay et al., 2004, 2005; Bitsch et al., 2004b; Tian et al., 2006). Recent evidence from studies in rats suggests that anthocyanins are rapidly absorbed from both the small intestine (Miyazawa et al., 1999; Tsuda et al., 1999; Talavera et al., 2003) and stomach (Passamonti et al., 2003; Talavera et al., 2003). In rats administered cyanidin 3-glucoside orally (0.9 mmol/kg body wt), the intact anthocyanin rapidly appeared in the plasma, but the aglycone of cyanidin 3-glucoside (cyanidin) was not detected, although it was present in the small intestine or jejunum (Tsuda et al., 1999). Protocatechuic acid, which may be produced by the degradation of cyanidin, was also present in the plasma at concentrations 8-fold higher than that of cyanidin 3-glucoside in the rat, but to our

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Table 10.2 Pharmacokinetics of anthocyanins following oral consumption in humans1

Reference

Source

Dose (mg)

Duration (h)

% Urinary Recovery

Cmax (nmol/L)

tmax (h) t1/2 (h)

Ohnishi et al., 2006 Kay et al., 2005 Felgines et al., 2005 Frank et al., 2005 Bitsch et al., 2004a Bitsch et al., 2004a Bitsch et al., 2004b Bitsch et al., 2004c Bitsch et al., 2004c Kay et al., 2004 Harada et al., 2004 Felgines et al., 2003 Frank et al., 2003 Frank et al., 2003 Nielsen et al., 2003 Nielsen et al., 2003 Mazza et al., 2002 Mülleder et al., 2002 Rechner et al., 2002 Wu et al., 2002 Wu et al., 2002 Bub et al., 2001 Bub et al., 2001 Bub et al., 2001 Cao et al., 2001 Matsumoto et al., 2001 Murkovic et al., 2001 Netzel et al., 2001 Miyazawa et al., 1999 Lapidot et al., 1998

cranberry chokeberry blackberry Hibiscus extract red grape juice red wine elderberry blackcurrant elderberry chokeberry purple sweet potato strawberries red wine red grape juice blackcurrant blackcurrant blueberry elderberry blackcurrant elderberry blueberry red wine de-alc red wine red grape elderberry blackcurrant elderberry blackcurrant mixed berries red wine

651 721 4312 147 283 280 3570 145 147 1300 311 77 280 283 1239 716 1200 1900 1000 720 690 68 56 117 720 2364 500 153 1624 218

24 24 24 7 7 7 5 7 7 24 24 24 7 7 4 4 4 6 7 24 6 6 6 6 24 8 – 5 – 12

5.00 0.15 0.16 0.018 0.23 0.18 0.053 0.04 0.37 0.048 0.02 1.9 0.23 0.18 0.07 0.05 0.003 0.03 0.039 0.08 0.004 0.03 0.03 0.03 0.05 0.11 0.05 0.03 – 3.3

– 96.1 – 7.62 222.72 95.52 – – – 592 0.00243 – 222.72 95.52 115.82,3 35.62,3 29.22 – – 97.4 – 1.4 1.73 2.83 97.4 60.0 – – 29.0 –

– 2.8 – 1.5 0.5 1.5 – – – – 1.5 – 0.5 1.5 0.7 0.7 4.0 – – – – 0.3 1.5 3.0 1.2 1.5 – – 1.0 –

– 1.5 – 2.6 1.8 2.0 – 1.7 1.7 – – – 1.83 1.52 – – – – – – – – – – 2.2 3.0 – – – –

1

Values represent total anthocyanins and include anthocyanin metabolites when identified. Molar equivalents derived from molecular weight of given anthocyanin. If multiple species listed in paper cyanidin glucoside (MW 449) was used to derive molar equivalents. 3 Data interpreted visually from a concentration vs. time graph (exact values were not identified in manuscript). 4 Dose derived from conversion of mg/kg to mg based on the reported average subject weight. Cmax derived from serum/plasma data. Tmax derived from serum/plasma data. – blank cells represent unavailable data in original manuscripts. C-3-gly, mixed cyanidin 3-glycosides; Cmax, maximum concentration; tmax, time to reach maximum concentration; t1/2, half-life of elimination. 2

knowledge protocatechuic acid has not been quantified in the plasma or urine of humans following consumption of anthocyanin. Also, in rats fed an anthocyanin-rich diet for two weeks, anthocyanins have been found in several tissues/organs, including the stomach, small intestine, liver, kidney and brain (Talavera et al., 2005; Felgines et al., 2006). In the brain, total anthocyanin content (blackberry anthocyanins and peonidin 3-O-glucoside) reached 0.25 ± 0.05 nmol/g of tissue. Table 10.2 presents absorption/bioavailability parameters of anthocyanins in human subjects from 30 published human studies. The parameters shown include maximal plasma

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concentration (Cmax), time to reach maximum concentration (tmax), and half-life of elimination (t1/2), and the values represent total anthocyanins, which include anthocyanin metabolites when identified. In the studies listed in Table 10.2, single doses of 56 to 3,570 mg anthocyanins were administered, generally in the form of berries, berry extracts, or concentrates. After such intakes, concentrations of anthocyanins measured in serum or plasma were very low, in the range 1.4–593 nmol/L. The time to reach Cmax ranged from 0.3 to 2.8 h (mean value 1.4 h) for plasma and from 1.5 to 3.0 h (mean value 1.8 h) for urine. Most of the studies reported low urinary excretion, ranging from 0.003 to 1.9% of the intake, although Ohnishi et al. (2006), Lapidot et al. (1998) and Felgines et al. (2003) reported higher levels of anthocyanin excretion (up to 5%) after the consumption of cranberry juice, red wine, and strawberry, respectively. Important features of the published results are: l l l l

anthocyanins appear to be absorbed as both intact glycosides and metabolites; anthocyanins are absorbed and eliminated rapidly; there is a considerable variability in the values of Cmax, tmax, and t1/2; and the maximum concentrations of anthocyanins (Cmax) in human blood and urine are very low.

10.3.1 Variability of absorption The reasons for the apparent variability of absorption/bioavailability of anthocyanins are discussed in detail in the following paragraphs and may be the result of several factors, including: (i) variations in dosage used in clinical studies; (ii) chemical structure of the anthocyanins used; (iii) food matrix in which they exist; (iv) age and/or gender of the human subjects; and (v) analytical methodology used for the extraction, separation, detection and identification of the anthocyanins in blood and urine. 10.3.1.1 Variations in dosage The doses of anthocyanins utilized in studies the listed in Table 10.2 ranged from 56 to 3,570 mg. A comparison of the corresponding values of Cmax, tmax, and t1/2 for the low vs. the high doses shows no decipherable pattern, which indicates that increasing the dose of anthocyanins may not always result in an increase in absorption. Additionally, the mechanisms of absorption and metabolism of polyphenols may rely on cofactors that are most probably saturatable. The theory of a saturatable transport mechanism involving a sodiumdependent co-transporter has been proposed for the absorption of quercetin glycosides, which are similar in structure to anthocyanins (Mizuma et al., 1994; Hollman et al., 1995; Wolffram et al., 1995; Hollman & Katan, 1998). Additionally, metabolic pathways involved with glucuronidation, methylation, and sulfation are limited by cofactors (Kay, 2006). Thus, it can be speculated that large (pharmacological doses) and small doses of anthocyanins may be absorbed and metabolized differently. For this reason, feeding studies that use a single dose of anthocyanins or flavonoids will not establish a complete representation of their absorption, metabolism and pharmacokinetics. More advanced multiple-dosing studies are required to establish a true understanding of the absorption and bioavailability of anthocyanins.

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10.3.1.2 Chemical structure of the anthocyanins Several human and animal studies have shown that aglycones and various sugar moieties and acylated derivatives have differing absorption efficiencies. For example, in humans and rats administered an extract of purple sweet potato (Ipomoea batatas cv Ayamurasaki), two acylated anthocyanins, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-d-glucopyranosyl)-βd-glucopyranoside)-5-O-β-d-glucopyranoside) and peonidin 3-O-(2-O-(6-O-(E)-caffeoylβ-d-glucopyranosyl)-β-d-glucopyranoside)-5-O-β-d-glucopyranoside), were detected in the plasma and urine by HPLC or liquid chromatography–mass spectrometry (LC–MS) (Harada et al., 2004). The plasma concentration of anthocyanins in humans reached a maximum 90 minutes after ingestion, and the recovery of anthocyanins in the urine was estimated as 0.01–0.03%. These results show that acylated anthocyanins can be selectively absorbed after ingesting food (Harada et al., 2004). A subsequent clinical study using purple carrots as the anthocyanin source reported that acylation of anthocyanins resulted in an 11–14-fold decrease in anthocyanin recovery in urine and an 8–10-fold decrease in anthocyanin recovery in plasma (Kurilich et al., 2005). Additionally, cooking was reported to increase the recovery of nonacylated anthocyanins but not acylated anthocyanins. When healthy volunteers were given a single oral dose of 150 mL of Hibiscus sabdariffa L. extract yielding 62.6 mg of cyanidin 3-sambubioside and 81.6 mg of delphinidin 3-sambubioside, the urinary excretion of cyanidin 3-sambubioside and delphinidin 3sambubioside was 0.016 and 0.021%, respectively. The corresponding dose-normalized Cmax estimates were 0.036 and 0.015 ng/mL/mg, respectively (Frank et al., 2005). The reason for the higher urinary excretion of cyanidin 3-sambubioside than delphinidin 3sambubioside is not apparent, but it suggests that absorbance efficiency was affected by structural differences in the anthocyanins. More recently, two studies on the bioavailability of black raspberry anthocyanins in pigs (Wu et al., 2006) and human subjects (Tian et al., 2006) reported higher total urinary excretion of more complex cyanidin anthocyanins (cyanidin 3-sambubioside, cyanidin 3-sambubioside-5-glucoside or cyanidin 3-rutinoside) compared to simple cyanidin 3-glucoside. In the study by Wu et al. (2006) weaning pigs fed a freeze-dried powder of marionberry, which contains cyanidin 3-glucoside (78%), cyanidin 3-rutinoside (20%), pelargonidin 3-glucoside (0.4%), and an unknown acylated cyanidin (1.5%), cyanidin 3-glucoside and cyanidin 3-rutinoside had similar apparent excretion rates relative to dose, whereas pelargonidin 3-glucoside had a much higher total urinary excretion than cyanidin-based anthocyanins. Pelargonidin 3-glucoside had an 8-fold higher apparent absorption rate than cyanidin 3-glucoside (Wu et al., 2004; Prior & Wu, 2006). A recent study on the effects of different aglycones, sugar moieties, and chemical structure on the absorption of different types of anthocyanins in caco-2 human intestinal cell monolayers revealed that delphinidin glucoside showed the lowest absorption efficiency, and malvidin glucoside showed the highest absorption efficiency. The results of this study indicate that more hydroxyl groups and fewer methoxy groups per molecule appear to decrease the bioavailability of anthocyanins. In addition, cyanidin glucoside showed significantly higher absorption/transport efficiency than cyanidin galactoside, and peonidin glucoside showed significantly higher absorption/transport efficiency than peonidin galactoside, thus indicating that glucose-based anthocyanins appear to have higher bioavailability than galactose-based anthocyanins (Yi et al., 2006). In fact, earlier studies

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indicated that anthocyanin absorption is delayed and even decreased with a concomitant administration of sugar, suggesting a competitive action of glucose and anthocyanidin glycosides on the hypothesized involvement of an SGLT1 (Bub et al., 2001; Mülleder et al., 2002). However, a recent examination of the effect of d-glucose, phloridzin, and quercetin 3-glucoside on the absorption of cyanidin 3-glucoside by mouse jejunum mounted in Ussing chambers, revealed that the presence of either d-glucose (10, 20, and 40 mmol/L) or phloridzin (50, 100, and 200 μmol/L) only resulted in a small but statistically insignificant inhibition of cyanidin 3-glucoside disappearance from the mucosal solution. But, when the flavonol quercetin 3-glucoside (50 μmol/L) was added to the mucosal solution together with the cyanidin 3-glucoside, quercetin 3-glucoside appeared to inhibit the absorption of cyanidin 3-glucoside (Walton et al., 2006a). This finding suggests that there might be a structurally dependent competitive inhibition, with preference toward flavonols. It is also possible that an absorption mechanism other than that mediated by the SGLT1 is involved (Walton et al., 2006a). It is obvious that anthocyanin form will affect absorption efficiency; however, we as well as others (Kroon et al., 2004) suggest that the difference in relative absorption efficiency of many anthocyanin forms as reported in the literature, such as that for rutanosides vs. glycosides, may largely be the result of increased stability rather than absorption efficiency. It is obvious that future degradation studies using labeled compounds are required to understand this matter. 10.3.1.3 Food matrix It is obvious that anthocyanins are seldom ingested on their own but rather as part of a fruit, vegetable, or meal containing various other phytochemicals. However, it is unclear whether anthocyanins are absorbed and metabolized in the same way when ingested in different foods. Indeed, many food constituents including fat, protein, carbohydrate and fiber will alter gastric emptying and subsequent absorption. Studies in our laboratory have shown significant differences in absorption rates of anthocyanins consumed either alone (2–3 h) (Kay et al., 2004, 2005) or with a high fat meal (4 h) (Mazza et al., 2002). Although matrix obviously affects absorbance rates, a study on the effects of a simultaneous intake of food or flavonoids on the absorption of anthocyanins in pigs showed that the addition of other food components to a dietary source of anthocyanins from blackcurrants delayed the absorption profile but did not decrease the total amount of anthocyanins absorbed (Walton et al., 2006b). Additionally, in a study by Bitsch et al. (2004a) the relative urinary excretion of total anthocyanins differed significantly between red wine (0.18%) and red grape juice (0.23%). The apparently higher absorption of the anthocyanins of red grape juice compared to red wine was attributed to a possible synergistic effect of the glucose content of the juice (Bitsch et al., 2004a). Furthermore studies as mentioned above indicate a possible absorption mechanism that is competitively inhibited by other flavonoids. 10.3.1.4 Analytical methodology Inappropriate analytical methodology for the extraction, separation, identification and/or quantification of anthocyanins and their metabolites in biological fluids and/or tissues will

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obviously lead to an under- or overestimation of absorption. Evidence presented by Murkovic et al. (2000) indicates that the analytical recovery of anthocyanins may be hindered as a result of binding with plasma proteins. In addition, it has recently been observed that anthocyanin metabolites may degrade under various storage conditions (Felgines et al., 2003) resulting in underestimating their bioavailability. Also, it is well known that different chemical forms of anthocyanins are present in equilibrium, depending on the pH, and this will affect identification when utilizing UV-detection techniques. In many studies cited in the past literature, analyses were performed with ultraviolet-visible light detection, on the basis of complete conversion of all of the chemical forms of anthocyanins into a colored flavylium cation upon acidification. However, it is possible that some forms existing at neutral pH would not be converted into the flavylium form because of possible binding to, or chemical reactions with, other components of the plasma or urine (Brouillard, 1982; Mazza & Miniati, 1993; Mazza et al., 2004). Not having a standard methodology has hindered our realization of anthocyanin bioavailability, and although there is no standard technique utilized at present, the most satisfactory method for mixture analysis is probably a multi-step method of separation, isolation, and quantification by LC with peak identification by MS and high-field NMR (Mazza et al., 2004). Also, it would be highly desirable in the future to conduct studies utilizing labeled anthocyanins for identification of all the metabolites.

10.3.2 Elimination Evidence indicates that post consumption, anthocyanins are rapidly absorbed, either through the gastric wall or stomach, and excreted in the urine. In a study on rats, Talavera et al. (2003) demonstrated that a high proportion (∼25%) of anthocyanin monoglycosides (glucoside or galactoside) were absorbed from the stomach. Similarly, Passamonti et al. (2003) presented data from an in-vivo experiment showing the involvement of the stomach in the absorption of grape anthocyanins in rats, and suggested that the capacity of anthocyanins to permeate the gastric mucosa may involve a bilitranslocase-mediated mechanism as the basis of the rapid rate of appearance of anthocyanins in the plasma of rats and humans. Recently, another rat study on the absorption and metabolism of red orange juice anthocyanins confirmed that anthocyanins are rapidly absorbed from both stomach and small intestine, and then excreted in the urine as intact and methylated forms (Felgines et al., 2006). The occurrence of this in humans under normal ‘dietary-dosages’ is undetermined. At the present time, there is no direct evidence for the absorption of anthocyanins from the stomach in humans, although from the values of tmax (time to reach maximum plasma concentration) it has been concluded that polyphenols fit into two groups – one with a tmax in the range of 5 to 6.5 h and the other with a tmax of 1.1 to 2.3 h (Williamson & Manach, 2005). In the group with the longer tmax values, such as isoflavones and rutinosides, their absorption occurs in the colon and/or they undergo enterohepatic recycling. The group with the faster absorption (i.e. with shorter tmax values) – such as anthocyanins, flavonol glucosides, catechins, and procyanidin dimers – are believed to be absorbed primarily from the small intestine (Williamson & Manach, 2005). In a recent human trial, we determined the pharmacokinetic parameters of anthocyanins after the administration of a 721-mg oral dose of cyanidin 3-glycosides from chokeberry

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extract to human subjects (Kay et al., 2004). We identified and quantified anthocyanins and anthocyanin metabolites in zero- to 7-h serum and zero- to 24-h urine samples. The cumulative concentration of total anthocyanins (parent and metabolites) detected in the serum was 376.6 nmol/h/L, and a maximum concentration (Cmax) of 96.1 nmol/L was reached within 2.8 h. Additionally, the total urinary excretion of anthocyanins (parent and metabolites) over 24 h was 1071.5 μg, reaching a maximum rate of excretion (Rmax) of 202.7 μg/h at 3.7 h. Also, Felgines et al. (2005) reported that the total urinary excretion of blackberry anthocyanins and metabolites was maximal between 2 and 4 h after a meal, but continued during the 24 h of the experiment. Additionally, Tian et al. (2006) examined human absorption and metabolism of black raspberry anthocyanins when administered at high doses (2.7 g/day) to humans for 1 week. The urinary excretion of anthocyanins and metabolites reached a maximum rate 4–8 h post ingestion (Williamson & Manach, 2005), which all seems to point primarily to absorption in the small intestine.

10.3.3 The concentration of anthocyanins in human blood and urine is very low The absorption of anthocyanins from food is limited and the concentrations found in human plasma are in the nM to low μM range (Morazzoni & Magistretti, 1990; Tsuda et al., 1999; Cao et al., 2001; Bub et al., 2001; Matsumoto et al., 2001; Felgines et al., 2002; Mülleder et al., 2002; Wu et al., 2002; Felgines et al., 2003; McGhie et al., 2003; Frank et al., 2003; Nielsen et al., 2003; Ichiyanagi et al., 2004; Bitsch et al., 2004c; Kay et al., 2004, 2005; Talavera et al., 2005; Felgines et al., 2006). In a recent study conducted in our laboratory, the total cumulative concentration of anthocyanins (parent and metabolites) detected in the serum over a 7-h sampling regime was 173.0 μg/h/mL, with a Cmax of 44.9 μg/mL occurring within 2.8 h (tmax) (Kay et al., 2004). Additionally, the total urinary excretion of metabolites and parent compounds over 24 h was 1071.5 μg, reaching an Rmax of 202.7 μg/h at 3.7 h. Most other studies have also reported low rates of urinary excretion, generally ranging from 0.003 to 1.9% of the intake (Table 10.2).

10.3.4 Suggested mechanisms of anthocyanin absorption At present, the mechanisms responsible for the gastrointestinal absorption of anthocyanins are not fully understood. Whether anthocyanins are absorbed through passive diffusion or via an active transport mechanism, such as the SGLT1, remains to be determined. Many studies have found that the glycosidic forms of anthocyanins are absorbed intact and appear in blood and urine (Milbury et al., 2002; Mülleder et al., 2002; Wu et al., 2002; Felgines et al., 2003; McGhie et al., 2003; Bitsch et al., 2004b; Cooney et al., 2004; Kay et al., 2004, 2005), whereas other studies have found anthocyanins to be present as metabolites (Felgines et al., 2003; Bitsch et al., 2004b; Cooney et al., 2004; Kay et al., 2004, 2005), as well as in animal tissues such as the liver, kidneys and brain (Passamonti et al., 2005; Talavera et al., 2005; Felgines et al., 2006). The potential mechanisms of anthocyanin absorption are shown in Fig. 10.3. For intact anthocyanin glycosides to be absorbed in the intestine, either they require a specific transporter, such as the intestinal SGLT1 to transport the glucoside across the basolateral

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Fig. 10.3 Potential mechanisms of anthocyanin absorption. Anth-3-gly, anthocyanin 3-glycoside; Anth-gluc, anthocyanin glucuronide; CBG, cytosolic beta-glucosidase; COMT, catechol-O-methyltransferase; LPH, lactate phlorizin hydrolase; SGLT, sodium-dependent glucose transporter; SULT, sulfotransferase; UDP-GT, UDP-glucuronosyltransferase. Adapted from Gee et al. (2000) and Day et al. (2000).

membrane and into the enterocyte, as suggested for other flavonoids (Hollman et al., 1999; Williamson et al., 2000), or they have to be hydrolyzed at the brush border by enzymes such as lactase phloridzin hydrolase, prior to passive diffusion of the aglycone (Hollman et al., 1999; Gee et al., 2000; Williamson et al., 2000; Manach et al., 2005). In addition, a portion of absorbed anthocyanins and/or other derivatives such as acylated anthocyanins may not be absorbed in the upper small intestine or stomach and may undergo bacterial metabolism in the lower intestine, where the anthocyanins will be deglycosylated and the released aglycones will be subjected to transport or further bacterial metabolism (Williamson et al., 2000; Keppler & Humpf, 2005; Manach et al., 2005). The impact of microbial metabolism in the lower GIT should not be overlooked as an important potential metabolic pathway for anthocyanins; unfortunately, this concept has yet to be sufficiently explored. The available evidence suggests that phenolic acid degradation products resulting from ring fission by the gut flora are likely to be the major metabolites in the lower GIT, but labeling studies are required to determine the extent to which this occurs in humans. The organic anion carrier bilitranslocase has also been proposed as a carrier protein for the transport of anthocyanin from the blood into the liver on the basis that its transporter activity is competitively inhibited by anthocyanins (Passamonti et al., 2002). However, the extent to which this transporter is involved in anthocyanin absorption needs further investigation. Finally, with most of the published studies reporting urinary excretion of anthocyanins in the range of 0.003 to 1.9% of the initial dose (Table 10.2), the question remains: what happens to the remainder of the anthocyanins ingested? Many believe that degradation (in vivo or in vitro) is a significant contributor to the reported low bioavail-

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ability of anthocyanins in the human body. However, as stated above, labeling studies will be required to identify potential degradation products. Until all degradation products of both parent compounds and metabolites are identified the extent of anthocyanin absorption and bioactivity will not be realized.

10.3.5 Structural transformations of anthocyanins Both in-vivo and in-vitro transformation of anthocyanins may account for a significant proportion of undetected anthocyanins in previous studies. Anthocyanins are positively charged compounds compared with the other, largely neutral, flavonoids, and pH has a marked influence on the color and stability of anthocyanins in aqueous media (Brouillard, 1982; Mazza & Brouillard, 1987a,b; Mazza & Miniati, 1993). It has been demonstrated that in an acidic or neutral medium the four primary anthocyanin structures exist in equilibrium: the flavylium cation AH+, the quinonoidal base A, the carbinol pseudobase B and the chalcone C (Fig. 10.4). For cyanidin 3,5-diglucoside for instance, the red cation (AH+) is the sole structure only when the pH of the solution is less than 0.5. With increasing pH its concentration decreases as hydration to the colorless pseudobases occurs, the equilibrium being characterized by a pKh value of 2.23 ± 0.10 when equal amounts of both forms exist. At this pH, however, small amounts of the colorless chalcones and the blue quinonoidal bases are also present, and the proportions of these and the carbinol forms increase with increasing pH at the expense of the red cationic form up to about pH 4.5. Between pH 4 and 6 very little color remains in the anthocyanin since the amounts of the colored forms AH+ and A are very small. The equilibrium between these species is characterized by pKa value of 3.38 ± 0.15 (Mazza & Brouillard, 1987b). The absence of color obviously makes quantification difficult when using conventional HPLC methods of detection. During the passage of anthocyanins through the GIT, they are exposed to different pH environments and therefore most likely to exist as different forms. The anthocyanin forms present in the different regions and tissues of the GIT are not known with certainty. However, it is known that the pH of the GIT rapidly changes throughout, from highly acidic in the stomach (pH 1–3, fasting) and gradually increasing throughout the GIT, reaching neutral pH in the terminal ileum (Fallingborg, 1999). Thus, we can safely say that the anthocyanin structures present in an healthy small intestine are carbinol pseudobases and the chalcones – cis- (CE) and trans-chalcones (CZ) (Fig. 10.4). Additionally, we also know that on heating anthocyanin solution above 30ºC, the equilibrium is driven toward the chalcone forms (C), and a concomitant decrease in colored forms (AH+ and A) occurs (Mazza & Brouillard, 1987a; Mazza & Miniati, 1993), thus further indicating that in-vitro storage conditions should be controlled when quantifying anthocyanins using conventional methods. The cis- (CE) and trans-chalcones (CZ), shown at the bottom of Fig. 10.4, are formed from the carbinol pseudobase by a fast ring-opening reaction and a slow isomerization process. Both chalcones differ from the usual chalcones by having their carbonyl function next to the B-ring, whereas in the normal chalcones the carbonyl group is adjacent to the A-ring. Using NMR spectroscopy and malvidin 3-glucoside, Cheminat & Brouillard (1986) were able to detect a small amount of the water 4-adduct. At sufficiently high pH values an anionic cis-chalcone appears. This slightly yellow species is believed to be in rapid equilibrium with the anionic water 2-adduct, a structure very difficult to identify.

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Fig. 10.4 Structural transformations of anthocyanins in water (Adapted from Cheminat & Brouillard, Tetrahedron Letters 1986, 273, 4457).

Both structures can also be regarded as canonical forms of a resonance hybrid, in which case identification would be impossible (Brouillard, 1988; Mazza & Miniati, 1993). Thus, at the pH and temperature of the GIT over 95% of the anthocyanins will most likely exist as carbinol pseudobases and chalcones – perhaps a major reason for the reported disappearance of the anthocyanins in the human body. In agreement with this supposition, a recent study on the stability and biotransformation of anthocyanins in vitro showed that incubation of malvidin, cyanidin, pelargonidin, delphinidin, and peonidin at 37°C and neutral pH led to nearly complete disappearance of the anthocyanidins after 60 min, and to the appearance of several new compounds, identified as dimers of two aglycone units (flavylium cation, hemiketal quinoid base and

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α-diketone) and phenolic acids derived from the B-ring of the anthocyanin skeleton (Fleschhut et al., 2006). Likewise, it has been shown that anthocyanidin glycosides are hydrolyzed by the intestinal microflora within 20 min to 2 h of incubation, and liberated aglycones degraded to phenolic compounds (protocatechuic acid, syringic acid, vanillic acid and phloroglucinol aldehyde) after only 20 min of incubation at neutral pH (Keppler & Humpf, 2005). Further chemical decay or further microbial metabolism of the phenolic compounds was accompanied by O-demethylation and the formation of unknown anthocyanidin intermediates. Thus a rapid and extensive degradation could be one major reason for the poor bioavailability of parent anthocyanins in the pharmacokinetic studies described so far in the literature. Further research is required to characterize the structures of these degradation products in vivo, along with their pharmacokinetic and bioactivity characteristics in humans.

10.4 Metabolism of anthocyanins 10.4.1 Human studies To date, metabolites of anthocyanins have been recovered from human urine as glucuronide(Wu et al., 2002; Felgines et al., 2003, 2005; Bitsch et al., 2004b; Cooney et al., 2004; Kay et al., 2004, 2005), methyl- (Wu et al., 2002; Kay et al., 2004, 2005; Felgines et al., 2005; Tian et al., 2006) and sulfo-conjugates (Felgines et al., 2003, 2005). Sulfo-conjugates have been reported in only two studies thus far and they were present at very low concentrations (Felgines et al., 2003, 2005), but glucuronidation and methylation have consistently been demonstrated to be major metabolic pathways of anthocyanins. The metabolites of anthocyanins have been shown to persist in the urine for up to 24 h, and may retain much of their basic anthocyanin structure (Kay et al., 2004, 2005). Pharmacokinetic evidence suggests that the concentration of the parent glycosides and their glucuronide derivatives are prominent in early blood samples (0–5 h), with increasing methylation occurring over time (6–24 h). This evidence suggests that anthocyanins bioactivity is probably altered over time as a result of metabolic transformation post consumption. One of the first reports to suggest the presence of metabolized anthocyanins in humans was published by Wu et al. (2002). In this study women consumed 12 g of elderberry extract (720 mg total anthocyanin). The researchers reported four metabolites characteristic of peonidin 3-glucoside, peonidin 3-sambubioside, peonidin monoglucuronide and cyaniding 3-glucoside monoglucuronide. A subsequent study by Felgines et al. (2003) demonstrated the presence of three monoglucuronides of pelargonidin, one sulfo-conjugate of pelargonidin, and pelargonidin itself in the urine of volunteers who had consumed a meal containing 200 g strawberries providing 179 μmol pelargonidin 3-glucoside. The total urinary excretion of strawberry anthocyanin metabolites corresponded to 1.8% of the pelargonidin 3-glucoside ingested. More than 80% of this excretion was in the form of monoglucuronides. Four hours after the meal, more than two-thirds of anthocyanin metabolites had been excreted, although urinary excretion of the metabolites continued until the end of the 24-h experiment.

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Fig. 10.5 Proposed pathway for the formation of anthocyanin metabolites in humans.

The capacity of human subjects to convert cyanidin 3-glycosides to glucuroconjugated and methylated conjugates was further demonstrated by Kay et al. (2004), who observed at least ten individual anthocyanin metabolites in the urine and serum of volunteers who had consumed approximately 20 g chokeberry extract containing 1.3 g cyanidin 3glycosides (899 mg cyanidin 3-galactoside, 321 mg cyanidin 3-arabinoside, 51 mg cyanidin 3-xyloside and 50 mg cyanidin 3-glucoside). A recent study by Cooney et al. (2004) following the consumption of boysenberry (Rubus loganbaccus × baileyanus Britt) extract containing cyaniding 3-sophoroside, cyanidin 3-glucoside, cyanidin 3-glucosylrutinoside and cyanidin 3-rutinoside identified urinary anthocyanins as monoglucuronides of peonidin, cyanidin and pelargonidin. A proposed pathway for the formation of anthocyanin metabolites in human urine and serum post consumption of cyanidin 3-glycosides is presented in Fig. 10.5. Additionally, Fig. 10.6 details the proposed sites of anthocyanin metabolism. The enzymes believed to be associated with the biotransformation of anthocyanins include: catechol-Omethyltransferase; S-adenosyl methionine; lactase-phlorizin hydrolase; β-galactosidase; uridine diphosphate glucuronosyltransferase; and cyotchrome P450. These enzymes are located in the small intestine, liver or kidney, and depending on the chemical structure, anthocyanins could exist as native forms or metabolites in blood and urine, whereas most other flavonoids are generally recovered primarily as metabolites (Prior & Wu, 2006). For a typical chromatogram of parent and metabolite anthocyanins in human urine post consumption of chokeberry extract refer to Fig. 10.7. In our study (Kay et al., 2004), the average concentration of anthocyanins and anthocyanin metabolites in the urine reached a level of 17.9 (range 14.9–20.9) nmol/L within 5 h post consumption and persisted in 24-h urine samples at an average level of 12.1 (range 1.1–13.0) nmol/L. In addition, the

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Fig. 10.6 Potential route of anthocyanin absorption, metabolism and elimination.

average total level of anthocyanins and anthocyanin metabolites detected in the serum was 591.7 (range 197.3–986.1) nmol/L within 2 h post consumption. Felgines et al. (2005), feeding whole blackberries as part of a meal, also found urinary anthocyanins mainly as methylated and/or glucuronidated conjugates. In addition to native cyanidin 3-glucoside, the authors identified methylated glycosides, glucuronides of anthocyanidins and anthocyanins, and sulfo-conjugates of cyanidin. Total urinary excretion of blackberry anthocyanin metabolites was 0.02% of the amount ingested. Additionally, monoglucuronides of anthocyanidins represented > 60% of this excretion. Lastly, a recent study feeding 45 g of freeze-dried black raspberries daily for 1 week

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Fig. 10.7 Typical chromatograms of human serum pre- and post consumption of chokeberry extract containing cyanidin 3-galactoside (C-3-gal), cyanidin 3-glucoside (C-3-glu), cyanidin 3-arabinoside (C-3-ara), and cyanidin 3-xyloside (C-3-xyl).

showed that anthocyanins were excreted in intact form and metabolized into methylated derivatives. The major urinary metabolites were a methylated derivative of cyanidin 3-rutinoside and two methylated derivatives of cyanidin 3-xylosylrutinoside. The methylated cyanidin 3-glucoside and sambubioside were present at very low concentrations (Tian et al., 2006). This study showed that methylation may be a major metabolic pathway for

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anthocyanins, and that the structures of the glycoside moieties affects the methylation pattern. The above human studies consistently indicate that anthocyanins are metabolized post consumption to various forms that involve the metabolic pathways associated with glucuronidation, methylation, and sulfo-conjugation (Felgines et al., 2003, 2005). Additionally, it appears that the primary metabolic pathway utilized depends on both the glycoside moieties present and the hydroxylation pattern of the anthocyanin species.

10.4.2 Animal studies 10.4.2.1 Rat studies Tsuda et al. (1999) found no cyanidin glucuronides in the livers and kidneys of rats after oral administration of cyanidin 3-glycoside, but found peonidin and protocatechuic acid, which was suggestive of cyanidin metabolism and degradation. More recently, cyanidin 3-glucoside and its four major metabolites (two monomethylated cyanidin 3-glucosides, a glucuronide of cyanidin and a methylated cyanidin) were detected in the plasma of rats after oral administration of cyanidin 3-glucoside (100 mg/kg of body mass) by Ichiyanagi et al. (2005a). The maximum plasma concentrations of the four metabolites were in the following order: monomethylated cyanidin 3-glucosides (or peonidin 3-glucoside) (21 nM) > monomethylated cyanidin 3-glucosides (20 nM) > glucuronide of cyanidin (8.5 nM) > methylated cyanidin (5 nM). When cyanidin 3-glucoside was directly injected into the neck vein, only the monomethylated cyanidin 3-glucosides were detected in the plasma, indicating that the glucuronide was likely produced during absorption from the GIT. Methylated anthocyanins as well as conjugated monoglucuronides were identified in the jejunum, liver, and kidney of rats by Talavera et al. (2005). These authors found that the proportions of anthocyanin derivatives differed among tissues, with the liver presenting the highest proportion of methylated forms. Additionally, the jejunum and plasma contained aglycone forms. In the brain, total anthocyanin content reached 0.25 nmol/g of tissue. Subsequently, Felgines et al. (2006) investigated the absorption of red orange anthocyanins in both the stomach and intestine using in-situ models and identified the red orange anthocyanins, cyanidin 3-glucoside and cyanidin 3-malonylglucoside, as well as their respective methylated derivatives, which were recovered in urine. This study indicated that red orange juice anthocyanins were rapidly absorbed from both stomach and small intestine, and then excreted in the urine as intact and methylated forms. Moreover, the absorption and metabolism of acylated anthocyanins and non-acylated anthocyanins appeared similar. El Mohsen et al. (2006) demonstrated that pelargonidin is absorbed and present in plasma following oral gavage to rats. In addition, the main structurally related pelargonidin metabolite identified in plasma and urine was pelargonidin glucuronide. Interestingly, p-hydroxybenzoic acid, a ring fission product of pelargonidin, was detected in plasma and urine samples obtained at 2 and 18 h post gavage, thus agreeing with the findings of Tsuda et al. (1999) on feeding cyanidin 3-glycoside. At 2 h post gavage, pelargonidin glucuronide was the major metabolite detected in kidney and liver, with levels reaching 0.5 and 0.15 nmol pelargonidin equivalents/g tissue, respectively. In addition, brain and

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lung tissues contained detectable levels of the aglycone, with the glucuronide also present in the lungs. Other tissues, including spleen and heart, did not contain detectable levels of pelargonidin or ensuing metabolites. Lastly, Matsumoto et al. (2006) reported that ingested delphinidin 3-rutinoside is primarily excreted to urine as the intact form and in the bile as the methylated form in rats. They detected small amounts of the metabolite 4′-O-methyl-delphinidin-3-rutinoside in the plasma, but they detected neither anthocyanidin (aglycone) nor glucuro- or sulfoconjugates. These results indicate that the tri-hydroxylated B-ring of delphinidin 3rutinoside might be metabolized differently from the di- and mono-hydroxylated cyanidin 3-glucoside and pelargonidin-3-glucoside. 10.4.2.2 Pig studies In a study by Wu et al. (2004), in which weaning pigs (7.9 ± 1.7 kg) were fed a freezedried powder of marionberry by stomach tube, the authors identified 11 metabolites and the four original anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, and one unknown acylated cyanidin-based anthocyanin) in the urine. The main metabolites were glucuronidated and/or methylated forms. In a follow-up study, the researchers (Wu et al., 2005) fed weanling pigs a single meal of either freeze-dried chokeberry, blackcurrant, or elderberry powder at a dose of 229, 140, or 228 μmol total anthocyanins/kg body weight, respectively, and demonstrated that the aglycone and sugar moieties altered the apparent absorption and metabolism of anthocyanins. Cyanidin anthocyanins were metabolized via methylation and glucuronidation as well as by the formation of both derivatives on the same anthocyanin molecule. Delphinidin anthocyanins were not metabolized to any measurable extent. Anthocyanins with either a di- or trisaccharide attached to them were excreted in the urine primarily as the intact forms. Additionally, over 80% of the anthocyanin compounds containing rutinose or sambubiose, which were excreted in the urine from blackcurrant, elderberry, or marionberry, were excreted as the intact compounds. The limited metabolism of these anthocyanins that did occur was via methylation. In a third study utilizing raspberries, Wu et al. (2006) found that the presence of complex sugars (sambubiose and rutinose) on cyanidin largely prevented its methylation or glucuronidation as observed with urinary metabolites. Of the total anthoycyanins (cyanidin 3-sambubioside, cyanidin 3-sambubioside-5-rhamnoside, and cyanidin 3-rutinoside) recovered in the urine, 92–100% was as the parent compounds, whereas only 41% of the total of cyanidin 3-glucoside recovered in the urine was as the parent compound. Pelargonidin was metabolized only to the monoglucuronide and was not methylated. Clearly, according to these pig studies, the anthocyanin aglycone and the glycoside have significant effects on the absorption and/or metabolism of anthocyanins. These results are largely in agreement with the results of comparable human studies discussed earlier.

10.4.3 Potential mechanisms of anthocyanin metabolism A proposed pathway for the metabolism of cyanidin 3-glycosides in humans is shown in Fig. 10.5, while their tissue specific metabolism is given in Fig. 10.6. Our findings (Kay et al., 2004, 2005) regarding the glucuronide conjugates and methylated forms of cyanidin-

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based anthocyanins in humans have been confirmed by subsequent human studies by Tian et al. (2006) and Felgines et al. (2005), and in studies utilizing the pig (Wu et al., 2004, 2005) and rat (Tsuda et al., 1999; Ichiyanagi et al., 2005; El Mohsen et al., 2006). Glucuronide conjugates and/or methylation of the catechol structure of the aglycone were the major types of metabolites of cyanidin-based anthocyanins indicated in the abovementioned studies. Methylation is a well-known pathway in the metabolism of flavonoids (Walle, 2004). The catechol-like structure of cyanidins makes them predictably prone to O-methylation by soluble catechol-O-methyltransferase (COMT). This reaction has been particularly well studied for quercetin both in vivo in hamsters and in-vitro experiments (Walle, 2004). The transformation occurs primarily in the liver and is catalyzed by COMT (Walle, 2004). The catechol structural unit is necessary for COMT activity. Using the pig as a model, Wu et al. (2004) identified both 3′-O-methyl and 4′-O-methyl esters of cyanidin in pig urine, demonstrating that both the 3′- and 4′-hydroxyl groups in the B-ring of cyanidin could be conjugated with a methyl group. However, the 3′-O-methyl ester was the predominant form observed (Wu et al., 2004). Since the catechol structure is necessary for COMT activity, it is not surprising that no methylated metabolites for pelargonidin, peonidin, and malvidin anthocyanins have been observed to date. Ichiyanagi et al. (2004) and Matsumoto et al. (2006) found methylation of delphinidin, which occurred only at the 4′-hydroxy group in the B-ring. No 3′ or 5′ methyl derivatives were detected in urine or liver. Glucuronic acid conjugates of anthocyanins in humans have been well documented (Wu et al., 2002; Felgines et al., 2003, 2005; Bitsch et al., 2004b; Kay et al., 2004, 2005; Tian et al., 2006). However, at the present time, neither the molecular site of glucuronidation nor the UDP-glucuronosyltransferase isoforms have been sufficiently characterized. Two pathways for the formation of cyanidin monoglucuronides have been proposed (Fig. 10.3). The first possibility is that the glucuronide is formed directly from cyanidin 3-glucoside by UDP-glucose dehydrogenase; the second possibility requires the hydrolysis of cyanidin 3-glycoside or peonidin 3-glycoside to aglycone followed by a rapid glucuronidation in the intestine. However, recent evidence from the ratio of cyanidin 3-glucoside to cyanidin monoglucuronide in the urine supports the second possibility as the most likely pathway (Wu et al., 2005). Also, as noted above, aglycones of cyanidin and peonidin were recently found in the plasma of rats (Talavera et al., 2005), which gives further support to the second possibility. A sulfo-conjugate of anthocyanidin (cyanidin sulfate) has been identified in human urine following the consumption of strawberries (containing mainly pelargonidin 3-glucoside) (Felgines et al., 2003) and blackberries (containing mainly cyanidin 3-glucoside) (Felgines et al., 2005). The formation of anthocyanidin sulfo-conjugates requires hydrolysis of glucoside to the aglycone and then sulfo-conjugation of the aglycone by sulfotransferases present in numerous tissues, including the intestine and liver (Runge-Morris, 1997). Sulfotransferase-catalyzed sulfation (or O-sulfonation) has long been recognized to play an important role in the detoxication of xenobiotics and endogenous hydroxylated products (Runge-Morris, 1997). Thus, sulfonation and glucuronidation of anthocyanins are believed to be important mechanisms in the excretion of these compounds from the body in the urine. A major difficulty in the assessment of the importance of sulfate conjugation

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of flavonoids in humans has been the inability to measure these conjugates in biological fluids (Walle, 2004), thus the limited number of reports of sulfo-conjugates may reflect limitations in the detection and quantification of these compounds using currently available analytical methodology.

10.5 Conclusions The preceding discussion provides evidence illustrating that anthocyanins have diverse effects in vitro that suggest potential health benefits in general. However, from the above treatment it is quite apparent that the basic aspects of anthocyanin absorption and metabolism are far from being fully understood. The reason for the low reported bioavailability of anthocyanins is also not readily apparent. It may be the result of multiple factors, including the likelihood that many of the known and/or unidentified metabolites may go undetected as a result of their low levels and/or the analytical methods used. Also, with the predominance of the colorless carbinol (75–80%) and chalcone (15–20%) forms of anthocyanins present in the blood and urine at neutral pH, it is highly likely that these chemical forms may escape detection, and/or be chemically bound to other components in the plasma or urine. These shortcomings could be overcome by using labeled anthocyanins in future studies. In addition, complete metabolic studies involving the collection of blood, urine, CO2, and feces are required. Until the complete absorption, metabolic fate, and bioactivity of anthocyanins and their metabolites in vivo are realized, it will not be possible to speculate on the magnitude of their biological importance to health and diseases. Future studies investigating the benefits of the consumption of anthocyanin-rich products should include identifying anthocyanin metabolites as well as their tissue distribution and bioactivities using molecular/cell biology and animal and human data.

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Chapter 11

Bioavailability, Metabolism, and Bioactivity of Food Ellagic Acid and Related Polyphenols Francisco A. Tomás-Barberán, Maria Teresa García-Conesa, Mar Larrosa, Begoña Cerdá, Rocio González-Barrio, Maria José Bermúdez-Soto, Antonio González-Sarrías and Juan Carlos Espín

11.1 Introduction Epidemiological studies have shown an inverse correlation between the intake of food of plant origin and mortality from chronic diseases such as cardiovascular and neurodegenerative diseases and cancer. These beneficial effects for health have been attributed to the dietary antioxidants present in plant food, as the oxidation process and free-radicals lie at the origin of these diseases (Arts & Hollman, 2005). In fact, consumers are increasingly coming to regard food antioxidants as anti-aging agents, and therefore there is a demand for antioxidant-enriched food. The food industry is aware of this trend as marketing studies identify the demand, leading to requests for new food products that fulfill these requirements from their production and R&D departments. All these facts have led to a big increase in the number of research projects and publications on food antioxidant constituents – particularly antioxidant polyphenols – over the last decade. A search of the scientific literature databases looking for either phenolic antioxidants or flavonoid antioxidants indicates that the number of publications under these keywords has increased exponentially during the last twenty years (Fig. 11.1). Most of these publications deal with the evaluation of antioxidant or free-radical scavenging activities in vitro, some of them ex vivo on LDL, but only a few aim at the evaluation of antioxidant activity in vivo. In addition, many of the studies evaluating the biological activity of polyphenols use concentrations that are much higher than those found under physiological conditions in vivo, and do not take into account the changes occurring in the gastrointestinal tract, the metabolism by human cells and by intestinal micro flora, and the final bioavailability of these metabolites and their distribution in different tissues. The lack of consideration of these issues dramatically reduces the validity of many of the studies carried out. It is quite common to find marketing claims in foods stating that a specific food product scored the highest in tests for the evaluation of antioxidant activity in vitro when compared with other powerful antioxidant foods, and the labels and marketing claims suggest Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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3000 2500 2000 Polyphenols Flavonoids

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Fig. 11.1 Number of publications about antioxidant activity of phenolics or flavonoids during the last twenty years.

that these or could be associated, directly or indirectly, with some health benefits related to the neutralization of oxidative stress. The present chapter shows that powerful antioxidant food products evaluated in vitro suffer extensive changes during digestion and metabolism, to be transformed into nonantioxidant metabolites that may be responsible for the biological activity associated with the intake of these products. We review the biological activity and bioavailability of food ellagitannins as an excellent model for understanding the biological activity of antioxidant polyphenols from food.

11.2 Ellagitannins and ellagic acid as examples of bioactive polyphenols Ellagitannins are combinations of hexahydroxydiphenic acid with glucose. Hydrolysis gives free hexahydroxydiphenic acid, which undergoes spontaneous cyclization to form ellagic acid through the formation of internal lactone bridges. The original ellagitannins have UV spectra quite unspecific but related to those of galloyl residues (maximum around 270 nm) while ellagic acid has a characteristic UV spectrum with two absorption maxima, one around 370 nm and the other around 250 nm. These phenolics are generally biosynthesized by controlled oxidation of the corresponding gallotannins, in which two different galloyl residues located in the vicinity are C–C linked (by the activity of oxidative enzymes) to produce the hexahydroxydiphenic groups. In some cases three galloyl units are C–C linked or O–C linked to give more complex molecules. Dimeric, trimeric or oligomeric forms of ellagitannins have also been reported, as well as the occurrence of C-glycosidic combinations. In a few cases, four galloyl residues are linked to give rise to complex compounds such as the ellagitannin punicalagin, characteristic of pomegranate. In this polyphenol, the ellagic acid structure is already present in the original ellagitannin, and is evident in its UV spectrum. These ellagitannins contain a large number of phenolic hydroxyls grouped in the ortho-position, by virtue of which they show a high antioxidant activity in vitro.

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Ellagitannins are present in significant amounts in many berries, including strawberries, red and black raspberries (Zafrilla et al., 2001) and blackberries, and nuts, including walnuts (Fukuda et al., 2003), chestnuts, oak acorns (Cantos et al., 2003) and pecans (Villarreal-Lozoya et al., 2007). They are also present in pomegranates (Gil et al., 2000), and muscadine grapes, and are important constituents of wood, particularly oak wood (Glabasnia & Hofmann, 2006). Thus they are relevant to several food products since ellagitannins migrate from wood to the food matrix during different aging processes (wines, whiskey, etc.). Ellagic acid has also been found in several honey types, and it has been proposed as a floral marker for heather honey (Ferreres et al., 1996).

11.3 Evaluation of antioxidant activity in vitro Ellagitannin-rich foods generally show a high free-radical scavenging activity when evaluated in vitro. The antioxidant activity of pomegranate juice is particularly strong, at twice that of red wine (Gil et al., 2000). This activity is due to the presence of ellagitannins extracted from the fruit husk during juice manufacturing (Gil et al., 2000). This remarkable antioxidant activity prompted research on the biological activity of these powerful antioxidants from pomegranate and was used by the industry to market pomegranate juice products as super-antioxidant food. Ellagitannins are also responsible for part of the antioxidant activity observed in strawberries (Hannun, 2004), raspberries (Zafrilla et al., 2001), blackberries, walnuts (Blomhoff et al., 2006) and pecans (Villarreal-Lozoya, 2007). The antioxidant activity of these food products can probably be related to the biological activity reported for them.

11.4 Biological activity associated with ellagitannin-rich food intake; clinical studies In parallel with studies of the antioxidant activity of ellagitannin-rich food, clinical studies have also shown biological activity that has been associated with these antioxidants, although no direct evidence of the biological activity of these polyphenols has been demonstrated. Several clinical studies have reported relevant biological activity after the intake of ellagitannin-rich foods, especially regarding the protective effect against cardiovascular diseases and cancer. Pomegranate juice, one of the richest food products in ellagitannins (Gil et al., 2000), showed protection against cardiovascular diseases, decreasing the effect of risk factors for atherosclerosis such as hypertension, platelet aggregation, blood lipid profiles and oxidative stress (Aviram et al., 2000; Fuhrman & Aviram, 2006). Pomegranate juice supplementation for three years to patients with carotid artery stenosis (CAS) led to decreased atherosclerotic lesion size, and the authors attributed these effects to the potent antioxidant characteristics of pomegranate juice (Aviram et al., 2002, 2004). Pomegranate juice consumption also reduced the systolic blood pressure of CAS patients after one year’s intake. In contrast, no significant effect on the patients’ diastolic blood pressure was observed. The serum angiotensin converting enzyme (ACE) activity was also significantly decreased

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after pomegranate juice intake (Aviran & Dornfeld, 2001). The authors suggest that this effect may be secondary to the ability of pomegranate juice-associated antioxidants, such as complex tannins, to inhibit ACE activity. Because ACE inhibitors are metabolized by cytochrome P-450 enzymes, serum ACE activity can be significantly affected by modulation of P-450 enzyme activity. In fact it has been demonstrated that pomegranate juice decreases the activity of different cytochrome P-450 enzymes by 20–60% (Aviram et al., 1999; Hidaka et al., 2005). In hypertensive patients treated with ACE inhibitors, the inhibitory effect of pomegranate juice consumption on cytochrome P-450 enzymes can possibly decrease P-450-mediated drug (ACE inhibitor) metabolism, hence serum ACE activity may be further decreased in these patients. The authors stated that as ACE activity is related to enhanced blood pressure and accelerated atherosclerosis, their data may suggest an additional important anti-atherogenic property of pomegranate juice. However, a recent publication has reported an association of rhabdomyolysis with pomegranate juice consumption, owing to cytochrome P-450 inhibition by the juice (Sorokin et al., 2006). A myopathy patient taking statins started drinking pomegranate juice after reading a report on its health benefits. After three weeks the patient presented in hospital with thigh pain, general malaise and brown urine, and his serum creatinine kinase was elevated. After statin and pomegranate juice were discontinued, the creatinin kinase levels decreased steadily to reach normal levels after 10 days. This report suggests that as pomegranate juice inhibits intestinal cytochrome P-450 enzymes, this effect can alter statin metabolism, increasing its absorption. Circulating human platelets play an important role in the development of atherosclerosis, and increased platelet aggregation is associated with enhanced atherogenicity. Furthermore platelet activation has been shown to be associated with oxidative stress. Following 2 weeks of pomegranate juice consumption a significant reduction (11%) in collagen-induced platelet aggregation was noted (Fuhrman & Aviram, 2006). The authors link the effects to an interaction of pomegranate juice constituents with the platelet surface binding sites for collagen or ADP, thus the ability of pomegranate juice to reduce platelet aggregation in humans was supposed to be a direct effect of the juice in vitro, although it may also be that the antioxidative properties of pomegranate juice constituents can attenuate oxidative stressinduced platelet activation. It has been proposed that pomegranate juice polyphenols can protect LDL against cellmediated oxidation via one or both of two pathways – direct interaction of polyphenols with the lipoprotein and indirect effects through the accumulation of polyphenols in arterial macrophages (Fuhrman & Aviram, 2006). All these antioxidative and antiatherogenic effects of pomegranate polyphenols have been demonstrated in vitro, and in vivo in humans and in atherosclerotic apolipoprotein E-deficient mice.

11.5 Questions arising after the demonstration of the large antioxidant activity in vitro and the biological activity associated with the intake of ellagitannin-rich food Although the antioxidant activity of ellagitannins from food products has been demonstrated in vitro, and the biological activity of dietary supplementation with these food products

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has been demonstrated in several studies, there are still some points that need to be clarified, and the purpose of our research has been to understand how ellagitannins can exert their biological activity in vivo. Thus we have investigated the following objectives: 1) Evaluation of the bioavailability and metabolism of ellagitannins in humans. 2) Evaluation of the distribution of the metabolites in different tissues. 3) Evaluation of the biological activity of the metabolites that are relevant in vivo, testing the proper metabolites at appropriate concentrations.

11.5.1 Bioavailability and metabolism of ellagitannins and ellagic acid and distribution of the metabolites in different tissues After the intake of ellagitannin-rich food (pomegranate juice and freeze-dried black raspberry), which also contains significant amounts of free ellagic acid, the acid has been detected in plasma and urine (Seeram et al., 2004; Stoner et al., 2005), although at relatively low concentrations. In these pharmacokinetic studies, ellagic acid started to be detected in plasma as soon as one hour after intake. In our studies with human volunteers and model animals (rats and pigs), however, we have not been able to detect free ellagic acid either in plasma or in urine. We have detected some ellagic acid methyl ether and dimethyl ether glucuronide in urine (Cerdá et al., 2006), a finding that has been confirmed by checking the metabolism of punicalagin and ellagic acid by colon cells (Larrosa et al., 2006a). This has also been found after the ingestion of an extract of pomegranate polyphenols (Mertens-Talcott et al., 2006). When pomegranate ellagitannins (from pomegranate husk) were given in large amounts to rats, no free ellagic acid was detected in their plasma and urine, while methyl-ellagic acid glucuronides were found in the urine (Cerdá et al., 2003a). In some cases, trace amounts of the original punicalagin were detected in plasma and urine, in spite of the large size of this molecule (Cerdá et al., 2003b). Although this is unusual in the light of classical bioavailability assumptions, under which it was thought that only small polyphenols could be absorbed, there is some recent evidence that large polyphenol molecules such as procyanidin trimers (MW, 866) or tetramers (MW, 1154) can be detected in plasma (Shoji et al., 2006), thus supporting our previous report on the absorption of punicalagin (MW, 1084). However, the main metabolites detected in plasma and urine associated with the ingestion of pomegranate ellagitannins were ellagic acid degradation products of the dibenzopyranone type, including urolithin A and B and related tri- and tetra-hydroxy derivatives, generally present as glucuronide and/or sulfate conjugates (Cerdá et al., 2003a, b). In this study the distribution of these metabolites in different organ tissues was also evaluated, and they were only detected in trace amounts in the rat liver and kidney. Iberian pigs have also been used as models to evaluate ellagitannin bioavailability, metabolism, and tissue distribution, as they provide an excellent model since their main dietary intake is oak acorns, a nut very rich in ellagitannins (Cantos et al., 2003). In addition, there is some evidence supporting a lower oxidation susceptibility of Iberian pigs’ meat and fat fed on acorns, compared to that of pigs fed on cereals, thus providing a good model for evaluating the antioxidant effects of acorn ellagitannins in vivo. After studying the luminal and tissue content of different portions of the intestine, and the contents of the gall bladder, plasma, and urine, several conclusions were reached regarding bioavailability

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Fig. 11.2 Disposition of metabolites of ellagitannins, ellagic acid, and related molecules in humans and pigs. (a) Dimethyl-glucuronide; (b) Glucuronides; (c) Methyl-glucuronides (Espín et al., 2007). (with permission from the American Chemical Society).

and metabolism. This study has revealed that ellagic acid is released from ellagitannins in the small intestine, probably owing to the physiological conditions. This agrees with the results observed in human cell-line cultures, in which punicalagin released ellagic acid that was then responsible for the biological activity observed (Larrosa et al., 2006a). The ellagic acid released from acorn ellagitannins in the pig is then metabolized by the intestinal microflora to render sequentially tetrahydroxy-dibenzopyranone derivatives, trihydroxydibenzopyranone derivatives, dihydroxy-dibenzopyranone (urolithin A) and monohydroxydibenzopyranone (urolithin B) (Fig. 11.2). In the jejunum, free ellagic acid and the tetrahydoxy and trihydroxy-dibenzopyranones are the main metabolites, while in the colon urolithin A and B are the main metabolites, showing that the metabolism of these compounds advances as they progress through the intestine. When analyzing the content of the intestinal tissues, it is clear that absorption increases as the number of phenolic hydroxyl groups per molecule decreases, and therefore ellagic acid is not observed in the tissues of the jejunum, while tetrahydroxy-dibenzopyranone, tri-hydroxy-dibenzopyranone, and urolithin A are detected in increasing amounts. In the tissues, the glucuronide conjugates are also observed together with the aglycones, showing that glucuronidation of these compounds already takes place in the intestinal wall. When the colon tissues were analyzed, urolithins A and B and their corresponding glucuronides were detected. Analysis of peripheral plasma showed the presence of only urolithin A and B glucuronides, while the tri- and tetrahydroxy-dibenzopyranone derivatives were not found.

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When the bile was analyzed a complex combination of metabolites was found that included tetrahydroxy-dibenzopyranone derivatives, trihydroxy-dibenzopyranone derivatives, and ellagic acid derivatives, mainly the methyl ethers of those molecules with orthodihydroxy groups susceptible to be substrates for catechol-O-methyl transferase (COMT) (ellagic acid, tetrahydroxy-dibenzopyranones and tri-hydroxy-dibenzopyranones), glucuronides and diglucuronides and sulfates. Some of them were also present as sodium salts. No urolithin B was detected in bile, showing that this metabolite was not retained in the liver and was directly excreted in urine as the glucuronide conjugate. The absence of tetrahydroxy-dibenzopyranone derivatives and tri-hydroxy-dibenzopyranone derivatives in peripheral plasma and urine, and their large amounts in bile and the lumen of the small intestine, indicate that there is extensive enterohepatic circulation of these metabolites, which only pass to peripheral plasma and to excretion in urine after extensive metabolism to urolithin A and B metabolites. These are not retained in the liver and are excreted in urine (Fig. 11.3). This explains the large clearing time for these metabolites, which are still being excreted in human urine as long as 72 hours after the ellagitannin intake from pomegranate, walnut, strawberry, raspberry, and oak-aged red wine (Cerdá et al., 2004, 2005). This also agrees with a recent report on the urinary excretion of urolithin A and B metabolites after the intake of pomegranate juice (Seeram et al., 2006). In a recent study using Iberian pig as an animal model (Espín et al., 2007), small amounts of ellagic acid dimethyl ether glucuronide and diglucuronide were detected in urine, showing that ellagic acid is absorbed and then extensively conjugated before excretion. When different tissues were analyzed to evaluate the distribution of these metabolites, they were not detected in lung, brain, heart, muscle, or fat, but were detected in trace amounts in liver and kidney, in agreement with what was found in rat tissues. Clinical intervention studies with healthy human volunteers or with volunteers with chronic obstructive pulmonary disease (Cerdá et al., 2006) have shown that after the intake of pomegranate juice only the urolithin A and B conjugates and ellagic acid dimethyl ether glucuronide were detected in urine, while in plasma only urolithins were detected. This means that although ellagic acid dimethyl glucuronides were expected to be found in plasma, if present they were there at concentrations below the detection limit (Cerdá et al., 2006). As a conclusion it can be stated that these antioxidant polyphenols present in food are extensively metabolized to non-antioxidant compounds (urolithins A and B) by the human colon microflora, and these metabolites are then absorbed, conjugated and excreted in urine (Cerdá, et al., 2005). This study shows that ellagic acid is released from ellagitannins in the small intestine and that the microbial transformation to urolithins occurs in the jejunum, where these metabolites start to be absorbed. These metabolites also accumulate in the bile and suffer enterohepatic circulation that extends their clearance time (Fig. 113). These bioavailability and metabolism studies indicate that the biological activity of ellagic acid and ellagitannins in food can be largely attributed to the metabolites produced by microflora and therefore, experiments were performed to evaluate their activity.

11.5.2 Evaluation of the biological activity of ellagitannin metabolites produced in vivo It seems clear from the bioavailability studies that ellagitannins and ellagic acid can exert important biological effects locally in the gastrointestinal tract, where they are present at

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Fig. 11.3 Proposed metabolism and disposition of ellagic acid and related molecules in humans and pigs.

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significant concentrations, either because they are naturally present in food products or because free ellagic acid is released under the physiological conditions of the pancreatic digestion in the small intestine. Under these conditions they can reach concentrations of up to 100 μM depending on the food source, the technological processing it has undergone, and the part of the gastrointestinal tract under consideration. The metabolites that are detected in peripheral plasma are urolithin A and B glucuronides (up to 5 μM), while in the portal vein plasma a much higher number of different metabolites should be present, as evidenced by the large amount of metabolites in bile. The concentration of these metabolites in urine can be much higher, but this mainly depends on the specific colonic microflora of individual people, which yields a high inter-person variability in the production of these microbial metabolites and gives rise to the so-called ‘low and high urolithin producers’ (Cerdá et al., 2005). The content of these metabolites in tissues is very small and generally below the detection limits. In the gastrointestinal tract the original ellagitannins, the released free ellagic acid and the bacterial metabolites are all present at much larger concentrations (up to 100 μM). Nevertheless in plasma only the metabolites are detected in significant amounts, generally as glucuronides, and at concentrations rarely above 5 μM. These findings should be taken into account when studying the biological activity of these molecules in vitro. Molecular models suggest that urolithins could display estrogenic and/or antiestrogenic activity. To check this, both urolithins and other known phytoestrogens (genistein, daidzein, resveratrol, and enterolactone) were assayed to evaluate the capacity to induce cell proliferation on the estrogen-sensitive human breast cancer MCF-7 cells as well as the ability to bind to α- and β-estrogen receptors. Both urolithins A and B showed estrogenic activity in a dose-dependent manner even at high concentrations (40 μM), without antiproliferative or toxic effects. Overall, urolithins showed weaker estrogenic activity than the other phytoestrogens. However, both urolithins displayed slightly higher antiestrogenic activity (i.e. antagonized the growth promotion effect of 17-β-estradiol in a dose-dependent manner) than other phytoestrogens including isoflavones and enterolactone. No estrogenreceptor binding was detected for resveratrol and enterolactone. Urolithins A and B entered into MCF-7 cells and were metabolized to yield mainly urolithin-sulfate derivatives. These results, together with previous studies regarding the absorption and metabolism of dietary ellagitannins and ellagic acid in humans, suggest that the gut microflora metabolite urolithins are potential endocrine-disrupting molecules that could resemble other ‘enterophytoestrogens’ (microflora-derived metabolites with estrogenic/antiestrogenic activity) (Larrosa et al., 2006b). As mentioned above, punicalagin spontaneously releases ellagic acid in cell culture medium (independently of the presence of cells). In the presence of cells, ellagic acid enters the cells and suffers methylation by the action of COMT, yielding dimethyl-ellagic acid. This first reaction blocks the most reactive ellagic acid moieties (ortho-dihydroxyl groups). Afterwards, dimethylellagic acid is glucuronidated and excreted to the medium (Larrosa et al., 2006a) (Fig. 11.4). This metabolic process is consistent with the presence of dimethylellagic acid-glucuronide in both plasma and urine (Cerdá et al., 2006; Seeram et al., 2006). This study by Larrosa et al. (2006a) showed that both punicalagin and ellagic acid provoked the same effects on Caco-2 cells, viz.: down-regulation of cyclins A and B1 and up-regulation of cyclin E, cell cycle arrest in the S phase, induction of apoptosis

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Fig. 11.4 Metabolism and apoptotic pathway proposed for punicalagin and ellagic acid in Caco-2 cells.

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via the intrinsic pathway (FAS-independent, caspase 8-independent) through bcl-XL downregulation with mitochondrial release of cytochrome c into the cytosol, and activation of initiator caspase 9 and effector caspase 3. An interesting result (not usually investigated in this type of study) was that neither ellagic acid nor punicalagin induced apoptosis in normal colon CCD-112CoN cells (no chromatin condensation and no activation of caspases 3 and 9 was detected). Therefore, in the case of Caco-2 cells, no specific effect can be attributed to punicalagin since it was hydrolyzed in the medium to yield ellagic acid, which entered into the cells and was metabolized to produce dimethyl-ellagic acid derivatives. In addition, our study suggested that the anticarcinogenic effect of dietary ellagitannins (at least in the easily hydrolyzable ones) could be mainly through their hydrolysis product ellagic acid, which induced apoptosis via the mitochondrial pathway in colon cancer Caco-2 cells but not in normal colon cells (Fig. 11.4; Larrosa et al., 2006a). To explore other putative biological activities, urolithins A (3,8-dihydroxydibenzo[b,d]pyran-6-one) and B (3-hydroxy-dibenzo[b,d]pyran-6-one) were synthesized and used to conduct gene-expression studies on human colon cancer Caco-2 cells. For the experiments, adhered subconfluent Caco-2 cells were challenged with each of the ellagic acid metabolites at 40 μM concentration (similar to the highest concentrations of these metabolites found in the intestine) for 48 h. The treatments cells were then directly lysed in the plates and total RNA was isolated. A preliminary search for potential candidate genes for which transcription levels may have been altered following exposure to ellagic acid metabolites was performed using microarray analysis. Fragmented biotin-labeled cRNAs prepared from control and treated cells were hybridized onto human Affymetrix HG-U133 Plus 2.0 GeneChip® arrays; the experiments and hybridizations were done in triplicate. Results from urolithin B-treated cells revealed significant transcriptional differences in over 700 probe sets, most of which were down-regulated. Typically, detected altered levels of gene expression were in the range 1.5–2.0-fold. Several selected genes that have been reported to be critically involved in cell signaling leading to control and regulation of cell growth and proliferation and cell cycle regulation are presented in Table 11.1. We are currently confirming those changes by RT-PCR. Treatment of Caco-2 cells with urolithin A also led to the modulation of several hundred probe sets, among which we detected the induction of several metallothioneins (Table 11.2). This result was striking, as increased intracellular metallothionein expression is found in many human and animal neoplasms. It is known that metallothioneins are intracellular proteins whose biological function is zinc and copper regulation as well as detoxification of toxic metals. Another function of these proteins is sweeping away free radicals. The induction of metallothionein synthesis is stimulated by such factors as metallic ions. We suspected that the urolithin-A test sample used in the cell experiments could contain traces of metals; indeed, copper itself was used during the chemical synthesis of the metabolite. We measured the residual levels of this metal in the samples containing urolithin-A, and found that they were about 5-fold higher than in the corresponding controls. Consequently, the observed induction of metallothioneins could not be unequivocally attributed to the metabolite and might have been produced by the metal contamination in the sample. This prompts a call for caution here for all researchers working on the evaluation of the biological activity of synthetic compounds, as traces of the chemical

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Table 11.1 Selected genes with altered expression levels in Caco-2 cells treated with urolithin B for 48 h. Accession number

Gene name

Gene symbol

Affyxa

Biological process

NM_002467

V-myc myelocytomatosis viral oncogene homolog (avian)

MYC

3.8

Overexpression is implicated in the etiology of a variety of hematopoietic tumors. Transcription regulation. Cell proliferation. Cell cycle arrest

NM_001964

Early growth response 1

EGR1

4.5

Transcription regulation. Studies suggest this is a cancer suppressor gene

NM_005252

V-fos FBJ murine osteosarcoma viral oncogene homolog

FOS

6.4

Forms the transcription factor complex AP-1. It has been implicated as a regulator of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death

NM_004985

V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

KRAS

0.68

Small GTPase mediated signal transduction. Regulation of cell cycle. Implicated in various malignancies, including colorectal carcinoma

NM_001946

Dual specificity phosphatase 6

DUSP6

5.9

MAP kinase phosphatase activity. Regulation of cell cycle. Tumor suppressor function

NM_201284

Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)

EGFR

0.4

Epidermal growth factor receptor signaling pathway. Negative regulation of cell cycle

NM_000499

Cytochrome P450, family 1, subfamily A, polypeptide 1

CYP1A1

24.0

Heme-thiolate monooxygenases. Involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics

a

, Fold up-/ down-regulation

Table 11.2 List of selected metallothioneins with altered expression levels in Caco-2 cells treated with urolithin A for 48 h. Accession number NM_005952 NM_005953 NM_005951 NM_005949 NM_176870

Gene name Metallothionein Metallothionein Metallothionein Metallothionein Metallothionein Metallothionein

1X 2A 1H 1E 1F 1M

Gene symbol

Affyxa

Biological process

MT1X MT2A MT1H MT1E MT1F MT1K

6.2 6.2 4.9 3.6 2.9 2.5

Metal ion binding Copper ion binding Metal ion binding Metal ion binding Cd and Cu ion binding Metal ion binding

a

, Fold up-/ down-regulation

catalysts (such as copper) may well be present in the final formulation, and dramatically influence the results. We have now further purified the metabolite by successive water-solubilization of the copper. Cells have been treated with the purified compound and gene expression analyses are currently in progress.

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11.6 Conclusion Ellagitannins are one of the latest groups of polyphenols to have their metabolism studied in humans. In spite of being considered as classical ‘anti-nutrients’, these compounds have attracted the attention due to their antioxidant and cancer chemopreventive activities in vitro and in vivo. In the light of the last studies carried out on their metabolism and biological activity, it may be most appropriate to investigate the antioxidant and anticarcinogenic effects of dietary ellagitannins, ellagic acid, and the colonic microflora metabolites (urolithins) in the gastrointestinal tract site, whereas the study of their systemic bioactivities (in distant organs) should be focused on the bioavailable urolithin derivatives. In this context, the study of the transciptomic profile changes in specific and appropriate human model cells will give us some essential information about the molecular mechanisms and main physiological pathways by which these metabolites may interfere with the cell machinery and modulate cell responses.

11.7 Acknowledgments This research has been funded by the Spanish CICYT (AGL2003-2195 and AGL200403989), by the ‘Fundación Séneca’ (PB/18/FS/02) and by the European Commission (FOODCT-2004-513960-FLAVO).

11.8 References Arts, I.C.W. & Hollman, P.C.H. (2005). Polyphenols and disease risk in epidemiologic studies. American Journal of Clinical Nutrition, 81, 317S–325S. Aviram, M., Kent, U.M. & Hollenbergh, P.F. (1999). Microsomal cytochrome P450 catalyzes oxidation of low density lipoprotein. Atherosclerosis, 143, 253–260. Aviram, M., Dornfeld, L., Rosenblat, M. et al. (2000). Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. American Journal of Clinical Nutrition, 71, 1062 –1076. Aviram, M. & Dornfeld, L. (2001). Pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduced systolic pressure. Atherosclerosis, 158, 195–198. Aviram, M., Dornfeld, L., Kaplan, M. et al. (2002). Pomegranate juice flavonoids inhibit low density lipoprotein oxidation and cardiovascular diseases: Studies in atherosclerotic mice and humans. Drugs under Experimental and Clinical Research, 28, 49–62. Aviram, M., Rosenblat, M., Gaitini, D. et al. (2004). Pomegranate juice consumption for three years by patients with carotid arterial stenosis reduces common carotid-media thickness, blood pressure and LDL oxidation. Clinical Nutrition, 23, 423–433. Blomhoff, R., Carlsen, M.H., Andersen, L.F. & Jacobs, D.R. (2006). Health benefits of nuts: potential role of antioxidants. British Journal of Nutrition, 96, S52–S60. Cantos, E., Espín, J.C., López-Bote, C., De la Hoz, L., Ordóñez, J.A. & Tomás-Barberán, F.A. (2003). Phenolic compounds and fatty acids from acorns (Quercus spp.), the main dietary constituent of free-range Iberian pigs. Journal of Agricultural and Food Chemistry, 51, 6248–6255. Cerdá, B., Llorach, R., Cerón, J.J., Espín, J.C. & Tomás-Barberán, F.A. (2003a). Evaluation of the bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from pomegranate juice. European Journal of Nutrition, 42, 18–28.

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Cerdá, B., Cerón, J.J., Espín, J.C. & Tomás-Barberán, F.A. (2003b). The repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is not toxic. Journal of Agricultural and Food Chemistry, 51, 3493–3501. Cerdá, B., Espín, J.C., Parra, A., Martínez, P. & Tomás-Barberán, F.A. (2004). The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolized into bioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microflora of healthy humans. European Journal of Nutrition, 43, 205 –220. Cerdá, B., Tomás-Barberán, F.A. & Espín, J.C. (2005). Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts and oak-aged wine in humans: Identification of biomarkers and individual variability. Journal of Agricultural and Food Chemistry, 53, 227–235. Cerdá, B., Soto, M.C., Albaladejo, M.D. et al. (2006). Pomegranate juice supplementation in COPD: a 5-week randomised, double blind, placebo-controlled trial. European Journal of Clinical Nutrition, 60, 245 – 253. Espín, J.C., González-Barrio, R., Cerdá, B., López-Bote, C., Rey, A.I., Tomas-Barberan, F.A. (2007). Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans’, Journal of Agricultural and Food Chemistry, 55, 10476–10485. Ferreres, F., Andrade, P., Gil, M.I. & Tomás-Barberán, F.A. (1996). Floral nectar phenolics as biochemical markers for the botanical origin of heather honey. Zeitschrift für LebensmittelUntersuchung und -Forschung, 202, 40 – 44. Fuhrman, B. & Aviram, M. (2006). Protection against cardiovascular diseases. In: Pomegranates. Ancient Roots to Moderm Medicine (eds N.P. Seeram, R.N. Schulman & D. Heber), pp. 63–89. CRC Taylor & Francis, Boca Raton, USA. Fukuda, T., Ito, H. & Yoshida, T. (2003). Antioxidative polyphenols from walnuts (Juglans regia L.). Phytochemistry, 63, 795 – 801. Gil, M.I., Tomás-Barberán, F.A., Hess-Pierce, B., Holcroft, D.M. & Kader, A.A. (2000). Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry, 48, 4581–4589. Glabasnia, A. & Hofmann, T. (2006). Sensory-directed identification of taste-active ellagitannins in American (Quercus alba L.) and European oak wood (Quercus robur L.) and quantitative analysis in bourbon whiskey and oak-matured red wines. Journal of Agricultural and Food Chemistry, 54, 3380 –3390. Hannum, S.M. (2004). Potential impact of strawberries on human health: a review of the science. Critical Reviews in Food Science and Nutrition, 44, 1–17. Hidaka, M., Okumura, M., Fujita, K. et al. (2005). Effects of pomegranate juice on human cytochrome P450 3A (CYP3A) and carbamazepine pharmacokinetics in rats. Drug Metabolism and Disposition, 33, 644 – 648. Larrosa, M., Tomás-Barberán, F.A. & Espín, J.C. (2006a). The hydrolysable tannin punicalagin releases ellagic acid which induces apoptosis in human colon adenocarcinoma Caco-2 cells by using the mitochondrial pathway. Journal of Nutritional Biochemistry, 17, 611–625. Larrosa, M., González-Sarrías, A., García-Conesa, M.T., Tomás-Barberán, F.A. & Espín, J.C. (2006b). Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic activities. Journal of Agricultural and Food Chemistry, 54, 1611–1620. Mertens-Talcott, S.U., Jilma-Stohlawetz, P., Rios, J., Hingorani, L. & Derendorf, H. (2006). Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum L.) polyphenols after ingestion of a standardized extract in healthy human volunteers. Journal of Agricultural and Food Chemistry, 54, 8956 – 8961. Seeram, N.P., Lee, R. & Heber, D. (2004). Bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate (Punica granatum L.) juice. Clinica Chimica Acta, 348, 63 – 68. Seeram, N.P., Henning, S.M., Zhang, Y., Suchard, M., Li, Z. & Heber, D. (2006). Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. The Journal of Nutrition, 136, 2481–2485.

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Shoji, T., Masumoto, S., Moriichi, N. et al. (2006). Apple procyanidin oligomers absorption in rats after oral administration: Analysis of procyanidins in plasma using the Porter method and high-performance liquid chromatography/tanden mass spectrometry. Journal of Agricultural and Food Chemistry, 54, 884 – 892. Sorokin, A.V., Duncan, B., Panetta, R. & Thompson, P.D. (2006). Rhabdomyolysis associated with pomegranate juice consumption. American Journal of Cardiology, 98, 705–706. Stoner, G.D., Sardo, C., Apseloff, G. et al. (2005). Pharmacokinetics of anthocyanins and ellagic acid in healthy volunteers fed freeze-dried black raspberries daily for 7 days. Journal of Clinical Pharmacology, 45, 1153 –1164. Villarreal-Lozoya, J.E., Lombardini, L. & Cisneros-Zevallos, L. (2007). Phytochemical constituents and antioxidant capacity of different pecan (Carya illinoinensis [Wangenh.] K. Koch) cultivars. Food Chemistry, 102, 1241–1249. Zafrilla, P., Ferreres, F. & Tomás-Barberán, F.A. (2001). Effect of processing and storage on the antioxidant ellagic acid derivatives and flavonoids of red raspberry (Rubus idaeus) jams. Journal of Agricultural and Food Chemistry, 49, 3651–3655.

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Chapter 12

Multiplicity of Phenolic Oxidation Products in Apple Juices and Ciders, from Synthetic Medium to Commercial Products Sylvain Guyot, Stéphane Bernillon, Pascal Poupard and Catherine M.G.C. Renard

12.1 Introduction Apple is one of the main fruits produced in the world (60 million tons in 2005) (ProdstatFAO-Database, 2006). Though apples are mainly used for the fresh market, a considerable volume is processed into foods and drinks (i.e. apple juices, concentrates, apple sauces and ciders). For instance, world production of apple juice concentrate for the market year 2003/2004 was estimated to be about 1.4 million tons (FAS/USDA Online Circular, 2006). The phenolic compounds in apple fruits have been extensively studied, and a lot of data concerning their characterization and their quantification in apple fruits, juices and ciders is now available (Spanos & Wrolstad, 1992; Nicolas et al., 1994). Apple phenolics (Fig. 12.1) are mainly hydroxycinnamic acids and flavonoids. 5-O-Caffeoylquinic acid (commonly called chlorogenic acid) is the major phenolic compound of the hydroxycinnamic acid class, while p-coumaroylquinic acid is usually present in low concentration. Catechins (mainly (−)-epicatechin and (+)-catechin) and procyanidins that correspond to their oligomeric and polymeric forms are the major components of the flavan-3-ol class. In apple procyanidins (−)-epicatechin is the major constituent and (+)-catechin is only found as a terminal unit. Procyanidins are located in the whole fruit and are the main class of polyphenols in apple fruits (Guyot et al., 2002). Being mainly located in the peel, flavonols and anthocyanins (mainly quercetin and cyanidin glycosides, respectively) are usually present in low level in juices and ciders. Finally, apple and apple products also contain dihydrochalcones, mainly phloridzin and phloretin xyloglucoside. During apple juice and cider making, apple phenolics undergo enzymatic oxidation. However, very little is known concerning the new phenolic molecules that are formed by oxidation when apples are processed. Depending on the technological practice, this group of phenolic derivatives may contribute to the overall quality of apple-based beverages (Lea & Timberlake, 1978). Basically, they are responsible for the color of juices and ciders Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Fig. 12.1 Main phenolic compounds in apple pulp.

and they may also be involved in sensory qualities such as bitterness and astringency. Moreover, as they still have a phenolic chemical structure, they may also contribute to the nutritional quality of ciders and juices. Thus, as do native polyphenols, oxidation products would contribute to the antioxidant capacity of these beverages. For example, dimerization reactions occurring in the course of polyphenol oxidation leads to the formation of oxidation products that significantly contribute to the overall radical scavenging activity (Hotta et al., 2002). Oxidation of apple phenolics takes place in the first stages of apple processing when fruits are crushed and pressed to produce the crude juice. This enzymatic phenomenon is well known and is generally defined by the generic term of enzymatic browning. However, this term is limiting by focusing only on the formation of colored products. Briefly, enzymatic oxidation of phenolic compounds occurs when the cellular organization between the plastidial polyphenoloxidase (PPO) and their vacuolar phenolic substrates is disrupted in the presence of dioxygen. In the case of apple processing, 5-O-caffeoylquinic acid is the preferential substrate of apple PPO (Nicolas et al., 1994). It is enzymatically converted into the corresponding o-quinone with the simultaneous reduction of dioxygen to water (Fig. 12.2a). Caffeoylquinic acid o-quinone is a very reactive species that is involved in several reaction pathways. First, as an oxidant, it is able to convert (−)-epicatechin into (−)-epicatechin o-quinone (Fig. 12.2a), which is then involved in various oxidative coupling reactions with other phenolic compounds (Fig. 12.2b). In the same manner, caffeoylquinic acid o-quinone is able to convert procyanidins into procyanidin o-quinones, which are thus also involved in oxidative coupling reactions (Lea, 1984). Of course, caffeoylquinic acid

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Fig. 12.2 Enzymatic oxidation of polyphenols in apple processing. (a) formation of primary and secondary o-quinones; (b) reactions of o-quinones.

o-quinone itself and secondary quinones formed by oxidation–reduction reactions have the capacity to react with other phenolic molecules, leading to the formation of coupling products (Fig. 12.2b). Considering all these coupling reactions, it is generally realized that enzymatic oxidation of polyphenols during fruit processing leads to the formation of polymerized oxidation products, which were called – as trivial and imprecise names – ‘brown pigments’ or ‘melanoidins’ with respect to their polymerized nature and their involvement in the color of juices and ciders. Nevertheless, the very polymeric nature of these compounds is far from having been established, and many of them are not colored. Many articles have been published concerning the characterization of phenolic oxidation products obtained in biomimetic models of wine, apple juice and cider making, using various phenolics as model compounds (i.e. caffeoylquinic acid, caftaric acid, caffeic acid, (+)-catechin, (−)-epicatechin) and different ways to carry out oxidation such as PPO extracts (Pierpoint, 1966; Cheynier et al., 1989; Richard-Forget et al., 1992; Guyot et al., 1996), chemical oxidants (Fulcrand et al., 1994) or autoxidation (Cilliers & Singleton, 1991; Oszmianski et al., 1996). Most of the compounds identified were colorless molecules corresponding only to the result of a dimerization reaction between simple phenolics such as catechins or hydroxycinnamic acids. As far as we know, structures corresponding to more highly polymerized products have never been clearly characterized. Of course, this may be owing to the limits of the analytical methods for the characterization of such polymeric molecules. However, another hypothesis is that the extensive oxidative polymerization postulated does not, in fact, take place to any significant extent. Besides, from a chemical point of view, it is unlikely that a polymerization pathway would lead to the generation of yellow compounds as a consequence of polyphenol oxidation during apple juice and cider processing. The formation of a yellow chromophore would rather be the result of successive oxidations and intramolecular rearrangements of the same phenolic molecule, leading to the formation of a quinonoid nucleus. This was clearly illustrated by the formation of the yellow dehydrodicatechin A (Weinges & Mattauch, 1971; Guyot et al., 1996) and more recently in our study dealing with the characterization of enzymatic oxidation products of phloridzin (Le Guernévé et al., 2004; Guyot et al., 2007), in which a yellow molecule resulting from sequential reactions (enzymatic oxidations, water

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addition and intramolecular nucleophilic additions) was identified as the major product of the enzymatic oxidation of phloridzin without any oxidative dimerization. In previous studies dealing with the oxidation of hydroxycinnamic acids and catechins in apple and grape must-like solutions (Cheynier et al., 1994; Richard-Forget et al., 1994), incubations were often carried out using a purified PPO or an acetone powder containing PPO activity as a catalyst of oxidation. Consequently, difficulties were encountered to clearly distinguish between the biochemical aspects of the oxidation phenomenon (i.e. enzymatic generation of o-quinone species) and the strictly chemical aspects of o-quinone reactivity (i.e. coupled oxidation–reduction reactions leading to secondary o-quinones, followed by oxidative coupling). In addition, owing to the presence of enzymatic proteins in the model solutions, tanning reactions with oxidation products may have occurred leading to partial inhibition of PPO activity (Le Bourvellec et al., 2004) and probably also to precipitation of some of the reaction products. Therefore, the use of a chemically generated o-quinone can be a real advantage for studying more specifically the reactivity of o-quinones in relation to the structure of the oxidation products. In contrast to studies in model solutions, the characterization of phenolic oxidation products that are really present in apple beverages is scarcely documented. Recently, a cider apple juice was analyzed by liquid chromatography–mass spectrometry / selected ion monitoring (LC-MS / SIM), showing evidence of several series of oxidation products that included caffeoylquinic acid in their structure (Bernillon et al., 2004). However, the juice in question had been obtained in hyper-oxygenated conditions, which do not precisely reflect the real conditions for apple juice and cider making. In the present chapter, we describe recent results illustrating our approach for characterizing the phenolic oxidation products formed in the course of apple processing. In particular, we show the benefit of LC-MS analysis for the characterization of (−)-epicatechin and 5-O-caffeoylquinic acid oxidation products from model mixtures to commercial apple juices and ciders.

12.2 Preparation and characterization of the caffeoylquinic acid o-quinone solution A methanolic solution of the o-quinone of caffeoylquinic acid was prepared by oxidation of caffeoylquinic acid (10 mM) using periodate immobilized on a resin according to a method adapted for the oxidation of caffeic acid (Fulcrand et al., 1994). The o-quinone was assayed by reversed phase high-pressure liquid chromatography (HPLC) on an aliquot of the solution after trapping the caffeoylquinic acid o-quinone in the form of its benzenesulfone derivative (Pierpoint, 1966). Thus, the caffeoylquinic acid o-quinone solution was obtained in 84% yield and no residual caffeoylquinic acid was present in the solution. Only traces of by-products were detected by chromatography. The difference was estimated to correspond primarily to a fraction of caffeoylquinic acid that would have been retained on the resin. The stability of the o-quinone solution was monitored by reversed phase HPLC: there was no significant degradation within 30 min when it was kept at 0°C. Thus, the caffeoylquinic acid o-quinone was considered pure and stable enough for its practical use in model solutions.

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12.3 Incubation of caffeoylquinic acid o-quinone in model solutions 12.3.1 Incubation of caffeoylquinic acid o-quinone with caffeoylquinic acid O-quinone (0.84 mM) and 5-O-caffeoylquinic acid (2 mM, 0.71 g/L) were incubated for 2 hours at 30°C at pH 4.0. These conditions were considered to be representative of apple juice processing. Depending on the apple variety, caffeoylquinic acid concentrations vary, usually from 70 to 200 mg/kg FW in dessert apples (van der Sluis et al., 2001) to up to 1.2 g/kg FW in some cider varieties (Sanoner et al., 1999). Considering the stoichiometry of the PPO enzymatic reaction (i.e. 1 mole of caffeoylquinic acid o-quinone is produced when 0.5 mole of O2 is consumed), the o-quinone concentration of 0.84 mM can be assimilated to 0.42 mmole/l of O2 consumed. This was consistent with the estimation of the quantity of oxygen that could be consumed by a cider apple must that we had obtained in a previous study (Le Bourvellec et al., 2004). After incubation, the mixture was analyzed by reversed-phase HPLC coupled with a UV-visible diode array detector (UV6000LP, Thermofinnigan) and an ion trap mass spectrometer (LCQ Deca ion trap, Thermofinnigan, San Jose, CA, USA) equipped with an electrospray ionization source. All MS experiments were carried out in the negative mode. Samples were separated on a Zorbax Eclipse XDB-C18 column (2.1 mm × 150 mm, 3.5 μm, Agilent Technologies) with a gradient of diluted formic acid and acetonitrile. The flow rate was 0.2 mL/min and the injection volume was 5 µL. Data were processed by Xcalibur® version 1.2 software. The model solutions and the commercial juice and cider were first analyzed in the Full-MS mode in the 50–2000 mass range and in the 240–600 nm UV-visible range. Then samples were analyzed in the LC-MS/MS mode by selecting the m/z values corresponding to the main pseudomolecular ions detected by the LC-Full-MS scans and also considering hypotheses for the formation of the oxidation products on the basis of literature data. The total ion current (TIC) chromatogram allowed the detection of a series of 16 resolved peaks numbered 1a–16a (Fig. 12.3) and listed in Table 12.1. Peak 5a was easily identified as residual caffeoylquinic acid according to its retention time, UV, mass spectrum and comparison with the commercial standard. All other peaks corresponded to oxidation products. The UV 280 nm chromatogram (Fig. 12.3) presented similarities with previously published chromatograms of caffeoylquinic acid oxidation mixtures obtained in the presence of polyphenoloxidase. Most of the products formed were eluted after caffeoylquinic acid, suggesting that they correspond to less polar compounds (Oszmianski & Lee, 1990). However, as previously observed by Richard-Forget et al. (1994), some products (1a–4a) eluted before caffeoylquinic acid. The UV spectra of the oxidation products were also in accordance with previous data. Thus, most of the oxidation products showed a maximum absorbance at or near 320 nm (Table 12.1) without significant absorbency in the 400 nm region, indicating that they were not colored products. Interestingly, peaks 2a, 6a, 7a and 8a absorbed mainly near 280 nm, suggesting that they corresponded to products in which

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Fig. 12.3 TIC and UV 280 nm reversed-phase HPLC chromatograms of the 5-O-caffeoylquinic acid o-quinone / 5-O-caffeoylquinic acid oxidation mixture.

Table 12.1 LC-UV-MS data of caffeoylquinic acid oxidation products. No.

RT (min)

λmax (nm)

[M–H]−

Main fragments on MS/MS spectrum

1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 15a 16a

9.6 10.4 11.7 12.6 13.3 14.1 15.0 15.9 16.8 18.2 18.8 21.1 21.5 23.2 23.7 24.5

280, 286 285, 290, 325 283 283 283 284, 327 327 289, 289, 293, 289, 293,

703 723 705 705 353 737 737 723 705 705 705 705 705 705 705 705

511 705 513 513 191 705 705 705 513 513 513 513 513 513 513 513

330 330 322

311

317 317 318 317 318

(100), 467 (16), 337 (38), 293 (20) (31), 585 (100), 513 (51) (100), 339 (6), 321 (5) (100), 339 (8), 321 (13) (100) (75), 513 (100) (100), 513 (94) (15), 585 (46), 549 (100), 531 (48), 339 (56) (89), 339 (11), 321 (100) (100) (100) (100) (100) (100) (100) (100)

Values in parentheses correspond to the intensities of the ions relative to the most intense (%).

the side-chain conjugation in the molecular structure of caffeoylquinic acid was lost, as observed earlier for caffeic acid oxidation products (Cheynier & Moutounet, 1992). According to the MS response, a major series of ten products (i.e. peaks 3a, 4a, 9a–16a on Fig. 12.3) were characterized by a pseudomolecular ion at m/z 705, and were thus assigned to caffeoylquinic acid dehydrodimers (MW, 706 Da). For most of them, the major MS/MS fragment corresponded to the loss of one quinic acid moiety (i.e. m/z 513; Table 12.1). Only peak 9a showed a clearly different MS/MS spectrum, with m/z 321 as the most intense fragment ion, corresponding to the loss of both quinic acid moieties. Considering the structural similarities between caffeoylquinic acid and caffeic acid, these products are probably analogs of caffeicins previously identified as the main oxidation

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products from caffeic acid autoxidation in a model solution (Cilliers & Singleton, 1991). In their study, these authors identified several caffeic acid dimers belonging to the class of neolignans characterized by a dihydrobenzofuran, a dihydrobenzodioxan or a dihydronaphthalene nucleus (Cilliers & Singleton, 1991). NMR results concerning our products (data not shown) indicated that products 10a and 11a were of the dihydrobenzofuran type whereas products 12a and 13a corresponded to dihydrobenzodioxan types. The oxidation product that eluted the earliest (1a) presented a pseudomolecular ion at m/z 703 (Table 12.1). This corresponded to a dehydrodimer that would have suffered an additional oxidation followed by an intramolecular addition, thus leading to a product having a molecular weight of 704 Da. This assignment was confirmed by the observation of the main fragment ion at m/z 511 on the MS/MS spectrum, corresponding to the loss of one quinic acid moiety (i.e. 192 amu). Basically, dehydrodimers still contain a free catechol moiety in their structure. For this reason, some of them may be reoxidized by an oxidation–reduction reaction leading to the formation of the intermediate o-quinones, which may further react intramolecularly leading to the formation of a stable oxidation product such as 1a. The short retention time of this product may be explained by a high rigidity of the molecular skeleton as a consequence of the intramolecular addition, leading to a reduction in the hydrophobic interactions with the chromatographic stationary phase. The third category corresponded to peaks 2a and 8a, with pseudomolecular ions at m/z 723 and UV absorbance only in the 280 nm region (Fig. 12.3 and Table 12.1). Actually, the LC-MS/MS chromatogram of m/z 723 allowed us to detect several additional peaks (data not shown) corresponding to this category of product. These were not visible on the TIC chromatogram probably because they co-eluted with other products giving more intense MS responses. These ions would correspond to dehydrodimers that have incorporated a molecule of water into their structure. Analogs of these products have been described by Fulcrand et al. in their study dealing with the oxidation of caffeic acid (Fulcrand et al., 1994). These molecules are characterized by a tetrahydrofuran nucleus having four asymmetric carbons, which may explain the multiplicity of the detected products. The lack of absorbance in the 320 nm region suggests that the side-chain conjugations of the caffeic acid moieties were completely lost in the course of the oxidative coupling, which is in agreement with the formation of the tetrahydrofuran nucleus. Finally, two products (6a and 7a) showing pseudomolecular ions at m/z 737 were detected on the TIC chromatogram (Fig. 12.3, Table 12.1). Their molecular mass (MW, 738 Da) would correspond to caffeoylquinic acid dehydrodimers (MW, 706 Da) that have incorporated two oxygen atoms (32 Da) into their structure in the course of the oxidation reaction. This could be the result of two successive sequences of oxidation, water addition and intramolecular rearrangement, such as has already been observed in the formation of phloridzin oxidation products (Le Guernévé et al., 2004). As a conclusion, the major products resulting from caffeoylquinic acid oxidation were dimeric forms at different oxidation levels, some of them having incorporated additional oxygen atoms as a result of the addition of water. An LC-MS/MS analysis was carried out by screening the m/z 1057 ions corresponding to the possible trimeric products that might have formed in the model solution (data not shown). No significant signal was observed, suggesting that extensive polymerization is not a favored route in the course of caffeoylquinic acid oxidation in model solutions mimicking apple processing.

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12.3.2 Incubation of caffeoylquinic acid o-quinone with (−)-epicatechin The major monomeric catechin found in apple and apple products is (−)-epicatechin (Nicolas et al., 1994). In dessert apples, its concentration in the flesh is usually in the range 30– 150 mg/kg FW (Tsao et al., 2003), and it may reach 1.4 g/kg FW in some French cider cultivars (Sanoner et al., 1999). Thus, the concentration of (−)-epicatechin that was used in the model solution (i.e. 2 mM, 0.58 g/l) was consistent with those found in apples. Although it is a substrate for polyphenoloxidase, (−)-epicatechin is mostly involved in the formation of oxidation products according to two mechanisms: first, it can be converted into its corresponding o-quinone by a coupled oxidation–reduction reaction with the caffeoylquinic acid o-quinone (Cheynier et al., 1989); second, it is a good target for o-quinone addition owing to the nucleophilic character of the phloroglucinol A ring (Fig. 12.1). Figure 12.4 presents the TIC and UV 280 nm chromatograms, showing the main oxidation products formed when (−)-epicatechin (2 mM, 0.58 g/l) is incubated for 2 hours at 30°C in the presence of caffeoylquinic acid o-quinone (0.84 mM) at pH 4.0. The conditions for HPLC-UV-MS and MS/MS were the same as described before for the oxidation of caffeoylquinic acid (section 12.3.1). Eleven peaks were clearly observed on the TIC chromatogram. The two main peaks (3b and 5b) were unambiguously identified as caffeoylquinic acid (MW, 354 Da) and residual (−)-epicatechin (MW, 290 Da), respectively, on the basis of their retention time, UV and MS spectra (Table 12.2). Caffeoylquinic acid was formed in the model solution by the reduction of its o-quinone in a coupled oxidation–reduction reaction with (−)-epicatechin. As a consequence, oxidation products corresponding to caffeoylquinic acid dimers (for instance ions at m/z 705 and 723 discussed above) were also formed and were detected as weak signals in the oxidation mixture containing (−)-epicatechin (data not shown).

Fig. 12.4 TIC and UV 280 nm reversed-phase HPLC chromatograms of the 5-O-caffeoylquinic acid o-quinone / (−)-epicatechin oxidation mixture.

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Table 12.2 LC-UV-MS/MS data of the main EC oxidation products in model solution. No.

RT (min)

λmax (nm)

[M–H]−

Main fragments on MS/MS spectrum

1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b

7.3 10.1 13.3 16.2 16.8 18.8 20.0 22.8 24.8 25.4 29.2

278 278 325 278 278 276, 386 274 274 279 278, 391 277, 385

863 577 353 577 289 863 577 577 577 575 575

845 559 191 559 245 845 559 559 559 531 531

(100), 819 (54), 801 (31) (100), 533 (40), 439 (30), 425 (9), 393 (24) (100) (100), 533 (40), 439 (30), 425 (54), 393 (84) (100), 205 (30), 179 (11) (12), 819 (23), 725 (100), 449 (22) (11), 533 (62), 439 (29), 425 (100), 289 (27) (64), 533 (100), 439 (89), 425 (42), 289 (3) (3), 533 (5), 439 (12), 425 (14), 289 (100) (19), 449 (100), 437 (28), 287 (12) (9), 449 (100), 437 (24), 287 (33)

Values in parentheses correspond to the relative intensities of the ions relative to the most intense (%).

The main series of oxidation products included five peaks (2b, 4b, 7b, 8b, 9b) showing pseudomolecular ions at m/z 577 and λmax in the 274–279 nm range (Table 12.2). These colorless products corresponded to dimeric catechins (namely dehydrodicatechins B) resulting from the oxidative coupling of two epicatechin molecules. Similar dimers resulting from enzymatic oxidation (Weinges & Huthwelker, 1970; Guyot et al., 1996), chemical oxidation (Young et al., 1987) or autoxidation (Sun & Miller, 2003) of (+)-catechin have been previously characterized. Noticeably, these compounds are isomers of native procyanidin dimers but differ by the nature and position of the interflavanic linkage. They show either biaryl or biaryl-ether linkages between the B-ring of one catechin unit and the A-ring of the other. The main fragments observed on the MS/MS spectra of these five products are listed in Table 12.2. Ions at m/z 559, 533, 439 and 425 were significantly present in all MS/MS spectra with different relative intensities. Ions at m/z 559 and 533 correspond to the loss of H2O and CO2, respectively. Ions at m/z 439 and 425 were typical of the retro-Diels–Alder fragmentation from the C ring of a catechin unit as described in the literature (Guyot et al., 1996; Sun & Miller, 2003). The fragment at m/z 393 was considered as specific for dehydrodicatechin, with a biaryl structure (Sun & Miller, 2003). This fragment ion was observed only for products 2b, 4b and for two minor products that were detected only on the MS/MS chromatogram of m/z 577 ions (data not shown). The other products of this series (7b, 8b and 9b) showed significant fragment ions at m/z 289, which was indicative of the presence of a biaryl ether moiety (Guyot et al., 1996). The second class of (−)-epicatechin oxidation products (peaks 10b and 11b) had pseudomolecular ions at m/z 575 and λmax at 391 and 385 nm, indicating that they were yellow products probably corresponding to the (−)-epicatechin isomers of dehydrodicatechins A, previously characterized in (+)-catechin oxidation mixtures (Weinges & Mattauch, 1971; Guyot et al., 1996). They resulted from the oxidation of a biarylic dehydrodicatechin B followed by an intramolecular addition. The main fragment ions at m/z 449 on the MS/MS spectra may be explained by the loss of a phloroglucinol moiety. Interestingly, products corresponding to dehydrotricatechins were also detected at m/z 865. However they appeared as numerous products giving weak and unresolved signals even on the MS/MS chromatogram (data not shown). In contrast, two products corresponding

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to peaks 1b and 6b on the TIC chromatogram were clearly detected with a pseudomolecular ion at m/z 863. They correspond to trimeric catechin structures resulting from oxidative coupling reactions including two intermolecular coupling reactions between 3 epicatechin molecules and one intramolecular addition, as was the case for dehydrodicatechin A. Therefore, the name of dehydrotricatechin A could be assigned to this family of oxidation products. Product 6b is yellow (λmax 386 nm) and showed a main MS/MS fragment at m/z 725, which corresponded to the loss of the A ring part (138 amu) from a catechin moiety according to a retro-Diels–Alder fragmentation. Moreover, the presence of a significant fragment ion at m/z 449 suggested some structural similarities with dehydrodicatechins. Product 1b was colorless (λmax 278) and highly polar. The main MS/MS fragments (m/z 845, 819, and 801) were attributed to the loss of H2O and CO2. Finally, although they did not appear as intense signals on the TIC chromatogram (Fig. 12.4), an additional series of oxidation products were clearly detected by screening the pseudomolecular ions at m/z 641 by MS/MS analysis. These pseudomolecular ions corresponded to heterodimers resulting from the oxidative coupling between a caffeoylquinic acid molecule and an epicatechin molecule. Figure 12.5 presents the extracted m/z 449-ion chromatogram from the LC-MS/MS analysis of the m/z 641 ions. Nine heterodimers, numbered 1c to 9c, with m/z 449 as main fragment ions were unambiguously detected. Such extracted chromatograms allowed more specificity by selecting only the ions corresponding to the loss of the quinic acid moiety (192 amu), which was a favored fragmentation as the quinic acid moiety was not involved in the oxidative coupling. To conclude, LC-MS analysis of the model solution resulting from incubation of caffeoylquinic acid o-quinone with (−)-epicatechin allowed the detection and characterization of a great diversity of oxidative coupling derivatives, some of them being yellow products. Several classes were distinguished: peaks giving intense signals on the TIC chromatogram corresponded mainly to dehydrodicatechins. However, two dehydrotricatechins and a series of caffeoylquinic acid/epicatechin dehydroheterodimers were also clearly detected.

Fig. 12.5 Extracted m/z 449-ion chromatogram of the m/z 641 LC-MS/MS analysis of the 5-O-caffeoylquinic acid o-quinone / (−)-epicatechin oxidation mixture.

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12.4 LC-MS analysis of oxidation products in commercial apple beverages A commercial apple juice and a commercial cider were purchased from the local market. After degassing, an aliquot of each beverage (1 ml) was diluted with 2 ml methanol/water (50/50) and filtered. Samples were then ready for HPLC-UV MS analysis under the same conditions as described for the analysis of model solutions (see section 12.3.1). Obviously, the TIC and UV 280 nm chromatograms (data not shown) showed major peaks that corresponded to apple fruit native phenolic compounds (i.e. 5-O-caffeoylquinic acid, p-coumaroylquinic acid, (−)-epicatechin, procyanidin B2, phloridzin and phloretin xyloglucoside), which have been extensively described and characterized in apple juices and ciders (Whiting & Coggins, 1975; Lea & Timberlake, 1978; Wilson, 1981; Spanos et al., 1990; van der Sluis et al., 2002; Guyot et al., 2003; Alonso-Salces et al., 2004; Kahle et al., 2005; Suarez & Palacios, 2005). These native phenolic compounds will not be further discussed as the present article concerns phenolic products that are formed in the course of the enzymatic oxidation occurring during apple juice and cider manufacture. The oxidation products that were first characterized in model solutions were searched for in the marketed products by using the potentialities of LC-MS, in particular its high selectivity and sensitivity in the MS/MS mode. Following LC-MS/MS analysis, the data were processed by extracting chromatograms corresponding to specific ions from the fullMS/MS data. Thus, Fig. 12.6 presents the chromatograms obtained for the m/z 513 extracted ions from LC-MS/MS analysis of the m/z 705 ions. These ions correspond to caffeoylquinic acid dehydrodimers resulting from oxidative coupling, as discussed in section 12.3.1. The ions at m/z 513 (i.e. loss of one quinic acid moiety of 192 amu) gave additional evidence that the detected products were caffeoylquinic acid dimers. Ten products were clearly detected both in the juice and in the cider. According to their retention time and MS/MS spectra (data not shown), they were unambiguously identified as products 3a, 4a, and 9a–16a previously observed in the model solution with caffeoylquinic acid o-quinone and caffeoylquinic acid. Compared with the model solution, the proportions of the various products were globally represented in the juice and in the cider, with product 10a and 11a as the most intense peaks. However, products 3a and 4a appeared as very intense ions in the juice and in the cider, although they appeared as minor products in the model solution. Products corresponding to hydrated oxidative dimers of caffeoylquinic acid were also detected in the beverages (data not shown). Both in the juice and in the cider, a series of intense signals was observed on the m/z 513-ion chromatogram extracted from the LCMS/MS analysis of the m/z 723 ions. In this case, the m/z 513 fragment ions corresponded to the loss of a water molecule (18 amu) and a quinic acid moiety (192 amu). Comparison of the retention times and the MS/MS spectra with those of the model solution allowed us to assign most of these signals. A series of nine oxidative coupling products corresponding to caffeoylquinic acid and (−)-epicatechin dehydrodimers were also clearly detected in the commercial apple juice and in the cider by LC-MS/MS screening of their pseudomolecular ions at m/z 641 (Fig. 12.7, b and c). As shown in Fig. 12.7, their retention times were similar to those of the corresponding dimers obtained in the model solution. The chromatographic profiles

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Fig. 12.6 Extracted m/z 513-ion chromatograms of LC-MS/MS analyses of m/z 705 ions. (a) model oxidation mixture of 5-O-caffeoylquinic acid o-quinone and 5-O-caffeoylquinic acid; (b) commercial apple juice; (c) commercial French cider. Numbers on the left correspond to the full-scale intensities.

Fig. 12.7 Extracted m/z 449-ion chromatograms of LC-MS/MS analyses of m/z 641 ions. (a) mixture of 5-O-caffeoylquinic acid o-quinone and (−)-epicatechin; (b) commercial apple juice; (c) commercial French cider. Numbers on the left are the full-scale intensities.

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of these products were very similar in the juice and in the cider, indicating that these oxidation products were not largely modified during the fermentation period of cider making. Of course, oxidation products in apple juices and ciders are not limited to the two series of coupling products corresponding to caffeoylquinic acid dehydrodimers and to epicatechin/caffeoylquinic acid dehydrodimers. Other native phenolics such as (+)-catechin, oligomeric and polymeric procyanidins and dihydrochalcones are probably involved in the formation of new phenolic structures as the result of the oxidation process during apple juice and cider making.

12.5 Conclusions LC-MS and particularly LC-MS/MS, as it allies high sensitivity, highly selective detection and a potential for structural information by interpretation of the main fragment ions in the MS/MS spectra, was an indispensable tool for this work. It allowed us to develop an approach that combined both the convenience of model solutions and the relevance of investigating the actual food products. Oxidation products were first tentatively identified from the LC-MS/MS injection of complex mixtures, then produced in large amounts in the model solutions. They could then conveniently be purified for complete structural elucidation after verification of their presence in the food products. We have confirmed the presence of multiple phenolic oxidation products – of which we have studied in detail only a few families – in actual beverages. Remarkably, the families of oxidation products that we detected and identified frequently result from intramolecular oxidation and rearrangements rather than intermolecular addition. Our results thus challenge the commonly held views on the polymeric nature of oxidation products of polyphenols. Indeed, an extended polymerization pathway is not needed to explain the complexity of the pool of new products that are formed in the course of enzymatic oxidation. Successions of oxidation, intramolecular rearrangements and the possibility of water addition, combined with the numerous asymmetric carbon atoms, suffice to explain the generation of numerous distinct molecules.

12.6 Acknowledgments Grants to PP and SB were respectively from the European Community, as part of the FLAVO project, and from INRA, Regions Bretagne and Pays de Loire and the CTPC as part of the ‘Cidre et polyphénols’ program.

12.7 References Alonso-Salces, R.M., Guyot, S., Herrero, C. et al. (2004). Chemometric characterisation of Basque and French ciders according to their polyphenolic profiles. Analytical Bioanalytical Chemistry, 379, 464 – 475.

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Bernillon, S., Guyot, S. & Renard, C. (2004). Detection of phenolic oxidation products in cider apple juice by HPLC electrospray ionisation ion trap mass spectrometry. Rapid Communications in Mass Spectrometry, 18, 939 – 943. Cheynier, V., Basire, N. & Rigaud, J. (1989). Mechanism of trans-caffeoyltartaric acid and catechin oxidation in model solutions containing grape polyphenoloxidase. Journal of Agricultural and Food Chemistry, 37, 1069 –1071. Cheynier, V., Fulcrand, H., Guyot, S., Oszmianski, J. & Moutounet, M. (1994). Reactions of enzymically generated quinones in relation to browning in grape musts and wines. ACS Symposium Series, 594, 130 –143. Cheynier, V. & Moutounet, M. (1992). Oxidative reactions of caffeic acid in model systems containing polyphenol oxidase. Journal of Agricultural and Food Chemistry, 40, 2038–2044. Cilliers, J.J. & Singleton, V.L. (1991). Characterization of the products of nonenzymic autoxidative phenolic reactions in a caffeic acid model system. Journal of Agricultural and Food Chemistry, 39, 1298 –1303. FAS/USDA Online Circular. (2006). World apple juice situation. http://ffas.usda.gov/htp/Hort_ Circular/2006/0506/Apple%20Juice%20Feature%20May%202006.pdf Fulcrand, H., Cheminat, A., Brouillard, R. & Cheynier, V. (1994). Characterization of compounds obtained by chemical oxidation of caffeic acid in acidic conditions. Phytochemistry, 35, 499–505. Guyot, S., Le Bourvellec, C., Marnet, N. & Drilleau, J.-F. (2002). Procyanidins are the most abundant polyphenols in dessert apples at maturity. Lebensmittel-Wissenschaft und Technologie, 35, 289 –291. Guyot, S., Marnet, N., Sanoner, P. & Drilleau, J.-F. (2003). Variability of the polyphenolic composition of cider apple (Malus domestica) fruits and juices. Journal of Agricultural and Food Chemistry, 51, 6240 –6247. Guyot, S., Serrand, S., Le Quéré, J.M., Sanoner, P. & Renard, C.M.G.C. (2007). Enzymatic synthesis and physicochemical characterisation of phloridzin oxidation products (POP), a new water-soluble yellow dye deriving from apple. Innovative Food Science and Emerging Technologies, 8, 443 – 450. Guyot, S., Vercauteren, J. & Cheynier, V. (1996). Structural determination of colorless and yellow dimers resulting from (+)-catechin coupling catalysed by grape polyphenoloxidase. Phytochemistry, 42, 1279 –1288. Hotta, H., Nagano, S., Ueda, M., Tsujino, Y., Koyama, J. & Osakai, T. (2002). Higher radical scavenging activities of polyphenolic antioxidants can be ascribed to chemical reactions following their oxidation. Biochimica Biophysica Acta, 1572, 123–132. Kahle, K., Kraus, M. & Richling, E. (2005). Polyphenol profiles of apple juices. Molecular Nutrition and Food Research, 49, 797 – 806. Le Bourvellec, C., Le Quéré, J.-M., Sanoner, P., Drilleau, J.-F. & Guyot, S. (2004). Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products. Journal of Agricultural and Food Chemistry, 52, 122–130. Le Guernévé, C., Sanoner, P., Drilleau, J.-F. & Guyot, S. (2004). New compounds obtained by enzymatic oxidation of phloridzin. Tetrahedron Letters, 45, 6673–6677. Lea, A.G.H. (1984). Farb- und gerbstoffe in englischen mostapfeln. Flüssiges Obst, 8, 356–361. Lea, A.G.H. & Timberlake, C.F. (1978). The phenolics of ciders: Effect of processing conditions. Journal of the Science of Food and Agriculture, 29, 484–492. Nicolas, J.J., Richard-Forget, F.C., Goupy, P.M., Amiot, M.J. & Aubert, S.Y. (1994). Enzymatic browning reactions in apple and apple products. Critical Reviews in Food Science and Nutrition, 34, 109 –157. Oszmianski, J., Cheynier, V. & Moutounet, M. (1996). Iron-catalyzed oxidation of (+)-catechin in model systems. Journal of Agricultural and Food Chemistry, 44, 1712–1715. Oszmianski, J. & Lee, C.Y. (1990). Enzymatic oxidative reaction of catechin and chlorogenic acid in a model system. Journal of Agricultural and Food Chemistry, 38, 1202–1204. Pierpoint, W.S. (1966). The enzymatic oxidation of chlorogenic acid and some reactions of the o-quinone produced. Biochemical Journal, 98, 567–580.

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Prodstat-FAO-Database. (2006). Online Database of Food and Agriculture Organization of the United nations. http://faostat.fao.org/site/567/default.aspx Richard-Forget, F., Amiot, M.J., Goupy, P. & Nicolas, J. (1994). Evolution of chlorogenic acid o-quinones in model solutions. ACS symposium Series, 594, 144–158. Richard-Forget, F., Rouetmayer, M.A., Goupy, P., Philippon, J. & Nicolas, J.J. (1992). Oxidation of chlorogenic acid, catechins and 4-methylcatechol in model solutions by apple polyphenol oxidase. Journal of Agricultural and Food Chemistry, 40, 2114–2122. Sanoner, P., Guyot, S., Marnet, N., Mollé, D. & Drilleau, J.F. (1999). Polyphenols profiles of French cider apple varieties (Malus domestica sp.). Journal of Agricultural and Food Chemistry, 47, 4847– 4853. Spanos, G.A. & Wrolstad, R.E. (1992). Phenolics of apple, pear and white grape juices and their changes with processing and storage – A review. Journal of Agricultural and Food Chemistry, 40, 1478 –1487. Spanos, G.A., Wrolstad, R.E. & Heatherbell, D.A. (1990). Influence of processing and storage on the phenolic composition of apple juice. Journal of Agricultural and Food Chemistry, 38, 1572 –1579. Suarez, B. & Palacios, V. (2005). Liquid chromatographic method for quantifying polyphenols in ciders by direct injection. Journal of Chromatography A, 1066, 105–110. Sun, W.X. & Miller, J.M. (2003). Tandem mass spectrometry of the B-type procyanidins in wine and B-type dehydrodicatechins in an autoxidation mixture of (+)-catechin and (−)-epicatechin. Journal of Mass Spectrometry, 38, 438 – 446. Tsao, R., Yang, R., Christopher, J., Zhu, Y. & Zhu, H.H. (2003). Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC). Journal of Agricultural and Food Chemistry, 51, 6347– 6353. van der Sluis, A.A., Dekker, M., De Jager, A. & Jongen, W.M.F. (2001). Activity and concentration of polyphenolic antioxidants in apple: effect of cultivar, harvest year and storage conditions. Journal of Agricultural and Food Chemistry, 49, 3606–3613. van der Sluis, A.A., Dekker, M., Skrede, G. & Jongen, W.M.F. (2002). Activity and concentration of polyphenolic antioxidants in apple juice. 1. Effect of existing production methods. Journal of Agricultural and Food Chemistry, 50, 7211–7219. Weinges, K. & Huthwelker, D. (1970). Oxidative Kupplung von Phenolen. III, 1. Isolierung und Konstitutionsbeweis eines 8.6′-verknüpften Dehydro-dicatechins (B4). Justus Liebigs Annalen der Chemie, 731, 161–170. Weinges, K. & Mattauch, H. (1971). Der chemische Konstitutionsbeweis des Dehydro-dicatechins A. Chemiker Zeitung, 95, 155 –156. Whiting, J.C. & Coggins, R.A. (1975). Estimation of the monomeric phenolics of ciders. Journal of the Science of Food and Agriculture, 26, 1833–1838. Wilson, E.L. (1981). High-pressure liquid chromatography of apple juice phenolic compounds. Journal of the Science of Food and Agriculture, 32, 257–264. Young, D.A., Young, E., Roux, D.G., Brandt, V. & Ferreira, D. (1987). Synthesis of condensed tannins. Part 19. Phenol oxidative coupling of (+)-catechin and (+)-mesquitol. Conformation of bis-(+)-catechins. Journal of the Chemical Society, Perkin Transactions, 1, 2345–2351.

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Chapter 13

Phytoestrogens in Drug Discovery for Controlling Steroid Biosynthesis Sampo Karkola, Annamaria Lilienkampf and Kristiina Wähälä

13.1 Introduction It has been estimated that 184,450 new cases of breast cancer will be diagnosed in 2008 in the USA (American Cancer Society, 2008). 26% of all diagnosed cancers in women are breast cancer, being the most frequently diagnosed cancer in women. Approximately 60% of breast cancer incidents are hormone dependent (estrogen-receptor positive). In men, prostate cancer is the most frequently diagnosed cancer, and an estimated 186,320 new cases will occur in the USA during 2008, contributing to 25% of all new cancers. The lower incidence of these cancers in East Asian countries has been connected to a high dietary intake of phytoestrogens. One approach to treat these hormone-dependent diseases is to block the production of the active sex steroids, estradiol and testosterone. These endogenous steroid hormones are involved in numerous physiological processes, including cell proliferation in tumor development. For example, estradiol binds to nuclear estrogen receptors in the cell and causes a cascade leading to cell division. Sex steroids are produced from cholesterol in a complex biosynthetic pathway. The final steps of sex hormone biosynthesis are catalyzed by the enzymes aromatase and 17β-hydroxysteroid dehydrogenase (17β-HSD) (Fig. 13.1). Aromatase catalyzes the conversion of the male androgens androstenedione and testosterone into female estrogens estrone (E1) and estradiol (E2), respectively. 17β-HSDs catalyze the conversion of E1 into E2 and androstenedione into testosterone, thus producing the active forms of the hormones from their precursors. The inhibition of these enzymes is an attractive target in developing drugs for hormone-dependent cancers. By inhibiting aromatase or 17β-HSD, the local production of estrogen or androgen is decreased, which may lead to arrested or reduced tumor growth. This chapter focuses on the inhibition of the key enzymes aromatase and 17β-HSDs, although the E2 and testosterone biosynthetic pathway includes several other enzymes.

13.2 Aromatase Human aromatase (CYP19A1, CYP450arom, EC 1.14.14.1, estrogen synthetase) is a cytochrome P450 enzyme bound to the endoplasmic reticulum in the cell (for a recent review, Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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Fig. 13.1 Reactions catalyzed by aromatase and isoenzymes of 17β-hydroxysteroid dehydrogenase (17β-HSD) in the final steps of sex hormone biosynthesis. Several 17β-HSDs also display other steroid dehydrogenase activities.

see Bulun et al., 2006). As already mentioned, it catalyzes the reactions in which the androgens testosterone and androstenedione are converted to estrogens estradiol and estrone, respectively (Fig. 13.1). The reaction mechanism includes three consecutive hydroxylation steps leading to the elimination of the C-19 carbon as formic acid and aromatization of the A ring of the steroid. The reaction requires three molar equivalents of oxygen and three moles of NADPH per mole of substrate. The catalytic complex consists of the aromatase enzyme with a catalytically crucial iron-binding porphyrin ring as a prosthetic group in the active site, and an NADPH-cytochrome P450 reductase, which provides the reaction with NADPH. Owing to the membrane bound character of the complex, the 3dimensional structure of aromatase has yet to be elucidated, but the structure is a target for crystallization and homology modeling studies. Aromatase is maily expressed in the ovaries of pre-menopausal women, in the placenta of pregnant women and additionally in the peripheral adipose tissue, breast tissue and brain (Simpson et al., 1994). It is overexpressed in or near breast cancer tissue and is therefore responsible for local estrogen production and proliferation of breast cancer tissue (Miller et al., 1997). The development of aromatase inhibitors has been an attractive target for pharmaceutical companies as well as academia. Both steroidal and non-steroidal inhibitors have shown high potency against aromatase, and a few of the developed molecules have reached the market for adjuvant or metastatic breast cancer treatment. These include aminoglutethimide, anastrozole (Arimidex® by AstraZeneca), letrozole (Femara® by Novartis),

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Fig. 13.2 Structures of the carbon skeletons of the phytoestrogens used in the inhibition studies.

exemestane (Aromasin® by Pfizer and Exemestane® by Roxane), and fadrozole (Novartis). Certain phytoestrogens have been found to possess anti-aromatase activity as well.

13.2.1 Natural phytoestrogens as aromatase inhibitors A wide range of natural phytoestrogens – the structures and numbering of the most important of which are presented in Fig. 13.2 – have been tested for aromatase inhibition activity. Kellis & Vickery (1984) were the first to study the effect of phytoestrogens in inhibiting aromatase activity. Since then, almost 100 natural phytoestrogens have been evaluated in human placental microsome-based, cell-based, or purified enzyme-based assays for their potency in inhibiting aromatase function. Most of the tested compounds are flavonoids, but isoflavonoids, lignans, chalcones, deoxybenzoins, coumestans and their derivatives have also been tested. Approximately a quarter of these compounds are racemic mixtures of two enantiomers, providing only a guideline to the potency of the enantio-pure, active compound. The biological data of selected natural phytoestrogens are listed in Table 13.1. 13.2.1.1 Anti-aromatase activity of phytoestrogens in human placental microsomebased assays Using human placental microsome (HPM)-based assay, flavonoids inhibit aromatase activity with IC50 values ranging from 0.07 μM to over 200 μM. α-Naphthoflavone was found to be the most active flavonoid tested, with IC50 values of 0.07 μM (Kellis & Vickery, 1984) and 0.18 μM (Stresser et al., 2000). Other sub-micromolar flavone inhibitors of aromatase are 7-hydroxyflavone, with IC50 values of 0.21 μM and 0.5 μM (Ibrahim & Abul-Hajj, 1990; Le Bail et al., 1998) and chrysin, with IC50 values of 0.50 μM (Kellis & Vickery, 1984) and 0.7 μM (Le Bail et al., 1998; Stresser et al., 2000). Phytoestrogen inhibitors with

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Table 13.1 The anti-aromatase activity of selected natural phytoestrogens. Compound Flavones 3′,4′-dihydroxyflavone 4′-hydroxyflavone 4′,5-dihydroxyflavone 4′,6-dihydroxyflavone 4′,7-dihydroxyflavone 5-hydroxyflavone 6-hydroxyflavone 7-hydroxyflavone

7-methoxyflavone 7,8-dihydroxyflavone

IC50, μM

Ki, μM

90 180 120 90 2 100 5.5 80 0.21 0.35 0.5 4 128 3.2 8 8.65 55 10

α-naphthoflavone

0.07 0.18 0.2 2.2

α-naphthoisoflavone apigenin

chrysin

100 0.18 1.2 2.9 3.33 20 0.5 0.5 0.7 0.7 4.33 7 2.6 1 2.4

flavone

8 10 48 375 > 100

galangin

12

22 95 isolicoflavonol kaempferide kaempferol luteolin

quercetin

0.1 80 11 61 1.2 1.7 25 12 > 100

27.2

4.8

Assay

Reference

HPM HPM HPM HPM HPM HPM JEG-3 HPM HPM JEG-3 HPM HAC HPM HPM HPM HPM

Ibrahim & Abul-Hajj, 1990 Ibrahim & Abul-Hajj, 1990 Ibrahim & Abul-Hajj, 1990 Ibrahim & Abul-Hajj, 1990 Ibrahim & Abul-Hajj, 1990 Ibrahim & Abul-Hajj, 1990 Saarinen et al., 2001 Ibrahim & Abul-Hajj, 1990 Le Bail et al., 1998 Saarinen et al., 2001 Ibrahim & Abul-Hajj, 1990 Sanderson et al., 2004 Jeong et al., 1999 Le Bail et al., 1998 Le Bail et al., 2000 Jeong et al., 1999 Chen et al., 1997 Kao et al., 1998 Kellis & Vickery, 1984 Stresser et al., 2000 Campbell & Kurzer, 1993 Kao et al., 1998 Kellis et al., 1986 Saarinen et al., 2001 Kellis & Vickery, 1984 Le Bail et al., 1998 Jeong et al., 1999 Sanderson et al., 2004 Saarinen et al., 2001 Kellis & Vickery, 1984 Stresser et al., 2000 Le Bail et al., 1998 Jeong et al., 1999 Sanderson et al., 2004 Kao et al., 1998 Edmunds et al., 2005 Campbell & Kurzer, 1993 Kellis & Vickery, 1984 Ibrahim & Abul-Hajj, 1990 Le Bail et al., 1998 Pelissero et al., 1996 Saarinen et al., 2001 Campbell & Kurzer, 1993 Saarinen et al., 2001 Kao et al., 1998 Lee et al., 2001 Saarinen et al., 2001 Saarinen et al., 2001 Wang et al., 1994 Le Bail et al., 2001 Saarinen et al., 2001 Wang et al., 1994 Kellis & Vickery, 1984 Saarinen et al., 2001

CHO HPM HPM AC CHO HPM JEG-3 HPM HPM HPM HAC JEG-3 HPM HPM HPM HPM HAC CHO RAr AC HPM HPM HPM HPM JEG-3 AC JEG-3 CHO HPM JEG-3 JEG-3 AC HPM JEG-3 AC HPM JEG-3

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Table 13.1 (Cont’d ) Compound Flavanones 7-hydroxyflavanone 7-methoxyflavanone 7,8-dihydroxyflavanone flavanone

hesperetin naringenin

IC50, μM

Ki, μM

Assay

Reference

5.1 0.3

HPM HPM HPM HPM HPM HPM HPM HPM HPM JEG-3 HPM HPM HPM CHO RAr

Le Bail et al., 2001 Le Bail et al., 1998 Le Bail et al., 1998 Ibrahim & Abul-Hajj, 1990 Le Bail et al., 2001 Jeong et al., 1999 Pelissero et al., 1996 Le Bail et al., 2001 Jeong et al., 1999 Saarinen et al., 2001 Stresser et al., 2000 Le Bail et al., 1998 Lee et al., 2001 Kao et al., 1998 Edmunds et al., 2005

RAr HPM HPM CHO AC CHO RAr HPM

Edmunds et al., 2005 Jeong et al., 1999 Le Bail et al., 2000 Kao et al., 1998 Campbell & Kurzer, 1993 Kao et al., 1998 Edmunds et al., 2005 Ibrahim & Abul-Hajj, 1990

HPM HPM HPM HPM

Ibrahim Ibrahim Ibrahim Ibrahim

& & & &

Abul-Hajj, Abul-Hajj, Abul-Hajj, Abul-Hajj,

50 50 50 50 34.6 > 50 30.6 > 50

HPM HPM HPM HPM HPM HPM HPM HPM

Le Le Le Le Le Le Le Le

et et et et et et et et

al., al., al., al., al., al., al., al.,

37 6 15 60 30 8.9 14 74 11

HPM HPM HPM AC HPM HEK HPM AC HPM

Adlercreutz et al., 1993 Adlercreutz et al., 1993 Adlercreutz et al., 1993 Wang et al., 1994 Adlercreutz et al., 1993 Saarinen et al., 2001 Adlercreutz et al., 1993 Wang et al., 1994 Adlercreutz et al., 1993

2.4 2.6 8 8 13.8 38.8 250 3.3 3.31 1.4 1.54 9.2 17

Isoflavones daidzein biochanin A

> 50 35.88 49

genistein isoflavone

> 200

Isoflavanones isoflavanone 4′-hydroxyisoflavanone 2-hydroxyisoflavanone 3′,4′-dihydroxyisoflavanone

120 160 170 > 200

Chalcones 2′-hydroxy-4′-methoxychalcone 2′-hydroxychalcone 2′,4-dihydroxychalcone 2′,4′-dihydroxychalcone 2′,4′,4-trihydroxychalcone 4-hydroxychalcone 4′-hydroxychalcone phloretin Lignans 3′-demethoxymatairesinol 4,4′-dihydroxyenterolactone DDMM enterodiol enterolactone

nordihydroguaiaretic acid

12.5 49 123 > 50

> > > >

7.3

14.4

Bail Bail Bail Bail Bail Bail Bail Bail

1990 1990 1990 1990

2001 2001 2001 2001 2001 2001 2001 2001

AC, adipose cells; CHO, Chinese hamster ovarian cells; DDMM, didemethoxymatairesinol; HEK, aromatase transfected human embryonic kidney cells (arom+HEK293); HPM, human placental microsomes; JEG-3, JEG-3 choriocarcinoma cells; RAr, recombinant aromatase.

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inhibition potency from 1.0 to 10 μM include apigenin, luteolin, naringenin (racemic), 4′,7-dihydroxyflavone, 7-hydroxyflavanone (racemic), 7-methoxyflavanone (racemic), naringin (racemic), 7-methoxyflavone, hesperetin (racemic), isogemichalcone C, 7,8dihydroxyflavone, 7,8-dihydroxyflavanone (racemic), flavanone (racemic), flavone and 4′hydroxyflavanone (racemic). Inhibitors with IC50 values from 10 μM up to sub-millimolar concentrations include (in descending order of inhibition potency) nordihydroguaiaretic acid (NDGA), quercetin, enterolactone, coumestrol, enterodiol, on-onetin, 3′-demethoxymatairesinol, biochanin A, 6-hydroxyflavone, 3′,4′-dihydroxyflavone, 4′,6-dihydroxyflavone, 5-hydroxyflavone, 2,4,4′-trihydroxydeoxybenzoin, 4′,5-dihydroxyflavone, flavan-4-ol (racemic), isoflavanone (racemic), 3-hydroxyflavone, 3′,4′-dihydroxyflavanone (racemic), 4′-hydroxyisoflavanone (racemic), O-demethylangolensin (ODMA, racemic), 2-hydroxyisoflavanone (racemic), 4′-hydroxyflavone, 3′,4′-dihydroxyisoflavanone (racemic), 4′hydroxyflavan-4-ol and isoflavone. Different groups of workers found remarkable differences in the IC50 values of flavanone and flavone. Pelissero et al. (1996) measured IC50 values of 375 μM and 250 μM for flavone and flavanone, respectively. Other groups have measured IC50 values from 8 μM to 48 μM for flavone (Kellis & Vickery, 1984; Ibrahim & Abul-Hajj, 1990; LeBail et al., 1998) and from 8 μM to 38.8 μM for flavanone (Ibrahim & Abul-Hajj, 1990; Jeong et al., 1999 and Le Bail et al., 2001). Pelissero et al. pointed out that the amount of protein they incubated was up to 100 times higher than in the studies reported by other groups. Therefore, when the amount of membrane-bound protein is increased, there is a concomitant increase in the membrane lipid content, which might cause the compounds to accumulate on the membranes, leaving less phytoestrogen in the aqueous fraction. Summarized on the basis of compound class, flavones are by far the most potent aromatase inhibitors compared to other phytoestrogens. Flavanones, lignans, coumestrol and deoxybenzoins exceed isoflavones and flavanols in inhibitory potency. The best isoflavonoid inhibitor, prunetin, has an IC50 value of 27.4 μM (Jeong et al., 1999) and the weakest, isoflavone, an IC50 value of > 200 μM (Ibrahim & Abul-Hajj, 1990). 13.2.1.2 Anti-aromatase activity of phytoestrogens in cell-based assays Several cell-based assays have also been used to test the anti-aromatase activity of natural phytoestrogens. These include adipose cells, H295R human adrenocortical carcinoma cells (HAC), Chinese hamster ovary (CHO) cells, aromatase transfected human embryonic kidney cells (arom+HEK293) cells, JEG-3 choriocarcinoma cells and MCF-7 cells. As in human placental microsome-based assays, α-naphthoflavone is the most potent inhibitor in cell-based assays, with Ki values of 0.2 μM (Campbell & Kurzer, 1993) and 2.2 μM (Kao et al., 1998). Chrysin shows a similar trend, with Ki values of 2.4 μM (Campbell & Kurzer, 1993) and 2.6 μM (Kao et al., 1998). Saarinen et al. (2001) observed high inhibition potency for chrysin using JEG-3 and Arom+HEK 293 cell lines, with an IC50 value of 0.5 μM (JEG-3). Apigenin was found to be the most potent inhibitor, with an IC50 value of 0.18 μM (JEG-3), surpassing 7-hydroxyflavone (IC50 value 0.35 μM, JEG-3) and chrysin. In contrast to HPM-based assays, when tested in cell-based assays flavones are not the only active compounds, as coumestrol – with a Ki value of 1.3 μM – and the lignan 3′demethoxy-3-O-demethylmatairesinol – with a Ki value of 5 μM (Wang et al., 1994) – are

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also active. Compared to HPM-based assays, most of the compounds show decreased potency. With some flavonoids (7-methoxyflavone, flavone, quercetin) the activity is lost when a cell-based assay is used. This might be owing to the inability of some of the compounds to penetrate the cell membrane (Wang et al., 1994). The isoflavone genistein is a weak inhibitor of aromatase, with a Ki value of 123 μM (Kao et al., 1998). Brooks & Thompson (2005) measured a significant decrease in estrone production when genistein was used as an inhibitor. In HAC cells, genistein showed no inhibition up to 100 μM, the highest concentration tested (Sanderson et al., 2004). Rotenone, a flavonoid derivative, was found to be the most potent inhibitor, with an IC50 value of 0.3 μM. 13.2.1.3 Anti-aromatase activity of phytoestrogens in recombinant-enzyme assays Edmunds et al. (2005) tested the ability of phytoestrogens to interact with aromatase directly using recombinant aromatase enzyme expressed in insect cell microsomes. For naringenin, the measured Ki value was 0.3 μM, and for chrysin it was 1.0 μM. As expected, the Ki values were lower than in cell-based assays, where the IC50 values for naringenin and chrysin were 5.1 and 2.6 μM, respectively (Kao et al., 1998). Notably, the Ki values for the isoflavones daidzein and genistein were > 50 μM, even in pure enzyme-based assays. 13.2.1.4 The anti-aromatase activity of various phytoestrogens Lignans such as enterolactone have been tested against aromatase activity using HPM, as well as adipose cells and arom+HEK293-based assays. Interestingly, the IC50 value for enterolactone in the arom+HEK293-based assay (8.9 μM; Saarinen et al., 2001) was lower than in the HPM-based assay (14 μM; Adlercreutz et al., 1993) or adipose cell-based assay (74 μM; Wang et al., 1994). Lignans with anti-aromatase activity in an HPM-based assay include 4,4′-dihydroxyenterolactone (IC50, 6 μM), nordihydroguaiaretic acid (IC50, 11 μM), didemethoxymatairesinol (DDMM; IC50, 15 μM), enterodiol (IC50, 30 μM) and 3′demethoxymatairesinol (IC50, 37 μM) (Adlercreutz et al., 1993). In an adipose cell-based assay, the IC50 values for DDMM and 3′-demethoxy-3-O-demethylmatairesinol were 60 μM and 84 μM, respectively (Wang et al., 1994). Brooks & Thompson (2005) found out that enterolactone significantly decreased the amount of E1 produced from androstenedione. Enterodiol was also able to inhibit aromatase. Chalcones are biosynthetic precursors of flavonoids lacking the flavonoid C-ring (Dixon & Steele, 1999). Le Bail et al. (2001) tested several chalcones and dihydrochalcones for their anti-aromatase activity. A hydroxy group at the 4′-position has a positive effect on aromatase inhibition activity as seen when comparing the IC50 values for 2′-hydroxychalcone (> 50 μM), 4′-hydroxychalcone (30.6 μM), 4,2′-dihydroxychalcone (> 50 μM) and 4,2′,4′trihydroxychalcone (isoliquiritigen, 34.6 μM). Superimposition of chalcone and flavone skeletons reveals that C-4′ in chalcones corresponds to C-7 in flavones, where a hydroxy group was found to have a high positive effect on aromatase inhibition. As with the C-7 in flavonoids, a methoxy group at the C-4′ in chalcones decreases the activity. The effect of the 4′-methoxy group can be seen in the inhibition percentages at low concentrations, although the IC50 values for 2′,4′-dihydroxychalcone and 2′-hydroxy-4′-methoxychalcone are both > 50 μM. Also, a 2′-hydroxy group alone decreases the anti-aromatase activity

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and possibly has a negative effect when combined with additional hydroxy groups. In contrast, a 6′-hydroxy group has a positive effect on the anti-aromatase activity (compare, e.g., 2′,6′-dihydroxy-4′-methoxychalcone, with an IC50 value of 14.3 μM, with 2′-hydroxy4′-methoxychalcone, whose IC50 is > 50 μM). The combination of 2′-, 4′- and 6′-hydroxy groups, however, generates a good inhibitor (e.g. 4,2′,4′,6′-tetrahydroxychalcone, IC50 2.6 μM, and 3,4,2′,4′,6′-pentahydroxychalcone, IC50 2.8 μM). The reduction of the double bond significantly decreases the anti-aromatase activity (e.g. 4,2′,4′,6′-tetrahydroxychalcone, IC50 2.6 μM and 4,2′,4′,6′-tetrahydroxydihydrochalcone, IC50 > 50 μM). To facilitate further conclusions about the effects of substituents in chalcones, the inhibitory data of chalcone itself and a few of its monohydroxylated derivatives should be tested. Jeong et al. (1999) measured the activity of flavonoid derivatives using an HPMbased assay. The tested compounds were hesperidin, naringin and silymarin. Hesperidin is hesperetin-7-O-rutinoside, a flavonoid conjugated with a disaccharide, naringin is naringenin-7-O-rutinoside and silymarin is a polyhydroxy flavanone derivative. Although these compounds are spatially large, they have IC50 values in the range of 3 to 67 μM. It should be noted, however, that the inhibition values reported by Jeong et al. differ from the results of other groups. Jeong et al. (1999) found an IC50 value of 128 μM for 7-hydroxyflavone, while e.g. Le Bail et al. (1998) reported an IC50 value of 0.21 μM for the same compound. It is possible that different inhibition mechanisms were functioning under the different conditions used by the two groups of workers. An extract from Broussonetia papyrifera was found to inhibit aromatase activity with an inhibition percentage of 74% at 80 μg/mL using an HPM-based assay (Lee et al., 2001). After separation and isolation of the compounds, five new and ten known aromatase-inhibiting phytoestrogens were identified, in addition to inactive phytoestrogens. The most active ones were isolicoflavonol, a prenyl derivative of kaempferol (IC50, 0.10 μM) and a furoflavanone (IC50, 0.10 μM). Other sub-micromolar inhibitors were abyssinone II (IC50, 0.40 μM) and a tetrahydroxy chalcone with an isoprenoid cinnamate ester side-chain (IC50, 0.50 μM).

13.2.2 The structure–activity relationship of phytoestrogens Flavonoids are the best aromatase inhibitors among phytoestrogenic compounds tested so far. Kellis & Vickery (1984), and Adlercreutz et al. (1993) measured the UV-VIS spectral changes that occur when phytoestrogens bind to aromatase enzymes. They observed that in the aromatase–phytoestrogen complex, the heme iron is converted from a highspin state into a low-spin state. This conversion is due to a ligand coordinating to the heme iron. Kellis & Vickery suggested that the flavones might occupy some alternative binding site and cause a change in the overall conformation of the protein. This modified conformation would have a decreased affinity for the ligand. Chen et al. (1997) and Kao et al. (1998) provided an atomic-level explanation of why flavonoids are better aromatase inhibitors than isoflavonoids, using computer modeling and site-directed mutagenesis studies. The hypothesis is that flavonoids coordinate to the heme iron via the 4-carbonyl oxygen. In flavonoids, the B-ring is at the 2-position of the chroman ring system whereas in isoflavonoids, the B-ring is at the 3-position. This difference in structure allows the 4-carbonyl in flavonoids to coordinate to the heme iron without the steric hindrance caused by a phenyl ring (Fig. 13.2). In the computational

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model, the superimposition of the flavonoid and androgen skeletons provides an orientation where the A- and C-rings of the flavonoid mimic the C- and D-rings of the natural substrate androstenedione. The 4-carbonyl group occupies space near the C-19 of the androgen. Additionally, Ibrahim & Abul-Hajj (1990) reported that reduction of the 4-carbonyl to a hydroxyl group (flavone or flavanone to flavan-4-ol) reduces the inhibition potency substantially, the IC50 values being 10 μM (flavone), 8 μM (flavanone), and 120 μM (flavan4-ol). This behavior is most likely due to the loss of coordination potential that results from reducing the carbonyl group to a hydroxyl. Similarly, Kellis et al. (1986) reported that elimination of the carbonyl function in α-naphthoflavone decreases the aromatase inhibition potency. This effect could also be seen when catechins were tested for antiaromatase activity (Sanderson et al., 2004). (+)-Catechin and (−)-epicatechin, which lack the 4-carbonyl functionality, had no inhibitory effect. From these studies it can be concluded that a sterically-unhindered carbonyl function is a necessary factor in the binding of phytoestrogens to aromatase enzymes. A C-7 hydroxy group in the flavonoid skeleton increases the inhibitory potency significantly. This can be seen when comparing the IC50 values of flavone (from 8 μM to 48 μM) and 7-hydroxyflavone (from 0.21 μM to 0.5 μM) (Ibrahim & Abul-Hajj, 1990; LeBail et al., 1998). A methoxy group in this position is less active than a hydroxy group (LeBail et al., 1998). As observed from the IC50 values for 5-hydroxyflavone (100 μM) and 6-hydroxyflavone (80 μM), a hydroxyl group at these sites reduces the inhibitory potency dramatically. 5-Hydroxyflavone forms an intramolecular hydrogen bond to the 4-carbonyl oxygen and weakens the coordination capacity of the carbonyl (Kao et al., 1998). However, such a dramatic effect was not observed when comparing 7-hydroxyflavone (IC50, 4 μM) and 5,7-dihydroxyflavone (chrysin; IC50, 7 μM) in measurements made by Sanderson et al. (2004).

13.2.3 Phytoestrogens as lead compounds for aromatase inhibition Phytoestrogens have been used as starting points to develop effective aromatase inhibitors. As the pioneers in testing natural phytoestrogens against aromatase, Kellis et al. (1986) were also the first to test the anti-aromatase activity of structural analogs and synthetic monohydroxy derivatives of α-naphthoflavone in an HPM-based assay. Although most of the tested compounds did not reach the activity of α-naphthoflavone, 9-hydroxyα-naphthoflavone (a metabolite of α-naphthoflavone) was shown to be a potent inhibitor of aromatase, with an IC50 value of 0.02 μM and a 3.5-fold increase in binding affinity compared to α-naphthoflavone. 10-Hydroxy-α-naphthoflavone was almost as active as αnaphthoflavone but the rest of the derivatives were less potent. Of the three structural analogs tested, none had nearly the potency of α-naphthoflavone. The dinaphthyl derivative had an IC50 value of 1.0 μM and a 16-fold decrease in activity compared to α-naphthoflavone. Modification of the chroman ring to a furan ring, and therefore the loss of the carbonyl group, produced an almost inactive compound with an IC50 value of > 100 μM. Also, a change of the B-ring position from 2 to 3, producing α-naphthoisoflavone, resulted in an almost inactive compound, with an IC50 value of 100 μM. Brueggemeier et al. (2001) created a benzopyranone-based library to test the effect of different substituents on the chroman ring system. The substituents were applied to positions 2, 6, 7 and 8. The C-2 substituents included phenyl, cyclohexenyl, chloropropyl

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and diethylacetal groups. The C-6 substituents were phenyl, chloro and methoxy groups, the C-7 substituents were hydroxy and methoxy, and the C-8 substituents were hydroxy, methoxy, and methyl groups. The aromatase activities were in the range of 55 to 120% at 1.0 μM inhibitor concentration. Chromones and xanthones with varying substitution patterns have also been investigated as molecular skeletons for aromatase inhibitors. Based on non-steroidal aromatase inhibitors similar to the marketed drug S-fadrozole, Recanatini (1996) created a comparative molecular field analysis (CoMFA) model, which has been enhanced (Recanatini & Cavalli, 1998) and used to design chromone- and xanthone-based aromatase inhibitors (Recanatini et al., 2001). It was deduced from the quantitative structure–activity relationship (QSAR) model that the functionalities required for binding were a heterocyclic ring (imidazole or 1,3,4-triazole) linked to an aromatic moiety and a H-bond acceptor (CN, NO2 or Br) at a suitable distance from the nitrogen in the heterocyclic ring. The most active inhibitors in an HPM-based assay were 2-(imidazol-1-ylmethyl)-4-nitroxanthone and 2(imidazol-1-ylmethyl)-4-cyanoxanthone, with IC50 values of 0.040 and 0.043 μM, respectively. It should be noted that in view of their IC50 values, these compounds surpass the commercial drug S-fadrozole (IC50, 0.052 μM). The weakest inhibitors in the series were 7-triazolylmethylchromone (IC50, 3.7 μM) and a reversed isomer of the second-best inhibitor, 2-cyano-4-imidazolylmethylxanthone (IC50, 2.3 μM). An interesting feature was found when studying the difference in activity between identically substituted imidazole and triazole compounds – compounds bearing an imidazole ring are more active than those with a triazole ring. Semi-empirical calculations showed that with a triazole group, the highest occupied molecular orbital (HOMO), and thus the electron density responsible for coordinating to the heme iron, was distributed throughout the xanthone skeleton. With the imidazole compound, the electron density was located at the imidazole ring, thus providing better opportunities for coordination. In general, xanthone was found to be a better core than chromone. Based on the QSAR model and biological data, the xanthone skeleton, an imidazolyl substituent and a strong H-bond acceptor were the best combination for good anti-aromatase activity. The QSAR model was further refined to include enantioselectivity. Chiral inhibitors were designed, synthesized and separated with chiral HPLC (Cavalli et al., 2005). The compounds were 3-benzyl-chromenones with an iron-coordinating imidazolyl group at the benzylic position. The R-enantiomers proved to be better inhibitors than the S-enantiomers, with an IC50 value of 0.0023 μM for the best inhibitor, (R)-3-[imidazol-1-yl-(4-nitrophenyl)methyl]-chromen-4-one. In the same study, an IC50 value of 0.017 μM was measured for the commercial drug S-fadrozole. Application of the model by Recanatini & Cavalli also led to optimization of a flavone lead compound, 3-(imidazol-1-ylmethyl)-7-methoxyflavone, with an IC50 value of 0.55 μM (Gobbi et al., 2006). In the tested compounds, the 7-substituent was a hydrogen atom or a methoxy group and the 4′-position was modified with a nitro, nitrile or methoxy group or bromine atom. The different substituents in the 4′-position did not improve the inhibition potency of the 7-methoxy series. However, a hydrogen at the 7-position and introduction of the different 4′-substituents mentioned above yielded a set of nanomolar range inhibitors. Most of the compounds were selective in respect of CYP17, 17α-hydroxylase/ C17,20-lyase, catalyzing the key step of androgen biosynthesis.

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The flavone skeleton has been used as a template to find substituents contributing to anti-aromatase activity (Sanderson et al., 2004). Different substitutions included monohalogenation or nitration of the flavone B-ring. In addition, a quinolinone skeleton, as such or halogen substituted, and α-, β-, and γ-naphthoflavones with various substituents were tested. Only a few of these compounds showed inhibitory activity in HAC cells. When the B-ring of the flavone was replaced with a pyridyl, no inhibitory effect was observed. Substituted naphthyl derivatives 2-(3- or 4-pyridyl)- α-naphthoflavone had IC50 values of > 30 μM and 5 μM, respectively, while 2-(3- or 4-pyridyl)-γ-naphthoflavones had IC50 values of 7 μM and > 30 μM, respectively. Despite the weak anti-aromatase activity of isoflavonoids, they have been used as a scaffold for potent aromatase inhibitors (Kim et al., 2004; Hackett et al., 2005; Su et al., 2005). In these inhibitors, the 4-carbonyl no longer serves as the iron-coordinating moiety. Instead, a hetero atom with a lone electron pair is introduced to the substituent at the 2position. Kim et al. designed and synthesized a library consisting of isoflavonoids with various substituents at the positions 2, 4′ and 7. Compounds with a bulky alkyl group or 2′-pyridylmethanethio group at C-2 did not inhibit aromatase activity. This is probably owing to the lack of a sterically-unhindered iron-coordinating atom in the molecule. When the position of the nitrogen in the pyridyl group was changed to 3′, IC50 values of 3.6 and 9.2 μM were measured for 2-(3′-pyridylmethyl)thio-7-hydroxyisoflavone and 2(3′-pyridylmethyl)thio-7-methoxyisoflavone, respectively. Nitrogen at the 4′-position provided even better inhibition, as seen from the IC50 values for 2-(4′-pyridylmethyl)thio7-hydroxyisoflavone (0.61 μM) and 2-(4′-pyridylmethyl)thio-7-methoxyisoflavone (1.6 μM). While keeping the unhindered coordinating moiety [(4′-pyridylmethyl)thio group] at C-2, modification of the substituents at C-4′ and C-7 produced other sub-micromolar inhibitors. Compounds with a C-7 hydroxy group and a C-4′ hydroxy or a C-4′ methoxy group had IC50 values of 0.22 μM (2-(4′-pyridylmethyl)thio-4′-methoxy-7-hydroxyisoflavone) and 0.28 μM (2-(4′-pyridylmethyl)thio-4′,7-dihydroxyisoflavone). The best inhibitor in the series was found to be a 7-benzyloxy derivative of (pyridylmethylthio)isoflavone, 2-(4′-pyridylmethyl) thio-7-benzyloxyisoflavone (IC50, 0.21 μM). This compound was synthesized after testing an intermediate in the synthesis route and observing that 2-(4′-pyridylmethyl)thio-4′-methoxy7-benzyloxyisoflavone, a precursor of its 7-hydroxy derivative, inhibited aromatase activity with an IC50 of 0.53 μM. The subsequent omission of the 4′-methoxy group yielded an even more active compound. Initially, the 7-benzyloxy group was thought to be too bulky to fit into the active site but eventually provided the strongest binder among the compounds under study. The hetero-ring modification of the compounds studied by Kim et al. (2004) produced further sub-micromolar inhibitors (Hackett et al., 2005). As in the previous experiment, the C-2, C-4′- and C-7 substituents of the isoflavonoid skeleton were modified. The substituents applied to C-2 were imidazolyl, triazolyl, thioimidazolyl, methylthioimidazolyl and thiotriazolyl groups. Substituents at C-4′ were a hydrogen or a methoxy group, and at C-7, a methoxy or a benzyloxy group. The strongest binder was found to be 2-(imidazol-1-yl)7-benzyloxy-isoflavone (IC50, 0.52 μM). As in the work of Kim et al., the non-methoxylated 4′-position enhanced inhibitory activity. This was proven by comparison of the IC50 values between 2-(imidazol-1-yl)-7-benzyloxy-isoflavone (0.52 μM) and 2-(imidazol-1-yl)4′-methoxy-7-benzyloxy-isoflavone (4.7 μM). Of the tested hetero-ring systems attached

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to C-2, and thus providing a coordinating atom for the heme iron, an imidazole was clearly the most potent one. Only imidazole and triazole ring systems produced inhibitors with IC50 values lower than 50 μM. An exceptional difference in IC50 values between identically substituted imidazolyl and triazolyl compounds was observed. 2-(Imidazol-1-yl)-7methoxy-isoflavone (IC50, 0.77 μM) was 23-fold more potent than its triazolyl counterpart 2-(1,2,4-triazol-1-yl)-7-methoxy-isoflavone (IC50, 18 μM). Following the approach by Recanatini et al. (2001), the HOMOs were calculated for both of the compounds, while the geometries were optimized using ab initio methods. As with Recanatini et al., the HOMO of the imidazole inhibitor was found to be located at the hetero ring, thus providing electron density for coordination to the heme iron. In case of the triazole compound, the HOMO was located throughout the isoflavone skeleton. To further investigate the coordination potency of imidazole groups, difference spectra of the bindings were measured. Both compounds showed that during binding, the concentration of the low-spin iron increases, providing evidence for azole ligation to the heme iron. The absorption minima were at 394 nm and 391 nm for the imidazole and the triazole compounds, respectively, and the maximum was at 410 nm with both compounds. The imidazole compound had a larger change in absorbance, thus providing further evidence for tighter binding compared to the triazole compound. In addition to modifications of the iron-coordinating hetero group at C-2 of the isoflavone core, Su et al. (2005) investigated the influence of the bulky substituent at C-7. The lead compound in the study was 2-(4′-pyridylmethyl)thio-7-benzyloxyisoflavone (IC50, 0.21 μM). While keeping the 4′-pyridylmethylthio group at C-2, it was observed that a 4′-biphenylmethoxy group at C-7 produced the best inhibitor, 2-(4′-pyridylmethylthio)-7(4′-biphenylmethoxy)-isoflavone, with an IC50 as low as 0.079 μM. α- and β-naphthylmethoxy substituents at C-7 increased the potency compared to the lead compound, with a small difference in IC50 values (0.112 μM and 0.090 μM, respectively). Kinetic measurements verified the results of dose–response studies and showed that the β-naphthylmethoxy substituent is slightly preferred over the α-naphthylmethoxy group. An aliphatic cyclohexylmethoxy group had decreased anti-aromatase potency, thus underlining the importance of an aromatic substituent at this position. Introduction of a nitro group at the 2′, 3′ or 4′ position of the C-7 phenyl ring increased the potency, but the position of the nitro group does not seem to have much effect. Together these findings suggest that beneficial π–π interactions take place upon binding to the aromatase active site. Further modifications, such as introduction of a methoxy group or a halogen to the C-7 aromatic ring, had a negative or no effect on the inhibition potency compared to the 7-benzyloxy compound. Replacement of the phenyl ring with a 2′-pyridyl hetero ring decreased the activity 2-fold.

β-Hydroxysteroid dehydrogenases and their 13.3 17β inhibition by phytoestrogens 17β-Hydroxysteroid dehydrogenases (17β-HSDs, EC1.1.1.62, 17β-hydroxysteroid oxidoreductases, 17β-HSORs) catalyze the NAD(H)/NADP(H)-dependent oxidation and reduction of hydroxy or keto groups at the C-17 position of estrogens and androgens. They are critical enzymes in many physiological processes, as they catalyze the synthesis or

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degradation of active sex hormones (see Fig. 13. 1). To date 14 mammalian isoenzymes of 17β-HSD have been identified with a different substrate and cofactor specifity, and tissue distribution (for recent reviews of 17β-HSDs see Lukacik et al., 2006; Moeller & Adamski, 2006). Most of the human 17β-HSDs belong to the short-chain dehydrogenase/reductase (SDR) superfamily, the only exception being 17β-HSD5, which belongs to the aldo-keto reductases (AKRs). 17β-HSDs have a relatively low sequence identity (20–25% inside the isoenzyme family) but they exhibit a common protein fold typical for SDR enzymes. The common features include, for example, Rossmann-fold motif, and conserved co-factor binding site and active site residues. 17β-HSD types 1, 2, 3, 4, and 5 as well as the fungal 17β-HSDcl have been shown to be target enzymes for phytoestrogen activity (Deluca et al., 2005). 17β-HSD1 is the main enzyme converting E1 into active E2 in an NADPH-catalyzed reduction reaction. It is mostly expressed in ovaries, placenta and breast tissue. Widely distributed 17β-HSD2 is responsible for NAD-dependent oxidation of E2 and testosterone into their inactive forms, and is thus an important enzyme in controlling levels of active steroids. 17β-HSD3 is almost exclusively expressed in testes and is the enzyme catalyzing the NADPH-dependent reduction of androstenedione to testosterone. Also 5α-androstenedione and estrone can be reduced by this isoform. Type 4 17β-HSD is an NAD-dependent oxidative enzyme involved in converting E2 into E1. 17β-HSD5 is highly expressed in testes and extragonadal tissues such as prostate, liver and adrenals. It catalyzes the NADPH-dependent reduction of androstenedione into active testosterone. The crystal structures of 17β-HSD1, 17β-HSD4, 17β-HSD5, 17β-HSD10, 17β-HSD11, and 17β-HSD14 have been resolved, making them attractive targets for structure-based drug design.

13.3.1 17β -HSD type 1 Mäkelä et al. (1994) were the first to report that the environmental estrogens coumestrol and genistein inhibit purified human placental 17β-HSD1. Since then several phytoestrogens have been tested with purified human placental 17β-HSD1 (Table 13.2) and on T47D breast cancer cell lines (Mäkelä et al., 1995, 1998). Coumestrol (1.2 μM or 0.12 μM) was found to be the most potent inhibitor of the purified enzyme with 18% or 49% conversion of E1 to E2, respectively. Flavones were better inhibitors than flavanones, which in turn were better than the corresponding isoflavones. Kaempferide, acacetin and fisetin at 1.2 μM concentration were the most potent inhibitors in a flavonoid series, with 27%, 39%, and 47% E1 conversion, respectively. The isoflavone genistein showed moderate activity at 1.2 μM concentration with 63% E1 conversion. Similar inhibitory activities, although less effective, were found in wild type T-47D and 17β-HSD1 cDNA transfected T-47D21 cells. It seems that a C-5 and C-7 hydroxy groups, and a C-4′ hydroxy or methoxy group are important for the biological activity. Zearalenone and diethylstilbestrol, both potent estrogens, did not show any inhibitory activity. No 17β-HSD1 enzyme activity was found in the MCF-7 cell line used in this study (enzyme concentrations below the limit of the immunoassay detection), hence no inhibitory activities were seen. Genistein and the mammalian lignan enterolactone have been reported to inhibit 17β-HSD1 activity in MCF-7 cancer cells (Brooks & Thompson, 2005). For enterolactone, 84% inhibition was observed

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Table 13.2 Phytoestrogens, their substitution patterns, and 17β-HSD1 activity. Substitution pattern* Compound Flavones Flavone 7-Hydroxyflavone 7-Methoxyflavone 7,8-Dihydroxyflavone 6-Hydroxyflavone Chrysin Apigenin Galangin Luteolin Quercetin Fisetin Kaempferol Pinostrobin Acacetin Kaempferide

3

Isoflavones Genistein Prunetin Biochanin A Daidzein Formononetin 3′,4′,7-tri-OH-Isoflavone Coumestans Coumestrol Medicarpin

6

7

OH OMe OH

8

2′

3′

4′

OH

OH

OH OH OH OH

OH

Flavanones Flavanone 4′-Hydroxyflavanone Naringenin 7,8-Dihydroxyflavanone 7-Hydroxyflavanone 7-Methoxyflavanone Eriodictyol Hesperetin Flavans Catechin Sativan

5

Inhibition†

OH OH OH OH OH OH OH OH OH

OH

OH OH OH

OH

OH OH OH

OH OH OH OH OH OH OH OMe OH OH

OH OH OH OMe OH OH OH OH OH OMe OH OH OH OH

OH OH OH OH

OH OH OH OH OMe OMe

E1→E2%

107.3 69.4 – – 95.6 96.0 57.7 59.1 111.4 123.0 47.2 58.0 93.1 38.5 26.7

± 19.6 ± 5.2 ± ± ± ± ± ± ± ± ± ± ±

17.9 23.9 18.4 6.9 14.6 11.8 9.4 20.8 11.0 3.8 7.6

NI 0.9 NI NI – 3.6 0.3 – 0.6 – – – – – – – – 15.0 NI 28 NI NI NI – 50

OH OH

OH OMe

103.9 ± 24.8 101.9 ± 17.3 53.0 ± 11.2 – – – – –

OH

OH OMe

120.6 ± 26.7 –

OH OH OMe OH OMe OH

62.6 – 91.3 68.1 97.2 –

OH OH OH

OMe

OH

IC50

± 29.9 ± 28.2 ± 10.3 ± 11.2

18.3 ± 7.5 –

1.0 > 50 4.9 10 > 50 5.2 0.2 > 50

* For the numbering of the flavonoid skeleton see Fig. 13.2. † The % conversion of estrone to estradiol at 1.2 μM inhibitor concentration (Mäkelä et al., 1995, 1998). IC50 values measured in human microsomal 17β-HSD1 (Le Bail et al., 1998, 2000, 2001). NI, no inhibition; –, not measured.

at 50 μM concentration, whereas enterodiol was inactive. In this assay, 17β-HSD1 activity was inhibited 59% or 24% by genistein at 50 or 0.1 μM concentration, respectively. Genistein (50 μM) was found to inhibit 17β-HSD1 activity in primary cultures of human granulosa-luteal cells in acute (4-h) or chronic (24-h) exposure (Whitehead et al., 2002). The inhibition was dose- and time-dependent. On the other hand apigenin, quercetin, biochanin A, and zearalenone showed no inhibitory effect on 4-h exposure at 10 μM concentration

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on FSH (follicle stimulating hormone)-induced 17β-HSD1 activity (Whitehead & Lacey, 2003). This may be explained by poor bioavailability or rapid metabolism of these compounds, or high enzyme activity and expression in these cells. It was shown that FSH priming significantly increases E2 production from E1 in human granulosa-luteal cells. In chronic exposure apigenin showed some inhibitory activity against 17β-HSD1. Flavonoids and coumestans have been shown to inhibit human placental microsomal 17βHSD1. Coumestrol, apigenin, luteolin and 7-hydroxyflavone were the most potent inhibitors, with IC50 values of 0.2, 0.3, 0.6 and 0.9 μM, respectively (Le Bail et al., 1998, 2000). In flavones, a 7-hydroxy group was shown to be essential for the inhibitory activity, as 7methoxyflavone is inactive. The isoflavones tested were less active than the corresponding flavones. The results from human placental microsomal assays are listed in Table 13.2. Also chalcones, lacking the heterocyclic C-ring, inhibit 17β-HSD1 in human placental microsomal preparations (Le Bail et al., 2001). The inhibitory activity of 12 chalcones with different hydroxylation patterns was measured. 4-Hydroxychalcone was the most potent inhibitor, with an IC50 value of 16.0 μM, while 4,2′,4′-trihydroxy- and 2′,4′-dihydroxychalcone showed moderate activity, with IC50 values of 33.8 and 34.6 μM, respectively. A series of synthetic 7-hydroxyflavones with various substituents at the 4′-position, as well as the genistein standard, failed to show any 17β-HSD1 inhibitory activity in human placental cytosol preparations at 100 μM concentration (Vihn et al., 2001). 13.3.1.1 Binding of phytoestrogens to 17β-HSD1 In-silico approaches have been used to investigate the binding of phytoestrogens to 17β-HSD1. The results from the inhibition studies by Mäkelä et al. (1995, 1998) have been further used to derive a pharmacophore model for the active site (Hoffrén et al., 2001). Coumestrol, apigenin and genistein were docked into the ligand-binding domain of 17β-HSD1 (PDB code 1FDT) using a flexible docking approach. Coumestrol superimposed well with E1 and seemed to adopt a uniform binding mode where the peripheral oxygens in both molecules are at a similar distance to Tyr155 and Glu282. However, the distances to Ser142 and His221 were longer than in E2. Genistein, although a more flexible structure, seemed to have a similar binding mode to coumestrol, with an additional bidentate interaction with Tyr218. Apigenin, as a flavone, had a different binding mode, probably owing to the different position of the aromatic B-ring. Apigenin seemed to lose the interaction to Ser142, but on the other hand His221 was closer than with genistein. An interaction with Tyr218 was also observed. When kaempferol was docked into the 17β-HSD1 crystal structure (PDB code 1EQU) it occupied the substrate (equilin) cavity in a binding mode where the 7-hydroxy group was towards the catalytic region (Tyr155, Ser142), and the 4-carbonyl and 5-hydroxy groups were facing Tyr218 and Ser222 (Kristan et al., 2005). The 4′-hydroxyl was close to Glu282 and His221, which was suggested as the explanation of why 4′-hydroxyflavones were the best flavonoid inhibitors.

13.3.2 17β -HSD types 2 and 4 Phytoestrogens have been found to inhibit oxidative 17β-HSD2 in PC-3 prostate cancer cells (Mäkelä et al., 1998). The most potent inhibitors of oxidation of E2 to E1 at 1.2 μM

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concentration were the flavones kaempferol, kaempferide and galangin, with E2 conversions of 27%, 32% and 49%, respectively. A C-3 hydroxy group seemed to be significant for their inhibitory activity, as its absence decreased inhibition. Coumestrol and genistein showed no significant inhibitory activity against 17β-HSD2 and thus selectivity towards 17β-HSD1 inhibition. In contrast, genistein has been shown to increase 17β-HSD2 activity and elevate levels of estrone in MCF-7 breast cancer cells (Brueggemeier et al., 2001). Similarly, genistein and coumestrol did not affect the oxidation of E2 in T-47D21 cells (Mäkelä et al., 1995). Phytoestrogens have been reported to inhibit oxidative human recombinant 17β-HSD4 (Adamski et al., 2000). The most potent inhibitors were quercetin, abietic acid and coumestrol, with IC50 values of 5, 40 and 50 μM, respectively.

13.3.3 Fungal 17β -HSDcl 17β-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is an NADPH-dependent enzyme that catalyzes the oxidation of 4-estren-17β-ol-3-one to 4-estrene-3,17-dione, and the reduction of 4-estrene-3,17-dione to 4-estren-17β-ol-3-one. 17β-HSDcl has been studied as a model enzyme for the SDR superfamily, although it possesses only 21.2% and 18.1% sequence identity with human 17β-HSD1 and 17β-HSD2, respectively. It has been suggested that this readily available enzyme could be used in preliminary screening of compounds, in a fast and low-cost spectrophotometric assay, without the need of radioactive ligands (Lanisnik Rizner et al., 1999). A total of 23 phytoestrogens and related compounds were tested against the oxidative and reductive pathways of recombinant 17β-HSDcl (Kristan et al., 2005). Most of the phytoestrogens inhibited the oxidation of 17β-hydroxy-4-estren-3-one at 100 μM concentration. The best inhibitors were 3-hydroxyflavone, 3,7-dihydroxyflavone, 5-methoxyflavone, 5,7dihydroxyflavone (chrysin) and 5-hydroxyflavone, with IC50 values of 0.4, 0.6, 0.7, 0.9 and 1.0 μM, respectively. Also flavone, quercetin, kaempferol, apigenin, coumestrol and biochanin A had IC50 values less than 10 μM. A 7-hydroxy substituent does not seem to be essential for the inhibition, as shown with 7-hydroxyflavone (IC50 value of 15 μM). The reductive pathway of 17β-HSDcl was inhibited to a lesser extent. 3-Hydroxyflavone, 5-methoxyflavone, coumestrol, kaempferol and 5-hydroxyflavone were the best inhibitors of the reduction of 4-estrene-3,17-dione, with IC50 values between 1.2 and 4.8 μM. Zearalenone showed selectivity by inhibiting only the oxidative direction, with an IC50 value of 12 μM, as did the isoflavone genistein by inhibiting the reduction with an IC50 value of 57 μM. When the structural requirements of phytoestrogens for the inhibition of 17β-HSDcl were compared to those of 17β-HSD1 and 17β-HSD2, similarities could be seen. The most potent inhibitors of the 17β-HSDcl oxidative pathway were flavones hydroxylated at C-3, C-4 or C-7. The same hydroxylation pattern is favored in the inhibition of oxidative 17β-HSD2. The most potent inhibitors of the reductive pathway were coumestrol, kaempferol and flavones hydroxylated at C-3, C-5, C-7 and C-4′. This substitution pattern was preferred in both reductive 17β-HSD1 and in oxidative 17β-HSD2. Trans-cinnamic acid esters and coumarin-3-carboxylic acid esters were tested against 17β-HSDcl because of their structural resemblance to flavonoids (Gobec et al., 2004).

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The most potent inhibitors of the two reverse enzymatic reactions were benzyl cinnamate and benzyl coumarin-3-carboxylate, with IC50 values of 0.7 and 7.0 μM for the reduction, and 2.7 and 8.0 μM for the oxidation, respectively. Phenyl cinnamate, 4-cyanophenyl cinnamate and 1-naphthyl cinnamate were found to inhibit the oxidative direction, whereas all the other substituted phenyl cinnamates tested were poor inhibitors in either direction. Cyclohexyl cinnamate was completely inactive, thus indicating the importance of the aromatic moiety. The benzyl ester of 3,4,5-trimethoxycinnamic acid was a less potent inhibitor than the unsubstituted benzyl cinnamate, with IC50 values of 11 μM for the oxidation and 144 μM for the reduction (Kristan et al., 2006). Similar results were observed with other 3,4,5-trimethoxycinnamic acid esters, the only exception being the phenyl ester of 3,4,5trimethoxycinnamic acid, which was a more potent inhibitor than the unsubstituted phenyl cinnamate. The corresponding cinnamic acid amides were found to be less active than their ester counterparts. 13.3.3.1 Binding of phytoestrogens to 17β-HSDcl A homology model for 17β-HSDcl has been created (Lanisnik Rizner et al., 2000) and verified by comparison with preliminary X-ray data of 17β-HSDcl (Cassetta et al., 2005). When kaempferol and chrysin were docked into the active site of the 17β-HSDcl model, as planar and rigid structures, each adopted two binding orientations where either the Aor the B-ring pointed towards the catalytic region residues Tyr167 and Ser153 (Kristan et al., 2005). When the binding of kaempferol to 17β-HSDcl was compared with the binding to human 17β-HSD1 (see section 13.3.1.1), it was shown that phytoestrogens occupied the steroid-binding pockets of both enzymes but seemed to bind in different orientations. 17β-HSDcl does not have the residues in the active site corresponding to Glu282 and His221 that seemed to be important for the selectivity of 17β-HSD1 towards 4′-hydroxyflavones. To further study the structural requirements of phytoestrogens and trans-cinnamates, the binding of these molecules to the active site of 17β-HSDcl has been investigated in a flexible docking and QSAR study (Sova et al., 2006). The possible binding modes of trans-cinnamates and flavonoids to 17β-HSDcl were examined. When the 30 docked conformations with lowest binding energy were superimposed, a relatively uniform binding mode and conformation were observed. For example in 3-hydroxyflavone, the 3-hydroxy group and the carbonyl group formed a hydrogen bond with Asn154. Aromatic π–π interaction between Tyr212 and the flavone B-ring and hydrophobic interactions with e.g. Val161 and Ala269 seemed to be important for the binding. The binding conformation differed from that found in the earlier study (Kristan et al., 2005), the B-ring being rotated out of the plane. Benzyl cinnamate bound to 17β-HSDcl in a similar manner, forming a hydrogen bond between the ester carbonyl and Asn154, and π–π interaction with Tyr212. Based on the docking results and biological data, a QSAR model was built, for both the reductive and oxidative pathways. The model had cross-validated r2 values of 0.69 and 0.63, respectively, and was able to predict the log(1/IC50) values of an external test set approximately within one order of magnitude. Recently, site-directed mutagenesis of several residues of 17β-HSDcl have been made to alter substrate specificity and cofactor requirements and improve catalytic activity of

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the enzyme (Kristan et al., 2007). Possible applications of these fungal mutant enzymes in the inhibition tests have yet to be studied.

13.3.4 17β -HSD type 3 Flavonoids and related compounds have been tested against crude human testes microsomal 17β-HSD3 (Le Lain et al., 2001). The crude enzyme was shown to have a similar substrate profile as recombinant 17β-HSD3. 7-Hydroxyflavone, baicalein and the isoflavone biochanin A were the most potent inhibitors, with IC50 values of 9.0, 9.3 and 10.8 μM, respectively. Diphenyl-p-benzoquinone (with an IC50 value of 2.7 μM) and phenyl-pbenzoquinone (with an IC50 value of 5.7 μM) were better inhibitors than flavonoids. The 7-hydroxycoumarins umbelliferone and 4-methylumbelliferone were found to inhibit 17β-HSD3 in the same assay, with IC50 values of 1.4 and 1.9 μM, respectively (Le Lain et al., 2002). 4-Hydroxy-, 7-methoxy- and 7-ethoxycoumarin had little or no activity against 17β-HSD3, thus indicating the significance of the 7-hydroxy group in these compounds.

13.3.5 17β -HSD type 5 Several dietary phytoestrogens have been tested on their capacity to inhibit human recombinant 17β-HSD5 (AKR1C3) in vitro (Krazeisen et al., 2001, 2002). Most of the 22 tested dietary hormones and related compounds had inhibitory effects on both the reductive and oxidative activity of 17β-HSD5 (Table 13.3). The most potential inhibitors of the reductive direction (androstenedione to testosterone) were zearalenone, coumestrol, quercetin, and biochanin A, with IC50 values of 4, 5, 9 and 14 μM, respectively. The natural estrogens daidzein and coumarin, as well as the antiestrogen tamoxifen, had no inhibitory

Table 13.3 Inhibition of oxidative and reductive directions of 17β-HSD5 by phytoestrogens with IC50 ≤ 20 μM (Krazeisen et al., 2001).

Compound Zearalenone Quercetin 7-Hydroxyflavone Biochanin A Kaempferol Abietic acid Naringenin Coumestrol Chrysin 3,7-Dihydroxyflavone Diethylstilbestrol 3-Hydroxyflavone Apigenin

IC50 (μM) for oxidation of androstanediol to androsterone 2 5 7 8 8 10 10 11 13 18 20 20

IC50 (μM) for reduction of androstenedione to testosterone 4 9 14 20 20 5 20 20

20

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effect. Surprisingly, the saponin 18β-glycyrrhetinic acid (IC50, 30 μM) inhibited the reduction of androstenedione but had no effect on the oxidative reaction. This could have been due to the pH difference (pH 9.0 for oxidation vs. pH 7.4 for reduction) in the activity measurements leading to possible deprotonation of His117 in the active site. The most potential inhibitors of the oxidative direction (androstanediol to androsterone) were zearalenone, quercetin, 7-hydroxyflavone, biochanin A and kaempferol, with IC50 values of 2, 5, 7, 8 and 8 μM, respectively. An increased degree of hydroxylation, resulting in increased hydrophilicity, seemed to improve the potency of the flavones. In addition, a 7-hydroxy group appeared to be important for the inhibition of 17β-HSD5. For both enzymatic pathways flavonoids were more potent inhibitors than isoflavonoids. The lack of the C-ring double bond in flavanones increased potency under oxidative conditions, but flavones were more potent inhibitors of the reductive direction than flavanones. When 5-hydroxyflavone was methylated into 5-methoxyflavone, the inhibition of the reductive direction was increased, but inhibition of the oxidative direction was decreased. In the isoflavone series biochanin A, with a methoxy group at the 4′-position, was a more potent inhibitor than genistein. The exact binding orientation of the inhibitors to 17β-HSD5 could not be determined from these data (Krazeisen et al., 2001, 2002), but it was suggested that these phytoestrogens bind to the hydrophilic cofactor-binding site rather than to the hydrophobic substrate-binding site. This was supported by the increased inhibitory activity with increased hydroxylation of the flavonoids, although the importance of a 7-hydroxy group also indicated binding to the substrate-binding site. A substrate or a water oxygen atom in a similar position has been found in several crystal structures of aldo-keto reductase (AKRs). Data derived from the crystal structure of 17β-HSD5 in a complex with flufenamic acid, a known inhibitor of the oxidative pathway, shows the importance of the carboxylic acid group interacting with the active-site residues Tyr55 and His117, and the cofactor nicotinamide ring (Lovering et al., 2004). These binding data combined with the activity data of flavonoids suggest that aromatic α,β-unsaturated carboxylic acids, the biosynthetic precursors of natural flavonoids, could be potential inhibitors. Cinnamic acid and its derivatives were tested against human recombinant 17β-HSD5 (Brozic et al., 2006). Cinnamic acid was found to have an IC50 value of 50 μM, whereas 3,4,5-trimethoxycinnamic acid and 3-(trifluoromethyl)cinnamic acid had similar activities, with IC50 values of 49 and 43 μM, respectively. The most potent compound in the series was α-methylcinnamic acid, with an IC50 of 6.4 μM. 4-Carboxy-, 3-hydroxy-, and 3,4-dihydroxycinnamic acids were only weak inhibitors of 17β-HSD5, indicating that polar substitution of the aromatic ring decreases the potency. Coumarin-3-carboxylic acid, a rigid derivative of cinnamic acid, was a poor inhibitor and 3-cyclohexylpropionic acid, the fully saturated derivative of cinnamic acid, failed to show any inhibitory activity. Cinnamic acids showed no inhibitory activity against fungal 17β-HSD, thus indicating specificity towards AKR enzymes.

13.3.6 Phytoestrogens as lead compounds for 17β-HSD inhibitors In addition to the previously mentioned compounds such as cinnamates, cinnamic acids and 7-hydroxyflavones, only a few drug-development projects have been reported that used phytoestrogens as lead compounds for 17β-HSD inhibition.

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Smith et al. (2001) have patented variously substituted 2-benzyltetral-1-ones as inhibitors of 17β-HSDs. These compounds can be considered to possess similar structural features to isoflavonoids. The inhibitory activity of these tetralones was tested against human testes microsomal 17β-HSD3. 4′-Chloro-6-hydroxy-2-benzyl-1-tetralone was the most active compound in the series, with an IC50 value of 1.8 μM. Corresponding 4′-fluoro and 4′-bromo derivatives showed reasonable activity, with IC50 values of 7.0 and 8.3 μM, respectively. The same compounds were also tested against 17β-HSD1. Tetralone derivatives were less potent inhibitors of type 1 enzyme, 4′-chloro-, 4′-fluoro- and 4′-bromo-2-benzyl-6hydroxytetral-1-one having IC50 values of 14.8, 21.0 and 14.6 μM, respectively. Yoshihama et al. (2000) have patented tetralone and benzopyranone derivatives as inhibitors of 17β-HSD. The best inhibitor in the series was 2-(3′,4′-dihydroxy)benzylidene substituted 7-hydroxybenzopyranone, with 58% inhibition of 17β-HSD1 activity in human placental microsomes (unspecified inhibitor concentration). The corresponding tetralone derivative showed 50% inhibition. Hydroxy substitution in the aromatic side-chain was not crucial for the activity, as 3′-hydroxy and 4′-hydroxy derivatives were only weak inhibitors of 17β-HSD1, and the unsubstitued 2-benzylidene-7-hydroxybenzopyranone showed 44% inhibition.

13.4 Conclusions Natural phytoestrogens possess anti-aromatase activities ranging from high nanomolar to high micromolar concentrations. The most potent natural phytoestrogen tested so far is α-naphthoflavone, with IC50 values of 0.07 μM in HPM-based assay and 2.2 μM in cellbased assay. Other active inhibitors include isolicoflavonol, apigenin, and 7-hydroxyflavone. All of the most active compounds are flavones, which as a compound class are superior to other phytoestrogens as aromatase inhibitors. Some of the tested natural phytoestrogens are racemic mixtures of two enantiomers. The biological data from these racemic mixtures should be applied with caution. These mixtures are seldom useful in lead generation or ligand-based drug discovery methods, since the process requires the exact 3D structures of the compounds under study. Comparison of the reported inhibition data between research groups should also be made with caution, owing to the various different experimental conditions. Phytoestrogens have successfully been used as lead compounds in developing potent aromatase inhibitors. Modification of the chromone ring system has led to the powerful inhibitor (R)-3-[imidazol-1-yl-(4-nitrophenyl)-methyl]-chromen-4-one, with an IC50 value of 0.0023 μM. The most active derivative of a natural phytoestrogen is a 2-(imidazol-1ylmethyl)-4′-nitroflavone, with an IC50 of 0.045 μM. Based on the studies presented here, it can be concluded that the following structural features in the chroman ring system results in effective aromatase inhibition: l

l l

an unhindered heteroatom with a lone electron pair (preferably an imidazole ring) capable of coordinating to the heme iron; an aromatic moiety at C-2 or C-3 in the chroman skeleton; and in isoflavone-based inhibitors, an aromatic moiety at C-7 and in flavone-based inhibitors, an unsubstituted C-7.

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Phytoestrogens and related compounds have been shown to inhibit multiple forms of 17β-HSD. The most-studied human isoforms so far have been 17β-HSD1 and 17β-HSD5. The biological activity of the most potent phytoestrogens is usually in the low to high micromolar range. Within all the isoforms, the most potent inhibitor is coumestrol against 17β-HSD1, with an IC50 value of 0.2 μM. Structural requirements of phytoestrogens for inhibition vary between the different 17β-HSD isoforms, suggesting some selectivity. For example, selectivity between reductive 17β-HSD1 and oxidative 17β-HSD2 is an essential property for a clinically valid candidate. Recently, 17β-HSDcl has been proposed as a model enzyme for inhibition studies. Compared to aromatase inhibitors, only a few efforts have been made to use phytoestrogens as lead compounds for 17β-HSD inhibitors. For 17β-HSD1 there are several crystal structures available and the binding of phytoestrogens to the active site has been studied with molecular modeling. In addition, biological data for diverse phytoestrogens are available. Together these data provide a good starting point for alteration of the structures for increased activity, selectivity, and pharmacokinetic properties. The estrogenic properties and lack of selectivity of many natural phytoestrogens, for example coumestrol and genistein, prohibit their use as such as inhibitors of estradiol biosynthesis. However, their chemical structures and biological activities make them attractive lead compounds in drug discovery.

13.5 Acknowledgments Funding by The Finnish Academy (grant 205781) is gratefully acknowledged.

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Recanatini, M. & Cavalli, A. (1998). Comparative molecular field analysis of non-steroidal aromatase inhibitors: an extended model for two different structural classes. Bioorganic and Medicinal Chemistry, 6, 377–388. Saarinen, N., Joshi, S.C., Ahotupa, M. et al. (2001). No evidence for the in vivo activity of aromatase-inhibiting flavonoids. Journal of Steroid Biochemistry and Molecular Biology, 78, 231–239. Sanderson, J.T., Hordijk, J., Denison, M.S., Springsteel, M.F., Nantz, M.H. & van den Berg, M. (2004). Induction and inhibition of aromatase (CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells. Toxicological Sciences, 82, 70–79. Simpson, E.R., Mahendroo, M.S., Means, G.D. et al. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews, 15, 342–355. Smith, H.J., Mason, P., Ahmadi, M., Nicholls, P.J. & Greer, V. (2001). Benzyl tetralins, formulations and uses thereof. PCT patent application WO0142181 Sova, M., Perdih, A., Kotnik, M. et al. (2006). Flavonoids and cinnamic acid esters as inhibitors of fungal 17β-hydroxysteroid dehydrogenase: A synthesis, QSAR and modelling study. Bioorganic and Medicinal Chemistry, 14, 7404 –7418. Stresser, D.M., Turner, S.D., McNamara, J. et al. (2000). A high-throughput screen to identify inhibitors of aromatase (CYP19). Analytical Biochemistry, 284, 427–430. Su, B., Hackett, J.C., Diaz-Cruz, E.S., Kim, Y.-W. & Brueggemeier, R.W. (2005). Lead optimization of 7-benzyloxy 2-(4′-pyridylmethyl)thio isoflavone aromatase inhibitors. Bioorganic and Medicinal Chemistry, 13, 6571– 6577. Vihn, T.K., Nicholls, P.J., Kirby, A.J. & Simons, C. (2001). Evaluation of 7-hydroxy-flavones as inhibitors of oestrone and oestradiol biosynthesis. Journal of Enzyme Inhibition, 16, 417–424. Wang, C., Mäkelä, T., Hase, T., Adlercreutz, H. & Kurzer, M.S. (1994). Lignans and flavonoids inhibit aromatase enzyme in human preadipocytes. Journal of Steroid Biochemistry and Molecular Biology, 50, 205 –212. Whitehead, S.A., Cross, J.E., Burden, C. & Lacey, M. (2002). Acute and chronic effects of genistein, tyrphostin and lavendustin A on steroid synthesis in luteinized human granulosa cells. Human Reproduction, 17, 589–594. Whitehead, S.A. & Lacey, M. (2003). Phytoestrogens inhibit aromatase but not 17β-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: Evidence for FSH induction of 17β-HSD. Human Reproduction, 18, 487–494. Yoshima, M., Nakakoshi, M., Nakamura, J. & Nakayama, S. (2000). Tetralone or benzopyranone derivatives and a method for producing them. US patent US6080781

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Chapter 14

Recent Advances in the Chemical Synthesis and Biological Activity of Phenolic Metabolites Denis Barron

14.1 Introduction The phenolic compounds that are contained in food are susceptible to extensive metabolism in the body. A precise characterization of the resulting metabolites is very important, since they represent the true forms to which the target organs and cells will be exposed. With the development of sensitive and high-performing chromatographic and spectrophotometric analytical tools (electrochemical detection, various LC-MS coupling techniques), the number of phenolic metabolites that have been detected in mammalian biological fluids has increased explosively over the last few years. However, whereas the mass-spectrometric identification of conjugates of ‘simple’ compounds with only one possible position of conjugation is straightforward, the situation is more complex in the case of conjugates of polyhydroxy compounds. In the flavonoid series, it is generally possible, on the basis of LC-MS analysis, to distinguish between conjugation on the A- and B-rings. In contrast, no distinction can be made between conjugation at positions 3′ and 4′ of the B-ring, or between positions 5 and 7 of the A-ring. Only access to fully identified standard conjugates would allow a clear distinction to be made between these position isomers. The availability of pure standard compounds is also mandatory when an exact quantification of the conjugates is required. Concerning the last point, it is noteworthy that in most of the studies, neither quantitative, nor qualitative analyses of the conjugates have been performed. In fact, plasma or urine samples are very commonly pre-treated with deconjugating enzymes and only the aglycones are analyzed. This situation arises from two main causes: (1) the absence of commercially available pure conjugates; and (2) the fact that the conjugates are extremely polar, and sometime unstable compounds (sulfates) that are difficult to extract from complex matrices like blood. Furthermore, it is already well established that some sulfate conjugates are resistant to hydrolysis with aryl sulfatase (Barron & Ibrahim, 1988a; Kern et al., 2003). On the other hand, isoflavone phosphates have been shown to be substrates of this enzyme (Kanakubo et al., 2001). Therefore assessment by chromatographic analysis of the nature of a conjugate on the sole basis of the disappearance of its peak after enzyme treatment could be debatable. Good access to a wide range of standard conjugates would be of great use for optimizing proper extraction Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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conditions, and for investigating in detail the substrate specificities of deconjugating enzymes. Beside all the problems already mentioned in relation to the reliable identification of conjugates, investigation of their biological properties is another area in which the availability of good standards is essential. Most biological studies require a minimum amount of compound, and adequate isolation or production of the conjugates – either by extraction from the biological fluids or by enzymatic synthesis – is rarely attainable. Although phenolic sulfate esters and glucuronides are naturally occurring plant products, the use of conjugates extracted from natural plant sources is usually not an option for mammalian studies because the most common positions of sulfation or glucuronidation in the plant kingdom often differ from those encountered in mammalian metabolites (Williamson et al., 2005). Thus only chemical synthesis of the conjugates would provide access to sufficient quantities of the compounds. The purpose of this chapter is to review, through a number of examples, the latest advances in the chemical synthesis of phenolic metabolites and in the knowledge of their biological activity. In this review, emphasis will be placed on the conjugated sulfated and glucuronidated forms of phenolic compounds. The metabolites resulting from the gut microbial transformations before conjugation will not be exhaustively covered. Selected examples will be assessed in more detail in order to develop specific themes.

14.2 The different sites of generation of phenolic metabolites 14.2.1 The saliva Surprisingly, little is known about the fate of flavonoid glycosides in the oral cavity. An early publication (Macdonald et al., 1983) reported that rutin, but not quercitrin (quercetin 3-O-rhamnoside) could be hydrolyzed by the human oral flora. The study, however, was performed on solutions of flavonol glycosides, which could be different from the real conditions in a food matrix. Incubation of a methanolic extract of onions with human saliva resulted in the deglucosylation of flavonoid glucosides (Hirota et al., 2001). The hydrolysis was faster with quercetin 4′-O-glucoside, but after 1 hour’s incubation less than 50% of the glucoside had been degraded. This hydrolysis could in practice be negligible, taking into account the usual chewing time in the mouth. However, the authors demonstrated that the flavonoids in onion soup bind to the epithelial tissues of the oral cavity, from where they are slowly released into the saliva. The hydrolysis of a more complete collection of flavonoid glycosides by human saliva has recently been carried out (Walle et al., 2005). Phloretin 2′-O-glucoside (phoridzin), genistein 7-O-glucoside (genistin), quercetin 3-O-glucoside (isoquercitrin), and quercetin 4′-O-glucoside (spiraeoside) were efficiently hydrolyzed, the latter being by far the best substrate. Rutin was hydrolyzed to a very slight extent, while quercetin 3-O-rhamnoside (quercitrin) and naringenin 7-O-rutinoside (naringin) were not hydrolyzed at all. This hydrolysis was mainly mediated by the oral bacterial flora. However a contribution by the oral epithelial cells contained in the saliva was also suspected. 17–24% of quercetin 4′-O-glucoside was hydrolyzed to quercetin after being held in the mouth for 5 min. The

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overall hydrolytic activity of human saliva was shown to be subject to large interindividual difference (Walle et al., 2005). Finally it has recently been demonstrated that some flavonoid glycosides, like genistein 7-O-glucoside or quercetin 4′-O-glucoside (but not quercetin 3-rhamnoside), are absorbed and hydrolyzed to their aglycones by oral squamous carcinoma SCC-9 cells (Browning et al., 2005). Based on the available data, it seems that hydrolysis of flavonoid glycosides in the mouth is limited to glucose conjugates, and that obviously this process is highly influenced by the nature of the food matrix, as well as by individual variations, as a result of which the importance of oral hydrolysis is difficult to assess.

14.2.2 The stomach Being acidic, the gastric milieu could potentially catalyze two main types of transformation, i.e. the depolymerization of procyanidins, and the hydrolysis of glycosides. The first report on the stability of procyanidins in simulated gastric conditions stated that procyanidin oligomers were cleaved to mixtures of monomers and dimers (Spencer et al., 2000). The study, however, was performed on procyanidin solutions and did not take into account the environment created by a food matrix. A further study (Rios et al., 2002), conducted on a cocoa beverage, concluded that on the contrary, procyanidins were stable in the environment of the human stomach. The latter result was confirmed by further studies in rats (Tsang et al., 2005). Similarly, although quercetin aglycone could be efficiently absorbed by the stomach this was not the case for its glycosides, and the latter were found not to be hydrolyzed in a gastric buffer of pH 4.5 (Crespy et al., 2002). A subsequent study confirmed the stabilities of quercetin 3-O-glucoside and 3-O-galactoside in the rat stomach (Chang et al., 2005).

14.2.3 The small intestine The small intestine is the primary site of absorption of some flavonoid glucosides. It has been shown that human lactase-phlorizin hydrolase (LPH) is able to hydrolyze glucosides of flavones, flavonols, flavanones and isoflavones (Nemeth et al., 2003). This enzyme is membrane bound and is located on the luminal surface of enterocytes. Thus the aglycone is released and immediately absorbed by the enterocyte, presumably by passive diffusion. An alternative route is possible but so far this has been mainly demonstrated for quercetin 4′-O-β-d-glucoside. This route involves the transport of the glucoside by the sodiumdependent glucose transporter SGLT1 (Hollman et al., 1999; Gee et al., 2000; Walgren et al., 2000a; Day et al., 2003), followed by its hydrolysis inside the enterocyte by the intracellular human cytosolic β-glucosidase. The substrate specificity of the latter enzyme is very broad (Berrin et al., 2002; Nemeth et al., 2003), but obviously its activity is limited to the glycosidic substrates that can be transported into the enterocyte. Furthermore quercetin and genistein glucosides are submitted to an intense intestinal efflux mechanism, probably mediated by the MRP-2 transporter (Walle et al., 1999; Walgren et al., 2000b), thus almost completely abolishing the intestinal absorption of these glucosides. The part of the quercetin that has been absorbed by the intestine is immediately conjugated. In man the

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intestine mainly catalyzes the glucuronidation and/or sulfation of quercetin. Using the insitu intestinal perfusion model in rats, quercetin was shown to be glucuronidated, methylated, or sulfated (Crespy et al., 1999). A later study confirmed that methylation and glucuronidation of quercetin take place in the intestine (van der Woude et al., 2004). Epicatechin and catechin were also shown to be methylated and/or glucuronidated by the jejunum part of the small intestine in rats. Presumably, O-methylation of the B-ring of catechin and epicatechin is catalyzed by catechol-O-methyltransferases (Kuhnle et al., 2000), which are known to be present in the enterocytes. However glucuronidation is believed to be the major conjugation process of the small intestine (Donovan et al., 2001). Curcumin glucuronide, sulfate and hexahydrocurcumin have been shown to be generated in the human small intestine (Ireson et al., 2001, 2002). In the synthesis of the latter two metabolites, the human phenol sulfotransferases SULT1A1 and SULT1A3, and alcohol dehydrogenase, could be involved.

14.2.4 The colon A number of compounds are too big to be absorbed by the small intestine, and/or cannot by degraded into smaller molecules by the enzymes that are present in the small intestine. Thus these compounds reach the colon, where they can be extensively modified by the colonic microflora. This is the case for flavonoid rhamnoglucosides like naringin or rutin, complex catechins from black tea, some hydroxycinnamic acid esters, or ellagitannins like punicalagin from pomegranate juice, to mention few examples. The structures of these metabolites are discussed in more detail in section 14.3.

14.2.5 The liver The liver further metabolizes the quercetin conjugates formed in the intestine, leading to a more complex conjugation pattern and accounting for the presence in the plasma of glucurono-sulfoconjugates of quercetin with possible methylation (Morand et al., 1998; Mullen et al., 2002, 2003; Graf et al., 2005). Using human hepatoma HepG2 cells, it was demonstrated that quercetin 7- and 3-glucuronides were either methylated on the B-ring, or deglucuronidated and further sulfated to quercetin 3′-O-sulfate (O’Leary et al., 2001, 2003). In man, the liver is a site of glucuronidation for quercetin and luteolin, but it appears that the regioselectivities of the UDP-glucuronosyl transferases differ between the liver and the intestine. In fact, incubation of quercetin with human liver microsomes resulted in the formation of quercetin 7-O-glucuronide as a major metabolite, while incubation with human intestine microsomes led to the formation of the 3′- and 4′-O-glucuronides as major products (Boersma et al., 2002). In man the liver acts on quercetin mainly as a site of methylation and glucuronidation, whereas in rats, it is a site of glucuronidation and sulfation (van der Woude et al., 2004). The liver is a site of extensive sulfation and additional methylation of catechin in rats (Donovan et al., 2001). After hydrolysis of the chlorogenic acids in the colon, the liver was shown to methylate the absorbed caffeic acid into ferulic and isoferulic acids (Mateos et al., 2006). The liver is also a major site of curcumin transformation (Ireson et al., 2001), leading to the reduced compounds hexahydrocurcumin and hexahydrocurcuminol.

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14.2.6 Metabolism at the target tissues or cells Very little information is currently available on this topic. It has been shown that isoflavones can be metabolized by, or in the vicinity of, tumor cells. Peterson et al. showed that breast cancer MCF-7 cells demethylate biochanin A (4′-O-methylgenistein) to genistein, and the latter was subsequently sulfated to genistein 7-O-sulfate (Peterson et al., 1996). On the other hand tumors are often invaded by inflammatory cells, generating a number of proinflammatory oxidants and leading to the formation of chlorinated (Boersma et al., 2001) or brominated derivatives of genistein (see also section 14.5.1.5).

14.3 Nature of the metabolites of phenolic compounds A plethora of reports is available on the evidence for the presence of phenolic conjugates in biological fluids using LC-MS technology. However, only in a very few cases have the metabolites been fully characterized, especially in terms of the position of conjugation; only those structures that have been unequivocally established are presented in this section.

14.3.1 Flavone and flavonol metabolites The structures of the flavone and flavonol conjugates that have been unambiguously identified are summarized in Fig. 14.1. Quercetin 3-glucuronide (2) has been identified as an intestinal metabolite in rats (Gee et al., 2000). Incubation of quercetin (1) or isorhamnetin (8) with human liver cell-free extracts in the presence of uridine 5′-diphosphoglucuronic acid (UDPGA) gave rise to four and three major glucuronides, respectively (Day et al., 2000). Quercetin 3-O-β-d-glucuronide (2) was identified by comparison with an authentic standard isolated from green bean. All the remaining glucuronides were identified by comparison with standards generated by incubation of quercetin or isorhamnetin with a crude microsomal extract of bovine UDP-glucuronosyltransferase. Although all the identifications were based on UV analysis coupled with the use of shift reagents only, quercetin 7-, 3′-, and 4′-glucuronides (3–5), as well as isorhamnetin 3-, 7-, and 4′-glucuronides (9–11) were identified. Subsequently, a more complete characterization of the conjugates resulting from the incubation of quercetin and luteolin with human microsomal preparations was carried out. The use of 1H NMR resulted in the identification of quercetin 3-, 7-, 3′-, and 4′glucuronides (2–5), and of luteolin 7-, 3′-, and 4′-glucuronides (21–23) (Boersma et al., 2002). Quercetin 4′-O-β-d-glucuronide (5) has also been identified in rat plasma, by comparison with a synthetic reference compound (Moon et al., 2001). The presence of quercetin 3′-O-sulfate (6) has been detected in human plasma, following consumption of onions, again by comparison with a synthetic standard (Day et al., 2001). The metabolites resulting from the incubation of quercetin or isorhamnetin with intestine or liver microsomal preparations from rats or humans have been identified in detail by 1H NMR spectroscopy (van der Woude et al., 2004). These were 3′-O-methylquercetin (isorhamnetin (8)), 4′-Omethylquercetin (tamarixetin (14)), quercetin 3′- and 7-O-sulfates (6 & 7), isorhamnetin 7- and 4′-O-sulfates (12 & 13), tamarixetin 7- and 3′-O-sulfates (18 & 19), and tamarixetin 3-, 7-, and 3′-O-glucuronides (15–17). The main metabolites present in human plasma

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OR2 R1O

O

OR3 R4

Gla = β-D-glucuronic acid

OH O 1: R1 = R2 = R3 = H, R4 = OH: Quercetin 2: R1 = R2 = R3 = H, R4 = OGla: Quercetin 3-O-β-D-glucuronide 3: R1 = Gla, R2 = R3 = H, R4 = OH: Quercetin 7-O-β-D-glucuronide 4: R1 = R2 = H, R3 = Gla, R4 = OH: Quercetin 3′-O-β-D-glucuronide 5: R1 = R3 = H, R2 = Gla, R4 = OH: Quercetin 4′-O-β-D-glucuronide 6: R1 = R2 = H, R3 = SO3–, R4 = OH: Quercetin 3′-O-sulphate 7: R1 = SO3–, R2 = R3 = H, R4 = OH: Quercetin 7-O-sulphate 8: R1 = R2 = H, R3 = Me, R4 = OH: Isorhamnetin 9: R1 = R2 = H, R3 = Me, R4 = OGla: Isorhamnetin 3-O-β-D-glucuronide 10: R1 = Gla, R2 = H, R3 = Me, R4 = OH: Isorhamnetin 7-O-β-D-glucuronide 11: R1 = H, R2 = Gla, R3 = Me, R4 = OH: Isorhamnetin 4′-O-β-D-glucuronide 12: R1 = SO3–, R2 = H, R3 = Me, R4 = OH: Isorhamnetin 7-O-sulphate 13: R1 = H, R2 = SO3–, R3 = Me, R4 = OH: Isorhamnetin 4′-O-sulphate 14: R1 = R3 = H, R2 = Me, R4 = OH: Tamarixetin 15: R1 = R3 = H, R2 = Me, R4 = OGla: Tamarixetin 3-O-β-D-glucuronide 16: R1 = Gla, R2 = Me, R3 = H, R4 = OH: Tamarixetin 7-O-β-D-glucuronide 17: R1 = H, R2 = Me, R3 = Gla, R4 = OH: Tamarixetin 3′-O-β-D-glucuronide 18: R1 = SO3–, R2 = Me, R3 = H, R4 = OH: Tamarixetin 7-O-sulphate 19: R1 = H, R2 = Me, R3 = SO3–, R4 = OH: Tamarixetin 3′-O-sulphate 20: R1 = R2 = R3 = R4 = H: Luteolin 21: R1 = Gla, R2 = R3 = R4 = H: Luteolin 7-O-β-D-glucuronide 22: R1 = R2 = R4 = H, R3 = Gla: Luteolin 3′-O-β-D-glucuronide 23: R1 = R3 = R4 = H, R2 = Gla: Luteolin 4′-O-β-D-glucuronide Fig. 14.1 Structures of the fully identified flavone and flavonol conjugates.

after consumption of onions were quercetin 3-O-β-d-glucuronide (2) and 3′-O-sulfate (6), and isorhamnetin 3-O-β-d-glucuronide (9) (Day & Williamson, 2001). As mentioned in section 14.2.4 the flavonol glycosides like rutin, which are not absorbed at the small intestine level, are degraded by the colon microflora. In fact, in-vitro degradation of rutin or quercetin by human fecal bacteria yielded 3,4-dihydroxybenzaldehyde (24), 3,4-dihydroxyphenylacetic (25), 3-hydroxyphenylacetic acid (26) (or its 4-isomer) and 3-hydroxyphenylpropionic acid (29) (or its 4-isomer) (Justesen et al., 2000; Rechner et al., 2004) (Fig. 14.2).

14.3.2 Flavanonone and flavanonol metabolites The biliary metabolites of orally administrated hesperetin (39) (Fig. 14.3) in rats have been identified as the 7-O-β-d-glucuronide (40), the 3′-O-β-d-glucuronide (41), the 3′-Oβ-d-glucuronide-7-O-sulfate (42), and the 7,3′-di-O-sulfate (43) of hesperetin (Abe et al., 1993). Hesperetin 7- and 3′-O-β-d-glucuronides (40 & 41) have been identified in rat plasma after oral administration of hesperidin (38) (the natural hesperetin 7-O-rhamnoglucoside present in Citrus) (Matsumoto et al., 2004). However most of the hesperidin is not absorbed in the small intestine and is fermented in the colon. The identified metabolites are close

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Fig. 14.2 Examples of metabolites resulting from the colon microbial degradation of flavonols, flavanones, flavan 3-ols, ellagic tannins, and lignans.

Fig. 14.3 Structures of various flavanone and flavanonol conjugates.

to those resulting from the fermentation of rutin, i.e. 3- (or 4-) hydroxyphenylpropionic acid (29), 3-hydroxy-4-methoxyphenylacetic acid (27), and 3-hydroxy-4-methoxyphenylpropionic acid (30) (Fig. 14.2) (Justesen et al., 2000). Silybin 20-glucuronide (45) (Fig. 14.3) has been identified in human plasma after consumption of capsules of silymarin, an extract rich in silybin (44). Identification was

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Fig. 14.4 Structures of (–)-epicatechin and its identified conjugates.

performed by comparison with a mixture of standard silybin glucuronides produced by the incubation of silybin with ovine liver glucuronyl transferase. All the conjugates were fully characterized by 1H- and 13C NMR spectroscopy (Kren et al., 2000).

14.3.3 Flavan 3-ol metabolites Three (−)-epicatechin metabolites have been purified from rat urine and characterized by NMR analysis (Fig. 14.4): 3′-O-methyl-(−)-epicatechin (49), (−)-epicatechin 7-O-β-dglucuronide (47), and 3′-O-methyl-(−)-epicatechin 7-O-β-d-glucuronide (50) (Natsume et al., 2003). The same study led to the isolation and identification of three (−)-epicatechin metabolites from human urine: (−)-epicatechin 3′-O-β-d-glucuronide (48), 4′-O-methyl(−)-epicatechin 3′-O-β-d-glucuronide (51), and 4′-O-methyl-(−)-epicatechin 5- or 7-O-βd-glucuronide. The corresponding compounds were also found in the blood of rats and humans. After administration of cocoa to healthy male subjects, a number of epicatechin- and 4′-O-methylepicatechin O-glucuronides have recently been identified in plasma using authentic standard compounds (Schroeter et al., 2006). More complex flavan 3-ols like epigallocatechin gallate of tea are not totally absorbed at the small intestine level and are in part fermented in the colon. Among the microbial metabolites, phenylpropionic acid (28), as well as 3-hydroxyphenyl-, 3,4-dihydroxyphenyl, and 3,4,5-trihydroxyphenylvalerolactones (31–33) (Fig. 14.2), have been identified (Li et al., 2000, 2001; Lee et al., 2002; Meng et al., 2002; Olthof et al., 2003).

14.3.4 Ellagic tannin metabolites Pomegranate juice is very rich in the antioxidant ellagitannin punicalagin. After consumption of pomegranate juice by humans, the presence of urolithin A (34) (Fig. 14.2) was detected in urine, while its glucuronide (35) was detected in plasma (Cerda et al., 2004). Although a structure was proposed for the conjugate, real evidence for the exact position of glucuronidation has yet to be obtained. Urolithin A is a product of metabolism by the human colon microflora, which after absorption gets glucuronidated.

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14.3.5 Lignan metabolites Plant lignans are converted into enterolactone (36) and enterodiol (37) (Fig. 14.2) by the colonic microbes (Heinonen et al., 2001). It has been shown that, after absorption these two colonic metabolites can undergo further oxidative metabolism, yielding small amounts of hydroxylated derivatives in rats (Niemeyer et al., 2000).

14.3.6 Isoflavone metabolites Following the discovery of the biological properties of equol, one of the main microbial metabolite of isoflavones, a lot of attention has been paid to the fermentation products of isoflavones. All the structures discussed in this section are displayed in Fig. 14.5. Incubation of daidzein (61) with human feces yielded dihydrodaidzein (54), benzopyran4,7-diol, 3-(4-hydroxyphenyl) (56) and equol (57). Similar incubation of genistein (62) only afforded dihydrogenistein (55) (Chang & Nair, 1995). In the rat, genistein is

Fig. 14.5 Structures of the isoflavone metabolites, including microbial products.

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extensively metabolized by the gut flora into dihydrogenistein (55), 6′-hydroxy-Odesmethylangolensin (60), and 2-(4-hydroxyphenyl)-propionic acid (52) (Coldham et al., 1999). In-vitro incubation of daidzein with rat liver microsomes resulted in the formation of a complex mixture of oxidized products, among which 6,7,4′-trihydroxy-, 7,8,4′trihydroxy-, 5,6,7,4′-tetrahydroxy-, and 6,7,3′,4′-tetrahydroxy-isoflavones (63, 64, 66 & 67) were the major compounds. Similar incubation of genistein yielded 5,6,7,4′- and 5,7,8,4′tetrahydroxyisoflavones (66 & 68) as major compounds (Kulling et al., 2000). Human liver microsomes produced 6-hydroxy- and 3′-hydroxyequol (58 & 59) from equol (57) (Ruefer et al., 2006). Compounds 63–65, 67, and 69 were produced from daidzein by human liver microsomes, and found in the urine of volunteers after soy intake. Compounds 66, 68, and 70 were formed from genistein by human liver microsomes and detected in urine (Kulling et al., 2001). In urine, these metabolites were likely to be present as glucuronides and/or sulfate conjugates, but no information on the structures of the real conjugated forms was given since all the samples were enzymatically hydrolyzed (by sulfatase and/or glucuronidase) before being analyzed.

14.3.7 Hydroxycinnamic acid metabolites The structures of these metabolites are shown in Fig. 14.6. Hydroxycinnamic acid esters are hydrolyzed, at least in part, by the colonic microflora (Plumb et al., 1999; Couteau et al., 2001; Rechner et al., 2002). In the case of 5-O-caffeoylquinic acid (71), the released quinic acid could be further aromatized into benzoic acid (Adamson et al., 1969), and after absorption of the latter and conjugation with glycine in the kidney (Quick, 1931), excreted in urine as hippuric acid (72). On the other hand the released caffeic acid (77) could be absorbed, or again metabolized by the microflora. The main transformations were

Fig. 14.6 Structures of the hydroxycinnamoyl derivatives and their metabolites, including microbial products.

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dehydroxylation at the 4′-position and reduction of the side-chain double bond, giving rise to a mixture of m-coumaric (78), dihydrocaffeic, 3-hydroxyphenylpropionic, and phenylpropionic acids (76, 75 & 74) (Rechner et al., 2004). After absorption of the two latter compounds by the colon, followed by β-oxidation and glycination in the liver, hippuric acid (72; Graefe & Veit, 1999) or m-hydroxyhippuric acid (73; Booth et al., 1957) are formed and excreted in human urine. Thus hippuric acid can be generated from either the quinic acid part or the caffeic acid part of chlorogenic acid, which explains why hippuric acid is the major metabolite of chlorogenic acid in human urine (Olthof et al., 2003). Of course the metabolites resulting from colonic microbial transformation are absorbed and conjugated in the liver (Rechner et al., 2002). Therefore they circulate in the plasma in conjugated forms with sulfate and/or glucuronic acid. The unambiguous structure determination of the conjugate is straightforward and can be directly carried out by mass spectrometric analysis only when one phenolic hydroxyl is available for conjugation, as for example in coumaric, ferulic, or 3-hydroxyphenylpropionic acids. A number of metabolites of rosmarinic acid (82) have been isolated and identified from the urine of rats fed with this compound. These were caffeic acid (77), m-coumaric acid (78), 3-hydroxyphenylpropionic acid (75), caffeic acid 4-O-sulfate (79), m-coumaric acid 3-O-sulfate (81), and ferulic acid 4-O-sulfate (80) (Nakazawa & Ohsawa, 1998).

14.3.8 Curcumin metabolites The structures are summarized in Fig. 14.7. After administration of curcumin (83) to mice, the presence of dihydrocurcumin and tetrahydrocurcumin glucuronides (86 & 87) was

Fig. 14.7 Structures of curcumin and its metabolites.

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Fig. 14.8 Structures of resveratrol and its conjugates.

detected in plasma by MS/MS analysis (Pan et al., 1999). Rat and human hepatocytes metabolized curcumin into two major compounds – hexahydrocurcumin (88) and octahydrocurcumin (hexahydrocurcuminol) (89) – and two minor ones – curcumin O-glucuronide (84) and curcumin O-sulfate (85). All these conjugates were also identified in rat plasma, together with hexahydrocurcumin glucuronide (although in the latter case the position of glucuronidation was not assigned). However here the curcumin conjugates were the most abundant, while all the reduced forms were detected in small amounts (Ireson et al., 2001).

14.3.9 Resveratrol metabolites The structures of resveratrol and its conjugates are shown in Fig. 14.8. Incubation of transresveratrol (90) with human liver microsomes in the presence of UDP-glucuronic acid led to the formation of the corresponding 3-O- and 4′-O-glucuronides (91 & 92) (Aumont et al., 2001). The compounds were identified by electrospray LC-MS and by mono- and two-dimensional 1H NMR experiments after individual isolation of the metabolites by HPLC. Human liver cytosol, in the presence of 3′-phosphoadenosine-5′-phosphosulfate, catalyzed the formation of trans-resveratrol 3-O-sulfate (93), 4′-O-sulfate (94), and 3,4′-di-O-sulfate (95) (Miksits et al., 2005). The conjugates were characterized by LC-MS and 1D- and 2D-NMR experiments. In addition to the above-mentioned sulfate conjugates, transresveratrol 3,5-di-O-sulfate (96), and 3,5,4′-tri-O-sulfate (97) were detected in Wistar rats fed with trans-resveratrol. The nature of the conjugates was determined by comparison with fully identified synthetic reference compounds (Wenzel et al., 2005).

14.4 The chemical synthesis of phenolic conjugates The subject of the chemical synthesis of flavonoid conjugates has been covered by two recent reviews (Barron et al., 2003; Williamson et al., 2005). Only the recent work will be covered in this section.

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Scheme 14.1 The synthesis of quercetin- and isorhamnetin glucuronides (adapted from Needs & Kroon, 2006).

14.4.1 The preparation of O-glucuronides The problems associated with the use of the commercial glucuronic acid donor methyl 2,3,4-tri-O-acetyl-α-d-glucopyranosyluronate bromide (100) (Scheme 14.1) are well known; it often gives very low glucuronidation yields. However one advantage of this reagent is its compatibility with partially protected precursors like 7,4′-di-O-benzylquercetin (98) and isorhamnetin (99), respectively. Although the 3, 5, and – in the case of O-benzylquercetin – the 3′-phenolic hydroxyls are free, when performed in appropriate conditions (Ag2O and dried CaSO4), glucuronidation affected position 3 with some selectivity (around 50%), yielding the protected 3-O-β-d-glucuronides (101) and (102) (Scheme 14.1). Thus, after removal of the benzyl groups by catalytic hydrogenation, and of the acetates by saponification, quercetin- and isorhamnetin 3-O-β-d-glucuronides (105 & 106) were isolated in 41% and 33% yields from their respective benzylated precursors (98 & 99) (Needs & Kroon, 2006). In contrast, the direct glucuronidation of resveratrol with methyl 2,3,4-tri-O-acetyl-α-d-glucopyranosyluronate bromide (100) was not regioselective and of poor yield, giving rise to a mixture of resveratrol 3- and 4′-O-β-d-glucuronides in 18% and 13% yields, respectively (Wang et al., 2004). On the other hand trichloroacetimidates are much more efficient glycosylation donors than acetobromosugars, but surprisingly, this kind of reagent has only been applied much more recently to glucuronidation reactions (Stachulski & Jenkins, 1998). Trichloroacetimidates are non-commercial reagents, but access to them is quite easy. In contrast to acetobromosugars, however, glucuronidation with trichloroacetimidates requires fully protected precursors, except at the position to be glucuronidated. In fact, direct glucuronidation

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Scheme 14.2

The synthesis of p-coumaric acid 4-O-β-d-glucuronide (adapted from Pearson et al., 2005).

of the partially protected 7,4′-di-O-benzylquercetin (98) with methyl 2,3,4-tri-O-acetylα-d-glucopyranosyluronate trichloroacetimidate (108), only resulted in the isolation of quercetin 3′-O-β-d-glucuronide in poor (11%) yield, despite the fact that neither 3- nor 5- competitive glucuronidations took place (Needs & Kroon, 2006). In contrast, glucuronidation of p-coumaric acid methyl ester (107) (Scheme 14.2), having only one available phenolic hydroxyl, gave rise to protected p-coumaric acid glucuronide (109) in very good (83%) yield, and to p-coumaric acid glucuronide (110) in 70% yield after saponification (Pearson et al., 2005). Methyl 2,3,4-tri-O-acetyl-α-d-glucopyranosyluronate trichloroacetimidate (108) has been applied to much more efficient regioselective syntheses of resveratrol glucuronides (Learmonth, 2003) (Schemes 14.3 and 14.4). In order to achieve the regioselectivity of the reactions, a total synthesis of the resveratrol skeleton was performed, starting from suitable precursors: 3,5-diacetoxystyrene (114) (Guiso et al., 2002) and 4-iodophenol (115) in the preparation of resveratrol 4′-Oβ-d-glucuronide (118) (Scheme 14.3), and 3,5-dihydroxyiodobenzene (121) (Deboves et al., 2001) and 4-acetoxystyrene (124) in the case of resveratrol 3-O-β-d-glucuronide (126) (Scheme 14.4). In the synthesis of the 4′-O-glucuronide (Scheme 14.3), glucuronidation of 4-iodophenol (115) took place with 66% yield. In the preparation of the 3-O-glucuronide (Scheme 14.4), glucuronidation of 3,5-dihydroxyiodobenzene (121), with two available hydroxyl groups, was, as expected, less efficient (35% yield). In both cases, formation of the resveratrol skeleton from the two iodobenzene and vinylbenzene precursors was carried out by a modified Heck reaction in acceptable yield. The usual deprotection reactions yielded the two desired resveratrol O-glucuronides, 118 and 126, in very good yield. Recently, a different approach was used in the preparation of resveratrol glucosides (Zhang et al., 2006). No total synthesis of the stilbene skeleton was undertaken. Instead, protected resveratrols were used as precursors. Of course the direct tert-butyldimethylsilylation

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HO

CHO

OH

TBSO

CHO

I 115: 4-Iodophenol

Acetylation

OTBS 112

OH

OAc

113

114: 3,5-Diacetoxy styrene

COOMe O OAc

OH

AcO

Ph3P=CH2

tert-Butyldimethyl silyl chloride

111: 2,4-Dihydroxy benzaldehyde

HO

O

CCl3

AcO 108 OAc

COOMe O

NH

BF3.OEt2, CH2Cl2, 0ºC to rt

O AcO OAc

I 116

66% OAc

Pd(OAc)2, BnEt3N+ClBu3N, DMF, 110ºC

COOH O

COOMe

O AcO

O OAc

HO OAc 118: Resveratrol 4’-O-β-D-glucuronide OH (84%)

331

OAc OAc 117: Acetyl resveratrol 4’-O-β-D-glucuronide (55%)

AcO

a) Aq. NaOH, 0ºC b) Amberlyst 15 H+ form

O AcO

OAc

Scheme 14.3 The regioselective synthesis of resveratrol 4′-O-β-d-glucuronide (adapted from Guiso et al., 2002; Learmonth, 2003).

Scheme 14.4 The regioselective synthesis of resveratrol 3-O-β-d-glucuronide (adapted from Deboves et al., 2001; Learmonth, 2003).

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Scheme 14.5

The products of the direct tert-butyldimethylsilylation of resveratrol (Zhang et al., 2006).

of resveratrol was not regioselective and gave rise to a mixture of 5 compounds, viz. the 3,5,4′-triprotected, the 3,4′- and 3,5-diprotected, and the 3- and 4′-monoprotected ones (Scheme 14.5; compounds 127–131). Although the purification of individual protected compounds was necessary and the recovered yields were poor (6–27%), this method could, in the future, provide faster access than total synthesis to suitable precursors for resveratrol glucuronidation.

14.4.2 The preparation of O-sulfates Methods for the synthesis of flavonoid sulfates have been published since the end of the 1980s (Barron & Ibrahim, 1987; Barron & Ibrahim, 1988a, 1988b). Using an appropriate combination of chemical and enzymatic methods, all the possible sulfated isomers of quercetin and other flavonoids could be prepared in mg amounts (for more details, consult the reviews by Barron et al., 1988 and Barron et al., 2003). Since this time, however, only a handful of publications have appeared. Recently a more direct route to quercetin O-sulfates has been described (Jones et al., 2005). The method made use of a sulfur trioxide–trimethylamine complex as a reagent in the direct sulfation of quercetin. Of course, in the absence of quercetin protection, a complex mixture of quercetin sulfates was obtained (Scheme 14.6) and purification of the individual compounds was required. Only quercetin 3-O-sulfate (132) was unambiguously identified by comparison with an authentic reference compound. The position of sulfation of the three other monosulfates (133–135) was only assigned on the basis of the proton NMR spectra, and three additional non-characterized quercetin disulfates were also present in the reaction medium. Furthermore, no elemental analysis of the final products was performed and thus no indication of the purity of the compounds was available. Knowing the challenges that are associated with the purification of sulfates conjugates, often contaminated with inorganic impurities (therefore non-UV absorbing), the relevance of this method in the preparation of standards for quantification might be questionable. In a more recent paper, the regioselective preparation of the human metabolite quercetin 3′-O-sulfate (134) was published (Needs & Kroon, 2006). 3,7,4′-Tri-O-benzylquercetin

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Scheme 14.6 The products of the direct sulfation of quercetin using a sulfur trioxide–trimethylamine complex (Jones et al., 2005).

Scheme 14.7

The preparation of quercetin 3′-O-sulfate (adapted from Jurd, 1962; Needs & Kroon, 2006).

(137) was used as precursor and sulfur trioxide–dimethylformamide complex as sulfating agent (Scheme 14.7). Interestingly, the sulfated groups appeared to be stable in the catalytic hydrogenation conditions used for removal of the benzyl groups, and quercetin 3′-O-sulfate was isolated in fair yield. Although the precursor compound (137) could be obtained in 66% yield from 7,4′-di-O-benzylquercetin (98), access to the latter compound was not that convenient. In fact, it first involved the acetylation of quercetin to its pentaacetate (136), followed by benzylation of this compound to give – after alkaline hydrolysis of the acetates – a mixture of 7-O-benzylquercetin and 7,4′-di-O-benzylquercetin, from which the latter compound had to be purified (Jurd, 1962). This, of course, accounts for the poor yield of recovery of 98 from 136 (Scheme 14.7).

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14.5 The biological properties of phenolic conjugates Information on the biological activities of plant phenolics has been largely collected from in-vitro studies. Thus, the question as to whether, after metabolic transformations, the metabolites will still retain the activity of the parent phenolic compound is a main concern for those in the scientific community working in this field. The data concerning the biological activity of phenolic metabolites are summarized in Table 14.1, emphasis being put on a comparison of the effects between a given phenol and its metabolites.

14.5.1 Antioxidant properties 14.5.1.1 Flavonol conjugates The antioxidant and radical-scavenging activities of polyphenols have been extensively studied and a wide literature is available on this subject. However the use of different reaction systems and assay conditions to evaluate activity has introduced some confusion and has sometimes even led to contradictory results. Thus many efforts have been made to standardize evaluation of antioxidant activity, especially in the case of food products. Flavonoids and phenolic acids were first classified according to their trolox equivalent antioxidant capacity (TEAC) (Rice-Evans et al., 1996). A recent review (Kim & Lee, 2004) has classified a large number of phenolics according to their vitamin C equivalent antioxidant capacity (VCEAC). The VCEAC of flavonoids depends not only on the number of hydroxyl groups present in the compound, but also on the pattern of hydroxylation, with a predominant effect of catechol substitution. The substitution of flavonoids by sugars induces lower VCEAC, presumably owing to the reduction in the number of free hydroxyl groups and/or the masking effect of the bulky sugar substituent, which causes steric hindrance (Kim & Lee, 2004). Considering these effects, it was of course expected that either methylation or glucuronidation of flavonoids would have a negative impact on the antioxidant activity of the conjugate. Indeed, 3′-O-methylquercetin (isorhamnetin) displayed lower ability than quercetin in preventing hydrogen peroxide-induced chromosomal damage to a lymphoblastoid cell line (Table 14.1, No. 1) (Saito et al., 2004). The capacity of methylated and glucuronidated conjugates of quercetin to delay the Cu2+-induced oxidation of human low-density lipoprotein (LDL) was studied by Manach et al. (1998). In fact, this resulted in a significant decrease in activity (Table 14.1, No. 2). However in that case it was impossible to draw any structure–effect relationships since the study was conducted on mixtures of conjugates. Later, quercetin 4′-O-β-d-glucuronide was shown to be almost inactive in scavenging the diphenylpicryl-hydrazyl (DPPH) radical and delaying the Cu2+-induced oxidation of human LDL (Table 14.1, No. 3) (Moon et al., 2001). The 3O-glucuronide experienced a much less important activity drop (Table 14.1, No. 3). This must be related to the fact that, again, the presence of a 3′,4′-dihydroxy or a 3′,4′,5′trihydroxy structure on the B-ring, and of a 3-hydroxyl group on the C-ring have been recognized by a number of studies (Bors et al., 1990; Kim & Lee, 2004) as important prerequisites for the radical-scavenging ability of flavonoids. However, between the

General activity

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

N°

1

2

3

4

5

6

7

Quercetin

Quercetin

Fe3+-induced lipid peroxidation Aqueous peroxy radicalinduced lipid peroxidation SIN-1-induced lipid peroxidation 15-LOX-induced lipid peroxidation

3-O-β-d-glucuronide

3-O-β-d-glucuronide

3-O-β-d-glucuronide

3-O-β-d-glucuronide 4′-O-β-d-glucuronide 7-O-β-d-glucuronide 3-O-β-d-glucuronide 3′-O-methyl

Moon et al., 2001

Janisch et al., 2004

Terao et al., 2001

Shirai et al., 2001

3-glucuronide: ↓ ∼ 34% 4′-glucuronide: ↓ ∼ 87% 3-glucuronide: ↓ ∼ 37% 4′-glucuronide: ↓ ∼ 87% 19% ↓ 73% ↓ 3% ↑ 76% ↓ Lycopene: 23% ↓ β-Carotene: 57% ↓ α-Tocopherol: 25% ↓ ↓ ∼ 73% ↓ ∼ 58% ↓ ∼ 29% ↓ ∼ 84% 3-glucuronide less potent than quercetin, which was active at 1 μM

10 –350 μM 5 μM

2 μM

50 μM

20 μM 20 μM 10 μM 20 μM 10 μM

Shirai et al., 2002

Manach et al., 1998

Important ↓ (at least half )

0.5 or 1.0 μM

Chemical Synthesis and Biological Activity of Phenolic Metabolites

Inhibition of the H2O2induced intracellular production of ROS

Quercetin

Quercetin

Delay the Cu2+-induced oxidation of human LDL

3-O-β-d-glucuronide 4′-O-β-d-glucuronide

Isorhamnetin. Mixtures of glucuronides of quercetin or isorhamnetin, obtained by enzymatic synthesis

Saito et al., 2004

Lower effect than quercetin

1–10 μM

Reference

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Effect on peroxynitriteinduced consumption of lipophilic antioxidants in human plasma LDL

Quercetin

Quercetin

Delay the Cu2+-induced oxidation of human LDL

3-O-sulfate 3′-O-methyl (Isorhamnetin)

Conjugate

Nature or magnitude of the effect as compared to the aglycone†

Active concentration of the conjugate*

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Scavenging of DPPH radical Delay the Cu2+-induced oxidation of human LDL

Quercetin

Aglycone

Inhibition of H2O2-induced chromosomal damage

Observed effect

Table 14.1 Comparison of the biological activities of phenols and their mammalian metabolites.

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General activity

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Antioxidant

8

9

10

11

12

13

14

15

16

Genistein Daidzein

Genistein

Free radical scavenging (ABTS)

Delay the Cu2+-induced oxidation of human LDL

Delay the Cu2+-induced oxidation of human LDL

(–)-Epicatechin (–)-Epigallocatechin (–)-Epigallocatechin gallate

(–)-Epicatechin

Delay of the Cu2+-induced oxidation of LDL

Lipid oxidation of canola oil

Eriodictyol

Luteolin

6′-hydroxy-Odesmethylangolensin 7-O-sulfate 4′-O-sulfate 7,4′-di-O-sulfate

8-Hydroxy 7-O-sulfate 4′-O-sulfate 7,4′-di-O-sulfate

4′-O-sulfate 7,4′-di-O-sulfate

3′-O-methyl 3′-O-methyl 3′-O-methyl

7-O-β-d-glucuronide 3′-O-β-d-glucuronide 4′-O-methyl-3′-O-β-dglucuronide

4′-O-sulfate 4′-O-methyl-7-O-sulfate

7-O-β-d-glucuronide

3′-O-β-d-glucuronide 7-O-β-d-glucuronide

3-O-β-d-glucuronide

Conjugate

Takamatsu et al., 2003 Natsume et al., 2004

Su et al., 2004

Rimbach et al., 2004

54% ↓ 42% ↓ Only about 3% ↓ ∼ 66% ↓ ∼ 64% ↓ ↓ ↓ ↓ 33% ↓ of TEAC value 97% ↓ of TEAC value

125 mM 0.5 μg/mL

Slightly smaller effect

5 μM

40% ↓ 70% ↓ 95% ↓

91% ↑ 20% ↓ 53% ↓ No activity

5 μM

0.5 mM

Turner et al., 2004

Turner et al., 2004

Matsuda et al., 2003b

Slight; 20% ↓

0.2 mM

Lu & Foo, 2001

Begum & Terao, 2002

No comparison

0.2 – 0.4 mM

33% ↓

10 μM

Reference

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Inhibition of the 2′,7′dichlorofluorescein diacetate

Diminution of advanced glycation end-products

Luteolin

Quercetin

Aglycone

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DPPH and superoxide anion radical-scavenging activities

Reduction of the impairment of erythrocyte deformability in the presence of cigarette tar extract

Observed effect

Nature or magnitude of the effect as compared to the aglycone†

Active concentration of the conjugate*

336

N°

Table 14.1 (Cont’d )

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Genistein

Daidzein

In-vitro oxidation of LDL (conjugated diene formation)

Delay the Cu2+-induced oxidation of human LDL

Free radical scavenging (DPPH) Free radical scavenging (DPPH)

Covalent binding to the DNA of HepG2 cells

Inhibition of angiotensin II-induced JNK activation

Inhibition of the phosphorylation of Akt

Inhibition of proliferation and induction of apoptosis in the human NCI-H209 lung cancer cell line

Modulation of COX-2 transcription in unstimulated Caco-2 cells

Modulation of COX-2 transcription in IL-1βstimulated Caco-2 cells

Antioxidant

Antioxidant

Antioxidant

Pro-oxidant

Interaction with signaling cascades

Interaction with signaling cascades

Interaction with signaling cascades

Enzyme inhibition and modulation of expression

Enzyme inhibition and modulation of expression

19

20

21

22

23

24

25

26

27

NR 33% ↓ 40% ↓ 90% ↓ No comparison with aglycone was made

25% ↑ 31% ↑ 50% ↑ 70% ↑ 67% ↑ No significant change

IC50 ≈ 1–10 μM 30 μM

0.5 –10 μM

10 μM

10 μM 3′-O-sulfate 3-O-β-d-glucuronide 3-O-β-d-glucuronide-3′-Omethyl

3′-O-sulfate 3-O-β-d-glucuronide 3-O-β-d-glucuronide-3′-Omethyl

Mixture of glucuronides prepared from the serum of rabbits

3′-O-methyl 4′-O-methyl 7-O-β-d-glucuronide

3-O-β-d-glucuronide

O’Leary et al., 2004

O’Leary et al., 2004

Yang et al., 2006

Spencer et al., 2003b

Yoshizumi et al., 2002

van der Woude et al., 2006

Chemical Synthesis and Biological Activity of Phenolic Metabolites

Quercetin

Quercetin

Quercetin

Quercetin

Quercetin

58% ↓

5 μM

3′- and 4′-O-methyl as a mixture

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Quercetin

38% ↑

0.33 mM

7-O-β-d-glucuronide

Kren et al., 2000

43% ↓

Ruefer & Kulling, 2006

77% ↑ EC50 40% ↓ EC50

Boersma et al., 2003

Ruefer & Kulling, 2006

98% ↓ trolox equiv 24% ↑ trolox equiv

3.2 fold increase

Turner et al., 2004

66% ↓ 73% ↓ 98% ↓

0.33 mM

25 μM

5 μM

20-O-β-d-glucuronide

3′-Chlorodaidzein

4′-O-methyl (Formononetin) 3′-hydroxy

4′-O-methyl (Formononetin) 3′-hydroxy

7-O-sulfate 4′-O-sulfate 7,4′-di-O-sulfate

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Silybin

Genistein

Scavenging activity of peroxyl radicals

Antioxidant

18

Equol

Delay the Cu2+-induced oxidation of human LDL

Antioxidant

17

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Enzyme inhibition and modulation of expression

Enzyme inhibition

Enzyme inhibition

Enzyme inhibition

Enzyme inhibition

Antitumor

Non-covalent protein binding

28

29

30

31

32

33

34

Binding affinity to HSA

Effect on angiogenesis

Inhibition of COX-2

Quercetin

Quercetin

Quercetin

Quercetin

Luteolin

Luteolin

Curcumin

Aglycone

7-O-sulfate 3-O-sulfate 7,4′-di-O-sulfate

3-O-sulfate 3-O-glucuronide

3′-O-sulfate

3-O-β-d-glucuronide 3-O-β-d-glucuronide-3′-Omethyl

7-O-β-d-glucuronide

7-O-β-d-glucuronide

O-sulfate Tetrahydrocurcumin Hexahydrocurcumin Hexahydrocurcuminol

Conjugate

NR

Dufour & Dangles, 2005

O’Leary et al., 2004

40% ↓

10 μM

∼ 43% ↑ of K1 ∼ 14% ↑ of K1 ∼ 89% ↓ of K1

Iwashita et al., 2001

No activity of the 2 conjugates at the active doses of quercetin (50 and 100 μM)

300 μM 300 μM; no inhibition at this concentration

Donnini et al., 2006

Yoshikawa et al., 1999

85% ↑ of IC50

NR

Quercetin and quercetin 3-glucuronide are antiangiogenic. On the other hand, quercetin 3′-sulfate is proangiogenic

Shimizu et al., 1984

4% ↓ of inhibition

10 μM

Upper nanomolar range

Ireson et al., 2001

45% ↓ 40% ↓ 33% ↓ NR

20 μM 20 μM 20 μM No significant activity at 20 μM

Reference

ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid; COX-2, cyclooxygenase-2; DPPH, diphenylpicryl-hydrazyl; GPDH, glycerol 3-phosphate deshydrogenase; HSA, human serum albumin; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; ROS, reactive oxygen species; TEAC, trolox equivalent antioxidant activity; 15-LOX, 15-lipoxygenase; PMA, phorbol 12-myristate 13-acetate.

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Inhibition of GPDH

Inhibition of lens aldose reductase

Inhibition of lens aldose reductase

Modulation of COX-2 expression in PMA-treated human colonic epithelial cells

Observed effect

Nature or magnitude of the effect as compared to the aglycone†

Active concentration of the conjugate*

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* The conjugates listed displayed activity at the mentioned concentration. However, no comment is made on its physiological significance. The reader is encouraged to find all the details in the corresponding reference. † A comparison of the observed effect with that of the corresponding aglycone is given. Again this is by no means an evaluation of the significance of the effect. NR, not relevant.

General activity

N°

Table 14.1 (Cont’d )

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3- and 4′-glucuronides, the 3-conjugate retained enough activity to play a role as antioxidant in blood plasma LDL, while the 4′ analog did not. A more detailed study on the effects of individual quercetin glucuronides on the modulation of LDL oxidation was conducted by Janisch et al. (2004) (Table 14.1, No. 4). Clearly substitution of the catechol B-ring was most detrimental to the activity. Quercetin 3-Oβ-d-glucuronide also displayed reduced activity, but again this effect was moderate as compared to catechol substitution. On the other hand quercetin 3-O-β-d-glucuronide displayed a moderately reduced ability to reduce the peroxynitrite-induced consumption of lipophilic antioxidants in human plasma LDL as compared with quercetin (Table 14.1, No. 5) (Terao et al., 2001). An additional study confirmed the still effective, but reduced antioxidant activity of quercetin 3-O-β-d-glucuronide as compared to the free aglycone quercetin (Table 14.1, No. 6) (Shirai et al., 2001). This study aimed to investigate lipid peroxidation, so in this case the lower antioxidant activity of quercetin 3-O-β-d-glucuronide may also have been due to the augmentation of its hydrophilicity, in addition to the loss of the 3-hydroxyl group. The effects of quercetin and of quercetin 3-O-β-d-glucuronide on the hydrogen peroxideinduced intracellular production of reactive oxygen species (ROS) in mouse fibroblasts was studied by Shirai et al. (2002). At 1 μM, quercetin inhibited intracellular ROS production, while quercetin 3-O-β-d-glucuronide could only inhibit it at a higher (10 μM) concentration (Table 14.1, No. 7), again confirming its reduced activity. Interestingly, when the cells were pre-treated with one of the two flavonoids before the addition of H2O2, quercetin 3-O-β-d-glucuronide, but not quercetin, suppressed the enhancement of intracellular ROS production. This was attributed to the fact that quercetin was appreciably metabolized by the cells into the ineffective 3′-O-methyl conjugate isorhamnetin, which lacks the catechol structure on the B-ring. In contrast, no metabolic conversion was observed with quercetin 3-O-β-d-glucuronide (Shirai et al., 2002). Since oxidative stress is involved in a number of pathologies, flavonoids, owing to their antioxidant and radical-scavenging properties, may indirectly exert a protective effect against these disorders. For example, cigarette tar extracts contain a number of ROS that could initiate peroxidative damage in erythrocyte membranes, as indicated by the impairment of erythrocyte deformability. This results in an increase in the viscosity of blood, with a negative impact on blood microcirculation. Thus flavonoids, as antioxidants, could exert a protective effect against this process. Begum & Terao (2002) demonstrated that both quercetin and its 3-O-β-dglucuronide decreased the impairment of erythrocyte deformability, including tests in which the erythrocytes were exposed to cigarette tar extract. It should be pointed out, however, that the effects of quercetin 3-O-β-d-glucuronide were reduced as compared to the aglycone (Table 14.1, No. 8). Flavonoids and their conjugates do not circulate in the plasma as free forms, but rather, they bind to human serum albumin. Recently it has been shown that the albumin-bound conjugates retain their ability to protect LDL from copper-induced oxidation. However the order of efficacy was slightly different: quercetin 7-glucuronide > quercetin 3-glucuronide > quercetin 4′-glucuronide > isorhamnetin 3-glucuronide > quercetin 3′-sulfate for the free forms, and quercetin 3′-sulfate > quercetin 7-glucuronide > quercetin 3-glucuronide > quercetin 4′-glucuronide = isorhamnetin 3-glucuronide for the bound forms (Janisch et al., 2004).

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14.5.1.2 Flavone conjugates The antioxidant activity of luteolin 3′-O-β-d-glucuronide was demonstrated by Okamura et al. (1994) but no comparison with the effect of luteolin was made in this study. Similarly, the antioxidant activities of luteolin 7-O- and 3′-O-glucuronides have been compared (Lu & Foo, 2001). The 7-glucuronide was a better DPPH and superoxide anion radical-scavenger than the 3′-glucuronide, confirming once again the role of the catechol B-ring (Table 14.1, No. 9). However in this case no comparison with the activity of luteolin was made. Diabetes is associated with a number of complications including the formation in the blood of advanced glycation end-products (AGEs), a process in which reactive oxygen species are involved. Thus, antioxidant flavonoids may reduce the formation of AGEs. Both luteolin and its 7-O-glucuronide inhibited the formation of AGEs, with a slightly reduced activity for the latter (Table 14.1, No. 10) (Matsuda et al., 2003b). 14.5.1.3 Flavanone conjugates Sulfation of eriodictyol at position 4′ led to an important decrease in its antioxidant properties (Table 14.1, No. 11) (Takamatsu et al., 2003), and this was probably owing to substitution on the catechol B-ring. Homoeriodyctiol 7-O-sulfate, whose catechol B-ring is methylated at position 4′, also displayed reduced antioxidant activity (Table 14.1, No. 11) (Takamatsu et al., 2003), although the diminution of the effect was slightly less pronounced than in the case of 4′-O-sulfation. All these effects are in accordance with the rules based on VCEAC calculations (Kim & Lee, 2004), especially concerning the role of catechol substitution. Also naringenin and its 7-O-sulfate were found to be inactive under the conditions of the oxidation of 2′,7′-dichlorofluorescein diacetate assay (Takamatsu et al., 2003). 14.5.1.4 Flavan 3-ol conjugates The activity of all the B-ring substituted epicatechin glucuronides was reduced (Table 14.1, No. 12) (Natsume et al., 2004). O-Methylation of either (−)-epicatechin, (−)-epigallocatechin, or (−)-epigallocatechin gallate at position 3′ led to a decreased potential against lipid oxidation of canola oil (Table 14.1, No. 13) (Su et al., 2004). 3′-O-methylepicatechin was shown to protect human erythrocytes from free-radical-induced hemolysis, even at a low – 2.5 μM – concentration (Zhu et al., 2005). The effects of catechin and its 3′- and 4′O-methyl conjugates on human LDL oxidation were investigated by Cren-Olive et al. (2003). 3′- and 4′-O-methylcatechin acted as amphiphilic chain-breaking antioxidants, and their scavenging properties implied an electronic transfer from the phenolate. However, taking into account the usual plasma concentration of catechin metabolites, the study concluded that the concentration needed to protect LDL against oxidation is much higher than in-vivo levels in plasma. Thus the protective effect of O-methylcatechins could be of little physiological significance. 14.5.1.5 Isoflavone conjugates Genistein was found to be a weaker antioxidant than quercetin. Genistein 4′-O-sulfate and 7,4′-di-O-sulfate were even less efficient (Table 14.1, No. 14) (Rimbach et al., 2004). In

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addition genistein 7,4′-di-O-sulfate was less effective than genistein 4′-O-sulfate, which was itself less effective than genistein at inhibiting collagen-induced platelet aggregation, nitric oxide production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (Rimbach et al., 2004). In another study, the effects of daidzein, genistein, their microbial metabolites and their sulfate conjugates on the oxidation of LDL were studied (Turner et al., 2004). While the activities of daidzein, genistein, and 6′-hydroxy-O-desmethylangolensin were similar, equol and especially 8-hydroxydaidzein (Table 14.1, No. 15) were more active. In contrast, as expected, all the sulfates exhibited reduced activity, with a more pronounced effect when sulfation was at position 4′ (Table 14.1, Nos 15–17). A recent study examined the antioxidant properties of isoflavones and their hepatic and microbial metabolites (Ruefer & Kulling, 2006). Both the radical-scavenging properties in a hydrophilic environment and the effects on lipid peroxidation (lipophilic environment) were investigated. Equol and the liver oxidative metabolites displayed high antioxidant activity, with 3′-hydroxygenistein being the most effective (Table 14.1, Nos 18 & 19). In contrast, formononetin, bearing a 4′-O-methyl, was the least active, confirming the fact that conjugation at this position is detrimental to isoflavone antioxidant activity (Table 14.1, Nos 18 & 19). Neither sulfate nor glucuronide conjugates were tested in this study. While isoflavones inhibit LDL oxidation, the concentrations that are necessary to observe this effect in vitro are much greater than the in-vivo doses attained after consumption of soy-containing products. This led to the hypothesis that isoflavones could have been metabolized into more active compounds. Since reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated in the modification of LDL and in the development of atherosclerotic lesions, it has been hypothesized that polyphenols would be able to interfere with this process. When genistein was incubated with HL-60 cells stimulated to undergo the respiratory burst in the presence of NO−2 , it was efficiently metabolized into mono- and dichlorogenistein, as well as nitrogenistein. Similar metabolism was observed when genistein was incubated with human neutrophils activated with phorbol 12-myristate 13 acetate (PMA) and again in the presence of NO−2 . Furthermore, 3′-chlorogenistein and 3′-chlorodaidzein were shown to be more potent inhibitors of the copper-mediated LDL oxidation than their respective precursors genistein and daidzein (Table 14.1, No. 20) (Boersma et al., 2003). The mechanism of this effect is not known. 14.5.1.6 The case of the 7- O-conjugates Silybin 20-O-β-d-glucuronide displayed reduced radical-scavenging capacity as compared to silybin aglycone (Table 14.1, No. 21) (Kren et al., 2000). This is owing to the fact that the main site for free radical reaction in silybin is the E-ring (Fig. 14.3), not the A-ring, owing to (1) intramolecular hydrogen bonding of the 5-hydroxyl with the 4-keto group; and (2) conjugation of the 7-hydroxyl with the 4-carbonyl, which favors deprotonation rather than one-electron transfer (Gyorgy et al., 1992). But surprisingly, the 7-glucuronide of silybin was a better radical-scavenging agent than silybin (Table 14.1, No. 21) (Kren et al., 2000). Subsequently, a similar situation was encountered in the case of rhamnetin (7-O-methylquercetin), which showed better superoxide radical-scavenging activity than

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quercetin (Kessler et al., 2002). Quercetin 7-O-β-d-glucuronide was also as effective as quercetin at delaying Cu2+-induced human plasma LDL oxidation (Table 14.1, No. 4) (Janisch et al., 2004). Again, the in-vitro antioxidant properties of (−)-epicatechin 7-O-glucuronide were found to be close to those of (−)-epicatechin in terms of superoxide-scavenging activity and ability to suppress induced LDL oxidation (Table 14.1, No. 12) (Natsume et al., 2004).

14.5.2 Pro-oxidant properties Under certain circumstances flavonoids, especially those with a B-ring catechol, can autooxidize even at physiological pH, and promote hydroxyl-radical generation in the Fenton reaction. On the other hand, reactive oxygen species, instead of being trapped, can metabolize the flavonoid into highly reactive products that can covalently bind to macromolecules like proteins or DNA (Walle et al., 2003; Kaldas et al., 2005; van der Woude et al., 2005). This could account in part for the toxicity of some flavonoids in vivo. The pro-oxidant activity is favored in the presence of Cu2+ and increases with flavonoid concentration (Cao et al., 1997). Structural requirements concerning pro-oxidant activity follow more or less the same rules as for antioxidant activity (Cao et al., 1997), and it has been suggested that examination of the antioxidant capacity expressed as TEAC values could be of help in predicting their pro-oxidant behavior (van der Woude et al., 2006). In the quercetin series, however, substitution at position 3 prevents auto-oxidation of the 3-OH and reduces the pro-oxidant activity (Kessler et al., 2002). When quercetin was placed in oxidative conditions such as exposure to peroxidase (Awad et al., 2000) or tyrosinase (Boersma et al., 2000), the compound was transformed into highly reactive quinone methide products, as revealed by the formation of adducts involving the A-ring of quercetin at positions 6 or 8, when glutathione (GSH) was present in the medium. The 3-OH, as well as the 2.3-double bond of quercetin, could be involved in the formation of the quinone methides that are precursors of A-ring adducts (Awad et al., 2001). This could explain in part why substitution of the 3-OH of quercetin diminishes the pro-oxidant activity, as mentioned above. However the mechanism of the formation of the adducts is still a matter of debate and an alternate mechanism, not involving the 3-hydroxyl, has been proposed (van der Woude et al., 2006). The regioselectivity of GSH adduct formation has been shown to be pH-dependent. Quercetin A-ring adducts were formed at low pH, B-ring adducts at neutral pH, and C-ring adducts at high pH (Awad et al., 2002). The formation of 2′-glutathionyl quercetin has been demonstrated in dermal fibroblasts exposed to quercetin (Spencer et al., 2003a). It is only very recently that the effects of methylation of the catechol group of quercetin on adduct formation have been studied (van der Woude et al., 2006). In fact tamarixetin and isorhamnetin have been shown to form adducts with GSH and with DNA. The formation of GSH adducts could only be detected in cells with a high level of oxidative enzyme activity, while DNA adducts formed in all cell types studied. However, the extent of DNA adducts was significantly lower in the case of 3′- and 4′-O-methylquercetin than in the case of quercetin itself (Table 14.1, No. 22) (van der Woude et al., 2006). This suggests that the pro-oxidant activity of quercetin derivatives is also linked to its electrondonating capacity, which confirms that the structural requirements for the radical-scavenging

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and pro-oxidant activities are closely linked. Finally the reduced pro-oxidant activity of the methylated catechins could explain the apparent lack of in-vivo carcinogenicity of quercetin.

14.5.3 Interaction with signaling cascades Oxidative stress induces in neurons a complex cascade of events involving activation of c-Jun N-terminal kinase (JNK), cleavage of procaspase-3, and increased caspase-3-like protease activity. All these events lead to neuron loss and 3′-O-methylepicatechin has been shown to be as effective as (−)-epicatechin in protecting neurons against oxidized lowdensity-lipoprotein-induced apoptosis (Schroeter et al., 2001). The mechanism of the neuroprotective action is still unknown, and the results of the study suggested that the catechins did not act through a reduction of the overall intracellular oxidative stress in neurons. Similarly, 3′-O-methylepicatechin has been shown to be as efficient as (−)-epicatechin in protecting human fibroblasts from oxidative-stress-induced cell death (Spencer et al., 2001b; Basu-Modak et al., 2003). Again, the mechanism is thought not to be directly related to the antioxidant properties of these flavan-3-ols, but rather to an adaptative response involving protein synthesis (Basu-Modak et al., 2003). Vascular smooth muscle cell (VSMC) hypertrophy is also induced by activation of JNK, and it has been prevented by quercetin 3-O-β-d-glucuronide through inhibition of this signaling pathway (Table 14.1, No. 23) (Yoshizumi et al., 2002). Quercetin is toxic to cortical neurons via inhibition of pro-survival protein kinase cascades (Akt/PKB and ERK). 3′- and 4′-O-methylquercetins are still neurotoxic, but to a lesser extent (Table 14.1, No. 24). Finally quercetin 7-O-β-d-glucuronide had no neurotoxic effect (Spencer et al., 2003b). However it could be that the latter compound, unlike the methylated quercetins, is innocuous because it cannot penetrate inside the neurons, owing to its highly hydrophilic character. Nitric oxide (NO) is implicated in a number of pathologies such as chronic or acute inflammation, and its production involves a complex cascade of events including NF-κB activation. The structural requirements of flavonoids for inhibition of NO production have been investigated (Matsuda et al., 2003a). While luteolin and quercetin displayed an inhibitory activity on NO production, luteolin 7-O- and kaempferol 3-O-glucuronides were totally devoid of activity. 7-O-methylation of quercetin to rhamnetin slightly reduced the activity, while 4′-O-methylation to tamarixetin enhanced it (Matsuda et al., 2003a). In the human lung cancer cell line NCI-H209, a mixture of quercetin glucuronides was shown to inhibit proliferation through G2/M arrest of the cell cycle, and to induce apoptosis via caspase-3 activation (Table 14.1, No. 25) (Yang et al., 2006). Cyclooxygenase-2 (COX-2) is involved in the synthesis of prostaglandin E2 and thus plays a role in inflammation and its associated diseases like cancer and cardiovascular diseases. The expression of COX-2 is inducible by the exposure of cells to, for example, cytokines. A number of flavonoids are known to inhibit COX-2, or to modulate COX-2 transcription. The ability of quercetin conjugates found in human plasma to inhibit COX-2 activity and transcription has been studied (O’Leary et al., 2004). In unstimulated Caco-2 cells, the effects of quercetin 3-O-β-d-glucuronide, quercetin 3′-O-sulfate, and isorhamnetin 3-O-β-d-glucuronide on COX-2 expression was dose-dependent, with a small

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activation of transcription at 0.1 μM (in contrast to quercetin, which already inhibited transcription at this concentration), while at 10 μM all the conjugates were inhibitory and usually more active than quercetin aglycone, isorhamnetin 3-O-β-d-glucuronide being the most potent (Table 14.1, No. 26). In IL-1β stimulated Caco-2 cells, again the three conjugates inhibited the cytokine-stimulated COX-2 expression at 10 μM. This time the quercetin glucuronide and sulfate were the most active and more efficient than quercetin aglycone (Table 14.1, No. 27). On the other hand, while quercetin 3-O-β-d-glucuronide, quercetin 3′-O-sulfate, and isorhamnetin 3-O-β-d-glucuronide were shown to down-regulate COX-2 transcription in human lymphocytes, these effects were not confirmed in vivo since the expression of COX-2 mRNA in lymphocytes was not affected by the consumption of high-quercetin onions (Pascual-Teresa et al., 2004). Curcumin metabolites have also been tested on phorbol ester-induced inhibition of COX-2 expression, as reflected by prostaglandin E2 (PGE2) levels in human colonic epithelial cells (Ireson et al., 2001). Curcumin aglycone decreased the induced PGE2 production down to almost pre-induction levels. The metabolites tetrahydrocurcumin, hexahydrocurcumin and curcumin sulfate were only weakly active. Octahydrocurcumin (hexahydrocurcuminol) was inactive (Table 14.1, No. 28).

14.5.4 Enzyme inhibition 14.5.4.1 Aldose reductase Aldose reductase catalyzes the reduction of aldoses to polyols, leading to their accumulation in the lens, and to the appearance of cataract. Shimizu et al. (1984) showed that luteolin 7-O-β-d-glucuronide and luteolin inhibit lens aldose reductase. The activity of the glucuronide was slightly lower than that of the aglycone (Table 14.1, No. 29), but this was an exception since in this study the flavonoid glycosides were generally found to be more active than their aglycones. Inhibition of aldose reductase by luteolin and its 7-O-β-d-glucuronide was confirmed by a further study, however with much more reduced activity (Table 14.1, No. 30) for the latter compound (Yoshikawa et al., 1999). A study on a wide range of potential inhibitors concluded that, indeed, conjugation of the 7- and/or one of the catechol positions of the B-ring leads to reduced inhibitory activity on aldose reductase (Matsuda et al., 2002). 14.5.4.2 β-glucuronidase Luteolin 3′-O-β-d-glucuronide was shown to be a potent competitive inhibitor of βglucuronidase, but no comparison with the activity of the aglycone was made (Narita et al., 1993). 14.5.4.3 Xanthine oxidase The level of xanthine oxidase increases during hepatitis and with the degree of brain edema. Thus the inhibition of xanthine oxidase might be relevant in the treatment of these diseases. This inhibition has been found to be highly dependent on the position of conjugation.

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While quercetin 3- and 7-glucuronides are much less efficient than quercetin, the activities of quercetin 3′- and 4′-glucuronides was found to be similar or slightly better than that of quercetin (Day et al., 2000). 14.5.4.4 Glycerol 3-phosphate dehydrogenase Adipocyte differentiation inhibitors can be of use in the prevention of obesity. Lipids accumulate in the process of differentiation and glycerol 3-phosphate dehydrogenase (GPDH) is one of the key enzymes in lipogenesis. While quercetin and isorhamnetin already inhibit GPDH activity at 50 μM concentration, quercetin 3-O-β-d-glucuronide was only slightly active at 300 μM and isorhamnetin 3-O-β-d-glucuronide was ineffective (Table 14.1, No. 31) (Iwashita et al., 2001). 14.5.4.5 Cyclooxygenase-2 Only quercetin and its 3′-O-sulfate were inhibitors of COX-2 activity, with a reduced effect for the sulfate as compared to the aglycone (Table 14.1, No. 32) (O’Leary et al., 2004). Furthermore the regular consumption of COX-2 inhibitors can cause a number of adverse effects, and the use of this class of therapeutic agent is more and more controversial. Today more attention is being focused on the development of compounds targeting the induction of COX-2 expression (see section 14.5.3) (Tsatsanis et al., 2006).

14.5.5 Effect on vascular function and angiogenesis Recently, the vascular effects of cocoa were clearly shown to be associated with the presence of epicatechin (Schroeter et al., 2006). Furthermore, taking into account the concentrations and the nature of the circulating flavan 3-ol metabolites, multivariate regression analysis identified both (−)-epicatechin and its 7-O-β-d-glucuronide as predictors of the vascular effects. Divergent effects on angiogenesis have been demonstrated for quercetin and two of its human metabolites, quercetin 3-O-glucuronide and quercetin 3′-O-sulfate. Quercetin and its 3-glucuronide have no effect on quiescent endothelium, while the 3′ sulfate stimulates quiescent endothelial cells. However, quercetin and its 3-glucuronide inhibit angiogenesis elicited by vascular endothelial growth factor (VEGF), whereas quercetin 3′-sulfate has no effect (Donnini et al., 2006). It should also be noted that quercetin and its 3-glucuronide displayed anti-angiogenic activity, while the 3′-sulfate displayed pro-angiogenic activity (Table 14.1, No. 33).

14.5.6 Non-covalent binding to proteins Quercetin and its 7-O-β-d-glucuronide, 3-O-β-d-glucuronide, 4′-O-β-d-glucuronide, 3-Oβ-d-glucuronide-3′-O-methyl, and 3′-O-sulfate conjugates were all shown to bind to human serum albumin (HSA) with values that predict that non-free metabolites are circulating in the plasma (Janisch et al., 2004). The binding of quercetin and some of its O-methyl and O-sulfate conjugates to bovine serum albumin (BSA) and HSA were recently investigated

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(Dufour & Dangles, 2005). On average, conjugation of quercetin lowered its affinity for albumin, but differences were found between BSA and HSA as well as between the type of conjugate. Quercetin 7-O-sulfate retained good affinity for both albumins, while additional sulfation at position 4′ considerably decreased the binding affinity (Table 14.1, No. 34). Methylation of the 3′-OH in isorhamnetin lowered the affinity for BSA while HSA was less affected, suggesting that the 3′-OH was important for BSA binding.

14.5.7 Activity of microbial and tissular metabolites Recently Cerda et al. showed that urolithin A, a metabolite resulting from the colonic fermentation of the ellagitanin punicalagin, displayed a considerably reduced antioxidant activity as compared to the latter compound. Furthermore, urolithin glucuronide was the actual circulating form of urolithin in plasma, and the conjugate was totally devoid of antioxidant activity (Cerda et al., 2004). On the other hand, tetrahydrocurcumin (THC), one of the metabolites of curcumin, showed a higher inhibitory effect than curcumin on the lipid peroxidation of erythrocyte membrane ghosts induced by tert-butylhydroperoxide, and the antioxidant effect was mainly related to the presence of the β-diketone moiety (Sugiyama et al., 1996). However, again one must realize that the major circulating form of tetrahydrocurcumin is THC glucuronide, and it is likely that the activity of the latter compound will be considerably reduced. Therefore the biological activity of these unconjugated metabolites might be of reduced physiological significance, unless of course deconjugation reactions take place later in tissues. For this reason, we decided that all the data concerning the activity of unconjugated forms were beyond the scope of this review. For example the activity of 3-hydroxyphenylpropionic acid, a microbial metabolite of caffeic acid derivatives, will not be covered in this chapter.

14.6 The cellular transport of phenolic conjugates 14.6.1 Cellular efflux Since a number of transporters are known to be present in polarized intestinal cells, their activity will have major consequences on the intestinal absorption of phenols, and thus on their bioavailability. For example, the phenolic compounds that have been conjugated after absorption by the intestine can be taken in charge by the MRP2 transporter, present at the apical side of the cell, and effluxed back into the lumen. In contrast, the activity of MRP1 and MRP3 transporters, both situated on the basolateral side, will push the conjugates toward the blood circulation (Fig. 14.9). Consequently, the efflux of compounds from cells has received much more attention than their uptake. In-situ perfusion of the jejunum and the ileum in rats with catechin solutions demonstrated that the effluent contained only native catechin, suggesting that no intestinal excretion of catechin glucuronides takes place (Donovan et al., 2001). In contrast, a significant proportion of quercetin glucuronides and sulfates is excreted in the intestinal lumen (Crespy et al., 1999). Using the Loc-I-Gut intestinal perfusion technique, it was shown that quercetin 3,4′-diglucoside, a naturally occurring constituent of onion, is rapidly deglucosylated and

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Fig. 14.9 A summary of the transporters that may be involved in the intestinal efflux of phenolic conjugates. BCRP, breast cancer resistant protein; MRP1 to 3, Multidrug resistance-associated proteins 1 to 3; P-gp, P-glycoprotein.

glucuronidated by human intestinal cells. Interestingly, quercetin 3′-O-glucuronide was the only conjugate that was effluxed back to the lumen, possibly under the mediation of the MRP-2 transporter. This could suggest that glucuronide conjugates on the B-ring at the 3′- or 4′-positions are excreted back into the lumen, while the 3- and 7-glucuronides are not, but are transferred into the blood circulation (Petri et al., 2003). However more data will be necessary to confirm this hypothesis. If this is the case, since positions 3′ and 4′ appear to be the preferred target of human intestinal UDP-glucuronosyl transferases (Boersma et al., 2002), this back efflux could represent a serious limitation to the intestinal absorption of quercetin. In addition even quercetin glucosides (Walgren et al., 2000b) and genistein glucosides (Walle et al., 1999) were found to be subject to an intense efflux mechanism, probably mediated by the MRP-2 transporter. This could considerably limit the intestinal absorption of, for example, the quercetin 4′-O-β-d-glucoside imported by the sodiumdependent glucose transporter SGLT1. Similarly, Caco-2 cells metabolize ferulic and caffeic acids into their corresponding sulfate conjugates, but these are rapidly excreted outside of the cells on the apical side (Kern et al., 2003). However the transporter involved in this efflux has not yet been characterized. The efflux of quercetin metabolites from HepG2 cells is likely to be MRP2-dependent (O’Leary et al., 2003). In HT-29 cell lines epigallocatechin gallate 4″-O-glucuronide is effluxed, probably by MRP (Hong et al., 2002, 2003). In intestinal Caco-2 cells, a number of conjugates have been shown to be substrates of MRP-2 (apical side), or MRP-1 and/or MRP-3 (both on the basolateral side). This is the case for epicatechin, probably as its sulfate conjugate (MRP-2) (Vaidyanathan & Walle, 2001); resveratrol – probably, again, as its sulfate conjugate (MRP-2 & MRP-3) (Kaldas et al., 2003); and epicatechin gallate (MRP-2 & MRP-1) (Vaidyanathan & Walle, 2003).

14.6.2 Cellular uptake In rats, the monocarboxylic acid transporter (MCT) has been shown to be involved in the absorption of ferulic acid by the stomach (Konishi et al., 2006). A similar transporter could be involved in the Caco-2 cellular uptake of p-coumaric (Konishi et al., 2003), m-coumaric (Konishi & Kobayashi, 2004a), caffeic (Konishi & Kobayashi, 2004b), ferulic (Konishi & Shimizu, 2003), and 3-hydroxyphenylpropionic acids (Konishi & Kobayashi, 2004a). However MCT1, the best-characterized isoform of the 14 known isoforms, was not the

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one implicated in phenolic acid transport (Watanabe et al., 2006). Ellagic acid has recently been shown to be transported by the organic ion transporter (OAT) family (Whitley et al., 2005), while epicatechin and epigallocatechin gallate are actively transported into Caco-2 cells under the mediation of the monocarboxylate transporter (Vaidyanathan & Walle, 2003). The uptake of quercetin 7- and 3-glucuronides by human hepatoma HepG2 cells has been shown to be mediated by active transport, but the exact nature of the transporter could not be determined. Evidence that chlorogenic acid could be absorbed by HepG2 cells has recently been obtained (Mateos et al., 2006). Catechin glucuronides are able to enter the hepatocyte, but it is not clear whether deglucuronidation must take place before absorption or not (Donovan et al., 2001). The presence of epicatechin- and 3′-Omethylepicatechin glucuronides has been detected in both the plasma and the brain of rats after ingestion of epicatechin, which suggests that epicatechin and its 3′-O-methyl ether could cross the blood–brain barrier. One possibility is that the epicatechin glucuronides formed systemically are hydrolyzed by tissue β-glucuronidases, releasing the aglycones that could, in fact, be the actual forms crossing the blood–brain barrier. After penetration into the brain, reconjugation with glucuronic acid would take place (Abd El Mohsen et al., 2002). Alternatively, the intact epicatechin glucuronides could cross the blood– brain barrier, as already shown in the case of morphine 6-O-glucuronide (Aasmundstad et al., 1995). More studies will be necessary to validate one of these hypotheses. On the other hand, epicatechin 7- and 5-O-β-d-glucuronides (Spencer et al., 2001a), as well as quercetin 7-O-β-d-glucuronide (Spencer et al., 2003a), were clearly shown to be unable to enter dermal fibroblasts. The OATP-B transporter has recently been shown to be involved in the apical uptake of estrone 3-sulfate by Caco-2 cells (Sai et al., 2006). However nothing is yet known about its possible contribution to the uptake of phenolic O-sulfates.

14.7 Conclusions Study of the data available up until now suggests that O-methylation, O-glucuronidation, and/or O-sulfation of mammalian metabolites of phenolic compounds rarely results in the complete disappearance of the biological activity of the compound before conjugation. However, especially in the case of sulfate and glucuronide conjugates, the biological activity is often considerably attenuated as compared to the native molecule. The case of O-methylation is more complex since on the one hand it results in a diminution in the number of available phenolic hydroxyls, and on the other hand, the concomitant enhancement of the lipophilic character of the compound might be advantageous in terms of, for example, cellular absorption by passive diffusion. But of course augmentation of the lipophilic character of O-methyl derivatives is almost completely counterbalanced by further sulfation or glucuronidation of the methylated metabolites. Thus to the question raised by Mulder in 1992, ‘Pharmacological effects of drug conjugates: is morphine 6-glucuronide an exception?’ (Mulder, 1992), one could be tempted to answer yes. It is true that one of the main physiological aims of metabolic conjugation is to decrease the hydrophobicity of compounds in order to prepare them for urinary excretion, and/or to lower the number of reactive hydroxyl groups for detoxification purposes. In fact, while resveratrol was found

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to be cytotoxic at concentrations ≥ 30 μM, neither resveratrol 4′-O-β-d-glucuronide nor resveratrol 3-O-β-d-glucuronide displayed any cytotoxicity at concentrations up to 300 μM (Wang et al., 2004). However, as pointed out by the 1992 article (Mulder, 1992), the situation might, in fact, be more complex than it appears: 1) The biological data have been collected from a very limited number of conjugates, owing to an unfortunate lack of scientific interest in the preparation of appropriate standards, and consequently a gap in the development of suitable methods of synthesis. 2) Considerable attention has been paid to the antioxidant properties of the conjugates, an aspect in which partial conjugation of phenolic hydroxyl groups is expected to have a dramatic negative impact on the compounds’ activity. In addition, even in the field of antioxidant activity, there are a few noticeable examples where conjugation (for example in position 7) results in the retention or even in the enhancement of antioxidant properties. Furthermore, when considering other activities like the inhibition of signaling cascades, the situation is very different, a number of conjugates even displaying higher activity than the aglycones. 3) Degradation by the colonic microflora of the compounds that have not been absorbed by the small intestine yields another group of metabolites, a number of which display similar and sometimes enhanced activity as compared to the original compounds. However, after absorption of these colon metabolites, further conjugation takes place in the liver and the main circulating forms would, once again, be conjugates with reduced activity. Therefore it is still extremely difficult to evaluate the trends concerning the positive or negative influences of conjugation on the biological activity of phenols. One of the main questions that will definitely need to be clarified in the future is how do the metabolites exert their activity at the target cells – extra cellular activation of signaling cascades (not necessarily requiring deconjugation), deconjugation before or associated with cellular absorption, or active uptake by cellular transporters? With regard to the last aspect, although a few studies on the uptake of phenols and their conjugates by intestinal or hepatic cells have been carried out, almost nothing is known about the mode of absorption in other types of cell. Another crucial question is: once phenolics have been imported into cells, what is the capacity of these cells to further metabolize them into more active compounds? In all cases, there will be an increasing need to obtain standard metabolites. Pure and properly characterized conjugates will be necessary to precisely quantify the circulating forms of absorbed phenolics and assess their bioavailability to tissues. Standards will also be required to study their transport in target cells, and to determine how they will be metabolized before, during, or after absorption by these cells. Fortunately, significant progress has recently been made in the synthetic preparation of O-glucuronides with the introduction of efficient glucuronidation reagents. In contrast, the preparation and especially the purification of O-sulfates to provide high-quality standards still present a challenge. Considering the increasing need for pure reference sulfated conjugates, one can hope that more attention will be paid in future to the development of better sulfation methods associated with appropriate purification processes.

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Chapter 15

Polyphenols and Gene Expression Uwe Wenzel and Hannelore Daniel

15.1 Introduction The physiological properties of plant polyphenols were initially recognized by the polyphenols’ ability to reduce blood capillary permeabilities, thus they were thought to be an essential food component and were named vitamin P. Numerous epidemiological studies performed over the last decades do indeed suggest that a high dietary intake of polyphenols reduces the risk of developing atherosclerosis and diverse cancers. These protective effects are mainly attributed to the high anti-oxidant capacities of polyphenols. However, the anti-oxidant activity has in most cases only been demonstrated in vitro at concentrations hardly reached in plasma or tissues on diets rich in vegetables and fruits. More recent studies suggest that other activities of polyphenols may be more important in understanding their structure-specific effects, including their ability to alter gene expression in target cells. Various polyphenols have been shown to alter protein functions including signaling molecules such as diverse kinases, thereby altering gene expression, whereas others have been shown to bind directly to nuclear receptors such as the pregnane X receptor (PXR) or aryl hydrocarbon receptor (AHR). Some of their effects may also come from interactions with electrophile response element/antioxidant response element (EpRE/ ARE) that alters gene expression. Expression profiling techniques applied to study the effects of polyphenols in mammalian cells, either by cDNA arrays at the transcript level or by proteome analysis, have revealed in all cases pleiotropic responses, with hundreds of targets changing simultaneously. In most cases, neither prime transcription factors nor response elements could be identified. We here summarize key signaling events by which polyphenols can affect gene transcription and then more specifically describe the effects of selected compounds on gene expression in the context of atherosclerosis and cancer prevention.

15.2 The effects of polyphenols on the expression of genes underlying detoxification mechanisms Epidemiological and preclinical evidence suggests that phytochemicals including polyphenolic compounds such as epigallocatechin gallate from tea, curcumin from curry, and soy isoflavones possess cancer-chemopreventive properties (Thomasset et al., 2007), in Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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particular for cancers affected by the diet like those of the mammary gland and the colorectum (Kim et al., 2004; Simopoulos, 2004; Yuan et al., 2006; Kumar et al., 2007). Individual genetic predisposition is a major contributor to overall cancer risk, and a variety of single nucleotide polymorphisms in genes for carcinogen activation and/or detoxification have been identified in association with the risk for cancer development. Polymorphisms in N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) have been related to the NAT activities of so-called ‘slow’, ‘intermediate’ or ‘fast acetylators’. Interestingly, the NAT2 fast acetylator genotype consistently revealed a higher risk of developing colon cancer when consuming larger quantities of red meat, which may reflect the greater ability of fast acetylators to activate aromatic amines within the colon mucosa (Roberts-Thomson et al., 1996; Chen et al., 1998). Glutathione-S-transferases (GST) have also been studied in detail with respect to cancer risk. GSTs are subdivided into the four classes alpha (A), pi (P), mu (M) and theta (T), and for each class various polymorphisms have been described. GSTM1- and GSTT1-null genotypes appear to confer a high risk for several types of cancer (Habdous et al., 2004). Considering the importance of these xenobiotic-metabolizing proteins in determining the concentrations of carcinogens in humans, it is interesting to note that some polyphenols, such as those present in coffee, have been shown to cause beneficial alterations in xenobiotic metabolism, including induction of GST and inhibition of NAT (Huber & Parzefall, 2005). The induction of enzymes of phase-I and phase-II metabolism is quite well understood, and therefore it seemed logical to investigate whether polyphenols affect the expression by targeting trans-acting elements, such as certain transcription factors, and/or cis-acting elements, such as response elements in the promoter regions of genes responding to polyphenols. Figure 15.1 summarizes the three prime pathways by which polyphenols have been shown to affect gene expression events in cell systems. In the HuH7 human hepatoma cell line it has been shown that dietary polyphenolic compounds such as quercetin target the AHR to induce the expression of paraoxonase-1, an enzyme mainly secreted by the liver that is considered to display a protective function with respect to cardiovascular diseases (Gouedard et al., 2004). Induction of the paraoxonase expression subsequent to the activation of the AHR by quercetin is finally mediated by a xenobiotic responsive element (XRE)-like sequence within the paraoxonase-1 promoter (Gouedard et al., 2004). It has to be stressed in this context that certain toxic compounds, such as the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene, are also inducers of paraoxonase-1, which suggests that polyphenols are regarded by cells as xenobiotics, and activate a mechanism that originally served the detoxification of deleterious agents (Gouedard et al., 2004). The binding of ligands to AHR initiates the expression not only of phase-I enzymes but also of phase-II enzymes, leading to their being regarded as bifunctional inducers (Burczynski & Penning, 2000). Classical bifunctional inducers, such as many PAHs, elevate phase-I enzyme expression via an XRE and are subsequently converted by the induced enzymes, such as cytochrome P450 1A1 (CYP1A1), to electrophiles that enhance gene transcription of phase-II enzymes via an EpRE/ARE (Burczynski & Penning, 2000). The electrophiles generated, however, in the case of PAHs are the prerequisite for their DNAadduct forming capacities and thus for their mutagenicity (Scates et al., 1996). Although

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PXR Nrf2

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XRE EpRE/ARE PRE

Xenobiotic efflux pumps Antioxidant stress proteins Detoxifying enzymes Fig. 15.1 Trans- and cis-acting elements of genes activated by polyphenols. Selected polyphenols have been shown to bind to the aryl hydrocarbon receptor (AHR) as well as to the nuclear factor E2-related factor (Nrf2), or the pregnane X receptor (PXR). Binding of polyphenols to the receptor entities leads to the binding of the complexes to the corresponding response elements in the promoter region of target genes with concomitant activation of gene transcription. XRE, xenobiotics response element; EpRE/ARE, electrophile response element/antioxidant response element; PRE, pregnane X receptor response element.

distinct polyphenols like quercetin induce CYP1A1 it needs to be emphasized that it effectively suppresses the induction that is caused by PAHs (Pohl et al., 2006). This strongly suggests that quercetin displaces PAHs from the AHR whereas its ability to activate it seems comparatively low. However, considering the dual effects of many phase-I enzymes, i.e. carcinogen activation and alleviation of excretion, compounds that selectively cause an induction of phase-II enzymes, so-called monofunctional inducers, are generally regarded as the better chemopreventive agents (Wilkinson & Clapper, 1997). Yannai et al. showed that, in AHR-defective cells, genistein and daidzein, the two major soy polyphenols, are able to induce phase-II enzymes directly (Yannai et al., 1998). GST represents a phase-II enzyme that can be induced by green tea polyphenols (Itoh et al., 1999) and by chlorogenic acid, one of the most abundant polyphenols in the human diet (Feng et al., 2005). These compounds lead to an activation of the basic leucine zipper transcription factor nuclear factor E2-related factor (Nrf2), which dimerizes with small Maf proteins (Itoh et al., 1999) and activates EpRE/ARE (Itoh et al., 1999; Feng et al., 2005). This finally then protects cells against toxic insults by the increased expression of phase-II enzymes and other genes of defense systems (Owuor & Kong, 2002). Similar to the XRE, the EpRE/ARE responds to cellular stress, and it has been suggested that a coordinated modulation of MAPK cascades by reactive oxygen species (ROS) is critical in the regulation of phase-II enzyme genes through the EpRE/ARE in cellular homeostasis (Owuor & Kong, 2002). Two mechanisms have been proposed to explain how Nrf2 senses oxidative stress. A cytosolic protein, Keap1, has been shown to suppress Nrf2

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transcriptional activity by binding and thereby retaining Nrf2 in the cytosol (Itoh et al., 1999). Upon activation by oxidative stress, the Keap1–Nrf2 complex dissociates through the phosphorylation of Nrf2, and Nrf2 then translocates into the nucleus to activate EpRE/ ARE (Itoh et al., 1999). However, through a series of studies examining both nuclear and cytosolic Nrf2, Owuor & Kong proposed an alternative mechanism in which most of the Nrf2 is localized in the nucleus and not in the cytosol before and during oxidative stress (Owuor & Kong, 2002). This could mean that depending on the external stimulus, different tissues differentially modulate Nrf2 localization and its activity as an oxidative stress sensor. The prime role of Nrf2 as a cellular stress sensor is demonstrated in Nrf2 −/− knock-out mice that lack, for example, the induction of GST (Itoh et al., 1997). Figure 15.2 summarizes the signaling pathways involving central Nrf2 as activated by ROS and electrophiles and for which it has been shown that they are affected by selected polyphenols. Besides phase-I and phase-II metabolism, the export of deleterious compounds from the cell is another mechanism that contributes to overall cellular defense. The export of xenobiotics from the cell was designated as phase III in the handling of xenobiotics (Xu et al., 2005). Phase-III transporters such as P-glycoprotein (P-gp), multidrug resistanceassociated proteins (MRPs), and organic anion transporting polypeptide 2 (OATP2) are expressed in many tissues including the liver, intestine, kidneys, and brain, and are usually induced along with phase-I and phase-II enzymes (Xu et al., 2005). This implies that the xenobiotic transporters may be regulated in a coordinated fashion with the detoxifying

Polyphenols Reactive oxygen species

Electrophiles

Cell Keap1 MAPKK

Actin

Nrf2 dissociation

MAPK small Maf

Nrf2 activation

DNA

small Maf

Nrf2

EpRE/ARE

Nucleus Antioxidant stress proteins increased expression Detoxifying enzymes

Fig. 15.2 Activation of the EpRE/ARE by a less well defined cross-talk of polyphenols, reactive oxygen species (ROS) and electrophiles. Binding of the compounds leads to the dissociation of Nrf2 from Keap-1, and in association with small MAfs enables Nrf2 to initiate the transcriptional activation of genes with EpRE/ARE in their promoters. It has also been suggested that a coordinated modulation of MAPK cascades by ROS is critical in the regulation of phase-II detoxifying enzyme genes through the EpRE/ARE in cellular homeostasis.

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enzymes for optimal protection from xenobiotic insult (Xu et al., 2005). Like the phase-I and phase-II enzymes, phase-III transporter-proteins can also be induced by polyphenols. This has been shown for example for hyperforin, which activates PXR, an orphan nuclear receptor acting as a key regulator of P-gp gene expression (Ekins & Erickson, 2002), causing in turn increased P-gp levels in the intestine (Matheny et al., 2004). PXR not only leads to increased expression of xenobiotic transporters when activated but is also involved in the regulation of phase-I enzymes, e.g. via binding to the respective response elements in the CYP3A4 promoter (Kluth et al., 2007). Whereas resveratrol has been shown to cause increased CYP3A4 promoter activity in a manner that is dependent on PXR, the major apple polyphenol quercetin has been shown to increase CYP3A4 promoter activity independently of PXR (Kluth et al., 2007). Moreover, resveratrol did not activate the isolated EpRE/ARE but enhanced the promoter activity of the phase-II gene gastrointestinal glutathione peroxidase (Kluth et al., 2007). It must be concluded from these results that individual polyphenols may affect phase-I, phase-II and probably also phase-III gene expression by distinct mechanisms (Kluth et al., 2007).

15.3 Polyphenols and the expression of genes underlying cancer-relevant processes Epidemiological, cell culture, animal and clinical studies performed in the last decade have shed light on the mechanisms by which dietary polyphenols interfere with the processes that underlie cancer development. The green tea polyphenol (−)-epigallocatechin gallate ((−)-EGCG), in particular, has received much attention, and convincing evidence for its protective action has been revealed (Chen et al., 2004). In cultured cells the tea polyphenols can potently induce apoptotic cell death and cause cell-cycle arrest in tumor cells but not in non-transformed normal cells (Ahmad et al., 2000; Chen et al., 2004). Recently-conducted meta-analysis, however, suggests that the preventive effects of tea polyphenols are more consistently associated with atherosclerosis protection than with cancers (Arts & Hollman, 2005; Kuriyama et al., 2006). The obvious discrepancies between experimental and epidemiological data are most likely due to the fact that in-vitro and in-vivo experiments are usually performed with test doses that cannot be achieved by diet. Nevertheless, even if obtained only at doses that may be regarded as pharmacological, the effects exerted by some of the polyphenols in cancer cells are impressive. Because cancers are, to a certain extent, genetically-mediated diseases that at a certain stage require mutations in proto-oncogenes, tumor suppressor genes or DNA-repair genes, analysis of the gene mutations (Yuan et al., 2005) and/or analysis of concomitant changes in gene expression by high-throughput technologies needs to be performed for comprehensive profiling (Mills, 2003). In addition to RNA-expression analysis using oligo or cDNA gene chips (Mills, 2003), protein-expression analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry are used for screening (Habermann et al., 2006). Moreover, analysis of post-transcriptional modifications or of the DNA-methylation status – both of which contribute to the phenotype of the cancer cells – can also be performed at a high throughput (Mills, 2003). These techniques are applied mainly for a better

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understanding of cancer initiation and progression, but will eventually also lead towards the development of new molecular-targeted drug therapies (Mills, 2003). Microarray-based mRNA analysis or proteome analysis techniques are widely used to profile tumor samples in a search for differences from normal cells. By employing gene microarray analysis, it has been shown that patterns of genes with altered transcript levels appear to predict their clinical behavior (Alizadeh et al., 2001). For example, gene expression analysis allowed the subdivision of human breast cancers into four major tumor groups (Perou et al., 2000). Most notably, ERBB2-positive tumors and the group of tumors with ‘basal-like’ characteristics (mixed ERBB2 status) were both found to be associated with a statistically significantly poorer prognosis. In addition, this study also identified two estrogen receptor-positive tumor subtypes, one of which showed a favorable prognosis and one of which showed a prognosis almost as poor as for ERBB2-positive tumors (Sorlie et al., 2001). Using a similar microarray gene expression profiling strategy the prognosis of stage II colon cancer patients could be quite well predicted (Barrier et al., 2006). In a clinical trial conducted to evaluate the safety and efficacy of a selective inhibitor of cyclooxygenase-2 (celecoxib) in hereditary non-polyposis colon cancer patients, it was shown that twentythree of twenty-five pairs of healthy colon biopsies, taken before and after celecoxib treatment, could be classified correctly by the pattern of gene expression (Glebov et al., 2006). The early steps of inflammatory reaction, cell signaling and cell adhesion, response to stress, transforming growth factor-beta (TGF-β) signaling, and regulation of apoptosis were identified as the main biological processes targeted by celecoxib, which provides an explanation for its activity as a chemopreventive agent (Glebov et al., 2006). Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines for which the chemosensitivity profiles of thousands of chemical compounds had been determined (Staunton et al., 2001). The aim of the experiment was to assess whether the gene expression signatures of untreated cells were adequate for the prediction of their chemosensitivity. Indeed the accuracy of chemosensitivity prediction was considerably better than would be expected by chance and the results suggest that – at least for a subset of compounds – genomic approaches for chemosensitivity prediction are feasible (Staunton et al., 2001). Overall, techniques of gene expression profiling are suitable for defining the biological responses in a cell or tissue and appear to be very helpful in improving diagnosis, prognosis, and therapeutic management. By analogy with defining genetic and transcriptional alterations in tumors, changes at protein level are characterized by proteome analysis approaches. Comparative proteome profiling of human breast cancer and adjacent normal breast tissues revealed 15 characteristic changes in 11 out of 12 breast cancer samples (Deng et al., 2006). Categorization of the proteins based on their function indicated pathological changes related to metabolism, promotion of proteolytic activity or a decline in activity of some enzyme inhibitors. Changes in expression levels of SMT3A, RPS12, and PSMA1 as new putative biomarkers were also first reported in this study (Deng et al., 2006). By surface-enhanced laser desorbtion/ionization (SELDI)-based proteome analysis the serum proteome of sixty-two colorectal cancer patients and thirty-one non-cancer control subjects was analyzed and proteins with diagnostic potential, such as complement C3a des-arg, alpha1-antitrypsin and transferrin were identified (Ward et al., 2006).

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Similarly, profiling methods have been employed for investigating the effects exerted by polyphenols in cancers. In F344 rats red-wine polyphenols (50 mg/kg body weight) administered with the diet for 16 weeks were shown to inhibit colon carcinogenesis induced by azoxymethane (Dolara et al., 2005). Global expression analysis of 5707 genes revealed an extensive down-regulation of genes involved in a wide range of physiological functions, such as metabolism, transport, signal transduction and intercellular signaling (Dolara et al., 2005). Pathway analysis tools allowed the identification of inflammatory response and steroid metabolism as two major pathways down-regulated in the colonic mucosa of polyphenol-treated rats (Dolara et al., 2005). In PC-9 human lung cancer cells NF-κBinducing kinase (NIK), death-associated protein kinase (DAPK 1), rho B and tyrosine protein kinase were identified by cDNA array analysis as down-regulated by EGCG (Fujiki et al., 2001). Since NIK activates the inhibitor of NF-κB kinase (IKK)α–IKKβ complex in the process of tumor necrosis factor-α (TNF-α) signaling, the down-regulation of the NIK gene by EGCG seems to be partly involved in inactivation of NF-κB signaling, resulting in growth inhibition of PC-9 cells (Fujiki et al., 2001). These results support previous findings showing that treatment with EGCG inhibits expression of the TNF-α gene and TNF-α release from the cells, and they also support an overall anti-inflammatory activity for EGCG (Fujiki et al., 2000; Suganuma et al., 2000). The author’s unit assessed the effects of flavone, a flavonoid that, owing to the absence of hydroxyl groups in the molecule, cannot be considered a polyphenol, on mRNA and protein levels in HT-29 human colon cancer cells. Flavone was shown to be a potent inducer of apoptosis in human colon cancer cells (Wenzel et al., 2000) but did not show any effects in the non-transformed human colonocyte cell line NCOL-1 (Wenzel et al., 2003) or primary mouse colonocytes (Wenzel et al., 2000). Analysis of changes in transcript levels by oligonucleotide arrays, using chips with around 10,000 oligonucleotide probes spotted, revealed numerous changes in gene expression related to cellular signaling, transcription and cancer development, but also of metabolism (Herzog et al., 2004a). Pathway analysis tools including the Bibliosphere software (Genomatix, Munich, Germany) revealed a complex interconnected network of genes derived from distinct signaling pathways (Fig. 15.3). PI-3-kinase-related pathways with various members of the insulin-signaling cascade changed upon flavone treatment, as also did members of the epidermal-growth factor- and G-protein coupled-receptor pathways and those of TGF-β- and p53-signaling. The transcription factor jun was identified as the center of regulation of both the Wnt-signaling and the MAP-kinase signaling pathways. Pathway analysis also revealed changes in the interleukin-6 (IL-6)-related mechanism, and regulation of several pathways of intermediary metabolism most likely linked to changes in enolase 2 (ENO 2). In contrast to mRNA-profiling, proteome analysis in its current state is less comprehensive and sensitive, as only those proteins present in high abundance can easily be identified. Consequently, proteome analysis applied to the same samples in which transcript profiling was performed, revealed only 20 protein spots with altered levels on treatment with flavone. These proteins were identified by MALDI-TOF mass spectrometry (Herzog et al., 2004a) and also submitted to pathway analysis. A network of interactions was identified with protein kinase C-beta (PKC-β) in the central position (Fig. 15.4). Six proteins – four of which are known to be involved in the removal of superoxide anions

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IF-β/IL-6 pathway

Insulin/AKT signaling Wnt/ MAP kinases

EGF signaling

JUN

Glycolysis Gluconeogenesis

TCA cycle

Fig. 15.3 Network of interconnected signaling pathways identified by cDNA-array analysis of HT-29 cells exposed to flavone. Key regulators of distinct pathways are indicated.

Insulin signaling TP53 signaling pathway

G-protein coupled receptors signaling

Pl3K-AKT signaling pathway IN

PKC-

APOA1 Hypoxia signaling

IN

ANXA6

M STIN IN

CAT

GNB2L1

IN

IN

PRDX4

PDIA3 Removal of superoxide radicals

Fig. 15.4 Network of interconnected signaling pathways with protein kinase C-beta (PKC-β) in the central position as identified by proteome analysis of HT-29 cells exposed to flavone.

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SDH TCA cycle PKC-β

NF-KB

Fig. 15.5 Predicted network based on the transcription factors that could have caused the alterations in the levels of the proteins that responded in HT-29 cells exposed to flavones with altered steady-state levels. Proteins identified by proteome analysis responsive to flavone are shown in purple boxes, transcription factors in unfilled boxes. From the analysis, PKC-β turns up as a prime regulator that could explain many of the observed changes in protein levels.

– displayed a direct or indirect interaction with PKC-β. Moreover, when transcription factors as targets of PKC-β were included in the analysis, it became evident that the same pathways could be affected by PKC-β at the protein level as already identified as being altered by flavone at the transcriptional level. It is worth noting that NF-κB is a target of PKC-β, and that treatment with flavone also affected succinate dehydrogenase (SDH) levels and other tricarboxylic acid cycle alterations (Fig. 15.5). This is in accordance with experimental findings in flavone-treated HT-29 cells that revealed an absence of the PKC-β protein (Herzog et al., 2004a) and an absence of NF-κB mRNA (Wenzel et al., 2000) associated with down-regulation of SDH at the protein level as well as other alterations of enzymes of intermediate metabolism (Herzog et al., 2004a). PKC-β was thus identified by various means as the prime target of flavone action in HT-29 cells, and it has been proposed as a valid biomarker in the development of colon cancer (Yu et al., 2003). The results obtained for the effects of flavone on apoptosis in colonic tumor cells, and in particular on the levels of metabolic enzymes, were mirrored by an impaired energy status of the cells (Herzog et al., 2004a). Qualitative changes in energy metabolism in HT-29 cells in response to flavone were observed with a characteristic time lineage. When cells were exposed to flavone for only 1 h, they released less lactate but showed increased lactate uptake into the mitochondria, leading to an enhanced flux of substrates through the respiratory chain associated with an accelerated production of superoxide anions, which in turn caused induction of apoptosis (Wenzel et al., 2005). When cells were exposed for

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24 h to flavone, down-regulation of the metabolic enzymes was observed, which may be taken as a measure of an adaptive response in a negative feedback manner to reduce the superoxide anion burden of the cells (Herzog et al., 2004a). Moreover, the observed up-regulation of superoxide anion detoxifying enzymes such as peroxiredoxin fits well into this model as another efficient mechanism whereby cancer cells escape apoptosis. In non-transformed colonocytes exposed to flavone only a few proteins showed alterations in the steady-state level, with only one related to intermediary metabolism (Herzog et al., 2004b). Normal cell metabolism seems therefore not to be affected by flavone, as the compound does not induce apoptosis in non-transformed cells (Wenzel et al., 2003). Figure 15.6 summarizes our current understanding of the effects of flavone on tumor-cell apoptosis. One process is driven by the metabolism, involving a huge increase in ROS production via enhanced mitochondrial respiration based on an increased influx of energy substrates into the mitochondria. Production of superoxide anions initiates gene expression of ROSdependent pathways and activates the apoptosis cascade via caspases. A second pathway involves altered gene expression of a variety of pathways centered around PKC-β, including genes that are also needed for altered energy metabolism, for example TCA-cycle and respiratory chain activity, which are considered to represent a compensation mechanism for reducing the ROS-burden again.

Fatty acids

cancer cell

flavone

Acyl-CoA

target gene groups

Carn

metabolic effects

TCA cycle Carn Pyruvate

Acetyl-CoA

EGF signaling Wnt signaling P53 signaling

Glucose

ATP Lactate

ATP

NADH2

TGF-β signaling respiratory chain

transcriptional effects

PKC-β

ERKs

–

O2 ROS ROS

Akt

NF- B

Survival

Caspases Apoptosis

flavone Fig. 15.6 Current model proposed for the modes of action of flavone in tumor cells. Increased supply of pyruvate/lactate to mitochondria together with increased expression of enzymes from tricarboxylic acid (TCA)-cycle leads finally to an increase in mitochondrial ROS production by an enhanced respiration rate. Moreover, production of ROS initiates gene expression of ROS-dependent pathways and activates caspases and the apoptosis cascade. A second mechanism involves altered gene expression in a variety of pathways centered around PKC-β, including genes coding for proteins of the TCA-cycle and respiratory chain that are needed for altered energy metabolism to enable a compensation mechanism to reduce the ROS-burden. Moreover, reduced PKC-β activity inhibits those pathways usually found to be activated in colon cancer, such as EGF-signaling and Wnt-signaling, and activates those with usually reduced activity in colon cancer, such as p53- or TGF-β-mediated signaling.

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The high selectivity of apoptosis-induction by flavone was not observed when cells were treated with quercetin, which was equally effective at inducing apoptosis in HT-29 and the non-transformed NCOL-1 colon cells (Herzog et al., 2004b; Wenzel et al., 2004). NCOL-1 cells grow quite well in culture, but this is not the case for normal intestinal colonocytes, emphasizing that NCOL-1 were selected because of a high proliferative capacity and that their phenotype should be regarded as pre-neoplastic; but based on a softagar assay they are classified as non-transformed (Deveney et al., 1996). Interestingly, in HT-29 and NCOL-1 cells quercetin also led to alterations in the levels of enzymes of intermediary metabolism (Herzog et al., 2004b; Wenzel et al., 2004). This may suggest that apoptosis-inducing flavonoids use a common signaling pathway to exert their effects, but the genetic make-up of the cells (transformed versus non-transformed) obviously determines the outcome of whether apoptosis is initiated or not. However, apoptosis-inducing activities in the normal human gut by quercetin may not be observed – at least not at the concentrations reached after a meal rich in fruits and vegetables.

15.4 The impact of polyphenols on the expression of genes underlying atherosclerosis-relevant processes Epidemiological studies most consistently reveal preventive effects of polyphenols in the pathogenesis of atherosclerosis (Arts & Hollman, 2005; Kuriyama et al., 2006). The polyphenol plasma concentrations to which a blood vessel is exposed are generally in the micromolar or even sub-micromolar range. For example, in Asians with a high intake of soy products the average plasma concentrations of the main isoflavones genistein and daidzein are around 2.5 μM (Izumi et al., 2000), with maximal physiologically reachable isoflavone plasma concentration – including the glucuronide and sulfate conjugates of the absorbed isoflavones – of 18.5 μM (Barnes, 1995; Shelnutt et al., 2002). With regard to the effects of polyphenols on the expression of genes involved in atherogenesis, most studies have addressed the red-wine polyphenols for defining the molecular basis of the ‘French Paradox’ (Delmas et al., 2005). Resveratrol was found to inhibit TNF-α-mediated as well as IL-6-induced ICAM-1 gene expression at the promoter, transcriptional and protein levels and this was taken as the prime mechanism of the proposed beneficial effects of resveratrol in endothelial responses to cytokines during inflammation (Wung et al., 2005). Moreover, red-wine polyphenols significantly inhibit the proliferation of human vascular smooth muscle cells, but not of human vascular endothelial cells, by reducing promoter activity and expression of the cyclin A gene (Iijima et al., 2002). Atherosclerosis is a consequence of a dysregulated cross-talk between different tissues and mediators and involves a large number of genes that control lipid metabolism, including those coding for the LDL-receptor, the receptor for oxidized LDL (CD36), the liver X receptor-α (LXR-α), the peroxisome proliferator activated receptors α and γ (PPARα and PPAR-γ) and c-myc (Kaul et al., 2004). Green tea polyphenols have also been assessed for their activity towards inhibition of the initiation and progression of atherosclerosis (Hofman & Sonenshein, 2003). They were shown to inhibit the oxidation of LDL (Xu et al., 2004), which blocks the up-regulation of transcriptional expression of sterol-response binding protein (SREBP), PPAR-γ, IL-8

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and apoprotein-E, all of which are known to be pro-atherogenic; this latter function involves centrally the oxysterol-receptor LXR-α (Kaul, 2001; Kaul et al., 2004). In this respect a seemingly vicious circle is disrupted via the reduced expression of PPAR-γ, since this transcription factor induces the expression of LXR-α, but also of pro-inflammatory factors such as IL-6, IL-8, TNF-α, and NF-κB (Bishop-Bailey, 2000). In addition, by preventing the oxidation of LDL the LXR-α-mediated down-regulation of a variety of apoptosis-preventing factors such as c-myc and bcl-2 is overcome (Kaul, 2001; Kaul et al., 2004). Finally, the up-regulation of PPAR-α by green tea polyphenols is beneficial, since PPAR-α activation has been shown to inhibit neutral lipid accumulation in macrophages, thereby contributing to a regression of atherosclerosis (Kaul et al., 2004). The cross-talk between mediators involved in lipid metabolism as influenced by polyphenols at the expression level is summarized in Fig. 15.7. As already described in the context of cancer-relevant processes, gene expression by polyphenols can occur via Nrf-2 activated EpRE/ARE. The concomitant changes in the expression of proteins acting in antioxidant defense play a role not only in cancer prevention but also in the inhibition of atherogenesis (Chen et al., 2006). In human aortic endothelial cells it has been shown that over-expression of Nrf-2 suppresses the expression of numerous pro-inflammatory genes when their transcription is activated by TNF-α. The Nrf2/EpRE/ARE pathway may therefore be considered as an endogenous atheroprotective system central to the suppression of redox-sensitive inflammatory genes (Chen et al., 2006). oxLDL

oxidation

LDL

Polyphenols

CD36

LDL-R expression

oxy-sterols

Lipid metabolism Nucleus

LxR-α

expression

Apo E IL-8 Bcl-2

PPARs C-myc

APOPTOSIS

IL-6 IL-8 TNF-α

Fig. 15.7 Genes that control lipid metabolism and the interplay of selected polyphenols with the pathways are shown (according to Kaul et al., 2004). For example, for green tea polyphenols it has been shown that they inhibit the oxidation of LDL, which impairs the up-regulation of transcriptional expression of the pro-atherogenic factors PPAR-γ, IL-8 and apoprotein-E, usually occurring via liver X receptor (LXR). A vicious circle is disrupted via the reduced expression of PPAR-γ, since this transcription factor induces the expression of LXR-α, but also that of pro-inflammatory factors such as IL-6, IL-8, TNF-α, and NF-κB. Moreover, the LXR-α-mediated down-regulation of a variety of factors that prevent apoptosis, such as c-myc and bcl-2, is overcome by polyphenols.

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In spite of studies showing polyphenol effects on selected genes involved in atherosclerosis, studies with comprehensive analysis of gene regulation by transcriptomics or proteomics tools are sparse. We have used transcript and proteome profiling techniques to assess the cellular functions of the soy isoflavones for which epidemiological studies showed that their intake is associated with multiple beneficial effects not only on breast and prostate cancers, menopausal symptoms, osteoporosis, and neurodegeneration, but also on atherosclerosis and stroke (Branca & Lorenzetti, 2005), leading to a recommendation of a minimum intake of 40–60 mg isoflavone aglycones/day, together with about 25 g of soy protein to decrease cardiovascular risk (US Department of Health and Human Services, FDA, 1999). We used proteomics and cDNA array technologies to identify genes that respond to treatment by genistein in endothelial cells. The transcriptional response to 2.5 μM genistein in primary human endothelial cells (HUVEC) was characterized by alterations in the expression of genes encoding for proteins centrally involved in the vascular tone such as endothelin-converting enzyme-1, endothelin-2, estrogen-related receptor alpha and atrial natriuretic peptide receptor A precursor (Ambra et al., 2006). At both the mRNA- and the protein level it was shown that genistein not only affects the expression of atherosclerosis-relevant genes but also possesses a high potency for preventing alterations at the expression level in an atherogenic direction that are caused by the endothelial stressors oxidized LDL or homocysteine (Fuchs et al., 2005a, 2005c). In immortalized human endothelial cells genistein was shown to completely block apoptosis when induced by ox-LDL (Fuchs et al., 2005c) and these effects were associated with changes in the steady-state levels of several apoptosis-relevant proteins. These findings suggest a putative atheroprotective function of genistein by effectively inhibiting apoptosis. Endothelial apoptosis is a crucial mechanism in atherosclerosis as it promotes thrombin aggregation (Bombeli et al., 1997) and monocyte adhesion to the endothelium (Huber et al., 2002). Genistein has also been shown to inhibit apoptosis in immortalized endothelial cells when initiated by homocysteine, and this was associated with reversion of most of the homocysteine-induced alterations of the cellular proteome (Fuchs et al., 2005b). Interestingly, quite different proteins and protein classes were affected by treating cells with either ox-LDL or homocysteine, suggesting that the two stressors – despite the fact that they both induce apoptosis – affect different signaling pathways in endothelial cells. Among the few proteins that were found to be regulated in the same direction by ox-LDL and homocysteine, and where in both cases genistein prevented the stress-induced alterations, we identified the transformation-sensitive protein IEF SSP 3521, the nudix hydrolase NUDT5, the triosephosphate isomerase and the thioredoxin-related protein (Huber et al., 2002; Fuchs et al., 2005b). Based on the known functions of these proteins, the thioredoxin-related protein appears most interesting as a novel endothelial stress marker. It obviously links the pro-apoptotic and pro-atherogenic effects of endothelial stressors with the protective functions of genistein. Thioredoxin-related protein exerts its effects by preserving NO-synthase activities in the presence of NO and has been shown to bind directly to the apoptosis signal-regulating kinase 1, thereby inhibiting the activation of stress-induced MAPK signaling cascades that eventually lead to apoptosis (Shao et al., 2002). The increase in expression of thioredoxin-related protein effected by genistein could involve the activation of EpRE/ARE in analogy to what has been shown for sulforaphane

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from broccoli on the expression of thioredoxin reductase, a protein similar to thioredoxinrelated protein (Hintze et al., 2005). However, EpRE/ARE is usually activated as a response to stressors but both ox-LDL and homocysteine decreased the levels of thioredoxin-related protein in endothelial cells (Huber et al., 2002; Fuchs et al., 2005b). In spite of that GSTP, a classical EpRE/ARE-activated stress response gene, revealed increased protein levels in cells treated with ox-LDL, homocysteine and genistein (Huber et al., 2002; Fuchs et al., 2005b). Taking into account that anti-atherogenic laminar flow and pro-atherogenic oscillatory flow in human aortic endothelial cells both induce the nuclear accumulation of Nrf2 to comparable levels, whereas Nrf2 binding to EpRE/ARE was significantly diminished in the case of oscillatory flow compared with that of laminar flow (Hosoya et al., 2005), a similar inhibition of Nrf2 activity at the level of DNA binding may be suggested with respect to its contribution to thioredoxin-related protein gene expression by ox-LDL or homocysteine. As there is an on-going discussion as to whether genistein and daidzein or the protein component and other constituents of soy are responsible for its protective functions, we also studied the effects of a soy extract on the expression of endothelial cell proteins. This comparative analysis revealed that quite different sets of proteins in cells stressed by ox-LDL were affected by a mixture of genistein and daidzein or the extract from which they were derived (Fuchs et al., 2007a). Only three proteins were identified as regulated in a common way, and for those the extract as well as the isoflavone mixture uniformly prevented their down-regulation caused by ox-LDL. These proteins were CMP-N-acetylneuraminic acid synthase, coproporphyrinogen oxidase, and transketolase. CMP-N-acetylneuraminic acid synthase controls the amount of sialylated glycoconjugates in the plasma membrane and could thereby reduce the uptake of LDL particles (Gorog & Pearson, 1984). This in turn could lead to a reduced mitochondrial ROS generation, as ox-LDL has been proven to induce ROS-formation in the mitochondria of endothelial cells (Zmijewski et al., 2005). We also observed an inhibition of mitochondrial ROS production in the presence of soy extract as well as the genistein and daidzein mixture in ox-LDL-treated cells, which may be most relevant for the anti-apoptotic activities of soy. Several studies have established that ox-LDL enhances arterial apoptosis via a mechanism dependent upon ROS production (Kotamraju et al., 2001). Mitochondrial dysfunction could additionally be fostered by the observed decline in coproporphyrinogen oxidase levels upon ox-LDL exposure of endothelial cells, which might be accompanied by reduced porphyrin levels needed, for example, for cytochrome b functionality. In fact, Senoo-Matsuda et al. showed that cytochrome b defects in complex II of the respiratory chain cause an overflow of superoxide anion production (Senoo-Matsuda et al., 2001). Increasing the activity of transketolase has been shown to prevent the development and progression of vascular damage in a mouse model by removing accumulating substrates for four major metabolic pathways known to be related to atherosclerosis and to be activated by glucose-induced ROS-generation (Hammes et al., 2003). In this context enhanced transketolase expression may substantially contribute to the isoflavones’ protection from endothelial damage caused by ox-LDL by counteracting on an increased polyol pathway flux, increased hexosamine pathway flux, increased formation of advanced glycation end-products, and finally by preventing the activation of PKC (Hammes et al., 2003).

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We also assessed changes in the proteome of peripheral blood mononuclear cells from postmenopausal women undergoing an eight-week intervention with soy extract-enriched cereal bars providing 50 mg isoflavones per day (Fuchs et al., 2007b). Proteome analysis revealed numerous proteins responding to treatment that could be linked to the genesis and progression of atherosclerosis. The observed changes of the marker proteins suggest that the soy extract provides some anti-inflammatory activity and also protects the fibrinolytic system. However, a general anti-inflammatory activity of the intervention based on numerous clinically relevant inflammation markers was not observed (Tikkanen et al., 1998). Taken together, polyphenols display a huge spectrum of different biological activities despite the fact that most of them are efficient antioxidants. Gene expression analysis as well as proteome profiling approaches to assess the molecular functions of selected polyphenols have revealed pleiotropic effects that in most cases involve changes in hundreds of target genes and target proteins. There is no unifying concept underlying the biological changes observed in mammalian cells exposed to polyphenols. Each compound has to be assessed individually with respect to its biological function and each read-out will depend on the concentrations used. Polyphenols can be considered as multifunctional compounds affecting gene expression by directly interacting with nuclear receptors that control transcription, and also by simultaneously targeting non-nuclear proteins, against which they act as inhibitors or activators. These proteins may then secondarily also transmit signals into the nucleus that alter gene expression. In this respect dedicated studies unraveling the action of polyphenols as a function of concentration, time and space are urgently needed. Although the high-throughput profiling techniques are helpful in revealing hints for biological activities, they currently serve more as a ‘gold-washing pan’ and provide only snap-shots of a cellular state.

15.5 References Ahmad, N., Gupta, S. & Mukhtar, H. (2000). Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells. Archives of Biochemistry and Biophysics, 376, 338 –346. Alizadeh, A.A., Ross, D.T., Perou, C.M. & van de Rijn, M. (2001). Towards a novel classification of human malignancies based on gene expression patterns. Journal of Pathology, 195, 41–52. Ambra, R., Rimbach, G., de Pascual-Teresa, S. et al. (2006). Genistein affects the expression of genes involved in blood pressure regulation and angiogenesis in primary human endothelial cells. Nutrition, Metabolism and Cardiovascular Disease, 16, 35–43. Arts, I.C. & Hollman, P.C. (2005). Polyphenols and disease risk in epidemiologic studies. American Journal of Clinical Nutrition, 81, 317S–325S. Barnes, S. (1995). Effect of genistein on in vitro and in vivo models of cancer. Journal of Nutrition, 125, 777S–783S. Barrier, A., Boelle, P.Y., Roser, F. et al. (2006). Stage II colon cancer prognosis prediction by tumor gene expression profiling. Journal of Clinical Oncology, 24, 4685–4691. Bishop-Bailey, D. (2000). Peroxisome proliferator-activated receptors in the cardiovascular system. British Journal of Pharmacology, 129, 823–834. Bombeli, T., Karsan, A., Tait, J.F. & Harlan, J.M. (1997). Apoptotic vascular endothelial cells become procoagulant. Blood, 89, 2429 –2442. Branca, F. & Lorenzetti, S. (2005). Health effects of phytoestrogens. Forum of Nutrition, 57, 100–111.

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The index is arranged in letter-by-letter sequence; page numbers in italics refer to figures; page numbers in bold refer to tables. A3GT see anthocyanidin 3-O-glycosyltransferase (A3GT) AATs see anthocyanin acyltransferases (AATs) ABC (adenosine 5′-triphosphate binding cassette), 149 ACE see angiotensin converting enzyme (ACE) N-acetyltransferase 1 (NAT1), polymorphisms, 360 N-acetyltransferase 2 (NAT2), polymorphisms, 360 acibenzolar-S-methyl (ASM), 220 adenosine 5′-triphosphate (ATP), 78 adenosine 5′-triphosphate binding cassette (ABC), 149 A-EpRE genes see antioxidant/electrophile-responsive element (A-EpRE) genes afzelechins, 187 aglycons, 20–1, 236–7 cellular metabolism, 73–4 conjugation, 72 diffusion, 8 solubility, 2 AHA10 gene, 120 expression, 122 AHR (aryl hydrocarbon receptor), 359, 360–1 aldehydes, 124 aldose reductase, inhibition, 344 alizarin, 9 allelochemicals, 7 allelopathy, use of term, 6–7 Alliaria petiolata (garlic mustard), 20 Allium cepa (red onion) 5-carboxypyranocyanidin, 178 cyanidins, 182 UGT73J1, 129 Allium schoenoprasum (chive), anthocyanin–flavone pigments, 188 Alstroemeria spp. (Peruvian lilies), anthocyanin glycosides, 181–2 Alternaria alternata (fungus), infection, 214 Anemone coronaria (poppy anemone), anthocyanins, 183 angiogenesis, phenolic conjugate effects, 345 angiosperms anthocyanins, 167 flavonoid biosynthesis, metabolic engineering, 142–9 flavonoid biosynthetic pathways, 139–40 angiotensin converting enzyme (ACE) inhibitors, 266 and pomegranate juice, 265–6 animals, innate immune response, 203

animal studies anthocyanin metabolism, 251–2 see also pig studies ANR (anthocyanidin reductase), 118 ANR gene, 118 expression, 121–2, 123 ANS see anthocyanidin synthase (ANS) ANS gene, 118 expression, 121–2 anthocyanic vacuolar inclusions (AVIs), 139, 149–52, 191–2 formation, 149 in petals, 150–2 occurrence, 150 use of term, 150 anthocyanidin 3-O-glycosyltransferase (A3GT), 140 sequencing, 145 anthocyanidin O-glycosyltransferases (AGTs), clones, 145 anthocyanidin reductase (ANR), 118 anthocyanidins, 168–78 classification, 228–9 equilibrium forms, 178–81 hydroxylation, 140 modification enzymes, 146 structure, 180, 181 see also proanthocyanidins (PAs) anthocyanidin synthase (ANS), 118–20, 140 recombinant, 120 roles, 190 anthocyanin acylglycosides, 183–6 anthocyanin acyltransferases (AATs), 147–8 aliphatic, 147 aromatic, 147 anthocyanin 3-O-coumaroyltransferases, 148 anthocyanin–flavone C-glycosides, 187–8 structure, 179 anthocyanin–flavone pigments, 187–8 anthocyanin galactosides, 128 anthocyanin glycosides, 181–3 anthocyanin glycosyltransferase (BpUGAT), 128 anthocyanin metabolites, 247 biosynthesis, 248–9 pathways, 248 anthocyanin-related regulatory proteins, 154 anthocyanins, 8, 132, 228–62 absorption, 72, 236–47, 249 analytical methodology, 241–2 mechanisms, 243–5 variability, 239–42 acylation, 145, 147–8 antiatherogenic activity, 233–4 antibacterial activity, 235

Recent Advances in Polyphenol Research, Volume 1. Edited by Fouad Daayf and Vincenzo Lattanzio © 2008 Blackwell Publishing Ltd, ISBN: 978-1-4051-5837-4

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anticancer activity, 234 anti-inflammatory activity, 233 antioxidant activity, 230–1, 232 in vitro, 231 antiviral activity, 235 applications, 181 flower pigments, 140, 144–9, 167, 228 bioactivity, 229–36 biosynthesis, 119, 189–91 regulatory mechanisms, 139, 152–7 blood concentration, 243 chromatograms, 250 color differences, 229 disaccharides in, 182–3 dosage variations, 239 elimination, 242–3, 249 in flavonoid complexes, 187–8 in food matrix, 241 gastric protective activity, 236 in health, 228 identification, 140 localization, in plant cells, 191–2 metabolism, 247–54 animal studies, 251–2 human studies, 247–51 mechanisms, 252–4 routes, 249 methylation, 148 modification enzymes, 146 molecular biology, 189–91 neuroprotective activity, 235 new, 174 obesity prevention, 235–6 occurrence, 169–73, 228 pharmacokinetics, 238, 242–3 prooxidant activity, 232 regulatory genes, 142–3 research publications, 167, 168 roles, 228 sources, 228 structural transformations, 245–7 in vitro, 246–7 in water, 246 structure, 175, 176, 185, 186 chemical, 240–1 classification, 228–9 research developments, 167–201 transport models, 149 urine concentration, 243 and vision improvement, 236 anthocyanoplasts, 150 Anthonomus grandis (boll weevil), 20 anthragallol, 9 anthraquinones, 9 anti-aging agents, antioxidants as, 263 antiatherogenic activity, anthocyanins, 233–4 antibacterial activity, anthocyanins, 235 antibiotic activity, tannins, 15 antibiotics, preformed, 11, 14–15 anticancer activity anthocyanins, 234 cocoa polyphenols, 105–9 epicatechins, 108–9 polyphenols, 359–60, 363–9 proanthocyanidins, 105–9 antifungal activity, 214–15 dihydrochalcones, 15 flavanones, 15

flavones, 15 flavonols, 15 and lipophilicity, 15 phenolic acids, 15 phenols, 15, 214–15 stilbenes, 15 anti-inflammatory activity, anthocyanins, 233 antimicrobial activity in vitro, 211–12 natural, 211 antioxidant activity anthocyanins, 230–1, 232 dietary flavonoids, 68, 70–1 ellagitannins in vitro, 265 issues, 266–74 flavonoids, 263–4 research publications, 264 phenolics, 263–4 and plant defenses, 222 research publications, 264 plant food, 263 polyphenols, in vitro, 359 pomegranate juice, 265 urolithins, 346 antioxidant/electrophile-responsive element (A-EpRE) genes, expression, 82–3 antioxidants and anti-aging agents, 263 marketing claims, 263–4 studies, in vitro vs. in vivo, 263–4 antirrhinum majus (snapdragon), aurones, 132 antiviral activity, anthocyanins, 235 apigenin, 130, 313 apigeninidin, 217 apigenin–xanthine oxidase complex, 75 apoptosis, 107 colon cancer cells, 365, 367– 9 induction, 271–3 apple beverages, oxidation products, liquid chromatography-mass spectrometry analysis, 288–90 apple juice phenolic oxidation products, 278–92 world production, 278 apples phenolics in, 278–81 world production, 278 apple skins, flavonoids, 128 Arabidopsis spp. (cresses), 46–7 defense modeling, 206 genomes, 50 ICS gene, 204 isoflavone synthase, 115 mutants, 205 proanthocyanidins, 121–2 salicylic acid, 203 Arabidopsis thaliana (thale cress) anthocyanidin synthase, 120 caffeoyl 3-O-glucoside, 127 glycosyl hydrolases, 123 phenylpropanoids, 6 salicylic-acid-dependent responses, 205–6 UGT72E1, 130 uridine diphosphate glycosyltransferases, 124, 130 arabinoxylans, acylation, 51 arbutin synthase, 127, 130

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aromatase catalysis, 293, 294 expression, 294 inhibition, 293–304, 312 by natural phytoestrogens, 295–300 phytoestrogens as lead compounds, 301–4 overexpression, 294 aromatase inhibitors, development, 294–5 Arrabidaea chica (puca panga), carajurin, 177 aryl hydrocarbon receptor (AHR), 359, 360–1 ASM (acibenzolar-S-methyl), 220 atherosclerosis and polyphenols, 359, 369–73 and pomegranate juice consumption, 266 ATP (adenosine 5′-triphosphate), 78 aureusidin, 8 aureusidin synthase (AUS), 132, 144 aurones, 8, 132, 140, 143–4 AUS (aureusidin synthase), 132, 144 auxin (indole-3-acetic acid), 10, 125 avenanthramides, 217 AVIs see anthocyanic vacuolar inclusions (AVIs) avocado fruit, preformed antifungals, regulatory mechanisms, 214–15 azoxymethane, 365 Aztecs, cacao use, 88 Babiaba stricta (baboon flower), anthocyanins, 183 BANYULS gene, 120 basal resistance see induced resistance BAs (benzoic acids), 125, 203–4 basic helix-loop-helix (bHLH), 153, 155 basic metabolism, use of term, 1 Bellis perennis (common daisy), anthocyanin glycosyltransferase, 128 benzoic acids (BAs), 125, 203–4 benzoquinones, 9 benzyl-protected building blocks and inter-flavan bond formation, 94–7 synthesis, 92–4 4-(benzylthio)catechin, 96 Berberis buxifolia (calafate), anthocyanins, 181 bHLH (basic helix-loop-helix), 153, 155 bilitranslocase, 244 biochanin A, 115, 129 biphenyls, 50 Blechnum novae-zelandiae (fern) anthocyanin glycosides, 182 anthocyanins, 186 Bombax malabaricum (silk cotton tree), aglycones, 168 Boolean operators, 206 Botrytis spp. (fungi), resistance to, 206 bovine serum albumin (BSA), 345– 6 BpUGAT (anthocyanin glycosyltransferase), 128 breast cancer, 105, 364 prevalence, 293 protective factors, 293 breast cancer cells apoptosis, 107 cell cycle analysis, 106, 107 flow cytometry, 108 necrosis, 107 proliferation, 107 Broussonetia papyrifera (paper mulberry), 300 brown rot, 215 BSA (bovine serum albumin), 345– 6

381

C1 gene, 121 C4H (cinnamate 4-hydroxylase), 140 cacao, early use of, 88 CA (trans-cinnamate), 203–4 Caco-2 cells, 271–4 model, 72 caffeic acid, 127, 215, 216 caffeic acid O-methyltransferase (COMT), deficiency in plants, 41, 44, Plate 2.3 caffeoyl 3-O-glucoside, 127 5-O-caffeoylquinic acid see chlorogenic acid caffeoylquinic acid caffeoylquinic acid o-quinone incubation, 282–4 oxidation products, 282–3 total ion current chromatograms, 282, 283 caffeoylquinic acid o-quinone, 279–80 characterization, 281 incubation, 282–7 with caffeoylquinic acid, 282–4 with (−)-epicatechin, 285–7 preparation, 281 total ion current chromatograms, 285 UV spectra, 282–3, 284 CAGT (coniferyl alcohol glycosyltransferase), 124 Caladium bicolor (caladium), transgenics, 143 Callosobruchus maculatus (cowpea weevil), 22 Campanula medium (Canterbury bells), anthocyanins, 184 CAN (carbonic anhydrase), 206–7, 220 cancer cell proliferation, 105–7 colon, 364, 365 lung, 365 see also anticancer activity; breast cancer; prostate cancer capsaicin, 127–8 carajurin, structure, 177 carajurone, structure, 177 carbonic anhydrase (CAN), 206–7, 220 5-carboxypyranocyanidin, structure, 178 5-carboxypyranopelargonidin, structure, 178 carnations, fusarium wilt, 214 caseins, 71 (−)-catechin, 7 (+)-catechin in apples, 278 as starting material, 92 catechin-4β-catechin, synthesis, 103–4 catechins, 187, 320 in apples, 278 epimerization, 90 oxidation, 281 regiochemistry, 98–9 structure, 90, 187, 279 catechol, 213 catechol-O-methyltransferase (COMT), 69–70 cyanidin methylation, 252 Catharanthus roseus (Madagascar periwinkle) anthocyanin glycosides, 181 CaUGT2, 127–8 ICS gene, 204 CaUGT2, 127–8 CCR (cinnamoyl-CoA reductase), 42–3 celecoxib, 364 cell-based assays, phytoestrogens in, anti-aromatase activity, 298–9 cell cycle analysis, breast cancer cells, 106, 107

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cells phenolic metabolite biosynthesis, 321 see also apoptosis cellular transport, phenolic conjugates, 346–8 Centaurea cyanus (cornflower) flower pigments, 144–5 protocyanins, 189 Centaurea maculosa (spotted knapweed), phytotoxins, 7 chalcone isomerase (CHI), 140 chalcones, 8, 140, 143–4 cis-chalcones, 244–5 trans-chalcones, 244–5 chalcones, anti-aromatase activity, 299–300 chalcone synthase (CHS), 140, 155 Charophyceae (algae), 4–5 CHD see coronary heart disease (CHD) Cheiranthus cheiri (wall flower), anthocyanins, 186 chemoreceptors, insects, 18 CHI (chalcone isomerase), 140 chlorogenic acid, 215, 216 in apples, 278, 279 UV spectra, 285 chocolate benefits, 88, 89 early use of, 88 chorismate, 205 chromones, anti-aromatase activity, 302 chrysin, binding, to fungal 17β-hydroxysteroid dehydrogenase cl, 309 CHS (chalcone synthase), 140, 155 CHS gene, expression, 156 Cicer arietinum (chickpea) isoflavonoids, 20 uridine diphosphate glycosyltransferase, 128 cider, phenolic oxidation products, 278–92 trans-cinnamate (CA), 203–4 cinnamate 4-hydroxylase (C4H), 140 cinnamic acids, structure, 126 cinnamoyl-CoA reductase (CCR), 42–3 Citrus aurantium (bitter orange), tangeretin, 17 c-Jun N-terminal kinases (JNKs) activation, 343 inhibition, 78–9 Cladosporium cucumerinum (fungus), 219, 221–2 resistance to, 218 Clitoria ternatea (butterfly pea), anthocyanins, 184 Cochliobolus carbonum (fungus), 216 Cochliobolus lunatus (fungus), 17β-hydroxysteroid dehydrogenase, 308–10 cocoa, early uses of, 88–9 cocoa-derived oligomeric epicatechins, synthesis, 88–112 cocoa polyphenols anticancer activity, 105–9 research, 88–9 cocoa procyanidins, chemical properties, 89–91 Colletotrichum circinans (fungus), and onion smudge disease, 15, 213 Colletotrichum gloeosporioides (fungus), resistance, 214–15 Colletotrichum lindemuthianum (fungus), and induced resistance, 220 Colletotrichum orbiculare (fungus), resistance to, 221 Colletotrichum sublineolum (fungus), 168 colon, phenolic metabolite biosynthesis, 320 colon cancer, 364, 365 colon cancer cells, apoptosis, 365, 367–9 combinatorial, use of term, 37

CoMFA (comparative molecular field analysis), 302 Commelina communis (Asiatic dayflower), commelinins, 188–9 commelinins, 188–9 comparative molecular field analysis (CoMFA), 302 Compositae (daisy family), 9 COMT see caffeic acid O-methyltransferase (COMT); catechol-O-methyltransferase (COMT) condensed tannins see proanthocyanidins (PAs) coniferaldehyde, 44–5 coniferyl alcohol glycosyltransferase (CAGT), 124 coniferyl alcohols biosynthesis, 36 diffusion, 39 coniferyl aldehydes, 124 glycosylation, 125 7-O-conjugates, antioxidant properties, 341–2 conjugation, phenolic metabolites, 317, 318 Convulvulaceae (bindweed family), anthocyanins, 185–6 Cornus alba (Siberian dogwood), anthocyanins, 182 Cornus suecica (Eurasia dwarf cornel), anthocyanins, 183 coronary heart disease (CHD) reduction, and red wine, 233–4 risk factors, 234 p-coumaric acid, 9, 126 p-coumaric acid glucuronides, synthesis, 331 coumarins, 126 roles, 10–11 structure, 125 p-coumaroylquinic acid, 278 p-coumaryl alcohols, biosynthesis, 36 coumestrol, 17β-hydroxysteroid dehydrogenase type 1 inhibition, 305–7 COX-2 see cyclooxygenase-2 (COX-2) cross-coupling, end-wise, 37, 39 Cruciferae (cabbage family), anthocyanins, 186 CSLF genes, 52 cucumbers, salicylic acid, 203–4 cucumerin A, 217 curcumin anticancer activity, 359–60 structure, 127 curcumin metabolites, 344 biosynthesis, 320 characterization, 327–8 structure, 327 curing, plants, 17 cyanidin 3-glycoside antioxidant activity, 230 antiviral activity, 235 metabolism, 248, 252–3 and obesity prevention, 235–6 structure, 179 cyanidins, 128, 140, 168 anticancer activity, 234 antioxidant activity, 231 methylation, 253 new, 182 cyanidin 3-sambubioside, 240 cyanidin sulfate, 253 cyanodelphin, 182 cyclin A gene, 369 cyclooxygenase-2 (COX-2) inhibition, 344, 345 roles, 343–4 cyclooxygenase isozymes, COX-2, 233

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CYP1A1, 360 inhibition, 79–80 CYP3A4, 363 CYP19A1 see aromatase CYPs see cytochromes P450 (CYPs) cytochromes P450 (CYPs) catalysis, 79 inhibition, 79–80 cytosolic multi-enzyme complexes, 149 daidzein, 113, 114 incubation, 325 sources, 115 in soy, 369, 372 daphnetin, 126 defense compounds induced, 11, 12 synthesis, 11–12 defense mechanisms, genetic control, 14 DEFICIENS protein, 156 dehydrodicatechin A, 280–1, 287 dehydrodicatechin B, 286 dehydrodicatechins, 286–7 DELILA, 153 delphinidin 3-rutinoside, 252 delphinidins, 128, 140, 168 anticancer activity, 234 biosynthesis, 143, 144, 145 occurrence, 182, 188–9 delphinidin 3-sambubioside, 240 Dendrobium × superbiens (orchid), anthocyanins, 184 3-deoxyanthocyanidin phytoalexins, synthesis, 12 3-deoxyanthocyanins, 190 Desmodium uncinatum (silverleaf ), leucoanthocyanidin reductase, 118 desoxyanthocyanidins, 168 3-desoxyanthocyanidins, 168–77 detoxification mechanisms, 359–63 DFR (dihydroflavonol 4-reductase), 140, 155 DFR gene, expression, 121–2, 132 DHCA (dihydroconiferyl alcohol), incorporation, into lignins, 42 DHFs (dihydroflavonols), 140 Dianthus caryophyllus (carnation), anthocyanic vacuolar inclusions, 191 dibenzodioxocins, 50 dietary flavonoids absorption, 72–3 antioxidant activity, 68 sites, 70–1 bioavailability, 67–8 binding processes in, 72–4 catabolism, 70 cellular metabolism, 73–4 conjugates, 69–70 conjugation, 72–3 gene expression, regulatory mechanisms, 80–3 interactions with digestive enzymes, 72 with food proteins, 71 with salivary proteins, 71–2 research issues, 67–8 tissue distribution, 73–4 transport, in plasma, 73 dietary phytoestrogens, 17β-hydroxysteroid dehydrogenase type 5 inhibition, 310–11 diferulate, 9

383

digestive enzymes, interactions, with dietary flavonoids, 72 dihydrochalcones antifungal activity, 15 in apples, 278 structure, 279 dihydroconiferyl alcohol (DHCA), incorporation, into lignins, 42 dihydroflavonol 4-reductase (DFR), 140, 155 dihydroflavonols (DHFs), 140 dihydroquercetin, 91 dirigent/replication hypothesis, 36, Plate 2.2 criticisms, 40–50, 53–4 issues, 37–8, 50, 52–3 and monomer substitution, 40–5 disaccharides, in anthocyanins, 182–3 disease resistance and phenolics, 15 see also induced resistance dormancy, 10–11 Drosophila melanogaster (fruit fly), genome, 1–2 drug discovery, phytoestrogens, for steroid biosynthesis control, 293–316flav EGCG see epigallocatechin gallate (EGCG) Eichhornia crassipes (water hyacinth), anthocyanin–flavone pigments, 188 electrophilic response element/antioxidant response element (EpRE/ARE), 359, 360–2, 370 activation, 371–2 ellagic acid apoptosis induction, 271–3 bioactivity, 264–5 bioavailability, in vivo, 267– 9 disposition, 270 food, 263–77 future research, 275 metabolism, 270, 271–3 in vivo, 267– 9 ellagic acid metabolites, tissue distribution, 267–9 ellagic tannin metabolites, characterization, 324 ellagitannin metabolites bioactivity, in vivo, 269–74 tissue distribution, 267–9 ellagitannins antioxidant activity in vitro, 265 issues, 266–74 bioactivity, 264–5 clinical studies, 265–6 in vivo, 267 issues, 266–74 bioavailability, in vivo, 267–9 biosynthesis, 264 future research, 275 metabolism, in vivo, 267–9 occurrence, 265 pig studies, 267–9 UV spectra, 264 enterodiol, 325 enterolactone, 325 anti-aromatase activity, 299 enzymatic browning, use of term, 279 epiafzelechins, 187 (−)-epicatechin in apples, 278 caffeoylquinic acid o-quinone incubation, 285–7 oxidation products, 285–7

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total ion current chromatograms, 285 UV spectra, 285 epicatechin-4α,8-epicatechin, synthesis, 102–3 epicatechin oligomers chromatograms, 95 structure, 90 synthesis, 104 epicatechins, 187, 215 anticancer activity, 108–9 epimerization, 90 methylation, 320 regiochemistry, 98–9 structure, 90, 324 epigallocatechin gallate (EGCG), 107 anticancer activity, 359–60, 363, 365 EpRE/ARE see electrophilic response element/antioxidant response element (EpRE/ARE) Erb2 gene, inhibition, 89 ERBB2-positive tumors, 364 eriodictyol, 191 sulfation, 340 ERK (extracellular signal-related kinase), 78 Erwinia amylovora (bacterium), 221 erythrose-5-phosphate, as intermediate, 13 Escherichia coli (bacterium), 191 esculetin, 126 estradiol, blocking, 293 estrogen synthetase see aromatase estrone, biosynthesis, 293 ET (ethylene), 205–6 β-ether frequency anomaly, 39–40 ethylene (ET), 205–6 Eustoma spp. (lisianthus), anthocyanic vacuolar inclusions, 150–2 Eustoma grandiflorum (lisianthus), anthocyanic vacuolar inclusions, 191 Evolvulus pilosus (blue daze), anthocyanins, 185–6 extracellular signal-related kinase (ERK), 78 F3H (flavone 3-hydroxylase), 140 F3H gene, 156 expression, 132 S-fadrozole, anti-aromatase activity, 302 ferrylmyoglobin, 71 ferulate dehydrodimerization, 51 mechanisms, 52 ferulic acid, 9 synthesis, 125 feruloyl pectin, roles, 9 flavan-3-ol, 24 flavan-3-ol conjugates, antioxidant properties, 339 flavan-3-ol metabolites, characterization, 324 flavanone conjugates, antioxidant properties, 339 flavanones, 73 antifungal activity, 15 IC50 values, 295–8 flavanonol metabolites, characterization, 322–4 flavanonone metabolites, characterization, 322–4 flavanyl acceptors, 91, 96–7 flavanyl donors, 91, 96–7 synthesis, 92, 104 flavone conjugates antioxidant properties, 339 structure, 321–2, 323 flavone 3-hydroxylase (F3H), 140 flavone metabolites, characterization, 321–2 flavones anti-aromatase activity, 302–3

antifungal activity, 15 binding, 73, 74 effects on colon cancer cells, 365, 367–9 in flower pigments, 140 hydroxylation, 8–9 IC50 values, 295–8 localization, 15 flavonoid biosynthesis angiosperms, metabolic engineering, 142–9 inhibition, 142 ornamental plants metabolic engineering, 139–66 molecular biology, 139–66 pathways, 113–14, 139–40, 141 regulatory mechanisms, 139 flavonoid complexes, with at least one anthocyanin subunit, 187–8 flavonoid glucosides, absorption, 319–20 flavonoid glycosides hydrolysis, 318–19 in saliva, 318 flavonoid–protein binding effects on human health, 74–83 processes, 67–87 prior to absorption, 71–2 flavonoid–protein covalent coupling, 69, 71 flavonoids, 2, 19 accumulation, 18–19 anti-aromatase activity, 300–1 antioxidant activity, 263–4 research publications, 264 antioxidant properties, 68 in apples, 278 applications, 5 flower pigments, 139, 140 ultraviolet protection, 6 binding, 67–87 chemical properties, biologically relevant, 68–71 gene expression redox regulation, 82 regulatory mechanisms, 80–3 glycosylation, 128–30 in host plant selection, 19–20 17β-hydroxysteroid dehydrogenase type 1 inhibition, 307 17β-hydroxysteroid dehydrogenase type 3 inhibition, 310 IC50 values, 295–8 pro-oxidant properties, 342–3 redox, 67 modulation, 76–7 roles, in health, 23, 74–83 structure, 129, 140, 141 and ultraviolet radiation, 6 see also dietary flavonoids; isoflavonoids flavonoid O-sulfates, chemical synthesis, 332–3 flavonol conjugates antioxidant properties, 334–9 structure, 321–2, 323 flavonol galactosides, 128 flavonol metabolites, characterization, 321–2 flavonol methyl ethers, localization, 15 flavonols antifungal activity, 15 binding, 73, 74 in flower pigments, 140 hydroxylation, 8–9 flavonol synthase (FLS), 118–20

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flavonones, binding, 74 flavopiridol, 105–6 flavylium cations, 179 flavylium compounds, structure, 179–81 floral binding protein2 gene, 156 flow cytometry, breast cancer cells, 108 flowering plants see angiosperms flower pigments anthocyanins, 140, 144–9, 167, 228 blue, engineering, 144–9 flavonoids, 139 future research, 157–8 genetic modification, 139 patterning, 153–5 stability, 140 yellow, engineering, 143–4 FLS (flavonol synthase), 118–20 food ellagic acid, 263–77 polyphenols, 263–77 see also plant food food matrix, anthocyanins in, 241 food proteins, interactions, with dietary flavonoids, 71 Fragaria × ananassa (garden strawberry), 5-carboxypyranopelargonidin, 178 French Paradox, 369 Freudenberg concept, 36, 50, 53 limitations, 53–4 Friedel–Crafts acylation, 91 fruit post-harvest resistance, 16–17 post-harvest treatment, 17 fungal hyphae, 218 fungal pathogens, 13–17 furofurans, 22 Fusarium oxysporum f. sp. dianthi (fungus), 214, 220–1 fusarium wilt, carnations, 214 fusion protein approach, 117 GA (gibberelic acid), 125, 156 galangin, 17β-hydroxysteroid dehydrogenase type 2 inhibition, 308 gastric protective activity, anthocyanins, 236 gastrointestinal tract (GIT), 237, 244 anthocyanin transformations, 245 gene discovery, in phenylpropanoid pathway, 113–38 gene expression flavonoids, regulatory mechanisms, 80–3 and polyphenols, 359–77 antiatherosclerosis mechanisms, 359, 369–73 anticancer mechanisms, 363–9 detoxification mechanisms, 359–63 urolithins, studies, 273–4 genistein, 113, 114, 129, 130 antioxidant activity, 340–1 atherosclerosis studies, 371–2 hydrolysis, 319 17β-hydroxysteroid dehydrogenase type 1 inhibition, 305–7 incubation, 325– 6 production, 117 sources, 115 in soy, 369, 372 genomes, plants, 1–2 Gerbera spp. (gerberas), pigmentation regulation, 156 Gerbera hybrida (gerbera), anthocyanidin synthase, 120, 190

385

gibberelic acid (GA), 125, 156 gingerol, 127–8 GIT see gastrointestinal tract (GIT) (1>3),(1>4)-β-glucans, biosynthesis, 51–2 β-glucuronidase, inhibition, 344 glucuronides, 268–9 O-glucuronides, 349 chemical synthesis, 328–32 glutathione (GSH), 73–4 glutathione S-transferases (GSTs), 149, 361–2 cancer risk studies, 360 glycerol 3-phosphate dehydrogenase (GPDH), inhibition, 345 glycosidases, 14 glycosylation, flavonoids, 128–30 glycosyl hydrolases, 123 glycosyltransferases (GTs), 140 in phenylpropanoid modification, 123–31 Glycyrrhiza echinata (Russian liquorice) UGT73F1, 128 GPDH (glycerol 3-phosphate dehydrogenase), inhibition, 345 green tea, polyphenols, 363, 369 –70 GSH (glutathione), 73–4 GSTs see glutathione S-transferases (GSTs) GTs see glycosyltransferases (GTs) hardwoods, lignin polymer models, Plate 2.4 HDL (high-density lipoprotein cholesterol), synthesis, 233–4 health anthocyanins in, 228 flavonoid–protein binding effects, 74–83 and plant food, 263 and plant phenolics, 22–4 Heliocoverpa armigera (cotton bollworm), 20 5-12-cis-heptadecenylresorcinol, 214 hesperetin, 73, 322 hesperidin, 322 anti-aromatase activity, 300 hexahydroxydiphenic acid, 264 Hibiscus sabdariffa (roselle), anthocyanins, 240 high-density lipoprotein cholesterol (HDL), synthesis, 233–4 hirsutidins, 181 hormone-dependent diseases, treatment, 293 horseradish peroxidase, 73 host plants recognition, 19 selection, 19–20 HPM (human placental microsome-based) assays, phytoestrogens in, anti-aromatase activity, 295–8 HRs (hypersensitive responses), 15–16 HSA (human serum albumin), 73, 345– 6 17β-HSDs see 17β-hydroxysteroid dehydrogenases (17β-HSDs) HT-29 cells, 367–9 human placental microsome-based (HPM) assays, phytoestrogens in, anti-aromatase activity, 295– 8 human serum albumin (HSA), 73, 345– 6 Hydrangea macrophylla (bigleaf hydrangea), metallocyanins, 189 3-hydroxyacetophenone, 214 3-hydroxyanthocyanins, 140 hydroxycinnamaldehydes, 44–5 incorporation, into lignins, 41–2

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hydroxycinnamic acid metabolites characterization, 326–7 structure, 326 hydroxycinnamic acids in apples, 278 isomers, 9–10 oxidation, 281 roles, 9–11 structure, 280 5-hydroxyconiferyl alcohol coupling, 44 substitution, Plate 2.3 4′-hydroxyflavonoids, 73–4 2-hydroxyisoflavone synthase, 114 17β-hydroxysteroid dehydrogenases (17β-HSDs), 293 catalysis, 294 fungal cl, 305, 308–10 phytoestrogen binding, 309–10 inhibition, by phytoestrogens, 304–13 type 1, 305–7 phytoestrogen binding, 307 type 2, 305, 307–8 type 3, 305, 310 type 4, 305, 307–8 type 5, 305, 310–11 Hypericum calycinum (great St John’s wort), 19 hypersensitive responses (HRs), 15–16 IAA (indole-3-acetic acid), 10, 125 ICS gene, 204 ICS (isochorismate synthase), 204–5 IFS see isoflavone synthase (IFS) IFS/chalcone isomerase, 116–17 IFS/CHI fusion gene, 117 IFS gene, 115–16 indole-3-acetic acid (IAA), 10, 125 induced plant defenses responses, 220–1 and salicylic acid, 202–10 types of, 211 induced resistance acibenzolar-S-methyl-mediated, 220 and phenolics, 202–3 and plant growth-promoting rhizobacteria, 220–1 and plant phenols, 219–20 responses, 220–1 and structural phenolics, 221–2 studies, transgenic plants, 205 use of term, 202, 216 induced systemic resistance (ISR) mechanisms, 202–3 use of term, 203 see also systemic acquired resistance (SAR) innate immune response, animals, 203 insecticidal activity, lignans, 22 insecticides, 22 insects chemoreceptors, 18 feeding deterrents, 20–1 host plant recognition, 19 host plant selection, 19–20 plant toxin sequestration, 18 see also phytophagous insects INTENSIFIER1 gene, 122 4,6-inter-flavan bonds, formation, 104–5 inter-flavan bonds formation, and benzyl-protected building blocks, 94–7

future research, 104–5 stereochemistry, 89, 97–102 IPL (isochorismate pyruvate-lyase), 205 Ipomoea batatas (sweet potato), anthocyanic vacuolar inclusions, 150 Ipomoea batatas cv Ayamurasaki (purple sweet potato), anthocyanins, 240 Iris hollandica (Dutch iris), anthocyanin 3-Ocoumaroyltransferases, 148 isochorismate pyruvate-lyase (IPL), 205 isochorismate synthase (ICS), 204–5 isoetin, 8–9 isoflavone conjugates, antioxidant properties, 339–41 isoflavone glucosides, 72 isoflavone metabolites, characterization, 325–6 isoflavones, 23, 73 anticancer activity, 359–60 antioxidant activity, 341 binding, 74 biosynthesis, 113–15 dietary sources, 115 functions, 113–15 metabolic engineering, 115–17 with artificial isoflavone synthase, 116–17 via ectopic expression of isoflavone synthase, 115–16 metabolism, 321 as phytoalexins, 115 phytoestrogenic activity, 114 in soy, 369 isoflavone synthase (IFS), 117 in isoflavone metabolic engineering, 115–17 isoflavonoids, 20 anti-aromatase activity, 303–4 biosynthetic pathways, 113–14 isolicoflavonol, 312 isorhamnetin glucuronides, synthesis, 329 isorhamnetins, 21 structure, 321–2 isosalipurposide, 8 ISR see induced systemic resistance (ISR) JA (jasmonic acid), 205–6 jasmonic acid (JA), 205–6 JNKs see c-Jun N-terminal kinases (JNKs) judaicin, 20 judaicin-7-O-glucoside, 20 kaempferide, 17β-hydroxysteroid dehydrogenase type 2 inhibition, 308 kaempferide triglycosides, 214 kaempferol, 20–1, 128, 130 binding, to fungal 17β-hydroxysteroid dehydrogenase cl, 309 17β-hydroxysteroid dehydrogenase type 2 inhibition, 308 Keap 1 protein, 83 4-ketones, synthesis, 102 lacinilene c, 217 lactase phlorizin hydrolase, 72 LAR gene, 118 LAR (leucoanthocyanidin reductase), 118 Lc gene, 122 LC-MS see liquid chromatography-mass spectrometry (LC-MS) LC-MS/MS see liquid chromatography-tandem mass spectrometry (LC-MS/MS)

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LDL receptor, 369–70 lectins, 71 Leguminosae (pea family), isoflavones, 115 Lens culinaris (lentil), anthocyanins, 183 leptostachyol acetate, 22 leucoanthocyanidin reductase (LAR), 118 leucoanthocyanidins, 118 leukotrienes, levels, 89 lignan metabolites, characterization, 325 lignans, 23 anti-aromatase activity, 299 insecticidal activity, 22 lignification, 36–66 as biochemical anomaly, 38–9, 51–2 β-ether frequency anomaly, 39–40 malleability, 43–5 mechanisms, 36 obligatory linkages, 40 roles, in defense, 218–19 lignification theories challenge hypothesis, 38 current, 37, Plate 2.1 criticisms, 38–40, 53–4 dirigent/replication hypothesis, 36 criticisms, 40–50, 53–4 issues, 37–8, 50, 52–3 issues, 36 lignin chains branching, 50 resinol units in, 49 lignin oligomers, sequencing, 45–6 lignin polymerization, theories, 36–54 lignin polymers, 36, 43, 53 branching, 50 models hardwoods, Plate 2.4 softwoods, Plate 2.4 repeating structures, 45 lignins biosynthesis, 36 branching, 49–50 dihydroconiferyl alcohol incorporation, 42 hydroxycinnamaldehyde incorporation, 41–2 monomer-independent sequences, 46–7 number of isomers problem, 48 polymer branching, 49–50 precursors, 127 primary structure, 36 polysaccharide, 51–2 sequencing, 47 well-defined, 45–7 racemicity, 47–8 repeating structures, 45 template replication, 38, 48–9 tyramine ferulate incorporation, 42–3 linoleic acid, 73 lipophilicity, and antifungal activity, 15 lipoxygenases, 71 Lippman, Marc, 88 liquid chromatography-mass spectrometry (LC-MS) advantages, 290 apple beverage oxidation products analysis, 288–90 liquid chromatography-tandem mass spectrometry (LC-MS/MS) advantages, 290 apple beverage oxidation products analysis, 288–90 caffeoylquinic acid studies, 282–4 liver, phenolic metabolite biosynthesis, 320

387

Lobelia erinus (edging lobelia), anthocyanins, 183 Lobelinin B, 183 Lobularia maritima (sweet alyssum), anthocyanins, 186 Lunaria annua (annual honesty), anthocyanins, 186 lung cancer, 365 luteolin, 8–9, 130 glucuronidation, 320 luteolin glucuronides antioxidant activity, 340 enzyme inhibition, 344 luteolinidin, 217 LXR-α, expression, 370 maackiain, 20 MADS protein, 156 malonic acid, 183 malonylawobanin, 189 malvidins, 128, 132, 140, 168 occurrence, 181, 183–4 mango fruit, Alternaria infection, 214 MAPKs (mitogen-activated protein kinases), inhibition, 78 MATE (multidrug and toxic compound extrusion) proteins, 149 Matthiola incana (hoary stock), anthocyanins, 121 MCT (monocarboxylic acid transporterT), 346–7 Meconopsis grandis (Himalayan blue poppy), metallocyanins, 189 Medicago sativa (alfalfa), IFS gene, 115–16 Medicago truncatula (barrel clover) ANR gene, 123 IFS gene, 115–16 UGT71G1, 129, 130–1 MeJA (methyl jasmonate), 205–6 metabolic engineering flavonoid biosynthesis angiosperms, 142–9 ornamental plants, 139–66 isoflavones, 115–17 in phenylpropanoid pathway, 113–38 strategies, 131–2 proanthocyanidins, in plants, 122–3 metabolites ellagic acid tissue distribution, 267–9 see also anthocyanin metabolites; curcumin metabolites; ellagitannin metabolites; phenolic metabolites; resveratrol metabolites; secondary metabolites metalloanthocyanins, 188–9 metallothioneins, 273–4 2-methoxyjudaicin, 20 7-O-methylapigenidin, 168 O-methylcyanidin, 168 7-O-methylcyanidin 3-galactopyranoside, 168, 181 methyl jasmonate (MeJA), 205–6 5-O-methylluteolinidin, structure, 177 metmyoglobin, 71 microarray-based mRNA analysis, 364 mitogen-activated protein kinases (MAPKs), inhibition, 78 Mitsunobu reactions, 92 molecular biology anthocyanins, 189–91 flavonoid biosynthesis, ornamental plants, 139–66 Monilinia fructicola (fungus), infection, 215 monocarboxylic acid transporter (MCT), 346–7 monolignols applications, 125

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biosynthesis, 36 dimerization, 37 substitution, 43–5 monomers, non-traditional, 44 monomer substitution and dirigent/replication hypothesis, 40–5 issues, 41–3 MtANR gene, 123 multidrug and toxic compound extrusion (MATE) proteins, 149 Muscari armeniacum (grape hyacinth), anthocyanins, 182 MUTABILIS, 153 MYB factors, 153, 156–7 Myb gene, 155, 156, 190–1 MYC, 153 myricetin, 128 Myrtus cauliflora ( jaboticaba), anthocyanins, 181 NADP+, reduction, 13 NADPH oxidation, 13 synthesis, 12–13 NahG (naphthalene hydroxylase), 205 naphthalene hydroxylase (NahG), 205 α-naphthoflavones, 312 anti-aromatase activity, 301 naphthoquinones, 9 naphthylphthalamic acid (NPA), 10 naringenin, 73, 191 naringin, 17 anti-aromatase activity, 300 NAT1 (N-acetyltransferase 1), polymorphisms, 360 NAT2 (N-acetyltransferase 2), polymorphisms, 360 NCOL-1 colon cells, 369 necrosis, 107 see also apoptosis neoplasias, 89 neuroprotective activity, anthocyanins, 234 N gene, 219 Nicotiana benthamiana, salicylic acid, 205 nitrobenzene, oxidation, 46–7 NMR (nuclear magnetic resonance), image interpretation, 43 NodD protein, 8 Nod factors, 8 nod genes, 115 expression, 8 non-host resistance, 14 NPA (naphthylphthalamic acid), 10 NPR1 protein, 220 Nrf2 (nuclear factor erythroid-2-related factor), 361–2, 370, 372 NtGT1s, 129–30 NtGT3, 129–30 nuclear factor eythroid-2-related factor (Nrf2), 361–2, 370, 372 nuclear magnetic resonance (NMR), image interpretation, 43 nyctinasty, 10 Nymphaea caerulea (African water lily), delphinidin, 182 OAT (organic ion transporter), 347 OATP-B transporter, 348 obesity prevention, anthocyanins, 235–6 olomoucine, 105–6 onion bulbs, resistance, 213

onion smudge disease, 15, 213 OPDA (12-oxophytodienoic acid), 205–6 Operophtera brumata (winter moth), feeding deterrents, 19 ORAC (oxygen radical absorbance capacity), 230 organic ion transporter (OAT), 348 Origanum vulgare (oregano) phenolics, 12–13 rosmarinic acid, 12–13 ornamental plants flavonoid biosynthesis metabolic engineering, 139–66 molecular biology, 139–66 Orychophragmus violaceus (hanadaikon), anthocyanins, 186 Oxalis triangularis (love plant) anthocyanin–flavone C-glycosides, 187–8 anthocyanins, 183–4 oxidative pentose phosphate pathway, 12–13 oxidative stress, neutralization, 264 ox-LDL, 372 12-oxophytodienoic acid (OPDA), 205–6 oxygen radical absorbance capacity (ORAC), 230 p53, phosphorylation, 108 PAHs (polycyclic aromatic hydrocarbons), 360–1 PAL (phenylalanine–ammonia lyase), 203–4 PAMP (pathogen-associated molecular patterns), 15 PAP1 gene, 123 Papilio xuthus (swallowtail butterfly), 20 PAs see proanthocyanidins (PAs) pathogen-associated molecular patterns (PAMP), 15 pathogenesis, mechanisms, 211 pathogen-related (PR) proteins, 202 pelargonidins, 128, 140, 168, 184, 187 absorption, 251–2 biosynthesis, 143 Penicillium digitatum (fungus), 17 5-pentadecylresorcinol, 214 peonidin, 140, 168 Perilla frutescens (green shiso), anthocyanidin synthase, 118 Peronospora parasitica (downy mildew), resistance to, 206 peroxidase, induction, 16 peroxisome proliferator-activated receptor-α (PPAR-α), activation, 370 peroxisome proliferator-activated receptor-γ (PPAR-γ), 81 expression, 369–70 petals, anthocyanic vacuolar inclusions, 150–2 petunidins, 140, 168, 181 PGPR (plant growth-promoting rhizobacteria), and induced resistance, 220–1 Phacelia campanularia (California bluebell), anthocyanins, 185–6 Phaseolus vulgaris (common bean), 22 induced resistance, 220 phenolic acids antifungal activity, 15 roles, 10–11 phenolic conjugates, 317, 318 antioxidant properties, 334–42 biological properties, 334–46 cellular efflux, 346–7 cellular transport, 346–8 cellular uptake, 347–8 chemical synthesis, 328–33

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effects on angiogenesis, 345 on vascular function, 345 enzyme inhibition, 344–5 future research, 349 interactions, with signaling cascades, 343–4 noncovalent binding, to proteins, 345–6 pro-oxidant properties, 342–3 phenolic glycosides, solubility, 2 phenolic hydroxyls, methylation, 91 phenolic metabolites analytical issues, 317–18 bioactivity, developments, 317–58 biological properties, 318 biosynthesis, sites, 318–21 characterization, 317, 321–8 chemical synthesis, developments, 317–58 detection, 317 future research, 348– 9 microbial, activity, 346 tissular, activity, 346 phenolic oxidation products in apple juice, 278–92 in cider, 278–92 phenolic phytoalexins, in plant defenses, 216–18 phenolics, 12–13 absorption, 3 antioxidant activity, 263– 4 and plant defenses, 222 research publications, 264 in apples, 278–81 biosynthesis, 3, 13, 16 chemical induction, and plant resistance, 221–2 classification, 2 defense-associated, biosynthetic origins, 212 and disease resistance, 15 dissolution, 3 and induced resistance, 202–3 as insect feeding barriers, 19 metabolism, 317 occurrence, 3 oxidation, 278–81 and plant–insect interactions, 17–22 as preformed defenses, 212–15 roles in fruit post-harvest resistance, 16–17 in health, 23–4 structural, and induced resistance, 221–2 structure, 213, 217 see also plant phenolics phenolic structural defenses, 218–19 phenols antifungal activity, 15, 214–15 bioactivity, 335–8 definition, 2 and plant defenses, roles, 222–3 and plant disease resistance, 211–27 radical coupling, 36, 37 phenylalanine, 205 phenylalanine–ammonia lyase (PAL), 203–4 phenylpropanoid alcohols, structure, 124 phenylpropanoid aldehydes, structure, 124 phenylpropanoid pathways angiosperms, 141 gene discovery, 113–38 metabolic engineering, 113–38 strategies, 131–2 phenylpropanoids

389

modification, via glycosyltransferases, 123–31 mutants, 6 phloretin xyloglucoside, in apples, 278 phloridzin in apples, 278 oxidation products, 280–1 phloroglucinols, 19 Phryma leptostachya (American lopseed), insecticidal activity, 22 phytoalexins, 14, 17, 202, 217–18 biosynthesis, 16, 217 characterization, 216 isoflavones as, 115 structure, 177 phytoanticipins, 14 use of term, 202 phytoestrogenic activity, isoflavones, 114 phytoestrogens, 271 anti-aromatase activity, 299–300 in cell-based assays, 298–9 in human placental microsome-based assays, 295–8 in recombinant-enzyme assays, 299 binding to fungal 17β-hydroxysteroid dehydrogenase cl, 309–10 to 17β-hydroxysteroid dehydrogenase type 1, 307–8 17β-hydroxysteroid dehydrogenase inhibition, 304–12 as lead compounds for aromatase inhibition, 301–4 for 17β-hydroxysteroid dehydrogenase inhibition, 311–12 natural, as aromatase inhibitors, 295–300 for steroid biosynthesis control, 293–316 structure, 295 structure–activity relationships, 300–1 phytophagous insects, 18 effects of tannins on, 22 salivary secretions, 21 Phytophthora infestans (fungus), 216 Phytophthora porri (fungus), resistance to, 206 phytotoxins, 7 Picea abies (Norway spruce), coniferyl alcohol glycosyltransferase, 124 Pieris napi oleracea (mustard white), 20 pigmentation patterning, 153–5 pigments plant phenolics, 8–9 see also flower pigments pig studies anthocyanin metabolism, 252 ellagitannins, 267–69 pinocembrin, 212 pinoresinol, coupling, 40 Pinus strobus (eastern white pine) aldehydes, 124 coniferyl alcohol glycosyltransferase, 124 pisatin, 216 PKC (protein kinase C), inhibition, 78 PKC-β (protein kinase C-β), 365–7, 368 plant cells, anthocyanins in, 191–2 plant defenses active, 215–16 digital approach, 206 early studies, 202 induced resistance, and phenolics, 202–3

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localized, 216–20 and phenolics, antioxidant activity, 222 and phenols, roles, 222–3 and plant phenolics, 3, 11–22 see also induced plant defenses plant disease resistance, and phenols, 211–27 plant food antioxidant activity, 263 and health, 263 plant growth, and plant phenolics, 9–11 plant growth-promoting rhizobacteria (PGPR), and induced resistance, 220–1 plant–insect interactions, and phenolics, 17–22 plant pathogens diversity, 211 types of, 211 plant phenolics applications, 3, 5–24 pigments, 8–9 signal compounds, 6–8 ultraviolet sunscreens, 6 classification, 3 functions, 3–24 and health, 22–4 and plant defenses, 3, 11–22 and plant growth, 9–11 secondary metabolites with diverse functions, 1–35 solubility, 2–3 structure, 3, 4 as waste products, 5 plant phenols, and induced disease resistance, 219–20 plant polyphenols chemical scaffolds, 113 definition, 2 physiological properties, 359 roles, 113 plant resistance and chemical induction of phenolics, 221–2 mechanisms, 11, 202 plants anthocyanin structures, research developments, 167–201 caffeic acid O-methyltransferase deficiency, 41, 44, Plate 2.3 curing, 17 genomes, 1–2 proanthocyanidins, metabolic engineering, 122–3 secondary metabolism, 1–5 ultraviolet exposure, 6 see also angiosperms; host plants; ornamental plants; tobacco plants plant secondary product glycosyltransferase (PSPG), 147 plant toxins, sequestration, 18 plumbagin, 9 Plumbago capensis (Cape plumbago), plumbagin, 9 Podosphaera xanthii (fungus), 217–18 PODs (polyphenol peroxidases), 21 polycyclic aromatic hydrocarbons (PAHs), 360–1 polyketide acetate/malonate pathway, 2 polymer branching, lignins, 49–50 polymerization, in lignification, 36 Polyommatus icarus (common blue), 18 polyphenol oxidases (PPOs), 21 polyphenol peroxidases (PODs), 21 polyphenol–protein complexes, origins, 7 polyphenols

anticancer activity, 359–60 antioxidant activity, in vitro, 359 definition, 2 enzymatic oxidation, 279–80 food, 263–77 and gene expression, 359–77 antiatherosclerosis mechanisms, 359, 369–73 anticancer mechanisms, 363–9 detoxification mechanisms, 359–63 protective effects, 359 use of term, 2 see also cocoa polyphenols; plant polyphenols polysaccharides, acylation, 51 pomegranate juice antioxidant activity, 265, 324 bioavailability, studies, 269 health effects, 265–6 metabolism, studies, 269 Populus deltoides (eastern cottonwood), pinocembrin, 212 post-harvest treatment fruit, 17 vegetables, 17 potatoes defenses, 216 salicylic acid, 203–4 PPAR-α (peroxisome proliferator-activated receptor-α), activation, 370 PPAR-γ (peroxisome proliferator-activated receptor-γ), 81 expression, 369–70 PPOs (polyphenol oxidases), 21 PR-10 protein, 12 pratensein, 115 preformed antifungals, regulatory mechanisms, 214–15 preformed defenses, phenolics as, 212–15 pregnane × receptor (PXR), 359 prevacuolar compartments (PVCs), 192 primary metabolism, use of term, 1 primin, 9 Primula obconica (German primrose), primin, 9 proanthocyanidin biosynthetic pathway gene discovery, 117–22 regulatory genes, 121–2 structural genes, 118–21 proanthocyanidins (PAs) anticancer activity, 105–9 biosynthesis, 119 metabolic engineering, in plants, 122–3 occurrence, 117–18 toxicity, 15 procyanidin B1, stereochemistry, 97–8, 99–100 procyanidin B2 O-benzylation, 100 O-methylation, 100 stereochemistry, 99–100 structure, 97–8, 99 procyanidins, 71–2 activity, 89 in apples, 278 cocoa, 89–91 cytotoxicity, 105, 106 structure, 279 synthesis, 89–105 benzyl-protected building blocks, 92–4 developments, 102–5 early studies, 91–2 prohexadione, 221

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proline, biosynthesis, 12–13 prooxidant activity, anthocyanins, 231 prostacyclin, levels, 89 prostaglandin F synthase–NADPH–rutin complex, 68, 70 prostate cancer prevalence, 293 protective factors, 293 protein kinase C (PKC), inhibition, 78 protein kinase C-β (PKC-β), 365–7, 368 protein kinases, inhibition, 78–9 proteins, noncovalent binding, to phenolic conjugates, 345–6 proteome analysis, 364, 365–9 protocatechuic acid, 213 protodelphin, 189 PR (pathogen-related) proteins, 202 Prunus mume (Japanese apricot), anthocyanins, 186 Pseudomonas aeruginosa (bacterium), and salicylic acid biosynthesis, 205 Pseudomonas fluorescens (bacterium), and salicylic acid biosynthesis, 205 Pseudomonas putida (bacterium), 205 Pseudomonas syringae (bacterium), and salicylic acid biosynthesis, 205 Pseudomonas syringae pv. syringae (bacterium), 222 PSPG (plant secondary product glycosyltransferase), 147 Pulsatilla cernua (pasqueflower), anthocyanins, 182–3 punicalagin, 264, 268 apoptosis induction, 271–3 fermentation, 346 metabolism, 271–3 structure, 324 purpurin, 9 puvalanol B, 105–6 PVCs (prevacuolar compartments), 192 PXR (pregnane X receptor), 359 Pycnogenol, 24 pyranoanthocyanidin, 168 pyranoanthocyanins, structure, 177–8 QSAR (quantitative structure–activity relationship) model, 302 quantitative structure–activity relationship (QSAR) model, 302 quercetin conjugates, metabolism, 320 quercetin-3-O-glucuronide, 78–9 quercetin glucuronides effects on angiogenesis, 345 enzyme inhibition, 345 synthesis, 329–30 quercetins, 9, 20–1, 71, 128, 130 antioxidant activity, 340–1 glucuronidation, 72–3, 320 hydrolysis, 318–19 oxidation, 76–7 pro-oxidant properties, 342–3 structure, 321–2 toxicity, 343 quercetin O-sulfates chemical synthesis, 332–3 effects on angiogenesis, 345 quinones, 9 structure, 126 o-quinones, 281

391

RA see rosmarinic acid (RA) Raphanus sativus (radish), seed germination, 11 rat studies, anthocyanin metabolism, 251–2 Rauvolfia serpentine (Indian snakeroot), arbutin synthase, 127, 130 Rc gene, 122 reactive oxygen species (ROS), 6 increased production, 368 production, enzyme inhibition, 74–5 recombinant-enzyme assays, phytoestrogens in, antiaromatase activity, 299 red wine anthocyanins, 237 and atherosclerosis, 369 and coronary heart disease reduction, 233–4 regulatory genes, for proanthocyanidin biosynthetic pathway, 121–2 regulatory proteins, anthocyanin-related, 154 resinols, softwoods, 40 resinol units, in lignin chains, 49 resistance mechanisms, 11 species-specific, 14 see also induced resistance; plant resistance resistance genes, 11 resveratrol, 369 t-butyldimethylsilylation, 330–2 resveratrol glucuronides, synthesis, 330, 331 resveratrol metabolites, 348–9 characterization, 328 structure, 328 R gene, 121 rhamnetin, antioxidant activity, 341–2 rhizobia, 7 rhizobia–legume symbiosis, 8 Rhizobium spp. (soil bacteria), genes, 7 Rhizoctonia spp. (fungus), 216 ribulose-5-phosphate, as intermediate, 13 robinin, 21 roots, communication, 7 ROS see reactive oxygen species (ROS) rosacyanin B, structure, 177–8 Rosa hybrida (rose), rosacyanin B, 177–8 roscovitine, 105–6 Rosea1 gene, 155 Rosea2 gene, 155 rosmarinic acid (RA), 12–13 metabolites, 327 rubrocampanin, 184 RUGT-5, 130 rutin, 318 SA see salicylic acid (SA) SABPs (salicylic acid binding proteins), 206–7, 219–20 salicylate glucosyltransferase, 125 salicylates, 19 salicylic acid binding proteins (SABPs), 206–7, 219–20 salicylic acid (SA) biosynthesis, 203–5 genetic approach, 204–5 in plant defenses, 16 early studies, 203 induced, 202–10 mechanisms, 205–7 synthesis, 125 saliva, phenolic metabolite biosynthesis, 318–19

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salivary proteins, interactions, with dietary flavonoids, 71–2 Salix spp. (willows), salicylates, 19 Salvia patens (gentian sage), metalloanthocyanins, 189 SAR see systemic acquired resistance (SAR) Sclerotinia sclerotiorum (fungus), 221 scoparone, 17 scopoletin, 17, 126 secondary metabolism mechanisms, 1 plants, 1–5 use of term, 1 secondary metabolites, 211 bioactivities, 1–2 classification, 2 with diverse functions, 1–35 transport, 5 seed germination, 10–11 Ser392, 108 sex steroids biosynthesis, 293 blocking, 293 SGLT1 see sodium-dependent glucose transporter 1 (SGLT1) shikimate/phenylpropanoid pathway, 2 shikimic acid pathway, 13 signal compounds, plant phenolics, 6–8 signaling cascades, interactions, with phenolic conjugates, 343–4 signaling pathways, analysis, 365 silicon, as defense modulator, 221 silybin, 323–4 silybin glucuronides, antioxidant activity, 341 silymarin, anti-aromatase activity, 300 sinapaldehyde, 44–5 sinapic acid, synthesis, 125 sinapyl alcohols, Plate 2.3 biosynthesis, 36 dehydrodimerization, 39 partial substitution, 44 sinapyl aldehydes, 124 glycosylation, 125 Sinningia cardinalis (sinningia), 3-deoxyanthocyanins, 190 small intestine, phenolic metabolite biosynthesis, 319–20 sodium-dependent glucose transporter 1 (SGLT1), 237, 241 roles, 243–4 softwoods lignin polymer models, Plate 2.4 resinols, 40 Sorghum bicolor (sorghum) 3-deoxyanthocyanidin phytoalexin synthesis, 12 3-desoxyanthocyanidin, 168 Sorghum caudatum, 7-O-methylapigenidin, 168 soy, polyphenols, 369, 372–3 soybeans, IFS gene, 116 species-specific resistance, 14 Sphagnum fallax (moss), p-coumaric acid, 126 Spodoptera exigua (beet armyworm), 20 Spodoptera frugiperda (fall armyworm), 20 Spodoptera littoralis (African cotton leafworm), 20 SREBP (sterol-response binding protein), 369–70 steroid biosynthesis control, phytoestrogens for, 293–316 sterol-response binding protein (SREBP), 369–70

stilbenes, 23 antifungal activity, 15 stomach, phenolic metabolite biosynthesis, 319 stone fruit, Monilinia infection, 215 structural genes, for proanthocyanidin biosynthetic pathway, 118–21 O-sulfates, 349 chemical synthesis, 332–3 syringaresinol, biosynthesis, 39 syringyl oligomers, 46 syringyl units, 45–6 coupling, 44–5 systemic acquired resistance (SAR), 16 mechanisms, 202–3 use of term, 203 see also induced systemic resistance (ISR) tangeretin, 17 tannins antibiotic activity, 15 effects on phytophagous insects, 22 toxicity, 15 see also ellagitannins; proanthocyanidins (PAs) TEAC (trolox equivalent antioxidant capacity), 230, 334 template replication, lignins, 38, 48–9 ternatin A1, 182 terpenoids, 2 testosterone biosynthesis, 293 blocking, 293 tetra-O-benzylepicatechin bromination, 102 formation, 92 oxidation, 93 tetrahydrocurcumin (THC), 346 tetrahydroxydibenzopyranones, 268–9 tetratriflates, synthesis, 100, 101 TFs see transcription factors (TFs) THC (tetrahydrocurcumin), 346 thioacidolysis, 46–7 thionocarbonates, synthesis, 100, 101 thioredoxin reductase, 372 TIC chromatograms see total ion current (TIC) chromatograms tissues, phenolic metabolite biosynthesis, 321 TMV see tobacco mosaic virus (TMV) tobacco mosaic virus (TMV), 204, 207 resistance to, 219 tobacco plants coumarins, 126 resistance, 219 salicylic acid, 203–4 transgenic, 117 TOGT, 126 tolerance mechanisms, 11 Torenia hybrida (snapdragon), malvidin, 132 total antioxidant potential (TRAP) assays, 230 total ion current (TIC) chromatograms caffeoylquinic acid, 282, 283 caffeoylquinic acid o-quinone, 285 (−)-epicatechin, 285 transcription factors (TFs), 81–2, 122–3, 156–7 activation, 78 in anthocyanin biosynthesis, 152–3 transgenic plants, induced resistance studies, 205 TRAP (total antioxidant potential) assays, 230 trichloroacetimidates, 329–30

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Tricyrtis formosana (toad lily) anthocyanins, 184 cyanidins, 182 trihydroxydibenzopyranones, 268–9 trolox equivalent antioxidant capacity (TEAC), 230, 334 TT2 gene, 121–2 TT8 gene, 121–2 TT12 gene, 120 TT19 gene, 120 TTG1 gene, 121 tumors, ERBB2-positive, 364 turgorins, 10 tyramine ferulate, incorporation, into lignins, 42–3 tyrosine kinase, inhibition, 89 UGT71C1, 130 UGT71C1 gene expression, 127 induction, 130–1 UGT71C4, 130 UGT71G1, 129, 130–1 UGT72E1, 124–5, 130 UGT72E2, 124–5 UGT73B2, 130 UGT73F1, 128 UGT73J1, 129 UGTs see uridine diphosphate glycosyltransferases (UGTs) ultraviolet (UV) sunscreens, plant phenolics, 6 umbel, 182 umbelliferone, 17β-hydroxysteroid dehydrogenase type 3 inhibition, 310 uridine diphosphate glycosyltransferases (UGTs), 129 active with non-flavonoid phenolics, 124–8 functional annotation, issues, 130–1 urolithins, 268, 269, 324 antioxidant activity, 346 estrogenic activity, 271 gene expression studies, 273–4 Utetheisa ornatrix (bella moth), 19 UV (ultraviolet) sunscreens, plant phenolics, 6

393

Vaccinium myrtillus (bilberry), anthocyanins, 233, 236 vascular function, phenolic conjugate effects, 345 vascular smooth muscle cell (VSMC) hypertrophy, 343 VCEAC (vitamin C equivalent antioxidant capacity), 334 vegetables, post-harvest treatment, 17 Venosa gene, 155, 156 Viburnum opulus (Guelder-rose), anthocyanins, 183 Vicia villosa (hairy vetch), 18 Vigna spp. (cowpeas), 20–1 Vigna luteola (hairypod cowpea), 21 Vigna marina (beach pea), 21 VIGS (virus induced gene suppression), 205 Viola cornuta (tufted pansy), pigmentation, 156 virus induced gene suppression (VIGS), 205 vision improvement, and anthocyanins, 236 vitamin C equivalent antioxidant capacity (VCEAC), 334 vitamin P, 359 use of term, 233 Vitis vinifera (grape vine) anthocyanic vacuolar inclusions, 191 leucoanthocyanidin reductase, 118 VvGT1, 131 VSMC (vascular smooth muscle cell) hypertrophy, 343 VvGT1, 131 WD-repeat (WDR) protein, 153, 155 WDR (WD-repeat) protein, 153, 155 wine see red wine xanthine oxidase catalysis, 75 inhibition, 23, 344–5 Xanthomonas spp. (bacterium), 222 xanthones, anti-aromatase activity, 302 xenobiotic responsive element (XRE), 360 xenobiotic transport, 362–3 XRE (xenobiotic responsive element), 360 zeatin, 125