Studies in Natural Product Chemistry, Volume 22: Bioactive Natural Products, Part C

Studies in Natural Products Chemistry Volume 22 Bioactive Natural Products (Part C ) Studies in Natural Products Chem...

Author: Atta-Ur-Rahman

77 downloads 1435 Views 40MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Studies in Natural Products Chemistry Volume 22 Bioactive Natural Products (Part C )

Studies in Natural Products Chemistry edited by Atta-ur-Rahman

Vol. 1 Stereoselective Synthesis (Part A) Vol. 2 Structure Elucidation (Part A) Vol. 3 Stereoselective Synthesis (Part B) Vol. 4 Stereoselective Synthesis (Part C) Vol. 5 Structure Elucidation (Part B) Vol. 6 Stereoselective Synthesis (Part D) Vol. 7 Structure and Chemistry (Part A) Vol. 8 Stereoselective Synthesis (Part E) Vol. 9 Structure and Chemistry (Part B) Vol. 10 Stereoselective Synthesis (Part F) Vol. 11 Stereoselective Synthesis (Part G) Vol. 12 Stereoselective Synthesis (Part H) Vol. 13 Bioactive Natural Products (Part A) Vo1.14 Stereoselective Synthesis (Part I) Vol. 15 Structure and Chemistry (Part C) Vo1.16 Stereoselective Synthesis (Part J) Vol. 17 Structure and Chemistry (Part D) Vol. 18 Stereoselective Synthesis (Part K) Vol. 19 Structure and Chemistry (Part E) Vol.20 Structure and Chemistry (Part F) Vo1.21 Bioactive Natural Products (Part B) Vo1.22 Bioactive Natural Products (Part C)

Studies in Natural Products Chemistry Volume 22 Bioactive Natural Products (Part C)

Edited by

Atta-ur-Rahman

H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

2000 ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon

- Singapore

- Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands 92000 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science Global Rights Department, PO Box 800, Oxford OX5 1DX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-maih [email protected]. You may also contact Global Rights directly through Elsevier's home page (http://www.elsevier.nl), by selecting 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WlP 0LP, UK; phone: (+44) 171 631 5555; fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2000 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for.

ISBN:

0 444 50588 1

Transferred to digital printing 2005 Printed and bound by Antony Rowe Ltd, Eastbourne

Foreword Plant and animal organisms offer a wide diversity of compounds which can serve as exciting new pharmacophores and which can reveal new mechanisms of action for controlling disease processes. Vol. 22 of Studies in Natural Products Chemistry comprises articles written on bioactive natural products by leading authorities in their respective fields. Robinson has reviewed the metabolism and pharmacology of alkaloids found in animals. Chemical ecology can be an attractive tool for identifying antifungal natural products and this area has been reviewed by Graham et al. The chapter by R ios and co-workers presents studies carried out on triterpenes which have shown anti-inflammatory activity. Malaria continues to be a major health problem in developing countries and there are a large number of deaths each year caused by it. The review by Kawanishi et al. presents the current status of work done in this field as well as on natural anti-diabetic compounds. Synthetic approaches involving intramolecular diyl trapping reaction are described by Little et al. for the synthesis of linearly fused tricyclopentanoids. The use of classical and biocombinatorial approaches to bioactive fungal natural products is discussed by Jiang et al. F010p has presented the chemistry of 2aminocyclopentanecarboxylic acid. Antioxidants continue to attract attention in medicine and many interesting flavonoids have been found in nature which possess antioxidant and pro-oxidant properties. These studies have been reviewed by Vanden Berghe and co-workers, while antioxidant activity found in South American plants is reviewed by Desmarchelier. Other interesting reviews include those on insect juvenile hormones in plants by Tobe, antiulcer and gastroprotective activity of flavones by Martin et al, biological activity of simple flavones by Tahara et al. Anti-convulsant plants by Raza and biological activity of anthracenones of the Karwinskia genus by PiSeyro-Lopez. A number of different species of plants of Polygonumspecies possess interesting biological activities. The bioactive compounds in such plants are reviewed by Adamczeski. Hypericum perforatum (St. Johns wort) is one of the extensively studied plants because of its wide range of biological activities, specially its use for the treatment of mild to moderate depression. The review by Erdelmeier describes the research carried out on this plant. Finally tropane alkaloids are reviewed by Christen in respect of their chemistry and biological activity. I would like to express my thanks to Dr. Shakil Ahmad and Dr. Durre Shahwar for their assistance in the preparation of the index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance. It is hoped that this volume, which represents the third volume of this series devoted to bioactive natural products, will be of great interest to organic chemists, medicinal chemists and pharmacologists.

Atta.ur.Rahman February, 2000

Ph.D. (Cantab), Sc.D. (Cantab)

This Page Intentionally Left Blank

Preface In his fascinating and beautifully written autobiography (For the Love of Enzymes, Harvard University Press, Cambridge, MA, 1989), Arthur Kornberg repeatedly emphasizes the central role of chemistry in understanding life processes. Nowhere is this connection clearer and more direct than in the field of "Natural Products" chemistry. From the early stereochemical studies on tartaric acid carried out by Louis Pasteur one-and-a-half centuries ago, through the subsequent pioneering work of chemists such as Emil Fischer, Otto Wallach, Robert Robinson, Vlado Prelog, R.B. Woodward, D.H.R. Barton .... (the list could go on and on), we have seen again and again how the careful study of naturallyoccurring compounds has, on the one hand, enriched our understanding of the science of organic chemistry itself, and on the other hand, provided deep insights into biological phenomena. Despite wide swings in the popularity or even "trendiness" of natural products research, the field continues to advance around the world. It is not a coincidence that natural products have played, and will continue to play, a seminal role in the discovery and development of pharmaceutically and agrochemically important agents. Three billion years of biological evolution have resulted in the development of metabolic pathways leading to the synthesis of hormones, pheromones, antibacterial, anti-fungal, anti-protozoan, and antiinsectan agents, as well as many other bioactive compounds that are of adaptive value in the lives of the organisms that produce them. The natural products chemists of the world (in some respects indistinguishable from "chemical ecologists") isolate these molecular entities, focussing chiefly on compounds with particularly interesting biological activities either from the human point of view or from that of the producing organism. They establish their structures and define their biosynthetic pathways. They study their mechanisms of action and their metabolic pathways. They devise synthetic methods which make novel target structures accessible for further research and for application. While much of this research is driven by the entirely worthy desire to obtain "useful knowledge," it is clear that scientists entering the field of natural products chemistry are often deeply motivated by their love of nature in general, of chemistry in particular, and by their fascination with understanding as much of life as possible at the molecular level. The "Studies in Natural Products Chemistry' series, now in its twenty-second volume, documents an incredible diversity of research. If we bear in mind the fact that for some of the most important groups of organisms (i.e. soil dwelling microbes; insects and other arthropods), most species have not yet been described, let alone subjected to chemical investigation, we can look forward eagerly to many future volumes in this series. With respect to the present volume, the reader can expect a veritable chemical feast.

Jerrold Meinwald

Cornell University Ithaca, NY 14853 USA


CONTENTS Foreword

V

Preface

vii

Contributors

xi

The Metabolism and Biochemical Actions of Alkaloids in Animals T. ROBINSON Using Chemical Ecology to Locate New Antifungal Natural Products STEPHANIE J. ECKERMAN AND KATE J. GRAHAM

55

Natural Triterpenoids as Anti-Inflammatory Agents J.L. RJOS, M.C. RECIO, S. MAlqEZ AND R.M. GINER

93

Current Status of the Chemistry and Synthesis of Natural Antimalarial Compounds and natural Substances used to Alleviate Symptoms of Diabetes (Aldose Reductase and A-Glucosidase Inhibitors) K. KAWANISHI AND N.R. FARNSWORTH 145 A Diradical Route to Bioactive Natural Products and their Analogs R. DANIEL LITTLE AND MICHAEL M. OTT

195

Bioactive Fungal Natural Products Through Classic and Biocombinatorial Approaches ZHI-DONG JIANG AND ZHIQIANG AN

245

The Chemistry of 2-Aminocyclopentanecarboxylic Acid FERENC FOLOP

273

Structure-Activity Relationship of Flavonoids as Antioxidant and Pro-Oxidant Compounds P. COS, M. CALOMME, L. PIETERS, A.J. VLIETINCK AND D. VANDEN BERGHE

307

Recent Advances in the Search for Antioxidant activity in South American Plants C. DESMARCHELIER, G. CICCIA AND J. COUSSIO

343

Insect Juvenile Hormones in Plants JACQUELINE C. BEDE AND STEPHEN S. TOBE

369

Antiulcer and Gastroprotective Activity of Flavonic Compounds" Mechanisms Involved M.J. MARTIN, C. ALARCON DE LA LASTRA, V. MOTILVA AND C. LA CASA

419

Simple Flavones Possessing Complex Biological Activity S. TAHARA AND J.L. INGHAM

457

Medicinal Plants with Anticonvulsant Activities MOHSIN RAZA, M. IQBAL CHOUDHARY AND ATTA-UR-RAHMAN

507

Chemistry, Structure and Biological Activity of Anthracenones of the Karwinskia Genus A. PllqEYRO-LOPEZ AND N. WAKSMAN

555

Bioactive Natural Products Derived from Polygonum Species of Plants: Their Structures and Mechanisms of Action NWAKA OGWURU AND MADELINE ADAMCZESKI

607

Hypericum Perforatum - St. John's Won:Chemical, Pharmacological and Clinical Aspects C.A.J. ERDELMEIER, E. KOCH AND R. HOERR

643

Tropane Alkaloids: Old Drugs used in Modem Medicine P. CHRISTEN

717

Subject Index

751

CONTRIBUTORS Madeline Adamczeski

Department of Chemistry, American Washington, D.C. 20016-8014, USA

University,

Zhiqiang An

Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, MA 02139-1562, USA

Atta-ur-Rahman

International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan

Jacqueline C. Bede

Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada

D. Vanden Berghe

Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium

M. Calomme


M. Iqbal Choudhary


P. Christen

University of Geneva, Laboratory of Pharmaceutical Analytical Chemistry, 20, Boulevard d'Yvoy, CH-1211 Geneva 4, Switzerland

G. Ciccia

C6tedra de Microbiologia Industrial y Biotecnologia, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina

P. Cos


J. Coussio

C~tedra de Farmacognosia, IQUIMEFA-CONICET, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Jun|n 956 1113 Buenos Aires, Argentina

C. Alarc6n de la Lastra

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcfa Gonz~lez s/n, 41012Seville, Spain

xii

C. Desmarchelier

C6tedra de Microbiologta Industrial y Biotecnologta, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina

Stephanie J. Eckerman

Chemistry Department College of St. Benedict/St. John's University 37 S. College Avenue, St. Joseph, MN 56374, USA

C.A.J. Erdelmeier

Dr. Willmar Schwabe GmbH & Co., Research and Development, Karlsruhe, Germany

N.R. Famsworth

Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Illinois, USA

Ferenc Ft~lOp

Institute of Pharmaceutical Chemistry, Albert SzentGy0rgyi Medical University, H-6701, Szeged, POB 121, Hungary

R.M. Giner

Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Avda. Vicent Andr/~s Estell~s s/n., 46100 Burjassot (Valencia), Spain

Kate J. Graham

Chemistry Department College of St. Benedict/St. John's University 37 S. College Avenue, St. Joseph, MN 56374, USA

R. Hoerr


J.L. Ingham

Department of Food Science and Technology, University of Reading, Whiteknights, P.O. Box 226, Reading RG6 2AP, England, U.K.

Zhi-Dong Jiang

Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, MA 02139-1562, USA

K. Kawanishi

Kobe Pharmaceutical University, Kobe, Japan

E. Koch


C. La Casa

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonzfilez s/n, 41012Seville, Spain

~176176

XIU

R. Daniel Little

Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA

S. M~iflez

Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Avda. Vicent Andr6s Estell/~s s/n., 46100 Burjassot (Val/mcia), Spain

M.J. Martin

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonz~ilez s/n, 41012Seville, Spain

V. Motilva

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonz~ilez s/n, 41012Seville, Spain

Nwaka Ogwuru

Department of Chemistry, American Washington, D.C. 20016-8014, USA

Michael M. Ott

Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA

L. Pieters


A. Pifleyro-Lopez

Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. del Valle, 66220, Garza Garcia, N.L. Mexico

Mohsin Raza


M.C. Recio

Departament de Farmacologia, Facultat de Farmhcia, Universitat de Valb,ncia, Avda. Vicent AndrOs Estell6s s/n., 46100 Burjassot (Valencia), Spain

J.L. Rios

Departament de Farmacologia, Facultat de Farmb.cia, Universitat de Valencia, Avda. Vicent AndrOs Estell~s s/n., 46100 Burjassot (Valencia), Spain

T. Robinson

Lederle Graduate Research Center, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst Box 34505, Amherst, MA 010034505, U.S.A.

University,

xiv

S. Tahara

Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan

Stephen S. Tobe

Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada

A.J. Vlietinck


N. Waksman

Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. del Valle, 66220, Garza Garcta, N.L. M~xico

Bioactive Natural Products


Atta-ur-Rahman (Ed.) Studies in Natural Pror Chemistry Vol. 22 9 2000 ElsevierScience B.V. All rights reserved

THE METABOLISM AND BIOCHEMICAL ALKALOIDS IN ANIMALS

ACTIONS OF

T. ROBINSON

Lederle Graduate Research Center Department of Biochemistry and Molecular Biology University of Massachusetts, Amherst Box 34505, Amherst, MA 01003-4505 U.S.A A B S T R A C T : The metabolism and pharmacology of naturally-occurring alkaloids are reviewed, with emphasis on work of the last ten years during which there have been many important advances. It is recognized that the final effects of alkaloids on animals may result from activity of their metabolic products rather than of the original substance. Therefore one section discusses how alkaloids are metabolized, first in terms of the general processes and then with many examples of the metabolism of particular types of alkaloid structures. The second section on pharmacology deals with detailed biochemistry of alkaloid action at the level of molecule-to-molecule, rather than describing behavioral or gross physiological effects. Modern molecular biological methods have revealed the intimate structures of neural receptors and other cellular molecules with which the alkaloids interact. In this section of the review the material is organized according to the processes being affected rather than according to types of alkaloid. Thus there are subsections for the various neural receptors, structural components of cells, enzymes, etc.

GENERAL INTRODUCTION The toxic and stimulatory effects of alkaloid-containing plants o11 the behavior of animals have been observed for many centuries; and since the early 19th Century it has been possible to attribute many of these effects to specific, chemically characterized substances, which became known as "alkaloids" because many of them formed salts with acids. Late in the 19th Century the action of certain alkaloids on specific physiological systems became clear. Since the middle of the 20th Century the availability of isolated and well-characterized biochemical systems has made possible still further refinement in our knowledge of how alkaloids act as they do in terms of membrane structures, enzymes, receptors, transporters, and so on. It is such systems of molecule vs. molecule that are the focus of this review. Behavioral effects, and even effects on gross physiological systems, have a vast literature that will be mostly passed over here. "Animals" is used here in a broad sense, to include insects, molluscs, and other lower life forms, as well as mammals. Microorganisms are, however, excluded. With the growing realization that some effects of alkaloids are, in fact, effects of their metabolic products rather than of the originally

4

T. ROBINSON

administered compound, it became essential to include information about how alkaloids are metabolized. METABOLISM

Introduction, Common Processes Animals have processes for dealing with foreign compounds, some of which are assimilated because of their nutritional importance, while others are of no value or even detrimental. Alkaloids fall into the latter group, but they are treated by processes that are not unique to them but have general roles in metabolism. Such processes as hydrolysis, oxidation, reduction, and conjugation are applied to alkaloids just as they are to such nutritionally important molecules as carbohydrates or proteins. The following sections review these processes, first in general and then as they apply to alkaloids. As a general rule, it appears that the metabolism of alkaloids in animals does not proceed to complete breakdown yielding carbon dioxide but that a few small modifications of the structure are produced.

A. Hydrolysis Several alkaloids contain ester groups, and an early step in their metabolism is the hydrolysis of the ester bond. Additional reactions may then occur to complete the metabolism. As will be shown in specific examples, esterases both in blood serum and the liver are active in different cases.

B. Oxidation Oxidative processes are well-known in animal metabolism; and they include pocesses that abstract hydrogen atoms from the substrate as well as those that add oxygen atoms to the substrate. For alkaloid molecules, though, oxygenation is more usual than dehydrogenation. This may be because the dehydrogenase enzymes are closely matched with the structures of their usual substrates, while oxygenases are less specific. Cytochrome P-450 Enzymes occur in all classes of organisms, and a single species may have several dozen different types, acting on hundreds of different substrates as dioxygenases. Some use flavoprotein as a reductant and some use cytochrome Bs. They have roles both in normal metabolism of steroids, eicosanoids, nitric oxide, etc. and also in oxidizing exogenous compounds, including certain alkaloids. Some exogenous compounds induce the formation of P-450 enzymes through a complex

ALKALOIDS IN ANIMALS

5

series of molecular events [1 ]. As well as introduction of oxygen atoms, the P-450 systems are also responsible for removal of N-methyl groups, converting them to carbon dioxide. Not all oxidations of alkaloids can be ascribed to microsomal P-450 systems. Mammalian liver contains a flavoprotein oxidase, first discovered in rabit liver and called "quinine oxidase". It is also an aldehyde oxidase, and its mechanism of action on heterocyclic nitrogen compounds probably involves addition of hydroxide ion to a suitable ring position, followed by dehydrogenation. Thus, the introduced oxygen atom comes from water rather than from molecular oxygen, in contrast to the P-450 oxygenases.

[2]. C. Conjugation Conjugation describes a process in which some exogenous molecule becomes joined to a common metabolite. Examples are the addition of acyl groups, amino acid residues, carbohydrate groups or sulfate groups. Conjugation may follow preliminary oxidative or hydrolytic processes that release hydroxyl groups suitable for the derivatization, there are also some cases of addition of methyl groups [3]. Besides metabolic process that degrade alkaloids, there are also process that produce alkaloids in animals. Some of these are straightforward condensations of amines with aldehydes or ketones as in the formation of isoquinolines or harman derivatives by condensation of an aldehyde or ketone with, respectively, dopamine or tryptamine [4, 5, 6]. More complex reactions must also occur to account for the formation of such complex structures as morphine, which is now well-established as an endogenous compound in animals, although at very low concentration [7, 8]. Amaryllidaceae Alkaloids

Galanthamine products found in human plasma and urine result from epimerization of the hydroxyl group and then dehydration to a ketone [9]. Caffeine and other Purines

Methylated xanthines like caffeine are degraded in humans and rats by oxidative removal of methyl groups [10]. Thus caffeine goes to 1,7dimethylxanthine and 1-methylxanthine [11]. After the first demethylation there is an alternate pathway producing 5-acetylamino-6-formylamino-3methyluracil. This pathway is more active in people with a more active acetylation system [ 12, 13]. There are individual, quantitative differences in the activity of this pathway in humans. In rats a major metabolite is

6

T. ROBINSON

1,3,8-trimethylallantoin, in which all three methyl groups are retained [ 14]. In rabbits the major urinary products are, in order, 1-methylxanthine, 1methyluric acid, 7-methylxanthine, and 1,7-dimethylxanthine [15]. Pretreatment of rats with caffeine increased the activities of the P-450 enzymes; so that demethylation and C-8 oxidation were doubled as compared with untreated rats [ 16]. Pretreatment with polycyclic aromatic compounds causes a similar increase in this P-450 activity [ 17]. Experiments with liver slices and cultured cell lines have corroborated this pathway of purine degradation. In human liver slices 16 xanthine derivatives were produced from caffeine by action of P-450 system. Demethylation at N-3 was the most prominent process [18, 19]. Comparison of cell lines from humans, hamsters, mice, and rats show some interspecies differences but all of them demethylated and oxidized caffeine [20]. Human liver cells give 1,3,7-trimethylurate as the major metabolite of caffeine, but also made were the intermediate products theobromine, theophylline, and paraxanthine [21]. Human liver microsomes convert theophylline to 1-methylxanthine, 3-methylxanthine, and 1,3-dimethyluric acid [22, 23]. Human kidney microsomes produced each of the three possible demethylated products as well as 1,3,70

CH3

0

(~H3 Caffeine

(~H3 Theophylline

1

O

O

CH3....,J~

iCH3

1,7-Dimethylxanthine

1

H

G 1-Methylxanthine

O

CH3~ ~

~I"[~C)C;H3 5-Acetylamino-6-formylamino-3-methyluracil

H Caffeine Metabolism


7

trimethyluric acid from caffeine [24]. It appears that two distinct P-450 systems are involved in these p r o c e s s e s - - - o n e responsible for demethylation and the other for oxidation at C-8 [16, 25, 26, 27]. In premature human infants the reverse of the demethylation process occurs, in that theophylline, which is 1,3-dimethylxanthine, becomes methylated at N-7 to make caffeine [28].

Cocaine and Other Tropane Alkaloids Because of its wide use as a drug of abuse, cocaine's metabolism has been studied more than that of other alkaloids of the tropane group. Isolation of breakdown products from the urine following administration of cocaine has indicated that processes of demethylation, ester hydrolysis, and hydroxylation of the aromatic ring account for the more than a dozen products that have been identified [29, 30, 31, 32]. Studies on the enzymology of these processes have localized these processes both histologically and, in some cases, intracellularly. Esterases that hydrolyze the two ester groups are widely distributed in tissues ~ for instance in serum, liver, and brain. It is thought that hydrolysis in the serum is slow and contributes little to the overall rate of degradation [33, 34, 35]. The enzyme in serum that is responsible for this hydrolysis is 2000 times more active in hydrolyzing the unnatural isomer (+)-cocaine than the natural (-)-cocaine, and this difference may account for the low pharmacological activity of the (+)-isomer [36]. An interesting sidereaction occurs in subjects (rats or humans) who consume ethanol at the same time as cocaine. The esterase present in the microsomal fraction of liver catalyzes a transesterification reaction that replaces the methyl ester /0-13 N~-..~

H %jL.;LX.X.,,~3

N ~ ~3

H (-)-Cocaine ~

H,,,j~2H5

R Cocaethylene drolysis

Ecgonine

8

T. ROBINSON

grouping with an ethyl ester, giving the compound known as cocaethylene [34, 37, 38]. Cocaethylene persists in the serum longer than cocaine itself and is more potent in some actions [39, 40]. Antibodies raised to a cocaine analog in its hydrolysis transition state were tested for hydrolytic activity, and one of them was found to be active [41 ]. Oxidation is the second major process in degradation of cocaine, and it occurs in the liver as a result of activity of microsomal cytochrome P-450 enzymes. Oxidative reactions account for hydroxylation of the aromatic ring of the benzoic acid moiety and for N-demethylation [30, 42, 43]. Specifically, it appears to be the 3A form of P-450 that is responsible, since feeding inhibitors of this isoform to mice inhibited the hepatotoxicity caused by the demethylated product [44]. There is less information about the metabolism of other tropane alkaloids; but for those that are esters (e.g. hyoscyamine, scopolamine) hydrolysis of the ester bond takes place to some extent, yielding bases, which may be further transformed [45, 46]. Rabbit serum has been found to have esterases that are relatively specific for esters of tropic acid, and this may account for the ability of rabbits to feed with impunity on leaves of Atropa belladona [47]. Mice transform released tropic acid to a glucuronide [48]. Oxidation also occurs, both in rats and humans, with the production of N-oxides and oxidative removal of the N-methyl group to make nor-derivatives [45, 46].

Colchicine Colchicine is demethylated i n the liver, forming 2-demethyl and 3demethyl products A microsomal P-450, NADPH-dependent system is responsible for this demethylation [49]

Cyclopeptide Alkaloids As might be expected, peptidases can catalyze hydrolysis of cyclopeptides ~ for example frangufoline [50].

CH3

Frangufoline


9

Ellipticine Group

CH3

Ellipticine

In the intact rat or liver microsomes ellipticine is hydroxylated at C-9 or C-7. The resulting hydroxyl derivative is then converted to a glucuronide or dehydrogenated to a keto derivative [51, 52]. 9Methoxyellipticine is demethylated and conjugated with glucuronic acid at the C-9 position or it is conjugated with glutathione at the C-10 position [52, 53]. 9-Hydroxy-2-methylellipticine is similarly conjugated, not only with glutathione but also with N-acetylcysteine [54]. Oxidation of elliptinium salts produces a quinone that reacts with amino compounds to form oxazoles [55].

Indole Alkaloids Serotonin is metabolized to bufotenin, and much of this is excreted in the urine as a glucuronide conjugate [56]. Vindoline and related alkaloids of C a t h a r a n t h u s roseus are extensively metabolized by mammals through the actions of esterases, peroxidases, and P-450 oxidases [57]. Oxidation of vindoline and vinblastine occurs in human serum, catalyzed by ceruloplasmin [58]. The major metabolite of yohimbine is l lhydroxyyohimbine, but the 10-hydroxy derivative is also produced [59]. Strychnine injected into rats gives as its major metabolite its 21,22epoxide, but other epoxidized and hydroxy derivatives are also prodced [60]. Harman is hydroxylated at C-6 by mice [61 ].

Isoquinoline and Morphinan Alkaloids Laudanosine is O-demethylated to various products by dogs, rabbits, and humans [62]; but 1,2,3,4-tetrahydroisoquinoline in human brain is Nmethylated [63]. Several isoquinoline alkaloids are O-methylated by the action of catechol O-methyl transferase [64]. It is now well accepted that small amounts of morphine and related alkaloids are not only present in many foods but are also synthesized normally by animals. Therefore the metabolism of exogenous morphine and related molecules calls for no processes outside of what must be usual metabolism [65, 66]. Normal human plasma has been found to contain

10

T. ROBINSON

80pg/ml of endogenous morphine [67]. The concentration is higher in anorexic and bulimic subjects [68]. The pathway for biosynthesis of morphine in animals is similar to that in plants with the final steps being" (R)-reticuline - : > ( + ) - s a l u t a r i d i n e codeinone > codeine > morphine

>-----> thebaine

>

The oxidative reactions of this sequence are catalyzed by the microsomal P-450 system [69, 70, 71]. A P-450 system from rat liver can also oxidize morphine [72]. One product of this oxidation is morphinone, a highly toxic electrophile that couples with thiol groups. The latter reaction may deplete glutathione and in other ways may account for the hepatotoxicity of morphine [73, 74]. The demethylation of codeine to morphine probably accounts for the analgesic action of codeine, and people with a defect in this demethylating system probably get no analgesia from codeine [75, 76]. Rats, too, show strain differences in the ability to demethylate codeine to morphine [77]. Quinidine, quinine, or sparteine inhibit the conversion of codeine to morphine, presumably by inhibiting the P-450 enzyme [71, 78]. While O-demethylation converts codeine to morphine, N-demethylation also occurs and produces norcodeine [79]. Conjugation with glucuronic acid is a major process for detoxification of codeine and morphine. For morphine both the 3- and 6-glucuronides are produced, but the ratio between them varies widely with the species. Generally the 3-glucuronide predominates; but in humans and guinea pigs there is also significant formation of 6-glucuronide, while rats make very little of that isomer [80]; but the unnatural isomer (+)-morphine is glucuronidated by rats more at the 6-position than the 3-position [81 ]. In the presence of low concentrations of ethanol more 6-glucuronide is made, but higher concentrations of ethanol inhibit the glucuronidation reaction [82]. Two enzymes that catalyze the glucuronidation have been characterized in liver microsomes [83, 84]. Along with the glucuronides as excretion products in the urine, the adduct of morphinone with glutathione can be prominent in some species [85]. Major excretion products from codeine are norcodeine, morphine, and codeine-6-glucuronide [86]. Nicotine

and

Related

Alkaloids

The metabolism of nicotine is a much-studied area in which metabolic products were first identified in the urine and later the mechanisms for their formation were characterized in detail. The major products obviously result from oxidations, some that introduce oxygen atoms, and some that remove methyl groups. The half-life of nicotine in blood can vary greatly. In mice a half life of only 6 minutes has been found [87], but in humans it is about 30 minutes [88]. Moreover there are strain to strain differences within a species [87] and even between males and females or between

11


smokers and nonsmokers [89]. While the major metabolic products are the same in all animals, there are quantitative differences from species to species.

HO.!~

H3CO"~

//~-----~N_CH3

3CO

(-)-Reticuline

HOOx O,

N'-CH3

_

~

H3CO O (+)-Salutaridine

o ~ . /~

.......

.~ O, ~

"-CH3

H3CO Thebaine

. HO N--OH

H

Codeinone

13-GIucosyl. N-'CH3 H Morphine-6-glucuronide Morphine-RelatedPathway

N

Codeine

HO.~ H

Morphine

12

T. ROBINSON

Generally found in significant amounts are nornicotine, cotinine, cotinine N-oxide, trans-3'-hydroxycotinine (and its glucuronide), quaternary N-methylated derivatives, nicotine l'-N-oxide, nicotine Nglucuronide, and some unchanged nicotine [90, 91, 92, 93, 94] . Interestingly, there are somewhat diferent patterns of metabolites depending on the route of nicotine administration [95] or on preadministration of ethanol or phenobarbital [96, 97, 98]. The natural form, S-(-)-nicotine gives rise to a different pattern of products than unatural R-(+)-nieotine [99, 100, 101 ].

CH 3

"~

H

Nornicotine

Nicotine

1

5'Hydroxynicotine

I

CH3

Cotinine

CH3

3-Hydroxycontinine

T

o

CH3

Cotinine-N-oxide

Nicotine Metabolism

At the subcellular level the importance of the P-450 system of microsomes in metabolizing nicotine has been clearly established [98, 102] 9The first step is oxidation by P-450 at C-5' to give the 1'-5'-iminium ion that is then oxygenated using water as the source of oxygen [103, 104]. A flavoprotein is implicated in this later step, or possibly also in an alternative pathway [ 104, 105, 106]. The N-demethylation and N-oxideforming reactions are also catalyzed in microsomes, but the details of these


13

processes are not characterized [98]. Formation of N-methyl quaternary ammonium derivative is catalyzed by a cytosolic enzyme from human liver but not from rat liver [ 107]. Quatemization abolishes the ability of nicotine to penetrate the blood-brain barrier [108].

Pyridines Arecoline is rapidly metabolized, the first step being hydrolysis of the ester group through action of carboxylesterase [ 109].

Pyrrolizidines The pyrrolizidine alkaloids are well-known as liver toxins. They not only poison livestock that eat plants containing them but have also been implicated in human cases where large amounts of certain medicinal herbs (e.g. comfrey) have been used. The reason for discussing them in this section is that the actual toxins are not the native alkaloids themselves but metabolic products of them produced in the liver by the action of microsomal enzymes. The esters may be hydrolyzed and N-oxides reduced, but it is dehydrogenation of the saturated pyrrolidine ring to a pyrrole that gives rise to the toxicity [1 10, 111]. For instance, monocrotaline in rat liver gives rise to monocrotalic acid and several pyrroles [112]. Retrorsine N-oxide is converted to dehydroretronecine [113]. In the liver these pyrroles react with thiol groups of essential molecules to form thioethers [114]. They may also be detoxified by conjugation with glutathione [ 115]. For instance dehydroretronecine is converted in rat liver to 7glutathionyldehydroretronecine. [ 116]. Dehydromonocrotaline, produced from monocrotaline is believed to be the active toxic metabolite. It reacts at its C-9" or C-7" position to alkylate the N-3 position of several purine nucleosides or various amino groups [117]; but it can be detoxified by conjugation with glutathione. Apparently an early action of the alkaloid is

t,. ~ I . ~

~CH2CH o

~H2CH v

o

Retrorsine-N-oxide

Dehydroretronecine

14

T. ROBINSON

a diversion of cysteine metabolism away from conversion to taurine and toward synthesis of glutathione [ 118]. Senecionine is converted to the Noxide not by a P-450 enzyme but by a flavin-containing oxidase in pigs and guinea pigs [ 119, 120]. An interesting sidelight on the metabolism of pyrrolizidines is that several butterfly and moth larvae that feed on plants containing these alkaloids metabolize them to products that serve the insects as pheromones or defensive substances. Thus the butterfly ldea leuconoe converts alkaloids from Parsonia laevigata to N-oxides [ 121 ]. The moth Creatonotos transiens makes its pheromone, R(-)-hydroxydanaidal from7(S)-heliotrine found in its host plant, Seneciojacobaea [122].

Quinolines Dictamnine fed to rats is demethylated and the ring system oxidized [123]. Quinidine is oxidized to 3-hydroxyquinidine and other products [ 124].

Quinolizidines The metabolism of sparteine by humans produces as the chief products 2and 5-dehydrosparteine as well as 2- and 7-oxo derivatives [124, 125]. Somewhere less than 10% of the Caucasian population have little ability to metabolize it at all [126, 127, 128]. Pachycarpine, the optical antipode of (-)-sparteine is converted in rats to (+)-(4S)-hydroxysparteine as the major product [ 129].

Tyramine Derivatives Cathinone is metabolized by humans to (R,S)-(-)-norephedrine and (R,R)(-)-norpseudoephedrine [130]. Mescaline is metabolized by mice to 3,4,5trihydroxyphenylacetaldehyde, which is oxidized to the corresponding acid by hepatic microsomes [ 131 ]. BIOCHEMICAL ACTIONS

Introduction As compounds that are hydrophilic and basic, it is no surprise that alkaloids interact readily with essential biochemical constituents such as proteins, polysaccharides, and nucleic acids, forming hydrogen bonds and ionic bonds. Less obvious reactions may also be relatively common. For example, essential thiol groups of proteins can undergo nucleophilic reactions with positively charged alkaloids, and pi complexes are possible


15

between aromatic rings of alkaloids and those of biological molecules. Metal ion coordination could play a role with some alkaloids that can complex such essential metals as iron or magnesium. Among the vast range of possibilities, some may have little to do with the actual, physiological effects of alkaloids; but others may provide exactly the mechanisms for the pharmacological effects. Biochemistry starts with knowledge of the nature of the reactants and their concentrations at the site of action. Access to this information is often difficult because administered alkaloids may be metabolically modified before becoming active, as discussed in the first part of this review. Moreover, physical barriers and complex transport systems may intervene between administration and action. The literature has many examples of drugs that at concentrations of 0.001M or higher have effects on purified biochemical systems in vitro, yet in vivo the effective concentration may be orders of magnitude lower. While injection can provide a more direct delivery of alkaloid to its site of action than can oral administration, alkaloids may become bound to serum proteins and thus be made less available to tissues. More and more it has become evident that subtle aspects of molecular shape and electron distribution must be considered in any explanation of drug action, and computer modeling has become an essential tool in visualizing molecular interactions, although it appears to have been applied more to the development of synthetic drugs than to naturally-occurring alkaloids [ 132]. In the following sections I have chosen to organize the information according to the type of biochemical system acted upon rather than according to structural types of the alkaloids. While there are arguments possible for either arrangement, I have been persuaded by observing that quite disparate structures can act on the same system; so that if they were treated separately, there would be much redundancy in describing the biochemical system each time that a different class of alkaloid was considered. Recent information on the toxicology of alkaloids can be found in [133]. CHOLINERGIC TRANSMISSION Transmission of nervous impulses by way of acetylcholine release and action is widespread, occurring not only in higher animals but also important in arthropods. In higher animals acetylcholine is the most important neurohormonal transmitter. It functions in the autonomic system, in motor nerves, and in some parts of the central nervous system. It functions not only in synapses between neurons but also on muscles or glands that are controlled by the neurons. After its action the acetylcholine is removed rapidly through hydrolysis by the enzyme acetylcholine esterase. Drugs, including some alkaloids, can interact with this process at several levels:

16

T. ROBINSON

1. As inhibitors of acetylcholine synthesis 2. As inhibitors or stimulators of acetylcholine release 3. As substances that mimic the action of acetylcholine (agonists) 4. As substances that block the action of acetylcholine (antagonists) 5. As blockers of ion channels that the agonist opened 6. As inhibitors of acetylcholine esterase One case of a natural alkaloid that affects acetylcholine synthesis is sanguinarine, which inhibits choline acetyl tranferase [134]. There are several alkaloids that affect release of acetylcholine, but they may not owe their primary pharmacological effects to this activity [135]. Among the agonists and antagonists of the cholinergic system there are many alkaloids, and several different structural types. To undertand their actions it is first necessary to understand that there are two main types of cholinergic receptors in the nervous system ~ nicotinic and muscarinic. Both respond to acetylcholine; but the nicotinic class has nicotine as a specific agonist, while the muscarinic class has the fungal alkaloid muscarine as its specific agonist. There are also different specific antagonists for the two classes. Nicotinic receptors respond faster than muscarinic ones and occur where fast responses are important to the organism. Molecular biology has now provided detailed information about the structures and reactions at the two types of cholinergic receptors; and the actions of alkaloids can be correlated with the nature of the receptors. Ion channel blockers and acetylcholinesterase inhibitors are considered in later sections of this review.

Nicotinic Receptor A symposium publication covers all aspects of nicotine pharmacology [136]. The nicotinic acetylcholine receptor, among other effects, comrols the passage of sodium and potassium ions across the membranes that contain it. It is composed of 2 or more alpha subunits and 2 or more other subunits arranged around the actual ion channel. The overall molecular mass of each subunit is approximately 50kDa. In the muscle receptor there are two identical alpha subunits and one each of the beta, gamma, and delta subunits. Receptors from other tissues also have similar structures, but the combination of subunits can be different and result in somewhat different responses to agonists and antagonists [137, 138]. The binding sites for acetylcholine are dependent on cysteine residues at or near positions 193 and 195 of the alpha subunit [ 139]. When acetylcholine becomes bound to two alpha subunits, the channel opens. The other subunits influence the affinity for acetylcholine, though not binding it themselves [140]. The prototypical antagonist at the nicotinic receptor is, of course, nicotine itself, which stimulates at low concentration but blocks the receptor at high concentration. Competitive binding experiments have shown that


17

acetylcholine and (-)-nicotine bind to the same high-affinity site [141 ]. The unnatural isomer, (+)-nicotine, is much less active [142, 143, 144]. The quinolizidine alkaloid (-)-cytisine and the piperidine alkaloid (-)-lobeline evidently bind to the same site as nicotine. It has been pointed out that all three of these alkaloids share in the relationship of a charged nitrogen atom to an aromatic ring and that while the cationic nitrogen associates with a nucleophilic cysteine side-chain in the high affinity site, the aromatic ring may interact with aromatic side-chains of the receptor [145, 146]. Arecoline, a piperidine derivative from the betel nut, is also an agonist [ 147]. Physostigmine, best-known as an inhibitor of acetylcholinesterase, is also an activator of the nicotinic receptor; but strictly speaking, it is not an agonist because it does not act at the same site as acetylcholine [ 148]. d-Tubocurarine, a powerful antagonist, is bound at two sites ~ a high affinity site using alpha and gamma subunits, and a site with 1/400 as much affinity using alpha and delta subunits [149]. By measuring the affinity of analogues, it was shown that the 12' and 13' hydroxylated positons of the alkaloid are important for binding [150]. Thermodynamic studies have shown that the binding is entropy-controlled [ 151 ]. Nicotine and alpha-bungarotoxin from cobra venom both bind in the region of residues 173-204. Nicotine becomes bound to tyrosine residue 198 of the Torpedo receptor [152]; but competitive antagonists are bound to tyrosine 190 and a neighboring cysteine [153]. The nor-diterpenoid alkaloids of Delphinium spp. are powerful antagonists, and they also compete with alpha-bungarotoxin, which, like them, prefers receptors made from 7 alpha subunits [ 138,154]. Indeed, the most potent, non-protein antagonist is the Delphinium alkaloid methyllycaconitine [155]. Structure-activity studies with the aconite alkaloids have revealed the importance of a C-3 hydroxyl group and an oxygen at C-8 [156]. (+)-Sparteine is an antagonist mostly competitive with acetylcholine but also to some extant a blocker of the open ion channel [157]. Despite the potency of these antagonists, they all show reversible binding [ 154]. H --...

C2H5N,,~, ~

,

jOC_~

l

I HaOCH3Aconitine C While some agonists and antagonists may bind to the acetylcholine site, others owe their activity to association with other s i t e s - - e v e n on different subunits. A pyrrolizidine from dendrobatid frogs blocks the ionic

18

T. ROBINSON

channels but does not compete with nicotine for a binding site in rat cerebral membranes [137]. Epibatidine, a chlorinated alkaloid from these frogs, has a structure resembling nicotine and acts as an agonist rather than antagonist [158]. Cocaine has myriad effects; and one of the less important ones comes from its action on the ion channel of the nicotinic receptor, thus antagonizing the effects of nicotine, though not binding at the same site [159]. Strychnine, known primarily for its antagonism of the glycine receptor, may also act on a regulatory site of the nicotinic receptor [ 160]. Physostigmine, known more for its inhibition of acetylcholinesterase, also acts on the nicotinic receptor at a site distinct from the acetylcholine site [ 148]. It and some other alkaloids seem to act as sensitizing modulators for the natural transmitter [ 161 ]. Many compounds structurally related to nicotine have been tested for activity with the nicotinic cholinergic receptor; but. as with the (+)- isomer of nicotine, they generally less activity as agonists, while showing other activities such as blocking the ion channel. Metabolic derivatives of nicotine may account for some of the actions attributed to nicotine, but not necessarily actions at the nicotinic receptor [162]. Nicotine does, indeed, act on other tissues; and some of these actions will be cited in other sections of this review. Although controlling ion channels is the best-known function of the nicotinic receptor, there are examples of other processes under control of this receptor. There is one, for instance, that mediates the release of dopamine or other catecholamines and is inhibited by ibogaine or strychnine [ 163, 164, 165].

Muscarinic Receptor The muscarinic cholinergic system has quite a different mode of operation in that the receptor is connected to the final action by a chain of events. Thus its response is slower than the nicotinic, where the receptor and ion channel are closely connected. Five distinct muscarinic receptors have been identified in mammals, based on anatomical location, genetic analysis, function, and amino acid sequence. All of them have seven transmembrane domains [166, 167, 168, 169]. The N- terminal domain outside the cell binds acetylcholine or other ligands at a site that includes an aspartate residue, while the C-terminal domain inside the cell is coupled to a socalled "G-protein", which is initially bound to guanosine diphosphate (GDP), but exchanges it for guanosine triphosphate (GTP) when activated by its transmitter. The activated G-protein then activates phospholipase C, which hydrolyzes phosphoinositides to release 1,4,5-inositol triphosphate [170]. The final action depends on which type of cell is involved; so that in some types ion channels are opened just as with the nicotinic receptor, but in other cases other processes are affected, for example the release of dopamine [171]. Since there are these differences


19

among receptors, it is not surprising that different alkaloids act upon them in different ways. Moreover, stimulation of the muscarinic receptor can make nicotinic receptors of the same cell more responsive to nicotine, perhaps by increasing the concentration of cytosolic calcium [172]. Pilocarpine is long-known as a muscarinic antagonist [173]. Imperialine, a steroid alkaloid, himbacine, a piperidine, and ebeinone, a quinolizidine, are all antagonists at M2 receptors [174, 175]. Quinidine appears to act not on the receptor itself but on the ion channel that is opened by the receptor [176, 177]. Veratridine, similarly, acts on sodium channels [178]. Cocaine, atropine (DL-hyoscyamine), and other tropane alkaloids are wellrecognized antagonists of the muscarinic receptor. Although (-)-cocaine is the naturally occurring form, (+)-cocaine was found to be the more potent with both M~ and M2 receptors. [ 179, 180, 181 ]. Tropanes may not all act in the same way, but atropine causes dissociation of the complex between receptor and its G-protein in heart membranes, and this may be its mechanism of action [182]. Although opioids have their own, specific receptors, they can also act on muscarinic cholinergic receptors [183]. Yohimbine, best-known as an adrenergic blocker, also acts on muscarinic receptors, stabilizing a non-permeable form of the sodium channel [ 184]. ADRENERGIC RECEPTORS The transmitter molecule in the adrenergic system is norepinephrine, but the overall structures and mechanisms of response are like those of the muscarinic cholinergic system [ 185]. That is, the receptor molecule spans the cell membrane with seven transmembrane domains. The outer segment of the protein is responsible for interacting with the transmitter, and the inner segment is associated with a G-protein, which in the inactive state is bound to GDP. When activated, the receptor converts the G-protein to a form that exchanges the GDP for GTP, and the G-protein then activates a particular effector such as an adenylate cyclase. Activation is terminated by hydrolysis of the bound GTP to GDP. Based on location and specific responses to particular agonists and antagonists the adrenergic receptors have been classified first into alpha and beta types, then further subdivided; so that there are two alpha and three beta types. Further, alpha 1 and alpha 2 both have several subtypes [186, 187]. The alpha l receptors activate phospholipase C, while the alpha 2 receptors inhibit adenylate cyclase. The beta receptors are coupled to G-proteins that stimulate adenylate cyclase [188, 189, 190]. This complexity means that no alkaloid can be described simply as "acting on the adrenergic system". Most information regarding the action of alkaloids relates to the alpha 2 receptor, where the indole alkaloids yohimbine and its isomer, rauwolscine, bind with high affinity and block the receptor [187, 191]. Dihydrocorynantheine, an indole alkaloid of similar structure preferentially binds to the alpha 1 receptor, as do some aporphines [192,

20

T. ROBINSON

193, 194]. For all of these alkaloids that bind to alpha receptors coplanarity of the A, B, C, and D rings is important. Then, specificity for 1 or 2 subtype depends on the conformation of groups on the E-ring [195]. The seventh hydrophobic domain of the receptor has been identified as the major determinant for alkaloid binding [ 189].

I

o-I Yohimbine

Protoberberines are also antagonists at alpha 2 receptors [134, 196]. Tetrandrine and dicentrine interact antagonistically with alpha 1 receptors [192, 197, 198]. One of cocaine's many actions may also be on an alpha receptor, since its vasoconstrictive action is antagonized by yohimbine [199]. PURtNERGIC RECEPTORS The class of purinergic receptors has been recognized more recently than other types of receptors, and it incorporates both the receptors known as "adenosine receptors" (P I) as well as receptors responding to adenine nucleotides such as ADP (P2). Although there is much overlap, by and large the P I group is antagonized by methylxanthines, like caffeine. The P2 group has a low affinity for methylxanthines [200, 201,202, 203]. The P l group of adenosine receptors is further subdivided into A l, A2 and A3 by their different preferences for different xanthines and synthetic drugs [204, 205]. A2 has been subdivided further into A2a and A2b. The adenosine receptors have been cloned and sequenced. All have the characteristic structure of receptors working through G-proteins ~ seven transmembrane segments with the N-terminal extracellular and the Cterminal intracellular. There are cysteine residues and carbohydrate on the extracellular loops. Histidine residues on transmembrane segments 6 and 7 are important for antagonist binding. Molecular modeling has suggested that the N-1 position of adenosine and the N-9 of xanthine antagonists occupy the same site on the receptor [206]. The A1 group finds its greatest expression in the central nervous system, but is also in other organs. The alkaloid best-known as an antagonist of adenosine receptors


21

is caffeine; b u t , in general, 8-substituted xanthines such as 1,7dimethylxanthine or theophylline, are active [203, 207, 208, 209]. Adenosine receptors regulate diverse functions, with downregulation of adenylate cyclase, calcium channels, and phospholipase A2, upregulation of potassium channels, and ambiguous efects on phospholipase C [210]. The inhibitory effects of the A l receptor being overcome by caffeine makes caffeine appear to be a stimulant of, for instance, the release of calcium. Tolerance to caffeine may come from upregulation or an increased number of adenosine receptors [210, 211 ]. Abrupt withdrawal of caffeine causes a fall in cyclic AMP and sensitization to adenosine [210]. There are some extremely complex interactions in this area. To follow just two examples: The A~ activation of phospholipase C causes increased hydrolyis of inositol lipids so that inositol-l,4,5-trisphosphate is produced and can induce calcium release from microsomes. Caffeine can directly inhibit opening of the calcium channel in the microsomes beyond any action it may have on the initial A l receptor [212, 213]. Caffeine acting on hippocampal A I receptors appears to stimulate release of acetylcholine, but it is actually blocking adenosine receptors that inhibit the release [214, 215]. The end-result is increased wakefulness produced by caffeine. While many effects of caffeine can be attributed to its antagonism at adenosine receptors, its effect on various ion channels is not always mediated by adenosine receptors, and caffeine will appear again in the section on ion channels. SEROTONERGIC RECEPTORS The identification of an active substance named "serotonin" as 5hydroxytryptamine in the mid-twentieth century has been followed by much progress in understanding the distribution and function of this substance in particular groups of neurons in the central nervous system, where it appears to function predominantly as an inhibitory transmitter. At this writing 14 distinct receptors for serotonin have been identified and some of them cloned and sequenced. They have been subdivided into 7 major types, with further subdivisions. All of them have seven membranespanning domains and are coupled with G-proteins [216, 217, 218, 219, 220]. Detailed information from one receptor that has been sequenced (5HT6) shows that a threonine residue at position 196 in the polypeptide chain is important for ligand binding. This threonine side-chain has been found to form a hydrogen bond with the N-1 of an indole ring in either serotonin itself or other, exogenous molecules. Substitution of alanine at this position greatly reduces affinity for agonists and the stimulation of adenylate cyclase, since the alanine side-chain lacks hydrogen bonding capability [220].

22

T. ROBINSON

The best-known of the agonists for serotonergic receptors are several ergot alkaloids, such as ergovaline, and their synthetic derivative LSD [221,222]. By testing a series of these compounds it has been found that a reduced five-membered indole ring increased specific binding to serotonin receptors [223]. A fiat conformation of the D-ring is essential for biological activity [224]. Alkaloids that act as serotonin antagonists include rauwolscine and its isomer yohimbine, which are also noted as adrenergic antagonists [225, 226, 227]. The non-ergot alkaloids asimilobine and lirinidine are also antagonists [228]. In addition to its receptors, serotonin has transporter molecules; and there are alkaloids that interfere with the transport and uptake process. DNA's for serotonin transporters of rat leukemia cells have been cloned and sequenced. One of them has a 653 residue protein with 12-13 transmembrane domains. Interestingly they show considerable homology with transporters of some other transmitter substances; and are sensitive to cocaine, reserpine and several other alkaloids [229, 230, 231,232]. Thus the inhibitory action of serotonin can be reduced not only by substances that act on its receptor but also by substances that hinder its access to the receptor [233]. DOPAMINE RECEPTORS AND TRANSPORTERS Dopamine, or 3,4-dihydroxytyramine is increasingly recognized as a neurotransmitter having a number of distinguishable receptors. It is also reactive with several other metabolites to produce alkaloidal products that may have significant physiological effects [4, 234, 235, 236]. There are at least five types of dopamine receptors in two families [237]. All of them have seven membrane-spanning alpha helices, and all but D3 are coupled to G-proteins. Dl and D5 activate adenylate cyclase. D2 inhibits adenylate cyclase. Within each type there are probably subtypes [238, 239, 240]. Genetic variants in the level of D2 receptors have important behavioral consequences. A low level leads to craving for substances that release more dopamine in the brain [241 ]. The binding of fifteen bisbenzylisoquinoline alkaloids to Dl and D2 receptors has been measured, and it was found that the most active ones had a 11,12'-ether bridge, as in thaligrisine. The configuration of chiral centers also makes a difference [242]. (-)Tetrahydropalmatine blocks brain dopamine receptors, while the (+)-form depletes dopamine levels [243, 244]. Tests of the activity of aporphines on D l and D2 receptors have shown that (R)- configured compounds had greater affinity than their (S)-antipodes because of the orientation of the lone pair on N-6. The most potent antagonists have hydroxyl at C-11, but a hydroxyl at C-10 makes an agonist rather than an antagonist [245]. Some of the ergot alkaloids interact with dopamine receptors that are not linked to adenylate cyclase [222, 246]. Reduction of the five-membered indole


23

ring results in decreased affinity for the receptor, in contrast to the effect with the serotonin receptor [223]. Another important aspect of dopamine neurochemistry is the role of dopamine transporters in moving dopamine across cell membranes. Techniques of molecular biology have permitted the manufacture of chimeric dopamine transporters having interchangeable segments of the molecule. In this way the requirements for specific sites or actions can be determined. From this, it appears that the amino terminus is important for ionic dependence and uptake mechanism, the carboxyl terminus for determining substrate affinity and stereoselectivity, and the middle section for interaction with inhibitors [247, 248]. Dopamine and cocaine probably bind to both shared and separate domains. A transporter from rat brain has 620 amino acid residues and 12 membrane-spanning domains. Both termini are intracellular. Segments 1-3 are important for both substrate and inhibitor interactions; segments 5-8 are involved only with inhibitors. [249, 250]. Another one from bovine brain has 693 residues [251 ]. Cocaine is the most-studied inhibitor of the dopamine transporter [252, 253]. It inhibits reuptake of dopamine at D l and D2 receptors so that action of the transmitter is prolonged and accentuated [229, 237, 254, 255, 256]. Testing of seven possible stereoisomers of cocaine showed that the natural form, (-)-cocaine, binds more than sixty times better than the next best isomer. Binding requires an aryl group connected directly or indirectly to C-3 and an ester group at C-2, both of them in the beta orientation [257]. Cocaine binds slowly to the carrier, while dopamine binds rapidly [258], and their binding is mutually exclusive [259]. Spermine inhibits the binding of cocaine [260]; and sodium ion concentration makes a difference [261 ]. A transporter from striatal synaptosomes has a binding site for ATP, which affects the structure but does not become hydrolyzed [262]. Reserpine acts on the dopamine transporter to cause release of the amine, so that free dopamine accumulates extracellularly. The binding appears to be non-covalent [247, 263]. (-)-Cathinone acts similarly [264]. Nicotine and, even more, some quaternary, N-methylated products of nicotine metabolism, inhibit dopamine uptake. This may help to explain why smoking relieves some symptoms of Parkinsonism [265, 266, 267]. In another way, nicotine acting on certain acetylcholine receptors evokes release of dopamine from rat striatal cells [268]. Veratridine has a similar effect [ 142]. THE OPIATE RECEPTOR The name "Opiate Receptor" has stuck even though it has been thought since the early 1970's that the endogenous transmitters acting on this receptor are oligopeptides, enkephalins and endorphins. It is also now accepted that morphine, the classical opiate, itself occurs naturally in animals and may act as an endogenous transmitter [66, 269, 270]. There

24

T. ROBINSON

are several different opiate receptors, distinguished by anatomical locations and by differential responses to selected agonists and antagonists [271 ]. As a rough simplification: mu receptors mediate the analgesic effect delta receptors mediate emotional effects kappa receptors mediate sedative effects sigma receptors mediate psychomimetic effects All of them have seven transmembrane, hydrophobic segments and are linked to regulatory G-proteins. Mu, delta, and kappa receptors have been purified, cloned, and sequenced [66, 272, 273]. The mu receptor is subclassified into two subtypes. Type 1 is a high affinity receptor for both morphine and enkephalins. Type 2 is the classic morphine receptor [66, 274]. The delta receptor has been reconstituted in lipid vesicles [275]. The enkephalins prefer delta receptors, and dynorphins prefer kappa receptors [276]. The synthetic drugs (R)-methadone and naloxone prefer mu receptors [277, 278]. The mu and delta receptors are associated with addiction and cause dopamine release, whereas kappa receptors cause aversion and decrease the release of dopamine [279, 280]. The morphine receptor in human lung has low affinity and perhaps does not fit into the established classification [281]. Cocaine indirectly raises the level of extracellular dopamine in the sustantia nigra by decreasing the number of kappa receptors [282. For binding to all receptors the free 3-hydroxyl group of morphine is important, so that codeine or morphine-3glucuronide have low affinity. The 3-glucuronide, lacking analgesic properties by itself, can nevertheless increase the analgesia produced by morphine. Thus it could be considered a regulator of morphine action [283]. The 6-glucuronide has affinity equal to that of morphine for mu receptors but higher for delta receptors and lower for kappa receptors [271,284]. Some actions attributed to morphine may, in fact, be due more specifically to the 6-glucuronide [66]. However, the 6-glucuronide penetrates poorly into brain capillary endothelial cells, except in the presence of vincristine, which inhibits P-glycoprotein that may bind the glucuronide [285]. Several alkaloids not structurally similar to morphine apparently act at opioid receptors. Harmaline binds to the delta receptor of rat synaptosomal membranes, whereas salsolinol binds preferentially to mu receptors [286]. Ibogaine and related indoles have low affinity for opioid receptors except moderate affinity for sigma2 [287]. Mitragynine appears from responses in Guinea pig ileum to act on an opioid receptor [288]. Some events following activation of opiate receptors are complicated because different concentrations of the agonist produce diffferent effects even on the same group of cells ~ in the release of catecholamines from chromaffin cells, for instance [269]. A transient increase in intracellular


25

free calcium caused by morphine results from specific stimulation of the delta receptors [289]. One way that morphine may decrease pain responses is that it inhibits release of the pain transmitter substance P from cells of the spinal cord [290]. On the other hand, it increases release of neuropeptide FF [291]. Symptoms of morphine withdrawal are accompanied at the cellular level with increased activity of phospholipase C and the various effects mediated by the product of this enzyme, inositol 1,4,5-triphosphate [292]. Morphine causes release of arachidonic acid from mouse macrophages; and since arachidonic acid is a precursor of prostaglandins and thromboxanes, there may be an indirect effect of morphine on processes affected by these hormones [293]. There are numerous results suggesting an interaction between morphine and adenosine receptors. Perhaps adenosine acts as a mediator of morphine effects [294]. AMINO ACID AND PEPTIDE RECEPTORS There are many small oligopeptides that serve as neurotransmitters and hormones. The enkephalins, endorphins, etc. have already ben mentioned in the section on opiate receptors. Two amino acids that serve in this way are glycine, an inhibitory transmitter, and glutamic acid, an excitatory transmitter, gamma-Aminobutyric acid (GABA) is less well established, but probably an inhibitory transmitter. Peptide hormones like vasopressin and somatostatin are made in the pituitary gland or hypothalamus and influence processes elsewhere in the body. Strychnine has been known for a long time as a blocker of a glycine receptor in the brain. There are also some glycine receptors insensitive to strychnine [295, 296, 297, 298]. A subunit of the glycine receptor that binds strychnine has been cloned and sequenced. It has sequence and structure homologies with the nicotinic cholinergic receptor; and the strychnine-binding sequence is around residue 200 of a 48kDa subunit, a region corresponding to the acetylcholine-binding region of the nicotinic receptor [299]. Tyrosine 197 and/or 202 is probably involved [300]. This site is not identical with the glycine site but interacts closely with it [301 ]. The binding of strychnine to this receptor is regulated by inorganic anions [302]. Corynine, another indole alkaloid, is also an inhibitor of the glycine receptor that acts at a site removed from the glycine-binding site [303]. Although not normally classed as alkaloids, the natural products kainic acid, quisqualic acid, and N-methyl-D-aspartic acid (NMDA) are established, specific agonists for glutamic acid receptors. They can be seen as conformationally restricted analogues of glutamic acid. At least six recognition sites have been recognized on this receptor [304]" glutamate site, glycine-modulatory site, magnesium-binding site, zinc-binding site, polyamine-modulatory site, cation channel.

26

T. ROBINSON

Ibogaine appears to be an antagonist with this receptor [305, 306]; and, in an indirect way, repeated administration of morphine causes increased expression of a glutamate receptor subunit [307]. There are also probably some glutamate receptors for which NMDA is not an agonist [304]. GABA receptors are sometimes refered to as benzodiazapine receptors because this synthetic drug is an agonist at the GABA receptor [308]. The best-known alkaloid inhibitor of the GABA receptor is bicuculline, which is a competitive antagonist of GABA [309, 310]. The securinine group of indolizidines is also active but less so than bicuculline [311 ]. Forty-five alkaloids structurally related to bicuculline have been tested for activity at this receptor on rat brain synaptic membranes. Both agonists and antagonists were found [312]. Some effects of morphine have been ascribed to the blocking of a GABA receptor, thus causing excitation; but this is a low affinity binding [313]. Both glycine and glutamic acid stimulate movement of chloride ion in hippocampal neurons; and caffeine inhibits this process, although it has no effect on the glycine-binding site of the NMDA receptor [314].

Bicuculline

There are a few cases of alkaloids that act at the receptors for peptide hormones. Spiroquinazoline inhibits the binding of substance P to its receptor on astrocytoma cells [315]. Chelerythrine and sanguinarine compete with vasopressin for receptors on rat liver cells [316]. Psycholeine is an antogonist at somatostatin receptors on cultured pituitary cells [317, 318]. Caffeine inhibits the binding of thyrotropinreleasing hormone to pituitary cells [319]. NUCLEIC ACIDS AND PROTEIN SYNTHESIS The much-studied systems of nucleic acid replication and transcription, followed by translation of the nucleic acid code into protein structure has many points that can be influenced by exogenous compounds, including some alkaloids. To begin with the structural integrity of stored


27

deoxyribonucleic acid, several alkaloids have been shown to bind to DNA in ways more specific than by the self-evident ionic attraction that would be expected between the anionic nucleic acid and the (usually) cationic alkaloid. Intercalation into the DNA double helix occurs with a variety of compounds and is a result of hydrophobic pi interactions between the stacked base residues of the DNA and appropriate functional groups of the foreign substance. Intercalation may follow an initial ionic attraction of the two molecules and can be measured by observing spectral changes [320, 321,322] or changes in chromatographic or electrophoretic mobility [323,324]. Binding can shift equilibria among the various conformations of the double helix and affect replication and transcription [325, 326]. Because of their promise as antitumor drugs ellipticine and derivatives are probably the most investigated alkaloids that interact with DNA. While it is the cationic form of ellipticine that binds, hydrophobicity rather than electrostatic attraction is the main driving force [327]; and intercalation at low concentration is accompanied by other binding modes if the concentration increases [328]. Ellipticine itelf intercalates by a noncooperative neighbor exclusion model without any specificity for base sequence [321]. The most active antitumor compounds in the ellipticine group are 9-hydroxy derivatives, and these are the ones that show the highest affinity binding to DNA. They have special afinity for a doublet sequence of two guanine-cytosine pairs [329]. A 9-methyl group is also important [323]. 9-Aminoellipticine interacts with single-stranded DNA, specifically at apurinic sites [330]. There are several other alkaloids that have been indicated by physical measurements to intercalate into the DNA double helix. Among them are melinone F and normelinone F, quaternary beta-carbolines from Erythrina melinoniana [331]. Aristolactam,N-beta-D-glucoside from Aristolochia indica has a very high affinity for intercalation [332]. Berberine and sanginarine intercalate, berberine preferentially at alternating adeninethymine stretches in the DNA chain [181,320, 333]. Hydrophobicity is believed to be the predominant driving force for the binding [322]. Several xanthine derivatives intercalate and cause swelling of DNA. The most effective are unnatural, synthetic compounds; but caffeine and theophylline have some effect, caffeine more than theophylline [326]. Fagaronine and nitidine appear to intercalate into initiation complexes, thus inhibiting the action of DNA polymerase [334]. The presence of a quaternary nitrogen in fagaronine is important for its activity [335]. Several sponge alkaloids containing the 2-aminoimidazole group intercalate into DNA but have other effects as well, such as inhibiting DNA polymerase [336]. In addition to the intercalation mechanism, other types of binding occur between DNA and alkaloids. Following administration to rats various isotopically labeled pyrrolizidines have been found to contribute covalently bound label to DNA of liver, lung, and kidney perhaps because

28

1". ROBINSON

their pyrrole metabolites alkylate nitrogen atoms of the bases [117, 337]. N-Acylated pyrrolidines from Chamaesaracha conioides bind to DNA with high affinity [338]. Camptothecin forms alkali-labile linkages with closed, supercoiled DNA. The active site on the alkaloid for this binding is the hydroxyl group at C-20 in the E-ring, but C-7 of the B-ring is also involved in an interaction with adenine residues [339]. Dictamnine forms adducts with DNA when irradiated [340]. Harmine may act similarly [341]. Budmunchiamines bind in an unknown way [342]. The steroidal alkaloids solasodine and O-acetylsolasodine apparently alkylate DNA by means of their spiro-aminoacetal group, which can open to make an electrophilic iminium species [343]. Isogravacridonchlorine from Ruta graveolens is a mutagen that has been shown to act as a frame shifter in Salmonella [344]. There is less information in the literature about binding of alkaloids to ribonucleic acid than there is for deoxyribonucleic acid; but such binding does occur. However, since ribonucleic acid is single-stranded, the intercalation mechanism does not apply. Binding of the purine alkaloids caffeine and theophylline follows the reverse order from their binding to DNA .... theopylline binds with 10,000 times the affinity of caffeine [345]. Elliptinium acetate, best-known for its intercalation into DNA, is easily oxidized to an A-ring quinone amine that binds to the 2'-oxygen atom of ribose units in RNA. The first product then cyclizes to a spiro derivative [346]. In addition to alkaloids that interact with already formed nucleic acids and thus inhibit replication and transcription, there are several that inhibit these processes by other mechanisms. Topoisomerases are essential enzymes in the replication process. They make a break in supercoiled DNA, allowing one segment of the DNA to pass through, and then reseal the break, thus permitting the DNA to uncoil and become accessible to polymerases. Two types of topoisomerase are recognized. Type I is monomeric, breaks a single strand, and is poisoned by camptothecin. Type II has multiple subunits, breaks both strands, and is poisoned by ellipticine. Both poisons act by prolonging the life of a transient intermediate in which a broken strand is held by the enzyme [347, 348]. As already noted, the ellipticines intercalate in DNA, but this alone may not be sufficient to inhibit topoisomerase action; an oxidizable phenolic group is necessary to prevent the enzyme from resealing breaks that it has made [349]. With camptothecin the topoisomerase I becomes linked to the 3' end of the broken DNA, so that the rejoining step is blocked [350]. Camptothecin interacts specifically with guanine residues [351 ], and in its presence cleavage occurs most frequently at the dinucleotide pair thymineguanine, whereas some sites that are normally cleaved are not cleaved when the alkaloid is present [352]. Interestingly the topoisomerase I of yeast is not inhibited by camptothecin, as a result of a different amino acid


29

sequence at its active site [353]. Fagarone and monomargine are other alkaloids that inhibit DNA topoisomerases [354, 355]. There are several others that inhibit DNA replication. Fagaronine and nitidine have ben mentioned previously as intercalating agents, and this intercalation evidently inhibits the action of DNA polymerase [334]. Several benzophenanthridines and protoberberines inhibit reverse transcriptase, which catalyzes the synthesis of DNA with an RNA template [356, 357]. Mimosine at the cellular level, rather than directly on the process, is an inhibitor of DNA synthesis because it chelates iron [358] and because it blocks the synthesis of thymidylate, an essential precursor of DNA [359]. Vincristine may also act as an inhibitor of thymidylate synthesis, perhaps because it alkylates an essential thiol group of an enzyme [360]. The translation step where proteins are assembled following the code of messenger RNA has a few instances of inhibition by alkaloids [361]. Emetine and tubulosine block peptide bond formation, acting similarly to the antibiotic cycloheximide by blocking translocation of the growing peptide chain from the A site to the P site of the ribosome. They evidently bind to a specific ribosomal site [362]. Homoharringtonine may act similarly [363]. Lycorine may act at the level of termination [364]. Narciclasine and related alkaloids of the Amaryllidaceae prevent binding of the 3' end of aminoacyl-tRNA to the peptidyl transferase site of the ribosome [365, 366]. Mescaline may act similarly [367]. Following the ribosomal process many proteins are modified further by being phosphorylated, and several alkaloids affect this phosphorylation step, among them reserpine and sanguinarine, which inhibit [368, 369]; veratridine, which stimulates [370]; and chelerythrine, which in some cases inhibits and in other cases stimulates [369, 371]. ENZYME INHIBITORS There are innumerable reports of enzymes being inhibited by alkaloids, and this should not be surprising because enzymes and alkaloids both have functional groups that could be expected to interact in various ways. Probably at some concentration almost any alkaloid could be found to inhibit almost any enzyme. The crucial question though is, "Does a particular enzyme-alkaloid interaction help to explain the larger physiological effect of the alkaloid?" In a few cases the answer is that it does, and in some other cases that it might. Perhaps the most famous instance of enzyme inhibition explaining a gross effect of an alkaloid is the inhibition of cholinesterase by (-)physostigmine (eserine). Since acetylcholine is such a widespread and essential neurotransmitter, an increase in its concentration at synapses has serious consequences; and physostigmine by inhibiting the hydrolysis intensifies and prolongs the action of acetylcholine on its receptors [372].

30

T. ROBINSON

Several derivatives of physostigmine have been tested for activity. The (+)-isomer had little activity, but (-)-N-methylphysostigmine was more active than the natural alkaloid [373]. Testing of many other alkaloids has found few with significant activity [374, 375]. Galanthamine, although less potent than physostigmine, is longer lasting [376]. Huperzine A, similarly, is slower to bind to the enzyme and slower to dissociate than physostigmine; but it is potent and has pharmacological use [377, 378, 379]. d-Tubocurarine, known best as an antagonist at the nicotinic cholinergic receptor, is also an inhibitor of acetylcholinesterase, where it appears to act at two different sites [380, 381]. A group of 3alkylpyridinium polymers from the sponge Reniera sarai has recently been found to have potent anticholinesterase activity [382]. Acetylcholinesterase binds its substrate at a site containing tryptophan and aromatic residues [383]. The catalytic site, as in other esterases, contains serine and aspartate residues. In its action the enzyme forms a transient intermediate in which the acetyl group of substrate is esterified with the serine residue, and then rapidly hydrolyzed. Physostigmine, in contrast, forms a carbamylated enzyme that is hydrolyzed very much more slowly. Several alkaloids owe their toxicity to specific inhibition of enzymes that catalyze the hydrolysis of glycosidic bonds in carbohydrate oligomers and polymers. Thus there are glucosidase inhibitors, mannosidase inhibitors, etc. [384, 385, 386]. Almost all of these alkaloids can be describd as indolizidines with a large number of hydroxyl groups in their structure, but there is at least one pyrrolizidine [387], one piperidine [388], and one tropane [389, 390]. Swainsonine and castanospermine are the best known alkaloids in this group. Swainsonine is an inhibitor of alpha mannosidase II of the Golgi apparatus. As a result of its action Nlinked oligosaccharides are made with extra mannose groups [391,392, 393]. Castanospermine inhibits beta-mannosidase I and several glucosidases [391 ].

slieR Swainsonine

The oligosaccharides affected are used in making glycoproteins, which are then impaired in their functions [392, 393]. Many other alkaloids have been reported to be enzyme inhibitors, but little detail is available about their action or pharmacological significance. They are listed in Table 1.


31

Enzyme Inhibition by Alkaloids

Table 1.

,

Inhibited Enzyme

Alkaloid

|m i

l

Reference

i

quinidine

P-450 oxidases

quinine

monoamine oxidase

quinine

phosphodiesterase

406

mannosidase

403

phosphodiesterase

407,408

xanthines

glutamic dehydrogenase

409

404 l

405 I

swainsonine

.

caffeine

t

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

beta-carbolines

dioxygenases

410

vinblastine, vincristine

lipoxygenase

411

vinblastine, vincristine

monoamine oxidase

412

papaverine

cAMP phosphodiesterase

413

chelerythrine

protein kinase C

414

beta-carbolines

monoamine oxidase

415

aldose reductase

416

sucrase

417

.

.

.

.

.

.

.

.

.

berberine, palmatine

.

.

castanospermine

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

xanthines

phosphodiesterase

408

berbamine

ATPase

418

allocryptopine

phosphodiesterase

419

nicotine, continine

3-OH-steroid dehydrogenase

420

O-methylase

421

.

cocaine .

.

.

.

.

.

.

.

purpurone

ATP-citrate lyase

422

bis-indoles

tyrosine kinase

423

harmaline

monoamine oxidase

424

bastadins

inosine-5'-phosphate dehydrogenase

425

protoberberines

tyrosine hydroxylase

426

chelidonine

monooxygenases

427

cerveratrums

cAMP phosphodiesterase

428

protoberberines

elastase

429

strychnine, brucine

lactic dehydrogenase

430

,

i

,

i

ii

CYTOSKELETON AND MEMBRANE DISRUPTORS

The cytoskeleton of cells is composed of several components that are responsible for maintaining the physical structure of the cell. It comprises

32

1".ROBINSON

the filamentous structures microtubules composed of the protein tubulin, microfilaments composed of the protein actin, and intermediate filaments that vary in composition. The cell membrane is a complex structure composed of proteins, lipids, and carbohydrates. All of these structures have been found to be adversely affected by particular alkaloids. Many detergent-like molecules are well known to destroy the orderly lipid structure of cell membranes. More subtle than gross effects on the membrane, there may be effects on transmembrane processes such as ion transport. The alkaloids that also show these effects are those with an amphipathic structure whose hydrophobic part associates with the membrane lipids, while the hydrophilic part resides in the aqueous medium. There are not many such alkaloids with these properties, and they are mainly glycosides of nitrogen-containing steroids that act specifically on sterol-containing membranes, where they form 1:1 complexes with the sterols. For this activity the alkaloid must be glycosylated at the 3-beta position, and additional specificity is conveyed by the nature of the glycosyl group and by the nature of the membrane sterol. The most active alkaloid in this respect is alpha-tomatine [421,422, 423]. Quinidine is a strong perturber of model lipid membranes that contain acidic phospholipids [424]. Synergism between alkaloids has been reported, and this may be important because plants that contain this type of compound usually have more than one of them [425]. Although the effects of these alkaloids on transmembrane ion transport may result from nonspecific membrane disruption, other effects may be more specific for certain ions; and they are therefore treated in the section on ion transport effects in this review. Oxidation of the lipid components of membranes is another process that is deleterious to membrane stability; and there are alkaloids that favor this oxidation and those that inhibit it. In the former group are sanguinarine [426, 427] and orellanine [428]. In the latter group are boldine [429], reserpine [368], colchicine and colchieeine [430] The effect of colchicine on inhibiting mitosis by disrupting microtubules has been known for many years, and now the detailed mode of action is becoming clear. The structural protein of tubules, tubulin, has alpha and beta subunits as well as subtypes of the beta foma. Colchicine binds specifically to the beta subunit, and kinetics of the binding change

0"t30 / ~ ~ 0 Colchicine

~3


33

somewhat with the beta subtype [431,432]. Following binding there is local unfolding of the beta-subunit helix in the carboxyl terminal region around arginine 390 [433]. By studying binding of derivatives of the alkaloid the requirements for binding have been characterized. Methoxy, or other oxygen-based, functional groups at C-1, C-2, and C9 are required; and the acetamido group must be at C-7 [434, 435, 436]. Binding is temperature-dependent and occurs in at least two steps initially with the trimethoxybenzene ring and later with the tropolone ring [437, 438, 439]. The B-ring is important for immobilizing the A and C rings [440]. Kinetically a fast, reversible binding is observed, followed by a slow conformational change [441 ]. In the binding interaction the ring A of colchicine becomes juxtaposed to the alpha-beta subunit surface [442], but the alkaloid molecule is bound covalently to the beta-subunit, perhaps making a bridge between two different regions of this subunit [443]. In the tubulin molecule residue 316 is directly involved in binding the trimethoxyphenyl ring. Tubulins have a hydrophobic residue at this location, either valine or isoleucine [444]. Residues 214-241 are also involved [445]. Colchiceine, which differs from colchicine by having a hydroxyl group instead of methoxyl on ring C, also binds to tubulin and inhibits assembly of microtubules; but, strangely, the two alkaloids bind at different sites and do not compete with each other [446]. Besides colchicine, there are other alkaloids that disrupt microtubules. The most studied are the dimeric indole alkaloids vinblastine and vincristine. They also bind to the beta subunit but not at the same site as colchicine [447, 448, 449]. Vincristine has the highest affinity in this group [450]. The related, monomeric alkaloids vindoline and catharanthine bind much more weakly [451 ]. The vinblastine high affinity binding site is at residues 175-213, while colchicine is nearby at 214-241, as well as at other loci [445]. In addition, there is a low-affinity binding site for vinblastine [452]. There are about 1.5 binding sites for vinblastine per molecule of tubulin (mol. wt. ca. 110kDa) [453]. Modifications of the vinblastine structure have permitted some inferences about the requirements for binding [454]. Magnesium and guanosine triphosphate at less than millimolar concentration increase the rate and stability of binding [455, 456]. Rather than causing unfolding, vinblastine apparently induces oligomerization of the tubulin [457]. Maytansine binds at a tubulin site where guanine nucleotides exchange and causes unfolding [457, 458]. Nonalkaloidal inhibitors such as paclitaxel and macrolides bind at different sites from those used by the alkaloids, but binding of the alkaloids prevents binding ofpaclitaxel [459, 460]. There is litle information about alkaloids that may act on other elements of the cytoskeleton. Reserpine binds to G-actin but not to Factin [461]. Nicotine at 10 micromolar causes disassembly of actin filaments [462].

34

T. ROBINSON

MISCELLANEOUS CHANNEL TRANSPORT EFFECTS In previous sections the membrane transport of inorganic ions controlled by receptors to endogenous transmitters has been discussed. There are many effects of alkaloids on ion transport where the normal mechanism of control is obscure or where the effect of the alkaloid appears to be directly on an ion channel rather than on a controlling receptor. Some of these effects are gathered together in this section. Ryanodine barely rates as an alkaloid, although it is often called one. It is a complex diterpenoid whose only nitrogen atom is in an esterified proline group, and it shows no basic properties. Since there is no known endogenous transmitter, the system that is acted on by ryanodine is called the "ryanodine receptor"; and as with the opiate receptor, it will probably continue to be called that even if an endogenous transmitter substance is discovered [463]. Ryanodine receptors have been most thoroughly studied in skeletal muscle, although they are also present (with some differences) in other tissues. In skeletal muscle ryanodine acts on a calcium release channel in the sarcoplasmic reticulum to provide intracellular calcium ions required for muscle contraction. There is a high affinity site where binding locks the channel in an open position and a low affinity site where binding closes the channel at high concentrations of the alkaloid [464, 465]. In both cases calcium must be present to open the channel and allow ryanodine to bind [466]. Isolation of the receptor protein from skeletal muscle, cloning the DNA for it, and deducing the amino acid sequence have all been acomplished; so that much is known about the mechanisms involved [467, 468, 469, 470, 471,472]. Adding up the masses of the 5037 residues in the sequence gives a monomeric molecular mass of 565,223Da. The ion channel is made from a symmetrical arrangement of four of these units. The purified receptor has been inserted into artificial membranes and responds there to ryanodine just as it does in vivo [473, 474, 475]. The receptor molecules are arranged so that both the N- and C-terminals are on the cytoplasmic side of the sarcoplasmic reticulum vesicles [476]. Each monomer of the receptor has a binding site for ryanodine, and binding to only one of the sites is inhibitory [477]. Partial digestion has narrowed the binding site down to a 135kDa fragment [478]. Several structural analogues of ryanodine have been tested to clarify the structural requirements for binding. The pyrrole group controls the orientation of binding, but the large, hydrophobic terpenoid moiety is also important [479, 480]. Spermine increases both the rate and affinity of ryanodine binding to the receptor [481 ]. Caffeine antagonizes the action of ryanodine, acting on the same pool but stimulating calcium ion release. It does not act at the same receptor site; and once the site has been blocked by ryanodine, caffeine is unable to unblock it, although excess calcium ion can [477, 482, 483,484, 485]. Caffeine increases the affinity for calcium of the calcium activator site [486]. The above discussion applies to the action of ryanodine on


35

skeletal muscle. There are differences observed in heart muscle, smooth muscle, brain, adrenal glands, and liver [487, 488, 489, 490, 491]. The release of calcium by caffeine leads to further effects that are dependent on calcium ion for example the activation of ATPase and oxidative phosphorylation [492, 493]. Caffeine is apparently influential on other ion transport process that are not under the control of adenosine receptors or affected by ryanodine. It increases the concentration of free calcium ion in pancreatic beta cells and also inhibits potassium channels [494]. Sparteine has a similar effect on the same cells [495]. In another example caffeine stimulates release of calcium from intracellular stores in liver cells and is not competitive with ryanodine [496]. Senecionine acts similarly [497]. Conversely, caffeine inhibits a calcium channel in rat cerebellar microsomes [212]. Quinine and quinidine block potassium channels, both calcium independent ones and calcium-activated ones in several types of membranes [498, 499, 500]. Quinine also has less specific effects on other ion channels ~ chloride as well as cations [501]. Blocking of potassium channels has a secondary effect of stimulating synthesis of phosphatidylserine and affecting other processes that are controlled by potassium concentration [289, 502, 503]. The bisbenzylisoquinoline alkaloid dauricine seems to act similarly to quinidine [504]. Some effects of quinine and quinidine, rather than showing actions on specific ion channels may come from more general effects on membrane structure. Quinidine does decrease the fluidity of liver plasma membrane [505]; and it does interact with lipid bilayers [506]. Some other alkaloids may have specific effects on ion channels or ion tranporters. Tetrandrine and hernandezine inhibit calcium entry into human vascular cells [507, 504, 508]. Tetrandrine binds directly to calmodulin [509]. alpha-Solanine inhibits active transport of calcium in rat intestine [510]. Certain diterpenoid alkaloids inhibit calcium-activated potassium channels in aorta muscle [511]. Hirsutine blocks voltagedependent calcium influx in aorta muscle [512]. Yohimbine and berberine inhibit ATP-sensitive potassium channels [513, 514]. Crambescidin is a calcium channel blocker that inhibits the cholinergic contraction of ileum muscles [515]. In contrast to these inhibitors of ion transport, veratridine and homobatrachotoxin activate sodium and calcium channels in several cell types [516, 517, 518, 519, 520]. Veratridine binds both a high affinity site and a low affinity site on fast sodium channels [521]. The toxicity of veratridine may result from calcium ions passing in through an open sodium channel [522]. A secondary effect of increased calcium concentration is increased phosphorylation of tyrosine residues that then modify the activity of several enzymes [370]. Caffeine stimulates chloride ion efflux from epithelial cells, independent of its activity on adenosine receptors [523]. Agonists of the mu opioid receptor activate inward conductance of potassium ions [524]. Cocaine causes loss of magnesium

T. ROBINSON

36

and calcium from smooth muscle cells [525, 526], but it has also been found to block sodium channels [537]. Harmaline inhibits a sodium/iodide symporter in thyroid cells [528]. Aside from inorganic ions, amino acids and other small organic molecules have transport systems to move them across membranes. Human erythrocytes have two different amino acid transporters, and both of them are affected by harmaline, though in different ways [529]. Caffeine and theophylline both inhibit glucose uptake by insulin-treated adipocytes [5301. Other alkaloids that appear to have effects on ion chanels may, more accurately, be general disrupters of membrane structure. For example alpha-solanine and alpha-chaconine affect ion channels, but are wellestablished as having general effects on membranes [531,532]. ABBREVIATIONS ATP ADP GTP GDP cAMP DNA RNA GABA

= = = = = = = =

Adenosine triphosphate Adenosine diphosphate Guanosine triphosphate Guanosine diphosphate Cyclic adenosine monophosphate Deoxyribonucleic acid Ribonucleic acid gamma-Aminobutyric acid

REFERENCES

[1] [2] [31 [41 [5] [61 [7] [8] [91 [lO] [ii]

Guengerich, F. P. Amer. Scientist ,1993, 81, 440-447. Stanulovi'c, M.; Chaykin, S. Arch. Biochem. Biophys.,1971,145, 27-34, 35-42. Collins, A. C.; Cashaw, J. L.; Davis V. E. Biochem. Pharmacol., 1973, 22, 2337-2348. Collins, M. A; Cheng, B. Y. Arch. Biochem. Biophys.,1988, 263, 86-95. Haber, H.; Roske, I.; Rottmann, M.; Georgi, M.; Melzig, M. F. Life Sci.,1997, 60, 79- 89. Breyer-Pfaff, U.; Wiatr, G.; Stevens, I.; Gaertner, H. J.; Mundle, G.; Mann, K. Life Sci.,1996, 58, 1425-1432. Cashaw, J. L. J. Chromatog.,1993, 613, 267-273. Kajita, M; Niwa, T.; Maruyama, W.; Nakahara, D.; Takeda, N.; Yoshizumi, H.; Tatematsu, A.; Watanabe, K.; Naoi, M.; Nagatsu, T. J. Chromatog. B 1994, 654, 263-269. Tencheva, J.; Yamboliev, I; Zhivkova, Z. J. Chromatog., 1987, 421, 396-400. Agfindez, J. A. G.; Luengo, A.; Benitez, J. Drug Metab. Dispos., 1992, 29, 343349. Stavric, B. Food Chem. Toxic., 1988, 26, 645-662.

ALKALOIDSIN ANIMALS [12]

[13] [14] [15]

[16] [17] [18] [19] [20] [21] [22] [23]

[24] [25] [26] [27]

[28] [29] [30] [31] [32] [33] [34]

[35] [36] [37] [38] [39]

37

Mellini, D. W.; Caporaso, N. E.; Issaq, H. J. J. Liq. Chromatog., 1993, 16, 1419- 1426. Miceli, J. N; Chapman, W. J. Liq. Chromatog., 1990, 13, 2239-2251. Arnaud, M. J.; Zvi, Z. B.; Yaari, A.; Gorodischer, R. Res. Commun. Chem. Pathol. Pharm., 1986, 52, 407. Beach, C. A.; Mays, D. C.; Sterman, B. M.; Gerber, N. J. Pharmacol. Exp. Therap., 1985, 233, 18-23. Berthou, F.; Goasduff, T.; Dr6ano, Y.; M~nez, J.-F.Life Sci. 1995, 57, 541-549. Campbell, M. E.; Grant, D. M.; Inaba, T.; Kalow, W. Drug Metab. Dispos., 1987, 15, 237-249. Berthou, F; Ratanasavanh, D.; Riche, C.; Picart, D.; Voirin, T.; Guiliouzo, A. Xenobiotica 1989, 19, 401-417. Berthou, F; Flinois, J. P.; Ratanasavanh, D.; Beaune, P.; Riche, C.; Guillouzo, A. Drug Metab. Dispos., 1991, 19, 561-567. Fuhr, U; Doehmer, J.; Battula, N.; W61fel, C.; Kudla, C.; Keita, Y.; Staib, A. H. Biochem. Pharmacol., 1992, 43, 225-235. Roberts, E. A.; Furuya, K. N.; Tang, B. K.; Kalow, W. Biochem. Biophys. Res. Commun., 1994, 201, 559-566. Rasmussen, B. B.; Nielsen, K. K.; Brasen, K. Anal. Biochem., 1994, 222, 9-13. Zhang, Z.-Y.; Fasco, M. J.; Kaminsky, L. S. J. Chromatog. B 1995, 665, 201208. Grant, D. M.; Campbell, M. E.; Tang, B. K.; Kalow, W. Biochem. Pharmacol., 1987, 36, 1251-1260. Tassaneeyakul, W.; Birkett, D. J.; McManus, M. E.; Tassaneeyakul, W.; Verpnesr M. E.; Anersson, T.; Tukey, R. H.; Miners, J. O. Biochem. Pharmacol., 1994, 47, 1767-1776. Tjia, J. F.; Colbert, J.; Back, D. J. J'. Pharmacol. Expt. Therap., 1996, 276, 912-917. Konishi, H.; Morita, K.; Yamaji, A. Biol. Pharm. Bull. Tokyo, 1996, 19, 593598. Brazier, J. L.; Ribon, B.; Desagr M.; Salle, B. Biomed. Mass. Spectrom., 1980, 7, 189-192. Jeffcoat, A. R.; Perez-Reyes, M.; Hill, J. M.; Sadler, B. M.; Cook, C. E. Drug Metab. Dispos., 1989, 17, 153-159. Jindal, S. P; Lutz, T. J. Pharm. Sci., 1989, 78, 1009-1014. Zhang, J. Y; Foltz, R. L. ,/. Anal. Toxicol., 1990, 14, 201-205. Roberts, S. M.; Munson, J. W.; James, R. C.; Harbison, R. D. Anal. Biochem., 1992, 202, 256-26 I. Sukbuntherng, J.; Martin, D. K.; Pak, Y.; Mayersohn, M. d. Pharmaceut. Sci., 1996, 85, 567-571. Brzezinski, M. R.; Abraham, T. L.; Stone, C. L.; Dean, R. A.; Bosron, W. F. Biochem. Pharmacol.,1994, 8, 1747-1755. Dean, R. A.; Zhang, J.; Brzezinski, M. R.; Boston, W. F. J. Pharmacol. Expt. Therap., 1995, 275, 965-971. Gatley, S. J. Biochem. Pharmacol., 1991, 41, 1249-1254. Katz, J. L.; Terry, P.; Witkin, J. M. Life Sci., 1992, 50, 1351- 1361. Boyer, C. S; Petersen, D. R. J. PharmacoL Exper. Therap.,1992, 260, 939-946. Xu, Y.-Q.; Crumb, W. J.; Jr.; Clarkson, C. W. J. Pharmacol. Expt. Therap., 1994, 270, 319-325.

38

[40] [41]

[42] [43] [44] [45] [46] [47]

[48] [49]

[50] [51] [52] [531 [54]

[551 [56]

[57]

[58] [59]

[6o] [61]

[62] [63]

[64] [65] [66] [67] [68] [69]

T. ROBINSON

McCance, E. F.; Price, L. H.; Kosten, T. R.; Jatlow, P. I. d. Pharmacol. Expt. Therap., 1995, 274, 215-223. Landry, D. W.; Zhao, K.; Yang, G. X.-Q.; Glickman, M.; Georgiadis, T. M. Science, 1993, 259, 1899-1901. Dalvi, R. R; Jones, R. H. Current Sci., 1986, 55, 558-561. Boelsterli, U. A.; Lanzotti, A.; GSldlin, C.; Oertle, M. Drug Metab. Dispos., 1992, 29, 96-101. Pellinen, P; Honkakoski, P.; Stenback, F.; Niemitz, M.; Alhava, E.; Pelkonen, O.; Lang, M. A.; Pasanen, M. Europ. J. Pharmacol., 1994, 270, 35-43. Elaev, N. R.; Evdokimov, L. D.; Ryzhakov, A. V. Biochemistry USSR 1986, 51, 561-565. Van der Meer, M. J.; Hundt, H. K. L.; MUller, F. O. d. Pharm. Pharmacol., 1986, 38, 781-784. Werner, G; Brehmer, G. Z. Physiol. Chem.,1967, 348, 1640-1642. Werner, G; Schmidt, K. H. Z. Physiol. Chem.,1968, 349, 677-691. Tateishi, T.; Soucek, P.; Caraco, Y.; Guengerich, F. P.; Wood, A. J. J. Biochem. Pharmacol., 1997, 53, 11 l-116. Suh, D.-Y.; Kim, Y. C.; Kang, Y.-H.; Han, Y. N.; Han, B. H. J. Nat. Prod., 1997, 60, 265-269. Paoletti, C.; Lecointe, P.; Lesca, P.; Cros, S.; Mansuy, D.; Datxuong, N. Biochimie, 1978, 60, 1003-1009. Lecointe, P; Puget, A. C. R. Acad. Sci.; Ser. III 1983, 296, 279-282. Braham, Y.; Meunier, G.; Meunier, B. C. R. Acad. Sci. Set. III, 1987, 304, 30 l- 306. Douyette, A. Biomed. Environ. Mass Spec., 1988, 15, 243-247. Gouyette, A.; Auclair, C.; Paoletti, C. Biochem. Biophys. Res. Commun., 1985, 131, 614-619. Raisanen, M.J. Life Sci. 1984, 34, 2041-2045. Rosazza, J. P. N.; Duffel, M. W.; EI-Marakby, S.; Ahn, S. H. J. Nat. Prod., 1992, 55, 269-284. Elmarakby, S. A.; Duffel, M. W.; Rosazza, J. P. N. J. Med. Chem., 1989, 32, 2158- 2162. Le Verge, R; Le Con'e, P.; Chevanne, F.; De Maindreville, M. D.; Royer, D.; Levy, J. J. Chromatog., 1992, 574, 283-292. Oguri, K.; Tanimoto, Y.; Mishima, M.; Yoshimura, H. Xenobiotica, 1989, 19, 171- 178. Tweedie, D. J.; Prough, R. A.; Burke, M. D. Xenobiotica, 1988, 18, 785-796. Canfell, P. C.; Castagnoli, N.; Jr.; Fahey, M. R.; Hennis, P. J.; Miller, R. D. Drug Metab. Dispos., 1986, 14, 703-708. Naoi, M.; Matsuura, S.; Takahashi, T.; Nagatsu, T. Biochem. Biophys. Res. Commun., 1989, 161, 1213-1219. Sekine, Y.; Creveling, C.; Bell, M.; Brossi, A. Helv. Chim. Acta,1990, 73, 426432. He, X; Tadi'c, D.; Brzostowska, M.; Brossi, A.; Bell, M.; Creveling, C. Helv. Chim. Acta, 1991, 74, 1399-141 I. Benyhe, S. Life Sci., 1994, 55, 969-979. Liu, Y.; Bilfinger, T. V.; Stefano, G. B. Life Sci., 1997, 60, 237-243. Marrazzi, M. A.; Luby, E. D.; Kinzie, J.; Munjal, I. D.; Spector, S. Life ScL, 1997, 60, 1741-1747. Amann, T; Zenk, M. H. Tetrahedron Lett., 1991, 32, 3675-3678.

ALKALOIDSIN ANIMALS

[70] [71] [72] [73] [74] [75] [761 [77] [78] [79]

[8o1 [81] [82] [83] [84]

[85] [86] [87]

[88] [89] [90] [911 [92] [93] [94] [95] [96] [97]

39

Ishida, T.; Yano, M.; Toki, S. Drug Metab. Dispos., 1991, 19, 895-899. Xu, B. Q.; Aasmundstad, T. A.; Bjomeboe, A.; Christophersen, A. S.; M~rland, J. Biochem. Pharmacol., 1995, 49, 453-460. Krowech, G.; Caldera-Munoz, P. S.; Straub, K.; Castagnoli, N.; Jr.; Correia, M. A. Chem.-Biol. Interactions, 1986, 58, 29-40. N agamatsu, K; Hasegawa, A. Biochem. Pharmacol., 1992, 43, 2631-2635. Skoulis, N. P.; James, R. C.; Harbison, R. D.; Roberts, S. M. Toxicology, 1989, 5 7, 287-302. Chen, Z. R.; Irvine, R. J.; Bochner, F.; Somogyi, A. A. Life Sci., 1990, 446, 1067- 1074. Chen, Z. R.; Somogyi, A. A.; Bochner, F. Lancet, 1988, H, 914-915. Mikus, G.; Somogyi, A. A.; Bochner, F.; Eichelbaum, M. Biochem. Pharmacol., 1991, 41, 757-762. Dayer, P.; Desmeules, J.; Leemann, T.; Stribemi, R. Biochem. Biophys. Res. Commun., 1988, 152, 411-416. Pawula, M.; Shaw, P. N.; Barrett, D. A. J. Chromatog. B, 1994, 653, 106-111. Aasmundstad, T. A.; Ripel, A.; Bodd, E.; Bjomeboe, A.; M~rland, J. Biochem. Pharmacol., 1993, 46, 961-968. Rane, A.; Gawronska-Szklarz, B.; Svensson, J.-O. J. Pharmacol. Exp. Therap., 1985, 234, 761-765. Aasmundtstad, T. A.; Lillekjendlie, B.; Morland, J. Pharmacol. Toxicol., 1996, 79, 114-119. Coughtrie, M. W. H.; Ask, B.; Rane, A.; Burchell, B.; Hume. R. Biochem. Pharmacol., 1989, 38, 3273-3280. Puig, J. F; Tephly, T. R. Molec. Pharmacol., 1986, 30, 558-565. Kumagai, Y.; Todaka, T.; Toki, S. J. Pharmacol. Exper. Therap., 1990, 255, 504- 510 Persson, K.; LindstrSm, B.; Spalding, D.; WahlstrOm, A.; Rane, A. J. Chromatog., 1989, 491, 473-480. Petersen, D. R.; Norris, K. J.; Thompson, J. A. Drug Metab. Dispos., 1984, 12, 725-731. Langone, J. J.; Franke, J.; VanVanakis, H. Arch. Biochem. Biophys., 1974. 164. 536-543. Kyerematen, G. A.; Owens, G. F.; Chattopadhyay, J. D.; deBethizy, J. D.; Vesell, E. S. Drug Metab. Dispos., 1988, 16, 823-828. Jacob, P.; II|, Shulgin, A. T.; Yu, L.; Benowitz, N. L. J. Chromatog., 1992, 583, 145-154. Kyerematen, G. A.; Taylor, L. H.; deBethizy, J. D.; Vesell, E. S. J. Chromatog., 1987, 419, 191-203. Rop, P. P.; Grimaldi, F.; Oddoze, C.; Viala, A. J. Chromatog., 1993, 612, 302309. Seaton, M. J.; Veell. E. S.; Luo, H.; Hawes, E. M. J. Chromatog., 1993, 621, 49-53. Zuccaro, P; Altieri, I.; Rosa, M.; Passa, A. R.; Pichini, S.; Ricciarello, G.; Pacifici, R. J. Chromatog., 1993, 621, 257-261. Benowitz, N. L.; Jacob, P.; III, Fong, I.; Gupta, S. J. Pharmacol. Exp. Therap., 1994, 268, 296-303. Rtldell, U.; Foth, H.; Kahl, G. F. Biochem. Biophys. Res. Commun., 1987, 148, 192-198. McCoy, G. D; DeMarco, G. J.Biochem. Pharmacol., 1986, 35, 4590-4592.

40

1". ROBINSON

[98]

McCoy, G. D.; DeMarco, G. J.; Koop, D. R.Biochem. Pharmacol., 1989, 38,

[99]

Nwosu, C. G; Crooks, P. A. Xenobiotica, 1988, 18, 1361-1372. Jacob, P.; III, Benowitz, N. L.; Copeland, J. R.; Risner, M. E.; Cone, E. J. J. Pharm. Sci., 1988, 77, 396-400. Pool, W. F.; Houdi, A. A.; Damani, L. A.; Layton, W. J.; Crooks, P. A. Drug Metab. Dispos., 1986, 14, 574-579. Nakayama, H.; Okuda, H.; Nakashima, T.; lmaoka, S.; Funae, Y. Biochem. Pharmacol., 1993, 5, 2554-2556. Peterson, L. A.; Trevor, A.; Castagnoli, N.; Jr. J. Med. Chem., 1987, 30, 249254. Obach, R. S; Van Vunakis, H. Biochem. Pharmacol., 1990, 39, R1-R4. Damani, L. A.; Pool, W. F.; Crooks, P. A.; Kaderlik, R. K.; Ziegler, D. M. Molec. Pharmacol., 1988, 33, 702-705. Williams, D. E.; Shigenaga, M. K.; Castagnoli, N.; Jr. Drug Metab. Dispos., 1990, 18, 418-428. Crooks, P. A; Godin, C. S. J. Pharm. Pharmacol., 1988, 40, 153-154. Oldendorf, W. H.; Stoller, B. E.; Harris, F. L. Proc. Nat. Acad. Sci. U. S., 1993, 90, 307-311. Patterson, T. A; Kosh, J. W. Gen. Pharmacol., 1993, 24, 641-647. Winter, C. K.; Segall, H. J.; Jones, A. D. Biomed. Environ. Mass Spec., 1988, 15, 265-273. Mattocks, A. R; Driver, H. E. Chem.-Biol. Interactions, 1987, 63, 91-104. Lam6, M. W.; Jones, A. D.; Morin, D.; Segall, H. J. Drug Metab. Dispos., 1991, 19, 516-524. Couet, C. E.; Hopley, J.; Hanley, A. B. Toxicon, 1996, 34, 1058-1061. Mattocks, A. R.; Jukes, R. Chem. Biol Interactions, 1990, 75, 225-239. Nigra, L; Huxtable, R. J. Toxicon, 1992, 30, 1195-1202. Mattocks, A. R.; Croswell, S.; Jukes, R.; Huxtable, R. J. Toxicon, 1991, 29, 409- 415. Niwa, H.; Ogawa, T.; Okamoto, O.; Yamada, K. Tetrahedron Lett., 1991, 32, 927- 930. Yan, C. C; Huxtable, R. J. Biochem. Pharmacol., 1996, 51, 321-329, 375-379. Williams, D. E.; Reed, R. L.; Kedzierski, B.; Ziegler, D. M.; Buhler, D. R. Drug Metab. Dispos., 1989, 17, 380-386. Miranda, C; Chung, W.; Reed, R. E.; Zhao, X.; Henderson, M. C.; Wang, J.L.; Williams, D. E.; Buhler, D. R. Biochem. Biophys. Res. Commun., 1991, 178, 546-552. Nishida, R.; Kim, C.; Fukami, H.; Irie, R. Agric. Biol. Chem. Tokyo, 1991, 55, 1787-1792. Nickisch-Rosenegk, E. v.; Schneider, D.; Wink, M. Z. Naturforsch., 1990, 45c, 881-894. Klier, B.; Eilert, U.; Schimmer, O. Planta Med., 1991, 57, A I03. Hoyer, G. L.; Clawson, D. C.; Brookshier, L. A.; Nolan, P. E.; Marcus, F. I. J. Chromatog., 1991, 572, 159-169. Guengerich, F. P. J. Med. Chem., 1984, 27, 1101-1103. Horai, Y; lshizaki, T.; Eichelbaum, M.; Hashimoto, K.; Chiba, K.; Dengler, H.J. Xenobiotica, 1988, 18, 1077-1084. Lennard, M. S. Pharmacol. Toxicol., 1990, 67, 273-283. Pospisil, J.; Patzelova, V.; M,'tca, B. Drug Metab. Dispos., 1992, 29, 330-332.

1185- 1188.

[~001 [loi]

[~02] [103]

[104] [1051 [106]

[107] [IOS] [109] [110]

[Ill] [112] [ll3] Ill4] [115] Ill6] [ll7] [ll8] [119] [120] [121] [122] [123] [124]

[125] [126] [127] [128]

ALKALOIDSIN ANIMALS

41

[129] Ebner, T.; Eichelbaum, M.; Fischer, P.; Meese, C. O. Arch. Pharm., 1989, 322, 399-403. [130] Mathys, K; Brenneisen, R..1. Chromatog., 1992, 593, 79-85. Watanabe, K.; Kayano, Y.; Matsunaga, T.; Yamamoto, I.; Yoshimura, H. Biol. Pharm. Bull. Tokyo, 1995, 18, 696-699. [1321 Rein, R.; Golombek, A., Eds. Computer-Assisted Modeling of Receptor-Ligand Interactions, A. R. Liss: New York, 1989. [133] Blum, M.; Ed. The Toxic Action of Marine and Terrestrial Alkaloids, Alaken: Fort Collins, CO1995. [134] Schmeller, T.; Latz-Br0ning, B.; Wink, M. Phytochemistry, 1997, 44, 257-266. [135] Alkadhi, K. A. Europ. J. Pharmacol., 1987, 138, 257-264. [136] Rand, M. J; Thurau, K., Eds. The Pharmacology of Nicotine, Oxford U. Press,1988. [137] Badio, B.; Shi, D.; Shin, Y.; Hutchinson, K. D.; Padgett, W. L.; Daly, J. W. Biochem. Pharmacol., 1996, 52, 933-939. [138] Brioni, J. D.; Kim, D. J. B.; O'Neill, A. B. Europ. J. Pharmacol., 1996, 301, 1-5. [139] Flores, C. M.; Rogers, S. W.; Pabreza, L. A.; Wolfe, B. B.; Kellar, K. J. Molec. Pharmacol., 1992, 41, 31-37. [140] Pedersen, S. E; Cohen, J. B. Proc. Nat. Acad Sci. U. S., 1990, 87, 2785-2789. [141] Martino-Barrows, A. M; Kellar, K. J. Molec. Pharmacol., 1987, 31, 169-174. [142] Rapier, C.; Lunt, G. G.; Wonnacott, S.J. Neurochem., 1988, 50, 1123-1130. [143] Rozental, R; Aracava, Y.; Scoble, G. T.; Swanson, K. L.; Wonnacott, S.; Albuquerque, E. X..1. Pharmacol. Exp. Therap., 1989, 251, 395-404. [144] Funayama, N.; Shinkai, M.; Takayanagi, I. Gen. Pharmacol., 1995, 26, 977981. [145] Barlow, R. B; Johnson, O. Brit. J. Pharmacol., 1989, 98, 799-808. [146] Flesher, J. E.; Scheffel, U.; London, E. D.; Frost, J. J. Life Sci. 1994, 54, 18831890. [147] Wolf-Pflugmann, M.; Lambrecht, G.; Wess, J.; Mutschler, E. Arzneim.-Forsch., 1989, 39, 539-544. [148] Okonjo, K. O.; Kuhlmann, J.; Maelicke, A. Europ. J. Biochem., 1991, 200, 671677. [149] Galzi, J.-L.; Revah, F.; Bessis, A.; Changeux, J.-P. Ann. Rev. Pharmacol. Toxicol., 1991, 31, 37-72. [150] Pedersen, S. E; Papineni, R. V. L..1. Biol. Chem., 1996, 270, 31141-31150. [151] Banerjee, B; Ganguly, D. K. Europ. J. Pharmacol., 1996, 310, 13-17. [~52] Lentz, T. Biochemistry, 1995, 34, 1316-1322. [153] Middleton, R. E; Cohen, J. B. Biochemistry, 1991, 30, 6987-6997. [154] Manners, G. D.; Panter, K. E.; Pelletier, S. W..I. Nat. Prod, 1995, 58, 863869. [155] Jennings, K. R.; Brown, D. G.; Wright, D. P.; Jr. Experientia, 1986, 42, 611613. [156] Mori, T.; Murayama, M.; Bando, H.; Kawahara, N. Chem. Pharm. Bull. Tokyo, 1991, 39, 379-383. [157] Voitenko, S; Purnin, S.; Omel'chenko, I.; Dyadyusha, G. G.; Zhorov, B.; Brovtsina, N.; Skok, V. Molec. Pharmacol., 1991, 40, 180-185. [~58] Fisher, M.; Huangfu, D.; Shen, T. Y.; Guyenet, P. G..1. Pharmacol. Expt. Therap., 1994, 270, 702-707. [159] Lerner-Marmarosh, N.; Carroll, F. I.; Abood, L. G. 1995, Life ScL 56, PL67-70.

42

T. ROBINSON

[160] Dar, D. E; Zinder, O. Naunyn-Schmiedeberg's Arch. Pharmacol., 1995, 352, 1116. [161] Storch, A; 13 Others Europ. J. Pharmacol., 1995, 290, 207-219. [162] Crooks, P. A; Dwoskin, L. P. Biochem. Pharmacol., 1997, 54, 743-753. [163] Schneider, A. S.; Nagel, J. E.; Mah, S. J. Europ. J. Pharmacol., 1996, 317, R IR2. [~64] Benwell, M. E. M.; Holtom, P. E.; Moran, R. J.; Balfour, D. J. K. Brit. 3,. Pharmacol., 1996, 117, 743-749. [165] Kuijpers, G. A. J.; Vegara, L. A.; Calvo, S.; Yadid, G. Brit..I. Pharmacol., 1994, 113, 471-478. [166] Hulme, E. C.; Kurtenbach, E.; Curtis, C. A. M. Biochem. Soc. Trans.., 1991, 9, 133-138. [167] Richards, M. H. Biochem. Pharmacol., 1991, 42, 1645-1653. [168] Brann, M. R.; Klimkowski, V. J.; Ellis, J. Life Sci., 1993, 52, 405-412. [169] Burgen, A. S. V. Life Sci., 1995, 56, 801-806. [170] Ross, E. M. 3'. Chem. Educ., 1988, 65, 937-942. [171] Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Ravard, A.; Deo, N.; Crooks, P. A. Europ. ,I. Pharmacol., 1995, 276, 195-199. [172] Forsberg, E. J.; Rojas, E.; Pollard, H. B. J. Biol. Chem., 1986, 261, 4915-4920. [173] Drake, M. V.; O'Donnell, J. J.; Polansky, J. R. J. Pharm. Sci., 1986, 75, 278279. [174] Eglen, R. M; Harris, G. C.; Cox, H.; Sullivan, A. O.; Stefanich, E.; Whiting, R. L. Naunyn-Schmiedeberg's Arch. Pharmacol., 1992, 346, 144-151. [175] Gilani, A. H.; Shaheen, F.; Christopoulos, A.; Mitchelson, F. Life Sci., 1997, 60, 535-544. [1761 Waelbroeck, M.; De Neef, P.; Roberecht, P.; Christophe, J. Life Sci., 1984, 35, 1069-1076. [177] Kurachi, Y.; Nakajima, T.; Sugimoto, T. Naunyn-Schmiedeberg's Arch. Pharmacol., 1987, 335, 216-218. [178] Mack, J. E; Matthews, J. C. Biochem. Pharmacol., 1987, 36, 991-994. [179] Sharkey, J.; Ritz, M. C.; Schenden, J. A.; Hanson, R. C.; Kuhar, M. J. J. Pharmacol. Exp. Therap., 1988, 246, 1048-1052. [180] Smith, T. D.; Annis, S. J.; Ehlert, F. J.; Leslie, F. M. J. Pharmacol. Exp. Therap., 1991, 256, 1173-1181. [~81] Schmeller, T.; Sporer, F.; Sauerwein, M.; Wink, M. Pharmazie, 1995, 50, 493495. [182] Matesic, D. F; Luthin, G. R. FEBS Lett., 1991, 284, 184-186. [183] Xu, H; Gintzler, A. R. Proc. Nat. Acad. Sci. U. S., 1992, 89, 1978-1982. [184] Zimanyi, I.; Lajtha, A.; Vizi, E. S.; Reith, M. E. A. Biochem. Pharmacol., 1988, 3 7, 641-645.

[185] Venter, J. C.; Fraser, C. M.; Kerlavage, A. R.; Buck, M. A. Biochem. Pharmacol., 1989, 38, 1197-1208. [186] Harrison, J. K.; Pearson, W. R.; Lynch, K. R. Trends Pharmacol. Sci., 1991, 12, 62-67. [187] Bylund, D. B; Eikenberg, D. C.; Hieble, J. P.; Langer, S. Z.; Lefkowitz, R. J.; Minneman, K. P.; Molinoff, P. B.; Ruffolo, R. R., Jr.; Trendelenburg. U. Pharmacol. Rev., 1994, 6, 121-136. [~88] Levitzki, A. Science, 1988, 241, 800-806. [189] Kobilka, B. K; Kobilka, T. S.; Daniel, K.; Regan, J. W.; Caron, M. G.; Lefkowitz, R. J. Science, 1988, 240, 1310-1316.


43

[190] Strosberg, A. D. Protein Sci., 1993, 2, 1198-1209. [1911 Convents, A.; Convents, D.; De Backer, J.-P.; De Keyser, J.; Vauquelin, G. Biochem. Pharmacol., 1989, 38, 455-463. [1921 Yu, S.-M; Ko, F. N.; Chueh, S. C.; Chen, J.; Chen, S. C.; Chen, C. C.; Teng, C. M. Europ. J. Pharmacol., 1994, 252, 29-34. [1931 Chuli~i S.; Noguera, M. A.; Ivorra, M. D.; Cortes, D.; D-Oc6n, M. P. Pharmacology, 1995, 50, 380-387. [1941 Chuli6, S.; Moreau, J.; Naline, E.; Noguera, M. A.; Ivorra, M. D.; D'Oc6n, M. P.; Advenier, C. Brit. J. Pharmacol., 1996, 119, 1305-1312. [195] lto, Y.; Yano, S.; Watanabe, K.; Yamanaka, E.; Aimi, N.; Sakai, S. Chem. Pharm. Bull Tokyo, 1990, 38, 1702-1706. [1961 Hui, K. K.; Yu, J. L.; Chan, W. F. A.; Tse, E. Life ScL 1991, 49, 315-324. [1971 Teng, C.-M; Yu, S. M.; Ko, F. N.; Chen, C. C.; Huang, Y. L.; Huang, T. F. Brit. J. Pharmacol., 1991, 104, 651-656. [198] Kwan, C. Y.; Chen, Y. Y.; Ma, M. F.; Daniel, E. E.; Hui, S. C. G. Life Sci. 1996, 59, PL359-364. [1991 Madden, J. A.; Konkol, R. J.;Keller, P. A.; Alvarez, T. A. Life Sci., 1995, 56, 679-686. [2001 Burnstock, G. Nucleosides & Nucleotides, 1991, 10, 917-930. [2011 Stone, T. W. Gen. Pharmacol., 1991, 22, 25-31. [202] Fredholm, B. B; Abbracchio, M. P.; Bumstock, G.; Daly, J. W.; Harden, T. K.; Jacobson, K. A.; Left, P.; Williams, M. Pharmacol. Rev., 1994, 46, 143-156. [203] Palmer, T. M; Stiles, G.L. Neuropharmacology, 1995, 34, 683-694. [2041 Clark, M; Post, R. M. Europ. d. Pharmacol., 1989, 164, 399-401. [2051 Sebasti~io, A. M; Ribeiro, J. A. Brit. J. Pharmacol., 1989, 96, 211-219. [206] Peet, N. P; Lentz, N. L.; Meng, E. C.; Dudley, M. W.; Ogden, A. M. L.; Demeter, D. A.; Weintraub, H. J. R.; Bey, P. d. Med. Chem., 1990, 33, 31273130. [207] Biaggioni, I.; Paul, S.; Puckett, A.; Arzubiaga, C. d. Pharmacol. Exp. Therap., 1991, 258, 588-593. [208] Rainnie, D. G.; Grunze, H. C. R.; McCarley, R. W.; Greene, R. W. Science, 1994, 263, 689-692. [209] Cooper, R. A; De Freitas, C.; Porreca, F., Eisenhour, C. M.; Lukas, R.; Huxtable, R. J. Toxicon, 1995, 33, 1025-1031. [2]o] Stiles, G. L. d. Biol. Chem., 1992, 267, 6451-6454. [2111 Ammon, H. P. T. Arch. Pharm., 1991, 324, 261-267. [212] Brown, G. R.; Sayers, L. G.; Kirk, C. J.; Michell, R. H.; Michelangeli, F. Biochem. J., 1992, 282, 309-312. [2131 Hirose, K.; lino, M; Endo, M. Biochem. Biophys. Res. Commun., 1993, 194, 726- 732. [214] Carter, A. J.; O'Connor, W. T.; Carter, M. J.; Ungerstedt, U. d. Pharmacol. Expt. Therap., 1995, 273, 637-642. [215] Porkka-Heiskanen, T; Strecker, R. E.; Thakar, M.; BjBrkum, A. A.; Greene, R. W.; McCarley, R. W.Science, 1997, 276, 1265-1268. [216] Shih, J. C.; Yang, W.; Chen, K.; Gallaher, T. Pharmacol. Biochem. Behavior, 1991, 40, 1053-1058,. [217] Hoyer, D; Clarke, D. E.; Fozard, J. R.; Hartig, P. R.; Martin, G. R.; Mylecharane, E. J.; Saxena, P. R.; Humphrey, P. P. A. Pharmacol. Rev., 1994, 46, 157-203. [218] Martin, G. R; Humphrey, P. P. A. Neuropharmacology, 1994, 33, 261-273.

44

T. ROBINSON

[219] Boess, F. G; Martin, I. L. Neuropharmacology, 1994, 33, 275-317. [220] Boess, F. G.; Monsma, F. J.; Jr.; Meyer, V.; Zwingelstein, C.; Sleight, A. J. Molec. Pharmacol., 1997, 52, 515-523. [221] Dyer, D. C. Life Sci., 1993, 53, PL223-PL228. [222] Eich, E.; Pertz, H. Pharmazie, 1994, 49, 867-877. [223] Ward, J. S; 6 Others ,I. Med. Chem., 1988, 31, 1512-1519. [224] Kidric, J.; Kocjan, D.; Hadzi, D. Experientia, 1986, 42, 327-328. [225] De Vos, H.; Czerwiec, E.; De Backer, J.-P.; De Potter, W.; Vauquelin, G. Europ. ,I. Pharmacol., 1991, 207, 1-8. [226] Winter, J. C; Rabin, R. A. ,I. Pharmacol. Exp. Therap., 1992, 263, 682-689. [227] Arthur, J. M.; Casaftas, S. J.; Raymond, J. R. Biochem. Pharmacol., 1993, 45, 2337-2341. [228] Shoji, N; Umeyama, A.;Saito, N.; luchi, A.; Takemoto, T.; Kajiwara, A.; Ohizumi, Y. J. Nat. Prod., 1987, 50, 773-774. [229] Uchimura, N; North, R. A. Brit. ,I. Pharmacol., 1990, 99, 736-740. [230] Hoffman B. J.; Mezey, E.; Brownstein, M. J. Science, 1991, 254, 578-579. [231] Hoffman, B. J.; Mezey, E.; Brownstein, M. J. Science, 1991, 254, 579-580. [232] Erickson, J. D.; Eiden, L. E.; Hoffman, B. J. Proc. Nat. ,4cad. Sci. U. S., 1992, 89, 10993-10997. [233] Izenwasser, S.; Rosenberger, J. G.; Cox, B. M. Life Sci., 1992, 50, 541-547. [234] Brossi, A. Planta Med., 1991, 57, $93-S100. [235] d'Ischia, M.; Constantini, C.; Prota, G. Tetrahedron Lett., 1993, 34, 3921-3924. [236] Baum, S. S; Rommelspacher, H. J. Chromatog. B 1994, 660, 235-241. [237] Caine, S. B; Koob, G. F. Science, 1993, 260, 1814-1816. [238] Civelli, O.; Bunzow, J. R.; Grandy, D. K.; Zhou, Q.-Y.; Van Tol, H. H. M. Europ. J. Pharmacol., 1991, 207, 277-286. [239] Civelli, O.; Bunzow, J. R.; Grandy, D. K. Ann. Rev. Pharmacol. Toxicol., 1993, 33, 281-307. [24o] Gingrich, J. A.; Jarvie, K.; Tiberi, M.; Fremeau, R. T.; Jr.; Caron, M. G. Dupont Biotech Update 1992, 7, 105, 108, 112, 118. [241] Blum, K.; Cull, J. G.; Braverman, E. R.; Comings, D. E. Am. Scientist, 1996, 84, 132-145. [242l Cortes, D.; Figadere, B.; Saez, J.; Protais, P. J. Nat. Prod., 1992, 55, 12811286. [2431 Guo-Zhang, J. Trends Pharmacol. Sci., 1987, 8, 81-82. [244] Chueh, F. Y.; Hsieh, M. T.; Chen, C. F.; Lin, M. T. Pharmacology, 1995, 51, 237-244. [2451 Schaus, J. M.; Titus, R. D.; Foreman, M. M.; Mason, N. R.; Truex, L. L. J. Med. Chem., 1990, 33, 600-607. [246] Spano, P. F; 7 Others Aging 23, Aging Brain and Ergot Alkaloids, Raven Press: NY,1983. [247] Schuldiner, S.; Liu, Y.; Edwards, R. H. J. Biol. Chem., 1993, 268, 29-34. [248] Giros, B; Wang, Y. M.; Suter, S.; McLeskey, S. B.; Pifl, C.; Caron, M. G. J. Biol. Chem., 1994, 269, 15985-15988. [249] Shimada, S.; Kitayama, S.; Lin, C.-L.; Patel, A.; Nanthakumar, E.; Gregor, P.; Kuhar, M.; Uhl, G. Science, 1991, 254, 576-578. [250] Reith, M. E. A.; Xu, C.; Chen, N.-H. Europ. J. Pharmacol., 1997, 324, 1-10. [251] Usdin, T. B.; Mezey, E.; Chen, C.; Brownstein, M. J.; Hoffman, B. J. Proc. Nat. Acad. Sci. U. S., 1991, 88, 11168-11171. [2521 Gawin, F. H. Science, 1991, 251, 1580-1586.

ALKALOIDSIN ANIMALS

45

[253] Faraj, B.A.; Olkowski, Z. L.; Jackson, R. T. Biochem. Pharmacol., 1995, 50, 1007-1014. [254] Imperato, A.; Obinu, M. C.; Demontis, M. V.; Gessa, G. L. Europ. J. Pharmacol., 1992, 229, 265-267. [255] Izenwasser, S.; Newman, A. H.; Katz, J. L. Europ. J. Pharmacol., 1993, 243, 201-205. [256] Self, D. W.; Barnhart, W.; Lehman, D. A.; Nestler, E. J. Science, 1996, 271, 1586-1589. [257] Carroll, F. I; Lewin, A. H.; Abraham, P.; Parham, K.; Boja, J. W.; Kuhar, M. J. J. Med. Chem., 1991, 34, 883-886. [258] H6ron, C.; Costentin, J.; Bonnet, J.-J. Europ. J. Pharmacol., 1994, 264, 391398. [259] Reith, M. E. A.; de Costa, B.; Rice, K. C.; Jacobson, A. E. Europ. J. Pharmacol., 1992, 22 7, 417-425. [260] Ritz, M. C.; Mantione, C. R.; London, E. D. Psychopharmacology, 1994, 114, 47-52. [261] Saadouni, S.; Refahi-Lyamani, F.; Costentin, J.; Bonnet, J.-J. Europ. J. Pharmacol., 1994, 268, 187-197. [2621 Cao, C. J.; Shamoo, A. E.;; Eldefrawi, M. E. Biochem. Pharmacol., 1990, 39, R9- RI4. [263] Rudnick, G.; Steiner-Mordoch, S. S.; Fishkes, H.; Stem-Bach, Y.; Schuldiner, S. Biochemistry, 1990, 29, 603-608. [264] Kalix, P. Neuropharmacology, 1986, 25, 499-501. [265] Dwoskin, L. P.; Leibee, L. L.; Jewell, A. L.; Fang, Z.-X.; Crooks, P. A. Life Sci., 1992, 50, PL233-PL237. [266] Yamashita, H; Kitayama, S.; Zhang, Y.-X.; Takahashi, T.; Dohi, T.; Nakamura, S. Biochem. Pharmacol., 1995, 49, 742-745. [267] Pontieri, F. E.; Tanda, G.; Orzi, F.; Di Chiara, G. Nature, 1996, 382, 255-257. [268] Nakamura, S.; Goshima, Y.; Yue, J.-L.; Miyamae, T.; Misu, Y. Europ. J. Pharmacol., 1992, 222, 75-80. [269] Epple, A.; Nibbio, B.; Spector, S.; Brinn, J. E. Life Sci., 1994, 54, 695-702. [270] Horfik, P.; Haberman, F.; Spector, S. Life Sci., 1993, 52, PL255-PL260. [271] Mignat, C.; Wille, U.; Ziegler, A. Life Sci., 1995, 56, 793-799. [272] Meng, F; Xie, G. X.; Thompson, R. C.; Mansour, A.; Goldstein, A.; Watson, S. J.; Akil, H. Proc. Nat. Acad. Sci. U. S., 1993, 90, 9954-9958. [273] Miyamae, T.; Fukushima, N.; Misu, Y.; Ueda, H. FEBS Lett., 1993, 333, 311314. [274] Ocafla, M.; Del Pozo, E.; Barrios, M.; Baeyens, J. M. Brit. J. Pharmacol., 1995, 114, 1296-1302. [275] Gomathi, K. G; Sharma, S. K. FEBS Lett., 1993, 330, 146-150. [276] Zhu, J.; Chen, C.; Xue, J.-C.; Kunapuli, S.; DeRiel, J. K.; Liu-Chen, L.-Y. Life Sci., 1995, 56, PL201-207. [277] Kristensen, K.; Christensen, C. B.; Christrup, L. L.Life Sci., 1995, 56, PL4550. [278] Young, A. M. J; Key, B. J. Neuropharmacology, 1984, 23, 1347-1350. [279] Adams, R. E; Wooten, G. F. Neurochem. Res., 1993, 18, 1041-1045. [2so] Herz, A. Shippenberg, T. S.; Bals-Kubik, R.; Spanagei, R. Arzneim. Forsch., 1992, 42-1,256-259. [2811 Cabot, P. J.; Dodd, P. R.; Cramond, T.; Smith, M. T. Europ. J. Pharmacol., 1994, 268, 247-255.

46

T. ROBINSON

[2821 Spangler, R.; Ho, A.; Zhou, Y.; Maggos, C. E.; Yuferov, V.; Kreek, M. J. Molec. Brain Res., 1996, 38, 71-76. [2831 Lipkowski, A. W.; Can', D. B.; Langlade, A.; Osgood, P. F.; Szyfelbein, S. K. Life Sci., 1994, 55, 149-154. [284] Bartlett, S. E; Smith, M. T. Life Sci., 1995, 57, 609-615. [285] Huwyler, J.; Drewe, J.; Klusemann, C.; Fricker, G. Brit. J. Pharmacol., 1996, 118, 1879-1885. [286] Airaksinen, M. M.; Saano, V.; Steidel, E.; Juvonen, H.; Huhtikangas, A.; Gynther, J. Acta Pharmacol. Toxicol., 1984, 55, 380-385. [2871 Bowen, W. D.; Vilner, B. J.; Williams, W.; Bertha, C. M.; Kuehne, M. E; Jacobson, A. E. Europ. ,I. Pharmacol., 1995, 279, R l-R3. [288] Watanabe, K.; Yano, S.; Horie, S.; Yamamoto, L. T. Life Sci., 1997, 60, 933942. [2891 Tang, R.; Novas, M. L.; Glavinovir M. I.; Trifaro, J.-M. Brit. J. Pharmacol., 1990, 99, 548-552. [290] Pang, I.-H; Vasko, M. R. Brain Research, 1986, 376, 268-279. [291] Payza, K.; Akar, C. A.; Yang, H.-Y. T. J. Pharmacol. Exp. Therap., 1993, 267, 88- 94. [292] Pellegrini-Giampietro, D. E.; Ruggiero, M.; Gianelli, S.; Chiarugi, V. P.; Moroni, F. Europ. J. Pharmacol., 1988, 149, 297-306. [293] Sergeeva, M. G.; Terentjeva, I. V.; Mevkh, A. T.; Varfolomeev, S. D. FEBS Lett., 1993, 323, 163-165. [294] Hancock, D. L; Coupar, I. M. Gen. Pharmacol., 1997, 28, 709-713. [2951 Harrison, B. L.; Baron, B. M.; Cousino, D. M.; McDonald, I. A. J. Med. Chem., 1990, 33, 3130-3132. [296] McDonald, J. W.; Penney, J. B.; Johnston, M. V.; Young, A. B. Neuroscience, 1990, 35, 653-668. [2971 Bristow, D. R.; Bowery, N. G.; Woodruff, G. N. Europ. J. Pharmacol., 1986, 126, 303-307. [298] Yadid, G.; Maor, G.; Youdim, M. B. H.; Silberman, M.; Zinder, O. Neurochem. Res., 1993, 18, 1051-I055. [299] Grenningloh, G; Pienitz, A.; Schmitt, B.; Methfessel, C.; Zensen, M.; Beyreuther, K.; Gundelfinger, E. D.; Betz, H. Nature, 1988, 328, 215-220. [3001 Ruiz-G6mez, A.; Morato, E.; GarcIa-Calvo, M.; Valdivieso, F.; Mayor, F.; Jr. Biochemistry, 1990, 29, 7033-7040. [3011 Marviz6n, J. C. G; Vfizquez, J.; Calvo, M. G.; Mayor, F., Jr.; G6mez, A. R.; Valdivieso, F.; Benavides, J. Molec. Pharmacol., 1986, 30, 590-597. [302] Marvizon, J. C. G; Skolnick, P. Molec. Pharmacol., 1988, 34, 806-813. [303] Leewanich, P.; Tohda, M.; Matsumoto, K.; Subhadhirasakul, S.; Takayama, H.; Aimi, N.; Watanabe, H. Europ. J. Pharmacol., 1997, 332, 321-326. [3041 Ikegami, F.; Kusama-Eguehi, K.; Sugiyama, E.; Watanabe, K.; Lambein, F.; Murakoshi, I. Biol. Pharm. Bull. Tokyo, 1995, 18, 360-362. [305] Popik, P.; Layer, R. T.; Skolnick, P. Pharmacol. Rev., 1995, 47, 235-253. [306] Layer, R. T.; Skolnick, P.; Bertha, C. M.; Bandarage, U. K.; Kuehne, M. E.; Popik, P. Europ. d. Pharmacol., 1996, 309, 159-165. [307] Carlezon, W. A.; Jr; Boundy, V. A.; Haile, C. N.; Lane, S. B.; Kalb, R. G.; Neve, R. L.; Nestler, E. J. Science, 1997, 277, 812-814. [308] Villar, H. O.; Davies, M. F.; Loew, G. H.; Maguire, P. A. Life Sci., 1991, 48, 593- 602.

ALKALOIDSIN ANIMALS

47

[309] Barolet, A. W.; Li, A.; Liske, S.; Morris, M. E. Can. J. Physiol. Pharmacol., 1985, 63, 1465-1470. [3101 Bartolini, A.; Giotti, A.; Giuliani, S.; Malmberg-Aiello, P.; Patacchini, R. Gen. Pharmacol., 1990, I, 277-284. [311] Beutler, J. A; Karbon, E. W.; Brubaker, E. M.; Malik, R.; Curtis, D. R.; Enna, S. J. Brain Research, 1985, 330, 135-140. [312] Kardos, J.; BlaskJ, G.; Kerekes, P.; Kovacs, I.; Simonyi, M. Biochem. PharmacoL, 1984, 3, 3537-3545. [3131 Jacquet, Y. F.; Saederup, E.; Squires, R. F. Europ. d. Pharmacol., 1987, 138, 285- 288. [314] Uneyama, H.; Harata, N.; Akaike, N. Brit. J. Pharmacol., 1993, 109, 459-465. [315] Barrow, C. J; Sun, H. H. d. Nat. Prod, 1994, 57, 471-476. [316] Granger, I.; Serradeil-le-Gal, C.; Augereau, J. M.; Gleye, J. Planta Med., 1992, 58, 35-38. [317] Gu~ritte-Voegelein, F; Sevenet, T.; Pusset, J.; Adeline, M.-T.; Gillet, B.; Beloeil, J.-C.; Gu6nard, D.; Potier, P. J. Nat. Prod, 1992, 55, 923-930. [318] Rasolonjanahary, R.; S6venet, T.; Voegelein, F. G.; Kordon. C. Europ. J. Pharmacol., 1995, 285, 19-23. [319] Karhap/i/t L; TSrnquist, K. Biochem. Biophys. Res. Commun. 1995, 210, 726732. [320] Debnath, D.; Kumar, G. S.; Nandi, R.; Maiti, M. Ind. J. Biochem. Biophys., 1989, 26, 201-208. [321] Zegar, I.; Gr/islund, A.; Bergman, J.; Eriksson, M.; Nord6n, B. Chem.-Biol. Interactions, 1989, 72, 277-293. [3221 Kumar, G. S.; Debnath, D.; Sen, A.; Maiti, M. Biochem. Pharmacol., 1993, 46, 1665-1667. [323] Malvy, C; Cros, S. 1986, Biochem. Pharmacol.;35, 2264-2267. [3241 Pezzuto, J. M; Che, C.-T.; McPherson, d. D.; Zhu, J.-P.; Topcu, G.; Erdelmeier, C. A. J.; Cordell, G. A. ,/. Nat. Prod, 1991, 54, 1522-1530. [325] Remers, W. A. ,1. Nat. Prod, 1985, 48,173-192. [326] Tornaletti, S.; Russo, P.; Parodi, S.; Pedrini, A. M. Biochim. Biophys. Acta, 1989, 1007, 112-115. [327] Dodin, G.; Schwaller, M.-A.; Aubard, J.; Paoletti, C. Europ. J. Biochem., 1988, 176, 371-376. [328] Monnot, M.; Mauffret, O.; Simon, V.; Lescot, E.; Psaume, B.; Saucier, J.-M.; Belehradek,J.; Jr; Fermandjian, S. FEBS Lett. 1990, 273, 71-74. [329] Schwaller, M.-A.; Aubard, J.; Auclair, C.; Paoletti, C.; Dodin, G. Europ. J. Biochem., 1989, 181, 129-134. [3301 Malvy, C; Bertrand, J. R. FEBS Lett., 1986, 208, 155-157. [331] Caprasse, M; Houssier, C. Biochimie, 1983, 65, 157-167. [3321 Chakraborty, S; Nandi, R.; Maiti, M.; Achari, B.; Saha, C. R.; Pakrashi, S. C. Biochem. Pharmacol., 1989, 38, 3683-3687. [333] Sen, A; Maiti, M. Biochem. Pharmacol., 1994, 48, 2097-2102. [334] Barret, Y; Sauvaire, Y. Phytotherap. Res., 1992, 6, 59-63. [3351 Pezzuto, J. M.; Antosiak, S. K.; Messmer, W. M.; Slaytor, M. B.; Honig, G. R. Chem.-Biol. Interactions, 1983, 43, 323-339. [336] Xu, Y.; Yakushijin, K.; Home, D. A. Tetrahedron Lett., 1992, 33, 4385-4388. [337] Candrian, U.; Ltithy, J.; Schlatter, C. Chem. Biol. Interactions, 1985, 54, 5769.

48

1".ROBINSON

[338] Chan, G. W; Berry, D.; DeBrosse, C. W.; Hemling, M. E.; MacKenzieLoCosto, L.; Often, P. H.; Westley, J. W..1. Nat. Prod., 1993, 56, 708-713. [339] Fukada, M. Biochem. Pharmacol., 1985, 34, 1225-1230. [3401 Fujita, H; Kakishima, H. Chem.-Biol. Interactions, 1989, 72, 105-11 I. [3411 Hudson, J. B.; Graham, E. A.; Fong, R.; Hudson, L. L; Towers, G. H. N. Photochem. Photobiol., 1986, 44, 483-487. [342] Pezzuto, J. M; Mar, W.; Lin, L.-Z.; Cordell, G. A.; Neszm~lyi, A.; Wagner, H. Phytochemistry, 1992, 31, 1795-1800. [343] Kim, Y. C.; Che, Q.-M.; Gunatilaka, A. A. L.; Kingston, D. G. I. J. Nat. Prod., 1996, 59, 283-285. [344] Paulini, H.; Popp, R.; Schimmer, O.; Ratka, O.; R0der, E. Planta Med., 1991, 57, 59-61. [345] Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science, 1994, 263, 14251429. [346] Pratviel, G.; Bemadou, J.; Ha, T.; Meunier, G.; Cros, S.; Meunier, B.; Gillet, B.; Guittet, E. d. Med. Chem., 1986, 29, 1350-1355. [347] Berry, D. E.; MacKenzie, L. J.; Busby, R. W.; Nasuti, C. A. d. Nat. Prod., 1992, 55, 401-413. [348] Froelich-Ammon, S. J; Osheroff, N. d. Biol. Chem., 1995, 270, 21429-21432. [3491 Auclair, C. Arch. Biochem. Biophys., 1987, 259, 1-14. [350] Hsiang, Y.-H. Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem., 1985, 260, 14873-14878. [351] Leteurtre, F.; Fesen, M.; Kohlhagen, G.; Kohn, K. W.; Pommier, Y. Biochemistry, 1993, 32, 8955-8962. [3521 Kjeldsen, E; Mollerup, S.; Thomson, B.; Bonven, B. J.; Bolund, L.; Westergaard, O. J. Molec. Biol., 1988, 202, 333-342. [353] Knab, A. M.; Fertala, J.; Bjomsti, M.-A. J. Biol. Chem., 1993, 268, 2232222330. [354] Larsen, A. K; Grondard, L.; Couprie, J.; Desoize, B.; Comoe, L.; Jardillier, J. C.; Riou, J. F. Biochem. Pharmacol., 1993, 46, 1403-1412. [3551 Mahmood, K; PaYs, M.; Fontaine, C.; Ali, H. M.; Haai, A. H. A.; Guittet, E. Tetrahedron Lett., 1993, 34, 1795-1796. [3561 Kakiuchi, N.; Hattori, M.; Ishii, H.; Namba, T. Planta Med., 1987, 53, 22-27. [357] Tan, G. T.; Pezzuto, J. M.; Kinghom, A. D.; Hughes, S. H. J. Nat. Prod., 1991, 54, 143-154. [358] Kulp, K. S; Vulliet, P. R. Toxicol. ,4ppl. Pharmacol., 1996, 139, 356-364. [3591 Lin, H; Falchetto, R.; Mosca, P. J.; Shabanowitz, J.; Hunt, D. F.; Hamlin, J. L J. Biol. Chem., 1996, 2 71, 2548-2556. [3601 Magnus, P.; Ladlow, M.; Elliott, J. J. Am. Chem. Soc., 1987, 109, 7929-7930. [361] Krayevsky, A. A. Bioorg. Chem. USSR 1978, 4, 853-878. [362] Leatherman, D. L; Middlebrook, J. L. J. Pharmacol. Exp. Therap., 1993, 266, 732-740, 741-748. [3631 Tujebajeva, R. M.; Gaifer, D. M.; Karpova, G. G; Ajtkhozhina, N. A. FEBS Lett., 1989, 257, 254-256. [364] Vrijsen, R.; Vanden Berghe, D. A.; Vlietinck, A. J.; Boey~, A. J. Biol. Chem., 1986, 261, 505-507. [365] Jimenez, J.; Sanchez, L.; Vazquez, D. FEBS Lett., 1975, 55, 53-56. [366] Jeven, M.; van den Berghe, D. A.; Vlietinck, A. J. Planta Med., 1983, 49, 109114.


49

[367] Datta, R. K.; Ghosh, J. A.; Antopol, W. Biochem. Pharmacol. 1974, 23, 16871692 [368] Chakrabarti, S.; Kumar, S.; Shankar, R. Biochem. Pharmacol., 1986, 35, 16111613. [369] Lombardini, J. B; Props, C. Biochem. Pharmacol., 1996, 51, 151-157. [370] Uezono, Y; Yanagihara, N.; Wada, A.; Koda, Y.; Yokota, K.; Kobayashi, H.; Izumi, F. Naunyn-Schmiedeberg's Arch. Pharmacol., 1989, 339, 653-659. [3711 Lombardini, J. B.Biochem. Pharmacol., 1996, 52, 253-257. [372] Gong6ra, J. L.; Sierra, A.; Mariscal, S.; Aceves, J. Europ. J. Pharmacol., 1988, 155, 49-55. [373] Brossi, A.; Sch0nenberger, B.; Clark, O. E.; Ray, R. FEBS Lett., 1986, 201, 190- 192. [374] Orgell, W. H. Lloydia, 1963, 26, 36-43, 59-66. [375] Mroue, M. A.; Euler, K. L.; Ghuman, M. A.; Alam, M. J. Nat. Prod., 1996, 59, 890-893. [376] Harvey, A. L. Pharmacol. Therap., 1995, 68, 113-128. [377] Kozikowski, A. P; Xia, Y.; Reddy, E. R.; Ttlekmantel, W.; Hanin, I.; Tang, Y. C. d. Org. Chem., 1991, 56, 4636-4645. [378] McKinney, M.; Miller, J. H.; Yamada, F.; Tuckmantel, W.; Kozikowski, A. P. Europ. J. Pharmacol., 1991, 203, 303-305. [379] Ashani, Y.; Peggins, J. O.; III; Doctor, B. P. Biochem. Biophys. Res. Commun. 1992, 184, 719-726. [380] Zorko, M; Pavlie, M. R. Biochem. Pharmacol., 1986, 35, 2287-2296. [381] Stojan, J; Pavlie, M. R. Biochim. Biophys. Acta 1991, 1079, 96-102. [382] Sepci'c, K; Guella, G.; Mancini, I.; Pietra, F.; Dalla Serra, M.; Menestrina, G.; Tubbs, K.; Macek, P.; Turk, T. J. Nat. Prod, 1997, 60, 991-996. [383] Dougherty, D.A. Science, 1996, 271. 163 [384] Elbein, A. D. Ann. Rev. Biochem., 1987, 56, 497-534. [385] Elbein, A. D. Plant Physiol., 1988, 87, 291-295. [386] James, L. F.; Elbein, A. D.; Molyneux, R. J.; Warren, C. D., Eds., Swainsonine and Related Glycosidase lnhibitors, Iowa S. U. Press: Ames, 1989 [387] Molyneux, R. J.; Benson, M.; Wong, R. Y.; Tropea, J. E.; Elbein, A. D. or. Nat. Prod, 1988, 51, 1198-1206. [388] Asano, N; Nishida, M.; Kizu, H.; Matsui, K.; Watson, A. A.; Nash, R. J. J. Nat. Prod, 1997, 60, 98-101. [389] Molyneux, R. J.; Pan, Y. T.; Goldmann, A.; Tepfer, D.; Elbein, A. D. Arch. Biochem. Biophys., 1993, 304, 81-88. [390] Nash, R. J.; Rothschild, M.; Porter, E. A.; Watson, A. A.; Waigh, R. D.; Waterman, P. G. Phytochemistry, 1993, 34, 1281-1283. [391] Bowlin, T. L; Sunkara, P. S. Biochem. Biophys. Res. Commun. 1988, 151, 859864. [392] Tulsiani, D. R. P.; Broquist, H. P.; James, L. F.; Touster, O. Arch. Biochem. Biophys., 1988, 264, 607-617. [393] Pan, Y. T.; Ghidoni, J.; Elbein, A. D. Arch. Biochem. Biophys., 1993, 303, 134- 144. [394] Koley, A. P.; Robinson, R. C.; Markowitz, A.; Friedman, F. K. Biochem. Pharmacol., 1997, 53, 455-460. [395] Mitsui, N.; Noro, T.; Kuroyanagi, M.; Miyase, T.; Umehara, K.; Ueno, A. Chem. Pharm. Bull. Tokyo, 1989, 37, 363-366. [396] Shute, J. K; Smith, M. E. Biochem. Pharmacol., 1985, 34, 2471-2475.

50

T. ROBINSON

[397] Toescu, E. C.; O'Neill, S. C.; Petersen, O. H; Eisner, D. A. ,1. Biol. Chem., 1992, 267, 23467-23470. [398] Ukena, D.; Schudt, C.; Sybrecht, G. W. Biochem. Pharmacol., 1993, 45, 847851. [3991 Prabhakaram, M; Singh, S. N. Biochem. Intern., 1989, 18, 217-225. [400] Eguchi, N.; Watanabe, Y.; Kawanishi, K.; Hashimoto, Y.; Hayaishi, O. Arch. Biochem. Biophys., 1984, 232, 602-609. [401] Maguire, M. H; Csonka-Khalifah, L. Biochim. Biophys. ,4cta, 1987, 921, 426436. [4021 Son, J.-K.; Rosazza, J. P. N.; Duffel, M. W. J. Med. Chem., 1990, 33, 18451848. [403] Mimaki, Y.; Takaashi, Y.; Kuroda, Y.; Sahida, Y.; Nikaido, T. Phytochemistry, 1996, 42, 1609-1615. [404] Herbert, J. M.; Augereau, J. M.; Gleye, J.; Maffrand, J. P. Biochem. Biophys. Res. Commun., 1990, 172, 993-999. [4051 Kemmerling, W. Z. Naturforsch., 1996, 51c, 59-64. [406] Nakai, N.; Fujii, Y.; Kobashi, K.; Nomura, K. Arch. Biochem. Biophys., 1985, 239, 491-496. [407] Trugman, G.; Rousset, M.; Zweibaum, A. FEBS Lett., 1986, 95, 28-32. [408] Xu, Y.; Liu, J.; Zhang, S.; Liu, L. Biochem. J., 1987, 248, 985-988. [409] Abu-Ghalyun, Y, Masaalmeh, A.; AI-Khalil, S. Gen. Pharmacol., 1997, 29, 621- 623. [41o] Meikle, A. W.; Liu, X. H.; Taylor, G. N.; Stringham, J. D. Life Sci., 1988, 43, 1845-1850. [411] de la Lande, I. S.; Parker, D. A. S.; Proctor, C. H.; Marino, V.; Mackay-Sim, A. Life Sci., 1987, 4 I, 2463-2468. [4121 Chan, G. W. J. Org. Chem., 1993, 58, 2544-2546. [4131 Chang, C.-J; Geahlin, R. L. d. Nat. Prod., 1992, 55, 1529-1560. [414] Fuller, R. W.; Wong, C. J.; Hemrick-Luecke, S. K.Life Sci., 1986, 38, 409-412. [415] Jaspars, M; Rali, T.; Laney, M.; Schatzman, R. C.; Diaz, M. C.; Sehmitz, F. J.; Pordesimo, E. O.; Crews, P. Tetrahedron, 1994, 50, 7367-7374. [416] Lee, M. K; Kim, H. S. Planta Med., 1996, 62, 31-34. [417] Neal, J. J. Phytochemistry, 1989, 28, 451-453. [418] Ori, K.; Mimaki, Y.; Sahida, Y.; Nikaido, T.; Ohmoto, T. Phytochemistry, 1992, 31, 3605-3607, 4337-4341. [419] Tanaka, T; Metori, K.; Mineo, S.; Hirotani, M.; Furuya, T.; Kobayashi, S. Planta Med., 1993, 59, 200-202. [420] Wade, T. G; Whitely, C. G. Biochem. Intern., 1981, 14, 189-197. [421] Keukens, E. A. J; de Vrije, T.; Jansen, L. A. M.; de Boer, H.; Janssen, M.; de Kron, A. J. P. M.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1996, 1279, 243-250. [4221 Keukens, E. A. J; de Vrije, T.; van den Boom, C.; de Waard, P.; Plasman, H. H.; Thiel, F.; Chupin, V.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1995, 1240, 216-228. [4231 Keukens, E. A. J.; de Vrije, T.; Fabrie, C. H. J. P.; Demel, R. A.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1992, 1110, 127-136. [4241 Surewicz, W. K. Biochim. Biophys. Acta, 1982, 692, 315-318. [4251 Roddick, J. G.; Rijnenberg, A. L.; Weissenberg, M. Phytochemistry, 1990, 29, 1513-1518.

A L K A L O I D S IN ANIMALS

51

[426] Arnason, J. T.; Guerin, B.; Kraml, M. M.; Mehta, B.; Redmond, R. W.; Scaiano, J. C Photochem. Photobiol., 1992, 55, 35-38. [4271 Tuveson, R. W.; Larson, R. A.; Marley, K. A.; Wang, G.-R.; Berenbaum, M. R. Photochem. Photobiol., 1989, 50, 733-738. [428] Oubrahim, H.; Richard, J.-M.; Cantin-Esnault, D.; Seigle-Murandi, F.; Tr6court, F.J. Chromatog. A 1997, 758, 145-157. [429] Speisky, H.; Cassels, B. K.; Lissi, E. A.; Videla, L. A. Biochem. Pharmacol., 1991, 41, 1575-1581. [430] Mourelle, M.; Fraginals, R.; RodrIguez, L.; Favari, L.; P~rez-Alvarez, V.Life Sci., 1989, 45, 891-900. [4311 Banerjee, A; Luduena, R. F. J. Biol. Chem., 1991, 266, 1689-1691. [432] Banerjee, A; Luduena, R. F. J. Biol. Chem., 1992, 267, 13335-13339. [433] SackeR, D. L; Varma, J. K. Biochemistry, 1993, 32, 13560-13565. [434] Floyd, L. J.; Barnes, L. D.; Williams, R. F. Biochemistry, 1989, 28, 85158525. [435] Medrano, F. J.; Andreu, J. M.; Gorbunoff, M. J.; Timasheff, S. N. Biochemistry, 1989, 28, 5589-5599. [436] Brossi, A. J. Med. Chem., 1990, 33, 2311-2319. [4371 Hamel, E.; Ho, H. H.; Kang, G.-J.; Lin, C. M. Biochem. Pharmacol., 1988, 37, 2445-2449. [438] Skoufias, D. A; Wilson, L. Biochemistry, 1992, 31, 738-746. [439] Engelborghs, Y.; Dumortier, C.; D'Hoore, A.; Vandecandelaere, A.; Fitzgerald, T. J. J. Biol. Chem., 1993, 268, 107-112. [440] Malty, S. N; Ray, K.; Banerjee, A.; Mukhopadhyay, K.; Roychowdhury, S.; Chaudhuri, G. G.; Biswas, B. B.; Bhattacharyya, B. Ind. J. Biochem. Biophys., 1988, 25, 585-589. [441] Diaz, J. F; Andreu, J. M. J. Biol. Chem., 1991, 266, 2890-2896. [442] Shearwin, K. E; Timasheff, S. N. Biochemistry, 1994, 33, 894-90 I. [443] Uppuluri, S.; Knipling, L.; SackeR, D. L.; Wolff, J. Proc. Nat. Acad. Sci. U. S., 1993, 90, 11598- l 1602. [444] Bums, R. G. FEBS Lett., 1992, 297, 205-208. [445] Rai, S. S; Wolff, J. J. Biol. Chem., 1996, 271, 14707-14711. [446] Hastir S. B.; MacDonald, T. L. Biochem. Pharmacol., 1990, 39, 12271-1276. [447] Raxworthy, M. J. Biochem. Educ., 1988, 16, 2-9. [448] Liliom, K.; Lehotzky, A.; Moln,Sr, A.; Ov,'tdi, J. Anal. Biochem., 1995, 228, 1826. [449] Smith, C. D; Zhang, X. J. Biol. Chem., 1996, 271, 6192-6198. [450] Lobert, S.; Vulevic, B.; Correia, J. J. Biochemistry, 1996, 35, 6806-6814. [451] Prakash, V; Timasheff, S. N. Biochemistry, 1991, 30, 873-880. [452] Jordan, M.A.; Margolis, R. L.; Himes, R. H.; Wilson, L. at. Molec. Biol., 1986, 187, 61-73. [453] Singer, W. D.; Jordan, M. A.; Wilson, L.; Himes, R. H. Molec. Pharmacol., 1989, 36, 366-370. [454] Borman, L. S; Kuehne, M. E. Biochem. Pharmacol., 1989, 38, 715-724. [455] Na, G. C; Timasheff, S. N. Biochemistry, 1986, 25, 6214-6222, 6222-6228. [456] Bowman, L. C.; Houghton, J. A.; Houghton, P. J. Biochem. Biophys. Res. Commun., 1986, 135, 695-700. [4571 SackeR, D. L. Biochemistry, 1995, 34, 7010-7019. [458] Huang, A. B.; Lin, C. M.; Hamel, E. Biochem. Biophys. Res. Commun., 1985, 128, 1239-1246.

52

T. ROBINSON

[459] Takahashi, M; Iwasaki, S.; Kobayashi, H.; Okuda, S.; Murai, T.; Sato, Y. Biochim. Biophys. Acta, 1987, 926, 215-223. [460] Horwitz, S. B. Trends Pharmacol. Sci., 1992, 13, 134-136. [461] Ohmi, K; Nakamura, S. Europ. J. Pharmacol., 1991, 207, 305-310. [462] Cheek, T. R; Burgoyne, R. D. ,1. Biol. Chem., 1987, 262, 11663-11666. [463] Sorrentino, V; Volpe, P. Trends Pharmacol. Sci., 1993, 14, 98-103. [454] McGrew, S. G.; Wolleben, C.; Siegl, P.; Inui, M.; Fleischer, S. Biochemistry, 1989, 28, 1686-169 I. [4651 Pessah, I. N; Zimanyi, I. Molec. Pharmacol., 1991, 39, 679-689. [466] Michalak, M.; Dupraz, P.; Shoshan-Barmatz, V. Biochim. Biophys. Acta, 1988, 939, 587-594. [467] Pessah, I. N.; Francini, A. O.; Scales, D. J.; Waterhouse, A. L.; Casida, J. E. J. Biol. Chem., 1986, 61, 8643-8648. [468] Campbell, K. P; Knudson, C. M.; Imagawa, T., Leung, A. T.; Sutko, J. L.; Kahl, S. D.; Raab, C. R.; Madson, L. J. Biol. Chem., 1987, 262, 6460-6463. [469] Inui, M.; Saito, A.; Fleischer, S. J. Biol. Chem., 1987, 262, 1740-1747. [4701 Takeshima, H; l0 Others Nature, 1989, 339, 439-445. [471] Marks, A. R; Tempst, P.; Hwang, K. S.; Taubman, M. B.; Inui, M.; Chadwick, C.; Fleischer, S.; NadaI-Ginard, B.; Proc. Nat. Acad. Sci. U. S., 1989, 86, 86838687. [472] McPherson, P. S; Campbell, K. P. J. Biol. Chem., 1993, 268, 13765-13768. [473] Hymel, L.; Inui, M.; Fleischer, S.; Schindler, H. Proc. Nat. Acad. Sci. U. S., 1988, 85, 441-445. [474] Hamilton, S. L; Alvarez, R. M.; Fill, M.; Hawkes, M. J.; Brush, K. L.; Schilling, W. P.; Stefani, E. Anal. Biochem., 1989, 183, 31-41. [4751 Lai, F. A.; Misra, M.; Xu, L.; Smith, H. A.; Meissner, G. J. Biol. Chem., 1989, 264, 16776-16785. [4761 Marty, I.; Villaz, M.; Arlaud, G.; Bally, I.; Ronjat, M. Biochem. J., 1994, 298, 743-749. [477] Hasselbaeh, W; Migala, A. Z. Naturforsch., 1992, 47c, 136-147. [478] Shoshan-Barmatz, V; Zarka, A. J. Biol. Chem., 1988, 263, 16772-16779. [479] Waterhouse, A. L.; Pessah, I. N.; Francini, A. O.; Casida, J. E. J. Med. Chem., 1987, 30, 710-716. [480] Welch, W.; Sutko, J. L.; Mitchell, K. E.; Airey, J.; Ruest, L. Biochemistry, 1996, 35, 7165-7173. [481] Zarka, A; Shoshan-Barmatz, V. Biochim. Biophys. Acta, 1992, 1108, 13-20. [4821 Hasselbaeh, W; Migala, A. Z. Naturforsch., 1988, 43c, 140-148. [4831 Barry, V. A; Cheek, T. R. Biochem. J., 1994, 300, 589-597. [484] Komori, S.; Itagaki, M.; Unno, T.; Ohashi, H. Europ. J. Pharmacol., 1995, 277, 173-180. [485] Chavis, P.; Fagni, L.; Lansman, J. B.; Bockaert, J. Nature, 1996, 382, 719-722. [4861 Pessah, I. N.; Stambuk, R. A.; Casida, J. E. Molec. Pharmacol., 1987, 31, 232238. [4871 Lindsay, A. R. G; Williams, A. J. Biochim. Biophys. Acta, 1991, 1064, 89-102. [488] Zhang, Z.-D.; Kwan, C.-Y.; Daniel, E. E. Biochem. Biophys. Res. Commun., 1993, 194, 1242-1247. [489] McPherson, P. S.; Campbell, K. P. J. Biol. Chem., 1993, 268, 19785-19790. [4901 Nakazato, Y.; Hayashi, H.; Teraoka, H. Brit. J. Pharmacol., 1992, 105, 597602.

ALKALOIDSIN ANIMALS

53

[491] Shoshan-Barmatz, V.; Pressley, T. A.; Higham, S.; Kraus-Friedmann, N. Biochem. J., 1991, 276, 41-46. [4921 Kunz, W. S.; Kuznetsov, A. V.; Gellerich, F. N.FEBS Lett., 1993, 323, 188190. [493] Khuchua, Z.; Belikova, Y.; Kuznetsov, A. V.; Gellerich, F. N.; Schild, L.; Neumann, H. W.; Kunz, W. S. Biochim. Biophys. Acta, 1994, 1188, 373-379. [494] Islam, M. S.; Larsson, O.; Niisson, T.; Berggren, P.-O. Biochem. J., 1995, 306, 679-686. [495] Paolisso, G; Nenquin, M.; Schmeer, W.; Mathot, F.; Meissner, H. P.; Henquin, J. C. Biochem. Pharmacol., 1985, 34, 2355-2361. [4961 McNulty, T. J; Taylor, C. W. Biochem. J., 1993, 291, 799-801. [497] Griffin, D. S; Segall, H. J. Biochem. l~armacol., 1989, 38, 391-397. [498] Mancilla, E; Rojas, E. FEBS Lett., 1990, 260, 105-108. [499] Freedman, J. E; Weight, F. F. Europ. J. Pharmacol., 1989, 164, 341-346. [5OOl Clark, R. B.; Sanchez-Chapula, J.; Salinas-Stefanon, E.; Duff, H. J; Giles, W. R. Brit. J. Pharmacol., 1995, 115, 335-343. [5011 G6gelein, H; Capek, K. Biochim. Biophys. Acta, 1990, 1027, 191-198. [5021 Sabath, D. E.; Monos, D. S.; Lee, S. C.; Deutsch, C.; Prystowsky, M. B. Proc. Nat. Acad. Sci. U. S., 1986, 83, 4739-4743. [5031 Pe|assy, C; Aussei, C. Pharmacology, 1993, 47, 28-35. [504] Wang, G; Lemos, J. R. Life Sci., 1995, 56, 295-306. [5051 Needham, L.; Dodd, N. J. F.; Houslay, M. D. Biochim. Biophys. Acta, 1987, 899, 44-50. [5061 Suwalsky, M.; Villena, F.; Bagnara, M.; Sotomayor, C. P. Z. Naturforsch., 1995, 50c, 527-534. [507] D'Oc6n, P.; Bl~zquez, M. A.; Bermejo, A.; Anselmi, E. J. Pharm. Pharmacol., 1992, 44, 579-582. [508] Low, A. M.; Berdik, M.; Sormaz, L.; Gataiance, S.; Buchanan, M. R.; Kwan, C. Y.; Daniel, E. E. Life Sci., 1996, 58, 2327-2335. [509] Xu, Y; Zhang, S. Biochem. Biophys. Res. Commun., 1986, 140, 461-467. [51o] Michalska, L.; Nagel, G.; Swiniarski, E.; Zydowo, M. M. Gen. Pharmacol., 1985, 16, 69-70. [5111 Knaus, H.-G; 11 Others Biochemistry, 1994, 33, 5819-5828. [512] Yano, S; Horiuchi, H.; Horie, S.; Aimi, N.; Sakai, S.; Watanabe, K. Planta Med., 1991, 57, 403-405. [513] Plant, T. D; Henquin, J. C. Brit. J. Pharmacol., 1990, 101, 115-120. [514] Wang, Y.-X.; Zheng, Y.-M.; Zhou, X.-B. Europ. J. Pharmacol., 1996, 316, 307- 315. [5151 Berlinck, R. G. S; Braekman, J. C.; Daloze, D.; Bruno, I.; Riccio, R.; Ferri, S.; Spampinato, S.; Speroni, E. J. Nat. Prod., 1993, 56, 1007-1015. [516] Rando, T. A.; Wang, G. K.; Strichartz, G. R. Molec. Pharmacol., 1986, 29, 467- 477. [517] Corbett, A. M; Vander Kok, M. A. Biochem. Biophys. Res. Commun., 1994, 199, 1305-1312. [518] Dumbacher, J. P.; Beehler, B. M.; Spandr T. F.; Garraffo, H. M.; Daly, J. W. Science, 1992, 258, 799-801. [519] Elenu, N.; Botana, L.; Espinosa, J. Biochem. Pharmacol., 1988, 3 7, 3523-3525. [520] Iwata, H.; Ohta, A.; Baba, A. Jap. J. Pharmacol., 1986, 41,293-297. [521] Kusaka, M; Sperelakis, N. Europ. J. Pharmacol., 1994, 271, 387-393.

54

T. ROBINSON

[522] Pauwels, P. J.; Van Assouw, H. P.; Peeters, L.; Leysen, J. E. J. Pharmacol. Exp. Therap., 1990, 255, 1117-1122. [523] Jacobson, K. A; Guay-Broder, C.; van galen, P. J. M.; Gallo-Rodriguez, C.; Melman, N.; Jacobson, M. A.; Eidelman, O.; Pollard, H. B. Biochemistry, 1995, 34, 9088-9094. [524] Koob, G. F; Bloom, F. E. Science, 1988, 242, 715-723. [525] Altura, B. M.; Zhang, A.; Cheng, T. P.-O.; Altura, B. T. Europ. J. Pharmacol., 1993, 246, 299-301. [526] He, G.-Q.; Zhang, A.; Altura, B. T.; Altura, B. M. d. Pharmacol. Exper. Therap., 1994, 268, 1532-1539. [527] Crumb, W. J.; Jr; Clarkson, C. W. d. Pharmacol. Expt. Therap., 1995, 274, 1228- 1237. [528] Kaminsky, S. M.; Levy, O.; Garry, M. T.; Carrasco, N. Europ. d. Biochem., 1991, 200, 203-207. [529] Young, J. D.; Mason, D. K.; Fincham, D. A. d. Biol. Chem., 1988, 263, 140143. [5301 Steinfelder, H. J; PethO-Schramm, S. Biochem. Pharmacol., 1990, 40, 11541157. [531] Blankemeyer, J. T.; Atherton, T.; Friedman, M. d. Agric. Food Chem., 1995, 43, 636-639. [532] Blankemeyer, J. T.; Stringer, B. K.; Rayburn, J. R.; Bantle, J. A.; Friedman, M. d. Agric. Food Chem., 1992, 40, 2022-2025.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22

55

9 2000 Elsevier Science B.V. All rights reserved

USING CHEMICAL ECOLOGY TO LOCATE NEW ANTIFUNGAL NATURAL PRODUCTS S T E P H A N I E J. E C K E R M A N and K A T E J. G R A H A M *

Chemistry Department College of St. Benedict~St. John's University 3 7 S. College Avenue St. Joseph, MN 56374, USA ABSTRACT: The quest for new antifungal drugs is critical for several reasons. Immune suppression causes susceptibility to fungal infections. The number of immune-suppressed individuals continues to rise as society is faced with an aging population, an increase in AIDS infected patients, and medical advances. Most drugs used to treat mycological infections have low bioavailability or are too toxic for prolonged use. Also, many new fungal strains are emerging with drug resistance as fungal pathogens are exposed to extended pharmaceutical treatment. As the need for new antifungal drugs continues to rise, chemical ecology appears to be an attractive tool for identification of such compounds. In competitive ecosystems, it is generally accepted that many organisms thrive because they produce secondary metabolites providing a selective advantage over competing organisms. Biorational criteria, in this context, meansusing the ecology of natural systems to reveal organic chemicals with specific bioactivities. By employing biorational criteria in selecting sources, potential drugs can be more effectively located. Therefore, biorationale predicts organisms encountering fungal competitors or pathogens will be a good source of fungistatic or fungicidal chemicals. Ecological clues point to a variety of sources which are expected to produce fungistatic secondary metabolites. Examples of such sources include antagonistic fungi, plants with fungal pathogens, and mycoparasites. Studies of antagonistic species can provide useful information for scientists interested in chemical ecology, but can also serve as a valuable complement to random, high-throughput screening for new bioactive compounds. THE N E E D F O R N E W A N T I F U N G A L D R U G S With the advent o f the antibiotic era, the possibility was raised that infectious diseases would be eradicated altogether. However, infections still remain the leading cause o f death worldwide [1]. N e w infectious diseases are continually being identified and, in addition, many k n o w n pathogens which were under control are again becoming health problems [1]. Elimination o f these infectious diseases has been hindered for several reasons [1 ]: 1) new human pathogens are being discovered and transmitted worldwide; 2) known microbes are mutating to form new, more virulent forms; 3) microbes are able to develop resistance to antibiotics; 4) new, stronger pharmaceutical agents have the side effect of decreasing the host's resistance; 5) the life o f an individual suffering from a chronic disease can

56

ECKERMAN and GRAHAM

be extended resulting in a suppressed immune system and 6) there is a lack of effective, non-toxic pharmaceutical treatments. Of particular interest are the last three reasons; the side effect of these aspects of modern medicine is a group of individuals who are more susceptible to infections. These individuals are usually immunocompromised and, thus, are susceptible to pathogens and even to microorganisms that would not typically attack a healthy individual. Microbes that are innocuous in the immunologically intact host can cause devastating infections in patients with compromised immune systems; organisms causing these infections are termed opportunists [ 1]. Opportunistic infections can be caused by all major groups of microbes, but opportunistic fungal infections assume particular medical significance because they are difficult to treat and can sometimes be fatal. The majority of mycoses in healthy individuals are infections of the skin and adjacent mucous membranes. Topical fungal infections are extremely common, highly refractory to therapy and tend to relapse, but these infections are rarely life-threatening. However, systemic opportunistic infections can be severe and life-threatening situations. These systemic opportunistic infections are becoming increasingly more frequent and, unfortunately, are difficult to treat due to a paucity of available antifungal drugs [2]. Many situations can lead to the development of an opportunistic fungal infection. Continued treatment with broad-spectrum antibiotics or hormones commonly causes an imbalance in the microecology of the respiratory and gastrointestinal tracts leading to fungal colonization. Immune suppression also causes susceptibility to fungal infections. The number of immune-suppressed individuals continues to rise as society is faced with an aging population, an increase in AIDS infected patients, and medical advances including tumor treatments and organ transplants. Cytotoxic therapies used in the treatment of malignant diseases have improved the prognosis of such patients; however, they have led to an increase in the frequency of invasive fungal infections by decreasing the host's resistance. In a traumatic accident resulting in an open wound or bum, the patient often has a reduced immune response and can develop a fungal infection. In addition, patients requiring treatment involving catheters, prostheses or other invasive devices are prone to fungal infections. These clinical situations have been reported throughout the medical literature [3-15]. Some of the causal agents of these opportunistic infections are wellknown opportunistic mycoses. However, an increasing number of mycoses are being caused by fungi not previously known in human medicine. These organisms include soil fungi, plant and insect pathogens, and saprophytic fungi. In fact, it appears that virtually any fungus can infect an individual with a compromised immune system. The numbers and types of opportunistic fungi isolated from patients are increasing

ANTIFUNGAL NATURAL PRODUCTS

57

rapidly. It is likely that this trend will continue as long as the numbers of immunocompromised patients continues to grow [ 16]. The most common fungus causing opportunistic infections is the yeast Candida [1,17,18]. The yeast's commensualistic relationship with humans enable it to multiply and replace much of the normal flora when environmental conditions are favorable. Yeast infections by Candida species have been increasing dramatically ill the last decade and there are now as many as seventeen different species known to cause infections in humans [17,18]. The most frequently isolated species are C. albicans, C. tropicalis, C. glabrata (formerly known as Torulopsis glabrata), C. parapsilosis, C. guilliermondii, and C. krusei [ 17-20]. Cryptococcus is the most common systemic fungal infection in AIDS patients and is usually in the form of meningitis due to C. neoformans. Cryptococcal meningitis, if untreated, progresses rapidly to death. Even with treatment, mortality rates remain unacceptably high at-~50% [21 ]. Other significant, but less frequent, opportunistic fungal pathogens that cause life-threatening disseminated mycoses include Aspergillus, Histoplasma, Coccidioides, and Fusarium. Aspergillosis can occur as a non-invasive infection of the respiratory tract in immunocompetent hosts; however, in immunosuppressed individuals, it can become an invasive disseminated infection which is often lethal. Histoplasma capsulatum and Coccidioides immitis are pathogens of immunocompetent hosts as well. Again, in immunosuppressed hosts, these pathogens can cause deadly systemic infections. Fusarium has only recently been noted as an opportunistic infection in immunosuppressed individuals and appears to have an intrinsic resistance to many antifungal agents [21 ]. In addition, the number of new pathogens and previously rare organisms being isolated from patients is increasing rapidly [17]. These organisms can vary greatly in pathology and in their response to current antifungal therapy. This situation poses considerable difficulty to medical practitioners who have had little training in mycology. As more eases are reported, physicians and the clinical microbiology laboratory must become better prepared to identify and treat fungal infections from a variety of new and emerging yeasts. Treatment of systemic mycoses is further complicated because of the discrepancy between the variety and severity of infections and availability of possible therapeutics. Until recently, there was a perception that lifethreatening fungal infections were too rare to require the attention of pharmaceutical companies. While this view has slowly been discounted, the number of antifungal drugs on the market remains limited. The development of antifungal drugs has also been hampered by the fact that fungi are eukaryotes, making it more difficult to develop drugs that inhibit fungal growth but are not toxic to the human patient. While the number of pathogens has been steadily increasing, there are still only four major classes of antifungal drugs currently available"

58

ECKERMAN and GRAHAM

polyene macrolides, azoles, fluoropyrimidines and allylamines [21-25]. The mode of action of these antifungal agents has been reviewed by Kerridge [26]. These classes of drugs are described in more detail below.

Polyene Macrolides The polyene macrolides, including Amphotericin B, 1, and Nystatin, 2, are fungicidal agents. These drugs have worked well for systemic mycoses, OH

s,,...~,~

~

HO~...,,,

OH

OH

OH

O

OH

H

OH

H2~

%, H O-

) ._

O

OH

OH

OH

OH

H3C--k QH

.o--X-x-A:y\ i H2N~

CO2H

'

but extensive attempts at chemical modification of the drug have been unable to reduce the nephrotoxicity of the polyene macrolides. There are over 200 antifungal agents which belong to this chemical class; all are structurally similar and have a common mechanism of action. Polyene antibiotics bind sterols in the fungal cell membrane resulting in disruption of cellular integrity [21]. Due to the severe toxicity of Amphotericin B, efforts have been made to utilize this drug in combination with other antifungal agents [21,22]. While these synergistic drug interactions are documented, so are many contraindications with other pharmaceutical agents such as cyclosporin, aminoglycoside antibiotics, digitalis glycosides, and neuromuscular blockers. Increased bioavailability and

ANTIFUNGALNATURALPRODUCTS

59

decreased toxicity of drugs of this class have also been achieved with either liposomal preparations or colloidal suspensions of the drug complexed with other constituents of cell membranes, thereby allowing intravenous administration in tolerable therapeutic doses. Unfortunately, new data suggests that resistance to Amphotericin B among new and emerging pathogens is becoming significant [23, 24]. Azoles

The synthetic imidazoles were introduced more than ten years ago as broad-spectrum antifungal agents for topical use [21-23]. The drugs of this class work by inhibition of the cytochrome P450-1inked monooxygenase component of C-14 lanosterol demethylase which catalyzes a key step in the biosynthesis of ergosterol, the main sterol in fungi. Ergosterol is an irreplaceable constituent in fungal cellular membrans and is, therefore, required for cell proliferation. Unfortunately, an isozyme of the cytochrome P450 is also a prerequisite in the synthesis of cholesterol, the main sterol in mammalian cells.

clO CI

i 3

S > OOCH3

0

\

60

ECKERMAN and GRAHAM

Ketoconazole, 3, is a member of the azole class of antifungals and was the first orally absorbed antifungal agent for treatment of mycoses. However, it was found to be mostly ineffective for disseminated mycoses in immunocompromised individuals. Structure-activity relationship studies led to the development of the newer, less toxic triazoles. The newer triazoles have a higher specificity for the fungal enzyme than the related human enzyme which leads to lower toxicity. In addition, these compounds have been designed to have a higher bioavailability. Fluconazole, 4, has been introduced on the US market for a number of years and is indicated for dermal and vaginal mycoses, cryptococcal meningitis and mucocutaneous candidiasis. Given orally, it exhibits little toxicity and is generally well tolerated. A large number of related triazoles are in various stages of development or clinical use. Itraconazole, 5, is the most recently approved azole derivative in clinical use and appears to be active against a number of disseminated mycoses for which fluconazole has had limited success such as aspergillosis, histoplasmosis, blastomycosis, coccidiodomycosis and sporotrichosis [27-29]. In addition, there are at least two new azole derivatives currently in clinical trials; Voriconizole, made by Pfizer, and Schering-Plough's SCH 56592 are both reported to be active against fluconazole-resistant infections.

5-fluoropyrimidines Flueytosine (5-fluoroeytosine), 6, is a synthetic nueleoside that is converted intraeellularly to 5-fluorouraeil which, consequently, interferes with protein synthesis [22]. Although this drug is indicated for disseminated cryptococcosis and disseminated candidiases, flucytosine is rarely used alone due to substantial resistance developed by many opportunistic fungal pathogens. It also has the side effect of suppressing bone marrow production which is particularly problematic in AIDS patients. Flucytosine is sometimes used in combination with amphotericin B to suppress the rapid development of resistance to the flueytosine, but the toxicity appears to increase dramatically in these circumstances [21 ].

O

F NH 2


61

Allylamines One of the first allylamine antimycotics reported, naftifine, 7, was shown to be a broad-spectrum antifungal agent. The allylamines inhibit the enzyme squalene epoxidase which catalyzes a key step in the ergosterol biosynthesis pathway. SARs of naftifine led to the development of terbinafine, 8 [22]. Terbinafine has found widespread clinical use in both oral and topical therapy of fungal infections of the skin, nails and hair. Terbinafine is active against a wide range of fungal pathogens, but is exceptionally potent against dermatophytes including B. dermatitidis, H. capsulatum, S. schenkii, and T. mentagrophytes [22]. In contrast, C. albicans is much less susceptible to in vitro doses of terbinafine. Terbinafine has generally shown poor activity for systemic mycoses, although there is some evidence that this drug may have application for treatment of some systemic mycoses [25]. Efficacy has been observed in sporotrichosis and there are some reports of successful therapy in cases of aspergillosis. An additional potential clinical use is in combination with other mycotics such as the azoles [25].

7

B

The increasing reports of fungal resistance to the available antifungal drugs described above is particularly troublesome in light of the variety and growing incidents of opportunistic infections [30-37]. With only a limited number of effective antifungal drugs to treat such infections, microbes are able to develop resistance mechanisms which undermine the utility of the currently available drugs. Recent studies have found examples of fungal resistance to agents such as flucytosine, amphotericin B and many of the azoles [30-37]. One current area of research is the investigation of fungal resistance mechanisms [30-32]. Several pharmaceutical companies are attempting to circumvent the resistance mechanisms by developing new agents with different modes of action.

62

ECKERMAN and

GRAHAM

DEVELOPMENT OF NEW ANTIFUNGAL DRUGS Progress is continuing in the study of existing agents and the development of new potential drugs. Further research in the biochemistry of fungi will provide novel molecular targets in the fungal cell and molecular bioassays for the identification of new lead compounds. One strategy used to develop new agents is to inhibit processes or enzymes unique to the fungus. Koltin [23] has reviewed some potential future antifungal targets. In addition, many pharmaceutical companies are exploring compounds that target enzymes and structures needed to make or maintain the fungal cell wall [29]. Pneumocandins and echinocandins compose a new class of cyclic lipopeptide antifungal compounds [38]. Echinocandin B, 9, inhibits 1,3-[3D-glucan synthase in C. albicans, a critical enzyme in the production of cell well components. Two semi-synthetic derivatives have entered clinical trials. Merck's L-724,872 has been well tolerated in phase I clinical studies and is currently in phase II trials in patients with HIV and candidiasis. However, it is not absorbed orally and must be delivered intravenously. Lilly's LY303366 is also currently in clinical trials with people with HIV and candidiasis. These compounds appear to have fungicidal activity yet are not as toxic as Amphotericin B [21, 29, 30].

H OH

.Q [i

H HH~.'~

/

/ - - \/

HO ~

OH ."

~r'-'- 0

, ~ ' ~ , , r I O "Ltn~176

3t::~0 H/

o.

,.Me

IT "X,,"X"OH -.

/ HO 9

Benanomicin A, 10, and B, 11, and Pradimicin A, 12, have recently been isolated from soil bacteria [39]. These compounds appear to bind calcium-dependent mannan causing disruption of the cell membrane.


63

Bristol-Meyers has developed 200 analogs of compounds in this class. Due to potential liver toxicity, the compounds have not yet entered clinical trials [21, 29, 30]. 3)CO2H

H I0 Benanomicin A I I Benanomicln B 12 P r a d i m i c l n A OH

R = OH R = NH 2 R = NHCH 3

OH

O

O

OH

O

The polyoxins and nikkomycins are products of soil Streptomycetes and appear to inhibit chitin synthetases 1 and 2 [40]. Chitin is an important structure in the fungal cell wall. Shaman Pharmaceuticals is currently exploring the development of Nikkomycin Z and related analogs, 13-15, [29]. Polyoxins and nikkomycins have not yet proven to be clinically useful drugs because the compounds are unable to penetrate fungi in the environment of the human body; however, structure-activity relationship studies of these classes of compounds or new inhibitors of chitin synthetase may still yield a successful antifungal drug. HO

OH

HNL

NH 2 HO

OH _CHO

R=

HN

HN

HN j

or

HO

OH

m

13 Nikkomycin Z

14 Nikkomycin X

1 5 Polyoxln C

64

ECKERMAN and GRAHAM

The overall situation is consequemly far from favorable, and there is an urgent need for further development of more classes of antifungal drugs. Obviously, these new drugs could reduce the incidence of resistance and provide feasible options for instances when such resistance arises. The ultimate goal is to develop non-toxic, well-tolerated antifungal medications with a high efficacy rate and no resistance. Moreover, a strong point should be made that efficacy and tolerance correlate inversely with the breadth of the spectrum of activity [2]. However, pharmaceutical companies appear to ignore this relationship and continue to develop antifungal compounds with as broad a spectrum of activity as possible so as to increase the use and money generated by their drug. Such a demand also ignores practical medical necessities, as mixed infections do not usually occur in systemic mycoses. The trend of future development in the field of antifungal agents should lead in the direction of narrow spectrum antifungal agents. SEARCH FOR NEW POTENTIAL ANTIFUNGAL LEADS Past and current research to develop new, clinically effective antifungal drugs has focused on four approaches [21 ]" 1) structure-activity relationship studies of known antifungal agents; 2) combination therapy of existing drugs; 3) delivery systems for existing drugs and 4) discovery of new prototype antibiotics. Only the last strategy is a potential source of new lead compounds. Given the need to identify new leads for effective antifungal drug development, natural products are a prime source for the discovery of new lead compounds [41]. It has been estimated that only 5-15% of higher plants have been systematically investigated for the presence of bioactive compounds, while even fewer marine and fungal sources have been explored [42, 43, 44]. A recent review article [41 ] estimated that the new approved antibacterial and anti-infective drugs reported between 1983 and 1994 are predominantly from natural sources or are modeled on a natural product prototype. The data reveal the essential role played by natural products in the discovery of drugs for a variety of purposes; discovery of novel antifungal compounds from natural sources is clearly a feasible and productive approach. In addition, natural products present a virtually limitless potential for secondary metabolic diversity. Several strategies and philosophies are available for the identification of bioactive compounds from natural sources. Different aspects of these strategies have been reviewed [44-47]. Several such approaches have been used previously for this purpose" 1) ethnobotany, 2) serendipity, 3) random sampling, and 4) exploitation of chemotaxonomic knowledge. More than 80% of the world's population use plants as their primary source of medicinal agents [45], leading to a well-established system of traditional medicine. Some of these remedies are well documented and are


65

either commercially available as the traditional preparation or have been exploited in the development of a pharmaceutical drug [41]. Recent interest has been shown in the preservation and collection of traditional medicinal lore, in light of the increasing loss of biodiversity and native cultures. The area of science which has been involved in the discovery of biologically active natural products from plants used in traditional medicine is ethnobotany. At the outset, ethnobotany requires intensive studies in epidemiology, traditional medicine, language, culture and ecology of a people and their environment. Then, botanists and medical doctors must work together in presenting specific disease descriptions and identifying the traditional healers' treatments. Once a plant and its medicinal preparation have been identified, a sustainable supply and policies for economic development of the potential drug must be established. Finally, the isolation and structure determination work can be done, followed by the clinical evaluation of the biologically active component [45, 47]. The ethnobotanieal strategy for uncovering bioactive compounds may eventually yield a novel antifungal compound, but the attempt can be a difficult and expensive endeavor. Some natural product chemists are interested in the isolation and characterization of novel compounds without any realistic expectation that the compounds will be developed for drug use. This approach, "serendipity", involves the selection of organisms to be analyzed by their potential to yield interesting chemicals; whether or not the organisms have known bioactivity is of little concern [46]. Typically, an exhaustive analysis of a plant sample for all types of secondary metabolites is performed. The result of this method is many new compounds with no known bioactivity. Biological activity is sometimes subsequently discovered in these libraries of purified chemicals. Many pharmaceutical companies resort to random sampling in the search for new structural types with potential biological activity [48]. This approach simply involves screening as many organisms as possible through a wide variety of biological assays. The goal is to acquire biodiversity in order to broaden the chemical diversity. For most pharmaceutical companies, the source organism is irrelevant until a lead has been identified. High-throughput screening can be a rapid and effective method for lead development for large companies that have automated systems and the ability to purchase thousands of samples on a regular basis. Typically, the rate of discovery of potential leads is very low so this method is not feasible for smaller labs. Once a chemical structural type has been identified with a specific biological activity, the compound must be isolated from the source or synthesized for further studies. If the lead compound is not available due to an ineffective synthesis or a limited source, then a search for alternative source organisms can be initiated. Organisms of similar taxa often produce a similar distribution of natural products. Exploiting this chemotaxonomic

66

ECKERMAN and GRAIIAM

knowledge can aid in the search for new sources of known compounds or in the attempt to augment the collection of structures that are currently available for analysis. However, in order for exploitation of chemotaxonomic knowledge to be effective, immediate access to several plant species is imperative [45, 46]. These approaches for identifying novel drug leads have all been shown to be effective. These efforts should and will continue. However, there are some benefits to be gained from the expansion of these approaches to include ecology-based searches for efficient location of biologically active molecules. CHEMICAL ECOLOGY As the need for novel antifungal compounds continues to rise, ecologybased strategies are becoming more prevalent. Chemical ecology is the study of chemical interactions between organisms and their environment, including other organisms. In competitive ecosystems, it is generally accepted that many organisms thrive because they produce chemicals which provide a selective advantage over competing organisms. Secondary metabolites are used by all species for such uses as deterring enemies, fending off pathogens, competing for nutrients, protecting against physical hazards of the environment and sexual selection. Chemical ecology is a multidisciplinary field that involves both isolation and characterization of chemicals that mediate environmental interactions and an understanding of the biological mechanisms for chemical signal recognition and transduction. Major progress has been made in chemical ecology in recent decades and ecology-based approaches have recently received attention as a potential strategy to search for biologically active compounds [49]. This reflects the highly improved chemical techniques for isolating and characterizing very small quantities. The increased sensitivity is particularly important because of the very low levels of secondary metabolites produced by most organisms. In addition, progress in sociobiology, ecology, and evolutionary biology has helped to provide new understandings of interspecies interactions. For example, biological monitoring of ecological systems has made it easier to study variation in source organisms. Given that chemical activities are often seasonal and sporadic, an understanding of an ecosystem would allow organisms to be chosen for chemical study under the correct circumstances and season. This approach should result in a more efficient survey of the potential secondary metabolites than that of a random screening program. For example, the leafcutter ant, Atta cephalotes, cultivates a fungus for food. The leafcutter ant has evolved to avoid leaves of Hymenaea courbaril, which produces a terpenoid that is toxic to the fungus [50]. However, the plant shows a dramatic decline in the production of this terpenoid during the latter half of the wet season just


67

before the dry season. This decline is probably due to a reduction in the synthesis of antifungal compounds in the dry season, when the risk of fungal attack is low. Random screening may not have led to the discovery of this potential antifungal agent, but an understanding of the ecology of the ant and its feeding patterns would have clearly indicated the season for collecting leaves that would contain reasonable quantities of antifungal compounds [50-53]. New advances in biochemistry and molecular biology may also promote the use of ecology-based searches for medicinal agents. Understanding the biochemical basis of an ecological interaction can lead to drug development as evidenced in an excellent review by Caporale [48]. For example, medicinal chemists are interested in locating inhibitors of the enzyme hydroxymethylglutaryl (HMG)-CoA reductase as inhibitors can lead to lower plasma cholesterol levels. HMG-CoA reductase catalyzes the conversion of acetyl-CoA to mevalonate, a key step in sterol biosynthesis. It is also known that mevalonate can overcome catabolite repression of gibberellin synthesis. Gibberellins are plant growth hormones produced by a phytopathogenic fungus, Gibberella fujikuroi. Thus, an inhibitor of HMG-CoA reductase may be involved in the regulation of the biosynthetic pathway of gibberellins. A chemical ecologist might have suggested screening such phytopathogenic fungi to locate inhibitors of HMG-CoA reductase [48,54]. Drug discovery targets can also be suggested by the study of organisms that have evolved the ability to regulate our biochemistry. Parasites and viruses often manipulate regulatory steps in host defenses [48, 55]. For example, some viruses are able to override cell death programs that otherwise eliminate virus-infected cells [48]. Understanding the biochemical control mechanisms of cell death can lead to the ability to save neurons in neurodegenerative diseases or kill cells in a tumor. Other organisms, such as leeches and ticks, are good sources of agents that selectively block the action of coagulation proteases [48]. Random screening may locate a compound, such as a coagulation protease inhibitor or an inhibitor of cell death, which has evolved to interact with the target of the screen. However, since diverse organisms share a great deal of biochemical structures and pathways, it is not surprising that small molecules from species as distant as plants, fungi, and bacteria can interact with macromolecules in humans. It may be that an interaction is coincidental; however, a bioactive natural product that interacts with a human protein may have evolved to interact with a homologous protein domain or active site in the organism in which it was made or an organism with which it is interacting. In this case, the definition of chemical ecology can be extended to include any study of molecular information transfer in biological systems. In an article on signal transduction by Clardy [56], it is argued that chemical ecology can include the effect that extracellular molecules can have on intracellular processes.

68

ECKERMAN and GRAHAM

Similarity in signaling pathways in different biological systems allows for the use of the secondary metabolites to probe cellular signaling. These studies can subsequently lead to the development of pharmaceutical agents [56]. For example, tetrandrine is a human L-type calcium channel blocker used to treat angina and hypertension isolated from a Chinese herb, Stephania tetrandra [57]. Evidence suggests that tetrandrine was produced to control the calcium channels within the original plant [48, 58]. The apparent homology of the receptors in animal and plant membranes suggests that knowledge of substrate-receptor interactions in other biological systems could lead to the development of new pharmaceuticals. Studying the diverse interspecies interactions revealed by chemical ecology may lead to the identification of key points of biochemical regulation. Molecular mechanisms have evolved that enlist and modify useful structures for new purposes. A clear understanding of the structure of a drug discovery target and its relationship to other proteins will lead to a more efficient search for potent and selective drugs. This understanding will come only from the detailed study of organisms in complex environmental settings and an increased attention to identifying the potential use of secondary metabolites as drugs. ECOLOGY-BASED

SEARCHES TO LOCATE SOURCES OF

ANTIFUNGAL AGENTS Evidence suggests that most secondary metabolites exist to protect an organism from pathogens or predators [47]. Thus, an understanding of chemical ecology appears to be a reasonable and efficient approach, particularly in the discovery of anti-infective or antifeedant compounds. Biorational criteria, using the ecology of natural systems to reveal organic compounds with antifungal bioactivities, is the underlying theme of this approach [55]. Ecological clues point to a variety of sources which are expected to produce fungistatic secondary metabolites and become an obvious target for antifungal research. Therefore, biorationale predicts organisms encountering fungal competitors or pathogens will be a good source of fungistatic or fungicidal chemicals. Studies of antagonistic species can provide useful information for scientists interested in chemical ecology, but can also serve as a valuable complement to random, high-throughput screening for new antifimgal compounds. NEW ANTIFUNGAL COMPOUNDS LOCATED BY CHEMICAL ECOLOGY

Several antifungal compounds which are capable of producing the above effects have been isolated and characterized using an ecology-based


69

approach. The following section is not intended to be an exhaustive list of antifungal drugs from natural sources but is simply a general overview of the applications of chemical ecology in the search for antimycotics.

Fungi Research has shown fungi to be excellent sources of novel bioactive metabolites which have been insufficiently explored [59, 43, 60]. Mechanisms of fungal antagonism and defense often include the production of biologically active metabolites by one species that exert effects on potential competitors and/or predators. Fungi chosen for investigation based on ecological considerations, such as evidence of interspecies antagonism, will likely be good sources of fungistatic or fungicidal chemicals [61-64]. In fact, the pneumocandins discussed earlier were isolated from the fungus Zalerion arboricola [38, 65]. These compounds appear to be the causative agents of antagonistic effects of Zalerion arboricola against a competing fungus on its natural substrate. Examples of antagonistic fungi include coprophilous [66, 67], aquatic [6870], lignicolous [71, 72], and saprophytic fungi [73, 74]. H CH

HO,,

HO HO

~

O

H

H3C

0". H. - 0

O

16

17

HO

v

18

H

HO

OH

H

OH 19

COOH OH NH2

E C K E R M A N and G R A H A M

70

Several reviews on the use of coprophilous fungi from competitive ecosystems as sources of antifungal natural products have been written by Gloer [61-63]. Coprophilous fungi are those which colonize the dung of herbivorous vertebrates. Many of these fungi are known to produce antifungal agents that inhibit the growth of competitors. Merck has reported several broad-spectrum antifungal agents from common coprophilous fungal species: australifungin, 16, from Sporormiella australis [75]; the sonomilides, 17, [76]; the zaragozic acids (e.g. 18), isolates from Sporormiella intermedia [77] and the sphingofungins, 19, from Paecilomyces variotti [78, 79]. Gloer's research group has also described several novel secondary metabolites isolated from coprophilous fungi with antifungal activity (e.g. 20-32) [80-89]. Given the high incidence of antimycotic activity in this category of fungi, it seems likely that this is a reasonable source of antifungal lead compounds. In fact, many of the compounds isolated from these fungi have shown potent effects against other coprophilous fungi, but only a few have shown activity against medically relevant fungi. i

H Oso. ~

~H..

~

21

20

.... t,

.OH

o,,:::

I

22 m u

OH

J'"

n ~

23

0

J "eH

,,iT' 24 25


71

OH

QH

"

oi~

26

27

Q....{ ~..~o 7;

~ CH3

|

~ CH3

28

~

H3

k o

OH H

OCH3

29

ao ~ OH .COOH

o ~.,."%~

o#'-,o. ~

31

ECKERMAN and GRAllAM

72

( /

T-o

0 31

OH m

OH

32 stachybotrin A 33 stachybotrin B

R = OH R = H

Studies of antagonistic aquatic fungi have also been revealed as a potential source of antifungal lead compounds. Review articles [68-70] discussing aquatic fungi indicate that very little research has been done on this category of fungi. Gloer has isolated a number of antifungal compounds from antagonistic aquatic fungi including stachybotrin A and B (32 and 33) [90], kirschsteinin, 34, [91] and anguillosporal, 35, [92]. The sesquiterpenedione culmorin, 36, [68] was isolated from the lignicolous marine fungus Leptosphaeria oraemaris and was identified as an agent responsible for interference competition between different marine fungi. A review by Shearer suggests that other lignicolous freshwater fungi could be sources of antifungal compounds [71 ].


73

OCH a

OH

0

OH

0

34

HO

H

HO-

OH

35

36

Antagonistic interactions between wood-rotting fungi have been studied because fungal decay can drastically reduce the market value of lumber. Fungitoxic compounds produced by antagonistic lignicolous fungi were isolated by Ayer , 37, [92, 93] and Strunz, 38, [94]. While these compounds are of interest for production of lumber, there is little evidence as yet that these compounds will be active against medically relevant fimgi. H

H

O

H3C OH 37' Trichodermal

38

Scytalidin

74

ECKERMAN and GRAHAM

Mycoparasitism involves the invasion of hyphae or sclerotia of one fungal species by another. As part of this process, some mycoparasites produce antifungal compounds which damage host cells. In particular, mycoparasites of the pathogenic fungus, Aspergillus sp. would be predicted to be potential sources of antifungal drugs [96]. Recent investigations of mycoparasitic fungi by Gloer have resulted in several known antifungal metabolites as well as two new tetraketides, three new monocillin analogs and some new substituted benzoate esters [96, 97]. In terms of saprophytic fungi, Choudhury [98] reported the isolation of two new secondary metabolites, 39 and 40, from Sporothrixflocculosa and Sporothrix rugulosa, fungicolous hyphomycetes found on sporocarps of other fungi. Other examples of antagonism between saprophytes and pathogens in the phyllosphere has been reviewed by Blakeman [99]. It appears that the antagonistic interactions of saprophytes and plant pathogens are usually based on competition for nutrients.

39

0 OH

Chemical ecology of dimorphic fungi also promises to be a source of potential antifungal lead compounds. A common feature of the majority of human pathogenic fungi is that they exhibit dimorphism. Pathogenicity in C. albicans appears to be linked to its ability to switch rapidly between morphological forms: yeast cells (single celled) and mycelia [100]. This ability of C. albicans to alter its phenotype rapidly appears to allow the fungus to adapt to different host environments [101-105]. Because an understanding of the biochemical regulation would be helpful in the design of new antifungal drugs, the molecular mechanism for control of filament formation is being studied. A genetic pathway that regulates morphological transition between blastospore and filamentous forms in C. albicans has been discovered [106-108]. Recently, a new model has been developed whereby a transcriptional repressor, TUP1, represses the genes responsible for initiating filamentous growth. This repression is lifted under inducing environmental conditions [ 109, 110]. Many environmental variables such as temperature, media and ratio of CO2 to 02 are known to influence the morphological state of C. albicans; however, no extracellular signal for the control of reversion has been identified [100]. Our preliminary data [111] indicate that a compound secreted by the


75

dimorphic fungus, Candida albicans, can trigger the dimorphic switch. These data suggest that biochemical control of fungal morphology is initiated by an extracellular signal as in the bacterial systems. An extracellular signaling compound responsible for biochemical regulation of dimorphism has been partially purified. Further purification and characterization is in progress. This finding could potentially lead to new antifungal drugs, and is a starting point for deeper understanding of the intracellular mechanism of dimorphism. This is a clear example of the response of an organism to chemical signals in the environment which could lead to the development of a potential pharmaceutical agent. Fungi have been shown to produce the requisite chemical diversity needed in the search for new drug leads. Other types of antagonistic fungi may well prove to be sources of drug leads [64, 112, 113]. More basic research in the area of ftmgal ecology and fungal biochemistry will clearly enable researchers to more effectively locate potential sources of new antifungal compounds. In addition, biorationale predicts fungal sources to be potential sources of other biologically active compounds such as insecticides, herbicides, pharmaceuticals from [61-63, 113]. Plants

Phytochemistry has proven to be fertile ground for antifungal drug discovery [42] and promises to continue to do so given the inherent chemical diversity and relevance of the chemical defense mechanisms in plants in response to fungal infections [114]. An overview of the research in this area is presented as much of this work has been reviewed many times. Plants synthesize and accumulate a broad variety of secondary metabolites to protect themselves from herbivores and infection from microbial pathogens [115, 116]. Compounds which inhibit the development of fungi and bacteria such as alkaloids, phenolics, or terpenoids are often accumulated on the external surface in leaf wax or trichomes [ 115, 116]. Cell walls of some plants have also been shown to contain antimicrobial proteins, such as thionins and defensins (see discussion later). Pathogens which do manage to penetrate these external barriers may encounter further deterrents, such as enzymes which can hydrolyze pathogen walls or biopolymers which restrict further pathogen development [ 115, 116]. It has been suggested that these enzymes, such as chitinases and glucanases, could be medically useful antifimgal drugs [117]. In addition, certain plant cells maintain high levels of antimicrobial secondary metabolites (constitutive chemicals) such as various phenols, flavenoids, alkaloids, coumarins, or cyanogenic glycosides [ 116, 118-120]. These products are often found in strategically located sites such as epidermal tissues or latex. Some of these products are activated by

76

ECKERMAN and GRAHAM

wounding; these compounds are often quickly oxidized to highly reactive antimicrobial phenols, quinones, and free radicals. After this rapid constitutive response to a pathogen, many plants begin to biosynthesize and accumulate low molecular weight, antimicrobial, lipophilic compounds (phytoalexins) in and around the site of infection. Screening different genotypes of one plant species with different degrees of resistance to a particular fungal pathogen has established a relationship between phytoalexin quantity and the extent of resistance [116, 121,122]. There are over 300 known phytoalexins with a striking amount of chemical diversity; phenolic acids, pyrones, flavenoids, pterocarpans, terpenoids and coumarins have all been reported as antimicrobial phytoalexins [118120]. In general, a plant may produce several phytoalexins in response to infection by a pathogen and many of these compounds are not otherwise found in the plant. Phytoalexins are not transported, so their protective effect is limited to the region of the infection and their synthesis is strictly regulated by the plant. Once the pathogen has been deterred, the phytoalexins are degraded; these compounds are thus transient and often do not show up in random screening [ 121,122]. Research on the chemical ecology of secondary metabolites in plants is highly relevant to the utilization of some of these substances in medicine. Over 250 antifungal metabolites originating from plants have been characterized since 1982 [118]. About half of these defense compounds are constitutive agents, while the other half are inducible phytoalexins. Antifungal compounds isolated from higher plants represent a wide variety of chemical classes including terpenoids, aromatics, aliphatics and alkaloids [118-120]. Since plants are known producers of antifungal compounds and because biorationale predicts that plants will produce defensive compounds against plant pathogens, continuing research on the characterization of phytoalexins produced in response to a fungal infection is likely to produce many novel antifungal compounds [123]. However, the success of drug discovery based on direct extraction of vegetative plant material is impacted by the availability of the plant source and the variability in phytochemistry due to genetic and/or metabolic regulation of secondary metabolism reflecting environmental conditions. The use of plant cell culture can address these issues by providing access to a broader array of phytochemicals from each plant species and creating a library of plant cell culture which can be grown and scaled up under controlled conditions whenever needed [124]. Genetic or environmental manipulation, hormones, or pathogen infection can be applied to the cell culture to induce phytochemical expression in the extract [125]. Recent developments in plant cell technology has allowed for the production of a number of plant products through cell culture. Shikonin, a red pigment produced by Mitsui and Co. from Lithosperm~m cells is a well known example of an industrial application of this approach [126].


77

In particular, Phytcra, Inc. has worked on developing protocols to establish and manipulate plant cell suspension cultures derived from native plants to locate antifungal drug leads. This work has led to the characterization of several compounds, 41-45, [127] with antifungal activity produced by from plant cell culture. In addition, this same company recently reported the characterization of sunillin, an antifungal compound which has never been isolated from the native plant [128].

oX

H a C O J J ~j~ OCH 3

OCHa 42 Chelerythrine

41 Chelfl

)

,~o macarpme

HO

i (HOH2C • ~

~

HO"

]

OH

O v ~ ~ O H

44 Verbascoslde

OH OH H~ H(>

OH OH

OH OH

45 Myricoside

78

ECKERMAN and GRAHAM

Sunilllin was isolated from an extract of a plant cell culture in which the host defense pathways had been triggered [128]. The development of sunillin as a marketable drug is currently being explored. Plants have been shown to be producers of antifungal compounds as a chemical defense system against fungal pathogens. In particular, exploring the chemical response of a plant (either the native plant or the plant cell culture) to a pathogen or fungal cell compounds is an ecology-based approach for location of transient, plant-derived antifungal compounds.

Marine Invertebrates and Microorganisms Marine natural products research has resulted in the isolation of numerous diverse and novel chemical structures with potent biological activities [44, 68, 69, 128]. Extensive screening of marine invertebrates for antifungal activity has been carried out over the last three decades [129-130]. More than 100 antifungal metabolites have been isolated from marine invertebrates and some have been developed for clinical use. While biorationale would predict that some of the soft-bodied invertebrates such as sponges need chemical defenses against predators [131 ] and microbial pathogens, most of the known antifungal compounds were discovered using high-throughput screening. Very little work has been done using chemical ecology of the marine organisms in their environment to locate antifungal drugs. In fact, it should be emphasized that some of the compounds isolated from marine invertebrates are believed to be produced by symbiotic microorganisms such as blue-green algae, bacteria or fungi [ 129]. Recent research in marine microbiology has focused on a search for pharmacologically active compounds [132], but these efforts have been hindered by a lack of understanding of the ecology of these complex systems. Current research on the chemical ecology of marine macroorganisms and associated microorganisms will increase the understanding of the role of antifungal compounds and facilitate the search for new antifungal agents [69, 13 l, 132]. In a review by Paul and Fenical, the chemical ecology of tropical marine algae was examined [133]. Consideration of the chemical ecology led to the use of ecologically relevant bioassays. In particular, antimicrobial assays were performed against known strains of marine bacteria and fungi as well as the usual terrestrial pathogenic microorganisms. The results of this study indicate that many of the algal metabolites have antifungal activity, and some are active against medically relevant fungi [133]. In particular, the sesquiterpenoid, 46, from Penicillus capitatus and two metabolites of Halimeda, 47 and 48, showed activity against Candida albicans. This approach should provide more information about the possible antimicrobial roles of secondary metabolites in the marine environment.


46

79

"OAc

Ac

Ac

OAc ~

~

CHO CHO~. CHO

H~ 48

Probably one of the most striking examples of the identification of antifungal compounds based on chemical ecology involves epibiosis. Marine microorganisms living on the surface of other organisms are called epibionts and play a significant role in the ecosystem [69]. Embryos of the shrimp Palaemon macrodactylus are resistant to infection by the fungus Lagenidium callinectes, a known pathogen of crustaceans. This resistance is due to the antifungal compounds 2,3-indolinone, 49, and tyrosol, 50 produced by bacteria isolated from the surface of the embryos [69, 134]. This observation suggest that aquatic plants and animals may be protected from pathogenic fungi by epibiontic bacteria. O

H

49

50

More research on the detailed interspecies interactions of marine and aquatic ecosystems are needed to improve our understanding of underwater habitats and to protect these ecosystems [135]. Recently, the coral reefs near Jamaica have been replaced by regions dominated by algae.

80

ECKERMAN and GRAIIAM

This change appears to be due to an imbalance of the ecosystem which allowed a pathogenic fungus to invade the natural reef population [135]. Somehow the natural antifungal protection was lost; the details of this development are unclear. Perhaps this protection was conferred by an epibiontic bacteria, although this explanation is speculative at this point. Only by preserving these ecosystems and this biodiversity can questions like this one be investigated. Bacteria

One of the first antifungal compounds to be used clinically was nystatin, 2 [ 136]. Nystatin was isolated by Brown and Hazen from the soil bacteria Streptomyces noursei. Since that time, more than 100 polyene macrolide antibiotics have been isolated from Streptomycetes, including Amphotericin B, 1, [136]. In addition, numerous other antifungal compounds have been discovered from soil bacteria and actinomycetes. For example, griseofulvin, 51, from Penicillium griseofulvum is used for treatment of topical mycoses. Cycloheximide, 52, is a powerful fungicide isolated from Streptomyces griseus which is too toxic for clinical use but has found utility as an agricultural fungicide. These bacteria continue to be valuable sources of chemically diverse antifungal agents, and major pharmaceutical companies continue to screen these bacteria as potential sources of antimycotics [137-141 ]. According to one study, at least one quarter of all actinomycetes produce antimicrobial metabolites [142]; it appears that the antagonistic interactions of the actinomycetes with soil fungi are usually based on competition for nutrients. Soil bacteria have been and continue to be among the most productive sources of diverse antifungal lead compounds, a fact which is not surprising given that an ecological niche must be shared with fungal competitors.

o.) . o

r 12

51

0

52


81

In a system similar to the marine epibiontic bacteria, rhizobacteria appear to confer disease resistance to the host plants. For example, treatment of sugar beets with the rhizobacterium Pseudomonas in nonsterile soil has been shown to result in enhanced plant growth and severely reduced fungal root colonization (up to 62%) [143]. Rhizobacteria clearly may be sources of antifungal compounds for pharmaceutical or agricultural use.

Insects

Insects are a largely unexplored source of pharmaceutical agents. However, biorationale predicts that many insects could benefit from antibacterial and antifungal compounds in order to protect food stores as well as eggs and larvae. Myrmicacin, 53, isolated from the metathoric glands of leaf-cutting ants Atta sexdens and Atta cephalotes, prevents sprouting of intruding fungal spores in the nest. This is an important strategy because these ants cultivate a particular strain of fungus as a food source for larvae, and therefore need to maintain a pure fungal culture. The same compound is apparently used to protect seed stores by the harvest ant Messor barbarus [ 144].

~COOH 53

Leaf-cutting ants cultivate fungi by storing leaves in the nest on which the fungus will grow. Consequently, these ants present interesting probes for broad-spectrum antifungal compounds because they will avoid food sources which may endanger their fungus culture [50-53]. There are strong indications that epiphylls which deter ,4tta cephalotes from feeding on their host plants actually do so by producing antimycotics which could endanger the ants' fungus garden [145]. Another instance in which an insect species potentially employs antifungal compounds is illustrated by the American burying beetle, Nicrophorus americanus. As the name suggests, burying beetles inter small animal or bird carcasses in the soil to feed their larvae, and the carcass is coated with secretions to retard decay [ 146]. Presumably these secretions contain antibacterial and antifungal compounds. Currently research on this insect and the secretions it employs is hindered by the

82

ECKERMAN and GRAHAM

beetle's endangered status, but this point underscores the need to preserve biodiversity if we are to exploit the vast advantage evolution has had in developing antifungal compounds over millions of years. Antifungal compounds in insects are not limited to small molecules. For example, the larvae of many insects have been found to contain antimicrobial proteins, notably in cecropia moths [147]. Because these types of compounds have been shown to be ubiquitous, occurring in vertebrates as well as plants and insects, their discussion is relegated to a subsequent section. Vertebrates

In the past decade a number of vertebrate species have been shown to produce antifungal compounds. The dogfish shark, Squalus acanthius, faces potential problems from fungal infections of its reproductive system because sharks lack placentas and must flush their wombs with microbeladen sea water to remove fetal waste. Research into this system revealed that the shark produces a potent antimicrobial steroid, squalamine, 54, which is highly effective against Candida [148]. Sharks also produce a limited number of antibodies when compared to other vertebrates, and it may be that this deficit is alleviated through the use of antimicrobials such as squalamine.

CH, CH

""oH

H2 54

Amphibians generally require a moist environment, as do fungi, and consequently many amphibian species may benefit from antifungal compounds. The observation that African Clawed Frogs with wounds rarely became infected, despite an environment conducive to microbial propagation, led to the discovery of the magainans [149-152]. Magainans are polypeptide antimicrobial compounds which have since been observed in a wide variety of frog skins [153, 154].


83

Antimicrobial Peptides There has been an explosion of recent interest in antimicrobial peptides, which have been shown to be ubiquitous in nature as evidenced in numerous review articles [155-159]. Frequently, these peptides have been located where microbial control is important, such as in mammalian intestines or trachea, in seminal fluid, on amphibian skin, as plant defense compounds or on insect larvae. Hence, such peptide antibiotics can be seen as a response to ecological conditions, specifically the propensity of microorganisms to thrive wherever they can gain access to a macroorganlsm. Antimicrobial peptides can be relatively low molecular weight compounds (3000 to 5000) such as cecropins, defensins and magainins, or higher molecular weight proteins such as lysozyme and serprocidins. Several of these compounds, including magainins, defensins, and the serprocidin, proteinase 3, have demonstrated potent antifungal activity. The diversity of these families of antimicrobials, which may stem from a need to overcome pathogen resistance, makes them a particularly bountiful source of pharmaceutical lead compounds. This area of research has garnered intense interest recently, with literally hundreds of articles being devoted to antimicrobial peptides in the last five years. CONCLUSION While chemical ecology will not replace other methods for locating sources of antifungal compounds, it is nevertheless an important and effective technique. Consideration of situations in which an organism would have a particular need to defend itself from fungi is a logical starting point from which to find leads for antimycotics. In several instances, questions about how an organism would cope with its environment have led directly to the discovery of compounds with antifungal activity which play a significant role in preserving the organism within its ecosystem. In this manner, the advantage that evolution has used in developing bioactive compounds can be exploited. REFERENCES [l] [2] [3]

DuPont, P., "Candida albicans, the Opportunist: a Cellular and Molecular Perspective", d. Am. Pod. Med. Assoc., 1995, 85(2) 104-115. Male, O., "The Significance of Mycology in Medicine", In Frontiers in Mycology, D.L. Hawksworth, Ed.; CAB International: Regensburg, 1991, Chapter 7. McCullough, M.; Ross, B.; Reade, P.; "Oral Candida albicans from patients infected with the human immunodeficiency virus and characterization of a

84

ECKERMANand GRAtlAM

genetically distinct subgroup of Candida albicans", Austr. Dent. J., 1995, 40(2) 91-97. [4] Cater, R.; "Chronic Intestinal Candidiasis as a Possible Etiological Factor in chronic Fatigue Syndrome", Med. Hypotheses, 1995, 44, 507-515. [5] Still, J.; Law, E.; Belcher, K.; Spencer, S.; "A Comparison of Susceptibility to Five Antifungal Agents of Yeast Cultures from Bum Patients", in Burns, Elsevier Science, Ltd.: Great Britain, 1995, 21(3) 167-170. [6] Burger, S.; Classen, D.; Burke, J.; Blatter, D.; "Candidal Brain Abscess Associated with Vascular Invasion", Clin. Infect. Diseases, 1995, 21,202-205. McCullough, M.; Ross, B.; Dwyer, B.; Reade, P.; "Genotype and phenotype of [7] oral Candida albicans from patients infected with the human immunodeficiency virus", Microbiology, 1994, 140, 1195-1202. Lim, E.; Stem, P.; "Candida Infection after Implant Arthroplasty", J. Bone Jt. [8] Surg., 1994, 68-,4(1), 143-145. Bozzette, S.; Gordon, R.; Yen, A.; Rinaldi, M.; Ito, M.; Fierer, J., "Biliary [9] Concentrations of Fluconazole in a Patient with Candidal Cholecystitis", Clin. Infect. Dis., 1992, 15, 701-703. [lo] Cooley, D.; Burnett, C.; "Fungal Infection in a Dissecting Aneurysm of the Thoracic Aorta", Texas Heart Institute Journal, 1993, 20 (1), 51-54. [11] Wessel, A.; Simon, C.; Regensburger, D.; "Bacterial and fungal infections after cardiac surgery in children", Eur. J. Pediatr., 1987, 146, 31-33. [12] Barber, G.; Miransky, J.; Brown, A.; Colt, D.; Lewis, F.; Thaler, H.; Kiehn, T.; Armstrong, D.; "Direct Observations of Surgical Wound Infections at a Comprehensive Cancer Center", Arch. Surg., 1995, 130(10), 1042-1047. [131 Lopez, P.; Bastida, M.; Martinez, J.; Ribas, J.; Jove, N.; "Acute Cholecystitis and Wound Infection due to Candida albicans", Eur. J. Clin. Microbiol. Infect. Dis., 1995, 14 (3) 253. [14] Docimo, S.; Kang, J.; Rukstalis, D.; Cotton, D.; Rukstalis, M.; DeWolf, W.; "Candida Epididymitis", Urology, 1993, 41 (3) 280-282. [15] Smith, L.; "Right Upper Quadrant Pain in Diabetic", Patient Care, 1994, 28 (12), 154. [16] Rinaldi, M.; "The Escalating Significance of Fungal Infections and the Need for New Antifungal Agents", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. [17] Hazen, K.; "New and Emerging Yeast Pathogens", Clin. Microbiol. Rev., 1995, 8(4), 462-478. Odds, F.; "Candida Species and Virulence", Am. Soc. Microbiol. News, 1994, 60(6), 313-318. [19] Banerjee, S.; Emori, T.; Culver, D.; Gaynes, R.; Jarvis, W.; Horan, T.; Edwards, J.; Tolson, J.; Henderson, T.; Marton, W.; "Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989", Am. J. Med., 1991, 91(Suppl. 3B) 86S-89S. [20] Badenhorst, L.; Botha, P.; Van Rensburg, M.; "The incidence of hospital fungal infections -- yeast fungaemia", South Afr. Med. J., 1991, 79, 302-303. [21] Clark, A.; "The Need for New Antifungal Drugs", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 1-19. [22] Richardson, K.; Marriott, M.; "Antifungal Agents", Ann. Rep. Med. Chem., Academic Press, Inc., 1987, Vol. 22, Chapter 16, 159-167.


85

Koltin, Y., "Targets for Antifungal Drug Discovery", Ann. Rep. Med. Chem., Academic Press, Inc., 1989, Vol. 25, Chapter 15, 141-148. [24] Cossum, P.; "Preclinical Evaluation of a Liposomal Nystatin Formulation", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, N J; Strategic Research Institute: New York, New York, 1995. [25] Ryder, N.; "Recent Advances with the Allylamine Antimycotic, Terbinafine", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. [26] Kerridge, D.; "Modes of Action of Antifungal Agents", Adv. Microb. Physiol., 1986, 27, 1. [27] Powderly, W.; "Fungal Infections", Journal of Physicians in AIDS Care, 1996, 2(10). [28] MacDougall, D.; "Opportunistic Infections: The Growing Challenge", Journal of Physicians in AIDS Care, 1996, 2(10). [29] Smart, T.; "ICAAC's Future Fungal Fighters", Gay Men's Health Crisis, 1996, 10(10), 10-12. [301 Stephenson, J., "Investigators Seeking New Ways to Stem Rising Tide of Resistant Fungi", d. Am. Med. Assoc., 1997, 277(1), 5-6. [31] Gallagher, P.; Bennet, D.; Henman, M.; Russell, R.; Flint, S.; Shanley, D.; Coleman, D.; "Reduced Azole Susceptibility of Oral Isolates of Candida albicans from HIV-positive Patients", d. Gen. MicrobioL, 1992, 138, 1901-1911. [32] Nguyen, M.; Morris, A.; Tanner, D.; Nguyen, M. ; Snydman, D. Wagener, M.; Rinaldi, M.; Yu, V.; "The Changing Face of Candidemia: Emergence of NonCandida albicans Species and Antifungal Resistance", Am. J. Med., 1996, 100. 617-623. [33] Kerridge, Marco and Wayman, "Drug Resistance in Candida albicans and Candida glabrata", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 245. [34] Scherer, S.; Magee, P.; "Genetics of Candida albicans", Microbiol. Rev., 1990, 54(3), 226-241. [35] Chakrabarti, A.; Ghosh, A.; Batra, R.; Kaushal, A.; Roy, P.; Singh, H.; "Antifungal Susceptibility Pattern of non-albicans Candida species and Distribution of Species Isolated from Candidaemia Cases", Indian J. Med. Res., 1996, 104, 171-176. [36] Hitchcock, C.; "Resistance of Candida Species to Azole Antifungal Agents", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, N J; Strategic Research Institute: New York, New York, 1995. [37] Sanglard, D. 1997. Cloning of Candida albicans Genes Conferring Resistance to Azole Antifungal Agents: Characterization of CDR2, a New Multi-drug ABC Transporter Gene", Microbiology, 1997, 143, 405-416. [38] Gordee, R.; Debono, M.; Parr, T.; "The Fungal Cell Wall-- A Target For Lipopeptide Antifungal Agents", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 46-63. [39] Oki, T.; "Pradimicin, A Novel Antifungal Agent", In New Approaches for Antifungal Drugs, P. Femandes, Ed., Birkhauser: Boston, 1992, 64-87. [40] McCullough, J.; "Importance of Chitin Synthesis for Fungal Growth and a s a Target for Antifungal Agents", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 32-45. [41] Cragg, G. M.; Newman, D. J.; Snader, K. M.; "Natural Products in Drug Discovery and Development", J. Nat. Prod, 1997, 60(1), 52-60. [23]

86

[42] [43]

[44] [45] [46] [47] [48] [49]

[50] [51]

[52] [53] [541

[55] [56] [57]

[581 [591

[60]

ECKERMAN and GRAHAM

Balandrin, M.; Kinghom, A.; Famsworth, N; In Human Medicinal Agents from Plants, Kinghom and Balandrin, Eds., ACS Symposium Series 534, American Chemical Society: Washington, D.C., 1993, 2-12. Hawksworth, D.; "The Fungal Dimension of Biodiversity: Magnitude, Significance, and Conservation", Mycol. Res., 1990, 95, 641. Rinehart, K; "Screening to Detect Biological Activity", In Biomedical Importance of Marine Organisms, Fautin, Ed., California Academy of Sciences: San Francisco, 1988, 13-22. Cordell, G.; "Changing Strategies in Natural Products Chemistry", Phytochemistry, 1995, 40(6), 1585-1612. Waterman, P.; "Searching for Bioactive Compounds: Various Strategies", J. Nat. Prod, 1990, 53(1), 13-22. Rouhi, M.; "Seeking Drugs in Natural Products", Chem. Eng. News, 1997, 75(14), 14-29. Caporale, L.; "Chemical Ecology: A View from the Pharmaceutical Industry", Proc. Natl. Acad. ScL, USA, 1995, 92, 75-82. Eisner, T.; Meinwald, J.; In Chemical Ecology: The Chemistry of Biotic Interaction, T. Eisner and J. Meinwald, Eds., National Academy Press: Washington, D.C., 1995. Hubbell, S.; Wiemer, D.; Adejare, A.; "An antifungal terpenoid defends a neotropical tree against attack by fungus-growing ants", Oecologia, 1983, 60, 321-327. Hubbell, S.; Howard, J.; Wiemer, D.; "Chemical Leaf Repellency to an Attine Ant" Seasonal Distribution Among Potential Host Plant Species", Ecology, 1984, 65(4), 1067-1076. Rockwood, L.; Hubbell, S.; "Host-plant selection, diet diversity, and optimal foraging in a tropical leaf-cutting ant", Oecologia, 1987, 74, 55-61. Rockwood, L; "Plant Selection and Foraging Patterns in Two Species of Leafcutting Ants", Ecology, 1976, 57, 48-61. Bruckner, B.; In Secondary Metabolites: Their Function and Evolution, Ciba Foundation Symposium 171, D. Chadwick and J. Whelan, Eds., Wiley: Chichester, U.K., 1992, 129-143. Eisner, T.; "Prospecting for Nature's Chemical Riches", Chemoecology, 1990, 1, 38-40. Clardy, J.; "The Chemistry of Signal Transduction", In Chemical Ecology: The Chemistry of Biotic Interaction, T. Eisner and J. Meinwald, Eds., National Academy Press: Washington, D.C., 1995. King, V.; Garcia, M.; Himmel, D.; Reuben, J.; Lam, Y.; Pan, J.; Han, G.; Kaczorowski, G.; "Interaction of Tetrandrine with Slowly Inactivating Calcium Channels", J. Biol. Chem., 1988, 263, 2238-2244. Graziana, A.; Fosset, M.; Ranjeva, R.; Hetherinton, A.; Lazdunski, M.; "Ca+2 Channel lnhibitors that Bind to Plant Cell Membranes Block Ca+2 Entry into Protoplasts", Biochemistry, 1988, 27, 764-768. Dreyfuss, M.M.; Chappella, I.; "Potential of fungi in the discovery of novel, low -molecular weight pharmaceuticals", In The Discovery of Natural Products with Therapeutic Potential V.P. Gullo, Ed., Butterworth-Heinemann: Boston, 1994, 49-80. Crittenden, P.; Porter, N.; "Lichen-forming fungi: potential sources of novel metabolites", TIB Tech., 1991, 9, 409-414.


[61] [62] [63]

[64] [65]

[66] [67]

[68] [69] [70] [71] [72] [73] [74]

[75]

[76]

[77]

87

Gloer, J., "The Chemistry of Fungal Antagonism and Defense", Can. J. Bot., 1995, 73(1), S 1265-S 1274. Gloer, J.; "New Antifungal and Anti-insectan Natural Products from Fungi: An Ecology-Based Approach", Rev. Latinoam. Quire., 1996, 24, 191-208. Gloer, J.; "Fungi from Competitive Ecosystems as Sources of Novel Bioactive Natural Products", Dev. Ind. Microbiol., 1993, 33, 1-11. Hussain, S.; Lane, S.; "Fungi .vs Fungi: A Potential Application of Antagonistic Properties", Mycologist, 1992, 6, 29-30. Schwartz, R.; Sesin, D.; Joshua, H.; Wilson, K.; Kerripf, A.; Goklen, K.; Kuehner, D.; Gaillot, P.; Gleason, C.; White, R.; Inamine, E.; Bills, G.; Salmon, P.; Zitano, L.; "Pneumocandins from Zalerion arboricola: Discovery and isolation", J. Antibiot., 1992, 45, 1853-1866. Webster, J.; "Coprophilous Fungi", Trans. Br. Mycol. Soc., 1970, 54, 161-180. Wicklow, D.; Hirschfield, B.; "Evidence of a competitive hierarchy among coprophilous fungal populations", Can. J. Microbiol., 1979, 25, 855-858. Liberra, K.; Lindequist, U.; "Marine Fungi -- A Prolific Resource of Biologically Active Natural Products?", Pharmazie, 1995, 50, 583-588. Konig, G.; Wright, A.; "Marine Natural Products Research" Current Directions and Future Potential", Planta Med., 1996, 62, 193-211. Shearer, C.; Zare-Maivan, H.; "In vitro hyphal interactions among wood- and leafinhabiting ascomycetes and fungi imperfecti from freshwater habitats", Mycologia, 1988, 80, 31-37. Strongman, D.; Miller, J.; Calhoun, L.; Findlay, J. ; Whitney, N.; " The biochemical basis for interference competition among some lignicolous marine fungi", Bot. Mar., 1987, 30, 21-26. Ginns, J.; Lefebvre, M.; Lignicolous Corticoid Fungi of North America: Systematics, Distribution, and Ecology, American Phytopathology Society: St. Paul, MN, 1993. Gains, W.; "The analysis of communities of saprophytic microfungi with special reference to soil fungi, In Fungi in Vegetation Science, W. Winterhoff, Ed., Norwell-Kluwer Academic Publishers, 1992, 183-223. Fokkema, N.; "Antagonism between fungal saprophytes and pathogens of aerial plant surfaces, In Microbiology of aerial plant surfaces, C. Dickinson, T. preece, Eds., Academic Press: New York, 1976, 487-506. Mandala, S.M.; Thornton, R.A.; Frommer, B.R.; Curotto, J.E.; Rozdilsky, W.; Kurtz, M.B.; Giacobbe, R.A.; Bills. G.F.; Cabeilo, M.A.; Martin, I.; Pelaez, F.; Harris, G.H.; "The Discovery of Australifungin, a Novel Inhibitor of Sphinganine N-acetyltransferase from Sporomiella australis I. Producing organism, fermentation, isolation and biological activity", d. Antibiot., 1995, 48(5), 349356. Morris, S.A.; Curotto, J.E.; Zink, D.L.; Dreikom, S.; Jenkins, R.; Bills, G.F.; Thompson, J.R., Vicente, F.; Basilio, A.; Liesch, J.M.; Schwartz, R.E.; "Sonomolides A and B: New, broad-spectrum antifungal agents isolated from a coprophilous fungus", Tetrahedron Lett., 1995, 36(50), 9101-9104. Bills, G.; Pelaez, F.; Polishook, J.; Diez-Matas, M.; Harris, G.; Clapp, W.; Dufresne, C.; Byme, K.; Nallin-Omstead, M.; Jenkins, R.; Mojena, M.; Huang, L.; "Distribution of Zaragozic Acids among filamentous ascomycetes", Mycol. Res., 1994, 98, 733-739.

88

[78]

[79]

ECKERMAN and GRAHAM

Horn, W.S.; Smith, J.L.; Bills, G.F.; Raghoobar, S.L.; Helms, G.L; Kurtz, M.B.; Marrinan, J.A.; Frommer, B.R.; Thornton, R.A.; Mandala, S.M.; "Sphingofungins E and F: novel serine palmitoyl inhibitors from Paecilomyces variotii", J. Antibiot., 1992, 45(10), 1692-1696. VanMiddlesworth, F.; Giacobbe, R.; Lopez, M.; Garrity, G.; Bland, J.; Bartizal, K.; Fromtling, R.; Polishook, J.; Zweerink, M.; Edison, A.; Rozdilski, W.; Wilson, K.; Monaghan, R.; Sphmgofungins A, B, C, and D; a new family of antifungal agents I. Fermentation, isolation and biological activity", J. Antibiot., 1992, 45, 861-867. Wang, Y.; Gloer, J.B.; Scott, J.A.; Malloeh, D.; "Appenolides A-C: three new antifungal furanones from the coprophilous Podospora appendiculata", J. Nat. Prod., 1993, 56(3), 341-344. Wang, Y.; Gloer, J.B.; Scott, J.A.; Malloch, D.; "Terezines A-D: new amino acid-derived bioactive metabolites from the coprophilous fungus Sporormiella teretispora", J. Nat. Prod., 1995, 58(1), 93-99. AIfatafta, A.A.; GIoer, J.B.; Scott, J.A.; Malloch, D.; "Apiosporamide, a new antifungal agent form the coprophilous fungus Apiospora montagnei", J. Nat. Prod., 1994, 57(12), 1696-1702. Weber, H.A.; GIoer, J.B.; "Interference competition among natural fungal competitors: an antifungal metabolite from the coprophilous fungus Pr'eussia fleischhakii", J. Nat. Prod., 1988, 51(5), 879-883. Weber, H.A.; Baenziger, N.C.; Gloer, J.B.; "Podosporin A" a novel antifungal metabolite from the coprophilous fungus Podospora decipiens (Wint.) Niessl.", J. Org. Chem., 1988, 53, 4567-4569. Weber, H.A.; Baenziger, N.C.; Gloer, J.B.; "Structure of Preussomerin A: an unusual new antifungal metabolite from the coprophilous fungus Preussia isomera",J. Am. Chem. Soc., 1990, 112, 6718-6719. Weber, H.A.; Swenson, D.C.; Gloer, J.B.; Malloch, D.; "Similins A and B: new antifungal metabolites from the coprophilous fungus Sporormiella similis", Tetrahedron Lett., 1992, 33(9), 1157-1160. Weber, H.A.; Gloer, J.B.; "The Preussomerins: novel antifungal metabolites from the coprophilous fungus Preussia isomera Cain", dr. Org. Chem., 1991, 56(14), 4355-4360. Lee, K.K.; GIoer, J.B.; Scott, J.A.; Malloch, D.; "Petriellin A" A novel antifungal depsipeptide from the coprophilous fungus Petriella sordida", J. Org. Chem., 1995, 60(17), 5384-5385. Whyte, A.; Gloer, K.; Gloer, J.; Koster, B.; Malloch, D.; "New antifungal metabolites from the coprophilous fungus Cercophora sordarioides", Can. J. Chem., 1997, 75(6), 768-772. Xu, X.; de Guzman, F.S.; Gloer, J.B." Shearer, C.A.; "Stachybotrins A and B" Novel bioactive metabolites from a brackish water isolate of the fungus Stachybotrys", J. Org. Chem., 1992, 57, 6700-6703. Poch, G.; GIoer, J.; Shearer, C.; "New bioactive metabolites from a freshwater isolate of the fungus Kirschsteiniothelia sp.", J. Nat. Prod., 1992, 55, 10901099. Harrigan, G.G.; Armentrout, B.L.; Gloer, J.; "Anguillosporal, a new antibacterial and antifungal metabolite from the freshwater fungus Anguillospora longissima", J. Nat. Prod., 1995, 58(9), 1467-1468. 9!

[80] [81]

IS2] [83] [84]

[85] [86]

[871 [88] [89]

[90] [91] [92]

9


89

[93] Ayer, W.; Miao, S.; "Secondary metabolites of the aspen fungus, Stachybotrys cylindrospora", Can. J. Chem., 1992, 71, 487-493. [94] Ayer, W.; Kawahara, N. "Lecythophorin, a potent inhibitor of blue-stain fungi, from the hyphomycetous fungus Lecythophora hoffmannii", Tetrahedron Lett., 1995, 36, 7953-7956. [95] Strunz, G.; Kakushima, M.; Stillwell, M.; "Scytalidin: A New Fungitoxic Metabolite produced by a Scytalidium Species", J. Chem. Soc. Perkin Trans. I, 1972, 2280-2283. [96] Joshi, B.; Gloer, J.B.; "Antifungal Metabolites from Mycoparasites of Aspergillus flavus Sclerotia", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy" Iowa City, Iowa, 1997. [97] Soman, A.G.; Gloer, J.B.; "New Bioactive Metabolites from the Mycoparasitic Fungi Mortierella vinaceae and Neocosmospora vasinfecta", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy: Iowa City, Iowa, 1997. [98] Choudhury, S.R.; Traquair, J.A.; Jarvis, W.R.; "4-methyl-7,1 l-heptadecadienal and 4-methyl-7,1 l-heptadecandienoic acid: new antibiotics from Sporothrix flocculosa and Sporothrix rugulosa", J. Nat. Prod., 1994, 57(6), 700-704. [99] Blakeman, J.; "Ecological succession of leaf surface microorganisms in relation to biological control", In Biological Control on the Phylloplane, C. Windels, S. Lindow, Eds., American Phytopathological Society: St. Paul, MN, 1985, 6-30. [100] Shepherd; "Pathogenicity of Morphological and Auxotropic Mutants of Candida albicans in Experimental Infections", Infect. Immun., 1985, ~.O.,54 I. [101] Mathews; "Pathogenicity determinants of Candida albicans: potential targets for immunotherapy?", Microbiology, 1994, 140, 1505. [102] Gow; Crombie; Gooday; "Polarized Morphogenesis of Candida albicans: Cytology, Induction and Control", in Candida and Candidamycosis, Tumbay, Ed., Plenum, New York, 1991, 13. [103] Shepherd; "Morphogenetic Transformation of Fungi", in Current Topics in Medical Mycology, McGinnis, Ed., Springer-Verlag, New York, 1985, ~., 278. [104] Scherer; Magee; "Genetics of Candida albicans", Microbiol. Rev., 1990, 54. 3, 226. [105] Odds, F.; "Candida Species and Virulence", Am. Soc. Microbiol. News, 1994, 6._0_0,313-318. [106] Liu; Kohler; Fink; "Suppression of Hyphal Formation in Candida albicans by Mutation of a STEl2 Homolog", Science, 1994, 266, 1723. [107] Kohler; Fink; "Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signalling components have defects in hyphal development", Proc. Natl. ,4cad. Sci. U.S.A., 1996, 93, 13223. [108] Leberer; "Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus, Candida albicans", Proc. Natl. Acad. Sci. U.S.A.,.1996, 93, 132217. [109] Braun; Johnson; "Control of Filament Formation in Candida albicans by the Transcriptional Repressor TUPI", Science, 1997, 277, 105. [110] Magee, P.T.; "Which Came First, the Hyphae or the Yeast?", Science, 1997, 277., 52. [l l l] Tasto, J.J.; Jensen, E.C.; and Graham, K.J., "Isolation of a Compound Responsible for the Biochemical Control of Dimorphism in Candida Albicans",

90

[112] [113] [114] [115] [116] [117] [118] [119] [120] [121]

[122] [123] [124] [125]

[126] [127]

[128]

ECKERMAN and GRAHAM

Annual Proceedings of the National Conference on Undergraduate Research, 1997, 5, 1704. Buczacki, S.T.; "A microecological approach to larch canker biology", Trans. Br. Mycol. Soc., 1973, 61, 315-329. Miller, J.D.; "Mycology, Mycologists and Biotechnology", In Frontiers in Mycology, D.L. Hawksworth, Ed., CAB International: Regensburg, 1991, Chapter 11,225-240. Harborne, J.; "Recent Advances in Chemical Ecology", Nat. Prod Rep., 1996, 83-98. Harbome, J.B.; "Role of secondary metabolites in chemical defense mechanisms in plants", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 126-139. Kuc, J.; "Compounds from plants that regulate or participate in disease resistance", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 213-229. Roberts, W.K.; Laue, B.E.; Selitrennikoff, C.P.; "Antifungal Proteins from Plants", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 141-151. Grayer, R.J.; Harborne, J.B. Harborne, "Anti fungal Compounds from Higher Plants", Phytochemistry, 1994, 37, 19. Clark, A.M.; Hufford, C.D.; "Antifungal Alkaloids", In The Alkaloids, Academic Press, 1992, Vol. 42, Chapter 2, 117-150. Wink, M.; "Allelochemical Properties or the Raison D'Etre of Alkaloids", In The Alkaloids, Academic Press, 1993, Vol. 43, Chapter 1, 1-118. Barz, W.; Bless, W.; Borger-Papendorf, G.; Gunia, W.; Mackenbrock, U.; Meier, D.; Otto, C.; Super, E.; "Phytoalexins as part of induced defence reactions in plants: their elicitation, function and metabolism", In Bioactive Compoundsfrom Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 140-156. Collinge, D.B.; Slusarenko, A.J.; "Plant gene expression in response to pathogens", Plant Molec. Biol., 1987, 9, 389-410. Bailey, J.A.; "Plant-pathogen interactions: a target for fungicide development", In Antifungal Agents: Discovery and Mode of Action, G.K. Dixon, L.G. Copping, D. Hollomon, Eds., BIOS Scientific Publishing: Bristol, 1995. Fowler, M.W.; "Industrial applications of plant cell culture", In Plant Cell Culture Technology, M. Yeoman, Ed., Blackwell: Oxford, 1986, 207-227. Fowler, M.W.; Cresswell, R.C.; Stafford, A.M.; "An economic and technical assessment of the use of plant cell cultures for natural product synthesis on an industrial scale", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 157-174. Fujita, Y.; "Industrial production of shikonin and berberine", In Applications of Plant Cell and Tissue Culture", Wiley: Chichester, 1988, 228-238. Siegel, S.A.; "Accessing and Exploiting Phytochemical Diversity for Antifungal Discovery -- A Natural Approach", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. Pazoles, C.J.; "Plant Cell Cultures as a Source of Chemical Diversity and Novelty for Drug Discovery", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy" Iowa City, Iowa, 1997.


91

[129] Fusetani, N.; "Antifungal Substances from Marine Invertebrates", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences' New York, 1988, 113-127. [1301 Molinski, T.F.; "Antifungal Marine Natural Products and Emerging Resistant Fungal Pathogens", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy: Iowa City, Iowa, 1997. [131] Garson, M.J.; "The biosynthesis of sponge secondary metabolites: Why it is important", In Sponges in Time and Space, R. van Soest, T. van Kempen, J. Braeka, Eds., Balkema: Rotterdam, 1994, 427-440. [132] Fenical, W.; Jensen, P.R.; "Marine Micro-organisms. A New Biomedical Resource" In Marine Biotechnology, D. Attaway, O. Zaborsky, Eds., Plenum Press: New York, 1993, 419-457. [133] Paul, V.J.; Fenical, W.; "Natural Products Chemistry and Chemical Defense in Tropical Marine Algae of the Phylum Chlorophyta", In Bioorganic Marine Chemistry, P. Scheuer, Ed., Springer-Verlag: Berlin, 1987, Vol. 1, 1-29. [1341 GiI-Turnes, M.S.; Hay, M.E.; Fenical, W.; "Symbiotic Marine Bacteria Chemically Defend Crustacean Embryos from a Pathogenic Fungus", Science, 1989, 246, 116-118. [135] Hughes, T.P; "Catastrophes, phase shifts and large-scale degradation of a Carribean reef", Science, 1994, 265, 1547-1551. [136] Omura, S.; Macrolide Antibtiotics: Chemistry, Biology and Practice, Academic Press: Newy York, 1984, Chapters 9-12. [137] Kohno, J.; Nishio, M.; Kawano, K.; Suzuki, S.I.; Komatsubara, S.; "TMC-34, a new macrolide antifungal antibiotic from a Streptomyces sp.", J. Antibiot., 1995, 48(10), 1173-1175. [138] Fujiu, M.; Sawairi, S.; Shimada, H.; Takaya, H.; Aoki, Y.; Okuda, T.; Yokose, K.; "Azoxybacilin, a novel antifungal agent produced by Bacilius cereus NR2991: Production, isolation and structure elucidation", J. Antibiot., 1994, 47(7), 833835. [139] Achenbach, H.; Muhlenfeld, A.; Fauth, U.; Zahner, H.; "The Galbonolides: Novel, Powerful Antifungal Macrolides from Streptomyces galbus ssp. eurythermus", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 128-140. [140] Inouye, Y.; Okada, H.; Nakamura, S.; "Hydroxamic Acid Antimycotic Antibiotics", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 180-182. [141] Kempf, A.J.; Hensens, O.D.; Schwartz, R.E.; Sykes, R.S.; Wilson, K.E.; Wichman, C.F.; Zink, D.L.; Zitano, L.; Mochales, S.; "L-660,631, a New Antifungal Agent", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 183. [142] Barbier, M.; Introduction to Chemical Ecology, Longman Group Limited: London, 1979. [143] Schroth, M.N.; Hancock, J.G.; "Disease-Suppressive Soil and Root-Colonizing Bacteria", Science, 1982, 216, 1376-1381. [144] Schildknecht, H.; Koob, K.; "Myrmicacin, The First Insect Herbicide", Angew. Chem. Internat. Edit., 1971, 10, 124-125. [145] Mueller, U.; Wolf-Mueller, B.; "Epiphyll Deterrence to the Leafcutter Ant Atta cephalotes", Oecologia, 1991, 59, 615-625.

92

ECKERMAN and GRAHAM

[146] Line, L.; "Microcosmic Captive Breeding Project Offers New Hope for Beleaguered Beetle", New Fork Times, 1996, September 17, BS-B6. [147] Boman, H.G.; "Cell-free immunity in insects", Ann. Rev. Biochem., 1987, 41, 103-126. [148] Stone, R.; "Deja Vu Guides the Way to a New Antimicrobial Steroid", Science, 1993, 259, 1125. [149] Zasloff, M.; "Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial eDNA sequence of precursor", Proc. Natl. ,4cad. Sci. USA, 1987, 84, 5449-5453. [150] Bevins, C.L.; Zasloff, M. "Peptides from frog skin", ,4nnu. Rev. Biochem., 1990, 59, 395-414. [151] Zasloff, M.; Martin, B.; Chert, H-C.; "Antimicrobial activity of synthetic magainin peptides and several analogues", Proc. Natl..4cad. Sci. USA, 1988, 85, 910-913. [152] Bechinger, B.; Zasloff, M.; Opella, S.J.; "Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy", Protein Sci., 1993, 2, 2077-2984. [153] Berkowitz, B.A.; Bevins, C.L.; Zasloff, M.; "Magainins: a new family of membrane-active host defense peptides", Biochera. Pharmacol., 1990, 39, 625629. [154] Barra, D.; Simmaco, M.; "Amphibian skin: a promising resource for antimicrobial peptides", TIB Tech., 1995, 13, 205-209. [155] Gabay, J.E.; "Ubiquitous natural antibiotics", Science, 1994, 264, 373-374. [156] Cociancich, S.: Bulet, P.; Hetru, C.; Hoffmann, J.A.; "The inducible antibacterial peptides of insects", Parasitol. Today, 1994, 10, 132-139. [157] Ganz, T.; Lehrer, R.I.; "Defensins", Pharmacol. ?'her., 1995, 66, 191-205. [158] Genz, T.; Lehrer, R.I.; "Antimicrobial peptides of leukocytes", Curt. Opin. Hematol., 1997, 4, 53-58. [159] Cammue, B.; De Bolle, M.; Schoofs, M.M.; Terra, F.; Thevissen, K. Osborn, R.W.; Rees, S.B.; Broekaert, W.F.; "Gene-encoded antimicrobial peptides from plants", In Antimicrobial peptides, J. Marsh, J.A. Goode, Eds., Ciba Foundation Symposium 186, John Wiley & Son's: Chichester, 1994, 9 l- 106.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistpy, Vol. 22

9:1


NATURAL TRITERPENOIDS AS ANTI-INFLAMMATORY AGENTS RIOS, J.L.*; RECIO, M.C.; MAI~IEZ, S. and GINER, R.M.

Departament de Farmacologia, Factdtat de Farmbcia, Universitat de ValEncia, Avda. Vicent Andrds Estellds s/n., 46100 Burjassot (Valbncia),

Spain

ABSTRACT" This chapter reviews the natural triterpenes with anti-inflammatory activity, including the traditional ones and the new compounds isolated over the last six years. Triterpenes are widely distributed in plants, and in many cases are the principles responsible for their anti-inflammatory effects. Many of these compounds are active in different in vivo experimental models such as hind paw edema induced by carrageenan, serotonin and phospholipase A2; ear edema induced by phorbol and daphnane esters, ethylphenylpropiolate, arachidonic acid and capsaicin; adjuvant arthritis and experimental models of allergy. Other effects have been studied in vitro, and some triterpenes are active against inflammatory enzymes like 5-1ipoxygenase, elastase and phospholipase A2. Others inhibit histamine, collagenase and interleukin release, lipid peroxidation and free radical-mediated processes, metabolism of endogenous corticoids, and complement and protein-kinase activities. In certain cases the mechanism of action depends on the skeleton type and/or substituents. For example, 13-boswellie acid (ursane-derived) and derivatives markedly inhibit 5-1ipoxygenase activity, whereas the principal mechanism of 1813-glycyrrhetinic acid (oleanane-derived) is the inhibition of endogenous corticoid metabolism. Some lanostanes are active against phospholipase A2 (e.g. ganoderic and dehydrotumulosic acids), and compounds with highly unsaturated rings can act as anti-peroxidatives (e.g. celastrol, a tetraunsaturated friedooleanane). FOREWORD Although certain kinds of larger molecules originating from the mevalonate synthetic pathway do in fact exist in plants, triterpenes represent the culmination of this metabolic route in the vast majority of botanical taxa. Thanks to the steadily increasing number o f scientific reports on new structures and interesting biological activities of this class of compounds in the recent years, our knowledge o f this area o f Phytochemistry and Pharmacognosy has expanded greatly. As might be expected, a large number of reviews focusing on occurrence and chemical aspects have been published, such as those from Mahato et al. [1-3] and Connolly and Hill [4-12]. The present review differs completely, however, both in aims and content, because it centres on the

94

R[OS et al.

description of the triterpenes for which novel anti-inflammatory properties have been reported from 1992 to 1997 ~ and on additional studies of well-known principles already considered active, such as glycyrrhetinic, ursolic and oleanolic acids, etc. Reports on triterpene glycosides, mostly saponins, are excluded as they are usually studied separately because of their particular physical and biological properties. Nevertheless, their importance is reflected in Pharmacognostic Background section. It should be pointed out that for the sake of coherence, most of the examples illustrating the chemistry section of this chapter are taken from the literature on terpenoids that have effects on the inflammatory process. Additionally, a section on the principles of inflammation and its pharmacology is included before the reports on the activity of triterpenes, which are classified according to the test models and mechanism of action. AN INTRODUCTION TO TRITERPENES

Concept The triterpenoids comprise a large group of diverse C30 natural secondary metabolites having relatively complex cyclic structures, usually tetra or pentacyclic, although acyclic or monocyclic skeletons can also be found. Most of them are alcohols, aldehydes, carboxylic acids or esters, and they are regarded as an important class of compounds in Phytochemistry. Modern isolation and analysis techniques have refined the structural elucidation of many already isolated and also newly found compounds. More than 40 skeletal types arising from the cyclisation and subsequent rearrangements of their biosynthetic precursor squalene can be distinguished. Most of the triterpenes, with the exception of those with hopane and gammacerane skeletons have a 3]3-oxygen function. The attachment of linear or branched sugar moieties to the triterpene framework, usually at the 3-hydroxyl position, results in the formation of a large number of naturally occurring saponins. Disubstitution or, less frequently, trisubstitution of the triterpenoid molecule with sugars is a regular feature for these compounds.

Occurrence Triterpenoids are widely distributed throughout the plant kingdom. Oleananes and ursanes, which often occur together, and lupanes are found *Authors' note: For complementary information see also the review of Safayhi, H.; Sailer, E.-R. Planta Med.,

1997, 63, 487.

NATURAL TRITERPENOIDS

95

in a wide range of families. Lanostanes are common in fungi and marine organisms and also occur in higher plants. The remaining skeletal types are more restricted in their natural occurrence. Cucurbitanes occur in the Cucurbitaceae and they have also been detected occasionally in at least five other families, namely Begoniaceae, Cruciferae, Desfontainiaceae, Elaeocarpaceae and Scrophulariaceae [13]. Dammaranes occur in several plant families, including Anacardiaceae, Rutaceae, Betulaceae and Rubiaceae, friedelanes in Celastraceae and Buxaceae, serratanes in Pinaceae and the Polipodiaceae fern [8], strictanes in lichens and fernanes in ferns. Hopanes are found in lichens, ferns, and certain higher plant families and can also be detected in geochemical samples. They are widespread among the prokaryotes, where they may perform the role of steroids in plants. Gammaceranes are also described in prokaryotes. The quassinoid nortriterpenoids are confined to the Simaroubaceae; the limonoids, by contrast, are found in this family and also, more abundantly, in the three related families, Rutaceae, Meliaceae and Cneoraceae [13].

Biological Significance The physiological role of triterpenoids is not yet wholly understood. However, their function in chemical defence has been established. This class of natural substances is involved in plant-animal and plant-plant interactions that occur in many ecosystems. Cucurbitacins represent the clearest example. These tetracyclie triterpenes of the Cueurbitaeeae normally repel insect feeders, and are therefore sequestered and stored by insects for defensive purposes. Cucurbitacin D, found in some Chrysomelidae species, is a potent kairomone to a phytophagous beetle that feeds specifically on these plants. Storage of this toxin protects against predation by mantids, which, however, have not learnt to avoid the adverse effect of ingesting these beetles. Another example of chemical protection occurs in the paper birch (Betula resinifera). The high content in papyriferie acid of the young internodes and twigs makes them unpalatable and deters grazing by the hare [14]. The tetracyclie triterpenoid quassin is an antifeedant to the aphid Myzus persicae [ 15], and the limonoids azadirachtins from Azadirachta indica are also potent insect antifeedants [ 13]. Ursolic acid has been identified as an allelopathic agent in Ceratiola ericoides and Calamintha ashei on competing sandhill grasses [16]. Lupeol, betulin, betulin aldehyde and betulinir acid from Melilotus messanenis possess potential allelopathic activity on dicotyledon species like Lactuca sativa and Lepidium sativum [17]. As a consequence of plantplant co-evolution, higher plants are able to form haustoria and thus become parasitic on their host plant. The oleanane triterpene soyasapogenol A isolated from the root of Lespedeza sericea, a

96

R|OS et aL

Leguminosae host of Agalinis species, has been described as a haustoriainducing compound [ 16]. Glycinoeclipin A is a pentanortriterpene which stimulates the parasitization of the soybean (Glycine max) by cyst nematodes [ 14]. As a result of plant-microbial parasite interaction, plants elaborate constitutive antifungal agents. Cucurbitacin I protects cucumber from Botrytis cinerea because it inhibits the induction of an enzyme involved in tissue damage and the spread of infection [18].

Pharmacognostic Background An overview on the traditional importance of anti-inflammatory triterpenoids in Pharmacognosy gives rather poor results unless saponins are included. Without them, the list would be reduced to glycyrrhetinic acid from liquorice, papyriogenins from Tetrapanax papyriferum, and triterpenoids from the resins of Commiphora species. However, diverse crude drugs containing triterpenoids in the combined form of saponins have been used extensively for their anti-inflammatory properties, not only in folk medicine but also in modem clinical therapeutics. The most important of these drugs is liquorice root (Glycyrrhiza glabra), which has been used since of the time of the ancient Greeks as an expectorant, antitussive and sweetening agent. In both Chinese and European medicine, liquorice decoctum is employed to treat throat inflammation. It contains about 6-13% glycyrrhizin, a glycoside from glycyrrhetinic acid. The extract and the aglycone have been shown to have corticosteroid-like effects, and are employed for the treatment of rheumatoid arthritis, Addison disease and other inflammatory processes. It has been reported that glycyrrhetinic acid has 1/8 of the anti-inflammatory potency of cortisol, reaching 1/5 of cortisol in the case of carbenoxolone, the sodium salt of glycyrrhetinie acid hemisuccinate [19]. The tincture of Aesculus hippocastanum seeds has been used successfully for haemorrhoids and venous congestion. Aesein, a mixture of oleanane triterpene saponins with a yield of about 13% relative to the crude drug weight, shows anti-inflammatory action, and it is administered orally for clinical use [19]. Among Oriental medicinal remedies there are many herbal drugs, such as ginseng or saiko, which contain triterpene saponins as their principal constituents and the ones that seem to be responsible for their efficacy [20]. Ginseng, the root of Panax ginseng has been well known in East Asian countries since ancient times as a panacea drug that favours longevity. It contains oleanolie acid and dammarane triterpene saponins called ginsenosides with diverse pharmacological properties including antiinflammatory activity [21]. Ginseng and its saponins are drugs that


97

normalise abnormal behaviour or symptoms caused for the most part by stress, but they cure no specific disease directly. The root of the Bupleurum species has been used in China as a traditional remedy for inflammatory diseases [20]. The main constituents of this drug are oligoglycosides of oleanane triterpenes called saikosaponin a-f. Saikosaponins cause a reduction in histamine secretion and enhance the anti-inflammatory actions of glucocorticoids [22]. Some of them have been shown to inhibit prostaglandin E2 production in the macrophage culture system [23]. It has recently been reported that the saikosaponins present in Heteromorpha trifoliata, which are structurally related to those of Bupleurum falcatum (saiko in Japanese), have anti-inflammatory activity. One in vivo study concluded that the isolated saikosaponins act by a mechanism close to that of steroids, but do not involve the glucocorticoid receptor [24]. Other crude drugs used as anti-inflammatory agents are the stem-bark of Akebia quinata, which provides saponins of oleanolie acid and hederagenin, roots of Astragalus membranaceus, from which the saponin astramembrainnin I has been isolated, and the roots of Platycodon grandiflorum with saponins called platycodins. Anti-inflammatory activity was also established with saponins from extracts of Phytolacca americana, Patrinia scabiosaefolia, Lamium album and Swertia cincta. In addition, saponins isolated from Eryngium planum, Hydrocotyle vulgaris, Polemonium coeruleum, Sanicula europaea and Thea sinensis have been shown to have anti-exudative activity [20,25,26]. Widespread reports indicate that, in addition to their anti-inflammatory role, triterpenes have other activities. Triterpenoids have been found to possess cytotoxic, antimicrobial and interesting effects on metabolism. Triterpenoids with antitumour activity include oleanane, lanostane, lupane, friedelane, hopane and quassionoid types. Glycyrrhetinic acid has been described as an antiviral, hypolipidemic and anti-atherosclerotic agent. Cucurbitacins B and E, and oleanolic acid possess a potent protective action on the liver, and ganoderic acid and its derivatives have been shown to be inhibitors of cholesterol biosynthesis. Lanostane derivatives, like suberosol, have also been found to inhibit HIV replication in H9 lymphocytes [1,3]. CHEMISTRY

Biosynthesis Triterpenes originate from squalene via mevalonic acid, which is formed from sequential condensation of three acetyl-coenzyme A units and subsequent reduction with NADPH to generate (3S)-3-hydroxy-3methylglutaryl-coenzyme A. The next ATP/Mg2+-dependent steps

R|OS et al.

98

convert mevalonate into (R)-5-diphosphomevalonic acid, and the latter into isopentenyl pyrophosphate (IPP), which is isomerised to dimethylallyl pyrophosphate (DMAPP). Both isomers represent the activated monomer building blocks for all terpenoids. Head to tail condensation of IPP with DMAPP generates geranyl diphosphate, and addition of a second IPP unit furnishes farnesyl pyrophosphate (FPP). Tail to tail condensation of two FPP units gives rise to squalene, which is oxydised to (3S)-2,3-oxidosqualene [27]. Oxidosqualene cyclase catalyses the conversion of (3S)-2,3-oxidosqualene to cycloartenol, the first cyclic precursor of tetracyclic triterpenes and phytosterols in higher plants and algae. In animals and fungi, oxidosqualene is cycled into lanosterol, the precursor of sterols. The formation of cycloartenol proceeds via the prec h a i r - b o a t - c h a i r conformation of (3S)-oxidosqualene, yielding a protosteryl C-20 cation that suffers a series of 1,2-methyl and hydride shifts with proton elimination, to give a cycloartene skeleton. Cyclization to pentacyclic triterpenes proceeds from the pre all-chair conformation of the substrate, yielding a tetracyclic dammarenyl C-20 cation. The following rearrangement leads to pentacyclic triterpenes via the baccharenyl, lupenyl and oleanyl cationic intermediates [28]. Squalene can also be cycled directly, without previous oxidation, to pentacyclic 3deoxytriterpenes, thus leading to hopane and gammacerane skeletons, as occurs in some bacteria and protozoa.

Structural Types Recent reviews classify triterpenoids according to chemical characteristics or biogenetic origin [3,11 ], but in the present review the emphasis is on activity and for this reason our classification is based only on antiinflammatory substances. T a b l e 1.

General

Skeletons of Anti-inflammatory Triterpenes Cited in this Review

29

30

28

23

24

~lcmimie

Ursane


99

(Table 1). contd.....

Taraxastane

Hopane

I"

Lupane

DtUlUIIWanf3

h O S t M C

Cucurbitane

100

R[OS et al.

The main kind of anti-inflammatory triterpenes isolated have oleanane, ursane, taraxastane, lupane and lanostane skeletons (Table 1). Some minor compounds such as hopane are included in other structural groups. Other anti-inflammatory triterpenes like the different cucurbitacins are not included in this review because of their high toxicity.

Oleanane Type Oleanane triterpenoids are the largest group within the triterpenes and encompass a huge number of active compounds. They are structurally classified as olean- 12-ene (Table 2) and 11-keto-olean- 12-ene (Table 3), directly derived from the oleanane skeleton. Other modifications give rise to the D:C-friedooleananes (Table 4), friedelanes (D:A-friedooleananes), 24-nor-D:A-friedooleananes (Table 5) and 24,30-dinor-D:Afriedooleananes (Table 6). The oleananes include glycyrrhetinic acid, probably the most widely studied triterpene. Table 2.

Anti-inflammatory Triterpene$ Derived from Olean-12-ene

128 )

R3 ,

,,,

,

16

RI6

R23

R24

R28

CH3

CH3

,,

p-Amyrin

OH[3

tt

CH3

[i-Amyrin acetate

OAc~

H

CH3

CH3

CH3

Erythrodiol

OHp

H

CH3

CH3

CH2OH

Oleanolic acid

OH~

CH3

CH3

COOH

Hederagenin

OH~

CH2OH

CH3

COOH

,

,

,

r

.,

acid

H

CH3

COOH

M~miladiol

'OH~ "

OHa

OH

CH3

CH3

CH3

Longispinogenin

OH[3

OH

CH3

CH3

CH2OH

.

J

....

.

.

.

.

.

.

CH3 .


Table 3.

101

Anti-inflammatory Triterpenes Derived from l l-Keto-olean-12-ene

'1'

R3 1813-Glycyrrhetinic acid

OH

.~

coo.

OH

Ha

COOH

HI3

COOEt

,,

,,

18a-Glycyrrhetinic acid .

.

.

.

.

.

.

.

.

.

.

.

.

.

,

H.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Ascorbic acid phosphate Na

Glycyrrhetinic phosphate vitamin C (GEPC) .

Anti-inflammatory Triterpenes Derived from D:C-Friedooleanane

Table 4.

D . . . .

R3

R7

R29

OHI3

H2

COOH

68

OCO(CH2)2cooK

H2

COOK

7-Oxo-isomultiflorenol

A8

OH[I

-O

CH3

3-Epibryononol

A8

OHa

H2

CH2OH

7-Oxo'dihydrokarounidiol

A8

OHa

=0

CH2OH

A7,9( 11)

OHa

H2

cH2oH

82

CH2OH

H2

CH2OH

Bryonolic acid " .

.

.

.

.

Bryonolic acid-3-O-succinate(K +)

.

.

.

.

.

.

.

m

.

Karounidiol Karounidiob3-O-i~enzoate

3-Epik~ounidiol

. . . . A7,9(11)

OcoPha .

A7,9(11) .....

OHIB

.

.

.

.

.

.

.

.

102

RIOS et a/.

Table 5.

Anti-inflammatory Triterpenes Derived from 24-nor-D:A-Friedooleanane

Ill

A

R2

R3 i

i

R6

R29

R7

ill m

Celastrol

A3,5,7,10(1)

Acetilcelastrol

=O

OH

A3,5,7,10(1)

=O

OAc

Pristimerin

A3,5,7,10(1)

=O

OH

Regeol B

A4

OHa

=O

OH~

Regeol C

A2,4,7,10(!)

OH

OH

=O

,

H

i

H

. . . . . . . .

. . . . .

CH3

OH

,,,

i

Anti-inflammatory Friedooleanane

Table 6.

H2

Triterpenes

Derived

H

H

ill

from

24,30-dinor-D:A-

R2

|

i

i

A i

|, ill

,,

R2 i

i,

,

I-'

'

,"

A3,5,7,]0(~)

Tingenin B

A2,4,10(1)

[ Regeoi A , i

'

,

=O OH ,

.....

i,ll

I II


103

Ursane Type

This structural group is divided into two subgroups, 3]3-hydroxy-urs-12ene (Table 7) and 3tx-hydroxy-urs-12-ene (Table 8). The ursane 13boswellic acid and its derivatives have been studied in vivo and in vitro as

anti-inflammatory agents. Anti-inflammatory Triterpenes Derived from 315-Hydroxy-urs-12-ene

Table 7.

= R20

[28 I

R2

R3

RI6

R20

H

H

|

ct-Amyrin .

.

.

.

R28 i

OH

.... c . ;

.

o~-Amyrin acetate

H

OAc

H

H

CH 3

a-Amyrin-linoleate

H

O-linoleate

H

H

" CH 3

ot-Amyrin-palmitate

H

O-palmitate

H

' CH 3

Uvaol

H .

.

.

Ursolic acid Brein Tormentic acid

.

.

. . . . . . H ....

OH .

.

.

.

H H

OH OH

OH

OH

H .

.

,L

.

.

H .

.

.

.

H OH

H H

'H

OH

CH2OH .

.

,.,

COOH CH 3 COOH

Anti-inflammatory Triterpenes Derived from 3t~-Hydroxy-urs-12-ene

Table 8.

R3~,,""

R3 , .=

,

,,, ,,,,,,

,

RII

,

[i-Boswellic acid Acetyl-I l-keto-lS-b0swellic acid (AKBA) I l-Keto-~-boswellic acid

OH

HE

OAc

-0

OH

=O

'

f04

RIOS et al.

Taraxastane Type The active taraxastane triterpenes are included in two g r o u p s - - t h e taraxast-20-ene, or taraxastene type (Table 9); and its isomer taraxast20(30)-erie, or ~-taraxastene type (Table 10). Table 9.

Anti-inflammatory Taraxastene Triterpenes

i

I

J

,

R ,,

,

,

__L_,

,

,

"

1 Taraxasterol Taraxasterol acetate T a b l e 10.

,,

"

_OH

"

OAc

Anti-inflammatory v-Taraxastene Triterpenes

"'*t R22 :16

R3

RI6

R22

R28

Illlll ,,

v-Taraxasterol

,

OH

H

H

CH3

OAc

H

H

CH 3

OH

OH

H

CH 3

Faradiol-myristate

O-myristate

OH

H

CH 3

Faradiol-palmitate

O-palmitate

OH

H

CH3

OH

OH

OH

CH2OH

Heliantriol C

OH

OH

H

CH3

Amidiol

OH

OH

H

u

acetate

Faradiol

,..

.

.

.

.

.

.

.

.

Heliantriol B0 .

.

.

.

.

_

.

.,

CH3


105

Lupane and Neolupane Types Betulin and betulinic acid are the best studied lupanes as antiinflammatory derivatives, and a great deal of research on their activity and mechanism of action has been published (Table 11). While the lupanes are widespread in plants, the occurrence of neolupanes is reduced to a small number of compounds and plants. Table 11.

Anti-inflammatory Lupane Triterpenes

28

RaV , , , e ~

~f

R3 I

I

iii

,,,,

RI6

R26

R28

H

CH3

CH3

,,

Lupeol

OH

Betulin

OH

CH3

CH2OH

Betulinie acid

OH

CH3

COOH

Calenduladiol

OH

OH

CH3

CH3

Heliantriol B2

OH

OH

CH2OH

CH3

........

L

Lupeoi-palmitate

O-palmitate

CH3

CH3

Lupeol-linoleate

O-linoleate

CH3

CH3

Lanostane and seco-Lanostane Types The lanostanes are the most relevant group of the tetracyclic triterpenes (Tables 12-13). Many of this type of compounds are described as antiinflammatories and their mechanism of action has been studied frequently. Cycloartenol and some 3,4-seco-lanostane derivatives have been reported to have activity also.

RiOS et al.

106

Anti-inflammatory Lanostane Triterpenes

Table 12.

~

R,,

,,,,-\

_.~R16

... m

R6

A

R6

R3 ,

,

ill,

....

RI6

,

A8

OAcl3

H

OH

Dchydropachymic acid

A7,9(11)

oAcp

H

OH

6a'Hydroxydehydropachymic acid

A7,9(I I)

OAcl] .....

OH

OH

Dehydroeburiconic acid

A7,9( I I)

---O

H

H

l) . . . .acid . . . . . . . . . .A7,9(1 . Dehydrotumulosic

0H~

H

OH

Pachymic acid

,

Anti-inflammatory Lanostane Triterpenes

Table 13.

R22 R21oo_ ~

~ oRI6

%

jR26 R27

~

Rls

i R,

I R, I s", S'6 I S~' ! S2~ I R26 I, Re'

A ,

.

,

,. . . .

i

A7,9(1 l) ' OFI[~

3]]-HYdroxylanosta-7,9(l 1),24trien-2 l-~ic acid .

.

.

.

.

.

3-O-Acetyl-I 6cthydroxytrametenolic acid

16ct-Hydroxytrametenolic acid .

.

.

.

H

H

"COOH

H

CH3

CH3

H

OHa

COOH

H

CH3

CH3

OHtx "'COOH

H

CH3

CH3

.......

oAc~)

AS '

AIi

OH~..........H ..


107

(Table 13). contd.....

•

,

','I

Masticadienoic acid*

A7

Masticadienolic acid (Schinol)* .

.

.

.

.

.

A7

.

.

.

.

.

.

.

.

H

H

CH3

H

OHo~

H

H

CH3

H

......

.

Pistacigerrimone A .

=O

CH3

COOH

CH3

COOH

.....

=0

A 1,7

H .

.

.

.

.

.

.

.

.

H

CH3

H

CH3

COOH

CH3

H

CH3

COOH

.

Pistacigerrimone D

A 1,5,8

=0

H

H

Ganoderic acid R

A7:9(11),24

OH~

H

H

CH3

OAc COOH

CH3

. . . . . . .

Ganoderic acid S

A7,9(11),24

=0

H

H

CH3

Ganoderic acid T

A7,9(I 1),24

OHo.

OH'

H

CH3

H

COOH

CH3

OAc COOH

CH3

r

*Compounds with 13oq14[~,!7o~-substitution

Hopa n e

Type

Of the hopane type only three compounds have been reported to be active

(Table 14). Table 14.

A n t i - i n f l a m m a t o r y H o p a n e Triterpenes

.....3 . r

R22

R

A Moretenol

A22(29i

Moretenol acetate

A22(29)

R3

CH3 OAc

Tetrahydroxybacterihopane (THBH) . . . . . . .

.

.

.

.

.

.

H2 .

.

R22

.

.

CH3 CH2(CHOH)3CH2OH '"

.

Isolation and Identification

Extraction, Separation and PreliminaryAnalysis Triterpenoids are usually non-polar compounds, but some oxygenated substituents, like earboxyl or hydroxyl, produce a moderate increase in polarity in the molecule. Apolar solvents such as dichloromethane and

108

R[OS eta/.

chloroform are usually needed to obtain an extract containing free triterpenes, but supercritical-fluid extraction (SFE) is being introduced into the isolation process used for natural products, including triterpenes [29,30]. There are different types of tests for detecting the presence of triterpenes in an extract. The Liebermann-Bouchard reagent is the one most frequent used to differentiate triterpenes and steroidal aglycones, whereas Zimmermann's test is appropriate for 3-oxo triterpenoids. Different reagents can be used to detect triterpenes in thin-layer chromatography (TLC) plates. Thionyl chloride, phosphomolibdic acid, silicotungstie acid [31], stannous chloride, arsenic chloride [32], antimonnium chloride, rhodamine 6G, and vanillin phosphoric acid [33] have all been described. It is possible to distinguish some compounds easily by derivatization in the plate. Oleanolic and glycyrrhetinic acids can be transformed into their methyl esters with methyl iodide, o~- and 13amyrin into acetyl derivatives with acetic anhydride/pyridine, and lupeol with acetyl chloride. The products of reaction are less polar and more easily separable from other natural products in TLC [33]. Silicagel impregnated with AgNO3has been used in TLC plates and flash columns to separate triterpenes [34]. Some of the anti-inflammatory triterpenes that have been described are not natural products, but artefacts. Using methanol during the extraction process may transform some kinds of glycosides, like saikosaponins, into 11-methoxy derivatives. Similarly, in the hydrolysis process a large number of glycosides may be transformed when mineral acids, like HCI or H3PO4 are used. This can be avoided by previous permethylation or enzymatic hydrolysis of original compounds. Separation methods vary depending on the type of compound. Free aglycones are easily separated by routine chromatographic systems preparative TLC (p-TLC), conventional column chromatography (CC), flash-CC, medium pressure liquid chromatography (MPLC), vacuum liquid chromatography (VLC), etc.---or by more complex techniques such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), droplet counter current chromatography (DCCC) or capillary-CC. Reverse-phase (RP)-HPLC is probably the best system for purifying triterpenoids, principally when mixtures of isomers are present [35]. Gunther and Wagner in 1996 [36] carried out the separation and quantification of active triterpenes from Centella asiatica employing an RP system with acetonitrile-water as mobile phase. Recently, Gaspar et al. [37] described the complete separation of a mixture of triterpenoid isomers from the fruit of A r b u t u s u n e d o by HPLC coupled to a mass spectrophotometer by means of a particle beam interface (HPLC-PBMS). The separation of different quassinoids from crude bark of Quassia amara was developed by Vitanyi et al. [38] using a reverse-phase HPLC-MS


109

with thermospray ionisation. Application of this technique to the study of triterpenes and their glycosides has been successful [39]. When Heinzen et al. [40] compared thermospray liquid chromatography (TSP-LC) and GCMS with electron impact ionisation (GC-MSEI) techniques to study triterpenes in different samples, greater selectivity and sensitivity were observed with TSP-LC. Determination of glycyrrhizin and glycyrrhetinie acid can be done in a capillary electrophoresis system. The great advantage of this technique is the running time, because the analysis of these two constituents can be completed in 10 min, whereas the HPLC analysis could take about 50 min [41]. Takaishi et al., [42] combined CC (silica gel and sephadex LH-20) and HPLC in the isolation of the anti-inflammatory compounds of Tripterygium wilfordii. Della Loggia et al. [43] isolated the topical antiinflammatory triterpenes mixture from Calendula officinalis using COz SFE, and the extract was purified using VLC and MPLC (silica gel) and HPLC (columns of silica impregnated with AgNO3, diol and RP). Later, Zittlerl-Eglseer et al. [44] isolated the anti-edematous triterpenes faradiol esters and v-taraxasterol from the same source using only silica gel CC and preparative-HPLC. Structural Elucidation

Diverse spectroscopic methods have been employed to characterise triterpenes. Ultraviolet (UV) and infrared (IR) spectroscopy are not very useful techniques in elucidating the structure of triterpenes, but the former gives information about compounds with conjugated double bonds and the latter may provide some information about substituents like the hydroxyl group, ester carbonyl group or t~,13-unsaturate carbonyl. Other physical data may be of interest to characterise new compounds, but the use of modem spectroscopic methods of nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are essential for the structural determination. NMR is the tool most widely used to identify the structure of triterpenes. Different one-dimension and two-dimension techniques are usually used to study the structures of new compounds. Correlation via H-H coupling with square symmetry (~H-~H COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), distortionless enhancement by polarisation transfer (DEPT), incredible natural abundance double quantum transfer experiment (INADEQUATE) and nuclear Overhauser effect spectroscopy (NOESY) allow us to examine the proton and carbon chemical shift, carbon types, coupling constants, carbon-carbon and proton-carbon connectivities, and establish the relative stereochemistry of the chiral centres.

110

RiOS et al.

Two excellent reviews describing the t3C NMR of pentacyclic triterpenes have been published in the last years. In 1992 Agrawal and Jain [45] published a compilation of the spectral data on 456 oleanane triterpenes and Mahato and Kundu [2] reviewed the spectral data of 393 compounds, including 43 types of structural groups. The use of MS together the NMR spectroscopy has been essential for the structural elucidation of triterpenoids. The review by Shiojima et al. [46] covers the mass spectra of saturated and unsaturated triterpenoids and explains each type of compound fragmentation. Bioassay-guided Isolation and Anti-inflammatory Studies

To isolate active compounds it is necessary to monitor the activity during fractionation and isolation. In the case of triterpenes as anti-inflammatory agents, different kinds of tests are employed. Subplantar administration of carrageenan in rats was commonly used in earlier studies to establish the anti-inflammatory activity of orally administered plant extracts or fractions, but a large amount of sample was necessary. Topical application of inflammatory agents is more useful in screening extracts and pure compounds, because the amount of sample is smaller than with oral application and it is easier to track the activity. The introduction of in vitro tests could be an alternative way to monitor the anti-inflammatory activity. Several examples of bioassay-guided isolation are given in the literature. Recio et al. [47] used a topical irritant agent to track the activity during the isolation of the anti-inflammatory triterpenes from Diospyros leucomelas, whereas Cu611ar et al. [48] employed a combined system of in vivo and in vitro experiments, consisting of the inhibition of an enzyme activity "Fig. (1)". Other authors prefer in vitro models. Ammon and Safayhi [49] used the inhibition of leukotriene formation in rat peritoneal neutrophils to isolate the boswellic acids of gum resin exudate of Boswellia serrata, whereas Takaishi et al. [42] studied the inhibition of interleukin-1 secretion, and Jain et al. [50] studied the in vitro inhibition of phospholipase A2 activity. The main results of these studies are summarised in the pharmacological activity section of this chapter. ANTI-INFLAMMATORY ACTIVITY OF TRITERPENES Introduction

Inflammation is characterised by redness, heat, pain, swelling and organ decreased function. This process is complex and involves different mechanisms attributable to a large variety of mediators, which are distributed in three phases.


111

Poria cocos

[ Concentratlon I.,II[,. ~Maceration: MeOH/H20 (7: in,

~, Extraction EtOAc/H20

t

l EtOAc extraci'I

t

I H2~ extract

I

Phytochemlcal analysis: TLC, HPIA2 {triterpene detection} Bloassay: TPA (acute ear edema}. PLA2 (in vitro Inhibition} _

IIIIII

I

I

II

_

_

IIII

I III

I

i

Active extract: EtOAc

Separation: CC (sephadex LH-20, dlol}, VI~ (SIO2) Bloassay-gulded Isolation: TPA edema and PLA2 activity

Selection of active principles Identification by spectroscopy

Pachymic acid

Dehydrotumuloslc acld

Anti-inflammatory activity: I n vivo a n d in vitro studies Mechanism of action Fig. (1). Scheme of isolation, identification and biological activity study of anti-inflammatory lanostanes from P o r i a c o c o s sclerotia.

The first one is an acute transient phase with local vasodilatation and increased vascular permeability. It is produced by vasoactive amines, such as histamine, which induce vascular permeability by acting on the H l receptors, and also has a variety of actions on inflammatory cells. Serotonin or 5-hydroxytriptamine (5-HT) is released from blood platelets and also contributes to vasopermeability. Plasmatic bradykinin (BK), the most important kinin, is a potent vasodilator and promoter of vascular

112

RIOS eta/,

permeability, and also stimulates the release of histamine from mast cells and the synthesis of prostaglandins (PGs). PGs constitute a group of compounds derived from the C-20 polyunsaturated arachidonic acid (AA), a component of the phospholipids of the cell membrane that is usually released by hydrolysis caused by phospholipase A2 (PLA2). Once AA is hydrolysed, it is converted through the cyclooxygenase (COX) pathway into prostaglandins (PGE2, PGD2 and PGF2,x), prostacyclin (PGI2) and thromboxane B2 (TXB2). PGs are potent vasodilators, with little effect on vascular permeability, and they potentiate the pain-producing effect of other mediators. The lipoxygenase (LOX) pathway produces leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). LTs are active as permeability-enhancing factors, although LTB4 is the most powerful endogenous chemotactic factor yet described "Fig. (2)".

Lyso-PAF

Ca 2+

Vascular Permeability Increase

Fig. (2). Release of lipidic mediators and their role in the inflammation.

A second delayed subacute phase is characterised by infiltration of leukocytes and phagocytic cells. The recruitment of inflammatory cells involves the concerted interaction of several types of chemotactic mediators such as complement factor C5a, platelet activating factor (PAF) and LTB4. Several cytokines, especially interleukin-1 (IL-1) and tumour


113

necrosis factor (TNF), appear to play an essential role. IL-1 comprises two distinct polypeptides (IL- ltx and IL-1J3) that produce similar biological responses, such as mobilisation and activation of polymorphonuclear leukocytes (PMNL), induction of COX and LOX enzymes, production of other growth factors and cytokines (IL-2, IL-6, IL-8, etc.). At the site of inflammation the stimulated PMNL are capable of producing reactive oxygen species such as the cytotoxic superoxide anion ('O2-) which can react with other molecules to produce the extremely reactive hydroxyl radical ('OH), considered the main initiator of lipid peroxidation. In addition, inflammatory cells are able to release large quantities of hydrolases, such as elastase and collagenase, that catalyse the hydrolysis of tissue components involved in the extracellular proteolysis of rheumatoid arthritis and other inflammatory states "Fig. (3)". If the noxious stimulus persists, the action of all these mediators contributes to the chronic proliferative phase of inflammation. It is in this third and last phase that tissue degeneration and fibrosis occur. Differentiation between the acute and/or chronic inflammation is traditionally based on the kind of inflammatory cells that predominate in the lesions. In fact, in acute injury Mast Cells

VASODILATATION

VASCULAR PERMEABILITY

Q

Cells

Enzymes Free radicals

] Hydrolytic

Complement system

Other Lymphoklnes

Lymphocytes

Fig. (3). Relationships between inflammatory mediators and some relevant cellular events.

I 14

RIOS et at

PMNL predominate, while in chronic lesions mononuclear phagocytic cells are the main ones present. In conclusion, pharmacological control of inflammation can be modulated by antagonising or preventing the release of mediators involved in initiating or amplifying inflammation, or by direct action on inflammatory cells. It is possible to act in four major areas" 1) inflammatory amine release and activity; 2) AA metabolism; 3) phagocytic and inflammatory cell functions, and 4) autoimmune processes. Antiinflammatory agents may therefore inhibit COX and/or 5-LOX pathways, block chemotactic factors and other mediators, inhibit activation of the complement system, oxidative phosphorylation, adhesion to vascular endothelium by leukocytes, scavenge free radicals, stabilise cell membranes, etc. "Fig. (4)". Membrane phosphollplds

I lllll

~I~'VASODILATATI(~N~ I ~ ~ ~ ~

VASCULAR

PERMIEAB!LITY

!

Chemotaxls --I _.'_. ' i

"'-| IL,'XVLt~, n

Mononuc|ear i . Cells n

-"

"-

. .

r--1 ..... ~ .......

_.._ . . . . . . . . . . .

tess " "

.............

i

,,nmune

9 IIII Fig. (4). Main targets of the anti-inflammatory drugs.

General Considerations About Research on New Anti-Inflammatory Drugs

In general, two major classes of antiphlogistic drugs are available for therapy: Non steroidal anti-inflammatory drugs (NSAIDs) which interfere


115

with the COX system (e.g. salicylates), and steroidal anti-inflammatory drugs such as glucocorticoids, which have the ability to inhibit both the COX and LOX pathways by interfering with the PLA2-reaction. Developing new anti-inflammatory agents requires the use of experimental models of inflammation that reflect, if possible, the whole set of symptoms and mechanisms normally present in the inflamed tissues. In practice, such a model is difficult to find, and it is necessary to combine experimental models in vivo with tests in vitro. Furthermore, the pharmacological response observed in experimental animals such as mouse or rat can not be the same as in humans. In vitro pharmacological studies can serve as an initial screening step in which the presumed effect on a particular aspect of an inflammatory process can be investigated. In vitro techniques are very reproducible and highly sensitive, but the usefulness of the results obtained is limited by the fact that they do not reflect what happens in vivo. They therefore can not replace the in vivo methods, but are a good complement for the conventional procedures. In any case, all potential therapeutics must ultimately be evaluated in in vivo models [51-

531.

The most readily available animal models generally reflect acute or subacute reactions because they are simple, reproducible and provide clues to the biochemical and cellular mechanisms involved in the transition from acute to chronic inflammation. The cardinal signs of inflammation are the end points for the measurement of anti-inflammatory drug action (erythema, hyperemia, edema or exudation).

Screening Methods The carrageenan-induced paw edema in rat or mouse is the method most commonly used to investigate the mechanism of the inflammatory process and to evaluate the anti-inflammatory effect of an agent. Few of the available anti-inflammatory drugs produce more than 60% inhibition of edema. The properties shown by carrageenan include granuloma formation, action on blood coagulation and the kinin system, and immunologic elicitation. The edema is expressed as the difference between the foot volume before and after the injection. On the other hand, paw edemas induced by substances like 5-HT or histamine are insensitive to conventional NSAIDs and could be considered experimental models in the search for anti-allergic compounds [54]. Among the models of inflammation in vivo, those that involve the skin have the particular advantage that the results are immediately and continuously observable. Models of skin inflammation are numerous and varied, ranging from acute and limited to chronic and tissue-destructive. Croton oil, different phorbol esters, principally 12-tetradecanoylphorbol13-acetate (TPA), AA and oxazolone, provide a range of skin inflammation models suitable for the evaluation of both topical and/or

116

RIOS et at

systemic anti-inflammatory drugs. The irritant is usually applied in the right ear while the left ear remains untreated and serves as a control [55]. On the other hand, rat adjuvant-induced arthritis is a model of chronic inflammation frequently reported in screening for anti-arthritic agents. Adult rats are the animals of choice because the disease does not occur in hamsters, mice or guinea pigs. The clinical and pathological changes in adjuvant disease are comparable to those of rheumatic arthritis [56]. Numerous medicinal plants and natural compounds have been tested for anti-inflammatory activities using these models, despite their having the disadvantage of variable biological response. In addition, large quantities of pure compounds must be administered to a large number of animals in order to construct dose response-curves. However, natural products are often isolated in small quantities, which makes it necessary to analyse their activity with in vitro tests, employing cells or enzymatic systems, where only 2-5 mg of the product is necessary. Since rat or human peripheral leukocytes, when stimulated, are able to produce prostanoids, LTs and PAF, they are good cell models for studying the activity of compounds on the synthesis of lipid mediators. Among the enzyme preparations, COX, LOX or complement fractions from different sources are commonly employed. In recent years, interest in PLA2 inhibitors has increased notably because extracellular PLA2s are implicated in the pathogenesis of several important inflammatory diseases. A substantial effort has been made to establish assay protocols for the screening and characterisation of specific competitive PLA2 inhibitors [50,57]. The present-day search for new natural anti-inflammatory drugs is based on the use of these models of inflammation in which products can interfere with any or all the systems involved in the inflammation processes. In the last few years, interest in triterpenes as anti-inflammatory agents and their mechanisms of action has also increased greatly. Oleanolic acid and ursolic acid have been recognised to have anti-inflammatory activity in carrageenan-induced paw edema in rats or mice, adjuvant-induced arthritis in rats, etc. Oleanolic acid, in addition, was able to suppress the delayed hypersensitivity reaction in mice induced by dinitrochlorobenzene (DNCB). These effects are attributable to different mechanisms of action ranging from the inhibition of histamine release to inhibition of complement activity [ 1,58]. The mechanism of action of boswellic acids from crude extracts of B o s w e l l i a serrata has been established [59] and it was reported recently that they inhibit the increased urinary excretion of LTE4 in astrocytoma patients in vivo and block leukotriene biosynthesis ex vivo [60]. Until now only a few natural terpenoids have been recognised as antiP LA2 compounds. However, it seems that acidic triterpenes such


117

pachymic, dehydrotumulosic, masticadienolic or masticadienoic acids are a new class of PLA2 inhibitors [48,50]. In the next sections the recent advances regarding the anti-inflammatory activity and mechanisms of action of triterpenes are presented and their chemical structure/pharmacological activity relationships are discussed. In vivo Anti-inflammatory Studies

Carrageenan-induced Edema Among the plant terpenoids with a lupane skeleton, lupeol is the simplest one and has recently been reported as the active principle of Crataeva religiosa (Capparidaceae). When it was tested on the habitual models of inflammatory response, it showed moderate activity on the carrageenaninduced rat hind paw and rat pleurisy [61 ]. A compound with a related structure is moretenol acetate, which was the most active of the triterpenes from Pluchea lanceolata (Asteraceae). When administered 50 mg/kg p.o., it inhibited by 55% the edema caused 3.5 h after carrageenan injection into rat hind paw [62]. At 100 mg/kg p.o. neolupenol, from the same plant, reached 55% inhibition at 2.5 h and 70% inhibition at 5 h [63].

9 9 I.,VA

L . t [ ~ l . , J t Jt V A

Taraxasterol acetate from the Ayurvedic drug Echinops echinatus (Asteraceae) reached 63% inhibition after 4 h, but at a high dose regime (200 mg/kg, p.o.), and this activity was not much improved when this product was given i.p. (68% inhibition) [64]. In a study on the anti-inflammatory properties of resins from Burseraceae species, mansumbinoic acid, an octanordammarane isolated from Commiphora incisa, induced, at 0.25 mmol/kg p.o., a 48% decrease in the area under the time-course curve obtained 6 h after induction of edema. This effect proved to be dose dependent, and the calculated ED50 was 0.15 mmol/kg. The closely related ketone mansumbinone was of minor potency

118

RIOS et al.

[65]. The same authors also undertook a search for the anti-inflammatory principles of Polygonum bistorta (Polygonaceae) and obtained positive results with friedelanol and glutin-3-one. Glutin-3-one induced 56% inhibition at 0.12 mmol/kg [66].

HOO

Mansumbinone

Mansumbinoic acid

Friedelanol

Some members of the lanostane class from Pistacia integerrima (Anacardiaceae) galls proved to be very potent. When administered at only 5 mg/kg i.p. they reduced carrageenan inflammation. Pistacigerrimone A (70%) and pistacigerrimone D (62%) were especially effective [67].

/

O/cloartenol


119

The cycloartenol localised in Crataegus monogyna (Rosaceae) constitutes the main component of a fraction that reduced the 3 h carrageenan paw edema at 40 mg/kg p.o. Moreover, this fraction almost abolished leukocyte infiltration in the mouse peritonitis caused by the same inflammatory agent [68]. In a comparative study on the anti-inflammatory activity of the Diospyros leucomelas (Ebenaceae) principles and other readily available triterpenoids administered at 100 mg/kg p.o., betulinic acid showed the highest activity (45% inhibition) 3h after carrageenan injection [47]. Erythrodiol had a more durable effect, inducing a 40% inhibition after 5 h [69]. After comparing the variable effects observed on this model, it was concluded that the presence of a primary alcohol or carboxylic acid function at C-27 or C-29 is clearly positive. Adjuvant Arthritis Apart from the acute anti-inflammatory activity described in the preceding section, taraxasterol acetate was also tested for chronic activity in the model of Freund's adjuvant-induced rat arthritis. When 80 mg/kg of this compound were administered i.p., a 57% decrease with respect to the control value for the injected limb was observed at 18 h. After 21 days, the secondary reaction, which is measured on the non-injected limb, was reduced by 78%. Given the fact that in this condition the injected limb suffered a comparable reduction of 67%, it seems that taraxasterol acetate did not act by modifying immune mechanisms [64]. Duwiejua et al. [66] also reported appreciable anti-arthritic activity for glutin-3-one: 44% and 74% reductions in the ipsilateral and contralateral paw swelling, respectively, after 10 days. In the last few years, Kweifio-Okai et al. [70-72] have been studying the anti-arthritic activity of some triterpenoids and their natural or semisynthetic fatty acid esters by applying the adjuvant model and evaluating the progression of the disease by means of blood cell counts and microscopic examination of bone joints. These authors established that the effect of orally administered (56 mg/kg) ct-amyrin palmitate on the acute phase (from days 11 to 19 after adjuvant administration) is that it leads to a suppression of the increase in the ipsilateral ankle swelling, o~-Amyrin palmitate reversed the increase in spleen weight by 50% and consistently reduced plasmatic hyaluronate and the number of granulocytes in blood and bone marrow. Histological results showed amelioration of joint damage expressed as synovial proliferation and erosion of subchondral bone [70]. A very close study carried out with esters of lupeol demonstrated that the 3-1inoleyl derivative had the strongest activity, with profiles similar to that reported for o~-amyrin palmitate, but that its effect lasted longer and was observable on day 42 [71,72].

120

RiOS et al.

To expand our understanding of the anti-inflammatory activity of the well-known oleanolic acid isolated from one of its best characterised sources, the seeds of Luffa cylindrica (Cucurbitaceae), a series of experiments were performed to correlate complement activity with the inflammation caused by both carrageenan and Freund's adjuvant, in presence and absence of oleanolic acid and ibuprofen, which were administered at 60 mg/kg i.p. It was concluded that the effect of this triterpenoid must be mediated by its anti-complementary activity [73]. An analogous conclusion was drawn regarding the activity of boswellic acids in the same tests [74]. Serotonin-induced Edema

The models based on intradermal or subcutaneous injections of 5-HT not only provide a good system for measuring the earliest inflammatory phase characterised by a rapid plasma extravasation, but are also utilised as the last step in certain experiments designed to establish whether a drug exerts its anti-inflammatory activity through interactions on glucocorticoid receptor (GCR) and on DNA or protein synthesis [75]. When a series of triterpenoids were studied under this scheme, the lupane derivatives betulin and betulinic acid, administered s.c. at 50 mg/kg, proved to be quite effective against 5-HT-induced edema in mouse hind paw, producing 76% and 63% inhibition, respectively. The effect of betulinic acid was greatly reduced by concomitant progesterone (GCR antagonist), actinomycin D (transcription inhibitor) or cycloheximide (ribosomal peptidyltransferase inhibitor), which suggests that this triterpenoid may act by a steroidal mechanism. Other compounds like ursolic acid and erythrodiol were also active, but to a lower degree, and they were not much affected by these corticoid inactivators [47,69]. In a later study with the two main lanostanes of the fungus Poria cocos (Polyporaceae), both pachymic and dehydrotumulosic acids were effective (62% and 79% inhibition, respectively). Progesterone and actinomycin D substantially reduced the activity of pachymic acid, none of the inhibitors affected the activity of dehydrotumulosic acid. Pachymic acid probably acts by a corticoid-related mechanism, while in the case of dehydrotumulosic acid the inhibition of PLA2 should be the main anti-inflammatory mechanism (see below) [76]. Ph ospholipase A z-induced Edema

The anti-inflammatory effect of pachymic and dehydrotumulosic acids was determined in vivo against mouse PLA2 paw edema. At a dose of 50 mg/kg i.p., both were active (54% and 57% inhibition, respectively). As it has been proved that this model involves a release of histamine and 5-HT, an antagonism with one or both of these amines might occur, although this


121

effect could arise from the previously known inhibitory effect on PLA2 in vitro [77,48]. In vivo Models o f Allergy

In an attempt to discover triterpenoids which, because of a structural relationship, might share the anti-inflammatory and/or anti-allergic properties of glycyrrhetinic acid without its adverse reactions, Tabata et al. [78] surveyed the activity of bryonolic acid on different models of experimental mouse allergy. This acid is a friedooleanene derivative characteristic of the Cucurbitaceae that was described as a new product in Bryonia dioica and later localised in many genera of the family such as Citrullus, Cucumis, Luffa and others. Bryonolic acid itself showed an IDs0 of 376 mg/kg when given i.p. to mice in order to relieve a passive cutaneous anaphylaxis (type I allergy), but certain semisynthetic derivatives such as 3-phtalate (2 K +) or diol 3,29-diphtalate (2 K +) were more potent, with IDs0 of 34.2 and 41.4 mg/kg, respectively. These esters and other closely related ones also proved to be very effective when given orally. Bryonolic acid-3-succinate (2 K +) was also active against sheep erythrocyte-induced Arthus reaction in mice (type III allergy) and picryl chloride-induced contact dermatitis (type IV allergy) [78]. Influence on ear edema in mice

The triterpene-enriched fraction of the supercritical CO2 extract of the dried flowers of Calendula officinalis (Asteraceae) inhibited the croton oilinduced ear edema in mice. Of the identified compounds, the faradiol monoesters, lupeol, ~F-taraxasterol and a mixture of taraxasterol/13-amyrin were tested for their anti-inflammatory activity. Faradiol, obtained by hydrolysis of the extract, was the most active compound. It showed a dose-dependent effect with a potency that equals that of indomethacin at 0.14 I.tmol/cm 2 (48% and 47% edema inhibition, respectively). The esterification of faradiol resulted in a reduction of more than 50% in the activity (only 31% inhibition was observed at 0.14 Bmol/cm2), whereas Wtaraxasterol, a C-1613 dehydroxylated derivative of faradiol, was less active (47% inhibition at a dose of 0.28 lamol/cm2) [43]. Given that the biological activity of the ester mixture from C. officinalis had already been studied, Zitter-Eglseer et al. [44] isolated, separated and identified the ester components in order to study their topical antiedematous properties using the croton oil-induced inflammation test in mice. The main compounds were identified as faradiol-3-myristic acid ester, faradiol-3-palmitic acid ester and qJ-taraxasterol. The two faradiolesters showed nearly the same dose dependent anti-edematous activity: 50% inhibition at 240 ~tg/cm2 and about 65% when the dose was double. These data confirm the previous observation made by Della Loggia et al.

122

RiOS et al.

[43], that the presence of the OH-group at C-16 enhances the activity while esterifieation at position 3 reduces it [44]. The same type of triperpenes has been found in the flowers of some Asteraceae plants: taraxasterol in Cynara seolymus and faradiol in Chrysanthemum morifolium. Both of these substances were examined for inhibitory activity against TPA-indueed ear edema in mice. The compounds were applied 30 min before TPA treatment and showed a strong inhibition of edema (IDs0 = 0.3 rag/ear for taraxasterol and 0.2 mg/ear for faradiol). In comparison with the standard drugs, the triterpenes were similar in potency to indomethaein (IDs0- 0.3 mg/ear), although all of them were less effective inhibitors than hydroeortisone (IDs0 = 0.03 mg/ear). In addition, faradiol was more effective than taraxasterol on tumour promotion in two-stage carcinogenesis in mouse skin [79]. The relationship between the hydroxylation of triterpenes and their inhibitory effect was again observed among the triterpenes isolated from the ligulate flowers of Chrysanthemum morifolium, Helianthus annuus (Asteraceae) and Taraxacum platycarpum (Asteraeeae) [80]. The compounds were identified as faradiol, heliantriol B0, heliantriol C and amidiol (taraxastane-type); ealenduladiol and heliantriol B2 (lupane-type), maniladiol and longispinogenin (oleanane-type), brein and uvaol (ursanetype). They were evaluated with respect to their anti-inflammatory activity against TPA-indueed ear edema in mice, and their effects were

Poricoic acid A HOOCt,o.. ~OH

.ooc-

y...

Poricoic acid B


123

compared with those of indomethacin and hydrocortisone. All the substances inhibited the edema generation, with IDs0 ranging from 0.03 mg/ear to 0.2 mg/ear. The anti-inflammatory activity of these triterpenes was stronger than that of indomethacin, which gave an IDs0 of 0.3 mg/ear on this test. Heliantriol B2, brein and heliantriol C showed a strong inhibitory effect (for the first two, IDs0 = 0.05 mg/ear; 0.03 mg/ear for heliantriol C) comparable to that of hydrocortisone activity. Whether the substance belongs to one or another triterpene type does not seem to be important for the activity, but there is a close relationship between the hydroxylation of triterpenes and the inhibitory effect. Thus, for the A2~ taraxastenes, faradiol showed a marked inhibitory effect (IDs0 = 0.2 mg/ear). Further hydroxylation of faradiol at C-22t~, which yields heliantriol C, substantially enhanced the effect. According to Della Loggia et al. [43], dihydroxy-triterpenes were stronger inhibitors than 313monohydroxy-triterpenes [80]. Eight lanostane-type triterpene acids and four 3,4-secolanostane-type triterpene acids were isolated from the MeOH extract of P o r i a c o c o s . The active principles were identified as pachymic acid, 3-O-acetyl-16o~hydroxytrametenolic acid, dehydropachymic acid, 313-hydroxylanosta7,9(11),24-trien-21-oic acid, dehydroeburiconic acid, and poricoic acids A and B. When topically administered 30 min before application of TPA their IDs0 ranged from 16 ~tg/ear (3-O-acetyl-16t~-hydroxytrametenolic acid) to 44 lag/ear (pachymic acid). In general, their activity against TPAinduced ear edema was similar to that of the reference drug hydrocortisone, and they were much more effective than indomethacin (IDs0 = 300 ~tg/ear) [81]. Nukaya et al. [82] obtained five compounds chemically related to pachymic acid from the MeOH extract of P. c o c o s . Among these compounds, 6ct-hydroxydehydropachymic and 16o~-hydroxytrametenolic acids inhibited the edema formation more potently than pachymic acid. Both 6tx-hydroxydehydropachymic and 16t~-hydroxytrametenolic acids at 5 Ixg/ear showed the same inhibitory effect as pachymic acid at 150 lag/ear. The anti-inflammatory activity of the hydroalcoholic extract of P. c o c o s against some acute and chronic inflammatory processes was established recently. It reduced the TPA-induced edema (80% inhibition at 0.5 mg/ear), but its effect was milder against the AA-induced ear edema (40% inhibition at 0.5 mg/ear). When the extract was assayed in chronic experimental model of inflammation, it caused a 53% reduction in ear thickness together with a 73% decrease in myeloperoxidase (MPO) activity, which shows that leukocyte infiltration into the inflammation site could be prevented by a repeated dose of 1 mg/ear. The main constituents isolated from the extract were identified as pachymic and dehydrotumulosic acids. Topical administration of these lanostanes inhibited the TPA-induced edema with IDs0 of 2.48 and 0.31 ~tg/ear, respectively. Reasons for the greater activity of dehydrotumulosic acid

124

R|OS eta/.

could be found in its structural characteristics: the presence of a free hydroxyl group at position 3, that is blocked by acetylation in pachymic acid, and the presence of a heteroannular 7-8,9-11-dien group, a character eonfering planarity to the molecule [83]. Yasukawa et al. [84] demonstrated the anti-inflammatory activity of some sterols and triterpenes on the TPA-induced ear edema test in mouse. Among them, karounidiol 3-O-benzoate, a D:C-friedooleanane triterpene, showed an interesting inhibitory effect (95%) of the edema formation at a dose of 2 mg/ear; its IDs0 was 0.4 l.tmol/ear. The D:C-friedooleanane triterpene type is the main kind of compound contained in the seeds of Trichosanthes kirilowii (Cucurbitaceae) a species employed in Chinese medicine as an anti-inflammatory agent. Some of the isolated compounds were evaluated for their topical anti-edematous activity against TPA-induced inflammation in mice. 3-Epikarounidiol, 7oxoisomultiflorenol and 3-epibryonolol gave IDs0 ranging from 0.2 to 0.6 mg/ear, and these values approach that of karounidiol 3-O-benzoate. In any case, the anti-inflammatory potency of these triterpenes was lower than that of hydrocortisone [85]. It has been reported that karounidiol and 7-oxo-dihydrokarounidiol have an inhibitory effect on TPA-induced inflammation [86]. Topically applied, they completely inhibited edema generation in a dose-dependent manner. The IDs0 of karounidiol and 7-oxo-dihydrokarounidiol were 0.4 and 0.3 mg/ear, respectively. 12-O-Hexadecanoyl- 16-hydroxyphorbol- 13-acetate (HHPA) was used at a dose of 2 ~tg/ear as an inducer of mouse edema to study the antiinflammatory activity of ursolic acid and some 4,4-dimethylcholestane derivatives. The compounds were administered 30 min before HPPA, and the maximum edema was reached 7 h after application. 200 Bg of ursolic acid reduced the edema by 49%. The functional groups of this triterpene are a hydroxyl group at position 3 and a carboxyl-group at C-28, but they are not essential for the activity. For example, cholesterol has a 31Jhydroxyl group but no carboxyl at C-28, and a structure similar to that of ursolic acid, but it not only was not active at a dose of 200 ~tg/ear, but even promoted inflammation. However, 4,4-dimethylcholesterol showed weak inhibitory activity at the same dose as cholesterol. These results suggested the importance of the 4,4-dimethyl group, common to all triterpene structures, for the anti-inflammatory activity. In this study the authors also observed that the oxidation of the 3-hydroxyl group to a 3oxo group increases the inhibitory activity, while epoxidation of the double bond at C-5 to a 5,6-~-epoxy group diminishes it [87]. The MeOH extract of Rosmarinus officinalis (Lamiaceae) contains 1620% of ursolic acid. Both the extract and the pure triterpene isolated from it were strong inhibitors of TPA-induced inflammation, ornithine decarboxylase (ODC) activity and tumour promotion in mouse skin. Some authors have suggested that the effects could be related to those of


125

steroidal anti-inflammatory compounds [88]. However, when ursolic acid isolated from Diospyros leucomelas was studied for its mechanism of action in vivo against serotonin-induced paw edema in mice, the triterpene activity appeared to have no relationship with that of glucocorticoids. There was no reduction in the anti-edematous effect in presence of the anti-glucocorticoid receptor progesterone and mRNA or protein synthesis inhibitors such as actynomicin D and cycloheximide [47]. Other mechanisms have already been proposed to explain the anti-inflammatory activity of ursolic acid, and they are discussed below. The inhibitory effect of ten triterpenes was studied on different in vivo models of acute inflammation in order to establish the possible relationship between their chemical structure and anti-inflammatory activity. Compounds belonging to the lupane (lupeol), oleanane (]3-amyrin, erythrodiol, hederagenin, oleanolic acid and oc- and 13-glycyrrhetinic acids) and ursane (o~-amyrin, uvaol and tormentic acid) series were assayed and the results compared with those previously reported for ursolic acid (ursane-type), betulin and betulinic acid (lupane-type). All the topically administered triterpenes were active against TPA-induced ear edema. Erythrodiol, tormentic acid, cz- and 13-glycyrrhetinic acids were the most active (over 80% inhibition). Erythrodiol and uvaol were the most effective compounds against ethylphenylpropiolate (EPP)-induced ear edema, in which the anti-inflammatory action is delayed. In general, the triterpene acids were the most active substances on the TPA test, while their effect was weaker against EPP-inflammation and did not depend on the presence of a carboxyl group. In this last method, the presence of a hydroxymethyl group at position 17 seems to increase the activity in the oleanane series, as occurs with erythrodiol [69]. In connection with the work on the relationship between chemical structure and anti-inflammatory activity, the effect of ursolic acid, betulin, betulinic acid and erythrodiol on a system of chronic dermal edema and cellular proliferation caused by repeated administration of TPA has recently been examined [89]. This experimental model of chronic inflammation has considerable selectivity for corticosteroids and leukotriene synthesis inhibitors. Erythrodiol and ursolic acid were significantly effective and also reduced the neutrophil infiltration detected by MPO activity. The lupane derivatives, betulin and betulinic acid, despite their possible steroid-like mechanism of action [47], were not effective in the chronic model. This result could mean that a six-member E ring of the pentacyclic structure is necessary for the activity against a multiple dose of TPA. The data confirm that a hydroxyl group at the C-28 position is important for the activity, as is also true in the case of erythrodiol, and it may explain the anti-inflammatory effect of this compound in each of the methods. A study was recently undertaken [90] to enlarge our understanding of the mechanism of action of triterpenes as topical anti-inflammatory agents

126

R|OS eta/.

by examining the effect of eleven triterpenes against mezerein-mediated ear edema in mice. Mezerein is an irritant diterpenoid present in some species of Thymelaceae with a certain selectivity for t~, 13 and T-isozymes of protein kinase C (PKC), enzymes involved in the edema formation induced by phorbol-esters. Of the triterpenes tested, erythrodiol was again the most active one (57% at 0.5 mg/ear), in a range similar to the standard drug indomethacin. Topical application of capsaicin on mouse ear causes a neurogenic edema, with a maximum at 30 min after the treatment. The release of 5HT, substance P (SP) and neurokinin-1 (NK-1), which interact with the respective receptors in the ear skin provoke the edema [91,92]. Dexamethasone, histamine H l and/or 5-HT and SP antagonists inhibited the edema, whereas AA metabolism inhibitors were not effective [93]. Glycyrrhetinic acid and three dihemiphthalate derivatives were recently examined for their anti-edematous activity against capsaicin-induced ear edema in mice [94]. Derivative compounds gave IDs0 values ranging from 41 to 53 mg/kg (p. o.); however, glycyrrhetinic acid was ineffective at 200 mg/kg (p.o.). In conclusion, although glycyrrhetinic acid is active in other experimental conditions of inflammation, it has no effect on vasodilation and plasma extravasation induced by the neuropeptides released by the action of capsaicin. In vitro Studies on Inflammatory Mediators

Modulation o f Lipoxygenase Activity

Leukotrienes, for which the 5-LOX is the key synthetic enzyme, are involved in initiating and maintaining a variety of inflammatory diseases such as psoriasis, bronchial asthma, chronic rheumatism and anaphylactic shock. In the family of leukotriene-type mediators, LTB4 is the most potent stimulator of leukocyte responses such as chemotaxis, cell adhesion, superoxide production, and release of hydrolytic enzymes. From the theoretical point of view, products with specific 5-LOX activity inhibition have a high therapeutic value. At the moment there are drugs caffeic acid, phenidone, 3-amino-(1-trifluoromethyl)pyrazoline (BW 755C), eicosatetrainoic acid, nordihydroguaiaretic acid (NDGA) and retinoids--- that are potent, but not specific inhibitors of 5-LOX activity. Since the 5-LOX activity is sensitive to general antioxidants, almost all of the first generation 5-LOX inhibitors belong to the redox type inhibitors. The gum resin of Boswellia serrata (Burseraceae) has been used for the treatment of inflammatory diseases in the traditional Ayurvedic medicine in India. The EtOH extract of the resin significantly decreased LTB4 production in rat peritoneal neutrophils in vitro [95]. The active principles of the gum resin exudate of B. serrata are pentacyclic triterpene acids identified as boswellic acids, whose chemical skeleton belongs to the


127

ursane type. The prominent form of boswellic acid is the 13-isomer, while the minor components are ot-boswellic acid and 11-keto-13-boswellic acid [49]. These substances have been studied in vitro to demonstrate that the mechanism of inhibition of the 5-LOX activity is not connected with antioxidative properties [96]. These authors therefore employed rat peritoneal PMNL, which when stimulated with Ca2+ ionophore produce mainly LTB4 and 5-HETE from endogenous AA. All the forms of boswellic acid inhibited 5-LOX activity in a concentration-dependent manner, with comparable IC50 values, ranging from 1.5 lxM for acetyl-11keto-13-boswellic acid (AKBA) to 7 IxM for acetyl-13-boswellic acid. At all the tested concentrations the boswellic acids decreased LTB4, its all-transisomers and 5-HETE levels simultaneously. This means that boswellic acids effects on the LTA4 hydrolase and/or glutathione peroxidase, which catalyse the conversion of LTA4 into LTB4 and of 5hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-HETE respectively, should be negligible. The results were compared with glycyrrhetinic acid, which despite its anti-inflammatory activity did not decrease the formation of LTB4 and 5-HETE. The reference drugs were hydrocortisone, which in this system exerted no immediate effects, and the redox type 5LOX inhibitor NDGA, which reduced the LTB4 formation with an IC50 of 0.5 I.tM. In washed human platelets, NDGA inhibited the formation of 12-LOX products as well as the COX product 12-hydroxyheptadecatrienoic acid (12-HHT), with an IC50 of 5 I.tM, while boswellic acids (mixture of o~- and 13-isomers) at concentrations up to 400 BM exerted no effect. In addition, in a cell-free system, the non-enzymatic peroxidation of AA by iron (II)ascorbate was not affected by boswellic acid at concentrations up to 400 BM, whereas NDGA abolished the peroxidation of AA in this test at a concentration of 10 laM. These results suggest that the activity of boswellic acids is selective in the 5-LOX pathway, and the mechanism of inhibition is different from that of the classic antioxidant 5-LOX inhibitors (NDGA, caffeie acid, quercetin) [96]. AKBA inhibited 5-LOX activity without modifying 12-LOX and COX-1 activities. Several in vitro experiments have been performed [59] to identify the mechanism of this novel inhibitor of the LTs synthesis. The activity of AKBA in the cell-free system and purified enzyme suggested that the point at which the inhibition of 5-LOX occurs is different from the AA substrate binding site, where the direct 5-LOX inhibitors (e.g. zileuton and AA-861) act. In contrast with AKBA, other pentacyclic triterpenes such as o~-amyrin or ursolic acid did not inhibit 5LOX activity in the cell-free system at comparable concentrations. In presence of increasing concentrations of the non-inhibitory triterpene amyrin, AKBA's inhibitory effect on 5-LOX activity was antagonised. In contrast with the functional antagonism, the effects of 5-LOX inhibitors from different chemical classes were not modified in presence of increasing

128

RiOS et al.

concentrations of amyrin. To study whether the antagonistic effects of inhibitory and non-inhibitory triterpenes were due to nonselective lipophilic interactions, the inhibitory activity of AKBA was studied in presence of tetracyclic steroids (e.g. cholesterol, cortisone, and testosterone). These four-ring compounds, at comparable concentrations, did not modify the 5-LOX activity or reverse the AKBA-induced inhibition of 5-LOX. In conclusion, AKBA is an enzyme-directed, non-redox based LT biosynthesis inhibitor that interacts with 5-LOX via a pentacyclic triterpene-selective binding site that is different from the AA substratebinding site [59]. The structural requirements of boswellic acid type 5-LOX inhibitors for selective bindings to the effector site and for enzyme inhibitory activity have recently been reported by Sailer et al. [97]. Saponification of AKBA yielded 11-keto-13-boswellic acid with a free 3o~-OH function. This deacetylation slightly diminished the 5-LOX inhibitory potency in intact cells and in a cell free system. A minor decrease in 5-LOX inhibitory activity was found to be caused by a reduction of the carboxyl function of 11-keto-13-boswellic acid to a primary alcohol function. This yielded an 11-keto diol-triterpene, which was still able to inhibit 5-LOX activity. In order to study whether the 11-keto function of pentacyclic triterpenes is enough to inhibit the activity, the compounds without a carboxyl group were assayed and proved to have no activity. 13-Boswellic acid, which lacks the keto function on C-11, only partially inhibited 5LOX activity. The reduction of the carboxyl function of 13-boswellic acid to an alcohol yielding 3~, 24-diol functions caused a total loss of inhibitory activity. However, the existence of an 11-keto group alone in the pentacyclic triterpene is not sufficient to inhibit the 5-LOX activity. It must be combined with an additional hydrophilic group in C-4 of ring A, as demonstrated by the action of AKBA, 11-keto-13-boswellie acid and 11keto diol corresponding to 13-boswellic acid. The data suggest that the binding of pentacyclic triterpenes to the effeetor site is mediated by the pentacyclic ring system, whereas defined functional groups, especially the 11-keto function, are required for inhibiting the 5-LOX activity [97]. Hopanoids are a group of unusual pentacyclic triterpenes that are present in species of bacteria and blue-green algae. Three hopanoids isolated from Zymomonas mobilis were evaluated for their ability to inhibit three AA-metabolising enzymes: 15-LOX from soybean, and human 5-LOX and COX. Only 15-LOX was inhibited by tetrahydroxybacterihopane (THBH) with an IC50 of 10 IxM, but not by the other hopanoids, THBH-glucosamine and THBH-ether. The latter two enzymes were not significantly inhibited at 25 or 50 IxM of THBH. The selective inhibition of 15-LOX by THBH indicates that potentially it has unique pharmacological properties, which are interesting, because most NSAIDs inhibit 5-LOX, 15-LOX and COX [98].


129

Flowers of Calluna vulgaris (Ericaceae) are used in folk medicine for their anti-inflammatory properties. Using a LOX activity test, extracts from this plant were screened to characterise the principle responsible for this effect. Ursolic acid was isolated from the acetone extract and its antiLOX activity was studied. Its IC50 was identical (about 0.3 mM) for both 5- and 15-LOX. Ursolic acid at a concentration of 1 IxM also inhibited LOX activity in mouse peritoneal macrophages as did NDGA, but ursolic acid was more specific. While NDGA diminished the level of COX metabolites, this enzyme was only slightly affected by the ursolic acid. Inhibition was also studied in human platelets and HL60 leukemic cells, and the results were compared with classic LOX and COX inhibitors. The effect of ursolic acid on macrophages was weaker than that of NDGA or BW755C, while the effect of the triterpene and BW755C on the formation of LOX and COX products was more pronounced than that of NDGA. In conclusion, the LOX inhibitory activity of ursolic acid appears to be dependent upon cell type (macrophage, platelet, granulocyte) [99,100]. In order to obtain more information on the chemical reactions involved in the LOX inhibitory effect of triterpenes, ursolic acid and its analogues (uvaol, oleanolic acid and methyl ursolate) were studied on LOX activity. The best inhibitors of soybean 15-LOX were ursolic and oleanolic acids, with IC50 values of 0.175 and 0.265 mM, respectively. These results showed that the carboxylic group in the position 28 in ursolie acid is implicated in the inhibition of LOX activity because methylation of this functional group abolished it. Other structural features of ursolic acid are also relevant for its inhibitory effect, for oleanolic acid is less active than ursolic acid, and yet they only differ in the position of one methyl group (at C-20 instead of C- 19) [101 ]. To establish the mechanism of action of amyrin acetates as antiinflammatory agents, ct-amyrin acetate, ~-amyrin acetate and 13-amyrin were tested for their effects on the synthesis of 5-LOX products in human neutrophils. None of the triterpenes had any effect on LTB4 synthesis but all reduced 5-HETE synthesis, tx-Amyrin acetate and 13-amyrin acetate inhibited neutrophil synthesis of 5-HETE to the same extent (about 30%). 13-Amyrin inhibited it by 58%, while equivalent concentrations of txamyrin only gave 27%. These data suggest a specific inhibition of glutathione peroxidase, ct-Amyrin, 13-amyrin and their acetates do not appear to affect the 5-LOX enzyme, which catalyses the conversion of AA to 5-HPETE and its subsequent conversion to LTA4, the unstable precursor of the LTs. The results are inconsistent with the greater antiarthritic/anti-inflammatory activity of 13-amyrin acetate in vivo, and for this reason, the authors propose that the selective anti-inflammatory activity of the acetates could be due to the relative effectiveness of the alcohols in inhibiting the 5-HETE synthesis [ 102]. t~-Amyrin and its palmitate and linoleate esters were also tested for COX and LOX activities. The anti-COX effect was assessed using the

130

RiOS et as

carrageenan paw edema in rats, whereas anti-LOX activity was evaluated by measuring the production of 5-HETE and LTB4 by human neutrophils. At a concentration of 50 IxM, t~-amyrin reduced 5-HETE production without affecting LTB4 synthesis, but t~-amyrin palmitate only reduced LTB4 while its linoleate ester had considerable anti-LOX effects. This effect may be due to the competition between the unsaturated fatty acid linoleate and AA in the formation of inflammatory products. The antiLOX effect of o~-amyrin linoleate is similar to that of methotrexate on human arthritic neutrophils; it reduces LTB4 formation by 50%, although o~-amyrin linoleate at 62 ~M caused twice the inhibition of 5-HETE by methotrexate [ 103]. These same authors have studied in vivo and in vitro the effects of the triterpenes lupeol, lupeol-3-palmitate and lupeol 3linoleate on the release of the anti-arthritic joint degradative enzyme collagenase and on the release of LOX inflammatory products by human neutrophils. These compounds hardly inhibited LTB4 formation, and lupeol linoleate was the most active out of the three, with a 30% inhibitory concentration of 27 ~tM. It seems that the mechanism for these effects may be a stabilisation of plasma membranes [71 ].

Human Leukocyte Elastase Activity and Other Hydrolytic Enzymes Human leukocyte elastase (HLE) is a serine protease produced and stored by PMNL and involved in the tissue destruction observed in many inflammatory diseases such as chronic arthritis. In fact, administration of exogenous elastase inhibitors could be a means of protecting tissues from proteolytic attack. Several plant triterpenes (ursolic, oleanolic and 1813-glycyrrhetinic acids) have been evaluated as competitive inhibitors of riLE in vitro [ 104]. Ursolic acid was the most active compound, interacting with the enzyme in a rapid, reversible manner. The authors concluded that the carboxyl group at position C-28 of ursolic acid is involved in binding to HLE, because the presence of a high concentration of NaC1 (1 lttM) in the assay system does not change the mode of inhibition but the Ki value rises from 4 ~tM to 13 IxM. This proves the contribution of electrostatic interactions involving this group. The results were compared with those of uvaol, a pentacyclic triterpene with a similar structure but with an OH group instead of COOH at position 28. Uvaol also proved to be a competitive inhibitor, with a Ki (16 ~tM) similar to that of ursolic acid in high salt solution. These results demonstrate that electrostatic interaction between the 28-carboxyl group and a positively charged group in the enzyme contribute to binding. This conclusion is also supported by the results obtained with other pentacyclic triterpenes belonging to the 13-amyrin series in which oleanolic acid is a stronger inhibitor than erythrodiol. It seems that the binding of triterpene acids to HLE is mediated by the formation of a salt bridge involving the COOH group of inhibitors. The


131

mechanism of inhibition can be understood if we compare ursolic acid activity with that of oleic acid and cis-unsaturated fatty acid analogues. The data suggest that the substrate-binding domain of HLE is able to bind hydrophobic structures. The boswellic acid derivative, AKBA, decreases the activity of riLE in vitro in a concentration-dependent manner, with an ICs0 value of 15 laM. This result has been compared with those obtained with ursolic acid, amyrin and 18]]-glycyrrhetinic acid, the last of which showed no inhibitory activity on HLE at concentrations up to 20 [tM [105]. Data analyses indicate different mechanisms for the inhibitory actions of the pentacyclic triterpenes AKBA and ursolic acid; inhibition is noncompetitive in the case of AKBA but competitive with ursolic acid. These results are in line with the hypothesis that pentacyclic triterpenes interact with the extended substrate-binding domain in HLE that can bind a variety of hydrophobic ligands [104]. Depending on the substrate length, HLE inhibition can be competitive (ursolic acid) or non-competitive (AKBA). In summary, HLE inhibition has been established for different compounds, but a dual HLE/5-LOX inhibitory property is characteristic of the pentacyclic triterpenes from the boswellic acid series. Although alternative mechanisms of action may contribute to the anti-inflammatory action of pentacyclic triterpenes, these compounds can serve as parent compounds for a new class of HLE inhibitors. Collagenase production and release are partly responsible for the joint destruction that characterises human rheumatoid arthritis. Triterpenes from the lupane and o~-amyrin groups have been studied in vitro to examine their effects on the release of the arthritic joint degradative enzyme collagenase using the rat osteosarcoma. This test and the rat synovial granuloma of adjuvant arthritis are similar; both models are based on connective tissue tumours with bone-invasive properties. The pentacyclic triterpenes assayed have been shown to possess general antiproteolytic effects that can explain the anti-arthritic effects in adjuvant arthritis in rats [71,103]. Effects on Phospholipase A 2A ctivity

PLA2 is an enzyme that is involved in the metabolism of phospholipids and, in consequence, inhibition of this enzyme could be useful for controlling certain inflammatory diseases. Anti-PLA2 activity against three different forms of PLA2 was reported by Jain et al. [50] for two lanostanes isolated from Schinus terebinthifolius (Anacardiaceae) and identified as masticadienoic and masticadienolic acids. According to the authors, these compounds have a novel pharmacophore that interacts with the catalytic site of the enzyme. The two substances differed significantly in their inhibitory potencies in relation to the PLA2 from different sources. Masticadienolic acid, also called schinol, at 0.016

132

RIOS et aL

mole fraction (mF) produced a 50% inhibition of the PLA2 from pig pancreatic enzyme, and a similar value was observed against PLA2 from bee venom. When it was assayed against the recombinant PLA2 from the human synovial fluid enzyme, the ICs0 was 0.05 mF. On the other hand, masticadienoic acid was active against PLA2 from pig pancreas and bee venom, but was ineffective on PLA2 from synovial fluid. These results were compared with those of pentacyclic triterpenes (ursolic and oleanolic acids) and the related lanostane derivatives from Ganoderma lucidum (Polyporaceae), called ganoderic acids [106]. Ursolic acid at 0.1 mF inhibited the activity of the bee venom enzyme by 50% without producing any significant effect on the enzyme from the two other sources, whereas oleanolic acid was totally inactive. Ganoderic acids presented different results depending on the substitution pattern of the rings. Ganoderic acids R and S were active against pig pancreas PLA2 (0.03 and 0.08 mF, respectively), while ganoderic acid T was able to inhibit all the enzymes regardless of the origin. Structure/activity correlation, based on the above mentioned results, indicated that the terpenoid nucleus, the substitutions on the tetracyclic ring, and the aliphatic acidic side chain are important for the activity. In addition, the results of kinetic studies suggest that the mechanism of inhibition was competitive, and that binding to the catalytic site was due to the carboxylic group because when it was methylated, the activity disappeared. The amphipathic character of both triterpenes could explain the minor potency of masticadienoic acid in relation to schinol: the ionised carboxylate group is probably at the surface whereas the tetracyclic nucleus must be in the hydrophobic zone of the bilayer [50]. Two other lanostane-type triterpenes, pachymic and dehydrotumulosic acids, were evaluated as PLA2 inhibitors in a polarographic assay carried out with PLA2 from Naja naja venom [48]. Dehydrotumulosic acid was the most active inhibitor, with an ICs0 of 0.845 mM, around three times lower than that of mepacrine (ICs0 = 2.16 mM), the reference drug used in this study. Pachymic acid exhibited a potency (ICs0 = 2.9 mM) in the same range as mepacrine. The results were in concordance with the structural features indicated above for triterpenes from Schinus but, in addition, it seems that the free carboxylic group present in the side chain can change its position along this chain. lnterleukin-1 Release

Celastrol, from Tripterygium wilfordii (Celastraceae), inhibited mouse IL-1 production in peritoneal macrophages, induced by lipopolysaccharide, and IL-2 production in splenic lymphocytes induced by concanavalin A [ 107]. This plant also furnished three novel tdterpenes named regeol A, B and C, belonging to the D:A-friedooleanane type and nine known triterpenes [42]. In rheumatoid arthritis, the degree of inflammation of the articular synovial membrane has to do with the production of IL-1. For this reason, the


133

triterpenes were evaluated for their inhibitory activity on IL-1 ct and IL-113 release from lipopolysaccharide-stimulated human peripheral mononuclear cells. Among the compounds, tingenin B, celastrol and derivatives of celastrol showed strong inhibitory activity (100% release inhibition), while regeol A only inhibited by about 50% and the other two new triterpenes were not active.

Lipid Peroxidation and Free Radicals Two of the more widespread triterpenes, oleanolic and ursolic acids, are inhibitors of lipid peroxidation (LP) in vitro, as demonstrated by Balanheru and Nagarajan [108] using rat hepatic microsomes with three different stimulator systems CC14,ADP/NADPH and iron/ascorbate. Pretreatment of the microsomal fraction with the assayed triterpenes always offered a higher degree of protection against peroxidation than did simultaneous and post-induction treatments. When using iron/ascorbate as inducers, both pre- and simultaneous treatment with oleanolic acid reduced malondialdehyde (MDA) production by 90%, a current parameter indicating oxidative rupture of fatty acid residues. The same percentage was obtained for pre-treatment with CC14 or ADP/NADPH. The highest value obtained for ursolic acid was a 61% reduction in the iron/ascorbate system [ 108]. Several well documented studies have shown the antioxidant properties of Glycyrrhiza glabra (Papilionaceae) saponins and sapogenins, and it has recently been reported that L-ascorbic acid-2-(2013-11-oxo-olean- 12-en-29oic acid ethyl ester-3[~-yl hydrogen phosphate) sodium salt (GEPC), a 13glycyrrhetinic acid ester conjugated to vitamin C, has the same properties. This chimaeric compound is a free radical scavenger that is much less active than ascorbic acid on the in vitro 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test, but it is an efficient inhibitor of free radical generation, particularly of hydroxyl generation by the Fenton-type reagent Clgi+/H202 monitored by electronic spin resonance (ESR). In addition, GEPC acts as an iron chelator and has no demonstrated prooxidant activity in the iron~leomycin DNA degradation system. With respect to its effects on LP, 1 mM GEPC strongly inhibited microsomal MDA production induced by Fe3+/ADP/NADPH, in contrast with the very limited effect of glycyrrhetinic acid [ 109]. Quite different are the chemical features of some newer antiperoxidative triterpenoids from Trypterigium wilfordii, because they have highly unsaturated A and B rings with a quinonoid-like structure over a friedooleanane skeleton. The main datum to be noted is that celastrol, the most representative compound of this series, inhibits mitochondrial LP, with an ICs0 = 7 ~tM. Its potency is then 15 times higher than that of t~tocoferol. Furthermore, it has been observed that while celastrol and its acetyl derivative affect the radical chain reaction in a biphasic manner, txtocoferol and pristimerin, a celastrol-methyl ester, do it monotonously.

134

R[OS eta/.

Considering that celastrol and pristimerin act as radical scavengers whereas acetylcelastrol is inactive, and that none of them affect oxygen radical generation, the 2-oxo-3-hydroxy grouping is surely essential for a free radical-related LP inhibition. When operating with mitoplasms (mitochondria deprived of their outer membrane) LP run monotonously in the presence of each triterpenoid, which indicates that acetylcelastrol should affect only the inner membrane. This is in fact confirmed by the increase in the ~ negative potential in neutral egg-yolk phosphatidilcholine liposomes, which brings with it an increase in the stability of the membranes [110]. Metabolism o f Endogenous Corticosteroids

This point probably constitutes one of the triumphs of the rational use, but also misuse, of triterpenoids in therapeutics, because both the antiinflammatory activity and potentially toxic mineralocorticoid side effect of liquorice products are known to depend on an inhibition of glucocorticoid metabolism. More precisely, they depend on the inhibition of 1113hydroxysteroid dehydrogenase (ll-HSD), a widely distributed key enzyme for the inactivation of cortisol---corticosterone in rodents--which catalyses the conversion of the 11-hydroxyl group into an 11-oxo group to give cortisone and 11-dehydrocorticosterone, respectively. This step is thought to be of great importance in the regulation of the access of aldosterone to the mineralocorticoid receptor (MCR), but it is also very important for the alternative access of glucocorticoids to both MCR or GCR and has thus led to the concept of "enzyme-mediated receptor protection" [ 111 ]. As a consequence, glycyrrhetinic acid has been shown to potentiate the slight anti-inflammatory effect of hydrocortisone in the skin; it displaces the dose/response plot to the left and increases the slope. Restraining the activity of 11-HSD is then a valuable goal if we want to increase the efficacy of both hormonal and exogenous corticoids in vivo, and even more so in the case of inflammo-proliferative skin diseases. In these diseases 11HSD is present in larger amounts, as was detected by immunohistochemistry of normal, psoriatic and eczematous human skin [1 12]. In a complete multi-organ screening, the inhibitory effect of glycyrrhetinic acid on 11-HSD in vivo was detectable in many other tissues 3h after i.p. administration, although it disappeared by 24 h. The most sensitive organs were kidney, liver, testis, thymus and spleen [ 113]. Whorwood et al. [1 14] have reported that after continuous administration of 75 mg/kg/day of glycyrrhizin, for 5 days in drinking water to rats, both 1 I-HSD activity and 11-HSD mRNA levels were significantly diminished in selected specific mineralocorticoid (distal colon, kidney) and glucocorticoid (liver, pituitary) tissues. It was further demonstrated that glycyrrhetinic acid had an effect not only on 11-HSD activity and 11-HSD mRNA levels in pituitary GH3 cells, but also on


135

prolactin (PRL) mRNA levels, which are indicative of hypophysial GCR activation. In this system, glycyrrhetinic acid reduced 11-HSD-related parameters at doses between 10-8-10.5 M, but failed to affect PRL-related parameters. This suggests that there is no direct interaction with GCR. In contrast, the glucocorticoid agonists dexamethasone and RU 28362 did not affect 11-HSD activity although they reduced, as was to be expected, the expression of the PRL gene. Simultaneous presence of corticosterone and glycyrrhetinic acid resulted in a substantial inhibition of PRL mRNA synthesis, and this effect was abolished by the glucocorticoid antagonist RU 38486 but not by the mineralocorticoid antagonist RU 26752. For this reason some authors have concluded that glycyrrhetinic acid protects GCR by means of 11-HSD inhibition, and that its actions do not arise from any direct steroid agonism [ 114]. More evidence of the importance of 11-HSD in the central nervous system feedback regulation of corticosteroid production is the diminished release of corticotrophin-releasing factor-41 (CRF-41) to hypophysial portal blood in the presence of glycyrrhetinic acid and stable levels of circulating corticosteroids. As this effect disappears in adrenalectomised rats, it is evident that glycyrrhetinic acid needs, and therefore acts through, the presence of corticosterone. Moreover, when dexamethasone, which is not extensively metabolised by 11-HSD, is added to the glycyrrhetinic acid treatment in adrenalectomised rats, CRF-41 levels fall, demonstrating the implication of the enzyme [115]. All these assumptions on the physiopathological role of 11-HSD and its modification by certain triterpenoids are well established, but the whole scenario is presently changing due to the recent description of two different isoenzymes: l l-HSD-1, which is bi-directional, NADPdependent, acts with little substrate specificity and has a Km in the mM range, and 11-HSD-2, which at first was considered unidirectional, collocalises with MCR, is NAD-dependent, acts with great substrate specificity and has a Km in the nM range. A further 11-HSD-3 has been characterised from the choriocarcinoma cell line JEG-3 [ 116]. 11-HSD-2 apparently performs the functions associated with the classic mineralocorticoid-like effects, while the widely distributed 11-HSD-1 must be responsible for certain hepatic metabolic processes, including the retro conversion of cortisone into cortisol, and of l l dehydrocorticosterone to corticosterone in rats. The latter conversion accounts for potential enzymatic anti-inflammatory effects based on an increase in these adrenal hormones. This was in fact recently demonstrated in glomerular mesangial cells stimulated with IL-113 and TNF-o~ to release PLA2, an event which is inhibited by 11-hydroxy-glucocorticoids. IL-113 and TNF-ct counterbalance their own pro-inflammatory effects by upregulating the reductase activity of 11-HSD-1, and therefore make 11keto-glucocorticoids appear to be active in this system [ 117].

136

RIOS a aL

Anti-complement A ctivRy

Some of the best known anti-inflammatory triterpenoids have been shown to have inhibitory activity on the complement cascade. A mixture of the aforementioned boswellic acids reduced in a dose-dependent manner the classic pathway activity by as much as 77%, and C3-convertase by 72% [118]. Oleanolic acid showed 85% and 71% inhibition, respectively, in the same tests, at a single dose of 100 l.tg/ml [ 119], and [3-glycyrrhetinic acid inhibited the classic human pathway with an ICs0 of 35 laM. The componem affected was C2. The o~ form of glycyrrhetinic acid was fairly inactive [ 120]. None of these three triterpenoids appreciably inhibited the alternative complement pathway. Histamine Release

In a search for the principles responsible for the histamine release inhibitory activity of Melaleuca leucodendron (Myrtaceae) fruits, some phenolics and triterpenoids were isolated. Ursolic acid was the only triterpenoid that had an effect. At a concentration of 1 mM, it was able to reduce by 95% the amount of liberated histamine from rat mast cells. Other triterpene aldehydes and acids from the same source were inactive [ 121 ]. In a similar biological preparation, the tetracyclic triterpenes penasterone and acetylpenasterol acted with IC50 of 1.5 lttM and 10 I.tM respectively [ 122]. Rather more specific was the work of Lee et al. [ 123], who studied the inhibitory activity of glycyrrhetinic acid on histamine synthesis in mouse mast cells, co-cultured with fibroblasts in order to approach the physiological phenotype of connective tissue mast cells. In these conditions, 50 IttM glycyrrhetinic acid inhibited histidine decarboxylase (HDC) by 80% and PKC-8-mRNA expression, thus suggesting that this isoenzyme regulates HDC activity. Protein Kinase C

One of the key points, possibly the most important one, in the inflammatory and tumour-promoter effects of TPA and related phorbols is the activation of PKC. For that reason many of the compounds biologically opposing TPA are tested for PKC inhibition. Glycyrrhetinic acid showed great homogeneity in the ICs0 for both inhibition of rat brain PKC (IC50 = 450 l.tM) and TPA binding to mouse epidermal membranes. This may mean that there is a causal relationship between these parameters. Glycyrrhizin was a much weaker inhibitor, causing a 23% reduction at 1 mM, in contrast with 90% for glycyrrhetinic acid at this dose [124]. In a later study covering the interaction with rat brain PKC and other protein kinases, the ICs0of glycyrrhetinic acid for PKC


137

inhibition was reported to be 121 IxM, whereas glycyrrhizin was inactive and betulinic, ot-glycyrrhetinic, oleanolic and ursolic acids had IC50 below 300 l.tM. The potencies were noticeably higher for the inhibition of the rat liver cAMP-dependent protein kinase, which led the authors to discuss whether this fact is useful in explaining previously reported biological effects of triterpenoids [ 125].

NO Synthesis Nitrogen monoxide (NO), widely misnamed "nitric oxide", is considered to have an important role in the regulation of inflammatory processes. When generated by constitutive NO synthase (NOS) it exerts protective and anti-inflammatory properties, in striking contrast with what occurs when inducible NOS activity dominates. For this reason, drugs capable of reducing or abolishing inducible NOS response are thought to be potential anti-inflammatory agents. Pristimerin, a friedooleanane which has been cited above as one of the main active constituents of the plant Trypterigium wilfordii, showed an inhibitory effect on lipopolysaccharideinduced NO production by cultured RAW 264.7 murine macrophages, measured in terms of L-arginine consumption and nitrite accumulation (IC50 = 0.2-0.3 IxM). This inhibition was proven not to depend on a direct enzyme inhibition but on a decrease in NOS induction, which occurred at a transcriptional level by a nuclear factor-kappa B (NF~zB)-mediated pathway. Furthermore, pristimerin 1 gM also reduced the NOS mRNA level, evaluated by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In contrast, no effect was observed on COX-2 mRNA levels, which are known to be also responsive to NF~cB [126]. ABBREVIATIONS AA

=

Arachidonic acid

Ag-Ab

=

Antigen-Antibody

AKBA

=

Acetyl-11-keto-13-boswellic acid

BK

=

Bradykinin

BW 775C =

3-Amino-( 1-trifluoromethy l)pyrazoline

COX

=

Cyclooxygenase

CRF

=

Corticotrophin-releasing factor

DAG

=

Diacylglycerol

DNCB

=

D initrochlorobenzene

138

RiOS a a/.

DPPH

=

1,1-Diphenyl-2-picryl-hydrazyl

EPP

=

Ethylphenylpropiolate

ESR

=

Electronic spin resonance

GCR

=

Glucocorticoid receptor

GEPC acid

=

L-Ascorbic acid-2-(20 13-11-oxo-olean- 12-ene-29-oic ethyl ester-313-yl hydrogen phosphate)sodium salt

HDC

=

Histidine decarboxylase

HETEs

=

Hydroxyeicosatetraenoic acids

HHPA

=

12-O-Hexadecanoyl- 16-hydroxyphorbol- 13-acetate

12-HHT

=

12-Hydroxyheptadecatrienoic acid

HLE

=

Human leukocyte elastase

5-HPETE

=

5-Hydroperoxyeicosatetraenoic acid

11-HSD

=

1113-Hydroxysteroid dehydrogenase

5-HT

=

Serotonin, 5-Hydroxytriptamine

IL-1

=

Interleukin- 1

/.p.

=

Intraperitoneally

IP3

=

Inositol triphosphate

LOX

=

Lipoxygenase

LP

=

Lipid peroxidation

LTs

=

Leukotrienes

MCR

=

Mineralocorticoid receptor

MDA

=

Malondialdehyde

mF

=

Mole fraction

MPO

=

Myeloperoxidase

NDGA

=

Nordihydroguaiaretie acid

NFKB

=

Nuclear factor-kappa B

NK-1

=

Neurokinin- 1

NOS

=

N O synthase

NSAIDs

=

Non-steroidal anti-inflammatory drugs

ODC

=

Omithine decarboxylase


PAF

=

Platelet activating factor

PGs

=

Prostaglandins

PKC

=

Protein kinase C

PLA2

=

Phospholipase A2

PLC

=

Phospholipase C

PMNL

=

Polymorphonuclear leukocytes

p.o.

per os

139

(Orally)

PRL

=

Prolactin

RT-PCR

=

Reverse transcriptase-polymerase chain reaction Subcutaneously

S.C. SP

=

Substance P

THBH

=

Tetrahydroxybacterihopane

TNF

=

Tumour necrosis factor

TPA

=

12-Tetradecanoylphorbol- 13-acetate

TXs

=

Thromboxanes

REFERENCES [1] [2] [3] [4] [5] [6] [7]

[8] [91 [~0]

[12] [13l [14] [15] [16] [17]

[18]

Mahato, S.B.; Nandy, A.K.; Roy, G. Phytochemistry, 1992, 31, 2199. Mahato, S.B.; Kundu, A.P. Phytochemistry, 1994, 3 7, 1517. Mahato, S.B.; Sen, S. Phytochemistry, 1997, 44, I185. Connolly, J.D.; Hill, R.A.; Boar, R. Nat. Prod. Rep., 1984, 1, 53. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1985, 2, I. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1986, 3, 421. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1989, 6, 475. Connolly, J.D.; Hill, R.A. In Methods in Plant Biochemistry. Terpenoids; Charlwood, B.V.; Banthorpe, D.V., Eds., Academic Press: London, 1991, Vol. 7, pp. 331-359. Connolly, J.D.; Hill, R.A.; Ngadjui, B.T. Nat. Prod. Rep., 1994, ! 1, 91. Connolly, J.D.; Hill, R.A.; Ngadjui, B.T. Nat. Prod. Rep., 1994, 11,467. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1995, 12, 475. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1996, 13, 15 I. Harborne, J.B.; Baxter, H. Phytochemical Dictionary, Taylor and Francis: London, 1993. Harbome, J.B. Nat. Prod. Rep., 1986, 3, 323. Harborne, J.B. Nat. Prod Rep., 1993, 10, 327. Harborne, J.B. Nat. Prod Rep., 1989, 6, 85. Macias, F.A.; Simonet, A.M.; Esteban, M.D. Phytochemistry, 1994, 36, 1369. Bar-Nun, N.; Mayer, A.M. Phytochemistry, 1990, 29, 787.

140

[19]

RiOSeta/.

Shibata, S. In New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical Activity; Wagner, H.; Wolff, P., Eds.; SpringerVerlag: Berlin, 1977; pp. 177-196. [20] Tang, W.; Eisenbrand, G. Chinese Drugs of Plant Origin. Chemistry, Pharmacology, and Use in Traditional and Modern Medicine, Springer-Verlag: Berlin, 1992. [21] Matsuda, H.; Samukawa, K.; Kubo, M. Planta Med., 1990, 56, 19. [221 Hashimoto, M.; Inada, K.; Ohminami, H.; Kimura, Y.; Okuda, H.; Arichi, S. Planta Med., 1985, 51, 401. [23] Ohuchi, K.; Watanabe, M.; Ozeki, T.; Tsurufuji, S. Planta Med., 1985, 51, 208. [24] Recio, M.C.; Just, M.J.; Giner, R.M.; Mfiflez, S.; Rios, J.L.; Hostettmann, K. d. Nat. Prod., 1995, 58, 140. [25] Hiller, K. In Biologically Active Natural Products; Hostettmann, K; Lea, P.L., Eds.; Clarendon Press: Oxford, 1987; pp. 167-184. [26] Rios, J.L.; Waterman, P.W. Phytother. Res., 1997, 11, 411. [27] Harrison, D.M. Nat. Prod. Rep. 1990, 7, 459. [28] Abe, I.; Rohmer, M.; Prestwich, G.D. Chem. Rev., 1993, 93, 2189. [29] Bevan, C.D.; Marshall, P.S. Nat. Prod. Rep., 1994, 11, 451. [301 Modey, W.K.; Mulholland, D.A.; Raynor, M.W. Phytochem. Anal., 1996, 7, I. [311 Chandel, R.S.; Rastogi, R.P. Phytochemistry, 1980, 19, 1889. [32] Rfos, J.L.; Sime6n, S.; Jim4nez, F.J.; Zafra-Polo, M.C.; Villar, A. Fitoterapia, 1986, 57, 153. [33] Jork, H.; Funk, W.; Fischer, W.; Wimmer, H. Thin-Layer Chromatography. Reagents and Detection Methods, VCH: Weinheim, 1990, Vol. la. [34] Li, T.S.; Li, J.T.; Li, H.Z.J. Chromatogr. A, 1995, 715, 372. [351 Shiao, M.S.; Lee, K.R.; Lin, L.J.; Wang, C.T. ACS Symposium Series, 1994, 547, 342. [36] Gunther, B.; Wagner, H. Phytomedicine, 1996, 3, 59. [37] Gaspar, E.M.S.M.; dasNeves, H.J.C.; Noronha, J.P. HRC-J. High Resol. Chromatogr., 1997, 20, 417. [381 Vitanyi, G.; Bihatsi Karsai, E.; Lefler, J.; Lelik, L. Rapid Commun. Mass Spectrosc., 1997, /1, 691. [39] Wolfender, J.L.; Maillard, M.; Hostettmann, K. Phytochem. Anal., 1994, 5, 153. [40] Heinzen, H.; de Vries, J.X.; Moyra, P.; Remberg, G.; Marttnez, R.; Tietze, L.F. Phytochem. Anal., 1996, 7, 237. [41] Chen, H.R.; Sheu, S.I.J. Chromatogr. A, 1993, 653, 184. [42] Takaishi, Y.; Wariishi, N.; Tateishi, H.; Kawazoe, K.; Nakano, K.; Ono, Y.; Tokuda, H., Nishino H.; Iwashima, A. Phytochemistry, 1997, 45, 969. [431 Della Loggia, R.; Tubaro, A.; Sosa, S.; Becker, H.; Saar, St.; Isaac, O. Planta Med., 1994, 60, 516. [44] Zitterl-Eglseer, K.; Sosa, S.; Jurenitsch, J.; Schubert-Zsilaveez, M.; Della Loggia, R.; Tubaro, A.; Bertoldi, M.; Franz, C. J. Ethnopharmacol., 1997, 57, 139. [45] Agrawal, P.K.; Jain, D.C. Prog. NMR Spectrosc., 1992, 24, 1. [46] Shiojima, K.; Arai, Y.; Masuda, K.; Takase, Y.; Ageta, T.; Ageta, H. Chem. Pharm. Bull., 1992, 40, 1683. [47] Reeio, M.C.; Giner, R.M.; Mfiflez, S.; Gu4ho, J.; Julien, H.R.; Hostettmann, K.; R|os, J.L. Planta Med., 1995, 61, 9. [48] Cu611ar, M.J.; Giner, R.M.; Recio, M.C.; Just, M.J.; M,'tflez, S.; Rios, J.L.d. Nat. Prod., 1996, 59, 977.

NATURALTRITERPENOIDS

[491

[50] [51] [52] [53] [54]

[55] [56] [57]

[58] [59]

[60] [61] [62] [63]

[64] [651 [661 [67] [681 [69]

[70] [71] [72] [73] [74] [75] [76] [77]

141

Ammon, H.P.T.; Safayhi, H.; Mack, T.; Sabieraj, J. J. Ethnopharmacol., 1993, 38, 113. Jain, M.K.; Yu, B.Z.; Rogers, J.M.; Smith, A.E.; Boger, E.T.A.; Ostrander, R.L.; Rheingold, A.L. Phytochemistry, 1995, 39, 537. Wagner, H. Planta Med., 1989, 55, 235. Cavey, D. In Pharmacology and the Skin; Hensby, C.N.; Lowe, N.J., Eds.; Karger: Basel, 1989, Vol. 2, pp.44-88. Bouclier, M.; Cavey, D.; Kail, N.; Hensby, C. Pharmacol. Rev., 1990, 42, 127. Davis, R.H.; Leitner, M.G.; Russo, J.M.; Byrne, M.E.J. Am. Pod. Med. Ass., 1989, 79, 263. Young, J.M.; De Young, L.M. In Pharmacological Methods in the Control of Inflammation; Spector, J.; Back, N., Eds.; Alan R. Liss: New York, 1989; pp. 215-231. Lewis, A.J.; Carlson, R.P.; Chang, J. In Handbook of Inflammation. The Pharmacology of Inflammation; Bonta, J.L.; Bray, M.A.; Parnham, M.J., Eds.; Elsevier Science: New York, 1985; Vol. 5, pp. 371-397. Pay~t, M.; Terencio, M.C.; Ferr~ndiz, M.L.; Alcaraz, M.J. Br. J. Pharmacol., 1996, 117, 1173. Liu, J. J. Ethnopharmacol., 1995, 49, 57. Safayhi, H.; Sailer, E.R.; Ammon, H.P.T. Mol. Pharmacol., 1995, 47, 1212. Heldt, R.M.; Winking, M.; Simmet, Th. Naunyn-Schmiedeberg's Arch. Pharmacol., 1996, 353 (suppl. 4), R142. Singh, S.; Bani, S.; Singh, G.B.; Gupta, B.D.; Banerjee, S.K.; Singh, B. Fitoterapia, 1997, 68, 9, Chawla, A.S.; Kaith, B.S.; Handa, S.S.; Kulshreshtha, O.K.; Srimal, R.C. Fitoterapia, 1991, 62, 441. Kaith, B.S. Int. J. Pharmacog., 1995, 1, 73. Singh, B.; Ram, S.N.; Pandey, V.B.; Joshi, V.K.; Gambhir, S.S. Phytother. Res., 1991, 5, 103. Duwiejua, M.; Zeitlin, I.J.; Waterman, P.G. ; Chapman, J. ; Mhango, G.J. ; Provan, G.J. Planta Med., 1993, 59, 12. Duwiejua, M.; Zeitlin, I.J.; Waterman, P.G.; Gray, A.I. Br. J. Pharmacol., 1993, 108, 236 P. Ansari, S.H.; Ali, M. Fitoterapia, 1996, 57, 103. Ahumada, C.; S~enz, T.; Garc|a, D.; De la Puerta, R.; Fernandez, A.; Marttnez, E.J. Pharm. Pharmacol., 1997, 49, 329. Recio, M.C.; Giner, R.M.; M,~flez, S.; Rtos, J.L. Planta Meal, 1995, 61, 182. Kweifio-Okai, G.; Bird, D.; Field, B.; Ambrose, R.; Carroll, A.R.; Smith, P., Vald~s, R. J. Ethnopharmacol., 1995, 46, 7. Kweifio-Okai, G.; De Munk, F.; Macrides, T.A.; Smith, P.; Rumble, B.A Drug Develop. Res., 1995, 36, 20. Kweifio-Okai, G.; Field, B.; Rumble, B.A.; Macrides, T.A., De Munk, F. Drug. Develop. Res., 1995, 35, 137. Kapil, A.; Sharma, S. J. Pharm. Pharmacol., 1995, 47, 585. Kapil, A.; Inflammopharmacology, 1994, 2, 361. Sugishita, E.; Amagaya, S.; Ogihara, Y. J. Pharm. Dyn., 1983, 6, 287. Rios, J.L.; Giner-Larza, E.M.; M~tflez, S.; Giner, R.M.; Recio, M.C. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 177. Giner-Larza, E.M.; Rios, J.L.; M~fiez, S.; Giner, R.M.; Recio, M.C. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 176.

142

[78] [79]

[80] [81]

[82] [83] [84]

[85] [86] [87]

[88] [89] [901 [91] [92] [93] [94] [95] [96] [97] [98] [99]

[~00] [101] [102] [103]

RiOSa a/. Tabata, M.; Tanaka, S.; Cho, H.J.; Uno, C.; Shimakura, J.; Ito, M.; Kamisako, W.; Honda, C. J'. Nat. Prod., 1993, 56, 165. Yasukawa, K.; Akihisa, T.; Oinuma, H.; Kaminaga, T.; Kanno, H.; Kasahara, Y.; Tamura, T.; Kumaki, K.; Yamanouchi, S.; Takido, M. Oncology, 1996, 53, 341. Yasukawa, K.; Akihisa, T.; Oinuma, H.; Kasahara, Y.; Kimura, Y.; Yamanouchi, S.; Kumaki, K.; Tamura, T.; Takido, M. Biol. Pharm. Bull., 1996, 19, 1329. Kaminaga, T.; Yasukawa, K.; Takido, M.; Tai, %; Nunoura, Y. Phytother. Res., 1996, 10, 581. Nukaya, H.; Yamashiro, H.; Fukazawa, H.; Ishida, H.; Tsuji, K. Chem. Pharm. Bull., 1996, 44, 847. Cu~llar, M.J.; Giner, R.M.; Recio, M.C.; Just, M.J.; M~tflez, S.; Rtos, J.L. Chem. Pharm. Bull., 1997, 45, 492. Yasukawa, K.; Takido, M.; Matsumoto, T.; Takeuchi, M.; Nakagawa, S. Oncology, 1991, 48, 72. Akihisa, T.; Yasukawa, K.; Kimura, Y.; Takido, M.; Kokke, W.C.M.C.; Tamura, T. Chem. Pharm. Bull., 1994, 42, l l01. Yasukawa, K.; Akihisa, T.; Tamura, T.; Takido, M. Biol. Pharm. Bull., 1994, 17, 460. Hirota, M.; Mori, T.; Yoshida, M.; Iriyr R. Agric. Biol. Chem., 1990, 54, 1075. Huang, M.T.; Ho, C.T.; Wang, Z.Y.; Ferraro, T.; Lou, Y.R.; Satauber, K.; Ma, W.; Georgiadis, C.; Laskin, J.D.; Conney, A.H. Cancer Res., 1994, 54, 701. M~tflez, S.; Recio, M.C.; Giner, R.M.; Rios, J.L. Eur. J. Pharmacol., 1997, 334, 103. Mb.flez, S.; Huguet, A.I.; Recio, M.C.; Giner, R.M.; Rios, J.L. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. ,4), 175. Inoue, H.; Nagata, N.; Koshihara, Y. Inflamm. Res., 1995, 44, 470. Inour H.; Nagata, N.; Koshihara, Y. Jpn. J. Pharmacol., 1995, 69, 6 I. Inoue, H.; Nagata, N.; Koshihara, Y. Br. J. Pharmacol., 1993, 110, 1614. Inoue, H.; Nagata, N.; Shibata, S.; Koshihara, Y. Jpn. J. Pharmacol., 1996, 71, 281. Ammon, H.P.T.; Mack, T.; Singh G. B.; Safayhi, H. Planta Med., 1991, 57, 203. Safayhi, H.; Mack, T.; Sabieraj, J.; Anazodo, M.I.; Subramanian, L.R.; Ammon, H.P.T.J. Pharmacol. Exp. Ther., 1992, 261, 1143. Sailer, E.-R.; Subramanian L.R.; Rail, B.; Hoernlein, R.F.; Ammon, H.P.T.; Safayhi, H. Br. J. Pharmacol., 1996, 117, 615. Moreau, R.A.; Agnew, J.; Hicks, K.B.; Powell, M.J.J. Nat. Prod., 1997, 60, 397. Simon, A.; Najid, A.; Chulia, A.J.; Delagr C.; Rigaud, M. Biochim. Biophys. ,4cta, 1992, 1125, 68. Najid, A.; Simon, A.; Cook, J.; Chable-Rabinovitch, H.; Delagr C.; Chulia, A.J.; Rigaud,M. FEBS Left., 1992, 299, 213. Es-Saady, D.; Najid, A.; Simon, A.; Denizot, Y.; Chulia, A.J.; Delage, C. Mediators Inflamm., 1994, 3, 181. Kweifio-Okai, G.; Macrides, T.A. Res. Commun. Chem. Pathol. Pharmacol., 1992, 78, 367. Kweifio-Okai, G.; De Munk, F.; Rumble, B.A.; Macrides, T.A.; Cropley, M. Res. Commun. Chem. Pathol. Pharmacol., 1994, 85, 45.


143

[104] Ying, Q.L.; Rinehart, A.R.; Simon, S.R.; Cheronis, J.C. Biochem. J., 1991, 277, 52 I. [105] Safayhi, H.; Rail, B.; Sailer, E.R.; Ammon. H.P.T.J. Pharmacol. Exp. Ther., 1997, 281,460. [1061 Hirotani, M.; Asaka, L.; Ino, C.; Furuya, T.; Shiro, M. Phytochemistry, 1987, 26, 2797. [107] L i, X.Y. Int. J. Immunother., 1993, 9, 181. [108] Balanheru, S.; Nagarajan, B. Biochem. Int., 1991, 24, 981. [109] Liu, J.; Mori, A.; Ogata, K. Res. Commun. Chem. Pathol. Pharmacol., 1993, 82, 151. [llO] Sassa, H.; Kogure, K.; Takaishi, Y.; Terada, H. Free Rad. Biol. Med., 1994, 17, 201. [lll] Funder, J.W.; Pearce, P.; Smith, R.; Smith, A.I. Science, 1988, 242, 583. [ll2] Teelucksingh, S.; Mackie, A.D.R.; Burt, D.; Mclntyre, M.A.; Brett, L.; Edwards, C.R.W. Lancet, 1990, 335, 1060. [ll3] Marandici, A.; Monder, C. Steroids, 1993, 58, 153. [ll4] Whorwood, C.B.; Sheppard, M.C.; Stewart, P.M. Endocrinology, 1993, 132, 2287. Ill5] Seckl, J.R.; Dow, R.C.; Low, S.C.; Edwards, C.R.W.; Flink, G. J. Endocrinol., 1993, 136, 47 I. [ll6] G6mez-S~nchez, E.P.; Ganjam, V.; Chen, Y.J.; Cox, D.L.; Zhou, M.Y.; Thanigaraj, S.; G6mez-SAnchez, C.E. Steroids, 1997, 62, 444. Ill7] Escher, G.; Galli, I.; Vishwanath, B.S.; Frey, B.M.; Frey, F.J.J. Exp. Med., 1997, 186, 189. Ill8] Kapil, A.; Moza, N. Int. J. Immunopharmacol., 1992, 14, 1139. [ll9] Kapil, A.; Sharma, S. J. Pharm. Pharmacol., 1994, 46, 922. [1201 Kroes, B.K.; Benkelman, C.J.; Van der Berg, A.J.; Wolbink, G.J.; Van Dijk, H.; Labadie, R.P. Immunology, 1997, 90, I 15. [121] Tsuruga, T.; Chun, Y.T.; Ebizuka, Y.; Sankawa, U. Chem. Pharm. Bull., 1991, 39, 3276. [122] Shoji, N.; Umeyama, A.; Motoki, S.; Arihara, S.; Ishida, T.; Nomoto, K.; Kobayashi, J.; Takei, M. J. Nat. Prod., 1992, 55, 1682. [123] Lee, Y.M.; Hirota, S.; Jippo-Kanemoto, T.; Kim, H.R.; Shin, T.Y.; Yeom, Y.; Lee, K.K.; Kitamura, Y.; Nomura, S.; Kim, H.M. Int. Arch. Allergy Immunol., 1996, ! I 0, 272. [1241 O'Brian, C.A.; Ward, N.E.; Vogel, V.G. Cancer Lett., 1990, 49, 9. [125] Wang, B.H.; Polya, G.M. Phytochemistry, 1996, 41, 55. [126] Dirsch, V.M.; Kiemer, A.K.; Wagner, H.; Vollmar, A.M. Eur. J. Pharmacol., 1997, 336, 211.



145


CURRENT STATUS OF THE CHEMISTRY AND SYNTHESIS OF NATURAL ANTIMALARIAL COMPOUNDS AND NATURAL SUBSTANCES USED TO ALLEVIATE SYMPTOMS OF DIABETES (ALDOSE REDUCTASE AND A-GLUCOSIDASE INHIBITORS) K. K A W A N I S H I * and N. R. F A R N S W O R T H

*Kobe Pharmaceutical University, Kobe , Japan Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Illinois, U.S.A. ABSTRACT" Atropine, camptothecin, cocaine, digitoxin, digoxin, morphine, pilocarpinr quinine, taxol, vinblastine and vincristine, among others, are important drugs obtained from higher plants and are used clinically. They have also served as lead compounds for the synthesis and modification of more effective and safer drugs, in many cases. In this chapter, drugs used as antimalarial compounds and for the complications of diabetes (aldosr reductasr and o~-glucosidase inhibitors) will be discussed. Natural product chemists have isolated as little as 1.0 mg of pure compounds from natural sources and have been able to determine their structures using high resolution instrumental techniques. Organic chemists have synthesized thousands of compounds to produce one new drug on the basis of natural product leads, and pharmacologists and biochemists have tested their biological activity. Recently chemists and pharmacologists have worked together to develop techniques for studying structure-activity relationships using computer graphics and have designed new drugs. Biochemists, molecular biologists and pharmacologists have identified many receptors on which drugs act. Thus, mechanisms of drug action at the molecular level are being identified. From the accumulation of these results structure-activity relationships will lead to the preparation of thousands of useful compounds. We must produce drugs in these ways, because we cannot rely on solely on the limited amount of active compounds produced naturally in plants, in many cases, for a number of reasons. However we need to employ plant extracts themselves, because there are millions of people who cannot buy expensive synthetic drugs in the world and these extracts are widely used by them. 1.0 N A T U R A L A N T I M A L A R I A L C O M P O U N D S It has been estimated that about 500,000 million cases o f malaria occur annually and about 1-2 million deaths due to Plasmodium falciparum are involved [1]. Most o f these cases occur in tropical countries and there appears to be little systematic research to discover novel antimalarial drugs. Perhaps one o f the reasons for this lack o f interest is that there is hope that an antimalarial vaccine is the answer, but this is not expected to be available in the near future, if ever.

146

KAWANISHI and FARNSWORTH

There are a few efforts to synthesize analogs of already effective antimalarial drugs, e.g. quinine and artemisinin and these will be discussed in this paper (vide infra). These studies are mainly targeted at finding less toxic entities and/or compounds that overcome the rapid resistance that the malarial parasite seems to acquire. One of the earliest systematic searches for antimalarial drugs from plants was published in 1947 [2], which mainly involved the injection of extracts into infected ducklings. Dichroa febrifuga, used in China for centuries as an antimalarial drug, was found to be active and the alkaloid febrifugine was determined to be the main active principle. However, febrifugine is well known to be too toxic for human use. The marine environment seems to be the area most studied in recent years for novel antimalarial agents [3-9] but a reasonable amount of effort seeking plantderived agents can also be identified [10-26] and activity has centered around the simaroubolides (quassinoids) [27-29], limonoids [30-31], bisbenzylisoquinoline alkaloids [1], as well as other minor classes of compounds.

1.1 Quinine and related compounds Quinine (1) has been known as the principal alkaloid in cinchona bark, which consists of various chemovars and hybrids of Cinchona species (Rubiaceae), such as C. ledgeriana, C. succirubra, C. calisaya, C. officinalis and others, grown and cultivated in tropical countries. A recent review has covered the distribution, cultivation, cytology, tissue culture methods, pharmacology and toxicology of Cinchona and it's alkaloids [32]. Cinchona bark was first mentioned to be used for the treatment of fever in Europe in 1643 and was included in the London Pharmacopoeia as "Cortex Peruanus" in 1677. About 35 quinine type alkaloids have been reported from Cinchona species. The content of quinine is highest in the bark of cultivated trees, while cinchonidine (3), cinchonine (4) and quinidine (2) are more abundant than quinine in C. succirubra and some other varieties of this genus. Quinine is schizontocidal and gametocidal in blood for P. vivax and P. malariae, but not for P. falciparum. Therefore, quinine has been used as a suppressive and therapeutic agent, but not as a prophylactic agent. Quinine, quinidine, cinchonidine and cinchonine contain a quinoline moiety attached through a secondary alcohol linkage to a quinuclidine ring having a vinyl group. Quinine and quinidine each contain a methoxy group, that is absent in cinchonidine and cinchonine. Based on the steric configuration of the secondary alcohol in the molecules, quinine and cinchonidine are (-)-isomers and quinidine and cinchonine are (+)-isomers. Differences in steric configuration do not result in significant changes in biological activity of these alkaloids. Quinine is less potent as an antimalarial but less toxic than quinidine. Cinchonamine

CHEMISTRY OF ANTIMALARIAL COMPOUNDS

147

(5), which contains a 5-vinyl-2-quinuclidyl group attached to an indole ring in addition to an ethanoyl group, has been isolated from H2

H3

H3CO~

~,~

~

8 quinine I

qulnldine 2

H2C~

HO

clnchonldine 3

"I

H2

I~ H

clnchonlne 4

clnchonamine

Remijia purdieana (Rubiaceae), but is devoid of antimalarial activity. Thus, the 5-vinyl-2-quinuclidyl group is not necessary for antimalarial activity. Quinine has been the lead compound for the development of a large number of antimalarial compounds. Quinacrine (6), 6-aminoacridine with 4-diethylamino-l-methylbutyl-, chloro- and methoxy- groups, was used as an antimalarial agent during the World War II. However, quinacrine is not useful for prophylaxis of malaria, is not gametocidal for P. vivax and is toxic. Following the synthesis of thousands of 4-aminoquinolines, chloroquine (7), 4aminoquinoline with attached 4-diethylamino-1-methylbutyl- and chlorogroups, was investigated and found to be an effective agent against the erythrocytic stage of P. vivax, P. ovale, P. malariae and certain strains of P. falciparum. Chloroquine suppresses P. vivax greater than does quinine but does not prevent relapses. Quinine also does not prevent relapses. Chloroquine is metabolized to monodesethylchloroquine (8), which is also highly active as an antimalarial. SN-6911 (9), a 4-aminoquinoline which

148

KAWANISHIand FARNSWORTH

contains 4-diethylamino-1-methylbutyl-, chloro- and methyl- groups, was developed as a more efficient derivative against human malarias.

~H3

( cH3

H N ~ N ~ [

~H3

CH3

: oH3

HN~N~CH3

oc~

el

el

quinacrlne6

chloroqulne 7

H3 H N ~ ~ v l

CI

~H3 ScH3

~

CI" monodesethyl chloroqulne8

v

H

~

: CH3 N

~

CH3

CH3 ~N"

SN 69119

Amodiaquin (10), 4-aminoquinoline which contains 3diethylaminomethyl-4-hydroxyphenylamino and chloro groups, was synthesized and found to be more potent against P. faleiparum than chloroquine. However it is more hepatotoxie than chloroquine and produces granuloeytosis. One of the N-ethyl substituents of chloroquine is 13-hydroxylated to produce hydroxychloroquine (11), which has similar efficient activity against faleiparum malaria, and is less toxic than chloroquine. This compound is preferred over chloroquine in the treatment of rheumatoid arthritis and lupus erythematosus. Many phenanthrenemethanol derivatives were also synthesized during World War II in a search for alternative drugs to quinine. Halofantrine (12) resulted from these studies, which is a 2-(dibutylamino)-ethyl derivative with trifluoromethyl and diehloro groups. Both the (+)- and (-)enantiomers ofhalofantrine are equally active against P. falr in vitro and against P. berghei in mice. The racemate of halofantrine is effective against the asexual erythrocytic stages of several Plasmodium species.

CHEMISTRY OF ANTIMALARIAL COMPOUNDS

149

Halofantrine is metabolized into N-desbutyl halofantrine (13) in humans, and it also has antimalarial activity. H3

~ CH3

CI hydroxychloroquine I I

amodlaquln 10

~

halofantrlne 12

C

H

s

H

N-desbutyl halofantrtne 13

Mefloquine (14), a 4-quinolinemethanol derivative with attached 2piperidyl and 2,8 bis-(trifluoromethyl) groups arose from a synthetic effort on 4-quinolinemethanol. This compound possesses activity against murine malaria and is clinically effective against chloroquine-resistant strains of P. falciparum. A study of the interaction of 4-quinolinemethanol derivatives with DNA produced two analogs of mefloquine, one (15) with a CONH2 in place of CF3 at position 2 of the ring and the other (16) with F in place of CF3 at position 8 of the ring. It was found that mefloquine was more active than either of these two compounds, suggesting that no relationship exists between the reaction with DNA and antimalarial activities [33]. These 4-quinolinemethanol derivatives include four optical isomers but they were tested as their racemates. Four optical isomers of 9phenanthrenemethanol with attached 2-piperidyl and 3,6bis(trifluoromethyl) groups (17) were all prepared and tested for antimalarial activity [34]. They were all active, indicating that stereospecificity was not required in the antimalarial activity.

150


HO

EONH 2

CF 3 CF3

CF3

mefloquine 14

F

15

F3(.,

16

CF 3

a-(2-plperldyl)-3,6-bls(trifluoromethyll-9-phenanthrenemethanol 17

Pamaquine (18), the first synthetic 8-aminoquinoline having a methoxy group, was found to have antimalarial activity. Additional analogs were prepared. Pentaquine (19), a 5-isopropylamino-pentylamino-derivative; isopentaquine (20), a 4-isopropylamino-l-methylbutylamino-derivative and primaquine (21), a 4-amino- 1-methylbutylamino-derivative were found to be more active and less toxic than pamaquine. Primaquine is most effective against the late hepatic stages and latent forms ofP. vivax and P. ovale, but is ineffective for suppression of P. vivax malaria. This drug is also active against the hepatic stages of P. falciparum, but not against the erythrocytic stages. Primaquine is used in combination with other drugs for the radical cure of relapses, as well as for prophylaxis. The 4-amino-l-methylbutylamino group in primaquine (21)was changed to a 1,4-pentanediamino group which became quinocide (22) and retained activity. 4-Methylprimaquine (23), which possesses a methyl group at position 4 of the quinoline ring of primaquine was also active and thus derivatives of methyl substitutions at position 4 in the quinoline derivatives were synthesized and tested [35]. Comparing the two derivatives with 3-aminopropylamino group (24) and 5-amino-l-methylpentylamino group (25) to primaquine (21) and 4-methylprimaquine (23),

CHEMISTRYOFANTIMALARIALCOMPOUNDS

H3CO~~NS~ ~

151

H3C _CH3

CH3 pamaquine 18

K~CH3 pentquine 19

H3o

HN~~,~..~

NH2

CH3 Isopentaquine 20

primaqulne

21

the 5-amino-l-methyl-pentylamino derivative (25) showed curative activity (3/5) at 160 and (5/5) at 640 mg/kg against P. berghei in mice, while the 3-aminopropylamino derivative (24) and 4-methylprimaquine (23) caused toxic death (5/5) at 640 mg/kg, and primaquine (21) did toxic death (5/5) at 320 mg/kg.

H 3 C O ~

H3C

H3 ,,~

NH2 (;Ha quinoclde 22

4-methylprimaquine

23

152

KAWANISH!and FARNSWORTH

H3C

~H3

~3

H3C

24

CH3

25

In the series of derivatives of 4-methylprimaquine, having an aryloxy or an alkoxy group at position 5 in the quinoline ring, WR-238,605 (26) with a 3-(trifluoromethyl)-phenoxy group at position 5 and a methoxy group at position 2, WR-242,511 (27) with a n-hexyloxy group, and WR-254,715 (28) with a phenylpentyloxy group at position 5 were promising for most blood schizontocidal activity [36]. However the two former compounds increased the activity at lower dosage range but produced toxicity at higher doses (160-640 mg/kg), although these were more effective than the parent derivative, primaquine (21) in both curative activity and toxicity. Thus, derivative (28) was an excellent candidate in this series.

c~

~H2CH2CH2CH2CH2CHa H3C

H

~

CH3

CH3

WR-238,60526

WR-242,511 27

NH2

CHEMISTRY OF ANTIMALARIALCOMPOUNDS

153

~

H2CH2CH2CH2CH2C6H5

3c

H~,,~~~~

NH2

CH3 WR-254,715 2 8

Quinine led to the development of chloroquine, which consists of an Nalkyl side chain in place of the quinoline ring in quinine. The N-alkyl side chain substituted at position 8 in the quinoline moiety was found to be as effective as the one substituted at position 4 or more. It is interesting that to develop more effective and less toxic drugs, the latest compounds developed possess substituents at positions 4,5,6 and 8 in the ring, such as WR-242,511 and WR-254,715, and in WR-238,605 even more substituents are attached, such as at positions 2,4,5,6 and 8. They were effective against a variety of stages in the life cycle of the parasite including the pre-erythrocytic stage, the drug-sensitive and drug-resistant asexual intra-erythrocytic stage, gametocytes and the intra-hepatic hypnozite stage for P. cynomlgii. In vivo WR-254,715 was more efficacious and less toxic than primaquine 1.2 Artemisinin (qinghaosu) and related compounds

The leaves and flowering tops of the wormwood plant, Artemisia annua (Compositae) have been used as a traditional Chinese medicine for the treatment of fever and malaria. A hot-water extract of this plant was not active in mice infected with P. berghei, but an ethyl ether extract was active. Artemisinin (29) was isolated from the petroleum ether extract of the plant and was found to be a novel sesquiterpene lactone with an endoperoxide group. The history of development of artemisinin and its analogues, as well as a review of the chemistry and pharmacology of this plant and its contained compounds, is available [37-39]. Arteannuin B (35), also isolated from A. annua, contains a ~,-lactone with a cadinane skeleton in the structure [40]. Artemisinin has demonstrated promising antimalarial activity against the erythrocytic stage of P. falciparum and is also active against both chloroquine-resistant and chloroquine-sensitive

154


strains of the parasite in vitro and in vivo. Artemisinin is inactive against the liver stages of P. falciparum. It is only poorly soluble in water or oil, which impairs its practical use as antimalarial agent. A search for improved analogs with more potency, water solubility, oral activity and better bioavailablity has been initiated. Hydrogenation of artemisinin gave two different compounds. One of the peroxide oxygen atoms of artemisinin was deoxylated to give the epoxide, deoxyartemisinin (30) by Pd/CaCO3. The lactone group of artemisinin was hydrogenated to the lactol, dihydroartemisinin (31 1, R = H 9major, 32 1, R = H 9minor) by sodium borohydride [41 ]. I]-Dihydroartemisinin (31 1, R = H) has been isolated from A. annua. The activity was greater for dihydroartemisinin (31 1, R = H) than for artemisinin, but not for deoxyartemisinin. Relative activities of artemisinin and deoxyartemisinin analogs were compared and the latter showed very little activity, while the former was active [42]. Artemisinin is metabolized to deoxyartemisinin (30), dihydrodeoxyartemisinin (33 1, R = H) and Crystal-7 (36) [43]. They were not active against P. berghei in mice. Dihydroartemisinin was dehydrated to obtain anhydrodihydro-artemisinin (37), with the double bond between positions 9 and 10, and an ether (38) [44]. The former was inactive but the latter was active. Ethers, esters and carbonates of dihydroartemisinin have been prepared to obtain novel and more active derivatives [45]. Atter examination of the ethers, including the epimers at position 10, artemether (31 2, R = C H 3 ) and arteether (31 3, R = CH2CH3), they were found to be about twice as active as artemisinin, but less active than dihydroartemisinin. Artemether has been isolated as a natural constituent ofA. annua. Arteether was found to be 34 times more active than chloroquine against the W-2 (Indochina) clone of P. falciparum (normally resistant to chloroquine) and three times less active against the D-6 (Sierra Leone) clone (normally resistant to mefloquine). Artemether was two times more active and eight times more active than mefloquine against the W-2 and D-6 clones, respectively. Both artemether and arteether are more oil soluble than artemisinin and are currently in clinical trials. Esters (32 4, R = C(=O)-alkyl or-aryl), of which ct-epimers were mainly obtained, were more active than the ethers. Sodium artesunate (32 5, R = C(=O)-CH2CH2COONa), the half succinic acid half-ester of dihydroartemisinin is water-soluble and shows potent antimalarial activity. Therefore, this can be administered intravenously. However it is uncertain whether this derivative is pharmacologically effective because of its sensitivity to hydrolysis. In considering this result new ether derivatives of dihydroartemisinin, which are stable and water soluble derivatives have been prepared [46]. Dihydroartemisinin was condensed with esters of aliphatic or aromatic carboxylic acids with hydroxy groups to produce mainly ethers with the t-configuration. Ethyl 2-(10dihydroartemisininoxy) acetate (31 4, R = CH2COOCH2CH3) and methyl

CHEMISTRYOF ANTIMALARIALCOMPOUNDS

155

p-[(10-dihydroartemisininoxy)methyl] benzoate (31 5, R = C H 2 - C 6 H 4 COOCH3) were active against the D-6 and W-2 strains, in vitro and they were as active as the parent compound, dihydroartemisinin. These two ethers, ethyl acetate and methyl benzoate were changed to potassium acetate (31 6, R = CH2COOK) and potassium p-methyl benzoate (31 7, R = CH2-C6H4-COOK) derivatives, respectively. The latter retained activity but the former did not. The potassium carboxylates were converted to the corresponding acids (31 8, R = CH2COOH and artelinic acid, 31 9, _CH3 5 .H ."~ H "." '

34 H

3

C

.CH3 H ~ H ~ '~

~

H3C

9. "'H H J95o~)

H

~

H 18

n 30

Attempts to decarbonylate 30 using Wilkinson's catalyst in refluxing 1,2-dichloroethane met with mixed success. A particularly annoying feature of the transformation was the fact than an olefin, isomeric with the desired product was also formed. For some unknown reason, the relative amounts of these materials varied from run-to-run. It was reasoned that the C-C ~ bond was participating and that it would be best to partially elaborate the A-ring before removing the functionality at C7 (Scheme 2). To this end, 18 was reduced with LAH, the resulting alcohol protected as the dimethyl tert-butyl silyl ether 31 and the n bond was then elaborated using borane in THF followed by oxidation with PCC. While it is usually assumed that reactions occurring at the bridgehead of a linearly fused tricyclopentanoid afford the more stable cis-, rather than trans-ring fusion, the presence of the bulky silyl ether assured the hydroboration would occur on the side away from it and guaranteed a cis-ring fusion. Cleavage of the silyl ether under standard conditions, followed by oxidation afforded ketoaldehyde 32 and set the stage for another attempt to remove the aldehyde. This time, treatment of the ketoaldehyde 32 with 1.2 equiv of Wilkinson's catalyst in refluxing 1,2-dichloroethane proceeded satisfactorily to afford ketone 33 in yields ranging from 76 to 91%. Unlike the decarbonylation of 25, no side reactions were detected.

BIOACTIVE NATURAL PRODUCTS

201

CO2CH 3 H

9

H

/

OTBDMS

"

H

u

I. LAH, Et20 (>95%) .

.

.

.

.

H

.

2. TBDMSCI, imidazole DMF (95%) H H 18

31

I. BH 3, THF; PCC (54-57%) 2. TBAF (89-93%) H

H

CHO (Ph3P)3RhCI. CICH2CH2CI reflux, 54 h {76-91%)

il H H

"r~ IIIH

O 33

0

H "~-V 32

S c h e m e 2. Tricyclopentanoid interconversions; hirsutene precursor.

The synthesis was completed by introducing the angular methyl group by using a standard blocking-alkylation-deblocking sequence illustrated below. Spectral data for ketone 35 matched those of an authentic sample [10]. Since 35 had been converted to hirsutene (1), the synthesis was complete.

.,"'~

H 1. NaOMe. EtOCHO 1 9 6 % 1 9n-BuSH, p-TsOH 182%} 33

/

'3. KOBu-t, t-BuOH. Mel (62%? ~ ~ ~ ~ ~ _

KOH. ethylene 9 glycol(51%1 ~

34 I

I

H

~

O I

I

35

I

Before closing this section it is worthwhile to point out that our initial plan calling for the use of an electron deficient diylophile in order to ensure cycloaddition and avoid diyl dimerization was overly conservative. Thus, as illustrated, the unactivated diazene 36 served admirably as a diyl precursor, leading to a respectable yield of the cis-anti tricyclopentanoid 37. The latter was easily converted to ketone 35, thereby culminating a much shortened synthesis of hirsutene (1) [ 11 ].

202

LITTLE and O T T

/~

_O.IMInTHF'

.~

36

~.~t +

37

38

Coriolin (3) and Hypnophilin (4) Coriolin (3) was first isolated from the mycelial cake of C o r i o l u s c o n s o r s by Umezawa and coworkers in 1971 [12], and has attracted widespread interest due primarily to its interesting molecular architecture and antitumor properties. Ten years later, Anke, Steglich and coworkers isolated and characterized the linearly fused tricyclopentanoid hypnophilin (4) [13]. It too is biologically active, displaying activity toward grampositive and gram-negative bacteria, fungi, and yeasts, as well as antitumor activity. We elected to showcase the diyl trapping reaction with the synthesis of both coriolin (3) and hypnophilin (4) [14]. H

3.,

,i

>

, 39

OH

4O

HO

I

~_~

BsIS

42 Scheme

H

3. Strategy toward coriolin (3) and hypnophilin (4).

41

OH

BIOACTIVE NATURALPRODUCTS

203

Commercially available dihydro-5-(hydroxymethyl)-4,4-dimethyl2(3H)-furanone (42) was chosen as the starting material since it incorporated the essential structural features that are present in the acyclic chain of diazene 41. To synthesize both coriolin (3) and hypnophilin (4), enone 39 was selected as a common intermediate. While 39 had previously been converted to coriolin (3), its conversion to hypnophilin (4) had not been accomplished. The furanone 42, although commercially available, could also b e obtained in large amounts by epoxidation of 3,3-dimethyl-4-pentenoic acid with 3-chloroperoxybenzoic acid (MCPBA) in chloroform at room temperature (84%). After protection of the primary alcohol 42 as a benzyl ether, the carbonyl unit was reduced with diisobutylaluminum hydride in ether at-78 ~ to afford the diastereomeric pair of lactols 43 in 97% yield and a ratio of approximately 2"1. The lactols were methylated with ptoluene sulfonic acid in methanol to provide the functionalized tetrahydrofurans in nearly quantitative yield. The benzyl group was removed by hydrogenolysis over palladium hydroxide on carbon to afford the alcohols 44 in 94% isolated yield; use of other catalysts, such as palladium on carbon, gave less reproducible results. OH ~

I. PhCH2Br, Ag20, DMF(76%}

~.

2: D~B~.. r~o (9Wo)

l'""t

HO...,ff'

1.

~.,

Bn 0 - - - ~

1%

42

MeOH,/>TsOH (>95~

2. H2, ed(OH}2 (94%)

I

I

I

I

0 "~ \ / )----/..,,

.o--/

|

"

44

43

III

~Me

I

I

II

The conversion of 44 to the corresponding aldehydes was carried out by using a Swem oxidation. Thus, treatment of 44 with oxalyl chloride in dimethylsulfoxide at-60 ~ followed by triethylamine, afforded aldehydes .

.

.

.

.

.

.

.

.

OMe I. (COCl)2, DMSO; Et3N O

HO

I. EN=NE

. . . . . . .

2. CpH, pyrrolidlne/AcOH, - (, % MeOH(55%, two s t e p s ) ~

$... 2. diimide (91%, 2 steps [l ~ "t E = CO2Me) ~

\\// 45

I

I Ill

OMe

I

l",,. ~ ""

/i ~ N/ E 4 6 , E = CO 2CH2CCl3 47, E = CO2Me

204

LITTLE and OTF

which were sufficiently pure to allow them to be used directly. Treatment with methanol, cyclopentadiene and pyrrolidine led to the formation of fulvenes 45 [5]. While after 24 minutes no starting material could be detected, the reaction was still incomplete. Instead, an intermediate formed and was transformed very slowly into the required fulvenes 45. Prolonged reaction times led to decomposition of the fulvene which had formed, so that it proved advantageous to work-up the reaction after 24 hours. Under the slightly acidic work-up conditions the intermediate was converted to the starting material, which was recycled. After several recyclings, the yield of fulvenes ranged from 40-45%. While attempting to improve the yield, it was discovered that the rate of fulvene formation increased dramatically when acetic acid was added to the reaction mixture. Thus, treatment of the aldehydes with 1 equiv of acetic acid, 2 equiv of pyrrolidine and 2.5 equiv of cyclopentadiene in methanol at room temperature gave, after 12 hours, the fulvenes 45 in 55% isolated yield over two steps (viz., Swern plus fulvene formation); no starting material could be recovered. With the fulvene unit destined to become the carbon framework of the diyl in hand, the next task called for preparation of the bicyclic skeleton of diazene 41. This was accomplished by carrying out a Diels-Alder reaction between the fulvenes 45 and either bis(2,2,2-trichloroethyl) azodicarboxylate in ether at 0 ~ for 1 hour, or dimethyl azodicarboxylate in ether at 0 ~ for 3 days. The endocyclic ~ bond of the resulting adduct was selectively hydrogenated using diimide to afford 46 and 47 in 82% and 91% isolated yield, respectively (two steps). Both 46 and 47 consisted of a mixture of diastereomers. However, this was of no importance with respect to the remainder of the sequence since the methyl ether bearing carbon was destined to be converted to an sp 2 hybridized center. In addition, from previous studies, there was evidence to suggest that the diyl would exist as a time-averaged planar intermediate and that the existence of diastereoisomers about the exoeyclic ~ bond (tether oriented toward the front or back) would not have any bearing upon the stereochemical outcome.[15] Thus, we were confident that each of the diastereomers would lead to the same products. Having assembled the bicyclic framework, attention was directed toward introduction of the diylophile. Deprotection of the masked aldehyde in 46 and 47 was most efficiently accomplished using 70% aqueous acetic acid at 50-60 ~ for 5 days (85% and 95% yield, respectively). The dimethyl dicarbamate was subjected to saponification with potassium hydroxide in refluxing ethanol for 1.5 hours, whereafter the in situ oxidation with potassium ferricyanide at 0 ~ gave rise to the dizaene 48 in yields ranging from 76-86%. We were gratified to observe that treatment of 48 with triphenylphosphonium methylide in THF at room temperature led to the desired diyl precursor 41 in 56-83% yield.


205

OH

I. AcOH 47

Ph3P=CH2

% 9

Ss

.,..~

,,

]]

THF (56-83

2. KOH, EtOH; K3Fe(CN) 6 (76-86%)

//N N 48

41

Ill

II

I I

II

The intramolecular diyl trapping reaction was studied in a variety of solvents (THF, MeOH, acetonitrile), and the diyl was generated both thermally and photochemically [ 14]. The photo-induced deazetation of 41 in methanol at -6 ~ afforded the desired tricyclopentanoid 40 in an excellent 84% yield. The transition state model portrayed by 49 nicely rationalizes the stereochemical outcome. The solvent study revealed that its choice had essentially no effect upon the product ratio at any given temperature. However, we did discover that methanol, a solvent which had not been utilized previously in intramolecular 1,3-diyl trapping reactions, was very useful for low temperature studies.

]\

MeOH

l OH

41

I

49

Ill

40

I

I Ill

III

Illl

To determine whether enthalpic or entropic factors were responsible for controlling the product distribution, the cycloaddition was conducted at several different temperatures. In analogy with previous results, there was reason to believe that both thermally and photochemically initiated extrusion of nitrogen would lead to the same 1,3-diyl, thereby allowing one to examine the chemistry over a reasonably large temperature range [15]. In practice, the product ratio (40: sum of minor products = EMnr)

206

LITTLE and OTF

varied from 4.7:1 in refluxing methanol, to 9.1:1 when the reaction was initiated photochemically at 6 ~ to 30" 1 when initiated photochemically at -60 ~ A plot of ln(40/EMnr) vs 1/T afforded a straight line from which we concluded that the variation in product ratio had its origins in enthalpic (AAH~t -2.19 kcal/mol), rather than entropic factors (AAS~-3.5 eu[ 14]). All attempts to convert 40 or its hydroxyl protected derivatives to the enone needed to complete the functionalization of the A-ring were unsuccessful. Most often, treatment of benzoate 50 with a variety of oxidizing agents (e.g., with Collins reagent) led to complex reaction mixtures wherein, in addition to small amounts of the desired enone 51, enone 52 was the major product. H

H

H

H

R :

h

OR

40, R = H 50, R = COPh

H

9

~)COPh

51

0

OCOPh

52

Fig (5). Isomeric enones formed during allylie oxidation.

Consequently, alternative modes of enone formation were examined. To set the stage for the use of a Rickborn-Crandall sequence to accomplish this objective [16], we treated alcohol 40 with MCPBA in methylene chloride, expecting to form a single epoxide. We were surprised to discover the formation of an 80/20 mixture of two adducts, 54a and 55a, in >90% yield. The ratio proved to be dependent upon the size of the -OR unit appended to C l l. When a bulky silyl ether was used (53), a single product formed, 55e. Desilylation afforded the minor epoxy alcohol 55a which was produced in the initial epoxidation of 40. X-ray analysis of the corresponding benzoate 55b, the major adduct derived from the epoxidation of 50, demonstrated conclusively the presence of a trans-fused A,B-ring system. Thus, even though the parent trans-fused bicyclo[3.3.0]octane is -~8 kcal/mol less stable than its cis-fused counterpart, such systems can be assembled, and in high yield. Furthermore, it is interesting to note that despite the fact that the C~l OR unit is formally on the o~-side of the tricyclic framework, the BC-ring fusion positions the substituent so that it is above the ~ bond of the A ring. Large R groups would, therefore, be expected to direct epoxidation to the opposite side, as observed. Heating the epoxides in THF with either (n-Bu)2NLi or LDA led to the expected diol 56 in 45% yield (two steps). All attempts to oxidize selectively the allylic alcohol met with failure; selective protection of the secondary hydroxyl group in 56 as a benzoate ester and oxidation using PCC afforded the desired enone 51 in 76% yield after crystallization.


T a b l e 1.

207

Epoxidation Ratios

H

H

H

H

H

H

MCPBA '#1 I

H

"

Na2CO3'

OR

40, 50, 53

0 ~

>90%

,

H

54a-c

Starting aikine ,,

O

" _ OR

"'"~)

H 55a-c

" " OR

R

Products

ratio

40

H

54a, 55a

80/20

50

COPh

54b,55b

SiMe2Bu-t

54c, 55c

,, ,,,,,

L

,

53 ,,,,

...... i' 27/73

0/100

,,

~ / ~ . . , , t t /

1. MC PBA. CHCI3. 0 ~ .,

_ H 40

. _ OH

'

2. (n-Bu)2NLi. THE (45%, two steps)

HO H 56

I

: OH

"'%

I. PhCOCI, pyr (89%) 2. P C C / C e l l t e (85%)

H

H

--.

o

H

51 ~llillli

li

II

i

I

i

O120 Ph i

We next addressed the problems posed by the addition of a methyl group to the hindered fl-carbon of enone 51, a carbon which is encumbered by being doubly substituted, and in a less obvious fashion, is hindered by virtue of the fact that the C-ring C ll methine hydrogen is pointed directly toward it. Thus, we were not surprised to discover that a simple Gilman reagent failed to produce the desired 1,4-addition product 57. In contrast, treatment of 51 with the higher order euprate, Me2CuCNLi2, in the presence of boron trifluoride etherate, converted it to the desired product 57 in a stellar 93% isolated yield [ 17].

208

LITTLE and OTF

H

H

Me2CuCNL|2, BF3, THF, -50 *C 193%1

H _

9 ~ -

OCOPh

~)COPh

51

57 II

IIII

To introduce the 7r bond and generate enone 58, the triquinane 57 was first converted into a 3:2 mixture of two regioisomeric trimethylsilyl enol ethers using LDA in THF at -78 ~ followed by the addition of TMSCI; the major isomer resulted from enolate formation toward Cr (note 57, below). Treatment with palladium acetate in acetonitrile converted it to the required enone 58, while the minor isomeric silyl ether was reconverted to the starting ketone 57. Enone 58, which has previously been synthesized and converted into the target molecule 59 by Koreeda and coworkers, was isolated in 40% yield, along with 50% recovered starting material; intermediate 58 proved to be identical in all respects to the material synthesized by the Koreeda group [ 18].

H

H

r

o11

: ~

I

lq "

ocom 57

H I. LDA.THF:::TMSCI = 2. Pd{OAc)2, CHsCN (40%, two steps)

" ~ O

bR 58, R = COPh 59, R=H

With a formal total synthesis of coriolin (3) accomplished, we turned our attention toward hypnophilin (4). In practice, it was found that treatment of enone 59 with LDA in THF at-78 ~ followed by trapping of the resulting enolate with formaldehyde at-30 ~ led to a mixture of the diastereomerie diols 60 in 85% yield (Scheme 4). The mixture was treated with tosyl chloride and pyridine in dichloromethane at room temperature; TLC analysis revealed that some dienone 39 was formed even under these reaction conditions. After 4 days, tosylation of the primary hydroxyl group was complete and the elimination reaction was


209

H LDA, THF.-78 ~

.CH20, . . .-30. .~

185~

"~~"

--

o

-\ 9-

.

-"

m

OH

OH

59

60

1. TsCI, pyr 2. DBU (80%) H

"

H H202. T H F / H 2 0

I1"

R

2c03

_._

:"

\ OH

4, hypnophlltn

39

Scheme 4. Completion of the hypnophilin (4) synthesis.

accomplished upon addition of 1,5-diazabicyclo[5.4.0]undec-5-ene (DBU). The dienone 39 was obtained in 80% yield and proved identical to the material synthesized previously by Schuda and Heimann [19]. Monoepoxidation of the endo-cyclic ~ bond in dienones similar to 39 has already been described. Thus, under reaction conditions applied by Danishefsky and coworkers [20], dieneone 39 was converted to hypnophilin (4) in 50% yield; some starting material (30%) was recovered. Bisepoxidation could also be detected, but that material decomposed during the column chromatographic purification on silica gel. Synthetic hypnophilin (4) displayed spectral data which were in full accord with those of natural material.

A9(12)-Capnellene (2) [21]. Bridged vs Fused Regioselectivity In 1974, a manuscript appeared describing the characterization of a sesquiterpene that was isolated from the methylene chloride extracts of the soft coral Capnella imbricata (Coelenterata, Octocorallia)collected off Sewaru, Leti Island, Indonesia [22]. The skeletal type was given the name capnellane, and the structure and absolute configuration of the compound, named A9~12)-capnellene-3fl,8fl,10a-triol (61), was secured by singlecrystal X-ray analysis. Over a period of several years, other capnellols were isolated from C. imbricata. In addition to these alcohols, two hydrocarbons, A9Cl2)-capnellene (2), the presumed biogenetic precursor of

210

LITTLE and OTF

the alcohols, as well as precapnelladiene (62) [23], the putative immediate precursor to the tricyclopentanoid skeleton found in 2, were also isolated from C. imbricata. H

H

HOll'"

61, R=OH

2

62

Fig. (6). Capnellenr (2) and other marine natural products.

It appeared as though the trapping reaction would be ideally suited for a total synthesis of A9(IE)-capnellene (2) [21]. The plan, illustrated in Scheme I, required construction of the bicyclic diazene 63, which was to serve as the direct precursor to the required diyl. From the outset we were aware of the potentially more stringent requirements that were to be imposed upon the present trapping reaction. Our immediate attention was directed to the fact that an unactivated diylophile was to be used and therefore that the diyl trapping reaction might be slowed sufficiently to allow dimerization of the diyl to compete with cycloaddition. Given the positive results obtained with the unactivated diazene 36, however, the prospects for success in the present instance also seemed high. H

.-

2, 691121-capnellene

63

Fig. (7). Diazene precursor to capnellene (2).

Diazene 63 was prepared in short order from readily available starting materials. Thus, acid 64, prepared in a standard manner from the isobutyric acid dianion and 3-methyl-3-butenyl p-toluenesulfonate, was first reduced with lithium aluminum hydride and then oxidized by using PCC/Celite to afford the expected aldehyde in 88% yield. In preparation for the Diels-Alder reaction that was to be used to assemble the bicyclic framework found in 63, the aldehyde was converted to fulvene 65. Initially, we attempted to accomplish this objective using methodology


211

developed by Freisleben [24]. Our past experience with the procedure provided very good yields of fulvenes that were otherwise often difficult to obtain without significant competing processes. In the present instance, treatment with cyclopentadiene and diethylamine in methanol at room temperature for 6 h led only to the recovery of unreacted aldehyde. Presumably, the neopentyl nature of the carbonyl carbon provides sufficient steric encumbrance so that the initial iminium ion forming step of the sequence does not occur at an appreciable rate under these conditions. Fortunately, since the aldehyde is devoid of ct-hydrogens, the problems were overcome by treating it with lithium cyclopentadienide in THF at room temperature [25]. In this way, fulvene 65 was obtained in yields ranging from 67-80% after chromatography on neutral alumina. A Diels-Alder reaction between 65 and dimethyl azodicarboxylate followed by selective reduction of the endocyclic ~ bond of the resulting adduct using diimide in dichloromethane provided 73-91% yields of the dimethyl dicarbamate precursor to diazene 63, which in turn, was obtained in 78% yield after saponification and oxidation using aqueous potassium ferricyanide.

I. LAH, Et20 2. PCC/Cellte, CH2C12 (88%, 2 steps) 3. CpLl, THF (67-80%)

64

65 I. MeO2CN=NCO2Me 2. KO2CN=NCO2K, AcOH (73-91%1 3. KOH, EtOH; K3Fe(CN)6 (78-86%) 63 I

I

I

The diyl trapping reaction did lead to the expected linearly fused product. However, cycloaddition was slowed to the point where it proved necessary to slowly add the diazene to a refluxing solution of THF to assure a low concentration of the diyl. Otherwise, it dimerized in a process that is characteristic of the triplet spin state [26]. Furthermore, the linear adduct was accompanied by the formation of nearly an equal amount of the previously unobserved bridged regioisomer. These materials proved difficult to isolate as hydrocarbons and were converted as a mixture to

212

LITTLE and O'IT

ketones 66 and 67 v i a hydroboration-oxidation (56% from 63). The structure of the bridged ketone 67 was confirmed by x-ray crystallographic analysis. Once separated and characterized, it proved a simple matter to convert the linearly fused ketone 66 to the natural product using a Wittig reaction in DMSO.

{a}THF, reflux, syringe p u m p (b) BH 3, THF; PCC 0

'N

:-

63 II

66

I

I

I

II

67 I

The most obvious difference between the present case and those discussed earlier is the existence of the gem methyl unit appended to the carbon adjacent to the diyl, and the placement of a methyl group on the internal carbon of the diylophile. As illustrated in 68, a reasonable transition state representation for a concerted cycloaddition leading to the linearly fused product, the positioning of these substituents sets up several energy raising nonbonded interactions. That illustrated between the gem methyl unit and the diyl ring hydrogen is alleviated if the alternative transition state formulation (69) leading to the bridged adduct is adopted. But what if these cycloadditions are not concerted, but instead occur in a stepwise manner? Then, the first step leading to the linearly fused material corresponds to a 5-exo-trig cyclization onto an alkene that is substituted with a methyl group (70 to 71a), while the bridged adduct results from a 6-endo-trig cyclization onto the opposite end of the alkene (70 to 71b). In analogy with monoradical chemistry [27], one notes that in cases such as these, the rate of the 6-endo-trig cyclization exceeds that of the alternative, though the rate differences are generally small.

68

H

Fig. (8). Transition state formulations.


(

9

213

" ca 2

70

"~

7 1 a (5-exo path} r

71b {6-endo path)

"~

Fig. (9). Monoradical-like cyclization modes.

In an attempt to sort out the relative importance of geminal substitution the nature of the substituent appended to the diylophile, we synthesized and examined the chemistry of the diyls derived from diazenes 72a-e, the first two having geminal substituents but no alkyl group on the diylophile, the third devoid of the geminal substituents but possessing a hydroxymethylene unit on the diylophile. To our surprise, geminal substitution played an imperceptible role. The only one of the three systems to provide a significant quantity of the bridged cycloadduct was 72e. vs

Table 2.

Effect of Substitution on Linear/Bridged Ratios a

R'

N

72a-c

R,

major product/comments

H

linear

|

a, CH 3 b, OCH3

c,H

,t,,

linear CH2OH

essentially !'1 bridge/linear

To address the possibility that both the singlet and triplet diradicals could be participants, we examined the chemistry of 72e in the presence of molecular oxygen. The reaction of TMM diyls with oxygen serves as a diagnostic for the intervention of triplet chemistry [28]. While in principle both the singlet and triplet diyl can react, a combination of the low oxygen concentration available under the reaction conditions, combined with the

214

LITTLE and O T [

short lifetime and rapid rate of intersystem crossing of the singlet to the triplet, allows only the triplet diyl to react. In the present case, bridged adduct formation was completely quenched, and the amount of linearly fused adduct 73 was reduced from 44 to 17%. These results indicate that all of the bridged and a portion of the linearly fused adducts are triplet derived. Table 3.

~

/~CH~OH

_~i I

CH2OH

CH3CN, reflux N

N 72c

73

no oxygen ,

,,,,

,,,

,,

|,

,,,

74

1"1.2 linear/bridge; 90% combined yield ,

with oxygen

,

no bridge & yield of linear decreases from 44 to 17%

I

The chemistry of diazenes 75a-e illustrates that linearly fused adducts are formed very efficiently when an electron withdrawing group is appended to either carbon of the diylophile. Product formation could not be quenched with molecular oxygen, indicating that these materials are singlet derived. The differing reactivity patterns can be understood using frontier molecular orbital theory, coupled with the Berson cascade mechanism [29]. According to the latter, when a diazene is heated or irradiated, the first formed interceptable intermediate is the singlet diyl. We were originally of the opinion that intramolecular cycloaddition would occur fast enough to render intersystem crossing (ISC) to the triplet manifold non-competitive. This does indeed seem to be the case when an electron withdrawing group is appended to the diylophile. But as evidenced by the findings described above, this is not always true; triplet chemistry can and does intervene. We suggest that the reason cycloaddition to the singlet diyl is rapid is related to the existence of a comparatively small diyl HOMO/diylophile LUMO energy gap, AE, the situation most likely to occur when an electron withdrawing group is appended to the diylophile. If AE is made larger, then that rate ought to decrease, thereby providing time for ISC to become competitive. Conceptually, this is an easy objective to achieve merely replace the electron withdrawing group with an alkyl substituent.


Table 4.

215

Influence of Electron Withdrawing Substituents on Product Ratio

EWG

EWG

N 75a-c

76a-c

77a-c

yield (%)

% linear

a, C O 2 C H 3

88

>95

b, C H O

93

88

98

91

EWG

c, C O C H 3 ,,,

Since the triplet diyl affords the bridged cycloadducts, we wondered whether it would be possible to optimize production of the triplet and simultaneously optimize the amount of bridged product formed. One motivation for so doing was the recognition that there are many bioactive natural products that possess the [3.2.1 ] subunit. If we were able to obtain these materials selectively, then the possibility of efficiently accessing the natural products would obviously exist. Once the triplet forms, it can choose either a stepwise 5-exo-trig or a 6endo-trig cyclization pathway for the initial carbon-carbon bond forming event. The former converts 80 to 81, an intermediate which will suffer progressively larger nonbonded interactions as the size of the diylophile substituent, Rf, increases. The alternative cyclization, 80 to 82, alleviates these, particularly if the radical site bearing Rf assumes either a planar or time-average planar geometry, thereby allowing it to maximize its separation from the remainder of the molecule. The preferences for 5- vs 6-membered ring formation are expected to be similar to those expressed in monoradical cyclizations, and this appears to be the case from the capnellene results described above. We reasoned therefore, that to optimize formation of the bridged cyeloadduct one should append a large alkyl group, Rf, to the internal carbon of the diylophile in 78. This will assure a large AE and ought to significantly tip the balance toward the selective formation of bridged cycloadducts. To add versatility and the possibility of functional group elaboration, a funetionalized alkyl group can be selected.

216

LITTLE and OTT Rf

D

N

,,-,.-,-,-,,.-.]m,-

Rf 79

78

5-exo,trlg

Rf

~ e

I' I I

--~

I

II i

Rf 6-endo,trig

80

Scheme 5. Cyclization modes - stepwise pathways.

The equation illustrated below demonstrates the dramatic influence the size of the alkyl group has on determining the bridged/linear regioisomer distribution. Thus, for the comparatively small CH2OH unit of 72e, a 1.2:1 ratio was obtained, while for the large dimethyl ketal found in 83, it increased to 16:1 (84/85) [30].

/"1 ~ L---~N~N

THF. reflux. ~ I raM. 3-4h

/ CtOMe)2CH3

83 _

"

.

.

.

.

.

CIOMel2CTIa

84 .

.

.

JI

85 I

I

I

_

/I .

.

.

.

.

.

I

The influence of both steric and electronic factors is illustrated in the chemistry of diazenes 86a and 86b[31 ]. The dimethyl ketal 86a affords the bridged adduct 87a to the near exclusion of its fused counterpart 88a. In stark contrast, the corresponding enone 86b provides the linearly fused adduct 88b nearly exclusively. From a detailed examination of these substrates, the following points have emerged: 1) The presence of geminal alkyl substituents adjacent to the first carbon exocyclic to the diyl ring has no apparent influence on the regiochemical outcome of intramoleeular diyl trapping reactions; 2) The regiochemical controlling factor turns out to be directly correlated with the


Table 5. H

217

Bridged Regioselection OCH 3 C O

3

~

~, OCH 3

.........

J

THF, reflux

----18

.,,

yield (%) ..

i

|

ii

.

,i

nature of the substituent appended to the diylophile. Electron withdrawing groups (EWG) attached to either the terminal or internal carbon results in the selective formation of linearly fused systems. In contrast, the presence of an alkyl group on the internal carbon of the diylophile leads to variable amounts of both linear and bridged adducts, depending upon the size of the substituent. The larger the group, the greater the preference for formation of the bridged material. This point is discussed in detail in the next section of this chapter. 3) Both the singlet and the triplet diyl play an important role, the former providing a linear adduct when an electron withdrawing group is appended to the diylophile, the latter being the precursor of the bridged isomer. In summary, the simple marine natural product A9(12)-capnellene (2) presented an unexpected challenge, one which ultimately forced us to revise our transition state model for the intramolecular diyl trapping reaction. While we were surprised to obtain the bridged adduct, its formation and discovery coupled with the spinnoff that has resulted from efforts designed to understand why it was produced, constitute what we believe to be the most important feature of our capnellene effort. This is one of those not-so-rare times when the unexpected proved of greater interest/importance than original objective(s). As a result of these studies we are now able to selectively form either the bridged or the linearly fused regioisomer by design. As the remainder of this chapter delineates, we are using this ability to gain access to aphidicolin (89) and taxol | analogs.

218

LITTLE and O'IT

Taxol (90), Baccatin III (91), and Target Analog 92 It is fair to say that taxol | (90) is one of the most important materials of this, or any century. Its successful use in the treatment of several forms of cancer is clear and well-established [32].

RO '""

6.

o

-: . . . ~ o

OBz

Ph

j

6A~

O

t.

BzHN

= R, taxol |

90

OH H

-

R, baccatln III, 9 1

Fig. (10). Taxoi | (90), baccatin III (91), and target structure 92.

While many ingenious approaches to the skeleton have been developed, only four total syntheses have been recorded.[33] As indicated recently by Danishefsky and coworkers, the availability of a renewable source of baccatin III (91) reduces the impact new total synthesis might have upon the supply problem [33h]. The development of general strategies that will allow the construction of a wide range of analogs whose bioactivity might equal or exceed that of the naturally occurring materials, clearly represents a laudable and noteworthy objective. Our target for application of the intramolecular diyl trapping reaction was tricycle 92 [34], a system possessing much of the functionality found in the B-ring of the natural product; the gem methyl unit is absent, though its role in defining bioactivity is unknown. Key to the successful implementation of our plan was the construction of aldehyde 97 and its subsequent conversion to the diazene 96 on a multigram scale; see Scheme 6. Scale, and the ability to scale up, are clearly important issues as they relate to the ability to utilize adaptations of this chemistry to construct useful amounts of bioactive materials. As indicated earlier in this chapter, the dioxolane subunit appended to the diylophile is critical to guarantee formation of the bridged cycloadduct, and is designed to serve as a synthon for the C1 hydroxyl group of the natural products [35]. Conversion of 95 to the target structure 92 was predicated upon the successful isomerization of the C-C ~-bond to the

BIOACTIVENATURALPRODUCTS

.... ~ ~ . , o

13~ .....~

[

219

~

~

r

F[~

r

9:$

92

94

F = functionality

H r

97

Scheme

96

95a, 9 5 b

6. Diylcycloadditionrouteto analogtaxol| 92.

tetrasubstituted position shown in 94. The remaining steps include alkylation of the enone 94, oxidative cleavage of the olefin followed by an intramolecular alkylation to afford the ABC ring system 92 common to taxoids. We believe that the route offers many opportunities for the construction of more elaborate materials. For example, an OR-unit positioned at the central carbon, Cr, of the tether that links the diylophile to the diazene in 96 becomes the important oxygen functionality found at C13 of the natural products. Furthermore, the use of a functionalized alkylating agent could incorporate the oxetane or provisional functionality to allow its construction at a later stasge. These are but two of many options. THF served as an inexpensive, readily available starting material. Ring opening using sodium iodide and benzoyl chloride in acetone [36], followed by alkylation of acetyl acetone, the addition of paraformaldehyde and deacylative methylenation in DMSO, afforded vinyl ketone 99 [37]. This material was most conveniently used without purification m a sequence consisting of ketalization, removal of the benzoate, and Doering oxidation to provide aldehyde 97 in a 60% yield overall [38].

220

LITTLE and O'IT Nal. BzCI THF

I~

Me2CO

OBz 98

O

O

{a}

DMSO. K2CO3 (b} {CH20)n

O

O

(a} HOCH2CH2OH, H*,CHaCH(OMe}2

O B

z

O

~

(b} KOH (c} SO3.pyr, DMSO, EtaN 99

97 Scheme 7. Assembly of tether and diyiophile.

Treatment of 97 with cyclopentadiene and diethylamine in methanol afforded fulvene 100 in a 90% yield. A Diels-Alder reaction with diethyl azodicarboxylate, followed by reduction of the A-5,6 g bond of the adduct using diimide, led efficiently (>95%) to the biscarbamate 101. The diazene linkage was unveiled in a customary fashion, to provide 20 grams of diazene 96 in an overall yield of 35%fi'om THF.

CpH. Et2NH. MeOH (90%}

100 (a} DEAD I {>95%} (b} N2H2

{a) KOH. EtOH (b} K3Fe(CN}e (83%} 98 1o~. E - COCt

Scheme 8. Entry to diyl procursor 96.


221

With substantial quantities of diazene 96 in hand, we were able to perform the diyl trapping reaction on a 20 gram scale, by far the largest ever used. The transformation proceeded superbly, affording 16 grams (80% isolated yield) of a 1:1 mixture of stereoisomeric bridged cycloadducts 95a and 95b [39]. In dramatic contrast to the first diyl trapping reactions where one typically used small quantities, sealed tubes, or found occasion to use a syringe pump, a simple dropping funnel was used to achieve a slow addition of diazene 96 to refluxing solvent. Many times intramolecular diyl trapping reactions are even easier to perform, simply calling for heating a solution of diazene in a given solvent until the starting material disappears [2]. Slow addition, as in the case of 96, is used when cycloaddition involves the triplet diradical to minimize or avoid entirely, dimerization of the diyl, a known triplet state process.

CH3CN, reflux __

{8o%} I0: I (bridged:linear}

@

\N~,~ N

102

96

~

ll,..

1

95a {a-H}, 95b ~-H}

The results are a clear and welcome validation of the hypothesis put forth earlier in this chapter. That is, given a choice between the 5-exo-trig or a six-endo-trig cyclization pathways portrayed below, the triplet diyl 103 prefers the latter to avoid energy-raising nonbonded interactions between vicinal substituents (see 104), and to allow the dioxolane unit to position itself as far from sterically demanding locations as possible, as is the case in structural representation 105. Here, the radical site to which R is bound can assume a planar or time-average planar geometry to minimize steric interactions. Isomerization of the double bond, in preparation for the oxidative cleavage designed to deliver the eight-membered ring, proved more challenging than anticipated. For example, attempts to do so using

222

LITTLEand O T r

5-exo,trlg @

! I !

R

I !

R= R 6-endo,trig 103 105 Scheme 9. Comparison of cyclization modes.

rhodium (III) chloride in refluxing ethanol afforded the di-substituted alkene 106 [40]. Since this reagent isomerizes double bonds to the thermodynamically preferred site, the results suggest that the tetrasubstituted olefin is not the most stable in this instance. Molecular mechanics calculations support this conclusion, consistently placing the di-substituted isomer 106 at lower energies than either the tri- and tetrasubstituted materials. Iio.-

I1,,-

RhCl 3

(

,

EtOH, reflux O 95a,b

106

Allylic oxidations were also explored using PCC, Collins reagent, and selenium dioxide. While the desired enone 108 formed, the less strained, less substituted olefin 107 was invariably the dominant product. Use of the Collins reagent afforded the best yields (40%), but produced the same inseparable mixture of regioisomers (3" 1, 1t)7:108). O

o_""i

107 III

I

I

CO ~~ 108


223

A clean reaction of 95a,b occurs with singlet oxygen, but affords an 85% yield of allylic alcohol 109. In this and each other instance, the less strained isomers were produced, prompting the exploration of a new strategy.

a. O2/TPP

('

b. N2H 2

185%1

95a,b iiiin

i

109 I lnnlllllllllll

I

II

I

Ill

Ideally, we imagined a regiospecific electrophilic oxyphenylselenation, placing the phenylselenyl unit at the bridgehead carbon of 95 (95 to 110). Subsequent oxidation of selenium, followed by a regiospecific syn elimination of the resulting selenoxide, could insert the double bond in the desired position, 111. However the literature suggested that the first step was likely to occur with the opposite regiochemistry [41 ].

llo*.

X PhSe-X -PhSeOH

I

110

III

Yet, as the following analysis shows, there was reason to believe that the process might occur in the desired manner. The first point to consider is the rt facial preference for selenonium ion formation. In this case, addition to 95 should occur syn to the ring junction hydrogen in order to assure formation of the significantly more stable cis-fused [3.3.0] subunit 112;[14] addition to the opposite face would afford the trans-fused counterpart 113.

224

LITTLE and OTI"

-

ph Set(~

.-" .. y

.,,,..

-

112

,,,..

% 9. . .

%

m

95 PhSe(~

%

'"'T-

-

-"

113 Scheme

I0. Selenonium ion formation- facial selectivity.

Assuming 112 is preferred, the question then becomes one of whether the nucleophile will attack at the bridgehead carbon Cx, or at Cy. The former would require the development of substantial carbocation character since a direct backside attack at Cx cannot occur (112 to 114, Scheme 11). Nucleophilic attack from the topside of the cation 114 would lead to an energetically unfavorable trans-fused [3.3.0] ring system and is unlikely to occur. Attack from the bottom ought to be retarded by sterie interactions with the phenylselenyl unit, and is also considered unlikely. What about a direct backside nucleophilic attack at the secondary center, Cy? This appears reasonable as it ought to encounter minimal steric inhibition and lead to a cis-fused [3.3.0] adduct, 115. Thus, despite literature precedent, we were hopeful that the reaction would occur in the desired manner.


225

SePh

c~

~

,,...

..--"-.

Coo" 112

y

I I I I I I I I I I

~

,,,.. |

~.o

114

I I I I I I I I I I I I I I

%

115 Scheme 11. Regioselective ring opening of selenonium ion.

While the addition did not occur using PhSeSePh, PhSeC1, or PhSeBr, the operation could be carried out conveniently and reproducibly via the addition of 95a to phenylselenyl trifluoroacetate, generated in situ from PhSeBr and AgOCOCF3 [42,43]. The trifluoroacetate unit hydrolyzed during workup to consistently afford a 93% yield of product 116. We were exceptionally pleased - the addition proved both regiospecific and efficient [44]. With the phenylselenyl unit in place, the oxidative syn elimination was examined. Compound 116 was oxidized with MCPBA and the reaction mixture added to refluxing carbon tetrachloride, but without success; the desired product could not be detected. To facilitate elimination, alcohol 1 1 6 was oxidized to the corresponding ketone 118, the thought being that the conjugative stabilization associated with formation of an enone might also lower the activation barrier. In practice, a Swem oxidation, followed by treatment with MCPBA at -78 ~ and addition of the reaction mixture to refluxing carbon tetrachloride, did lead to the formation of enone 94 as a single product in near quantitative yields. The overall transformation from the

226

LITTLE and OTF

?"

|lle-

a. "PhSeOzCCF3" b. K O H / E t O H (93%)

116

95a

a. MCPBA b. CCI 4, reflux OH

?N 1% o 117

bridged system 95a to the enone 94 could now be accomplished consistently in 68-78% overall yield, and on scales greater than 10 grams. PhS_e / ~ )

O

b. CCI 4, reflux

~.~0

..

118 I

III

I

The viability of the proposed route to the eight membered ring was confirmed by the ozonolytie cleavage of silyl ether l19b. This material was readily available from enone 94 via a selective 1,2-reduction using DIBAL-H, and protection of the resulting allylie alcohol l19a as a silyl ether. Verification of the structure was obtained by NMR using HMQC TOCSY [45]. A particularly noteworthy observation was that the chemical shift difference between the methylene protons, H~ and H,, was 1.9 ppm. This remarkably large separation suggests that the material exists in a conformation wherein the earbonyl units are oriented as shown, with syn proton, Hs, resonating at 3.76, and the anti, Ha, at 1.89 ppm. A similar


227

difference was observed for the methylene protons located at C3 (taxol | numbering), one appearing at 1.89, the other deshielded considerably, appearing at 3.83 ppm. Notice also that in structure 120, the C-He bond is oriented to allow overlap of the carbonyl ~* orbital with incipient negative charge. This allignment is needed to assure the appropriate stereoelectronics for the enolate forming step that is to be utilized in each of the efforts described to append ring C to the [5.3.1 ] core structure. {a} DIBAL-H 94

{b)'" t-BuPh;SiCl

/

I

t

~'"'/"v~

~- ( ~q,s k

OP

)

{a}

0 3.

-78 *C

. . . (b} . Me2S (7o%}

(77%1

l19a, P - H l19b, P = SIPh2Bu-t

i

~ OslPh2Bu't

120 Scheme 12. Oxidative cleavage leading to the [5.3. I] ring system.

o,s

120, R= CCH3I-OCH2CH20-}

The enolate of enone 94 was alkylated regioselectively using 4-iodo-1to afford a 2.5"1 mixture of diastereomers 121a and 121b in 77% yield. The mixture of diastereomers were separated and the major product was carried forward. Decoupling

(tert-butyldiphenylsiloxy)butane

228

LITTLE and OTF

O 1

LDA, I{CH2)40"rBDPS r

-78 ~ to RT {77%} 2.5: I. 1 2 1 a : 1 2 1 b

121a (~-H) 121b {~-H}

94 II

I

II

IIII

III

III

and NOE experiments were used to determine that the ct-alkylated material 121a was the major product. Saturation of Ha produced enhancements in the vicinal syn and anti methylene protons, Hb (8%) and Hr (5%), respectively. The key result was that He showed a 5% enhancement, suggesting that it was on the same face of the molecule as Ha. Molecular mechanics calculations were in full accord, consistently placing 121a at a slightly lower energy than its 13-alkylated stereoisomer 12lb.

,,../~~ /'

\ \ '_JL/'""\

He

.

/

~--~NOE ""

O OTBDPS 121a Fig. (11). Overhauser effects.

Enone 121a was reduced with DIBAL-H and the resulting allylic alcohol protected using benzyl bromide to afford 122 in an 80% yield overall. The major diastereomer was oxidatively cleaved and the silyl group removed, thereby affording alcohol 123 in 70% yield (two steps). Conversion to the corresponding iodide 124 was accomplished using a one step procedure involving the addition of 123 to a stirred solution of triphenylphosphine, iodine and imidazole in THF/MeCN. The stage was set for intramolecular alkylation; a single product 125 formed immediately (70%) when iodide 124 was added to LDA at-78 ~ Unfortunately, deprotonation and cyclization occurred from Ca rather than Cb. Similar results were obtained independent of the counterion (e.g., NaHMDS,


229

OBn

\ i~~/..,,,\ '"V

I. DIBAL-H

>

~

II ."

2. BnBr {80~

"',1|~

O~

HO 124

123

~OBn

t

I LDA 170%1

I • 'OBn llt

125

230

LITTLE and OTT

A simple solution to the problem would be to eliminate the unwanted deprotonation at Ca thereby leaving only the opportunity for cyclization to occur in the desired sense. To do so we elected to maintain the carbonyl oxidation state at Ca by protecting the ketone as the corresponding dioxolane 126. In this manner, the oxidative cleavage of the double bond in 126 leads to an eight-membered ring devoid of an abstractable proton at Ca (see 127). Alkylation, should it occur, is thereby forced to occur in the desired sense.

Q

, IOl

I

I

III

II

II III

I

I

I

Ull

II

While conceptually simple, this approach appeared flawed when

ozonolytic cleavage of the ~ bond in the model substrate 129 failed miserably [46]. Fortunately, ruthenium tetraoxide, generated in situ from excess sodium periodate and 10 mol % ruthenium dioxide, worked splendidly [47]. After 10 minutes the reaction was complete, and the dione 130 was isolated in an 80% yield. o

Ill,.u

,v

94 5% TsOH, HOCH2CH2OH Phil. reflux (65%}

:

Oa, DMS

.......

,

_

CH3CN-COI4-H20

(80%1 Scheme 13. Ruthenium tctroxidr as an important alternative to ozonolysis.


231

Buoyed by these results, we moved to the more demanding target structure, 92. It proved advantageous to use the mixture of diastereomers 121a,b in the ketalization step, since the use of either of the pure forms met with epimerization. Several conditions were examined, but the original 5 mol% TsOH proved optimal (50% ketal 126, 20% recovered starting material). Each isomer independently afforded the same 2.5" 1 ratio of isomeric ketals, suggesting that the 2.5" 1 ratio obtained in the initial alkylation step (94 to 121) reflected a thermodynamic distribution.

0

,,,..

\_-6

HOCH2CH2 OH Phil. TsOH. reflux

121a,b

126 I 1. RuO 2. NaIO4 (80%) 2. TBAF (95%)

nllo..~ 127

The critical ruthenium tetroxide oxidative cleavage of the g bond in 126 occurred rapidly and with consistently good yields (70-80%). However, it was important to carefully monitor the reaction to avoid overoxidation. Removal of the silyl ether with TBAF afforded a mixture of alcohols 127 (2.5"1, still reflecting the mixture resulting from the alkylation step) which could be separated by column chromatography. The major diastereomer 127a was oxidized to provide aldehyde 131, a substrate seemingly well-suited for the use of an intramolecular aldol condensation to complete the addition of the third ring [48]. When a methanolic solution of 131 was heated gently in the presence of a variety

232

LITTLE and O'I'F

of equilibrating bases (e.g., KOH; K2CO3; Na2CO3, NaOH), it was smoothly transformed into a single UV active product in a 70% yield. Characterization revealed that it did not correspond to the desired adduct 132, but instead to enal 133, the product resulting from closure of the aldehyde enolate onto the ketone carbonyl, rather than the reverse. 0

o::::on( 127a

) 131

t

~

base

0/-.7

t ,

0

"|ll''/~ 133

Scheme

14. Aldol condensation charts its own course.

At this point, it appeared that enolate formation toward the pro C3 carbon (see 92 for numbering) might not be a viable proposition. Given a choice, either it did not occur in that direction or if it did, then the enolate equilibrated to an alternative form that led to 125 in the case of the attempted alkylation of iodide 124, or to 133 in the attempted aldol cyclization of 131. We eventually elected to explore the intramolecular alkylative cyclization of keto iodide 134. To this end, mesylation of 127a followed by displacement with NaI led to 134 (80%). Treatment of this iodide with excess LDA in THF at-78 ~ followed by warming to room temperature, produced a single product. Workup, isolation, and characterization indicated that the desired adduct 92 had indeed been produced, and in a reasonable 63% yield.


a,,..

233

O I. MsCl, NEt 3 ,

,

2. Nal, a c e t o n e

{8o%} I

134

127a

5 eq LDA THF -78 ~ (63%)

~

, II

0

H

*.

92

Molecular mechanics calculations place the t r a n s fused B,C ring system at considerably lower energies than its cis counterpart. The literature suggests that base-induced cis-to-trans isomerization is easily accomplished [49]. So, to ensure that the t r a n s ring fusion had been obtained, 92 was treated with K O B u - t / t - B u O H (8 h, room temp.)[49a]. The starting material was recovered unchanged, strongly supporting the notion that isomerization did not occur and that 92 does indeed possess the requisite stereochemistry. SUMMARY A functionalized taxol analog 92 has been synthesized from THF (Scheme 15). Substantial quantities of diazene 96 were synthesized in a convenient eight step sequence. The regioselective diyl trapping reaction, 96 to 95, consistently produced high yields of the key intermediate on scales >20 grams. A regioselective oxyphenyselenation reaction added the phenylselenyl unit to the bridgehead carbon of 95 and proved critical in efforts to isomerize the initially formed disubstituted double bond, and ultimately leading to enone 94. Cleavage of the tetrasubstituted olefin provided the [5.3.1] ring system present in taxoids. A regioselective

234

LITTLE and OTT

intramolecular alkylation served to append the side chain and form the core A-B-C ring system present in the natural products. This first generation synthesis of the taxol | skeleton provides ample opportunity to add key functionality in our ongoing second generation efforts designed to produce bioactive materials.

dlyl trap

., (8 steps)

~ |

|

~

r

0 N /

98

(3 steps) "

('"'

'"'

o

- " _",. ~---0

Scheme 15. Overview of route used to access analog 92.

It is now a relatively simple matter to selectively assemble either bridged or linearly fused tricyclie systems. The chemistry just described illustrates one of the materials toward which we have applied the methodology. In the next section, we focus upon its application to another important natural product, viz., the challenging anticancer agent, aphidieolin (89).

Aphidicolin (89) Aphidicolin (89) is a diterpenoid tetraol produced by the mold Cephalosporium aphidicola. In 1972, Hesp and co-workers reported its isolation and structure [50]. Aphidieolin (89) displays marked activity against Herpes simplex type I virus in cultures of human embryonic cells, as well as antifeedant properties. Furthermore, aphidicolin (89) exhibits antitumor activity in the C6 mouse colon and B 16 mouse melanosarcoma screens and has been shown to inhibit the growth of leukemic T- and Blymphocytes [51]. Such results have stimulated interest in clinical investigations of the effectiveness of 89 against human tumors.


235

Development of 89 as an antitumor agent has been limited by its poor water solubility. However, reports of enhanced antitumor activity associated with the more water-soluble compounds such as aphidicolin17-glycinate HCI salt (135) and 16-fluoroaphidicolin (136) have revived interest in aphidicolin (89) and its analogs as potential therapeutic agents [52]. The potent activity of aphidicolin (89) is presumed to arise through its activity as a specific reversible inhibitor of DNA polymerase-a [51 ]. R2N I

al ~16

8

H

aphldlcolin (89}; R l = OH, R2 = H aphidicolin-17-glycinate HCI salt (135); R l - O H , R 2 = COCH2NH2-HCI

.

,~

HO

~

OH

16-flouroaphidicolin (136); R l = F, R2 = H

Fig.(12). Aphidicolin (89) and derivatives.

These biological properties along with its structural complexity have made 89 an attractive synthetic target. Aphidicolin (89) possesses an interesting array of ring systems, including both spiro and fused substructures, as well as the ever-challenging vicinal quaternary stereogenic centers [53]. The first two syntheses were reported independently by Trost and by McMurry in 1979. Many followed, including the first enantioselective synthesis reported by Holton in 1987. As is portrayed in Scheme 16, Fukumoto's approach to aphidicolin (89) used an intramolecular Diels-Alder reaction to both form and simultaneously fuse rings A and B onto a pre-existing CD unit (139 to

140) [54]. The route mirrors that conceptualized by this research in the early 1980's, the major difference being that in our route, an intramolecular diyl trapping reaction was to be utilized to assemble the [3.2.1] core (see Scheme 17). Thereafter, an intramolecular Diels-Alder reaction was planned in much the same manner as that implemented by the Fukumoto group. Nearly as soon as we realized that the diyl trapping reaction could be used to construct the [3.2.1 ] unit, aphidicolin (89) emerged as a target structure of interest. The problem, however, was that for many years it was not possible to use the diyl trapping reaction to form the bridged materials selectively, or in substantial quantity. That we are now able was in no small part motivated by the desire to synthesize the natural product [21.

236

LITTLE and OTF

~

~

O

0 H

{CH2}20H

o H

v

HO 137

139

138 ~ O

I 220 ~

140 Scheme 16. The Fukumoto route to aphidicolin (89).

We set our sights on a plan designed to converge with Fukumoto's Diels-Alder triene precursor, viz., with keto alcohol 138. We reasoned that if the intramoleeular diyl trapping reaction could be used to synthesize the bisketal 143, then conversion to 138 should be accomplished by oxidative cleavage of the lr bond to afford diol 142, selective protection of the primary alcohol, and the insertion of unsaturation between Cs and C:3. The latter operation is designed to allow use of both of the epimers that result from the diyl eyeloaddition step, as the Cs stereogenie center is removed when the sp 2 hybridized center is created. A least hindered side delivery of dihydrogen to the A-8,13 7r bond ought to position the Cs side chain on the alpha face, as needed. Removal of the alcohol protecting group and the selective conversion of the side chain ketal to a methyl ketone ought to afford 138, our convergent point with Fukumoto. To test the proposed route we capitalized upon our ability to use the intramoleeular diyl trapping reaction to synthesize 95, the mono-ketal analog of 143. Ozonolytie cleavage of the C-C 7r bond of 95 followed by reduction with NaBH 4 afforded diol 145 in an 85% yield. The primary alcohol was selectively protected as a silyl ether, and the secondary alcohol oxidized with PCC to provide ketone 146 as a 1:1 mixture of diastereomers.


"~

237

~o

~o o o.

" , t ~ OH

9

:

HO

HO

HO

142

141 89

o~O I

/-t~, N 144

143

Scheme 17. The diyl trapping route to aphidicolin (89).

i(O,,..

/

b. N~BH4

Co

\

|,,,~.....~OH ,,..X.-"5~

Co

95

1

OH

145

I

II

2. PCC

/

\

i,,..~0 ,,..X.--"g~

Co

1

OTBDI~

146

III

Methodology developed by Ireland and coworkers was the first applied to insert a double bond between carbons 8 and 13 [55]. Unfortunately, treatment of enol phosphate 147 with lithium in liquid ammonia afforded a complex mixture of products. Though signals were present in the vinyl region of the IH NMR spectrum, none of the purified materials corresponded to the desired product 148.

238

LITTLEand 0 1 T

(a) LDA (b) (EtO)2POCI

,,.._

, 0

147

148

/~ LI/N]-I3

,,,,. ~ . ~ ~ . ~ ~ . . ~ O P

~.~0 I

148

II

The Shapiro reaction appeared to offer a reasonable alternative protocol. To this end, ketone 146 was converted to the corresponding tosylhydrazone 149 in reasonable yield (70%, in addition to recovered starting material). Both diastereomers of 149 were then subjected to a variety of standard, literature procedures [56]. In no case was product observed, only recovered starting material. Careful examination of the i,,..

..

O

NNHTs

i

TsNHNH2 D. EtOH

( | ~ ~ ~ ~ H ~

(70%)

PO

149

146, P = TBDPS

0~,0

n-BuLl, TMEDA reflux (85%)

/Ill,.

,i

compare (~O " ~

'

H

138

OH

PO

(a) dilmlde ~

TsOH TBAF (75%) OH 3 steps 150

Scheme 18. Convergencewith Fukumoto; a model system.

148

BIOACTIVENATURALPRODUCTS

239

literature reveals that the Shapiro reaction is, in fact, not very effective when there is branching adjacent to the carbonyl. We were, however, able to achieve the desired transformation by using TMEDA as the reaction solvent. Thus, at-78 ~ five equivalents of n-BuLi were added to a stirred solution of tosylhydrazones 149 in TMEDA. The reaction mixture was then refluxed for 8 h and upon workup, the desired olefin 148 was isolated in an 85% yield. It is important to note that both diastereomers were converted to olefin 148. This contrasts with our taxol | (90) related effort wherein only one of the diastereomers produced in the diyl cycloaddition proved effective in the oxyphenylselenation process. The sequence to 150 was completed as planned, by a least hindered exo face reduction of the olefin in 148 using diimide generated in situ, and deprotection of the ketal and silyl groups. OVERVIEW While the sequence just outlined has been carried out using the monoketal 95, rather than the bisketal 143, we believe that it will also be applicable to the latter. In fact, we have managed to utilize the intramolecular diyl trapping reaction to synthesize 143 and have, in preliminary experiments, demonstrated that it is possible to selectively convert the exocyclic ketal to a methyl ketone 151, as is needed to synthesize aphidicolin (89) [57]. At this time we are perfecting our route to diazene 144 and anticipate that the natural product is finally within our grasp.

CHaCN, reflux 12 h (80%)

N//N

143 144

PPTS, acetone, H20 ~ 0

~ [90%1

151 I

I

Ill

I

LITTLE and OTr

240

ACKNOWLEDGEMENTS We are exceptionally grateful to both the National Institutes of Health (National Cancer Institute), and the National Science Foundation for their support of our research programs. REFERENCES

[1] [2] [31

[4] [5] [6]

[7] [8] [91

[10] [11]

[12] [13] [14] [15] [16] [17]

[18] [19]

Paquette, L. A.; Doherty, A. M. Polyquinane Chemistry Springer-Verlag: Berlin, 1987. (a) Little, R. D. Chem. Rev. 1986, 86, 875. (b) Little, R. D. Chem. Rev. 1996, 96, 93. Little, R. D. In Comprehensive Organic Synthesis, Trost, B. M., Fleming, I., Eds.; Pergamon: Oxford, 1991; Vol. 5, Chapter 3.1, and references therein. Little, R. D.; Bukhari, A.; Venegas, M. G. Tetrahedron Lett. 1979, 305. (a) Stone, K. J.; Little, R. D. J. Org. Chem. 1984, 49, 1849. (b) McLoughlin, J. I.; Little, R. D. J. Org. Chem. 1988, 53, 3624. (a) Little, R. D.; Carroll, G. L. J. Org. Chem. 1979, 44, 4720. (b) Meehan, S.; Little, R. D. J. Org. Chem. 1997, 62, 3779. (c) Rastetter, W. H. J. ,4m. Chem. Soc. 1976, 98, 6350. (d) Gassman, P. G.; Schenk, W. N.J. Org. Chem. 1976, 42, 918. (e) Schwaebe, M. K.; Little, R. D. Electrochemica Acta, 1997, 42, 2201. Little, R. D.; Muller, G. W. J. Am. Chem. Soc. 1981, 103, 2744. (a) Little, R. D; Venegas, M. G. Org. Synth., Coll. Vol. VII ; Wiley: New York, 1990, 56. (b) Little, R. D.; Bregant, T. M. In Encyclopedia of Reagents for Organic Synthesis, Paquette, L. A., Ed.; Wiley: New York, 1995; Vol. 1, p. 572. With a two-carbon tether connecting the diyl to the diylophile, evidence strongly supports a stepwise mechanism, even when the diylophile is activated by an electron withdrawing group. Campopiano, O.; Little, R. D.; Petersen, J. L. J. Am. Chem. Soc. 1985, 107, 3721. Hudlicky, T.; Kutchan, T.; Wilson, S. R.; Mao, D. T. J. Am. Chem. Soc. 1980, 102, 6351. Little, R. D.; Higby, R. G.; Moeller, K. D. J. Org. Chem. 1983, 48, 3139. (a) Kunimoto, T.; Umezawa, H. Biophys. Acta 1974, 298, 513. (b) Ishizaka, M.; Iinuma, H.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1972, 25, 320 Kupka, J.; Anke, T.; Giannetti, B. M.; Steglich, W. Arch. Microbiol. 1981, 130, 223. (b) Giannetti, B. M.; Steffan, B. Steglich, W. Tetrahedron 1986, 42, 3587. (c) Steglich, W. Pure Appl. Chem. 1981, 53, 1233. (a) Van Hijfie, L.; Little, R. D. J. Org. Chem. 1985, 50, 3940. (b) Van Hijtte, L.; Little, R. D.; Petersen, J. L.; Moeller, K. D. J. Org. Chem. 1987, 52, 4647. Stone, K. J.; Little, R. D. J. Am. Chem. Soc. 1985, 107, 2495. Kissel, C. L.; Rickbom, B. J. Org. Chem. 1972, 37, 2060. (a) Lipshutz, B. H.; Parker, D. A.; Kozlowski, J.; Nguyen, S. L. Tetrahedron Lett. 1984, 25, 5959. (b) Lipshutz, B. H.; Wilhelm, R. S.; Kozlowski, J. Tetrahedron 1984, 40, 5005. (c) Lipshutz, B. H. ,4cc. Chem. Res. 1997, 30, 277. Koreeda, M.; Mislankar, S. G. J. Am. Chem. Soc. 1983, 105, 7203. Schuda, P. F.; Heimann, M. R. Tetrahedron 1984, 40, 2365.


[20] [21] [22] [23] [24]

[25] [26]

[27]

[28] [291 [301 [31] [32]

[33]

[34]

241

(a) Danishefsky, S.; Zamboni, R.; Kahn, M.; Etheredge, S. J. J. Am. Chem. Soc. 1980, 102, 2097. (b) Danishefsky, S.; Zamboni, R.; Kahn, M.; Etheredge, S. J. J. Am. Chem. Soc. 1981, 103, 3460. Little, R. D.; Carroll, G. L.; Petersen, J. L. d. Am. Chem. Soc. 1983, 105, 928. Kaisan, M.; Sheikh, Y. M.; Durham, L. J.; Djerassi, C.; Tursch, B.; Daloze, D.; Braekman, J. C.; Losman, D.; Kadsson, R. Tetrahedron Left. 1974, 2239. Birch, A. M.; Pattenden, G. Tetrahedron Lett. 1982, 23, 991. Freisleben, W. Angew. Chem. 1963, 72, 576. Stembach, D. D. In Strategies and Tactics in Organic Synthesis, Lindberg, T. Ed.; Academic Press: San Diego, 1989; Vol. 2, Chapter 12. Platz, M. S.; Berson, J. A. J. Am. Chem. Soc. 1976, 98, 6743. (a) Beckwith, A. L. J.; Ingold, K. U. In Rearrangements in Ground and Excited States; deMayo, P., Ed.; Academic Press: New York, 1980; Vol.l, Chapter 4 and references therein. (b) Lusztyk, J.; Maillard, B.; Deycard, S.; Lindsay, D. A.; Ingold, K. U. J. Org. Chem. 1987, 52, 3509. Little, R. D.; Losinski-Dang, L.; Venegas, M. G.; Medic, C. Tetrahedron Lett. 1983, 24, 4499. Berson, J. A. In Diradicals; Borden, W. T., Ed.; Wiley: New York, 1982; Chapter 4. (a) Masjedizadeh, M. R.; Dannecker-Doerig, I.; Little, R. D.; J. Org. Chem. 1990, 55, 2742. (b) Little, R. D.; Masjedizadeh, M. R.; Moeller, K. D.; DanneckerDoerig, I. Synlett 1992, 107. Masjedizadeh, M., Postdoctoral Research Report, UCSB. (a) Taxane Anticancer Agents; Georg, G. I.; Chert, T. T.; Ojima, I.; Vyas, D. M.; Eds.; American Chemical Society: San Diego, 1995; Vol. 583. (b) Nicolaou, K. C.; Dai, W. M.; Guy, R. K. Angew. Chem. Int. Ed. Engl. 1994, 33, 15. (c) Swindell, C. S. Org. Prep. Proced. Int. 1991, 23, 465. (a) Nicolaou, K. C.; Zang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claibome, C. F.; Renaud, J.; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J. Nature 1994, 357, 630. (b) Nicolaou, K. C.; Nantermet, P. G.; Ueno, H.; Guy, R. K.; Couladouros, E. A.; Sorensen, E. J. J. Am. Chem. Soc. 1995, 117, 624. (c) Nicolaou, K. C.; Liu, J. J.; Yang, Z.; Ueno, H.; Sorensen, E. J.; Claibome, C. F.; Guy, R. K.; Hwang, C. K.; Nakada, M.; Nantermet, P. G. J. Am. Chem. Soc. 1995, 117, 634. (d) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Nantermet, P. G.; Claibome, C. F.; Renaud, J.; Guy, R. K.; Shibayama, K. J. Am. Chem. Soc. 1995, 117, 645. (e) Nicolaou, K. C.; Ueno, H.; Liu, J. J.; Nantermet, P. G.; Yang, Z.; Renaud, J.; Paulvannan, K.; Chadha, R. J. Am. Chem. Soc. 1995, 117, 653. (f) Holton, R. A.; Somoza, C.; Kim, H. B.; Liang, F.; Biediger, R. J.; Boatman, D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. S.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1597. (g) Holton, R. A.; Kim, H. B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. S.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1599. (h) Masters, J. J.; Link, J. T.; Snyder, L. B.; Young, W. B.; Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 1995, 34, 1723. (i) Wender, P. A.; Badham, N. F.; Conway, S. P.; Floreancig, P. E.; Glass, T. E.; Houze, J. B.; Krauss, N. E.; Lee, D. S.; Marquess, D. G.; McGrane, P. L.; Meng, W.; Natchus, M. G.; Shuker, A. J.; Sutton, J. C.; Taylor, R. E.; J. Amer. Chem. Soc. 1997, 119, 2757. Ott, M. M.; Little. R. D. d. Org. Chem. 1997, 62, 1610.

242

[35]

[36] [37]

[38] [39]

[40]

[41] [42] [43] [44]

[45] [46] [47] [48]

[49]

[50] [5~] [52] [531

LITTLE and 01T The corresponding dimethyl ketal is too labile to be useful, particularly in large scale operations. Its relatively facile hydrolysis complicates the diyl cycloaddition since the diazene-ketone leads preferentially to the linearly fused rather than the desired bridged adduct. The ethylene glycol ketal provided an ideal solution to the problem. Nystr0m, J. E.; McCanna, T. D.; Helquist, P.; Amouroux, R. Synthesis 1988, 56. The sequence leading to 99 was originally explored and developed by Mr. Scott Meehan of UCSB. The use of DMSO allowed the chemistry to be conducted at room temperature, made the process very convenient to conduct, and led to significantly improved yields. Parikh, J. R.; Doering, W. E. J. Am.Chem. Soc. 1967, 89, 5505. The identity of these materials was confirmed by conversion to and comparison of the spectral properties with with the known ketones; see reference 30. (a) Grieco, P. A.; Nishizawa, M.; Marinovic, N.; Ehmann, W. J. J. Am. Chem. Soc. 1976, 98, 7102. (b) Mehta, G.; Murthy, A. N. J. Org. Chem. 1987, 52, 2875. Raucher, S. J. Org. Chem. 1977, 42, 2950. Reich, H. J. J. Org. Chem. 1974, 39, 428. (a) Sharpless, K. B.; Lauer, R. F. J. Org. Chem. 1974, 39, 429. (b) Reich, H. J.; Wollowitz, S.; Trend, J. E.; Chow, F.; Wendelbom, D. F. J. Org. Chem. 1978, 43, 1697. In principle, both stereoisomers 95a and 95b can be converted to the same tetrasubstituted olefin. Unfortunately, and despite much effort, only 95a proved useful in the oxyphenylselenation step. We thank Dr. Andre D'Avignon of Washington University for performing the HMQC-TOCSY experiments. (a) Blechert, S.; Muller, R.; Beitzel, M. Tetrahedron 1992, 48, 6953. (b) Galatsis, P.; Manwell, J. J. Tetrahedron 1995, 51,665. (a) Carlsen, P. H. J.; Katsuki, T.; Martin, V, S,; Sharpless, K. B. J. Org. Chem. 1981, 46, 3936. (b) Mehta, G.; Krishnamurthy, N. J. Chem. Soc., Chem. Commun. 1986, 1319. (a) Marshall, J. A.; Greene, A. E. J. Org. Chem. 1972, 37, 982. (b) Marshall, J. A.; Greene A. E., Ruden, R. Tetrahedron Lett. 1971, 855. (r Marshall, J. A.; Greene A. E. Tetrahedron Lett. 1971, 859. (d) Heathcock, C. H.; Trice, C. M.; Germroth, T. C. J. Am. Chem. Soc. 1982, 104, 6081. (e) Crimmins, M. T.; Gould, L. D. dr. ,4m. Chem. Soc. 1987, 109, 6199. (a) Blechert, S.; Kleine-Klausing, A. Angew. Chem., Int. Ed. Engl. 1991, 30, 412. (b) Magnus, P.; Ujjainwalla, F.; Westwood, N.; Lynch, V. Tetrahedron Lett. 1996, 3 7, 6639. Bundret, K. M.; Dalziel, W.; Hesp, B.; Jarvis, J. A.; Neidle, S. J. Chem. Soc., Chem. Commun. 1972, 1027. Pedrali-Noy, G.; Belvedere, M.; Crepaldi, T.; Focher, F.; Spardari, S. Cancer Res. 1982, 42, 3 810. Hiramitsu, T.; Mouri, A.; Suzuki, H. (Nippon Mektron Ltd.). Japan Patent Kokai Tokkyo Koho JP 5-310621. (a) Trost, B. M.; Nishimura, Y.; Yamamoto, K.; McElvain, S. S. J. Am. Chem. Soc. 1979, 101, 1328. (b) McMurry, J. E.; Andrus, A.; Ksander, G. M.; Musser, J. H.; Johson, M. A. J. Am. Chem. Soc. 1979, 101, 1330. (r Corey, E. J.; Tius, M. A.; Das, J. J. Am. Chem. Soc. 1980, 102, 1742. (d) Ireland, R. E.; Godfrey, J. D.; Thaisrivongs, S. J. Am. Chem. Soc. 1981, 103, 2446. (e) van Tamelen, E. E.; Zawacky, S. R.; Russell, P. K.; Carlson, J. G. J. Am. Chem. Soc. 1983, 105, 142.


[54] [551 [561

[57]

243

(f) Bettolo, R. M.; Tagliatesta, P.; Lupi, A.; Bravetti, D. Helv. Chim, Acta. 1983, 66, 1922. (g) Tanis, S. P.; Chuang, Y. H.; Head, D. B. Tetrahedron Lett. 1985, 26, 6147. (h) Holon, R. A.; Kennedy, R. M.; Kim, H. B.; Kraft, M. E. J. Am. Chem. Soc. 1987, 109, 1597. (i) Rizzo, C. J.; Smith, III, A. B. J. Chem. Soc., Perkin Trans. 1991, 1, 969. (j) Tanaka, T.; Murakami, K.; Okuda, O.; Inoue, T.; Kuroda, T.; Kamel, K.; Murata, T.; Yoshino, H.; Imanishi, T.; Kim, S. W.; Iwata, C. Chem. Pharm. Bull. 1995, 43, 193. Toyota, M.; Nishikawa, Y.; Fukumoto, K. Tetrahedron Lett. 1995, 36, 5379. Ireland, R. E.; Pfister, G. Tetrahedron Lett. 1969, 2145. (a) Dauben, W. G.; Lorber, M. E.; Vietmeyer, N. D.; Shapiro, R. H.; Duncan, J. H.; Tomer, K. J. Am. Chem. Soc. 1968, 90, 4762. (b) Chamberlin, A. R.; Stemke, J. E.; Bond, F. T. J. Org. Chem. 1978, 43, 147. (c) Stemke, J. E.; Chamberlin, A. R.; Bond, F. T. Tetrahedron Lett. 1976, 34, 2947. (d) Lipton, M. F.; Shapiro, R. H. J. Org. Chem. 1978, 43, 1409. Unpublished research with Dr. Joachim Dickhaut, UCSB; postdoctoral research report, 1994.


Atta-er-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22

245


BIOACTIVE FUNGAL NATURAL PRODUCTS THROUGH CLASSIC AND BIOCOMBINATORIAL APPROACHES Z H I - D O N G JIANG* AND Z H I Q I A N G AN

Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge MA, 02139-1562 ABSTRACT" The kingdom of fungi is an important source of bioactive natural products, which have been a driving force in the development of modem pharmaceutical industry. Fungal natural products have provided revolutionary pharmaceuticals against various diseases, and have provided unique and inspirational chemicals for innovative drugs. Fungi are essentially an untapped source of drugs in spite of many remarkable therapeutic agents discovered from them so far. Of approximately 1.5 million species of fungi, to date only about 70,000 species have been described. Among these 70,000 species, only a small fraction can be isolated from nature and fermented in laboratory media for drug screening. The vast majority of slow-growing and unculturable fungi have received little attention for drug discovery due to technical limitations. Therefore, the potential for discovering bioactive agents from slow-growing and unculturable fungi is even greater than that from the species that we have explored. With the maturing technology of genetic engineering, it is now possible to express genes from unculturablr organisms in laboratory strains for secondary metabolism study. Today, mankind is not only still facing the challenge to treat untamed diseases, but is also fighting newly recognized diseases, and diseases that once were subdued but are developing resistance to the current therapeutic regimes. In this review, we will discuss recent developments and progress of several important fungal metabolites and their derived products as examples of drugs produced by natural isolates. We will also discuss the current progress of biocombinatorial drug discovery by genetic engineering approaches, as well as possibilities and strategies of exploring genetic diversity from unculturable fungi for drug discovery. The vast fungal kingdom, which consists of an estimated 1.5 million species, is of interest to the pharmaceutical industry for its production of many important secondary metabolites. Since more than one half century ago, when the fungal metabolite penicillin was first partially purified and used for treating bacterial infections, bioactive fungal metabolites have strongly influenced the development o f the modern pharmaceutical industry. Mevinolin, cyclosporin A, and [3-1actam antibiotics are examples of revolutionary pharmaceuticals that have a fungal origin. In addition, the diverse and unique chemical structures of fungal metabolites have served as an important source o f inspiration for structural motifs to synthetic chemists [1-2]. Fungal metabolites o f various biosynthetic origins have produced breakthrough pharmaceutical and agricultural products during the last

246

ZHI-DONG JIANG AND ZHIQIANG AN

decade. In this review, we describe recent progress of several fungal metabolites of pharmaceutical and agricultural importance. This by no means includes all the structures, rather we aim to demonstrate the potential of discovering new chemical structures from fungal sources and the value of fungi as an important source of compounds. Readers are encouraged to study recent reviews and databases that have extensively covered fungal natural products [3-5, 133]. In spite of the remarkable therapeutic agents discovered so far, fungi are essentially an untapped source of active metabolites. Only a small fraction of the fungal taxa can be and have been fermented in laboratory media for drug discovery. In this chapter, we also briefly discuss the potential of using biocombinatorial approaches to tap into the genetic diversity of fungi for drug discovery. FUNGI Fungi are nutrition-absorptive eukaryotic organisms found in every ecological niche. The kingdom Fungi comprises more than 1.5 million species of organisms [6]. It is difficult to generalize the characteristics of fungi due to the tremendous ecological, physiological, and morphological diversity within the fungal kingdom. The classification of fungi is consistently evolving as new information emerges related to fungal evolution and systematics. Currently, there are four phyla in the fungal kingdom: Chytridiomycota,Zygomycota,Ascomycota,and Basidiomycota. Figure 1 illustrates the current classification scheme for fungi. i. . . . Basidiomycota '-Ascomycetes I

Fun~---

I_

I

Ascomycota -----

Zygomycota Chytridlomycota

Deuteromycetes

Fig. (1). Phylogenetic Relationships of Major Groups in the Fungi Kingdom.

Chytridiomycetes is the only class in the phylum Chytridiomycota. Fungi in this group produce motile cells at some stage in their life cycle. Chytrids are present in both aquatic habitats and soils. This group of fungi has not been widely used for natural products screening because they are extremely small and difficult to isolate and culture. Since chytrids occur in very competitive ecological niches, access to this group of fungi might yield interesting secondary metabolites. The phylum Zygomycota comprises two classes: Zygomycetes and Trichomycetes. Many fungi in the class Zygomycetes produce a thick-

BIOACTIVE FUNGAL NATURAL PRODUCTS

247

walled resting spore known as zygospore. Species of Zygomycetes can be isolated from a variety of ecosystems. Some Mucorales fungi have been extensively used for production of various secondary metabolites, but in general, this group of fungi has not been well explored for natural products drug discovery. Fungi in the class Trichomycetesare obligately associated with living arthropods. Because they can not be easily cultured, they have not been subjected widely to drug discovery. Fungi in the phylum Ascomycota are grouped into two, classes: ascomycetes and deuteromycetes. Ascomycetes produce asci, which are saclike structures containing sexual ascospores. Deuteromycetes are asexual ascomycetes. Fungi in Ascomycota occur in a broad range of ecosystems and consist of species greatly diverse in morphology and habitat. Ascomycetes are well known and extensively investigated for their ability to produce a large number of secondary metabolites. Many of these metabolites are effective therapeutics. Drugs developed from natural products isolated from ascomycetes and other fungi are summarized in Table 1. Table 1.

Some of Fungal Significance

Metabolites

with Pharmaceutical

Producing organism

Fungal metabolite

Related industrial products

,, ,,,,,i

,

,

and Agricultural

i

,

i

Peniciilins

Penicillium sp.

Penicillin antibacterial agents

Cephalosporin C

Cephalosporium acremonium

Cephalosporin antibacterial agents

Mevinolin

Aspergillus sp.

Lovastatin, simivastatin, lipitor and other HMG-CoA inhibitors

Cyclosporins

Tolypocladium inflatum

Cyclosporin A, immunosuppresant

Mycophenolic Acid

Penicillium sp.

CellCept, immunosuppresant

Griseofulvin

Penicilllium griseofulvum

Griseofulvin, antifungal agent

Strobilurins

Strobilurus tenacellus

Amistar and kresoxim methyl, agricultural fungicides

Fusidic Acid

Fusidium coccineum

Fusidic acid, antibacterial agent

Ergot alkaloids

Claviceps purpurea

Ergotamine for migraine Ergonovine for obstetrics

Gibberellins

Fusarium moniliforme Fusarium graminearum

Zearalenone ,

ill

Gibberellins, plant growth hormone Zearalenone, growth promoter in cattle ,,,,,

The fungi in the phylum Basidiomycota are called basidiomycetes, which are distinguished from other fungi by the production of sexual basidiospores. Basidiomycetes consist of fungi commonly known as mushrooms, puffballs, rusts, and smuts.

248


Antifungal Agents Lipopeptides The number of cases of systemic fungal infections has grown in recent years due to the increased number of immune compromised individuals including patients with acquired immunodeficiency syndrome (AIDS), and patients receiving such therapeutics as anti-neoplastic agents, immunosuppressants, or broad-spectrum antibiotics. Currently available chemotherapies for systemic fungal infections such as amphotericin B and fluconazole have limited uses because of their toxicity and/or insufficient therapeutic efficacy. Although more advanced liposomal formulations of amphotericin B have significantly improved its efficacy and reduced its toxicity, reports of fungal resistance to this drug are increasing [7-9]. New antifungal drugs with improved activity are greatly needed. Fungi have been a very productive source of potential antifungal agents and have yielded two classes of promising cyclic peptides with two novel mechanisms of antifungal activity. Echinocandins and pneumocandins, produced by Aspergillus species and Zalerion arboricola respectively, belong to the same class of cyclic lipopeptides whose characteristic long chain acyl groups connect to the cyclic peptide ring through an amide bond. As fungicidal agents, they inhibit ~-l,3-glucan synthase and prevent the formation of this class of glucan, an essential component of the fungal cell wall in many fungi [ 101 1]. Echinocandins are active in animal models against Candida sp., Pneumocystis carinii, and some Aspergillus sp.. They are not active against Cryptococcus sp. which contain mostly o~-glucan [ 12]. Two derivatives of echinocandin and pneumocandin, L-743872 by Merck and LY-303366 by Eli Lilly, are currently in clinical trials [127].

Aureobasidins Aureobasidins, produced by Aureobasidium pullulans, are another class of cyclic peptides [13-15]. Aureobasidin A, the most abundant component, is a fungicidal agent with strong activities against Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum. Its fungicidal activities in mice with candidiasis are more effective than fluconazole and amphotericin B, and it is well tolerated by mice [ 15]. The molecular target for aureobasidin A was identified recently as inositol phosphorylceramide synthase (IPC synthase), an enzyme that catalyzes a key step in sphingolipid biosynthesis, which is essential for yeast growth and viability. Aureobasidin A tightly binds to IPC synthase with an IC50 of 0.2 nM [16].


249

Strobilurins and Oudemansins

In the agricultural antifungal area, two novel fungicides have been developed from the structural motif of strobilurins and oudcmansins. Strobilurins and oudemansins are fungicidal fungal metabolitcs isolated from Strobilurus tenacellus, Oudemansiella mucida, and several other basidiomycetes, as well as the ascomycete Bolinea lutea [17-20]. Strobilurin A is potently active against a wide range of plant pathogenic fungi through inhibiting the cytochrome bcl complex [21-23]. However, the producing fungi S. tenacellus is resistant to strobilurin A by a mutation of single amino acid at position 127 in its cytochrome bc 1 [24].

CH 3

CH3

Aurcobasidin A

\

Ho/

HO

F

o

~N-x / o

o ~ O H

~Ott -

-

Echinocandin

B

250


~

CH 3

0

~

Strobilurin A

Me

O

Amistar (Azoxystrobln)

CN

CH3

IOCH3 0 Oudemansln A

Kresoxin methyl

The potent in vitro antifungal activities of strobilurin A were not maintained in the field due to the labile nature of its triene system. Several agricultural firms are engaged in synthetic efforts to produce stable and more potent analogues as potential products. In 1996, two products, amistar and kresoxim methyl, were approved as agricultural fungicides in the U.S. after more than a decade of research and development.

Anti-hypercholesterolemia Agents H M G - C o A Inhibitors

During the past decade, the most important development in the field of fungal natural products may have been the discovery and successful commercialization of HMG-CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase) inhibitors. Their huge success in the pharmaceutical market reflects the fact that coronary heart disease has become one of the top killers in the industrialized nations (in the U.S. alone, more than 600,000 people die of heart disease each year) [25]. Hypercholesterolemia has been identified as an important cause for this disease. In the cholesterol biosynthetic pathway, HMG-CoA reductase is a rate-limiting


251

enzyme that catalyzes the condensation of acetoacetyl-CoA with acetylCoA to form 3-hydroxy-3-methylglutaryl-CoA. Since Endo and his colleagues discoved compactin, a specific HMG-CoA reduetase inhibitor, this biological target has been a focus of international hypercholesterolemia research for more than 20 years, and there is no sign that interest in developing new inhibitors is waning [26]. After compactin was first isolated from Penicillium citrinum [27-28], several derivatives of compactin were isolated from Monascus and other filamentous fungi including Aspergillus, some species of Penicillium, Phoma, and Trichoderma [29]. The clinically important metabolite mevinolin was isolated independently by two groups of scientists at Sankyo and Merck from Monascus ruber and Aspergillus terreus, respectively [30-31 ]. The biosynthesis of mevinolin in Aspergillus terreus involves two polyketide chains, C 18 and C4, each synthesized from acetate units. The two methyl groups at 6 position and on the side chain were derived from methionine. Propionate was not incorporated [34]. Pravachol is a 6~-hydroxy acid form of compactin and can be produced by microbial transformation [32]. Pharmacologically, the active form of mevinolin is the ~l-hydroxyacid form; the lactone form is inactive. Chemical modification of the ester side chain revealed that its stereochemistry was not important but aeyl moiety was essential. Removal of the side chain or side chain ether analogues resulted in marked loss of potency [33]. Extensive SAR studies have led to the development of simvastatin, a 2,2-dimethylbutyrate derivative of mevinolin. Simvastatin, with the dissociation constant Ki of 0.2 nM, is about 3 times more potent than mevinolin and 11 times than pravachol and has been clinically proven to be more effective than either mevinolin or pravachol.

~

Rl

R2

O

Mevlnolln

CH 3

I R2

I O

CH3

Compactln

H

RI~~" 0

252

ZHI-DONGJIANG AND ZIlIQIANG AN

H0 ~ , ~

H O~ COOH H

H Paravachol

COOH

u ,m

CHa

0 Llpltor (atorvastatln)

The discovery of mevinolin, the first HMG-CoA reductase inhibitor approved to market, is a good example of how dramatic and competitive it can be to develop an innovative drug. Although Merck was the first pharmaceutical company to market a drug in this class, Merck was not the first to test this class of compounds in the clinic. In 1976, researchers at Sankyo first isolated compactin (mevastatin), which entered Phase I clinical trials in Japan and several other countries in 1978. It was a promising agent to significantly reduce the cholesterol levels with very few serious acute side effects. A year later, scientists at Merck isolated a related compound, mevinolin, which was put to Phase I clinical trials in early 1980, only to be suspended shortly thereafter. Prompting the suspension was the news that compactin caused intestinal lymphomas in 50 percent of the dogs treated with the agent. However, in 1984, Merck restarted the Phase I clinical trials with mevinolin after several physicians discovered that the drug dramatically reduced the cholesterol levels in patients whose severe hypercholesterolemia did not respond to existing therapies. Remarkably, mevinolin caused very few side effects. In November 1986, mevinolin was approved by the Federal Food and Drug Administration for marketing. In the meantime, scientists at Sankyo


253

discovered another HMG-CoA reductase inhibitor, pravachol, which was subsequently licensed to Bristol-Meyers Squibb. Interestingly, pravachol was initially detected as a urinary metabolite of compactin in dogs and was the 6-~-hydroxy-l]-hydroxyacid form of compactin. Pravachol possessed cholesterol-reducing activity similar to mevinolin and was approved for marketing in the U.S. in 1991 [35]. However, the pharmaceutical industry's race for a slice of the huge market for HMG-CoA reductase inhibitors did not stop there. It was recently estimated that the worldwide HMG-CoA inhibitor market will reach $13 billion in the next few years [36]. This vast potential market for HMG-CoA reductase inhibitors has prompted large synthetic efforts to generate clinically more effective compounds. Studies have shown that the hydronaphthalene structure of natural inhibitor served only as a holder of the lactone and therefore could be replaced with other structural templates. One such compound produced by varying the templates is lipitor. Clinically, under the defined conditions, lipitor is more effective by several parameters in reducing lipids level. In three separate studies, lipitor has been shown to be superior to mevinolin, pravachol, and simvastatin in lowering the triglycerides by significant margins [37-39]. In 1996, nine years after mevinolin was first marketed, Parke-Davis introduced lipitor, a new synthetic HMG-CoA inhibitor developed from mevinolin structural motif, and it is quickly becoming one of the best selling drugs [40].

Squalene Synthase Inhibitors Squalene synthase is an enzyme catalyzing the formation of squalene from farnesyl diphosphate which is a committed step in the cholesterol biosynthetic pathway. Therefore, squalene synthase is considered a better target than HMG-CoA reductase because farnesyl pyrophosphate, a downstream product of HMG-CoA reductase, is needed for prenylation of proteins and for the biosyntheses of ubiquinone and dolichol (Fig. 2). Before squalestatins and zaragozic acids were discovered, a number of squalene synthase inhibitors were synthesized that showed respectable inhibitory potencies in vitro, but none were successful in animal testing [41]. It was the discovery of squalestatins and zaragozie acids that renewed interest in this biological target, and at picomolar potencies they were the most active inhibitors of squalene synthase. Squalestatins and zaragozir acids were isolated from several fungi including Phoma sp. C2931, Sporormiella intermedia, and Leptodontium elatius. Squalestatins and zaragozic acids possess the unusual structural feature of a highly functionalized bicyclic core with three earboxylic groups. Zaragozic acids differ from one another by varying side chains and long chain acyl groups [42-44].

254

Z H I - D O N G J I A N G AND Z H I Q I A N G AN

.oo~.

'~oo~

"

"

R2 Squalestatln 1 R l

CH 3 |

Squalestatln 2 R l

=

Squalestatln 3 R l

~ i O

=

|

Rl

-

Z a r a g o z l c acid C

Rl

=

R2

_

/o.

= l O

R2

-

H2C

R2

_-

H2C

R2

=

H2C

H

%CH 3

O Rl

=

0

H

Z a r a g o z l c acid B

R2 !

., o...

Z a r a g o z l c acid D

=

=

]

OH


255

Acetyl CoA

.~etoaeetyl CoA

HMG CoA

Isopentenyl tRNA

~tatin

IIMGCoAReductase Mevalonle Acid

i I !

Isopentenyl Pyrophosphate ~

FamesylaUon of P r o t e l n s ' q [ - - - ' - - F a m e s y l P y r o p h o s p h a t e ~

Sualene Synthase ~

~

Dlmethylallyl

- - - ~

Prophosphate

Ublqulno a n d Dollchol

Squalestatins

Squalene

Lanosterol

! I I

Cholesterol Fig.

(2).

Cholesterol Biosynthesis and Utilities of Isoprenyi Intermediates.

Biosynthetically, squalestatin 1 was derived from mixed precursors. The major portion of the molecule was formed from two polyketide chains made of acetate units. The other portions were derived from benzoic acid (or phenylalanine), succinate, and methionine. Five of the oxygens were derived from atmospheric oxygen; the oxygens at the two ester carbonyls were derived from acetate (Fig. 3). Based upon this biosynthetic pathway, several biosynthetic analogues were isolated through feeding

256

Table 2.


Biosynthetic Analogs of Squalestatin through Precursor Feeding

/

/ o~__~ ....\

\ o

oil,.

oH

H

O

O

C

~

R il

H O O C ~ X-" o il

,=

,

OH

COOH ,

i

ICso(ng/ml)

|',

I

0.2 Squalestatln I R = H 2 C - ~ 0.4

F Analogue I R = H 2 C ~

____/

0.3

F

Analogue!! R = H z C - ' ~ 0.2 AnalogueIII R = H 2 C - ~ ~

F 0.4

AnaloguelV R = H 2 C ~ 0.2 AnalogueV R =

H2C

~S 0.1

AnalogueVI R - H2C~'ff~

experiments. Several of them were shown to be as potent as squalestatin 1 [45-48] (Table 2, Fig. 3).


#

i~b-.-

,"-'k ,. *

ox,

2"o t OH

O

O"

0 ~~ t " " ' ~ " 6 OH

H~176

257

.

COOH

Benzoic acid Acetate Succlnate NH 2 Methyl of methlonlne *,~

S

COOH

Fig. (3). The Biosynthetic Precurosrs of Squalestatin A.

Recently, zaragozic acids were shown to inhibit Ras farnesyltransferase, which involves posttranslational famesylation of all Ras proteins. Zaragozic acids blocked Ras processing in Ras transformed tumor cell lines at concentrations as low as 10 nM. Therefore, they potentially have anticancer applications [49].

Immunosupressive Agents Cyclosporin, Rapamycin, and FK506 Cyclosporin A, an immunosuppressant well known for its clinical property in organ transplantation [131, 132], was initially isolated as an antifungal agent from several fungi, including Tolypocladium inflatum [50]. Rapamycin and FK506, relatively new immunosuppressive agents used in the clinic, are metabolites of Streptomyces sp. Recently, it was revealed that the three agents had intriguing mechanisms of biological activities. Chemically, cyclosporin A is a cyclic peptide of eleven amino acid residues, very different from FK506 and rapamycin. FK506, although slightly smaller, resembles rapamycin, and their structures are partly identical. Surprisingly, it is FK506 and cyclosporin A that share almost

258


\

"[ T

" H301-]~"f

[it

z

Y ,

O

~

pH3y

~,,.~

O

T -T ~

'11111~ '0

H

Cyclosporln A

~

H,

CH3 O

I H

OCH3

OH

H3 O

H $,~

J~

H3Co O

HO H3C_ft,./

~"~0

CH3

O"

~

"Cl 13

"OCH3

~H

v .~

"vOCH3

q,,

FK 506

Rapamycln

identical biological effects, not rapamycin and FK506. Furthermore, FK506 and rapamycin first bind to the same cytosolic binding protein, FKBP12, to form two different complexes, while cyclosporin A binds to a distinctly different protein, cyclophilin A. However, cyclosporin Acyclophilin A complex and FK506-FKBP12 complex inhibit the same target (calcineurin), while rapamycin-FKBP 12 complex inhibits a different target called FRAP. Both FKBP12 and cyclophilin A are peptidylproline cis-trans isomerases and are potently inhibited by FK506 and cyclosporin A respectively with subnanomolar IC50 values. The small molecules must bind their binding proteins to form the complexes that are specific inhibitors to calcineurin and FRAP; the small molecules alone


259

exert no effects on calcineurin. Cyclosporin A and FK506 are highly specific inhibitors of their respective binding proteins [51-56]. Mycophenolic acid

The study of mycophenolic acid (MPA) has a long history. MPA was first observed in 1896 [57] and its structure determined in 1952, which was confirmed by a X-ray study in 1972 [ 129]. A wide range of biological activities were observed in MPA including antifungal, antiviral, and antitumor [58-59]. In the 1970s, attempt was made to develop this compound as an antitumor drug, but it was later withdrawn due to its toxicities [60]. In 1972, scientists at Eli Lilly firmly elucidated the mode of action of MPA to be a potent inhibitor of inosine monophosophate dehydrogenase (IMPDH) with the dissociation constant Ki values in the nanomolar range against IMPDH(s) from several biological sources [61 ]. MPA selectively inhibits the de novo pathway of guanosine nucleotide synthesis in vivo,. Because the proliferation of T- and B-lymphocytes require the de novo synthesis of purines, whereas other cell types can utilize salvage pathways, MPA selectively inhibits the proliferative responses of T- and B-lymphocytes to both mitogenic and allospecific stimulation. To develop MPA as an immunosuppressant, the prodrug mycophenolate mofetil was synthesized to improve the pharmacokinetic profile of MPA [62]. When mycophenolate mofetil is administered orally, it is rapidly absorbed and metabolized to MPA, the active metabolite. In 1996, one hundred years after MPA was discovered, mycophenolate mofetil was approved by the FDA for renal transplant rejection. The development of this drug shows how a compound that may be toxic in one disease area may be useful in other therapeutic areas.

Ho•H3

H

\ /0

0

Mycophenollc Acid

O

CH3

Mycophenolate Mofetil

CH 3

260


A n t i c a n c e r Agents

llludin S and Hydroxymethylacylfulvene Illudins are unique sesquiterpenes produced by the fungus Omphalotus illudens [63-65] The compounds are extremely toxic and have caused poisoning when Omphalotus is mistaken for edible mushrooms [66-67].

OH .-

HO~'"

OH .~

]

9

CH2OH

HO~,"

O

O

llludln S

llludln M

2OH

0

9

HO~'" 0 Hydroxymethylacylfulvene

~176

0 Dehydrotlludtn M

llludin S and illudin M are reactive cytotoxic compounds that alkylate thiols through Michael addition reaction to form stable aromatic adducts [68-70] (Fig. 4). The antitumor activities of these two compounds were studied in a variety of rodent tumor models in the 1980s when they were found to be potent anticancer agents. However, because their cytotoxicity was not highly selective, illudins were not safe to use as anticancer agents [71]. Evidently, the ttl3-unsaturated ketone and cyclopropylmethyl carbinol were the key features of illudins required in the alkylation reaction. But in order to make illudins safer, analogues had to be less reactive to thiols and more selective to tumor cells. A series of compounds was prepared that resulted in the discovery of hydroxymethylacylfulvene, which caused complete tumor regression in metastatic lung carcinoma (MV 522) in xenograft animals and exhibited outstanding activities against breast, colon, and skin cancers. This compound entered Phase I clinical trials in 1995 [72-74].


261

- H2

_OH ".,, HO ~'"

CH2OH

H+~tt~'~~SR

"CH20 H

OH OH

"%' H20H OH

SR

Fig. (4). Reaction of thiols with llludin S.

Fumagillin and A GM-14 70 Angiogenesis is a complex morphogenic process that forms new blood vessels. It is a rare event in healthy individuals and is a tightly regulated essential process involved in the normal growth and wound healing. However, in a variety of pathological states such as solid tumor growth, psoriasis, diabetic retinopathy, and ophthalmic diseases, it becomes an uncontrolled process [75]. The growth of solid tumor relies heavily on the formation of new blood vessels to supply nutrients to the tumor. Therefore, angiogenesis inhibitors can potentially be used as antitumor agents. One such compound is fumagillin, a well-known fungal metabolite [76] whose antiangiogenesis activity was discovered serendipitously in Folkman's lab when the producing organism, Aspergillus fumigatus, contaminated the endothelial cell culture in his lab [130]. The antiangiogenesis activity of fumagillin was identified at Tekeda in the late 1980s. Another natural product, ovalicin, structurally similar to, but more stable than fumagillin, exhibited similar bioactivity, and was synthesized enantioselectively by Corey's group[128]. There are several in vivo assay models for angiogenesis, including the shell-less chicken chorioallantoic membrane (CAM) assay, the cornea assay, and the endothelial cell assay. The CAM assay is most frequently used assay method because it is easy to use and less costly. In this assay, an inhibitor's effect on the shell-less chicken embryo culture can be observed directly, although quantitating the result is subjective. The mouse cornea assay is widely favored; because the cornea is an avascular tissue, any blood vessel development unequivocally demonstrates the lack of antiangiogensis activity [77-79]. However, all of these in vivo assay

ZIII-DONG JIANG AND ZHIQIANG AN

262

models share a common shortcoming" their low throughput is a major impediment to large-scale screening efforts. Fumagillin is a potent angiogenesis inhibitor that inhibits endothelial cell proliferation in vitro, ncovasculation in CAM assay, and tumor angiogenesis in the mouse [80]. At l0 ug, it exhibited 75% inhibition of blood vessel formation in the CAM assay [81-82]. In the late 1980s, Takeda Chemical Industries undertook synthetic effort to discover analogues of fumagillin that were more stable and more potent. As a result, AGM-1470 (formerly TNP-470) was synthesized to inhibit in vitro the proliferation of human umbilical vein endothelial cells at 50 times the potency of fumagillin. AGM-1470 reduces the growth rate of Lewis Lung carcinoma and B 16 melanoma in mice. In 1997, the molecular target of AGM-1470 and ovalicin was elucidated by Griffith and Su to be the bifunctional enzyme methioninc aminopcptidasc (type 2) (MctAP2) [83]. AGM-1470 potently and specifically inhibits MctAP2 with ICs0 values of 1.0 nM by covalcntly binding to the protein. AGM-1470 neither inhibits the type 1 methionine aminopeptidase (MetAP1) nor affects the other function of MctAP2, inhibition of elF-2oc phosphorylation. This finding suggests that MetAP2 may play an important role in the proliferation of endothelial cells and therefore may become a highthroughput screening target for new antiangiogenic agents.

O CH3

0 -CH3

!

"../H 9

-

1 .,~

_

"~ OH 0

O Fumagfllln

0

AGM-1470 -0 .CH3

y

"~ 0 Ovalicin


263

Tapping Fungal Diversity for Drug Discovery Using Genetic Engineering Even though fungi are a proven source of drugs, they have been barely explored for drug discovery. Of approximately 1.5 million species, to date only about 70,000 species have been described. Of the 70,000 described species, only a small fraction has been screened for drug discovery; fungi that have been selected for drug screening are usually those that can be easily isolated from nature and easily cultured and maintained in laboratory media. However, culturable fungi constitute only a small portion of the fungal world. In contrast, unculturable and slow-growing fungi have received little attention for drug discovery due to technical limitations of studying them in the laboratory. No doubt many new agents will continue to be discovered from culturable fungi, but there is a clear need to expand the potential fungal drug source to include unculturable organisms. It is certain that novel metabolites exist in tmculturable and slow-growing fimgi. Since fungal secondary metabolites are encoded by genes, these genes can be cloned from unculturable fungi and introduced into fast-growing heterologous hosts. The heterologous hosts will then express the secondary metabolite-encoding genes from unculturable fungi to synthesize novel secondary metabolites. Recent studies have shown that genes involved in secondary metabolite biosynthesis in microorganisms are often clustered. For example, the genes involved in the biosynthesis of 13lactam antibiotics from three different eukaryotic organisms (Penicillium chrysogenum, Cephalosporium acremonium, A. nidulans) and two prokaryotes (Nocardia lactamdurans, Streptomyces clavuligerus) are all clustered. The three genes required for melanin biosynthesis of the filamentous fungus Alternaria alternata are clustered within a 30 kb fragment of genomic DNA. The genes required for the production of the polyketide antibiotics frenolin and nanaomycins by Streptomyces roseofulvus are clustered within a 10 kb DNA fragment. The entire set of actinorhodin biosynthetic genes from Streptomyces coelicolor is clustered within a 26 kb DNA fragment. More recently, genes involving sterigmatocystin biosynthesis in A. nidulans were defined within a 60 kb DNA fragment. The cyclic peptide siderophore biosynthetic genes in Ustilago maydis are also clustered (Sally Leong, personal communication). Based on this evidence, it is possible that a gene cluster for a particular secondary metabolite will be included when large pieces of DNA from unculturable fungi, as exemplified by cosmid clones (35-45 kb), yeast artificial chromosome clones (> 100 kb), and bacterial artificial chromosome clones (> 100 kb), are introduced into a recipient laboratory strain. The recipient strains for expressing genes from unculturable fungi should be easy to manipulate genetically; numerous fungi can serve as

264


recipient hosts. The availability of multiple-recipient laboratory strains increases the chance for heterologous expression of foreign genes. One of the assumptions underlying this genetic approach is that genes or clusters of genes from unculturable fungi can be expressed in laboratory strains because transcription and translation control sequences from one organism often function in closely related organisms, and sometimes even in distantly related organisms [97,120]. For example, the fungal transformation vector pAN7-1 [118], which has the Escherichia coli hygromycin phosphotransferase gene (hph)under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter and trpC terminator, efficiently confers hygromycin B resistance to a variety of fungal species. More than 40 fungal species have been transformed with this vector, ranging from the basidiomycetes Schizophyllum commune [115] and Laccaria laccata [84] to various ascomycetous species [97]. The A. niger glucoamylase gene promoter functioning in U. maydis [121] is another example of an ascomycete promoter that functions in a basidiomycete. Even genes from plants have been shown to be transcribed in Saccharomyces cerevisiae. An example of this is the maize storage protein zein gene [108]. A few Aspergillus genes have been isolated by complementation of S. cerevisiae mutant strains [119]. All this evidence suggests that genes of foreign origin may be expressed in laboratory strains. However, one must select a donorrecipient combination cautiously; not all transcription control sequences from one fungus function in other fungal hosts [ 121 ]. Several strategies, such as mRNA analysis, reporter gene analysis, and complementation of auxotrophic markers, can be applied to test gene expression. In summary, by exploring genetic diversity from unculturable fungi and by creating biocombinatorial diversity, we improve the likelihood of discovering novel secondary metabolites. Because the recipient strains are fast-growing, industrial organisms, once novel-drug producing transformants are identified, scale-up fermentation for commercial production can be quickly implemented.

Synthesizing Unnatural Natural Products Using Biocombinatoriai Approaches Recent advances in molecular genetics of secondary metabolite biosynthesis have made it possible to develop a whole new concept of drug discovery known as biocombinatorial production of synthetic natural products. The promise of this genetic engineering/chemistry hybrid approach for developing novel drugs and recent progress in the combinatorial biosynthesis of novel bacterial polyketides have led scientists to explore the potential of novel biocombinatorial fungal secondary metabolites as therapeutic agents. The use of fungi as sources of natural genes and as hosts for expressing engineered chimeric genes is


265

especially timely. Until now, most work in this area has focused on bacteria, yet fungi are known to be prolific producers of biologically active secondary metabolites. The pathways of several classes of fungal secondary metabolites, such as polyketides and non-ribosomal peptides, are suited for biocombinatorial manipulations. Here we use the genetic engineering of bacterial polyketides as an example to illustrate the concept and challenges of this approach. We will also speculate the advantage and potential of engineering fungal polyketides and other secondary metabolites. Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and 13-ketoaeyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of I]-carbons, e.g., ~-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. Aromatic PKSs (or type II) are composed of several separate, largely monofunctional proteins, whose active sites are used iteratively for the assembly and functional-group manipulation of the polyketide chain. At least 13 different sets of aromatic polyketide PKSs have been cloned from Streptomyces and Saccharopolyspora species [100]. Best studied among all PKSs is the PKS for the benzoisochromanequinone antibiotic actinorhodin [109, 113, 114]. Genes for actinorhodin biosynthesis are designated actI-VIl. ActI encodes 3 different active sites: KS, AT, and chain-length-determining factor (CLF). ActllI encodes KR. ActI and actlII constitute the minimum PKS. Actll is responsible for transcriptional regulation of the act genes and for actinorhodin export. ActlV-VII encode several post-synthetic modifying functions, e.g. cyclization (VII), aromatization, and subsequent chemical tailoring. Other aromatic PKSs share the same basic architecture with minor structural differences [98, 100]. Molecular genetic analysis of PKS genes has confirmed earlier biochemical and chemical findings that the structural diversity of

266


polyketides is a result of the different numbers and types of acyl units involved [109]. This body of research also supports the idea that novel polyketides can be produced by manipulating the sequence and specificity of enzyme-mediated reduction, dehydration, cyclization, and aromatization [89, 95, 99-101, 104-106, 112-114, 117, 126]. The era of rational design of novel antibiotic structures was ushered in by early successes synthesizing complex polyketides. The modular PKSs for complex polyketides contain a unique active site for each enzymecatalyzed reaction in the pathway, giving rise to final structures that are determined by the numbers and types of active sites. Donadio et al. demonstrated in the early 1990s that analogues of the erythromycin polyketide backbone could be generated by eliminating active sites within the PKS [90-92]. By repositioning a chain-terminating cyclase domain from the C-terminus of module 6 of DEBS3 to the C-terminus of module 2 of DEBS 1, Cortes et al. were able to construct a multienzyme unit that catalyzed only the first two rounds of polyketide chain extension [89]. The mutant produced a triketide lactone structure without any trace of erythromycin, the wild-type polyketide, indicating premature chain termination and cyclization. By expressing the entire DEBS gene cluster in a heterologous host, substantial quantities of 6-deoxyerythronolide B, the aglycone of the macrolide antibiotic erythromycin, were produced [101]. In contrast, aromatic PKSs contain a single set of iteratively used active sites, although basic organization of the two types of PKS is similar. Their characteristics make it difficult to predict the structure of the polyketide that will be produced after a PKS gene is modified. Great advances in research on the combinatorial biosynthesis of novel aromatic polyketides have been made by Khosla, Hopwood, and their collaborators. Hybrid aromatic polyketides have been generated by transferring partial or complete biosynthetic gene clusters between different polyketide producers and screening for new structures. For example, mederrhodin A, a novel aromatic polyketide, was made by a medermycin-producing Streptomyces strain transformed with the actVA gene, that strain normally produces only native mederrhodin [99]. This approach of generating novel polyketides was largely empirical, and required the activity of enzymes for late tailoring steps. During the last few years, tremendous progress has been made in the understanding of genetic programming of aromatic PKSs; consequently, the rational design of novel aromatic polyketides is also advancing. Several novel compounds have been generated by constructing and expressing recombinant PKSs in Streptomyces sp. [95, 106, 112-114]. Based on their work and the work of others, McDaniel et al. summarized 6 strategies for rational design of novel aromatic polyketides. They are chain length, ketoreduction, cyclization of the first ring, first ring aromatization, second ring cyclization, and additional cyclization.


267

Fungi, filamentous fungi in particular, are an especially rich source of polyketides [ 116]. To date, nucleotide sequences have been reported for 7 fungal PKS genes, all of which encode type I PKSs. The C. heterostrophus PKS1 gene is responsible for the biosynthesis of T-toxin [124]. The MSAS1 gene ofPenicillium urticae [123] and P. patulum [85] encodes 6-methylsalicylic acid synthase (MSAS), which catalyzes synthesis of the tetraketide 6-methylsalicylic acid, the precursor of a mycotoxin, wA gene ofAspergillus nidulans encodes a PKS of 1,986 amino acids, whose product is a component of green pigment; pksA of A. parasiticus encodes a polypeptide required for the production of a yellow pigment and for biosynthesis of aflatoxin B-l, a potent carcinogen [88]; pksL1 of A. parasiticus encodes 2,109 amino acids and is required for production of several related aflatoxins and their biosynthetic precursors [93]; pksST ofA. nidulans encodes a polypeptide of 2,181 amino acids required for the production of the polyketide-derived mycotoxin sterigmatocystin [125]; PKS1 of Colletotrichum lagenarium encodes a polypeptide of 2,187 amino acids required for production of the pentaketide precursor of melanin [ 122]. Similar principles and roles for genetic engineering of bacterial polyketides could be applied to combinatorial synthesis of fungal polyketides and other fungal secondary metabolites, such as nonribosomal peptides, but specific protocols need to be developed for fungi since fungal gene regulation and structure are very different from bacteria. For example, fungal genes contain nontranslated DNA known as introns. The concept of making new metabolites by chimeric genes is rather simple and obvious, but the challenge of constructing a number of chimeric gene clusters is great. This approach, though very promising, has yet to be proven practically. CONCLUSION As the pharmaceutical and agricultural industries continue their endeavor to find small molecular agents for a variety of diseases, natural products have become an important source of bioactive compounds. Many natural products have been used directly or have provided structural templates for synthetic analogues for these diseases. Economically, natural products and their related compounds account for a significant portion of worldwide pharmaceutical sales. Among the billion-dollar drugs for 1995, approximately 40% were small molecule natural products or drugs that contained structural motifs of natural products. Recent advances in molecular biology and genetics and their application to biocombinatorial synthetic natural prodcuts will generate unprecedented novel natural products. New separation and detection technologies will accelerate the process of discovering drugs from natural products. With the help of these novel approaches, natural products will play a even greater role as a

268


source chemical diversity for the pharmaceutical and agricultural industries. ACKNOWLEDGEMENT

The authors gratefully acknowledge Ms. Louise Gachet for editing this manuscript. REFERENCES

[1] [z]

Cragg, G.M.; Newman, D.J.; Snader, K.M.J. Nat. Prod., 1997, 60, 52. Beese, K. In Phytochemical Diversity, Wrigley, S.; Hayes, M.; Thomas, R.; Chrystal, E. Ed.; The Royal Society of Chemistry: Cambridge, UK, 1997, pp. 129-140. [3] (a) Frisvad, J.C.; Thrane, U. In Chromatography of Mycotoxins, Betina, V. Ed.; Elsevier: Amsterdam, 1993, pp. 253. (b) Jong, S.C.; Gantt, M.J. ,4 TCC Catalogue of Fungi~Yeasts, 17th ed. With an index to the industrial applications. American Type Culture Collection, Rockville, MD, 1987. [4] (a) Jong, S.C.; Donovick, R. Adv. Appl. Microbiol., 1989, 34, 183. (b) Gloer, J. In Developments in Industrial Microbiology Series, Gullo, V.P.; HunterCevera, J.C.; Cooper, R.; Johnson, R. Ed.; Society of Industrial Microbiology: 1993, Vol. 33, pp. 1-12. [5] (a) Demain, A.L. Ciba Found. Symp., 1992, 171, 3. (b) Cole, R. J.; Cox, R. H. Handbook of Toxic Fungal Metabolites, Academic Press: New York, 1981. [6] Hawksworth, D. L.; Rossman, A. Y. Phytopathology, 1997, 87, 888. [7] Kelly, S.L,; Lamb, D.C.; Kelly, D.E.; Manning, N.J.; Loeffler, J.; Hebart, H.; Schumacher, U.; Einsele, H. FEBS Lett., 1997, 400, 80. [8] Nolte, F.S.; Parkinson, T.; Falconer, D.J.; Dix, S.; Williams, J.; Gilmore, C.; Geller, R.; Wingard, J.R. Antimicrob. Agents Chemother., 1997, 41, 196. [9] Sandven, P.; Nilsen, K.; Digranes, A.; Tjade, T; Lassen, J. Antimicrob. Agents Chemother., 1997, 41, 1375. [10] Mizoguchi, J.; Saito, T.; Mizuno, K.; Hayano, K. J. Antibiotics, 1977, 30, 308. [ll] Baguley, B.C.; Rommele, G.; Gruner, J.; Wehrl, W. Eur. J. Biochem., 1970, 97, 345. [12] James, P.G.; Cherniak, R.; Jones, R.G.; Stortz, C.A.; Reiss, E. Carbohydr. Res., 1990, 198, 23. [13] Takesako, K.; Ikai, K.; Haruna, F.; Endo, M.; Shimanaka, K.; Sono, E.; Nakamura, T.; Kato, I. Yamaguchi, H. J. Antibiotics, 1991, 44, 919. [141 Yoshikawa, Y.; Ikai, K.; Umeda. Y.; Ogawa, A.; Takesako, K.; Kato, I.; Naganawa, H. J. Antibiotics, 1993, 46, 1347. [15] Takesako, K.; Kuroda, H.; Inoue, T.; Haruna, F.; Yoshikawa, Y.; Kato, I.; Uchida, K.; Hiratani, T.; Yamaguchi, H. J. Antibiotics, 1993, 46, 1414. [16] Nagiec M.M.; Nagiec E.E.; Baltisberger, J.A.; Wells, G.B.; Lester, R.L.; Dickson, R.C.J. Biol. Chem., 1997, 272, 9809. [17] Musilek, V. British Pat. 1965, GB 1163910.


[18] [19]

[20] [El]

[22]

[23] [24]

[25]

[26] [27] [28] [29]

[3o] [31] [32] [33]

[34] [35] [36] [37] [38]

[39] [40]

[41]

[42]

[43] [44] [45] [46]

269

Vondracek, M.; Capkova, J.; Slechta, J.; Benda, A.; Musilel, V.; Cudlin, J. British Pat., 1967, GB 1181892. Bauerle, J.; Anke, T. Planta Med., 1980, 39, 195. Anke, T.; Besl, H.; Mocek, U.; Steglich, W. J. Antibiotics, 1983, 36, 661. Becker, W.F.; yon Jagow, G.; Anke, T.; Steglich, W.; FEBS Lett., 1981, 132,

329. Von Jagow, G.; Becker, W.F. Bull. Mol. Biol. Med. 1982, 7, !. Von Jagow, G.; Gribble G.W.; Trumpower, B.L. Biochemistry 1986, 25, 77578O. Kraiczy, P.; Haase, U.; Gencic, S.; Flindt, S.; Anke, T.; Brandt, U.; Von Jagow, G. Eur. J. Biochem., 1996, 235, 54. Manson, J.E.; Tosteson, P.H.H.; Rider, P.M.N. Eng. J. Med., 1992, 21, 1406. Endo, A.; Hasumi, K. Nat. Prod. Report, 1993, 541. Endo, A.; Kuroda, M.; Tsujita, Y. J. Antibiotics, 1976, 29, 1346. Endo, A.; Kuroda, M.; Tanzawa, K. FEBS Lett., 1976, 72, 323. Juzlova, P.; Martinkova, L.; Kren, V. J. Industrial Microbiol., 1996, 16, 163. Endo, A.; J. Antiobiotics, 1979, 32, 852. Alberts, A.W.; Chen, J.; Kuron, G. Proc. Natl. ,4cad. Sci. USA, 1980, 77, 3957. Serizawa, N.; Serizawa, S.; Nakagawa, K.; Furuya, K.; Okazaki, T.; Terahara, A. J. Antibiotics, 1983, 36, 887. Sliskovic, D.R.; Roth, B.D. In Biological Inhibitors, Choudhary, M.I. Ed.; Harwood Academic Publishers: 1996, pp.87-160. Moore, R.N.; Bigam, G.; Chan, J.K.; Hogg, A.M.J. Am. Chem. Soc., 1985, 107, 3694. Scherer, F. Pharmaceutical R & D: Costs, Risks and Rewards, Government Printing Office: 1994, pp. 74-75. O'Reilly, B. Fortune, 1997, October 13. Bertolini, S.; Bon, G.B.; Campbell, L.M.; Famier, M.; Langan, J.; Mahla, G.; Pauciullo, P.; Sirtori, C.; Egros, F.; Fayyad, R.; Nawrocki, J.W. Atherosclerosis, 1997, 130, 191. Dart, A.; Jerums, G.; Nicholson, G.; d'Emden, M.; Hamilton-Craig, I.; Tallis, G.; Best, J.; West, M.; Sullivan, D.; Braes, P.; Black, D.; Am. J. Cardiol. 1997, 80, 39. Alaupovic, P. Heinonen, T.;I Shurzinske, L. Black, D.M. Aeherosclerosis, 1997, 133, 123. B W Health Wire, October, 23, 1997. Abe, I.; Tomesch, J.C.; Wattanasin, S.; Prestwich, G.D. Nat. Prod. Reports, 1994, 279. Dawson, M.J.; Farthing, J.E.; Marshall, P.S.; Middleton, R.F.; O'Neill, M.J.; Shuttleworth, A.; Stylli, C.; Tait, R.M.; Taylor, P.M.; Wildman, H.G.; Buss, A.D.; Langley, D.; Hayes, M.V.J. Antibiotics, 1992, 45, 639. Bergstrom, J.D. et al, Proc. Natl. Acad. Sci. U S d, 1993, 90, 80. Tanimoto, T.; Hamano, K.; Onodera, K.; Hosoya, T.; Kakusaka, M.; Hirayama, T.; Shimada, Y.; Koga, T.; Tsujita, Y. J. Antibiotics, 1997, 50, 390. Chen, T.S.; Petuch, B.; MacConnell, J.; White, R.; Dezeny, G.; Arison, B.; Bergstrom, J.D.; Colwell, L.; Huang, L.; Monaghan, R.L.J. Antibiotics, 1994, 47, 1290. Carol, A.J.; Sidebottom, P.J.; Cannell, R.J.P.; Noble, D.; Rudd, B.A.M., J. Antibiotics, 1992, 45, 1492.

270

[47] [48]

[49] [50] [51] [521

[531 [541 [551 [56] [57]

[58] [59]

[60] [61] [62] [63]

[64] [65] [66] [67] [68] [69] [70] [711 [72] [73] [74] [75] [76]


Cannel, R.J.P.; Dawson, M.J.; Hale, R.S.; Hall, R.M.; Noble, D.; Lynn, S.; Taylor, N.L.d. Antibiotics, 1994, 47, 247. Tanimoto, T.; Hamano, K.; Onodera, K.; Hosoya, T.; Kakusaka, M.; Hirayama, T.; Shimada, Y.; Koga, T.; Tsujita, Y.J. Antibiotics, 1997, 50, 390. Qian, Y.; Sebti, S.M.; Hamilton, A.D. Biopolymers, 1997, 43, 25. Ruegger, A.; Kuhn, M.; Lichti, H.; Loosli, H.-R.; Huguenin, R.; Quiquerez, C.; Von Wartburg, A. Helv. Chim. Acta, 1976, 59, 1075. Rahfeld, J.-U.; Schierhom, A.; Mann, K.; Fisher, G. FEBS Lett., 1994, 343, 65. Wiederrecht, G.; Brizuela, L.; Elliston, D.; Sigal, N.; Siekierka, J. Proc. Acad. Sci. USA, 1991, 88, 1029. Tropschug, M.; Barthelmess, I.; Neupert, W. Nature, 1989, 342, 953. Liu, J.F.; Lane, J.D.; Friedman, J.; Weissman, I.; Schreiber, S.L.; Cell, 1991, 66, 807. Liu, J.; Albers, M.; Wandless, T.J.; Luan, S.; Alberg, D.A.; Belshaw, P.J.; Cohen, P.; MacKintosh, C.; Klee, C.B.; Sehreiber, S.L. Biochem., 1992, 31, 3896. Brown, E.J.; Albers, M.W.; Shin, T.B.; Ichickawa, K.; Keith, C.T.; Lane, W.S.; Schreiber, S.L. Nature, 1994, 369, 756. Go sio, B. Rivista Igiene Sanita Publica Ann., 1896, 7, 825. Noto, T.; Sawada, M.; Ando, K.; Koyama, K. d. Antibiotics, 1969, 22, 165. Ando, K.; Suzuki, S.; Tamura, G.; Arima, K. J. Antibiotics, 1968, 21, 649. Sweeney, M.J.; Gerzon, K.; Harris, P.N.; Holmes, R.E.; Poore, G.A.; Williams, R.H. Cancer Research,1972, 32, 1795. Sweeney, M.J.; Hoffman, D.H.; Esterman, M.A. Cancer Research, 1972, 32, 1803. Nelson, P.H.; Gu, C-L.L.; Allison, A.G.; Eugui, E.M.; Lee, W.A. US Patent 4,753,935, 1985. Nakanishi, K. Nature, 1963, 197, 292. McMorris, T.C.; Anchel, M.J.d. Am. Chem. Soc., 1963, 85, 831. Nakanishi, K.; Ohashi, M; Tada, M.; Yamada, Y. Tetrahedron, 1965, 21. 123 !. Maretic, Z.; Russell, F.; Golobic, V. Toxicon, 1975, 13, 379. French, A..L.; Garrettson, L.K. Clin. Toxic., 1988, 26, 81. McMorris, T.C.; Kelner, M.J.; Wang, W.; Moon, S.; Tactic, R. Chem. Res. Toxicol. 1990, 3, 574. Tanaka, K.; lnoue, T.; Tezuka, Y.; Kikuchi, T. Chem. Pharm. Bull., 1996, 44, 273. McMorris, T.C.; Kelner, M.J.; Wang, W.; Estes, L.A.; Montaya, M.A.; Taetle, R.J. Org. Chem., 1992, 5 7, 6876. Kelner, M.J.; McMorris, T.C.; Beck, W.T.; Zamora, J.M.; Tactic, R. Cancer Res., 1987, 47, 3186. MacDonald, J.R.; Muscoplat, C.C.; Dexter, D.L.; Mangold, G.L.; Chen, S.F.; Kelner, M.J.; McMorris, T.C.; Von Hoff, D.D. Cancer Res., 1997, 57, 279). McMorris, T.C.; Kelner, M.J.; Wang, W.; Yu, J.; Estes, L.A.; Taetle, R. d. Nat. Prod, 1996, 59, 896. McMorris, T.C.;Kelner, M.J.; Wang, W.; Diaz, M.A.; Estes, L.A.; Taetle, R. Experientia, 1996, 52, 75. Folkman, J. Adv. Cancer Res., 1985, 43, 175) (Folkman, J.; Klagsbrun, M. Science, 1987, 235, 442. Tarbell, D.S.; Carman, R.M.; Chapman, D.D.; Huffman, K.R.; McCorkindale, N.J.d. Am. Chem. Soc., 1960, 82, 1005.

BIOACTIVE FUNGALNATURALPRODUCTS

[77]

[78] [79]

[80] [81] [82] [83] [84]

[85] [86]

[871 [88] [89] [90] [91]

[92] [93] [94] [95] [96]

[97]

[98] [991

[100] [~0~] [~021 [103] [104] [105]

271

Langer. L.; Conn, H.; Vancanti, J., Maudenschild, C.; Folkman, J.; Proc. Natl. Acad. Sci., USA,, 1980, 77, 4331. lngber, D.E.; Folkman, J. J. Cel. Biol., 1989, 109, 317. Zimrin, A.B.; Villeponteau, B.; Maciag, T. Biochem. Biophys. Res. Commun. 1995, 213, 630. Ingber, D.E.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.; Breem, H.; Folkman, J. Nature, 1990, 348, 555. Folkman, J.; Fujita, T.; Ingber, D.; Kanamaru, T. U.S. Patent, 1992, 5135915. lngber, D.E.; Folkman, J. d. Cel. Biol., 1989, 109, 317. Griffith, E.C.; Su, Z.; Turk, B.E.; Chen, S.; Chang, Y.; Wu, Z.; Biemann, K.; Liu, J.O. Chem. Biol., 1997, 4, 461. Barrett, V.; Dixon, R. K.; Lemke, P. A. Appl. Microbiol. Biotechnol., 1990, 33, 313. Beck, J.; Ripka, S.; Siegner, A.; Schiltz, E.; Schweizer, E. Eur. J. Biochem., 1990, 192, 487. Bibb, M. J.; Sherman, D. H.; Omura, S.; Hopwood, D. A. Gene, 1994, 142, 31. Brown, D. W.; Yu, J.-H.; Kelkar, H. S.; Fernandes, M.; Nesbitt, T. C.; Keller, N. P.; Adams, T. H.; Leonard, T. J. Proc. Natl. Acad. Sci. USA, 1996, 93, 1418. Chang, P. K.; Cary, J. W.; Yu, J.; Bhatnager, D.; Cleveland, T. E. Mol. Gen. Genet. 1995, 248, 270. Cortes, J.; Wiesmann, K. E. H.; Roberts, G. A.; Brown, M. J. B.; Staunton, J.; Leadlay, P. F. Science, 1995, 268, 1487. Donadio, S.; Katz, L. Gene, 1992, 111, 51. Donadio, S.; MeAlpine, J. B.; Sheldon, P. A.; Jackson, M. A.; Katz, L. Proc. Natl. Acad. Sci. USA, 1993, 90, 7119. Donadio, S.; Staver, M. J.; McAlpine, J. B.; Swanson, S. J.; Katz, L. Science, 1991, 252, 675. Feng, G. H.; Leonard, T. J. J. Bacteriol., 1995, 177, 6246. Fernandez-Moreno, M. A.; Martinez, E.; Boto, L.; Hopwood, D. A.; Malpartida, F. d. Biol. Chem., 1992, 267, 19278. Fu, H.; McDaniel, R.; Hopwood, D. A.; Khosla, C. Biochemistry, 1994, 33, 9321. Hawksworth, D. L.; Rossman, A. Y. Phytopathology, 1997, 87, 888. Hondel, C. A. M. J. J. v. d.; Punt, P. J.; Gorcom, R. F. M. V. Heterologous gene expression in filamentous fungi. In More gene manipulations in fungi, J. W. Bennett, and L. L. Lasure, Eds.; Academic Press, Inc.: San Diego, 1991, pp. 396-428. Hopwood, D. A.; Khosla, C. In Secondary metabolites: their function and evolution, C. F. Symposium, Ed.; Chichester: Wiley, 1992, Vol. 17, pp. 88112. Hopwood, D. A.; Malpartida, F.; Kieser, H. M.; Ikeda, H.; Duncan, J.; Fujii, I.; Rudd, B. A. M.; Floss, H. G.; Omura, S. Nature, 1985, 314, 642. Hutchinson, C. R.; Fujii, I. Annu. Rev. Microbiol.,1995, 49, 201. Kao, C. M.; Katz, L.; Khosla, C. Science, 1994, 265, 509. Katz, L.; Donadio, S. dnnu. Rev. Microbiol., 1993, 47, 875. Keller, N. P.; Adams, T. H. SAAS Bull Biochem. Biotechnol., 1995, 8, 14. Khosla, C.; Ebert-Khosla, S.; Hopwood, D. A. Mol. Microbiol., 1992, 6, 3237. Khosla, C.; McDaniel, R.; Ebert-Khosla, S.; Torres, R.; Sherman, D. H.; Bibb, M. J.; Hopwood, D. A. d. Bacteriol., 1993, 175, 2197.


272

[1o6] Kim, E.; Cramer, K. D.; Shreve, A. L.; Sherman, D. H. J. Bacteriol., 1995, 177, 1202.

[107] Kimura, N.; Tsuge, T. Journal of Bacteriology, 1993, 175, 4427. [los] Langridge, R.; Eibel, H.; Brown, J. W. S.; Feix, G. The EMBO Journal, 1984, [109] [110] [Ill] [112] [ll3] [114]

IllS] [116]

[117] [118] [119] [120] [121] [1221 [123] [124] [125] [126] [1271 [1281 [1291 [130] [131] [1321 [133]

3, 2467. Malpartida, F.; Hopwood, D. A. Nature, 1984, 309, 462. Martin, J. F. J. Ind. Microbiol., 1992, 9, 73. Mayorga, M. E.; Timber lake, W. E. Mol. Gen. Genet., 1992, 235, 205. McDaniel, R.; Ebert-Khosla, S.; Fu, H.; Hopwood, D. A.; Khosla, C. Proc. Natl. Acad. Sci. USA, 1994, 91, 11542. McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Science, 1993, 262, 1546. McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Nature, 1995, 375, 549. Mooibroek, H.; Kuipers, A. G. J.; Sietsma, J. H.; Punt, P. J.; Wessr J. G. H. Mol. Gen. Genet., 1990, 222, 4 I. O'Hagan, D. The polyketide metabolites, J. Mellor, Ed.; Ellis Horwood: Chichester, 1991. Pieper, R.; Luo, G.; Cane, D. E.; Khosla, C. Nature, 1995, 378, 263. Punt, P. J.; Oliver, R. P.; Dingemanse, M. A.; Pouwr P. H.; Vanden Hondel, C. A. M. J. J. Gene, 1987, 56, 117. Rambosek, J.; Leach, J. CRC Crit. Rev. Biotech., 1987, 6, 357. Romanos, M. A.; Scorer, C. A.; Clare, J. J. Yeast, 1992, 8, 423. Smith, T. L.; Gaskell, J.; Berka, R. M.; Yang, M.; Henner, D. J.; Cullen, D. Gene, 1990, 88, 259. Takano, Y.; Kubo, Y.; Shimizu, K.; Mise, K.; Okuno, T.; Furusawa, I. Mol. Gen. Genet., 1995, 249, 162. Wang, I.; Reeves, C.; Gaucher, G. M. Can. J. Microbiol., 1991, 37, 86. Yang, G.; Rose, M. S.; Turgeon, B. G.; Yoder, O. C. Plant Cell., 1996, 8, 2139. Yu, J.; Leonard, T. J. J. Bacteriol., 1995, 177, 4792. Yu, T.; Hopwood, D. A. Microbiology, 1995, 141, 2779. Krishnarao, T.V.; Galgiani, J.N. Antimicrob. Agents Chemther., 1997, 41, 1957. Coey, E.J.; Guzman-Perez, A.; Noe, M.C.J. Am. Chem. Soc., 1994, 116, 12109. Harrison, W.; Shearer, H.M.M.; Trotter, J. J. Chem. Soc. Perkin II, 1972, 1542. Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.; Brem, H.; Folkman, J. Nature, 1990, 348, 555. Borel. J. F. Pharmacol. Rev., 1990, 41, 259. Thomson, A.W.; Whiting, P.H.; Simpson, J.G. Agents Actions, 1984, 15, 306. Pearce, C. Adv. Appl. Microbiol., 1997, 44, 1.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22 9 2000 ElsevierScience B.V. All rights reserved

273

THE CHEMISTRY OF 2-AMINOCYCLOPENTANECARBOXYLIC ee

ACID

ee

FERENC FULOP

Institute of Pharmaceutical Chemistry, Albert Szent-GyOrgyi Medical University, H-6701 Szeged, POB 121, Hungary, ABSTRACT: The syntheses, transformations and some of the biological features of 2aminocyclopentaneearboxylie acid are reviewed. The (IS,2R) enantiomer (cispentaein) was recently isolated from different natural sources, c i s - 2 - A m i n o e y c l o p e n t a c e e a r b o x y l i c acid is also a component of the antibiotic amipurimyein. The paper discusses the syntheses of the racemic compounds, resolutions of the racemates and enetioselective syntheses of the title compounds. The transformations to heteroeyelic compounds, applications in peptide syntheses, and biological effects are reviewed.

INTRODUCTION [~-Amino acids, although of less importance than their o~-analogues, are also present in peptides and different heterocycles, and their free forms and derivatives exhibit interesting pharmacological effects [ 1]. A number of syntheses and transformations have been performed on their stereoisomeric alicyclic analogues (e.g. 1-3). Until recently, the investigations were mainly of academic interest since no naturallyoccurring compounds were known. Among the 13-amino acid derivatives of cycloalkanes, one of the most exciting is (1R,2S)-2-aminocyclopentanecarboxylic acid (cispentacin), an antifungal antibiotic, recently isolated independently by two Japanese groups from Bacillus cereus [2] and Streptomyces setonii [3]. cis-2-Aminocyclopentanecarboxylic acid (cis-2-ACPC) is a component of the antibiotic amipurimycin reported by Goto et al. Amipurimycin (4) contains a nucleic base attached to the anomeric carbon of a branched-chain deoxy sugar. The chain is extended by a dipeptide containing the cis-2ACPC moiety. Amipurimycin, isolated from S t r e p t o m y c e s novoguineensis, is strongly active both in vitro and in vivo against Pyricularia oryzae, responsible for rice blast disease. It is also active in vitro against Alternaria kikuchiana and Helminthosporium sigmoideum var. irregulare [4-13]. Although the absolute configuration of the cis-2ACPC moiety has not been determined, it is probably similar to that of naturally-occurring cispentacin.

274

FERENC FIDLOP

~iOOH 1

~ vC

H~.

- NO H20 H

2

COOH

~ 8

NH2

oon NH

H O_

H0

H~O H

N~

NH2

4

Chart 1.

Besides the pharmacological importance of the alicyclic I]-amino acids, they can be used as building blocks for the preparation of modified (unnatural) analogues of biologically active peptides. By insertion of an alicyclic 13-amino acid in place of an t~-amino acid of a naturally-occurring pharmacologically active peptide, the activity or the effect can be modified. By means of such an exchange, the stability of the natural peptides can be increased. The difference in the ring size allows modification of the conformations of the peptides. Such investigations are applied for determination of the fine structures of receptors. Due to the natural occurrence and the novel biological activity, interest in investigations of alicyclic 13-amino acids has been aroused. A number of new enantioselective syntheses have been developed and protected by patents [ 14-26]. The writing of this review was prompted by the renewed interest in the title compound 1. The primary focus was its synthesis and some of its transformations. Besides 1, other alicyclic [3-amino acids, such as cis- and trans-2-aminocyclohexanecarboxylic acid (2), 2,3-diendo- and 2,3-diexo-3-aminobicyclo[2.2.1 ]heptane-2-carboxylic acid (3) and some of their partially unsaturated analogues and derivatives will be mentioned. The biological properties of these and related compounds will also be discussed. CISPENTACIN. ISOLATION AND CHARACTERIZATION In 1989-90 two Japanese research groups independently isolated a simple, unique [3-amino acid having the chemical structure (-)-(1R,2S)-2-ACPC (5). In the course of antifungal screening, cispentacin was isolated from the culture broth of a Bacillus cereus strain by Oki et al. [2, 27]. In parallel with that investigation, Hashimoto et al. isolated an identical substance

2-AMINOCYCLOPENTANECARBOXYLIC ACID

275

from Streptomyces setonii No.7562 [3, 28]. The isolation and purification were in both cases based on ion-exchange chromatography. C,O O H

Chart 2.

S

NH2

Cispentacin has an amphoteric character; it is readily soluble in water, slightly soluble in methanol, and insoluble in acetone or ethyl acetate. It gives a positive ninhydrin test. Its accurate mass spectrum shows that cispentacin has the molecular formula C6HIINO2. The most important spectral data are as follows: IR (KBr) cm -l, 295, 2870, 2680, 2550, 2200, 1645, 1550, 1415, 1335, 1310, 1170, 1070, 840 [2]. ~H NMR (400 MHz, D20) ~5, 1.70-1.89 (4H, m), 2.04-2.15 (2H, m), 2.87 (1H brq, J = 6.2 Hz), 3.73 (1H, brq, J = 6.2 Hz) [2]. ~3C NMR (100 MHz, D20) ~5, 22.0 (t), 28.8 (t), 30.2 (t), 48.4 (d), 53.7 (d), 181.7 (s) [2]. The absolute configuration was first described from a derivative. Cispentacin was treated with 1-(3-methylaminopropyl)-3-ethylcarbodiimide to give the corresponding [3-1actam, which exhibited a positive Cotton effect at 214 nm, indicating the (1R,2S) configuration of the antibiotic [2]. The absolute configuration was later proved by X-ray diffraction of its phenylalanine derivative [28]. A simple HPLC method was developed for the separation and identification of the (1S,2R), (1R,2S), (1S,2S) and (1R,2R) enantiomers of 2-ACPC by using pre-column derivatization with the chiral derivatizing reagents 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (Marfey's reagent) and 2,3,4,6-tetra-O-acetyl-~-D-glucopyranosyl isothiocyanate [29]. Analogue and homologue derivatives can also be detected by this method [30-33]. The two enantiomers of cispentacin can be determined in rat urine by reverse-phase HPLC after derivatization with Marfey's reagent [34]. Physicochemical data such as dielectric increments [35], partial molar volumes [36] and protonation and complex formation constants [37, 38], were earlier determined for racemic cis- and trans-2-ACPC. SYNTHESES OF 2-AMINOCYCLOPENTANECARBOXYLIC ACID

Syntheses of the Racemic Compound Racemic cis-2-ACPC was first prepared by Plieninger and Schneider, by Hofmann degradation of cis-2-carbamoylcyclopentanecarboxylic acid 10

276

FERENCFOLOP

with sodium hypobromite [39]. The synthesis of cis-cyclopentane-l,2dicarboxylic anhydride 9, necessary for the preparation of carboxamide 10, starts from the ethyl tetracarboxylate 6 by sodium ethylate ring closure, followed by hydrolysis and decarboxylation. In acetic anhydride, isomerization and ring closure take place, resulting in the cis-anhydride 9 (Scheme 1) [40, 41]. Anhydride 9 can be prepared alternatively by the reaction of acetic anhydride with cis-cyclopentane-1,2-dicarboxylic acid 8, prepared from cyclopent- 1-ene- 1,2-dicarboxylic acid [42, 43]. OOEt COOEt Na, EtOH COOEt COOEt 6

~ ~OOEt H2SO4 ~._ ~ i ( ~--COOEt ~.~--COOEt AcOH, H20,A COOEt

COOH OOH

7 A I Ac20

~COOH "~NH2 11 Scheme

_.~ H~ degrad.

~COOH ~'CONH2 I0

NHaOH ~'~ 9

O

1.

Ammonolysis of alicyclic anhydrides followed by Hofmann degradation is one of the most frequently used methods for the synthesis of alicyclic 13-amino acids, since many anhydrides are commercially available cheap substances produced by Diels-Alder addition of maleic anhydride and the corresponding dienes [44]. By this method, cis- and trans-2-aminocyclohexane- and -cyclohexenecarboxylic acids, cis-2aminocycloheptanecarboxylic acid [45], 3-endo-aminobicyelo [2.2.1 ]heptane-2-endo-carboxylic acid, 3-endo-aminobicyclo[2.2.1]hept-5ene-2-endo-carboxylic acid and some analogue ]3-amino acids have been prepared [44, 46-48]. For partially unsaturated alicyclic compounds, a modified Hofmann degradation is used, sodium hypochlorite being applied to avoid bromine addition to the double bond [48, 49]. Since Hofmann degradation affords a crude product containing a large amount of inorganic derivatives, ion-exchange chromatography is an excellent technique for desalting. The reaction of chlorosulfonyl isocyanate (CSI) and cyclopentene at -78 ~ yielded 2-chlorosulfonyl-2-azabicyclo[3.2.0]heptan-3-one (13), which was transformed to azetidinone 14 with potassium bisulfite. The

2-ANIN l OCYCLOPENTANECARBOX ACI YLD IC

277

resulting 13-1actam was hydrolysed with concentrated hydrochloric acid to give the cis amino acid hydrochloride 15 (Scheme 2) [43]. Although the free amino acid 11 was prepared from 15 on treatment with a large excess of silver oxide [43], ion-exchange chromatography was later found to be more suitable [2], use of the expensive silver oxide thereby being avoided.

(~ 12

CSI ,,

~

o

~ (

NaHSO3

(

NS02CI

13

14

a

~ C NO HO 2H 11 Scheme2.

I' C(iiilC . 15

~ C ONOHEt2 16

With ethanolic hydrogen chloride, the 13-1actam 14 gave the ethyl ester 16 [501. The 1,2-dipolar cycloaddition of chlorosulfonyl isocyanate to different cycloalkenes has become a well-known route for the synthesis of cycloalkane-fused 13-1actams, and for alicyclic 13-amino acids, after hydrochloric acid treatment. The addition takes places regio- and stereospecifically, in accordance with the Markovnikov orientation rule [51-55]. In this manner, a number of homologue and analogue alic.yclic [3amino acids have been prepared, such as c i s - 2 - a m l n o - 2 methylcyclopentanecarboxylic acid [56], cis-2-amino-2-methylcyclohexanecarboxylic acid [56], (1R*,2S*,4S*)2-amino-4-tert-butylcyclopentanecarboxylic acid [55, 58], 3-exo-aminobicyclo[2.2.1]heptane-2-exocarboxylic acid [59, 60] and 3-exo-aminobicyclo[2.2.1 ]hept-5-ene-2-exoearboxylie acid [61 ]. The following relatively long procedure also results stereospecifically in the racemic cis amino acid 11. The commercially available 5-hexen-1-ol (17) was transformed into the aldehyde 18 by Collins oxidation, and subsequent N-benzylhydroxylamine treatment gave the nitrone 19. Cyclization resulted only in the cis-fused isoxazolidine 20. Catalytic hydrogenolysis with Pd/C in acetic acid was effected by cleavage of the NO bond and removal of the protecting benzyl group (Scheme 3). Boc protection, Jones oxidation and removal of the protecting group gave the

278

FERENCFOLOP

desired 11 [62]. Although the yields of the individual steps are high, the overall yield of this process is rather low in consequence of the length of the process.

24% for quercetin aglycone > 17% for quercetin rutinoside [31 ]. Consequently, humans absorb considerable amounts of quercetin, but the absorption depends on the glycosidic nature of the flavonoid [31 ].

314

V A N D E N B E R G H E et as

ANTIOXIDANT ACTIVITY OF FLAVONOIDS

Free Radical Scavenging Activity Free radicals are reactive and therefore short-lived as a consequence of containing one or more unpaired electrons. However, there is still a considerable difference in their reactivities and lifetimes [70]. 02 ~ reacts quickly with only a few molecules, whereas OH" is so unstable that it reacts at the site of formation. ROO 9 and RO 9 have intermediate reactivities [40]. Their relatively short lifetimes and migration distances complicate the measurement of free radicals. Free radicals can be generated in vitro either enzymatically, e.g. by the xanthine/XO system, or nonenzymatically, e.g. by the action of transition metals or ionizing radiation. Flavonoids can interfere both with the formation and propagation reactions of free radicals [27]. To act as an antioxidant, a free radical scavenger should produce a more stable and therefore less harmful compound after reaction with a free radical. In general, flavonoids are excellent hydrogen- or electron-donors and the resulting flavonoid radical is relatively stable due to electron delocalization and intramolecular hydrogen bonding [71]. 02 ~ Scavenging Activity

02*- is a ROS produced in vivo by a number of enzyme systems, by autooxidation reactions, and by non-enzymatic electron transfers [33]. Hydroperoxyl radical (HO2o), which is much more lipid-soluble than O2o-, can be formed in acid medium by protonation of 02 ~ [35]. Many studies demonstrated the O2"- scavenging activity of flavonoids and tried to determine structure-activity relationships. In most studies either the phenazine methosulfate-NADH system [72-76] or the xanthine/XO system [16, 77-79] was used to produce 02"-. The non-enzymatic phenazine methosulfate-NADH system [80] generates a myriad of free radicals and other ROS, including OH', H202, and 02 ~ [81]. Consequently, this method is not recommended [35]. Another source of O2~ is the enzyme XO that catalyzes the oxidation of hypoxanthine and xanthine to uric acid. During re-oxidation of XO, molecular oxygen acts as electron acceptor, producing 02"- and H202 [82]. These reactions can be written as follows (Eq. 4-5) [78]: xanthine + 202 + H20 xanthine + 02 + H20

XO = uric acid + 202 ~ + 2H § (Eq. 4) XO ~ uric acid + H202 (Eq. 5)

STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS

315

It is important to consider that flavonoids can inhibit XO. Those containing C-5 and C-7 OH groups are especially involved, so that an overestimation occurs for the 02"- scavenging activity of these compounds [83-86]. This can be checked by measuring the formation of uric acid (spectrophotometrically or by HPLC) [38]. To detect O2"- most studies used the indirect NBT assay [16, 72-75]. In this method O2"-reduces the yellow dye nitro-blue tetrazolium (NBT 2§ to produce the blue formazan, which is measured spectrophotometrically at 560 nm [87]. Cotelle et al. [77, 79] and Cos et al. [78] investigated the O2"- scavenging activity of flavonoids using respectively chemiluminescence with lucigenin and the nitrite method. Only Sichel et al. studied the 02"- scavenging activity of flavonoids by a direct measurement of the reduction of the 02"- electron spin resonance (ESR) signal intensity, which was generated by the addition of acetone to H202 in alkaline medium [88]. This method has the disadvantage of using media with high and therefore with a nonphysiological pH. All studies conclude that an ortho-dihydroxy (catechol) structure in the B-ring is important for a high O2"- scavenging activity [ 16, 72, 74-79, 88]. Baumann et al. [72] and Huguet et al. [74] found that the scavenging activity of flavones and flavanones decreased in the following order: an ortho-dihydroxy function in the B-ring > a 4'-OH substitution > no OH groups in the B-ring. According to Huguet et al. [74], this was not the case for flavonols, since he found - in contrast to Baumann et al. [72] and Cos et al. [78] - that galangin was a better O2"- scavenger than quercetin. Huguet et al. also demonstrated that a resorcinol moiety in ring A increased the scavenging activity and that flavones were slightly more active than flavanones [74]. Cotelle et al. observed that the O2"- scavenging activity was favored by the presence of a 6-OH group [77, 79]. Interestingly, there is no unanimity about the role of the C-3 OH group regarding the 02"- scavenging activity. Sichel et al. [88] and Cos et al. [78] found a higher activity for flavonols compared to related flavones, whereas Huguet et al. [74] found the opposite. According to Baumann et al., there was no relationship between the presence of a C-3 OH group and the 02"scavenging activity [72]. Replacing the active OH groups by one or more methoxy groups decreased the activity [72, 74, 77], whereas the activity increased by introducing a gallate structure [76]. The presence of a sugar moiety attached to the flavonoid nucleus affects the activity. Aglycones were more active than their glycosides, but for quercetin and rutin (quercetin 3-rutinoside) contradictory results have been reported [73, 74, 88]. Yuting et al. [75] and Huguet et al. [74] described a higher activity for rutin, whereas Sichel et al. [88] demonstrated a higher activity for quercetin. Robak and Gryglewski [73] and Baumann et al. [72] observed no significant difference in activity between these compounds.

316

VANDEN B E R G H E et al.

OH" Scavenging A cavity The most common method to produce the highly reactive OH" is the socalled Fenton reaction, which was first observed by Fenton in 1894 (Eq. 6) [89]. Fe 2§ + H202 --> Fe 3§ + OH- + OH" (Eq. 6) However, there is still doubt whether some or even all of the OH" produced from the Fenton reaction may remain bound to the iron center, either as the FeOH 3+ (Eq. 7)or the FeO 2§ intermediate (Eq. 8) [90-91]. Fe 2+ + H202 "-~ FeOH 3§ + OH- (Eq. 7) Fe 2+ + H202 ~ FeO 2+ + H20 (Eq. 8) The Fenton reaction was used by Yoshiki et al. [92] and Puppo [93], whereas Husain et al. [94] generated OH" by UV photolysis of H202. It should be considered that several flavonoids chelate transition metals and therefore inhibit the production of OH- generated in the Fenton reaction. Different methods were applied to detect OH'. Husain et al. [94] spin trapped the OH" by 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and measured these radicals by the highly sensitive HPLC-electrochemical detection method [95]. The technique of spin-trapping involves the addition of a free radical to a diamagnetic compound (the spin-trap) to form a more stable radical adduct [96]. The intensity of chemiluminescence was observed by Yoshiki et al. [92] as a measure for OH. scavenging activity, whereas Puppo [93] detected the OH. either by the deoxyribose assay (see also section pro-oxidant activity of flavonoids) or by aromatic hydroxylation of phenylalanine. In aromatic hydroxylation OH-attack a specific detector, in this case phenylalanine, resulting in the production of three isomeric tyrosines. These hydroxylated products can be separated by HPLC equipped with a highly sensitive electrochemical detector [97]. Only a few studies are published concerning the OH- scavenging activity of flavonoids. In accordance with the 02 ~ scavenging activity, three studies reported an increase of the OH" scavenging activity with an increasing number of OH groups in the B-ring [92-94]. However, this was not confirmed by Hanasaki et al. [16] and Laughton et al. [32], who observed that e.g. quercetin could enhance OH" production and could therefore act as a pro-oxidant (see also section pro-oxidant activity of flavonoids). Apparently, the OH" scavenging activity varies with the method applied. The introduction of a C2-C3 double bond and/or a C-3 OH group had no significant effect on the activity [92, 94], whereas according to Husain et al. [94] the presence of a carbonyl function at C-4 appeared to play an important role. Husain et al. reported that the most


317

active flavonoids (myricetin and quercetin) were better OH" scavengers than ethanol and 1-butanol, but less active than DMSO [94].

RO0" Scavenging Activity ROO 9are formed in vivo during the major chain-propagation step in lipid peroxidation, but they can also be formed from DNA and proteins [37]. The ROO 9scavengers can be divided into water-soluble scavengers, e.g. scavengers of ROO 9resulting from DNA or proteins, and lipid-soluble scavengers, e.g. chain-breaking antioxidants [37]. Torel et al. [98] studied the ROO 9scavenging activity of flavonoids from the inhibition of the formation of hydroperoxide isomers during auto-oxidation of linoleie acid and methyl linoleate [99]. The level of hydroperoxides was evaluated by measuring the UV-absorption of conjugated dienes at 234 nm. The ROO ~ scavenging activity increased in the following orders" cateehin < quercetin < rutin = luteolin < kaempferol < morin for linoleic acid; rutin < catechin < morin = kaempferol for methyl linoleate [98]. A common method to generate ROO" is the use of azo-initiators (R-N=N-R), which decompose at a temperature-controlled rate, yielding molecular nitrogen and two carbon radicals (R~ (Eq. 9) [ 100]. The R ~ can react rapidly with molecular oxygen to produce ROO ~ (Eq. 10). R-N=N-R --) R~ + N2 + R ~ (Eq. 9) R 9+ 0 2 ~

R O O ~ (Eq.

10)

The solubility and the rate of decomposition both depend on the structure of R [100]. 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) is a water-soluble radical generator, whereas 2,2'-azobis(2,4dimethylvaleronitrile) (AMVN) is lipid-soluble. The oxygen radical absorbing capacity (ORAC) assay is based on the generation of ROO ~ by AAPH and the detection of the chemical damage to the indicator protein ~l-phycoerythrin (]3-PE), which is monitored by a decrease in its fluorescence emission [101-102]. The inhibition of the fluorescence loss, which is expressed in ORAC units, is related to the antioxidant ability to protect ~-PE against free radical damage. One ORAC unit is equivalent to the protection provided by 1 IxM Trolox, a watersoluble vitamin E analog [101 ]. Flavones with a single OH group had a lower activity than Trolox, whereas kaempferol, quercetin, and myricetin had respectively ORAC values of 2.7, 3.3, and 4.3 [29]. Methylation of all the OH groups of luteolin resulted in an undetectable ORAC value [29]. Cao et al. concluded that a catechol moiety on ring B is essential for a high ROO 9 scavenging activity and that this activity is proportional to the number of OH groups [29].

318


Wang and Zheng [103] studied the ROO 9scavenging activity of several flavonoids by AAPH-initiated auto-oxidation of linoleic acid in cetyl trimethylammonium bromide micelles. The activity decreased in the following order: {x-tocopherol (a well-known chain-breaking antioxidant) > morin > rutin > quercetin. Naringin (naringenin 7-O-neohesperidoside) and hesperidin (hesperetin 7-O-rutinoside) had no significant activity. According to Wang and Zheng, this was due to the blockade of the 7-OH group - which is most likely the ROO 9target site - with a glycoside [103]. Terao et al. [104] and Ioku et al. [105] investigated the ROO ~ scavenging activity of flavonoids by measuring the inhibition of an azo compound-induced peroxidation of methyl linoleate in a solution of nhexane, isopropanol, and ethanol [106]. According to Terao et al., the rate constants for the ROO ~ scavenging activity decreased in the following order: o~-tocopherol > quercetin > epicatechin and epicatechin gallate [104]. loku et al. demonstrated the effect of glycosylation on the ROO 9 scavenging activity [105]. All quercetin monoglucosides had a lower ROO ~ scavenging activity than the aglycone. However, it appeared that the position of the glucosyl moiety on the flavonoid was important for both the ROO 9scavenging activity and the antioxidant mechanism. The 7-OH position appeared to affect the stability of trapped radicals, resulting in a decrease of chain-breaking activity, whereas an ortho-dihydroxy structure in the B-ring and to a lesser degree the 3-OH group were essential for the ROO 9scavenging activity of flavonoids [ 105]. Terao et al. [104] and Ioku et al. [105] also examined the hydroperoxide formation from liposomal phosphatidylcholine, which contained the chelator diethylenetriaminepenta-acetic acid (DETAPAC) for preventing the pro-oxidant effect of traces of iron, by exposure to the water-soluble radical initiator AAPH. Terao et al. demonstrated that - in contrast to the rate constants for the ROO 9scavenging activity - the effectiveness of epicatechin and epicatechin gallate in membrane phospholipids was comparable to that of quercetin, but much higher than observed for {xtocopherol, o~-Tocopherol is located within the membranes, whereas flavonoid aglycones interact with the polar head of phospholipid bilayers, being located near the surface of membranes [107]. This location may be favorable for scavenging of ROO" originating from the aqueous phase [104105].

DPPH Scavenging A ctivity Several studies described the interaction of flavonoids with the 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical [76, 79, 107-108] (Fig. (3)). DPPH is a "stable" free radical and is frequently used in ESR studies [109]. The DPPH assay provides information on the reactivity of flavonoids with a stable free radical [79]. Because of its odd electron, DPPH gives a strong absorption band in ethanol at 517 nm. As this

STRUCTURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS

319

electron becomes paired off, the absorption vanishes and the degree of resulting decolorization is stoichiometric with respect to the number of N{C6Hs}2 I *N O2N " ~y NO2

NO2 Fig. (3). Structure

of the I,l-diphenyl-2-picrylhydrazyl (DPPH) free radical.

electrons taken up [109]. Cotelle et al. demonstrated that a catechol or a pyrogallol type moiety on the B-ring was essential for the DPPH radical scavenging activity [79]. Ratty et al. studied the flavonoid-DPPH interaction in Tris/NaCl buffer (pH=7.4) and in liposomal suspension [107]. The DPPH scavenging activity decreased in the following orders: OH

OH

OH

OH HO

~

O

HO

i"i~

/ OH

",s OH

OH OH I C~-O

(-)-Epicatechln

Ho.2 OH

OH

OH Gallic acid

X

HO

OH ,,,pOH

OH OH

OH

(-)-Eplcatechin gallate

H O i_i i i ~l ( ~_0.

~

OH OH

o

--

"*e

OH

OH (-}-Eplgallocatechln Fig. (4). Structures

OH

{-}-EplgaIlocatechin gallate of some flavanols and flavanol-gallate esters.

-\

OH

320

VANDEN BERGHE et at

quercitrin = quercetin > morin > rutin = myricetin > taxifolin > catechin for the buffer, and morin > quercetin = rutin > myricetin > quercitrin > catechin > taxifolin for the liposomal suspension [107]. Rekka and Kourounakis examined the interaction of five hydroxyethyl rutosides and quercetin with the DPPH free radical and found quercetin and 7-monohydroxyethyl rutoside to be more active than di-, tri- and tetrahydroxyethylated rutosides [108]. According to Chen and Ho, the DPPH radical scavenging activity of some flavanols (see Fig. (4) for structures) decreased in the following order: epigallocatechin gallate > epicatechin gallate > epigallocatechin > epicatechin [76]. Free Radicals and Pulse Radiolysis

Pulse radiolysis is a unique technique to generate specific radicals and to study the kinetic parameters for the primary radical attack, the stability of the secondary formed radicals, and the redox potentials of these secondary formed radicals [110]. In pulse radiolysis a short pulse of ionizing radiation will generate excited states, ions, and radicals in a sample solution [111]. In aqueous solutions a short electron pulse will result within 1 nanosecond (10 -9 sec) in the formation of the following primary radicals (Eq. 11) [112]:

H20 - - - / ~ - - - -

e-aq + H" + OH* (Eq. 11)

The radicals e-aq (hydrated electron) and H. (hydrogen atom or hydrogen radical) are reducing species, whereas the OH* is an oxidizing agent I112]. It is possible to 'select out' a particular radical by alterations of pH and addition of various compounds [35]. One method of generating almost exclusively OH* is irradiation of an aqueous solution, saturated with nitrous oxide gas (N20), converting e-aq into extra OH* (Eq. 12-13) [112]. e-aq + N 2 0 --4 N2 + O -

(Eq. 12)

O-+ H + - ) OH-(Eq. 13) In an oxygenated formate (HCOO-) solution the following reactions occur to produce 02" (Eq. 14-17) [35, 112]. e-aq + 02 --) 02"- (Eq. 14) H" + 02 --) 02"- + H + (Eq. 15) OH" + HCOO- --) CO2"- + H20 (Eq. 16) CO2 ~ + 02 ---) C O 2 + 02 ~

(Eq. 17)


321

Azide radicals (N3") are produced by reaction of radiolytieally generated OH" with azide anions (N3-) (Eq. 18) [ 113]. N3- + OH" --r N3~+ OH- (Eq. 18) The application of N3~ in pulse radiolysis studies includes several advantages: (1) the low absorption of N3 ~ facilitates optical pulse radiolysis experiments, (2) the non-ionic character of N3" excludes effects of charge and dependence on ionic strength, and (3) in contrast to OH', N3~ react primarily via electron transfer, thereby simplifying the secondary chemistry [113]. Bors et al. used these electrophilic N3" to produce flavonoid aroxyl radicals, whose generation rates and stability were determined [114-117]. The stability of the flavonoid aroxyl radical prevents radical chain reaction and defines the extent of flavonoid activity as antioxidant [116]. It was shown that quercetin and kaempferol have good radical scavenging activities, but only the quercetin aroxyl radical decays slowly enough (more than 40 times slower compared to kaempferol) to act as a potential antioxidant [ 116]. According to Bors et al., three structural elements are required for optimal radical scavenging activity [ 115-117]: (1)

(2) (3)

An ortho-dihydroxy structure (catechol moiety) in the B-ring, which is the radical target site for flavonoids with a saturated C-2 and C-3 double bond, confers a high stability to the flavonoid aroxyl radical and participates in electron delocalization. A 2,3-double bond in conjugation with a 4-oxo function is responsible for electron delocalization from the B-ring. The 3- and 5-OH groups are, together with the 4-oxo function, essential for maximal radical scavenging activity. In the absence of a C2-C3 double bond, the presence of an aliphatic 3-OH group does not contribute to the radical scavenging activity [ 117].

Bors et al. also investigated the reaction rate constants of flavonoids with different radical species and found for quercetin the following values: 43xl 08 M -1 sec-I with OH', 0.9x105 M -! see-1 with 02"- and ranging from 107 until 108 M -! sec-l with different ROO- [ 116, 118]. Jovanovic et al. studied the acid-base and redox properties of the flavonoid aroxyl radicals by pulse radiolysis of aqueous solutions [119]. The flavonoid aroxyl radicals were generated by bromide radical ion (Br2"-) induced oxidation of flavonoids (F-OH) (Eq. 19), followed by a rapid loss of a proton to form the neutral flavonoid aroxyl radical (F-O') (Eq. 20). F-OH + Br2"- --->F-OH "+ + 2Br-(Eq. 19) F-OH .+ --->F-O. + H + (Eq. 20)

322

V A N D E N B E R G H E et a2

The absorption spectra of the flavonoid aroxyl radicals resembled those produced by the action of N3 9[114-117]. The dissociation constants (pKa) of the 3',4'-dihydroxyflavonoid aroxyl radicals were similar to those of the 3,4-dihydroxybenzoate and the 3,4-dihydroxycinnamate radicals, namely ranging from 4 to 5. Consequently, at physiological pH the 3',4'dihydroxyflavonoid aroxyl radicals are negatively charged which can hinder their passage through the cell membranes. The reduction potentials of flavonoids depend strongly on the electron-donating properties of the substituents of the B-ring. Hesperidin aroxyl radical had a reduction potential of 0.72V, whereas the aroxyl radicals of catechin and rutin had reduction potentials of respectively 0.57V and 0.60V. According to the low reduction potentials of the flavonoid radicals, it is expected that they can efficiently scavenge ROO 9and 02 ~ [119]. Their rate constants with 02 ~ were determined by kinetic conductivity at pH=10 and by optical pulse radiolysis at pH=7. At pH=10, the flavonoids were ionized, in either the A- or B-ring, and their reaction with 02 ~ resulted in the reduction of 02" to 022- and the formation of the flavonoid aroxyl radical (Eq. 21). O2"- 4- F-O- --> 022- + F-O ~ (Eq. 21)

The rate constants were in the following increasing order: 8.8xl 02, and 5.1• M -1 sec -l for respectively galangin, kaempferol, quercetin, and rutin [119]. These values confirm the importance of a catechol moiety for O2o- scavenging activity (see also section O2~ Scavenging Activity). According to Jovanovic et al., the reactivity of flavonoids with O2~ depends on their charge. Uncharged catechin reacted at pH=7 four times faster than negatively charged catechin at pH-10. Nevertheless, flavonoids can scavenge O2"- at pH values ranging from 7 to l0 [119]. Trichloromethylperoxyl radicals (CC1302~ are frequently used to study the ability of compounds to scavenge ROO ~ [ 120]. CC1302 ~ were generated by radiolysis of an aqueous mixture of propan-2-ol and CCI4. Flavonoids showed variable rate constants: 108 M -l see -~ for fisetin and morin, 107 M -l sec -! for myricetin and quercetin, and 106 M -l see -l for catechin and epicateehin [ 121 ].

2.4x103, 4.7•

Free Radicals and Electron Spin Resonance (ESR) ESR is a spectroscopic technique for studying paramagnetic molecules or molecules with unpaired electrons, which include free radicals and many transition metals [ 122]. An unpaired electron can align itself in an external magnetic field either parallel or antiparallel to that field, yielding two possible energy levels. Application of electromagnetic radiation of the proper energy will induce transitions and an ESR signal will appear. The


323

basic ESR line of an electron can "split" into two or more "hyperfine" lines as a consequence of the interaction of the unpaired electron with nuclei having magnetic moments (e.g. IH, 2H and 13C). The distance between two lines in the ESR spectrum is called the hyperfine coupling constant. Hyperfine splitting can greatly enhance the identification of free radicals through analysis of their ESR spectrum [122]. Van Acker et al. investigated the antioxidant activity of flavonoids by quantum chemical calculations together with experimental parameters, such as half peak oxidation potentials (Ep/2) and ESR data [123]. Calculations of the geometry of several flavonoids indicated that the structure of a flavonol aglycone was planar, i.e., complete conjugation, in contrast to the flavones and the flavonol derivative rutin. The lack of a 3OH group in a flavone and the resulting loss of the corresponding hydrogen bond caused a slight twist (+ 20%) between the B-ring and the rest of the molecule. The introduction of a sugar moiety on the 3-OH group of a flavonol caused the loss of eoplanarity of the B-ring with the rest of the molecule and could therefore explain the lower scavenging activity of rutin compared to quercetin [123]. Consequently, the planarity of a flavonoid is related to its scavenging activity and a free 3-OH group is required for high activity. Actually, a free 3-OH group interacts with the B-ring through a hydrogen bond, holding the B-ring in the same plane as the rest of the molecule. The difference in heat of formation (AAHf) between the flavonoid molecule and its corresponding radical is a suitable parameter to describe the abstraction of a H 9from an O-H bond and is well correlated for the flavonols with their Ep/2. Studies of the spin distributions of quercetin and taxifolin showed that most spins remained in the B-ring. More precisely, 84% of the spins were located on the 4'-oxygen. However, spin densities and ESR hyperfine coupling constants both measured and calculated indicated that the delocalization was larger in the flavonol quercetin than in the dihydroflavonol taxifolin [ 123]. Cotelle et al. [77, 79] and Kuhnle et al. [124] investigated by ESR the ability of flavonoids to form stable radicals. The flavonoid radicals were generated by aerial, alkaline oxidation of the corresponding flavonoids. It was shown that flavonoids with a pyrogallol or catechol moiety in the Bring gave rise to semiquinone or pyrogallol-type anion radicals which were stable enough to be detected by ESR.

Trolox Equivalent Antioxidant Capacity (TEAC) Miller and Rice-Evans have recently developed a method to measure the total antioxidant capacity of solutions of pure compounds, as well as plasma, serum and other body fluids, plant extracts, beverages, etc. [125126]. This method is based on the inhibition of the absorbance of the 2,2'azinobis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS o§ by antioxidants. Although the chromophore ABTS o§ has

324

VANDEN BERGHE et as

characteristic absorption maxima at 417, 645, 734, and 815 nm, the suppression of the absorbance is quantified at 734 nm, because at this wavelength myoglobin and many other compounds do not interfere [ 127]. The relatively long-lived radical cation ABTS ~ is formed through the interaction of ABTS with the ferrylmyoglobin radical ('X-Few=O) (Eq. 23) [ 126], which is derived from the oxidation of metmyoglobin (HX-Fe m) by H202 (Eq. 22) [128]. HX-Fe m + H202 ~ "X-Fdv=o + H20 (Eq. 22) 9X-Few=O + ABTS ~ HX-Fd n + ABTS "+ (Eq. 23) The Trolox Equivalent Antioxidant Capacity (TEAC) assay reflects the relative ability of antioxidants ( h y d r o g e n - o r electron-donating compounds) to scavenge the ABTS o§ generated in the aqueous phase, compared to the antioxidant standard Trolox [127]. The TEAC is defined as the millimolar concentration of Trolox having an antioxidant capacity equivalent to 1 mM of the test compound [127]. However, according to Strube et al. the decrease in absorbance of ABTS o+measured at a fixed time could be originated from a scavenging effect and/or a decrease in the rate of ABTS -+ formation [129]. It was shown that Trolox scavenges ABTS "+, whereas quercetin acts by both mechanisms [129]. By using the TEAC assay, Rice-Evans et al. established a structureantioxidant activity relationship of flavonoids [28, 130-134]. The antioxidant activity of a flavonoid is determined by the following structural elements [28, 130-134]:

(1)

(2)

(3)

A catechol moiety in the B-ring is essential for a high antioxidant activity, whereas the presence of a pyrogallol moiety or a single OH group in the B-ring reduces the activity. The TEAC values were 4.72, 3.12, 2.55, and 1.34 mM for respectively quercetin, myricetin, morin, and kaempferol. The C2-C3 double bond enhances the activity by stabilization of the flavonoid aroxyl radical through electron delocalization across the molecule. Taxifolin (dihydroquercetin) had a TEAC value of 1.9 mM compared to 4.72 mM for quercetin. In the absence of a catechol moiety, the C2-C3 double bond has no significant influence on the antioxidant activity. The TEAC values of kaempferol (1.34 mM) and dihydrokaempferol (1.39 mM) were quite similar, because of the low hydrogen-donating capacity of a monophenolic B-ring. A free 3-OH group is required to get the maximal antioxidant activity out of a flavonoid with a catechol moiety in the B-ring and a C2-C3 double bond, such as quercetin. Blockade of the 3-OH group by introducing a glycoside reduces the antioxidant activity, which was showed by a TEAC value of 2.4 mM for rutin compared to 4.72


(4)

(5)

325

mM for quercetin. Removing the 3-OH group from quercetin led to a lower TEAC value of 2.1 mM for luteolin. The 3-OH group has no influence on the activity when there is no ortho-dihydroxy structure in the B-ring present. The TEAC values were quite similar for apigenin (1.45 mM) and kaempferol (1.34 mM). The 5,7-dihydroxyphenolic A-ring contributes to the antioxidant activity with a TEAC value ranging from 1.35 to 1.5 mM. Glycosylation of the 7-OH group has a negative influence on the TEAC values, like naringenin (1.5 mM) and hesperetin (1.37 mM) compared to respectively their glycosides narirutin (naringenin 7-Orutinoside) (0.76 mM) and hesperidin (1.08 mM). The flavanol catechin, which lacks the C2-C3 double bond together with the 4-oxo function in the C-ring, had a TEAC value of 2.4 mM, whereas epigallocatechin had a TEAC value of 3.82 mM. It appears that when there is no conjugation between the A- and B-ring via the C-ring, the insertion of a third adjacent OH group in the B-ring enhances the antioxidant activity. Another possibility to increase the antioxidant activity is the introduction of a gallic acid moiety by ester linkage via the 3-OH group (epicatechin gallate) giving a TEAC value of 4.93 mM.

These results are in agreement with the structure-activity relationship proposed by Bors et al., who used pulse radiolysis to study the antioxidant activity of flavonoids [116-118].

Quenching of Singlet Oxygen Singlet oxygen ( I O 2 ) is an excited state of 0 2 where spin restriction is removed. There are two types of singlet oxygen, namely the very shortlived sigma singlet oxygen (lEg§ and the, because of its longer life-time, biologically more important delta singlet oxygen (IAgO2) [35]. In IAgO2 the two outer electrons occupy the same orbital, whereas in leg+ they occupy separate orbitals [42]. IO2 is formed in vivo via photosensitized reactions or by chemical excitation reactions which do not require light excitation [35]. Flavonoids can interact with IO2 in two different ways: they can combine chemically with ~O2 or they can transfer the excitation energy of tOE to the flavonoid molecule, which then enters an excited state. The latter phenomenon is called quenching [35]. Flavonoids have been reported to quench singlet oxygen [ 17, 135-137], but only Toumaire et al. tried to establish a structure-activity relationship [17]. Sorata et al. studied the suppression of photosensitized hemolysis of human erythrocytes by the flavonols quercetin and rutin [135]. At lower concentrations, the effect of quercetin was more important than that of rutin [135]. According to Takahama et al., quercetin suppressed JOE-dependent photobleaching of

326

VANDEN B E R G H E et al

crocin in the presence of rose bengal as photosensitizer [136]. This was accompanied by the transformation of quercetin into a compound with an absorption maximum at approximately 330 nm. Devasagayam et al. studied the protective role of flavonoids in inhibiting single-strand breaks in plasmid pBR322 DNA induced by IO2, which was generated by thermal dissociation of the endoperoxide of 3,3'-(1,4naphtylene)dipropionate [137]. In this assay, all flavonoids tested protected the DNA mainly by chemical reaction or quenching of IO2, whereas quercetin also might react with DNA. The protective ability of the flavonoids decreased in the following order: myricetin > catechin > rutin > fisetin > luteolin > apigenin > quercetin (which showed no protection) [137]. At equimolar concentration (100 ~tM) myricetin showed a better protective effect than ct-tocopherol. However, increasing the concentration of myricetin did not increase its protective effect [137]. Toumaire et al. produced ~O2 by photosensitization with rose bengal [17]. The rate constants of the chemical reaction of flavonoids with IO2 and their rate constants of ~O2 quenching were determined by kinetic measurements and near-IR luminescence of IO2 [ 17]. Flavones were much more reactive than the corresponding flavonols, whereas flavanones, dihydroflavonols, and flavanols were chemically inert towards IO2 [17]. These results were explained by the dioxetane formation of IO2 with the C2-C3 double bond of the flavonoid molecule [17]. However, it is important to consider that a five-membered cyclic peroxide intermediate was proposed for the chemical reaction of 3-hydroxyflavone with IO2 [138]. The dioxetane formation of a flavonoid is activated by an electrondonating substituent in position 3 [35]. Dioxetanes are unstable and decompose into two carbonylated fragments. In conclusion, the efficiency of the chemical reactivity between flavonoids and IO2 is greatly affected by the structure of ring C" the presence of a OH group in the 3-position increases the reactivity of the C2-C3 double bond towards IO2. This was confirmed by the fact that glycosylation of the 3-OH (e.g. rutin compared to quercetin) decreased the chemical reactivity [17]. The degree of quenching of ~O2 decreased in the following order: catechin > fisetin > quercetin = rutin > luteolin > taxifolin [ 17]. Catechin was the most efficient quencher with a rate constant of 5.8xl 06 M -l see-~, being 3 to 5 times higher than the other flavonoids measured. This might be due to the absence of a carbonyl group in ring C of catechin, resulting in a less planar structure [17]. The most important structural element for efficient IO2 quenching by flavonoids is the presence of a catechol moiety on ring B [17]. Chelation of Transition Metals

The transition metals iron and copper are essential cofactors of several enzymes which are involved in oxygen metabolism. Approximately two-


327

third of the human body's iron is found in hemoglobin, with smaller amounts in myoglobin, various enzymes (e.g. catalase, cytochrome P450, ...), the iron carder proteins transferrin and lactoferrin, and the iron storage proteins ferritin and hemosiderin [39]. Copper is present in several enzymes, such as Cu, Zn-SOD and dopamine-I]-hydroxylase. In plasma it is mainly attached to ceruloplasmin, a large protein containing six or seven copper ions per molecule, and to a minor extent to the plasma protein albumin [139]. When these transition metals are present in free state in biological systems, they can catalyze free radical reactions. For example, iron and copper act as catalysts in the generation of OH" through the Fenton reaction (see section OH" Scavenging Activity) and the HaberWeiss reaction. The Haber-Weiss reaction or superoxide-driven Fenton reaction is an interaction between H202 and 02 ~ in the presence of traces of iron or copper, and results in the formation of OH" (Eq. 24-26) [35]. Fe 3+ + O2"- ~ Fe 2+ +

02

(Eq. 24)

Fe 2§ + H202 ~ Fe 3§ + OH- +OH" (Fenton reaction) (Eq. 25)

O2"- + H202 ---) 02 § OH- + OH ~ (Haber-Weiss reaction) (Eq. 26) Traces of transition metals accelerate also auto-oxidation reactions and the decomposition of LOOH to LOO o, LO o, and cytotoxic aldehydes [139]. Agents that complex these transition metals decrease their biological effects dramatically [36]. Flavonoids possess the ability to form a complex with Cu 2§ ions [140141]. Hudson and Lewis investigated this Cu2§ activity by studying their UV spectra [140]. The 4-carbonyl group in cooperation with either the 3-OH group or the 5-OH group was responsible for the complexation of Cu 2§ with flavones and flavanones. This was confirmed by the formation of a weak complex with the flavanol catechin [140]. According to Thompson et al., flavonoids chelated Cu 2§ ions mainly at neutral and high pH values, and to a minor extent at low pH values [ 141 ]. Actually, the degree of Cu 2§ complexation - but not the stability - is related to the basicity of the flavonoid molecule. Quercetin was found to form the most stable complex with Cu 2§ 5-Hydroxyflavone formed a slightly more stable complex than 3-hydroxyflavone. In fact, the sixmembered chelate ring of the former had a higher stability compared to the five-membered ring of the latter [141 ]. The relative chelating capacity of a series of flavonoids was studied spectrophotometrically by measuring the ability to release Fe 2§ ions from a Fe2§ complex and to chelate Fe 2§ ions [27]. The flavonoids were classified into four groups" (1) flavonoids, such as apigenin, that could release Fe 2§ from this complex were ranked as good chelators, (2) flavonoids, such as quercetin, that could not remove Fe 2§ from the

328

V A N D E N B E R G H E et al.

complex but chelated Fe 2§ (3) flavonoids, such as galangin, that could not remove Fe 2§ from the complex and chelated Fe2§ to a minor extent, and (4) flavonoids, such as naringin, that did not chelate Fe 2§ [27]. In conclusion, a 3-OH group and a catechol moiety are more important for iron chelation than a 5-OH group [27]. Afanas'ev et al. demonstrated the ability of rutin to form a stable complex with Fe 2§ at physiological pH [142]. The absorption spectrum of the Fe2§ complex did not change during 8 hours [142]. Morel et al. investigated radiochemically the capacity of three flavonoids (catechin, quercetin and diosmetin) and desferrioxamine - a powerful chelator of Fe 3§ - to remove Fe 3§ from iron-loaded hepatocytes [18, 143]. Nitrilotriacetic acid - a low affinity iron-chelator- was used to maintain Fe 3§ in a soluble state. The iron-chelating ability decreased in the following order: desferrioxamine > catechin > quercetin > diosmetin (which had a very low activity) [18, 143]. PRO-OXIDANT ACTIVITY OF FLAVONOIDS Nowadays, there is a growing interest in the pro-oxidant effects of flavonoids. It is of great importance to understand that a compound cannot just be classified as an antioxidant on the basis of one antioxidant experiment, because it can act as a pro-oxidant in another system. For example, diethylstilboestrol is an inhibitor of lipid peroxidation in vitro [144], but can accelerate oxidative DNA damage in vivo [145]. It is therefore recommended to use a battery of test systems involving DNA, lipids, proteins, and carbohydrates to determine both the antioxidant and pro-oxidant properties [ 146]. Some of the pro-oxidant effects of flavonoids have been attributed to the fact that they can undergo auto-oxidation reactions when they are dissolved in aqueous buffers. The rate of auto-oxidation can be determined by measuring the oxygen consumption. Hodnick et al. studied the inhibition of succinoxidase in isolated mitochondria and found that in a series of flavonols with various B-ring OH substitution patterns the strongest enzyme inhibitors were those with a pyrogallol (myricetin) or catechol moiety (quercetin) [ 147-148]. The flavonols myricetin, quercetin, and quercetagetin (6-hydroxyquercetin) were found to undergo autooxidation, resulting in the production of 0 2 " - a n d H202 during mitochondrial respiratory bursts. According to Hodnick et al., 02"-was involved in the auto-oxidation of myricetin, since addition of SOD inhibited this process, in contrast to the auto-oxidation of quercetagetin [147]. Myricetin, quercetin, and quercetagetin had oxidation potentials significantly lower than those that did not undergo auto-oxidation. Consequently, inhibition of succinoxidase by the three flavonols appears to be associated with their ability to participate in redox reactions [ 148].


329

In agreement with the results from Hodnick et al. [ 147-148], Canada et al. [149] found myricetin underwent auto-oxidation more rapidly than quercetin, but this process was prevented by glycosylation of the 3-OH group (rutin). Virtually no auto-oxidation was detected for quercetin at physiological pH, whereas the rate of auto-oxidation increased considerably with increasing pH. Both the addition of iron for the flavonols quercetin and myricetin, and the addition of iron followed by SOD for quercetin resulted in an increase of the auto-oxidation rate, whereas the addition of SOD to the two flavonols reduced the autooxidation rate [149]. Consequently, 02 ~ might be involved in the autooxidation of myricetin [ 149] - a flavonol with a pyrogallol moiety in the Bring - as indicated for the auto-oxidation of pyrogallol [150]. The ROS produced by auto-oxidation were identified as OH" and H202 by ESR [149]. The deoxyribose assay is a simple test tube assay to determine the antioxidant or pro-oxidant properties of test compounds against carbohydrates [ 151-152]. In the deoxyribose assay, OH" are produced from the reaction of H202 with Fe 2+ (Fenton reaction), where the latter is formed by the ability of ascorbic acid to reduce ferric iron (Fe 3§ to ferrous iron (Fe 2+) (Eq. 27). Fe 3§ + ascorbate ---) Fe 2+ + semidehydroascorbate (Eq. 27) Deoxyribose attacked by OH- produces malondialdehyde (MDA); following heating in acidic conditions, MDA forms with thiobarbituric acid (TBA) a pink chromogen that can be quantified spectrophotometrically at 532 nm (Fig. (5)). H

~,~N~

OH +

~HO

T~ ~.~

~H2

H+

"~

"~

OH

OH

SH

CHO OH

TBA

MDA

|TBA)2-MDA adduct (pink chromogen)

Fig. (5). Detection of malondialdehyde (MDA) by the thiobarbituric acid (TBA) test.

Two different antioxidant actions can be measured using this method. Firstly, the addition of Fe 3§ as Fe3+-EDTA to the assay results in free OH- in solution and therefore a compound can inhibit the deoxyribose damage by scavenging these OH*. Secondly, the addition of free Fe 3§ instead of Fe3+-EDTA, will result in the binding of some of these Fe 3+ to deoxyribose, followed by a reduction to Fe 2§ which then stimulates OH" generation at the site of damage [153]. The ability of a compound to inhibit the site-specific deoxyribose degradation is due to its iron chelating

9

330


activity, thereby preventing the iron ion to catalyze the Fenton reaction. Potential pro-oxidant properties of test compounds can be determined when ascorbic acid is omitted from the test system. A potential prooxidant compound will reduce Fe 3§ to Fe2§ resulting in the generation of OH" [33]. A disadvantage of this easy-to-use assay is the restriction to water-soluble compounds, because organic solvents, like ethanol and DMSO are powerful OH" scavengers. Laughton et al. [32] and Puppo [93] investigated the pro-oxidant activity of flavonoids with the deoxyribose assay. According to Laughton et al., the flavonols quercetin and myricetin accelerated at 100 IxM the generation of OH" from a mixture of H202 and Fe3+-EDTA [32]. This specific pro-oxidant activity was higher for myricetin than for quercetin. Interestingly, the addition of SOD prevented their pro-oxidant effect, which excludes a simple reduction of Fe 3+ to Fe 2§ by the flavonols. Consequently, Laughton et al. suggest therefore that Fe3+-EDTA induces an oxidation of the flavonols, yielding O2o-, which is responsible for the reduction of Fe3§ to Fe2§ These pro-oxidant effects were not demonstrated with Fe 3§ alone or with the physiologically more relevant Fe3§ diphosphate (ADP) or Fe3§ complexes. The pro-oxidant results were explained by the action of EDTA altering the redox potential and iron solubility [154]. Puppo found that the OH" generating effect of flavonoids decreased in the following order: myricetin > quercetin > catechin > morin > kaempferol; flavone had no effect [93]. Changes in UV/VIS absorbance, which were due to the oxidation of the flavonoid molecule, were related to their pro-oxidant activity and were faster with myricetin than with quercetin. These results, together with the observation that the addition of SOD inhibited the pro-oxidant activity, support the pro-oxidant hypothesis of Laughton et al. [32]. In the presence of the biologically more relevant chelators ATP and citrate, the flavonoids did not act as pro-oxidants. Consequently, iron chelators influence the pro-oxidant or antioxidant capacities of flavonoids. Laughton et al. investigated also the possible pro-oxidant effects of quercetin and myricetin on DNA with the bleomycin assay [32]. Bleomycin is an antitumor antibiotic that binds both iron ions and DNA. A bleomycin-Fe 3§ complex will degrade DNA in the presence of 02 and a reducing agent (e.g. ascorbic acid) or H202 [33, 155]. In addition, deoxyribose - a sugar originating from DNA - degrades to form MDA, which can be measured with TBA (see also deoxyribose assay) [155]. Laughton et al. found that quercetin and myricetin accelerated DNA damage, most likely by reducing the Fe3+-eomplex to a Fe2+-complex [32]. Hanasaki et al. [ 16] investigated the antioxidant and pro-oxidant activities of flavonoids with a method [156] based on the oxidation of DMSO by OH" generated by the Fenton reaction - to form the stable compound methanesulfinic acid (MSA) as shown in Eq. 28. -


331

OHH3C---S----CH

3 +

OH 9

~

C H 3" +

DMSO

H3C---S

OH

(Eq. 2 8 )

MSA

MSA is derivatized with a diazonium salt to produce a diazosulfone, which is quantified by HPLC [157]. The flavonoids catechin, epicatechin, 7,8-dihydroxyflavone, and rutin showed a OH" scavenging activity 100 to 300 times higher than mannitol, whereas baicalein (5,6,7trihydroxyflavone), quercetin, myricetin, and morin enhanced the OHproduction [16]. The pro-oxidant activity of morin increased with an increasing concentration of morin, whereas an increasing concentration of the other three flavonoids did not enhance their pro-oxidant activity. Hanasaki et al. suggested that these pro-oxidant flavonoids produce H202 during their auto-oxidation, which then stimulates the Fenton reaction [16]. Cao et al. investigated the possible pro-oxidant activity of flavonoids by the ORAC assay [29]. Depending on their concentration, flavonoids scavenged OH- in a CuE+-n202 system. However, at higher flavonoid concentrations the activity declined with increasing concentration. In the presence of Cu 2+ without H202, the flavonoids acted as pro-oxidants rather than as antioxidants [29]. The pro-oxidant activity decreased in the following order: myricetin > quercetin > kaempferol > taxifolin [29]. Flavone and 6-hydroxyflavone showed no significant pro-oxidant activity. Clearly, these results demonstrate the importance of a C2-C3 double bond and the number of OH substitutions regarding the Cu2+-initiated prooxidant activity of flavonoids [29]. Several flavonoids were investigated in one assay for both the inhibition of XO and the O2"- scavenging activity, measured spectrophotometrically S u m m a r y of the Classification of Flavonoids into Six Categories According to their Inhibition of Xanthine Oxidase (XO) and Superoxide

Table 3.

Radical (02*-) Scavenging Activity [78] Category

Inhibition of XO

02 ~ Scavenging Activity i

Example i

A

o

+

(-)-epigallocatechin

B

+

o

baicalein

C

+

+

myricetin

D

+

-

galangin

E

o

-

7-hydroxyflavanone

F

o i

o: No effect; +: Effect; -: Pro-oxidant effect

O

naringenin

332

FANDEN BERGHE et aL

and with the nitrite method, respectively [78]. The relative concentrations of uric acid (as a measure of the inhibitory activity on XO) and 02"- were displayed as a function of the concentration of the flavonoid tested (Fig. (6)). According to these two activities, the flavonoids were classified into six categories (Table 3). Since a reduction in uric acid production and consequently an inhibition of XO results automatically in an equivalent reduction in 02"-- which is the result of the XO activity - a flavonoid with a complementary pro-oxidant activity will not show an equivalent reduction of 02"- due to complementary 02"- production. The 02"- curve will be situated above the uric acid curve. Galangin, chrysin, apigenin, and luteolin were classified as XO inhibitors with an additional pro-oxidant effect on the production of 02"- (category D) (Fig. (6)), whereas 7hydroxyflavanone had a marginal effect on XO, but a pro-oxidant effect on the production of 02"- (category E) (Fig. (6)) [78]. 02"- Scavengers without inhibitory activity on XO and XO inhibitors with an additional 02"- scavenging activity were classified into categories A and C, respectively (Fig. (6)) [78]. category A

(%)120' 110

~.

superoxld'e.=.~ m,,

90.

8o~ 70 ~ 60 ~ 50: 40: 3o-

2O ~

~o~ 0

. . . . . . . .

.I

i

. . . . . . . .

,

120(%) " uric acid 110 100 90 .80 "70 "60 "50 "4O -30 "20 .10 0 -

-

- J ~ - - -

I I0 lO0 concentration of taxffolin Qd~ ~tegory C

(%)12G

120{%)

Superoxlde~

uric acld [ llO 100

90 ~

90 ~' 80 p

80~ 7O~

60~ 30 ~

~ 60 "50 p ~ 4o 30

20 ~

,2o

5o ~ 4o ~

:~o

IO: 0

,

.1

.

.

.

.

.

.

.

.

~JL,

.-.

. . . .

concentration of fl~e~in (/dUO

0 100


{%} 120

11o;r

(Fig. 6). contd .....

category D

~

superoxld~

333

120{%}

uric acid llO I00

9o; 8o ~ 7o ~

'80

6o.

"60

50' 40" 30 ~ 2O ~ ]0~

o!1

(%} 120~_~

140 "30 "2O

I I0 I00 concentration of galangin {glD category E 0--- superoxide

II0' O0 =

90 ~

soi

70" 6O; 5O ~ 4O ~ 30 ~ 20"

120(%} uric acld

II0 I00 9O 8O 70 6O 5O 4O 3O 2O I0

0

0

.I I I0 I00 concentration of 7-hydroxTfiavanone Fig. (6). The concentration of flavonoid vs. the relative superoxide amount detected and the uric acid production. The results are expressed in % = (Ain the presence of flavonoid / Ain the

absence of flavonoid) x 100 [78].

CONCLUDING REMARKS According to the studies discussed above, it can be concluded that flavonoids act as antioxidants through different mechanisms. Most studies propose three structural elements as essential for optimal antioxidant activity: (1) a catechol or pyrogallol moiety in the B-ring, (2) a free 3-OH group, and (3) a C2-C3 double bond. Flavanols are exceptions to the rule, since they do not possess a C2-C3 double bond. The flavonols quercetin and myricetin meet these conditions and are therefore good candidates for antioxidant use. However, similar flavonoids can act as pro-oxidants,

334

V A N D E N B E R G H E et aL

possibly promoting oxidative damage to biological molecules. Consequently, there is a need for more detailed studies to elucidate the mechanism of the pro-oxidant effect and to determine its relevancy in vivo. There is also a lack of studies concerning the in vivo antioxidant actvity of flavonoids. Some studies demonstrate that humans absorb considerable amounts of flavonoids, but the absorption and metabolism of most flavonoids are not known. Many techniques currently used in the antioxidant research are highly specialized and the value of the obtained results depends often on the applied techniques. Consequently, there is a need for collaborative studies to standardize these methods. First, in most studies the purity of flavonoids is not mentioned and this can mask their activity. Second, most flavonoids are not readily water-soluble and are therefore dissolved in organic solvents. But some organic solvents, like DMSO and ethanol, are powerful OH" scavengers. Third, measuring flavonoids at millimolar concentrations is not relevant, because such plasma levels are never obtained. Some flavonoids show interesting activity at micr0molar or lower concentrations. Research should be focused on the pharmacology of such molecules. A lot of these flavonoids exhibit not only antioxidant activity but also other biological activities (anti-inflammatory, antiviral, ...) so that these molecules could be very useful in the future for the treatment of some diseases due to their combined activities. ABBREVIATIONS AAPH ABTS ABTS o+

= = =

AMVN CC1302 ~

= = =

2,2'-azobis(2-amidinopropane) dihydrochloride 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonic acid) 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonie acid) radical cation 2,2'-azobis(2,4-dimethylvaleronitrile) bromide radical ion trichloromethylperoxyl radical

Cu,Zn-SOD

=

copper- and zinc-containing superoxide dismutase

DETAPAC

=

diethylenetriaminepenta-acetic acid

DMPO

=

5,5-dimethyl- 1-pyrroline N-oxide

DPPH

=

1,1-diphenyl-2-picrylhydrazyl (free radical)

e-aq

=

hydrated electron

Eva EDTA

=

half peak oxidation potential

=

ethylenediaminetetra-acetic acid

Br2"-


ESR

=

electron spin resonance

GSH

=

reduced glutathione

GSHPx

=

glutathione peroxidase

GSSG

=

oxidized glutathione

H-

=

hydrogen atom or hydrogen radical

HCOO-

=

formate

HO2 ~

=

hydroperoxyl radical

H202

=

hydrogen peroxide

HOC1

=

hypochlorous acid

HX-FeIII

=

metmyoglobin

MDA

=

malondialdehyde

Mn-SOD

=

manganese-containing superoxide dismutase

N3"

=

azide radical

N3-

=

azide anion

NBT

=

nitro-blue tetrazolium

IO 2

=

singlet oxygen

IAgO2 leg+

=

delta singlet oxygen

=

sigma singlet oxygen

02"-"

=

superoxide radical

03

=

ozone

OH"

=

hydroxyl radical

ORAC

=

oxygen radical absorbing capacity

13-PE

=

13-phycoerythrin

R 9

=

carbon radical

RO-

=

alkoxyl radical

ROO 9

=

peroxyl radical

ROS

=

reactive oxygen species

SOD

=

superoxide dismutase

TBA

=

thiobarbituric acid

TEAC

=

trolox equivalent antioxidant capacity

oX.Few=O

=

ferrylmyoglobin radical

XO

=

xanthine oxidase

335

336

VANDEN BERGHE et as

REFERENCES

[I] [2] [3]

[4] [s] [6] [7]

[8] [9] [101 [11]

[12] [13]

[14] [15] [16] [17]

[is] [19]

[20] [21] [22]

[23] [24]

De Meyer, N.; Haemers, A.; Mishra, L.; Pandey, H.-K.; Pieters, L.A.C.; Vanden Berghe D.A.; Vlietinck, A.J.J. Med. Chem., 1991, 34, 736-746. Vanden Berghe, D.A.R.; Haemers, A.; Vlietinck, A.J. In Bioactive Natural Products: detection, isolation and structural determination; Colegate, S.M., Molyneux, R.J., Eds.; CRC Press: London, 1993; Chapter 17, pp. 405-440. Edenharder, R.; von Petersdorff, I.; Rauscher, R. Murat. Res., 1993, 287, 261274. Calomme, M.; Pieters, L.; Vlietinck, A.; Vanden Berghe, D. Planta Med., 1996, 62, 222-226. Mora, A.; Paya, M.; Rios, J.L.; Alcaraz, M.J. Biochem. Pharmacol., 1990, 40, 793-797. Sanz, M.J.; Ferrandiz, M.L.; Cejudo, M.; Terencio, M.C.; Gil, B.; Bustos, G.; Ubeda, A.; Gunasegaran, R.; Alcaraz, M.J. Xenobiotica, 1994, 24, 689-699. Shimoi, K.; Masuda, S.; Shen, B.; Furugori, M.; Kinae, N. Murat. Res., 1996, 350, 153-161. Shimoi, K.; Masuda, S.; Furugori, M.; Esaki, S.; Kinae, N. Carcinogenesis, 1994, 15, 2669-2672. Lasure, A.; Van Poel, B.; Cimanga, K.; Pieters, L.; Vanden Berghe, D.; Vlietinck, A.J. Pharm. Pharmacol. Lett., 1994, 4, 32-35. De Bruyne, T.; Van Driessen, G.; Van Poel, B.; Laekeman, G.; Vlietinck, A.J. Phytochemistry (Life Sci. Adv.), 1992, 11, 63-66. Middleton, E.; Kandaswami, C., Biochem. Pharmacol., 1992, 43, 1167-1179. Panthong, A.; Kanjanapothi, D.; Tuntiwachwuttikul, P.; Pancharoen, O.; Reutrakul, V. Phytomedicine, 1994, 1, 141-144. Hertog, M.G.; Feskens, E.J.; Hollman, P.C.; Katan, M.B., Kromhout, D. Lancet, 1993, 342, 1007-1011. Knekt, P.; Jarvinen, R.; Reunanen, A.; Maatela, J. Brit. Meal J., 1996, 312, 478481. Hertog, M.G. et al. Arch. Intern. Med., 1995, 155, 381-386. Hanasaki, Y.; Ogawa, S.; Fukui, S. Free Radic. Biol. Meal., 1994, 16, 845- 850. Tournaire, C.; Croux, S.; Maurette, M.-T.; Beck, I.; Hocquaux, M.; Braun, A.M.; Oliveros, E. J. Photochem. Photobiol. B: Biol., 1993, 19, 205-215. Morel, I.; Lescoat, G.; Cillard, P.; Cillard, J. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 437443. Laughton, M.J.; Evans, P.J.; Moroney, M.A.; Hoult, J.R.C.; Halliwell, B. Biochem. Pharmacol., 1991, 42, 1673-1681. Hoult, J.R.S.; Moroney, M.A.; Paya, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 443- 455. Siess, M.H.; Leclerc, J.; Canivenc-Lavier, M.C.; Rat, P.; Suschetet, M. Toxicol. Appl. Pharmacol., 1995, 130, 73-78. Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165-2170. Lindahl, M.; Tagesson, C. Inflammation, 1993, 17, 573-582. Gil, B.; Sanz, M.J.; Terencio, M.C.; Ferrandiz, M.L.; Bustos, G.; Paya, M.; Gunasegaran, R.; Alcaraz, M.J. Life Sci., 1994, 54, 333-338.


[25] [26] [27] [28] [29] [301 [31] [32] [33] [34] [35] [36] [37]

[38] [39] [40] [41]

[42] [43] [44] [45]

[46]

[47] [48] [49]

[50] [51] [52] [53] [541

[551 [561 [571

[58] [59]

[60]

337

Middleton, E.; Kandaswami, C. In The Flavonoids: Advances in research since 1986; Harbome, J.B., Ed.; Chapman & Hall: London, 1993; Chapter 15, pp. 619-652. Middleton, E. Trends Pharmacol. Sci., 1984, 5, 335-338. Van Acker, S.A.B.E.; van den Berg, D.-J.; Tromp, M.N.J.L.; Griffioen, D.H.; van Bennekom, W.P.; van der Vijgh, W.J.F.; Bast, A. Free Radic. Biol. Med., 1996, 20, 331-342. Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Free Radic. Biol. Med., 1996, 20, 933-956. Cao, G.; Sofic, E.; Prior, R.L. Free Radic. Biol. Med., 1997, 22, 749-760. Hollman, P.C.H.; van Trijp, J.M.P.; Mengelers, M.J.B.; de Vries, J.H.M.; Katan, M.B. Cancer Lett., 1997, 114, 139-140. Hollman, P.C.H.; de Vries, J.H.M.; van Leeuwen, S.D.; Mengr M.J.B.; Katan, M.B. Am. J. Clin. Nutr., 1995, 62, 1276-1282. Laughton, M.J.; Halliwell, B.; Evans, P.J.; Hoult, J.R.S. Biochem. Pharmacol., 1989, 38, 2859-2865. Aruoma, O.I. JAOCS, 1996, 73, 1617-1625. Halliwell, B. Free Rad. Res., 1996, 25, 57-74. Halliwell, B.; Gutteridge, J.M.C. Free Radicals in Biology and Medicine; Clarendon Press: Oxford, 1989. Ahmad, S. Oxidative Stress and Antioxidant Defenses in Biology; Chapman & Hall: New York, 1995. Halliwell, B. Biochem. Pharmacol., 1995, 49, 1341-1348. Halliwell, B. Free Rad. Res. Comms., 1990, 9, 1-32. Gutteridge, J.M.C.; Halliwell, B. ,4ntioxidants in Nutrition, Health, and Disease; Oxford University Press: New York, 1994. Halliwell, B. Annu Rev. Nutr., 1996, 16, 33-50. Krinsky, N.I. Proc. Soc. Exp. Biol. Med., 1992, 200, 248-254. Sies, H. Oxidative Stress; Academic Press: London, 1985. Demple, B.; Harrison, L. Annu Rev. Biochem., 1994, 63, 915-948. Antunes, F.; Salvador, A.; Pinto, R.E. Free Radic. Biol. Med, 1995, 19, 669677. Grune, T.; Reinheckel, T.; Joshi, M.; Davies, K.J.A.J. Biol. Chem., 1995, 270, 2344-2351. Harris, E.D. Faseb J., 1992, 6, 2675-2683. Cerutti, P.A. Lancet, 1994, 344, 862-863. Wiseman, H.; Halliwell, B. Biochem. J., 1996, 313, 17-29. Jenner, P. Lancet, 1994, 344, 796-798. Witztum, J.L. Lancet, 1994, 344, 793-795. Halliwell, B. Haemostasis, 1993, 23, 118-126. Halliwell, B.; Gutteridge, J.M.C.; Cross, C.E.J. Lab. Clin. Med., 1992, 119, 598-620. Taylor, A. Ann. N.Y. Acad. Sci., 1992, 669, 111-123. Wiseman, H. Nutritional Biochemistry, 1996, 7, 2-15. Gutteridge, J.M.C. Free Rad. Res. Comms., 1993, 19, 141-158. Gaziano, J.M. Nutrition, 1996, 12, 583-588. Gordon, M.H. Nat. Prod. Rep., 1996, 13, 265-273. Willett, W.C. Science, 1994, 264, 532-537. Block, G.; Patterson, B.; Subar, A. Nutr. Cancer., 1992, 18, 1-29. Byers, T.; Perry, G. dnnu. Rev. Nutr., 1992, 12, 139-159.

338

[61] [621 [63]

[64] [65] [66] [67] [68]

[69] [70] [71]

[72] [73] [74] [75]

[76] [77] [78] [79]

[80] [81]

[82] [83] [84]

[85] [86]

[87]

[88]

[89] [90] [911


Diplock, A.T. MoL Aspects Med., 1994, 15, 293-376. Havsteen, B. Biochem. PharmacoL, 1983, 32, 1141-1148. Harborne, J.B. The Flavonoids: Advances in Research since 1986, Chapman & Hall: London, 1993. Kandaswami, C.; Middleton, E. Adv. Exp. Med. BioL, 1994, 366,351-376. Pierpoint, W.S. Prog. Clin. BioL Res., 1986, 213, 125-140. Hollman, P.C.H. Eur. d. Clin. Nutr., 1997, 51, $66-$69. Ktlhnai, J. Wld. Rev. Nutr. Diet., 1976, 24, 117-191. Hertog, M.G.L.; Hollman, P.C.H.; Katan, M.B.; Kromhout, D. Nutr. Cancer, 1993, 20, 21-29. Das, N.P. Biochem. Pharmacol., 1971, 20, 3435-3445. Pryor, W.A. Ann. Rev. PhysioL, 1986, 48, 657-667. Shahidi, F.; Wanasundara, P.K.J.P.D. Crit. Rev. Food Sci. Nutr., 1992, 32, 67103. Baumann, J.; Wurm, G.; von Bruchhausen, F. Arch Pharm., 1980, 313, 330337. Robak, J.; Gryglewski, R.J. Biochem. PharmacoL, 1988, 37, 837-841. Huguet, A.I.; Manez, S.; Alcaraz, M.J.Z. Naturforsch., 1990, 45c, 19-24. Yuting, C.; Rongliang, Z.; Zhongjian, J.; Yong, J. Free Radic. Biol. Med., 1990, 9, 19-21. Chen, C.-W.; Ho, C.-T. d. Food Lipids, 1995, 2, 35-46. Cotelle, N.; Bernier, J.L.; Henichart, J.P.; Catteau, J.P.; Gaydou, E.; Wallet, J.C. Free Radic. BioL Med., 1992, 13, 211-219. Cos, P.; Ying, L.; Calomme, M.; Hu, J.P.; Cimanga, K.; Van Poel, B.; Pieters, L.; Vlietinck, A.J.; Vanden Berghe, D. d. Nat. Prod., 1998, 61, 71-76. Cotelle, N.; Bernier, J.-L.; Catteau, J.-P.; Pommery, J.; Wallet, J.-C.; Gaydou, E.M. Free Radic. BioL Med., 1996, 20, 35-43. Nishikimi, M.; Appaji Rao, N.; Yagi, K. Biochem. Bioph. Res. Co., 1972, 46, 849-854. Richter, H.W.; Fetrow, M.A.; Lewis, R.E.; Waddell, W.H.d. Am. Chem. Soc., 1982, 104, 1666-1671. Terada, L.S.; Left, J.A.; Repine, J.E. In Methods in Enzymology; Packer, L., Glazer,A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 651-656. Hayashi, T.; Sawa, K.; Kawasaki, M.; Arisawa, M.; Shimizu, M.; Morita, N. jr. Nat. Prod, 1988, 51, 345-348. Rastelli, G.; Constantino, L.; Albasini, A. Eur. d; Med. Chem., 1995, 30, 141146. Rastelli, G.; Constantino, L.; Albasini, A. Eur. d. Med. Chem., 1996, 31, 693699. Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165-2170. Ponti, V.; Dianzani, M.U.; Cheeseman, K.; Slater, T.F. Chem.-Biol. Interact., 1978, 23, 281-291. Sichel, G.; Corsaro, C.; Sealia, M.; Di Bilio, A.J.; Bonomo, R.P. Free Radic. BioL Med., 1991, 11, 1-8. Koppenol, W.H. Free Radic. Biol. Med., 1993, 15, 645-651. Sutton, H.C.; Winterbourn, C.C. Free Radic. Biol. Med., 1989, 6, 53-60. Lloyd, R.V.; Hanna, P.M.; Mason, R.P. Free Radic. BioL Med., 1997, 22, 885888.

STRUCFURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS [92] [93] [94] [95] [96] [97] [98]

[99]

[loo] [1ol] [102]

[103] [104] [1o5] [1061

[107] [lOS] [109] [llO] [Ill] [1~2] [113] Ill4] [~15] [116] [117]

[118] [119] [12o]

339

Yoshiki, Y.; Okubo, K.; Onuma, M.; Igarashi, K. Phytochemistry, 1995, 39, 225-229. Puppo, A. Phytochemistry, 1992, 31, 85-88. Husain, S.R.; Cillard, J.; Cillard, P. phytochemistry, 1987, 26, 2489-2491. Floyd, R.A.; Lewis, C.A.; Wong, P.K. In Methods in Enzymology; Packer, L., Ed.; Academic Press: New York, 1984, Vol. 105, pp. 231-237. Finkelstein, E.; Rosen, G.M.; Rauckman, E.J. Arch. Biochem. Biophys., 1980, 200, 1-16. Aruoma, O.I. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 67-82. Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25, 383-385. Porter, N.A.; Weber, B.A.; Weenen, H.; Khan, J.A.J. Am. Chem. Soc., 1980, 102, 5597-5601. Niki, E. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, 100-108. Cao, G.; Alessio, H.M.; Cutler, R.G. Free Radic. Biol. Med., 1993, 14, 303311. Cao, G.; Verdon, C.P.; Wu, A.H.B.; Wang, H.; Prior, R.L. Clin. Chem., 1995, 41/42, 1738-1744. Wang, P.-F.; Zheng, R.-L. Chem. Phys. Lipids, 1992, 63, 37-40. Terao, J.; Piskula, M.; Yao, Q. Arch. Biochem. Biophys., 1994, 308, 278-284. Ioku, K.; Tsushida, T.; Takei, Y.; Nakatani, N.; Terao, J. Biochim. Biophys. Acta, 1995, 1234, 99-104. Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. J. Biol. Chem., 1984, 259, 41774182. Ratty, A.K.; Sunamoto, J.; Das, N.P. Biochem. Pharmacol., 1988, 37, 989- 995. Rekka, E.; Kourounakis, P.N.J. Pharm. Pharmacol., 1991, 43, 486-491. Blois, M.S. Nature, 1958, 181, 1199-1200. Fomi, L.G.; Willson, R.L. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press" New York, 1990, Vol. 186, pp. 179-188. Adams, G.E.; Wardman, P. In Free Radicals in Biology: Volume III; Pryor, W.A., Ed.; Academic Press: New York, 1977; Chapter 2, pp. 53-95. Butler, J.; Land, E.J. In Free Radicals: A practical approach; Punchard, N.A., Kelly, F.J., Eds.; IRL Press: Oxford, 1996; Chapter 4, pp. 47-61. Alfassi, Z.B.; Schuler, R.H.J. Phys. Chem., 1985, 89, 3359-3363. Erben-Russ, M.; Bors, W.; Saran, M. Int. J. Radiat. Biol., 1987, 52, 393-412. Bors, W.; Saran, M. Free Radical Res. Com., 1987, 2, 289-294. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 343355. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Antioxidants in Therapy and Preventive Medicine; Emerit, I., Ed.; Plenum Press: New York, 1990; pp. 165170. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 420429. Jovanovir S.V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M.G.J. Am. Chem. Soc., 1994, 116, 4846-4851. Aruoma, O.I. Food Chem. Toxicol., 1994, 32, 671-685.

340


[121] Aruoma, O.I.; Spencer, J.P.E.; Butler, J.; Halliwell, B. Free Rad. Res., 1995, 22, 187-190. [122] Swartz, H.M.; Swartz, S.M. In Methods of Biochemical Analysis, Glick, D., Ed.; John Wiley & Sons: New York, 1983; Volume 29, pp. 207-323. [123] van Acker, S.A.B.E.; de Groot, M.J.; van den Berg, D.-J.; Tromp, M.N.J.L.; Donn6-Op den Kelder, G.; van der Vijgh, W.J.F.; Bast, A. Chem. Res. Toxicol., 1996, 9, 1305-1312. [124] Kuhnle, J.A.; Windlr J.J.; Waiss, A.C.J. Chem. Soc. (B), 1969, 613-616. [125] Miller, N.J.; Rice-Evans, C.; Davies, M.J.; Gopinathan, V.; Milner, A. Clin. Sci., 1993, 84, 407-412. [1261 Rice-Evans, C.; Miller, N.J. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 279-293. [127] Miller, N.J.; Rice-Evans, C.A. Redox Rep., 1996, 2, 161-171. [128] Giulivi, C.; Cadenas, E. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 189-202. [1291 Strube, M.; Haenen, G.R.M.M.; van den Berg, H.; Bast, A. Free Radical Res., 1997, 26, 515-521. [130] Rice-Evans, C.A.; Miller, N.J.; Bolwell, P.G.; Bramley, P.M.; Pridham, J.B. Free Radical Res., 1995, 22, 375-383. [131] Salah, N.; Miller, N.J.; Paganga, G.; Tijburg, L.; Bolwell, G.P.; Rice-Evans, C. Arch. Biochem. Biophys., 1995, 322, 339-346. [1321 Rice-Evans, C.A.; Miller, N.J. Biochem. Soc. T., 1996, 24, 790-795. [133] Paganga, G.; AI-Hashim, H.; Khodr, H.; Scott, B.C.; Aruoma, O.I.; Hider, R.C.; Halliwell, B.; Rice-Evans, C.A. Redox Rep., 1996, 2, 359-364. [134] Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Trends Plant Sci., 1997, 2, 152159. [135] Sorata, Y.; Takahama, U.; Kimura, M. Biochim. Biophys. Acta, 1984, 799, 313317. [136] Takahama, U.; Youngman, R.J.; Elstner, E.F. Photobiochem. Photobiophys., 1984, 7, 175-181. [137] Devasagayam, T.P.A.; Subramanian, M.; Singh, B.B.; Ramanathan, R.; Das, N.P.J. Photochem. Photobiol. B: Biol., 1995, 30, 97-103. [138] Studer, S.L.; Brewer, W.E.; Martinez, M.L.; Chou, P.-T. J. Am. Chem. Soc., 1989, 111, 7643-7644. [139] Halliwell, B.; Gutteridge, J.M.C. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 1-85. [140] Hudson, B.J.F.; Lewis, J.l. Food Chem., 1983, 10, 47-55. [141] Thompson, M.; Williams, C.R.; Elliot, G.E.P. Anal. Chim. Acta, 1976, 85, 375-381. [142] Afanas'ev, I.B.; Dorozhko, A.I.; Brodskii, A.V.; Kostyuk, V.A.; Potapovitch, A.I. Biochem. Pharmacol., 1989, 38, 1763-1769. [1431 Morel, I.; Lescoat, G.; Cogrel, P.; Sergent, O.; Pasdeloup, N.; Brissot, P.; Cillard, P.; Cillard, J. Biochem. Pharmacol., 1993, 45, 13-19. [144] Wiseman, H.; Halliwell, B. FEBS Lett., 1993, 322, 159-163. [145] Roy, D.; Liehr, J.G. Cancer Res., 1991, 51, 3882-3885. [146] Aruoma, O.I. Free Radic. Biol. Med., 1996, 20, 675-705. [147] Hodnick, W.F.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 35, 2345-2357. [148] Hodnick, W.F.; Milosavljevic, E.B.; Nelson, J.H.; Pardini, R.S. Biochem. Pharmacol., 1988, 37, 2607-2611.


341

[149] Canada, A.T.; Giannella, E.; Nguyen, T.D.; Mason, R.P. Free Radic. Biol. Med., 1990, 9, 441-449. [150] Marklund, S.; Marklund, G. Eur. J. Biochem., 1974, 47, 469-474. [151] Halliwell, B.; Gutteridge, J.M.C.; Aruoma, O.I. Anal Biochem., 1987, 165, 215-219. [152] Aruoma, O.I. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 57-66. [153] Aruoma, O.I.; Chaudhary, S.S.; Grootveld, M.; Halliwell, B. J. Inorg. Biochem., 1989, 35, 149-155. [154] Grootveld, M.; Halliwell, B. Free Radical Res. Com., 1986, 1, 243-250. [155] Evans, P.J.; Halliwell, B. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 82-92. [156] Babbs, C.F.; Steiner, M.G. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press" New York, 1990, Vol. 186, pp. 137-147. [157] Fukui, S.; Hanasaki, Y.; Ogawa, S. d. Chromatogr., 1993, 630, 187-193.


Atta-ur-Rahman (Ed.) Studies in Natw'al Products Chemistry, Vol. 22

343


R E C E N T A D V A N C E S IN T H E S E A R C H FOR A N T I O X I D A N T A C T I V I T Y IN S O U T H A M E R I C A N PLANTS C. DESMARCHELIER I, G. CICCIA l, and J. COUSSIO 2 t Cdtedra de Microbiologia Industrialy Biotecnologia, 2 Cdtedra de Farmacognosia, IQUIMEFA-CONICET; Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina.

Abstract: Over the past two decades, researchers have turned to many of the traditional

folk medicines used in the Far East, Europe and North America in order to uncover the presence of new natural occurring compounds with antioxidant activity. However, in recent years, this interest has also awoken for plants in South America. The existence of geographic areas with highly diverse flora, the unique ecological and physiological conditions under which many of these plants grow and the presence of human societies with a strong tradition in the use of plant resources as medicinal agents, makes this region extremely interesting for this purpose. The present paper reviews the advances in the search for antioxidant activity in plants used as medicinal agents in different areas of South America.

FREE RADICALS, NATURAL ANTIOXIDANTS AND THEIR ROLE IN HUMAN HEALTH It has been determined that active oxygen molecules such as superoxide (O2, OOH.), hydroxyl (OH.) and peroxyl (ROOH o) radicals play an important role in oxidative stress related to the pathogenesis of different diseases such as Alzheimer, Parkinson and Hodgkin's disease, cataracts, acute liver toxicity, inflammation processes and DNA damage that leads to carcinogenesis. These free radicals and other related compounds are generated in (a) mitochondria, which produce the superoxide radical and hydrogen peroxide; (b) phagocytes, generators of nitric oxide and hydrogen peroxide during the "respiratory burst" that takes place in activated phagocytic cells in order to kill bacteria after phagoeytosis; (c) peroxisomes (microbodies), which degrade fatty acids and other substances yielding hydrogen peroxide; and (d) citocrome P-450 enzymes, responsible for many oxidation reactions of endogenous substrates [1 ]. An elaborate cellular defence system against oxygen-free radical toxicity exists, which includes, in a first line, factors that prohibit the fomlation of, or scavenge primary initiators of the lipid peroxidation process, such as metal-ion-binding proteins or the enzymes superoxide dismutase (SOD),

344

DESMARCHELIER et aL

catalase and selenium-dependent glutathione (GSH) peroxidase [2]. Vitamins E and C as well as 13-carotene may also scavenge oxygen radicals. These antioxidants are natural occurring compounds which also include flavonoids, phenolic acids and nitrogen compounds such as alkaloids and chlorophyll derivatives [1,3,4]. Recent epidemiological data suggest that the consumption of polyphenol rich items in the diet is associated to an increase in plasma antioxidant concentrations, thus reducing the exposure to cellular oxidative stress [5,6,7,8,9]. Moreover, it has been determined that the intake of essential antioxidants such as vitamin C, vitamin E and various carotenoids is inversely related to the risk of coronary heart disease and certain forms of cancer [ 10]. Plants, therefore, have become a rich source of potential antioxidant agents that can be useful to mankind. The aim of the present review is to describe the advances in the search for antioxidant compounds and antioxidant activity in medicinal plants used for different purposes in South America. E C O L O G Y AND E T H N O B O T A N Y IN THE S E A R C H FOR NATURAL ANTIOXIDANTS

The search for new active natural compounds such as antioxidants is most productive when plant-surveying takes place in areas where biological diversity is high [ 11 ], such as tropical and subtropical ecosystems, due to the unique ecological characteristics of these regions. For example, it has been stated that some flavonoids, with their strong absorption in the 300400 nm UV region, act as internal light filters for the protection of chloroplasts and other organelles from UV damage. Leaves of nearly all species in high-UV environments, such as arctic, alpine and tropical latitudes, have very low epidermal UV transmittance. The light-filtering ability of these compounds may reinforce their antioxidant effects to provide a high level of protection against damaging oxidants generated either thermally or by light [ 12]. There is evidence that alkaloids also occur with more frequency in species of tropical origin, where UV and UV-B intensities are much higher than in temperate regions [ 13]. Although light intensities also vary with altitude, there is little data on alkaloids concentration as a function of altitude, except for the case of Lycopersicon species growing at high elevations in Peru, which contain higher concentrations of tomatine, a steroidal alkaloid, than related species from lower elevations [14]. Coevolutionary biochemical interactions of plants with their natural predators could contribute to uncover medicinally useful chemicals such as antioxidants [15]. For example, many flavonoids possess allelochemical properties by warding off microbial or animal pathogens as phytoalexins or as antifeedants, and the phytoalexin response is frequently accompanied by an oxidative burst in the cell [ 16]. Moreover, it has been demonstrated that root flavonoids, mainly flavones, isoflavones and some

SEARCH FOR ANTIOXIDANT ACTIVITY

345

chalcones, participate in the process of the induction of nodulation genes during the Rhizobium-legume symbiosis that occurs in root nodulation in Leguminoseae [ 17] a highly represented family in tropical and subtropical areas. Gathering information on the traditional knowledge of the use of medicinal plants by local inhabitants in these regions may also increase the chances of success, since this information could suggest the presence of biologically active principles in the plant, thus serving as a guide that may help in discovering active compounds [ 18]. Although medicinal plants will be rarely used as "antioxidants" in traditional medicine, the therapeutic properties of many active principles present in them could be due, in part, to their capacity of scavenging oxygen free radicals which may be involved in many diseases. For example, plants used to treat inflammatory diseases could act by reducing the oxidative stress that takes place in cells undergoing this process [19]. Likewise, several plants used for hepatoprotective purposes have shown to be active as antioxidants [20,21,22]. Thus, the search for antioxidant activity should centre its attention in plants used traditionally in oxidative stress-related pathologies. "CHACO" AND THE SOUTHERN LOWLANDS This region occupies large areas of south Brazil, north-east Argentina, south-east Bolivia and the territories of Uruguay and Paraguay. The main ecological systems found in the region are the subtropical rainforests, gallery forests, xerofitie deciduous forests ("Chaco"), pyrogenir savannahs, halophylous shrubby steppes, palm forests ("palmares") and herbaceous prairies formed by different Gramineae ("pampas"). The presence of indigenous human groups with an ethnopharmacologieal legacy is only important in the areas of Argentina, Bolivia and Paraguay. Different flavonoids and phenolic compounds have been isolated from medicinal plants of this region, many of which have shown antioxidant properties. A variety of experiments indicate that selected flavonoids and polyphenols possess antialergie, anti-inflammatory, antiviral, anticarcinogenie and antioxidant activities [23]. This latter activity has been attributed to the presence of phenolic groups in their chemical structure. It has been shown that lipid peroxidation can be inhibited by flavonoids, which act as strong 02- scavengers [24] and singlet oxygen (IO2) quenchers [25], and that this activity is closely associated with their chemical structure, especially the number of hydroxyl groups linked to the basic skeleton and also to their configuration [26]. It has also been proposed that these compounds act as H-atom donors to the peroxyl radical, thus inhibiting the autoxidation of fatty acids by means of chain radical termination [27].

346

DESMARCilELIER et aL

OH H HO

OH

O

Fig. (1). Quercetin from Achyrocline satureioides and Pterocaulon polystachium.

Achyrocline satureioides (Lam.) DC. (Compositae), known as "marcela" or "yatei carl", is a medicinal plant widely used in this region by its choleretic, antispasmodic and hepatoprotective properties. Phytochemical analysis of this species has confirmed the presence of flavonoids quercetin, Fig. (1), and its derivatives, and caffeic, chlorogenic and isochlorogenic acids, Fig. (2), in the aerial parts [28]. The high content of polyphenolic compounds in A. satureioides has led us to study its antioxidant properties, in order to determine the free radical scavenging activity and capacity to reduce lipid peroxidation and iron (II)-dependent DNA damage [29]. The inhibition of luminol-enhanced chemiluminescence induced by an azo-bis initiator (a compound that decays spontaneously to generate peroxyl radicals), allowed the determination of the TRAP (total reactive antioxidant potential) and TAR (total antioxidant reactivity) indices [30] for the aqueous and methanolic extracts, using Trolox, a vitamin E hydrosoluble synthetic analog, as a standard. A reduction in iron (II)-dependent DNA damage was also observed, indicating that the extracts studied are capable of reducing the in vitro oxidation of DNA deoxyribose. Lipid peroxidation, on the other hand, was assessed using two different methods: hydroperoxide-initiated chemiluminescence (CL) and the production of thiobarbituric acid-reactive substances (TBARS) in rat liver homogenates. As expected, the extracts of A. satureioides were effective in reducing lipid peroxidation, and the results obtained are summarised in Table 1.

of~~~ HO0 0H

ogr..o o.

oH

OH O E ~ OH OH Cafeic acid

Chlorogenic acid

Isochlorogenic acid

Fig. (2). Caffeoylquinic acids in Achyrocline satureioides and Pterocaulon polystachium.


T a b l e 1.

A n t i o x i d a n t Activity in Different Extracts of

TRAP

Extract

TAR

Achyrocline satureioides

CL

TBARS

DNA damage

(~tg/mi)

(~tg/ml)

225 (404-139)

> 1000

NA

ND

> 1000

ND

0tg/ml) | ',,,"

Aqueous Methanolic

Ill

,

,,

J

,

91-F 15

1537 ~ 148

128 4" 20

1910 4- 171

347

II ill

IIIl~

ill

i

Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) are expressed in (M Trolox equivalents. CL and TBARS are expressed as IC50 ((g/mi), and include 95% confidence intervals. NA: not active; b/D: not determined due to lack of dose dependence.

Another related species, Achyroclineflaccida (Weinm) DC., has also shown to contain different polyphenolic compounds, the most interesting being the flavanone 7,4" dihydroxy 5-methoxy flavanone, and the corresponding chalcone, Fig (3), [31 ]. The latter was highly effective in reducing hydroperoxide-initiated chemiluminescence in rat liver homogenates [32]. Recent studies have also demonstrated that different extracts of this plant exhibited chain-breaking antioxidant activity in FeE+/ascorbate-induced lipid peroxidation in rat liver microsomal fractions and scavenged peroxyl radicals in an aqueous assay system [33]. These activities, together with an inhibition in neutrophil functions, were also observed in Pterocaulon polystachium DC. (Compositae), another medicinal plant widely used in the region. The antioxidant properties 0

H HO

HO

OMe

O

Fig. (3). 7,4" Dihydroxy 5-methoxy flavanone and 4,2',4'-trihydroxy 6'-methoxychalcone from

dchyrocline flaccida. OH

I

O Fig. (4). Rhamnetin from

Pterocaulon polystachium.

H

348

DESMARCtlELIER et

observed were attributed to the presence of quercetin, rhamnetin, Fig. (4), caffeic, chlorogenic and isochlorogenic acids, all of these previously identified in this species [34,35]. Baccharis coridifolia DC. (Compositae) is a sympatric shrub of ,4. satureioides, commonly known as "mio-mio" or "romerillo". The infusion of aerial parts such as leaves is used as an hepatoprotective, and topical use of these parts is also extended in order to treat inflammation. Toxicity of the plant to cattle and other domestic animals has been claimed in this region, probably due to the presence of macrocyclic tricothecens, which show high toxicity towards eucariotic organisms [36]. The involvement of free radical-mediated cell damage in major hepatic diseases has led us to study the in vitro antioxidant activity of the aqueous extract of B. coridifolia [37]. As expected, the extract significantly reduced CL and the production of TBARS in rat liver homogenates, due to the presence of different flavonoids and hydrosoluble polyphenolic compounds in the plant l, thus confirming its antioxidant properties. Other species which were active in these assays are included in Table 2 [38]. Although the active principles in ,4canthospermum australe (Loefl.) have not been identified to the moment, chlorogenic and isochlorogenic acids have been isolated from B. crispa Spr Ok. and P. purpurascens DC. [34]. Table 2.

Antioxidant Activity of Argentine Aqueous Plant Extracts: IC50 and 95% C o n f i d e n c e Interval for Inhibition of H y d r o p e r o x i d e - i n i t i a t e d Chemiluminescence (CL) and the Production of Thiobarbituric Acidreactive Substances (TBARS) in Rat Liver Homogenates i

ii

i

.

ii

IC50 and 95% confidence interval (pg/ml) Botanical name CL ',

",,

,,"

L, ,

i

TBARS i

i

i

i,

Acanthospermum australe (Locfl.)Ok.

123 (183-84)

767 (2953-352)

Baccharis coridifolia DC.

141 (251-84)

556 (1213-320)

B. crispa Spr.

544 ( 1018-340)

554 (2286-247)

Pterocaulon polystachium Malme

462 (776-306)

> 1000

P. purpurascens DC.

376 634-247)

> I000

CL: chemiluminescence. TBARS" thiobarbituric acid reactive substances.

1Bianchi, N.; Sebold, D. Ensayo de toxicidade excessiva e screening fitoquimico de algumas esp~cies do g~nero Baccharis L. (Asteraceae). In: XIV Simp6sio de Plantas Medicinais do Brasil. Florian6polis, September, 1996 (abstract M 004).


349

The methoxylated flavonols 5,7,3',4'-tetrahydroxy-3,6,8-trimethoxy flavone, 5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone and 5,3',4'trihydroxy-3,6,7-trimethoxyflavone have been isolated from aerial parts of Plucchea sagittalis (Lam.) Cabrera (Compositae), Fig. (5), locally known as "cuatrocantos" or "lucero" [39]. Although the biological activity of these compounds has not been determined, these could be responsible for the peroxyl radical scavenging activity observed in different extracts of this plant. Recent studies have also demonstrated that taraxasteryl acetate, also present in this species, is active against topical inflammation, as well as on reactive oxygen species and stress protein production 2. Other studies indicated that 5,6,3'-trihydroxy-7,4'-dimethoxyflavone and pedalitin, Fig. (6), identified in another medicinal plant of the region, namely Eupatorium inulaefolium H.B.K. Hier. (Compositae) [40], were capable of reducing lipid peroxidation in mouse liver homogenates, measured as a reduction in rat liver CL [32]. OH OC H3

H3CO"

~

"~ OH

H

CH3 O

5,7,3',4'-tetrahydroxy-3,6,8-trimethoxy flavone

OH

OH

O

R I -- H; R2 = CH3; 5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone R 1 -" CH3; R2 - H; 5,3",4"-trihydroxy-3,6,7-trimethoxyflavone Fig. (5). Flavonols from Plucchea sagittalis.

2p~rez-Garc|a, F.; Matin, E." Adzet, T.; Cafligueral, S. Taraxasteryl acetate, an active principle from Pluchea sagittalis: effects on inflammation, free radicals and stress protein synthesis. In: II World Congress on Medicinal and Aromatic Plants for Human Welfare (WOCMAP II). Mendoza, Argentina, November,

1997. (abstract O 02 I).

350

DESMARCHELIER eta/.

OH

R MeO,

O

HO

(1) R = Me; 5,6,3"-trihydroxy-7,4"-dimethoxyflavone (2) R = H; pedalitin Fig. (6). Flavonoids from Eupatorium inulaefolium. Table 3.

Effects of Polyphenols Isolated from Argentine Medicinal Plants on tertbutyl H y d r o p e r o x i d e - i n i t i a t e d Chemiluminescence of Mouse Liver Homogenates* ,

i

i

ii

Polyphenol ,

IC50 (gM) ,,

i

Catechin

3

Eriodictyol

Myricetin 4,2',4'-Trihydroxy-6'-methoxychalcone 3,4-Dicafeoylquinic acid

20

Isochlorogenic acid

30

Cafeic acid

50

5,6,3'-Trihydroxy-7,4'-dimethoxyflavone

5O

Cynarin

50

Chlorogenic acid

150

Apigenin

150

Quercetin

200

Pedalitin

200

Sylimarin

200

Quercetin-3-methyl ether

200

7,4'-Dihhydroxy-5-methoxyflavonone

500

Kaempferol-3,7-dirhamnoside

500

Quercitrin i

*Taken from Fraga et al., 1987, [32].

900 ii,

1

SEARCtl FOR ANTIOXIDANT ACTIVITY

351

Different flavonoids and polyphenols isolated from Argentine medicinal plants were assayed in vivo as liver chemiluminescence inhibitors to determine their protection against oxidative stress produced by CC14, a potent stimulator of lipid peroxidation [32]. Previously, a large number of polyphenols was screened in vitro for their abilities to inhibit CL in liver homogenates (Table 3). The highest activities were observed in catechin, Fig. (7), and eriodyctiol, Fig. (8), suggesting that these compounds are highly effective as water-soluble protectors against lipid peroxidation and other free radical-mediated cell injury. H HO

H

Fig. (7). Catechin from Heisteria pallida. OH H HO

OH

O

Fig. (8). Eriodyctiol from Argentine medicinal plants.

ANTIOXIDANT ACTIVITY IN MEDICINAL PLANTS FROM THE ANDES The Andes is a mountain range system extended along the Pacific coast of South America, from northern Venezuela down to the far south of Chile and Argentina. The average altitude of the system is 3,500 m, and with an extension of 8,500 km, also occupies large areas of Colombia, Ecuador, Peru and Bolivia. The remarkable variations in altitude that occur along the Andes are the source of unique biological communities to particular and small areas, sometimes harbouring many endemic plant species [41 ]. As in other regions of the Continent, the traditional use of plants in healing practices is very extended among the inhabitants throughout this region [e.g. 42]. The antioxidant capacity of closely related lignans isolated from Chilean medicinal plants was assessed by their effects upon the rate of rat

352

DESMARCHELIER et aL

Moo/

XoMo

RI = H; R2 = H; dihydroguayaretic acid RI = H; R2 = OMe; isopregomisin R I = OMe; R 2 = OMe; guayacasin Fig. (9). Lignans from Porlieria chilensis.

brain homogenate autoxidation [43]. The structurally similar lignans dihydroguayaretic acid, guayacasin and isopregomisin, Fig. (9), isolated from Porlieria chilensis Johnst. (Zygophyllaceae), commonly known as "guayacfin" and traditionally used as an antirheumatic, proved to be powerful antioxidants with activities similar to that of propyl gallate. The results obtained (Table 4) suggest that the free radical scavenging activity in these compounds can be explained in terms of their highly substituted phenolic structures and to the number of methoxyl groups present in their structure. The flavonoids 3"-methoxycalicoptedn and 7"-methylsudachitin, Fig. (10), from Baecharis incarum Wedd. (Compositae), also tested under this method, were considerably less active than the studied lignans or quercetin, isolated from Aristotelia chilensis (Mol.) Stuntz. (Elaeocarpaceae), another medicinal plant of the region commonly known as "maqui". Table 4.

Antioxidant Capacities of Lignans and Flavonoids from Chilean Medicinal Plants* ii

Compound

(IC50)TBAR 0tM)

Dihydroguayaretic acid

2.8

Guayacasin

1.1

Isopregomisin

0.7

Propyl gallate

1.0

Quercetin

!.] (0.8) !

3~

64.0 (48.0) 1

7"-Methyisudachitin

60.0 (51.0) 1 ,

I IC50 determined through luminescence measurements. *Taken from Faur~ et al., 1990, [43].

i

lll l

L


353

MeO o.e

Meo- .I .fL OH

O

R -- OMe; 3"-methoxycalieopterin R = H; 7"-methylsudanehitin Fig. (10). Flavonoids from Baccharis incarum.

The leaf decoctions of Tessaria integrifolia R. et P. and Mikania cordifolia Willd. (Compositae) are used in traditional medicine of the Peruvian Andes as anti-inflammatory agents. Three caffeoylquinic acids isolated from these species were tested for their activities on monoeyte migration and superoxide anion production [44]. 3,5-Di-O-caffeoylquinic and 4,5-Di-O-caffeoylquinic acids exhibited an appreciable antiinflammatory activity in vitro, while the tricaffeoyl derivative was inactive. The compounds in study also decreased the zymosan induced liberation of O2-by macrophages, which was dependent on the concentration of the inhibitory molecules. These results indicate that the caffeoylquinic acids isolated from the leaves of T. integrifolia and M. cordifolia could affect migration and 02-secretion in activated human macrophages. The aerial parts of Eupatorium articulatum L. (Compositae) are widely used in the Andes region of Ecuador for the treatment of inflammatory diseases. The methanolic extract of this species exhibited a significant in vivo anti-inflammatory activity, when tested for its ability to reduce carrageenan-induced inflammation in rat paw oedema, thus supporting the traditional use claimed for this plant. Since the inflammation process is associated to the generation of reactive oxygen species and the induction of lipid peroxidation, the antioxidant properties of the extract were also studied [45]. Significant activity was observed when tested for its ability Table 5.

Percentage Inhibition in C a r r a g e e n a n Induced I n f l a m m a t i o n , M i c r o s o m a i Lipid Peroxidation, Superoxide Generation and Xanthine Oxidase Production in the Presence of the Ethanolic Extract of E. artlculatum*

Lipid peroxidation

inflammation

i

I00 (g/ml I

3hi

592 i

0 2 9generation

xanthine oxidase

70.6 (I.I

81.2 (2.1

i

5h

I

7h

....

42.3 I 53.6 I I

*Taken from de las Heras et al, 1997, [45].

.

I

61.7 (I.1 .

.

.

DESMARCHELIER eta/.

354

to inhibit non-enzymatic lipid peroxidation in rat liver microsomes induced by FeCl3-ascorbate and in scavenging superoxide anions by the inhibition xanthine oxidase activity (Table 5). Alkaloids and other nitrogen compounds of higher plants have also shown to exert antioxidant effects in various systems. These effects include inhibition of peroxidation in rat liver homogenates and physical quenching of superoxide radicals [3]. Boldine, Fig. (11), is an alkaloid present in the leaves of Peumus boldus Mol. (Monimiaceae), a widely distributed evergreen tree native to Chile. Boldine-containing boldo leaf extracts and infusions are used in traditional medicine in the treatment of a variety of conditions, among which liver complaints and dysfunctions are generally mentioned [46]. Anti-inflammatory and antipyretic effects of boldine have been previously described [47], and recent studies have shown that boldine behaves as a very potent antioxidant in biological systems undergoing lipid peroxidation, thus providing a possible rationale for the hepatoprotective properties attributed to boldo .[48,49,50,51,52]. HO CH30

CH30"

y OH

Fig. (11). Boldine from Peumus boldus.

Leaf extracts of another Chilean medicinal plant, Psoralea glandulosa L. (Fabaceae), have shown to possess anti-pyretic and anti-inflammatory activities [53]. A major component of this plant is the meroterpenoid bakuchiol, Fig. (12), which has been claimed as the active principle, responsible for the therapeutic properties described [54]. Although bakuchiol was demonstrated to be a natural anti-inflammatory agent able to control leukocyte functions such as eicosanoid production, migration and degranulation in the inflammatory site, it only showed a weak inhibition in the generation of superoxide. Future studies will be necessary in order to determine if this compound is capable of reducing lipid peroxidation and other free radical induced cell damage.

HO Fig. (12). Bakuchiol from Psoralea glandulosa.


355

BRAZILIAN AMAZON The region of the Amazon forest in Brazil is roughly delimited by latitudes 0 ~ and 12~ and longitudes 48 ~ and 74~ and has an average elevation of 100 m and an area of 3.4 million km 2 [55]. Besides high indices of botanical diversity and endemism, this large and heterogeneous territory contains a large number of new undescribed species [56], and is inhabited by different native groups which have extensive experience with medicinal plants. Palm trees are extensively distributed in the Amazon and in other tropical ecosystems of South America. It has been determined that oil palm (Euterpe spp., Arecaceae) is the best reported source of tocotrienols, Fig. (13), which have been regarded as better than ct-tocopherol and vitamin E for their antioxidant activity [57]. Tocotrienols are natural Vitamin E analogues which differ from tocopherols in the number of methyl groups besides having an unsaturated chain [58]. Another Brazilian palm, Elaeis guineensis Jacq. (Arecaceae), has also been stated as an excellent source of 13-carotene and tocotrienols [59]. Carotenoids such as 13-carotene, "t-carotene and lycopene have shown to act in vitro as antioxidants at low oxygen concentrations by forming a stabilised radical after addition of peroxyl radicals [60], and by reducing the in vivo production of pentane, an oxidation product of fatty acids during lipid peroxidation [61 ]. HO

Fig. (13). y-tocotrienol from Euterpe, Elaeis, lryanthera and Virola spp.

Different species of the rainforest trees Virola and lryanthera (Myristicaceae) contain a large number of chemically different substances with antioxidant activity, including tocotrienols [62]. For example, two tocotrienols isolated from the ethanolic fruit extract of Iryanthera grandis Duke. have demonstrated in vitro antioxidant activity. Tocotrienol-8 antioxidant capacity measured as the inhibition of spontaneous brain autoxidation was 10 times higher than Vitamin E in the MDA assay and 8 times higher in the chemiluminescence assay 3. Bioassay guided fractionation also showed that two flavonoids, 3-0-rhamnosil-kaempferol and 3-0-rhamnosil-quercetin, together with a novel dimmeric dihydrochalcone, Fig. (14), are responsible for the inhibition of the spon-

3Barros, S. Higher plants as sources of antioxidants for the treatment of oxidative stress mediated disease. In: International Symposium: Oxygen Radicals in Biochemistry, Biophysics and Medicine. Buenos Aires, March, 1994 (abstract $8).

356

DESMARCIIELIER et aL

OH HO

OH

O

R = H; 3-0-rhamnosil-kaempferol

R = OH; 3-0-rhamnosil-quercetin

O

HO,

OH

Me 1000

3 (! 7-0.01)

561 (1239-337)

C. lechleri 2

935

NA

NA

161 (912-48)

ND

200 (950-154)

H. pallida 1

3850

12

NA

636 (1201-397)

ND

NA

U. tomentosa I

84

56 (36-83)

889 (2415.469)

17 (50-I)

NA

I methanolic extract, 21iophylized latex. Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) are expressed in (M Trolox equivalents. CL and TBARS are expressed as IC50 ((g/ml), and include 95% confidence intervals. NA: not active; ND: not determined due to lack of dose dependence.

"Sangre de drago" is a red viscous latex obtained from the bark of Croton lechleri Muell. Arg. (Euphorbiaceae) and other species of Croton. Cicatrizant, anti-inflammatory, antiviral and antitumour properties have been claimed for "sangre de drago" by indigenous populations in many parts of the westernmost part of the Amazon valley, including Colombia, Ecuador, Peru and Bolivia (83). In addition to the alkaloid taspine [84] and the lignan 3",4-O-dimethylcedrusin [85], responsible for the antiinflammatory, antiviral, antitumour and wound healing properties, the latex of C. lechleri was found to contain several proanthocyanidins as major constituents, which account for up to 90 % of the dried weight. These include catechin, epicatechin, gallocatechin, epigalloeatechin, Fig. (20), and five novel dimmers and trimmers of these compounds [86], Fig. (20). The implication of antioxidation in the wound healing process, together with the participation of oxygen free radicals in inflammation and carcinogenesis, has lead us to study the free radical trapping capacity of "sangre de drago" [87]. The capacity of the lyophilised latex from C. lechleri to scavenge peroxyl radicals derived from thermolysis of ABAP was determined by monitoring the intensity of luminol enhanced chemiluminescence. Although "sangre de drago" was highly effective in scavenging free radicals at high concentrations, the additive incorporation of lower concentrations of the latex yielded an instantaneous increase in chemiluminescence, suggesting prooxidant activity. DNA sugar damage induced by Fe (II) salts was also used to determine the capacity of the latex to suppress hydroxyl radical-mediated degradation of DNA. As in the case of luminol enhanced chemiluminescence, "sangre de drago" was

362

DESMARCHELIER et aL

Epieatechin OH

HO

~H

,,,( H Galloeateehin

OH

HO

,,,( ~H '~'t3H Epigallocateehin

Fig. (20). Proanthocyanidins from Croton lechleri.

highly effective in reducing oxidation of DNA at higher concentrations, but showed an increase in the production of TBARS at lower doses, compared to the control. Lipid peroxidation was also inhibited in the presence of the latex, as determined by a reduction in the production of TBARS in rat liver homogenates, both in vitro and in vivo. The results obtained are summarised in Table 8. Uncaria t o m e n t o s a (Willd.) DC. is a vine belonging to the family Rubiaceae that grows in the Amazon forests of Bolivia and Peru, and is locally known as "ufia de gato". The aqueous extract and decoction of the bark of this plant are widely used in traditional Peruvian medicine for the treatment of cancer and as a potent anti-inflammatory agent. Antioxidant activity in extracts of U. tomentosa was determined using different bioassays, and the free radical scavenging capacity was observed both against lipid-peroxidation and DNA damage in methanolic extracts of bark


363

and roots (Table 8) [88]. Since U. tomentosa extracts and fractions previously showed significant anti-inflammatory activity in rat paw oedema [89] and antimutagenic effects against induced photomutagenesis [90], it may be proposed that their efficacy could be partially due to their free radical scavenging activity. Although the compounds responsible for the antioxidant activity observed in U. tomentosa have not yet been identified, the presence of flavonoids kaempferol and dihydrokaempferol, Fig. (21), has been previously reported in the bark of the related species U. guianensis, also known as "ufia de gato" [91 ].

HO

Kaempferol

OH HO

Dihydrokaempferol Fig. (21). Flavonoids from Uncaria guianensis.

FUTURE The search for new active natural products such as antioxidants in South American plants has, in many aspects, only just begun. The poorly known flora of the drylands of"Patagonia", located in the far south of Argentina; the xerofitic vegetation of the "Caatinga" region, which covers almost 1 million km 2 in north-eastern Brazil and is extensively used in local popular medicinal practice, particularly among the African derived populations which inhabit the region; the flora of the "Pantanal", a swamp ecosystem in west centre Brazil, which is highly preserved and with scarce registered ethnobotanical data; and the Chocoan rainforests in north-west Colombia, all enclose, with no doubt, pharmacological secrets that are waiting to be unveiled in the future.

364

DESMARCHELIER eta/.

ACKNOWLEDGEMENTS Studies on antioxidant activity in South American medicinal plants are funded by the International Foundation for Science (IFS), in Stockholm, Sweden (Grant agreement No. F/2628-1), BID-CONICET (grant PMTSID0370) and the University of Buenos Aires (grant UBACYT FAO12/J). REFERENCES [~] [21 [31 [4] [51 [6] [71 [8] [91 [10] [ll] [121 [131 [14]

[151 [161 [171 [18]

[19] [201

[21] [22] [23] [24]

Gordon, M. Nat. Prod. Rep., 1996, 265. Bast, A.; Goris, R. Pharm. Weekbl. [Sci.], 1989, 11, 199. Larson, R. Phytochemistry, 1988, 27, 969. Haslam, E., J. Nat. Prod., 1996, 59, 205. Kinsella, J.; Frankel, E.; German, J.; Kanner, J. J. Food Technol., 1993, 85. Kinsella, J.; Frankel, E.; German, J.; Parks, E., Kanner, J. Lancet, 1993, 341, 454. Maxwell, S.; Cruickshank, A; Thorpe, G. Lancet, 1994, 344, 193. Serafini, M.; Ghiselli, A.; Ferro-Luzzi, A. Lancet, 1994, 344, 626. Kondo, K.; Matsumoto, A.; Kurata, H.; Tanahashi, H.; Koda, H., Amachi, T.; Itakura, H. Lancet, 1994, 344, 1152. Rice-Evans, C. Biochemist, 1996, 17, 8. Cox, P; Balick, M. Sci. Am.,1994, June, 82. Caldwell, M.; Robberecht, R.; Flint, S. Physiol. Plant., 1983, 58, 445. Levin, D. Am. Nat., 1976, 110, 261. Larson, R.; Marley, K. Phytochemistry, 1984, 23, 2351. Gentry, A. In Human Medicinal Agents from Plants, Kinghorn, D.; Balandrin, M., Eds.; ACS Symposium Series, Maple Press, York, P.A., 1993; pp. 13-24. Bors, In Handbook ofAntioxidants; Cadenas, E.; Packer, L., Eds.; Marcel Dekker Inc., N.Y., 1996; pp. 409-466. Phillips, D. In Recent Advances in Phytochemistry. Vol. 26. Phenolic Metabolism in Plants, Stafford, H., Ibrahim, R., Eds.; Plenum Press, N.Y., 1992; pp. 201231. Soejarto, D.; Gyllenhaal, C.; Ashton, P.; Sohmer, S. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Eds.; Columbia University Press, N.Y, 1996; pp. 284-310. Cross, C.; Halliwell, B.; Borish, E.; Pryor, W.; Ames, B.; Saul, R.; McCord, or. Ann. Intern. Med., 1987, 107, 526. Lin, J.; Lin, C.; Chen, M.; Ujiie, T.; Takada, A. J. Ethnopharmacol., 1995, 47, 33. Seivam, R.; Subramanian, R.; Gayathri, R.; Angayarkanni, N. J. Ethnopharmacol., 1995, 47, 59. Sultana, S.; Perwaiz, S.; Iqbal, M.; Athar, M. J. Ethnopharmacol., 1995, 45, 189. Middleton, E. Int. J. Pharmacognosy, 1996, 34, 344. Baumann, J.; Wurm, G.; Bruchhausen, F. Arch. Pharm., 1980, 313, 330.


365

[25] Sorata, Y.; Takahama, U.; Kimura, M. Biochem. Biophys. Acta, 1984, 799, 313. [26] Yokozawa, T.; Dong, E.; Wu Liu, Z.; Shimizu, M. Phytother. Res., 1997, 11, 446.

[27] Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25,383. [281 Broussalis, A.; Ferraro, G.; Gurni, A.; Coussio, J. Acta Farm. Bonaerense, [29]

[30] [31] [32] [33] [34]

[35] [36] [37]

[38] [39]

[40] [41] M2] [43]

[44] [45] [461 [471

[48] [49] [50] [51] [52]

1989, 8, 11. Desmarchelier, C.; Coussio, J.; Ciccia, G.; Braz. J. Med. Biol. Res. 1998, 31, 1163. Lissi, E.; Pascual, C.; del Castillo, M. Free Rad. Res. Com. 1992, 17, 299. Norbedo, C.; Ferraro, G.; Coussio, J. or. Nat. Prod., 1982, 45, 635. Fraga, C.; Martino, V.; Ferraro, G.; Coussio, J.; Boveris, A. Biochem. Pharmacol., 1987, 36, 717. PayS, M.; Silla, M.; Vay~, E.; Alcaraz, M.J.; Coussio, J.; Ferraro, G.; Martino, V.; Hnatyszyn, O.; Debenedetti, S.; Broussalis, A.; Muschietti, L. Phytother. Res., 1996, 10, 228. Debenedetti, S.; Palacios, P.; Wilson, E.; Coussio, J. Acta Hort., 1993, 333, 191. Debenedetti, S.; Palacios, P.; Wilson, E.; Coussio, J. Fitoterapia, 1994, LXV, 188. Jarvis, B.; Midiwo, J.; Bean, G.; AbouI-Nasr, B.; Barros, C. (1988) J. Nat. Prod., 1988, 51,736. Mongeili, E.; Desmarchelier, C.; Rodriguez Talou, J.; Coussio, J.; Ciceia, G. J. Ethnopharmacol. 1997, 58, 157. Desmarchelier, C.; Novoa Bermudez, M.J.; Coussio, J.; Ciccia, G.; Boveris, A. Int. d. Pharmacognosy 1997, 35, 116. Martino, V.; Ferraro, G.; Debenedetti, S.; Coussio, J. ,4cta Farm. Bonaerense, 1984, 3, 141. Ferraro, G.; Martino, V.; Coussio, J. Phytochemistry, 1977, 16, 1618. Gentry, A. In Conservation Biology; M.E. Soule, Ed., Sinauer Associates, Sunderland, Mass., 1986, p. 78. Girault, L. Kallawaya: curanderos itinerantes de los Andes. UNICEF-OPSOMS" Paris, 1987. Faur6, M.; Lissi, E.; Torres, R.; Videla, L. Phytochemistry, 1990, 29, 3773. Peluso, G.; de Feo, V.; de Simone, F.; Bresciano, E.; Vuotto, M.L.J. Nat. Prod. 1995, 58, 639. de las Heras, B.; Slowing. K.; Bened, J.; Carretero, E.; Ortega, T.; Toledo, C.; Bermejo, P.; Iglesias, I.; Abad, M.J.; Serranillos, P.G.; Liso, P.A.; Villar, A.; Chiriboga, X. J. Ethnopharmacol. 1998, 61, 161. Speisky, H.; Cassels, B.; Nieto, S.; Valenzuela, A.; Nuflez-Vergara, L. J. Chromatogr., 1993, 612, 315. Backhouse, N.; Delporte, M.; Guivemau, M.; Cassels, B.; Speisky, H. Rev. Med. Chile, 1994, 31, 114. Bannach, R.; Valenzuela, A.; Cassels, L.; Nuflez-Vergara; Speisky, H. Cell Biol. Toxicol., 1996, 12, 89. Campos, A.; Lissi, E. Bol. Soc. Chil. Quire., 1995, 40, 375. Speisky, H.; Cassels, B.; Lissi, A.; Videla, L. Biochem. Pharmacol., 1991, 41, 1575. Speisky, H.; Cassels, B. Pharmacol. Res. 1994, 29, 1. Valenzuela, A.; Nieto, S.; Cassels, B.; Speisky, H. J. Am. Oil Chem. Soc., 1991, 68, 935.

366

[53] [54]

[551

[56] [571

[58] [59]

[60] [611 [62] [63]

[64] [651 [66] [67] [68]

[69] [70] [71] [72] [73]

[74] [75] [76] [77]

[78] [79]

[80] [81]

DESMARCHELIER et aL

Backhouse, N.; Delporte, C.; Negrete, R.; Salinas, P.; Pinto, A.; Aravena, S.; Cassels, B. Int. d. Pharmacognosy, 1996, 34, 53. Ferr~ndiz, M.L.; Gil, B.; Sanz, M.; Ubeda, A.; Erazo, S.; Gonzfilez, E.; Negrete, R.; Pacheco, S.; Pay~t, M.; AIcaraz, M.J.J. Pharm. Pharmacol., 1996, 48, 975. Souza Brito, A. R. M.; Souza Brito, A. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Ed.; Columbia University Press, N.Y, 1996; pp. 386401. Gentry, A. In Biological Diversification in the Tropics; Prance, G., Ed.; Columbia University Press, N.Y. 1982; p., 714. Duke, J.A.; Vasquez, R. Amazonian Ethnobotanical Dictionary, CRC Press: Boca Rat6n, 1994. Traber, M. In Handbook ofAntioxidants; Cadenas, E.; Packer, L., Eds.; Marcel Dekker Inc., N.Y., 1996; p. 43-61. Melo, M.; Mancini, J. Rev. Farm. Bioquim. Univ. Sao Paulo, 1989, 25, 147. Burton, G.; Ingold, K., Science, 1984, 224, 569. Allard, J.; Royall, D.; Kurian, R.; Muggli, R.; Jreejeebhoy, K. Am. J. Cli. Nutr., 1994, 59, 884. Dugall, S.; Kartha, A. Indian J. Agric. Sci., 1956, 26, 391. Barros, S.; Teixeira, D.; Aznar, A.; Moreira Jr, J.; lshii, I.; Freitas, C. Cienc. Cult. Sao Paulo, 1996, 48, 114. Desmarchelier, C.; Mongelli, E.; Coussio, J.; Ciccia, G. Braz. d. Med. Biol. Res. 1997, 30, 85. Kijjoa, A.; Giesbrecht, A.; Akisue, M.; Gottlieb, O.; Gottlieb, H. Planta Med., 1980, 39, 85. Gustafson, K.; Cardellina, H.; McMahon, J.; Pannell, L.; Cragg, G.; Boyd, M. J. Org. Chem. 1992, 57, 2809. Desmarchelier, C.; Repetto, M.; Coussio, J.; Ribeiro Latorre, L.; Kato, M.; Barros, S.; Ciccia, G., Planta Meal, 1997, 63, 561. Van Acker, S.; Koymans, L.; Blast, A. d. Free Radic. Biol. Med., 1991, 15, 311. Houghton, P. J. Alt. Compl. Med., 1995, I, 131. Tyler, V. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Eds.; Columbia University Press, N.Y, 1996; pp. 3-10. Desmarchelier, C.; Gurni, A.; Ciccia, G.; Giulietti, A. d. Ethnopharmacol., 1996, 52, 45. Lambelet, F.; Saucy, F.; Loliger, J. Experientia, 1985, 41, 1384. Serbinova, E.; Kagan, V.; Han, D.; Packer, L. Free Radical Biol. Med., 1991, 10, 263. Rifici, V.; Khachadurian, K. J. Am. Coll. Nutr., 1993, 12, 631. Koch, H. Deut. Apoth. Ztg., 1988, 128, 2493. Akhtar, M.; Khan, Q.; Khaliq, T. J. Pak. Med. Ass., 1985, 35, 207. Simopoulos, A.; Norman, H.; Gillaspy, J.; Duke, J. Journal of the American College of Nutrition, 1992, 11,374. Burton, G.; Ingold, K., J. Am. Chem. Soc., 1981, 103, 6472. Gupta, M. (Ed.) 270 plantas medicinales iberoamericanas. CYTED, Santaf6 de Bogota, 1995. Desmarchelier, C.; Repetto, M.; Coussio, J.; Llesuy, S.; Ciceia, G. Int. d. Pharmacognosy 1997, 35, 288. Desmarchelier, C.; Coussio, J.; Ciccia, G.; Phytother. Res. 1997, 11, 460.

SEARCHFOR ANTIOXIDANTACTIVITY

[82] [83] [84]

[85] [86] [87]

[88] [89] [90] [91]

367

Ferrfindiz, M.; Alcaraz, M. Agents and Actions, 1991, 32, 283 Anonymous. Plantas medicinales amaz6nicas: realidad y perspectivas. Tratado de Cooperaci6n Amaz6nica, Secrctaria Pro-Tempore, Lima, 1995. Vaisberg, A..; Milla, M.; Planas, M.; Cordova, J. de Agusti, E.; Ferreyra, R.; Mustiga, M.; Carlin, L.; Hammond, G. Planta Med., 1988, 55, 140. Picters, L.; de Bruyne, T. Claeys, M.; Vlietinck, J. J. Nat. Prod., 1993, 56, 899. Cai, Y.; Evans, F.; Roberts, M.; Phillipson, J.D.; Zenk, M.; Gleba, Y.; Phytochemistry, 1991, 30, 2033. Desmarchelier, C.; Witting Schaus, F.; Coussio, J.; Ciccia, G.; J. Ethnopharmacol. 1997, 58, 103. Desmarchelier, C.; Mongelli, E.; Coussio, J.; Ciccia, G. Phytother. Res. 1997, 11,254. Aquino, R.; De Feo, V.; De Simonr F.; Pizza, C.; Cirino, G. J. Nat. Prod. 1991, 54, 453. Rizzi, R.; Re, F.; Bianchi, A.; de Feo, V.; de Simone, F.; Bianchi, L.; Stivala, L.A.J. Ethnopharmacol. 1993, 38, 63. Lock de Ugaz, O.; Callo, N. Revista de Quimica, 1991, 5, 47.



369


INSECT JUVENH E HORMONES IN PLANTS JACQUELINE C. BEDE and STEPHEN S. TOBE*

Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada. A s Gregor Samsa a,:.,oke one morning from uneasy dreams he found hanaelf transformed ln hls bed lnto a glgantie lnsect. -

en "The

ea.n xpho a" E t )

ABSTRACT: In the defence against insect herbivory, plants may produce compounds which interfere with the insect endocrine system [2, 3, 4]. The focus of this review is to provide an overview of plant secondary metabolites which act either as insect juvenile hormone (JH) mimics or interfere with JH biosynthesis. Juvenile hormones are

sesquiterpenoids which are involved in the regulation of developmental processes such as metamorphosis and reproduction in most insect species [5]. The first section of this review provides an overview of insect JHs, including their physiological action and biosynthesis in hemimetabolous insects such as the cockroach, Diplopterapunctata, and their general chemistry. In the next part, examples of phytochemicals which act as JH mimics (juvenoids) or which inhibit JH biosynthesis will be presented, illustrating the diversity of compounds which are able to interfere with the endocrine system of the insect. Finally, a unique example of the production of insect juvenile hormone III (JH III), methyl-1OR,11-epoxy-3,7,1l-trimethyl 2E,6E-dodeeadienoate, in the sedge Cyperus iria L. will be presented and the possible biological role(s) of this compound discussed. INTRODUCTION One defensive strategy of plants against insect herbivory is the production of secondary metabolites which interfere with the physiology of the insect [6, 7]. It is thought that targeting the insect endocrine system makes it difficult for the insect to evolve counteradaptive strategies [3]. Insect juvenile hormones (JHs) are sesquiterpenoids which are involved in the regulation of insect metamorphosis and reproduction [5, 8]. In some insect species, these hormones are also involved in the regulation of other physiological processes including diapause and behaviour [8]. Plants have been shown to contain compounds which can either mimic JH activity or act as antagonists by inhibiting JH biosynthesis. The purpose of this review is to highlight some of these phytochemicals which are known to

370

BEDE and TOBE

interfere with the endocrine system of insects, specifically with the function of insect JH. Although there are many examples of plant secondary compounds which interfere with the physiology of insects [6, 7], there is debate as to the nature of the selective forces responsible for the evolution of these compounds [9, 10]. The argument has been made that although it appears that these secondary metabolites serve to protect the plant against insect herbivory today, it is likely that they were initially selected as a response to vertebrate herbivory [ 11 ]. However, phytochemicals which specifically interfere with the insect endocrine system possibly represent compounds which have evolved as protection against insect attack and therefore represent plant adaptations to insect herbivory. INSECT JUVENILE HORMONES To date, six JHs have been identified and their structures elucidated (Fig. (1)). Structurally, they share a sesquiterpenoid skeleton, a methyl ester O OMe

JH O

0 OMe

4-methyl J H I

O JH I

OMe O J H II OMe O J H Ill OMe

O J H III blsepoxide

OMe Fig. (1). Structures of insect juvenile hormones (JHs).

JUVENILE HORMONES

371

moiety on C-1 and an epoxide function. Juvenile hormone III (JH llI), methyl- 1OR, 11-epoxy-3,7,11-trimethyl 2E,6E-dodecadienoate, is the most common of these hormones and has been isolated from Lepidoptera (moths and butterflies), Coleoptera (beetles), Hymenoptera (sawflies, wasps, ants and bees), Orthoptera (grasshoppers, locusts, katydids, crickets), Dictyoptera (cockroaches) and Isoptera (termites) [ 12, 13]. In most of these insect orders, JH III is the only JH present. In the Lepidoptera, four other homologs have been isolated in addition to JH III. The ethyl branched homologs, juvenile hormone I (JH I), methyl- 1OR, 11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, and juvenile hormone II, methyl-10R, 1 1-epoxy-3,7,11-trimethyl-2,6tridecadienoate, and their corresponding acids predominate in this order [ 14, 15, 16]. Two additional homologs, juvenile hormone 0 (JH 0), 1OR, 11-epoxy-3,7-diethyl-11-methyl-2,6-tridecadienoate, and 4-methyl JH I have been isolated from embryos of the tobacco homworm, Manduca sexta; JH 0 has also been isolated from males of the silkworm, Hylophora cecropia [ 17, 18, 19]. In higher Diptera (flies), a unique JH bisepoxide, methyl-6,7,10,11-bisepoxy-3,7,11-trimethyl 2E-dodecenoate, has been isolated and appears to be the principle JH of some species in this order [20]. CHEMISTRY OF JUVENILE HORMONE HI Chemical data for JH III is summarized in Appendix I, Table 1. In the infrared spectrum, the band at 1720 cm -~ is characteristic of an ester carbonyl group (C=O) and the band at 1650 cm -I is reflective of the alkene nature of the molecule (C=C) [ 14, 21 ].

Mass Spectroscopy Electron impact mass spectroscopy routinely uses a high energy beam of 70 eV; however, in the fragmentation pattern of JH III (Appendix I, Table 2), important high mass ions are also observed in the low energy resolution spectrum (15 eV) [22, 23, 24]. The observed peak at m/z 248 results from the migration of two hydrogen atoms to the epoxide oxygen and the subsequent loss of water. Mass ions at m/z 234 and 206 characterize the methyl ester moiety; m/z 234 represents the loss of the methyl ester group (CH3OH) and m/z 206 represents the loss of the methyl ester and the C=O from the acid. Fragmentation cleavage patterns give rise to mass ions at m/z 195, 114, 81 and 71 [22]. Low mass ion fragments (m/z 135, 114, 81, 71 and 43), which are detected in both the high and low energy resolution spectra, arise from hydrogen migration patterns suggestive of McLafferty-type rearrangements [25]. Scheme I illustrates the hydrogen

372

BEDE and TOBE

m/e 266

1 §

o~

§ o

o§

m / e 43

mle 71

Scheme I. Mass spectroscopy fragmentation pattern of juvenile hormone III [23] (reprinted with kind permission from the American Chemical Society).

rearrangement which generates an ion of m/z 71 and demonstrates the relationship between fragments produced at m/z 71 and 43 [23]. The base peak ion (m/z 81) results from two carbon-carbon bond cleavages and the transfer of one hydrogen atom [23] and can be generated through mass ions of m/z 195, as shown by Trost [22], or m/z 153 [23]. The mass ion m/z 135 is generated through the fragmentation of ions at m/z 153 and 195 and also m/z 163 [23] (Lietke). Lastly, the intense ion peak at m/z 114, which is present in the spectrum of JH III and its biosynthetic precursor methyl famesoate (MF), may occur by hydrogen transfer through a McLaffertytype rearrangement or by the migration of a hydrogen from C-4 to the carbonyl oxygen followed by transfer of another hydrogen from C-8 or C8' to C-4 [23-26].

JUVENILE HORMONES

373

§

~

9

m/e 266

H

m/e 195

OH * -H

CloHI5+

Of"

~

+ m/e 81

CH3OH

* - {CO+CH3OH) ~

CIoH15 +

~

* -

m / e 135

~

m/e 135

CIIHI5O+

CO

m/e 163

II. Mass spectroscopy fragmentation pattern of juvenile hormone III [23] (reprinted with kind permission from the American Chemical Society). Scheme

Nuclear Magnetic Resonance Proton Nuclear Magnetic Resonance Spectrum of Juvenile Hormone III Table 3 compares the assignment of chemical shifts based on three reports in the literature (Appendix I) [21, 22, 27]. The singlet at 8 3.72 represents the three protons on the methyl ester. The vinyl proton on C-2 is denoted by a signal at 8 5.75. The sharp doublet at 8 2.18 represents a vinyl methyl group which is cis and 13 to the carbonyl group. The vinyl proton on C-6 is indicated by ~55.22. The methyl on C-7 is shown by a singlet at ~i 1.65 The unusual chemical shift at 8 2.74 represents the epoxide proton on c - J 0 and the singlets at 8 1.27 and ~i 1.32 are due to the two methyl groups on C-11. The remaining protons on C-4, C-5, C-8 and C-9 are represented by bands at ~i 1.7 and ~i 2.1.

374

BEDE and TOBE

13CNuclear Magnetic Resonance Spectrum of Juvenile Hormone III The 13C nuclear magnetic spectrum assignment for JH III is listed in Table 4 (Appendix I) [28].

Chirality of Juvenile Hormone III

The absolute configuration of the epoxide group of insect JHs was determined by spectroscopy and circular dichroism [29]. In the first method, perchloric acid was used to catalyze the hydrolysis of the epoxide in the presence of H2180. The resulting vic-diol was analyzed by mass spectroscopy and definitively demonstrated the 10R,11S chirality of the epoxide [30]. The chirality was confirmed by circular dichroism. The epoxide was hydrolyzed by sulfuric acid and tetrahydrofuran and the circular dichroism cotton effect of the resulting acyclic glycol was measured in the presence of tris(dipivaloylmethanato)praseodymium [31]. General Chemistry of Juvenile Hormone III

Juvenile hormones are lipophilic molecules and relatively insoluble in aqueous solutions. The solubility of JH I is approximately 3 x 10.2 mM in 0.2 M Tris-HC1 buffer, pH 7.5 [32]; changes in pH, buffer and ionic strength had no effect on solubility. The addition of proteins such as bovine serum albumin (BSA) and immunoglobulin G increases the solubility of JH I; for example, in 5% BSA, a 1 mM solution of JH I can be made. Sonication also increases the solublity of the hormone by producing stable, finely dispersed micelles. Juvenile hormone III is more hydrophilic than JH I; in 5 mM Tris-HCl, pH 8.3, the solubility of JH III is greater than 2 x 10-1 mM [33]. However, its limited solubility must be recognized when preparing solutions. The epoxide group on insect JHs is particularly susceptible to hydrolysis through a SN2-type mechanism, generating the transdihydrodiol [27, 34, 35]. Therefore, protic acids, in aqueous or methanolic solutions readily convert the JH to its diol or 11-methoxy-10-hydroxy derivative, respectively [36, 37]. This reaction can be used to quantify JHs in insect haemolymph by generating derivatives which can either be analyzed directly or derivatized further and analyzed by gas chromatography-mass spectroscopy (GC-MS) (See Appendix I, Table 5 for references). Oxidation of the epoxide with neutral alumina or silica or heating to temperatures above 150oC results in an allylic alcohol which will undergo cyclization at higher temperatures (180oC)to a tetrahydrofuran derivative [27, 38]. Therefore, in the past, the direct analysis of JH by GC was contraindicated by the tendency for thermolysis at the temperatures required for volatization [38, 39].

JUVENILE HORMONES

375

However, this can now be overcome through the use of a cool-on-line injector and programming of the oven after sample injection (P. Teal, personal comm.). Cyclization of JH III to mono- and bicyclic products also occurs readily in the presence of boron trifluoride or phosphoric acid (H3PO4) [34]. Transition metals may catalyze similar reactions in aqueous solutions [12]. Inorganic salts, such as ferric chloride and zinc and magnesium sulfate, react with JH I, producing undetermined products [35]. The methyl ester function on C-1 of JH III is resistant to saponification by strong base [14]. One must also be aware of the binding affinity of JHs to different substrates. Juvenile hormones strongly adsorb to many commonly used plastic materials such as polystyrene, Millipore filters PSAC 02510, polyvinylchloride, polyethylene and plexiglass and to a lesser extent, glass and teflon [32]. Therefore, if plastics are used, it is recommended that they should be tested to determine the degree of JH affinity. Glassware should be treated with siliconizing agents or polyethylene glycol (PEG) to block JH binding sites [32]. Prior to this treatment, glassware should be washed with a nonionic detergent, rinsed in distilled water and heated to at least 200oC for 5 hours [27]. The use of an acid, such as chromic acid, in the washing of glassware is contraindicated because of the susceptible nature of the epoxide group to acid hydrolysis [27, 35]. Then the glassware can be treated with PEG or a siliconizing agent. Traces of acid catalyst in the PEG (Carbowax) may need to be removed before coating of the glassware, again because of the susceptibility of the epoxide ring to hydrolysis in the presence of acid. The solvents used should be of the highest quality and glass distillation is recommended in some cases [27]. Pure JHs are oils which can be stored for a number of years at -20oC or lower [ 12]. Solutions of JH, which are made up in aprotic, relatively nonvolatile solvents such as hexane or iso-octane, can also be stored at this temperature [12, 40]. In both these situations, the solution should be stored under an inert gas such as N2 to prevent the oxidation of the double bonds. Extraction and Quantification of the Juvenile Hormones

Methods for the extraction and quantification of physiological levels of insect JHs by chromatographic techniques (thin-layer chromatography, high performance liquid chromatography, gas chromatography-mass spectroscopy (GC-MS), immunological techniques (radioimmunoassay)) and the radiochemical assay will not be reviewed here but key references are listed in Appendix I, Table 5. Analysis by electron impact GC-MS requires an initial derivatization(s) due to the susceptibility of the epoxide group to thermolysis, followed by detection through electron capture or single ion monitoring. This treatment is not necessary with chemical ionization GC-MS.

376

BEDE and TOBE

Chemical Synthesis of Juvenile Hormone III The chemical synthesis of JH IIl will not discussed in this chapter; references on the synthesis are included in Appendix I, Table 5 and reviewed in the following [29, 41, 42]. ~

IOLOGICALROIEOF INSl ~ I ' ~ H O R M O N I ~

Metamorphosis Insect growth is discontinuous and comprised of discrete periods of metamorphosis, the change of form, which includes redifferentiation of body tissue and the shedding of the old cuticle. Pterygote (winged) insects undergo either complete metamorphosis (holometabolous, such as Lepidoptera which show larval (caterpillar), pupal and adult stages) or incomplete (partial) metamorphosis (hemimetabolous, such as the cockroach in which the larval or juvenile form resembles the adult form). The regulation of this metamorphosis by insect hormones, the ecdysones and JHs, differs in holometabolous and hemimetabolous insects. This chapter will focus on the latter. In the events leading up to metamorphosis, neurosecretory cells secrete prothoracicotropic hormone which acts on the prothoracic gland to stimulate the synthesis and release of the moulting hormone, 20hydroxyecdysone [5]. Ecdysone and its metabolites are responsible for the induction of the moulting process whereas the titre of JH in the insect haemolymph in the interval prior to the moult determines the characteristics. In hemimetabolous insects such as the Pacific beetle cockroach, Diploptera punctata, the presence of JH III is necessary for the maintenance of juvenile characteristics: JH III biosynthesis by the corpora allata (CA) is relatively high throughout immature stages and there is a drop in the rate of production during the penultimate and final nymphal stages prior to the moult to an adult (Fig. (2)) [43].

Gonadotrophic Cycle In adult females of most insect species, JHs are involved in the regulation of reproduction and oocyte maturation. Juvenile hormone-dependent processes include the synthesis of vitellogenin, the precursor of the yolk protein, by the fat body and the release of vitellogenin and its subsequent uptake and incorporation into the oocyte [44, 45]. Most insects are oviparous, laying eggs externally which undergo subsequent embryogenesis and development. However, there are also examples of viviparous insects, in which the fertilized egg undergoes early development within the female and derives nourishment from her, and

JUVENILE HORMONES

Penultimate instar 3.5 -mm

=3

377

Last instar 3.5-

Ecdysis

2.5

3.02.5-

l

2.0

2.0-

1.5

1.5

1.0

1.0

is

3.0

", S 0.5 0.0

~

0.5o

g

1'o

2'o

0.0

0

5

1'0

..... 1'5 270

Age (days) Fig. (2). Rates of juvenile hormone III (JH III) biosynthesis by the corpora ailata (CA) of the penultimate and final instars of the cockroach, Diploptera punctata [43]. The rate of biosynthesis, which is closely correlated to haemolymph titers, was measured by an in vitro JH III radiochemical assay [45, 46, 47]. Each point represents the mean of 3-12 determinations. Arrows indicate approximate times of ecdysis (reprinted with kind permission from Academic Press).

ovoviviparous insects, in which the eggs are incubated within the reproductive tract of the female. In these last two situations, there is a period of gestation within the insect. These three categories are represented within the order Dietyoptera (cockroaches), allowing comparison of the relationship between reproductive strategies and JH III titres in these insects [51, 52]. In the oviparous brown-banded cockroach, Supella longipalpa, females produce successive oothecae (eggs and protective easing) at five to seven day intervals. Each cycle of maturing oocytes is closely associated with a cycle of JH III biosynthesis by the corpora allata (CA) (Fig. (3)) [48]. Interestingly, mating is not necessary for the production and release of JH III and a similar trend is observed in virgin females [48]. This can be compared to JH III titres in another oviparous insect, the American cockroach, Periplaneta americana. In this species, mated adult females produce batches of eggs in rapid succession and the vitellogenir cycle encompasses two asynchronous egg-laying periods. In this situation, there is continuous JH III biosynthesis and no time when the hormone is completely absent [53, 54].

378

BEDE and TOBE

30 t 0

o,,,,4

m

Mating

25

20

=i "=1

& 0'

-

0

| . . . . . . .

5

1'0

1'5

2'0

Age (days) Fig. (3). Juvenile hormone III (JH III) release from the corpora allata (CA) of the oviparous cockroach, Supella longipalpa [48]. Juvenile hormone release was measured by the in vitro radiochemical assay [49, 50] and values represent the means of 5-12 determinations. Approximate times of mating and oviposition are indicated by arrows (reprinted with kind permission from Elsevier Scientific).

In both ovoviviparous and viviparous species of cockroaches, JH III biosynthesis is associated with vitellogenic growth of oocytes followed by periods of ovarian arrest and reduction of JH III biosynthesis during gestation [55, 56]. In mated females of the viviparous cockroach, Diploptera punctata, JH III titre is closely correlated to oocyte development (Fig. (4)) [57]; high rates of biosynthesis correspond to the rapid growth of oocytes as compared with the low synthesis observed during pre- and post-vitellogenic periods [52]. In adult virgin females, only basal JH III biosynthesis is observed. Similar results are seen in the ovoviviparous cockroach, Nauphoeta cinerea [58.]. BIOSYNTHESIS OF INSECT JUVENILE HORMONE III In D. punctata, JH III is biosynthesized in retrocerebral endocrine organs, the corpora allata (CA) [45]. The sesquiterpenoid skeleton of this compound is formed through the terpenoid biosynthetic pathway from acetyl-CoA. The early steps of this pathway involve the sequential condensation of three acetyl-CoA molecules (3 x 2C) to form the biosynthetic intermediate mevalonate (MVA, 6C). 3-Hydroxy-3methylglutaryl CoA reductase, which catalyzes the formation of MVA, is thought to be the rate-limiting enzyme in this pathway [59], although the

JUVENILE HORMONES

379

1800 1600 1400 1200 Q; *I,U

Oviposition

1000 800 600 400 200 0

-

o

....

....... 5"

--.,.,,~~

6'....... 7'

8"

lO

Age (days) Fig. (4). Juvenile hormone III (JH III) haemolymph titres in mated adult females of the viviparous cockroach, Diploptera punctata [57]. Haemolymph titre was measured by derivitizing JH III and subjecting the resultant methoxyhydrin to gas chromatography-mass spectroscopy [37]. Arrows indicate approximate time of oviposition (reprinted with kind permission from Birkhaeuser Pubi.).

regulatory importance of this enzyme has recently been questioned [60]. In the next series of steps, MVA undergoes three phosphorylations (net two) and a decarboxylation to generate the important isoprene intermediate, isopentenyl diphosphate (IDP, 5C); isomerization of this compound forms dimethyl allyl diphosphate (DMADP, 5C). Through head-to-tail covalent linkages of DMADP and IDP, prenyl diphosphate intermediates are formed which give rise to the terpenoid classes, such as the monoterpenes (10C), sesquiterpenes (15C), diterpenes (20C) and sterols. However, insects lack the enzymes to synthesize higher terpenoids [61] and JH III is synthesized from the fifteen-carbon farnesyl diphosphate (FDP). In vitro labelling of JH III in the CA of D. punctata has confirmed that the C-15 skeleton is derived from 3 moles of MVA [62]. In these studies, 6 moles of [2-14C]acetate were incorporated per molecule of JH III which reflects the expected incorporation of three acetate molecules, followed by the loss of a labelled carbon dioxide (14CO2) for every isoprene synthesized. From FDP, a phosphatase or pyrophosphatase catalyzes the removal of the pyrophosphate group, generating farnesol (Fig. (5)). The next two steps, oxidation of this alcohol to an aldehyde (farnesal), then a carboxylic acid (farnesoic acid), are catalyzed by one or two NAD+-dependent dehydrogenase(s) [63]. In the cockroach, D. punctata, methylation of

380

BEDE and TOBE

famesoic acid forms methyl famesoate (MF), followed by epoxidation at C 10,C 11 producing JH III [64, 65].

Farnesyl diphosphate

Phosphataseor pyrophosphataseI

Famesol

Famesoldehydrogenase,NAD+ 1

Farnesal

Farnesaldehydrogenase,NAD+ I

Farnesoic acid

JUVENILE HORMONES

381

(Fig. (5) contd .....

Methyl transferase S-adenosyl-methlonlne

!

Methyl farnesoate

Epoxidase 0 2, NADPH + H +

I

0 J u v e n i l e H o r m o n e HI Fig. (5). Biosynthetic pathway of juvenile hormone III in the cockroach, Diploptera punctata [65, 71].

The enzyme which catalyzes this methylation reaction, S-adenosylmethionine: famesoic acid o-methyltransferase (EC 2.1.1.-), is a cytosolic enzyme in the CA of adult female locusts, Locusta migratoria, and cockroaches, D. punctata [66, 67] and adult female tobacco homworms, M. sexta [68]. In a developmental profile, o-methyltransferase activity paralleled JH III biosynthesis in the CA of final larval instars and adult females of D. punctata [43, 57, 67]. The subsequent epoxidation reaction is catalyzed by a methyl farnesoate reduced flavoprotein: oxygen oxidoreductase (EC I. 14.14.-), which is associated with microsomal fractions in CA homogenates from the cockroach, Blaberus gigantus, and the locust, L. migratoria [66, 69, 70]. In Locusta, this enzyme was further shown to be a NADPH-dependent cytochrome P450 monooxygenase with an apparent Km of 7.7 x 10 -6 M, although the solubility of the substrate (methyl famesoate) limited the rate of the reaction. This pathway or variations of it occur in other insect species. The ethyl branches of the higher JH homologs found in the Lepidoptera (Fig. (1)) are derived from isoleucine and valine which are first metabolized to propionate and then incorporated, instead of acetate, in the early steps of biosynthesis [72, 73]. The sequence of the final steps of JH biosynthesis may also be different in these insect species. In the adult female CA of some Lepidoptera, epoxidation of farnesoic acid to the JH acid occurs before the final methylation step [64, 65, 71]. Interestingly, in larval stages of the tobacco hornworm, M. sexta, and adult males of the silkworm, Hyalophora cecropia, the CA synthesize and release JH acids into the

382

BEDE and TOBE

haemolymph which undergo the final methylation step in the imaginal disks or the accessory sex glands, respectively [74, 75]. In D. punctata, the haemolymph titre of JH is determined by the rate of biosynthesis and release from the CA and its degradation and clearance from the haemolymph by tissue uptake and excretion [ 13, 76]. The two primary routes of metabolism are through the cleavage of the methyl ester group by haemolymph esterases or the hydration of the epoxide by tissueassociated epoxide hydrolases. In the CA of final instars and in adult females of D. punctata, the low JH III titre in the haemolymph corresponds to low rates of biosynthesis by the CA and high esterase activity [43, 57, 77]. PLANT JUVENILE HORMONE MIMICS From the previous section, it is evident that JH III titers in insect haemolymph are precisely regulated during development. During the final stadium, there is a drop in haemolymph titer [43]. For example, in the penultimate stadium of the cockroach, D. punctata, the rate of JH III biosynthesis by the CA is between 1 to 3 pmol/hour per pair, but falls to undetectable levels later in the final stadium (Fig. (2)). Application of JH at this stage results in the inappropriate retention of juvenile characteristics at the next moult. The morphological effects are dependent on the dose of hormone, the age of the larvae and the sensitivity of the insect. In susceptible insects, application of JH to final stadium larvae may produce "adultoids" at the next moult or supernumerary larvae. In the latter case, the insect moults into a giant immature stage retaining juvenile features, without reaching sexual maturity. Therefore, application of synthetic JH analogues to insects in their final larval stages could be useful in the control of insects which are pests in their adult stages such as mosquitos and fleas. In adult female D. punctata, JH III is involved in the regulation of vitellogenin synthesis during ovarian development [44, 45, 78]. After the final moult and subsequent mating, the total amount of JH III in whole body extracts rises from 32.1 ng to its highest value on day 4 (196 ng), then falls to 12.8 ng over the next three days and remains at this low level [57]. Application of JH or synthetic JH analogues to insect eggs disrupts embryonic development. This can range from immediate ovicidal effects to delayed developmental effects [7]. Application to eggs result in apparently normal larvae which ultimately moult into supernumerary sixth-instars instead of adults [79]. Interestingly, the male linden bug, Pyrrhorocis apterus, treated with JH analogue, can transfer it to the female during mating, resulting in her sterility [80]. Therefore, application of JH or its synthetic analogues to preoviposition adult females or eggs may result in the disruption of embryogenesis.

JUVENILE HORMONES

383

One defensive strategy of plants against insect herbivory is the production of secondary metabolites which mimic JH activity [3]. Many of these compounds have been extracted from diverse plant species and are termed "juvenoids" based on their activity in vitro. However, a number of criteria should be met before these compounds are defined as JH mimics: Application of this compound should not block the insect's development but rather cause retention of juvenile characteristics in at least a single insect species [81 ]. The compound should demonstrate the ability to mimic JH at the three principle stages of insect development: the eggs, the juvenile and the adult (female). Therefore, observation of ovicidal effects is suggestive ofjuvenoid activity but not definitive. In the assessment of efficacy, dose response curves are necessary and allow the comparison of the activity of different compounds. These effects should represent the biological situation. For example, many of the studies described below have been performed using topical application of the putative juvenoid; this may not reflect the insect's natural contact with the chemical. The plant must be considered; for example, the amount of compound in the tissues must be sufficient to produce a biological effect on the test insect. In the evaluation of this, it should be remembered that many plants have specialized tissues which contain high levels of secondary metabolites. For example, many conifer species contain specialized secretory structures, such as resin ducts, which contain high localized concentrations of terpenoids [82]. There may also be dynamic variations in the amounts of these compounds in the plant, resulting from seasonal fluxes or synthesis induced upon stress. Lastly, other possible biological activities of the compound must be considered.

Juvabione, the "Paper Factor" The best known example of a plant-derived JH mimic is juvabione or the "paper factor" which was identified after it was observed that fifth instars of the linden bug, Pyrrhocoris apterus, from Europe, failed to mature into normal adults and instead metamorphosed into sixth instar supernumerary intermediates when reared in the United States [83]. The apparent cause of this was traced to the paper towels used in the rearing jars. Extracts from these paper towels Were only active against the Hemipteran insect, P. apterus, but inactive against pupae of the silkworms, Hylophora cecropia, H. gloverL Antheraea mylitta and Samia cynthia, and two other Hemipteran insects, Rhodnius prolixus and Oncopeltus fasciatus. Therefore, it appeared that only insects in the family Pyrrhocoridae are sensitive to this JH analogue.

384

BEDE a n d T O B E

In the search for the paper factor, extracts from several gymnosperms were assayed on P. apterus fifth instars (Table 1) [83, 84, 85]. Extracts of wood and bark of a number of these conifers were also injected into pupae of the wax moth, Galleria mellonella; a localized scaleless patch was observed in the pupal cuticle at the site of injection with these extracts. However, no abnormal effects on development were observed [86]. The authors speculated that this absence of activity may be attributable to the dilute concentration of active ingredients in the extracts. T a b l e 1.

Juvenoid

"

"

Activity of Gymnosperm

'"

"

'|

Plant

,

Extracts

"'

1

Insect

Stage

[ Balsam fir

i I ,

Ables balsamea (L.) Miller

.

,

Pyrrhorocis apterus

5th instar larvae

_,

,

!

eggs

Galleria mellonella i

,

Fir

..

|

adult females

I

. . . . . . .

t

pupae

i

'~

Ref

Topical

+++

83

Topical

+++

84

Topical

+++

84

.

.

Injection

86 i

P. apterus

5th instar larvae

Topical

+++

85

5th instar larvae

Topical

+++

85

i

i

'"

Response

l

A. nordmanniana (Steven) Spach

Douglas Fir

i

'

Method of application

|

Pseudotsuga men-ziesii (Mirb.) France

P. apterus

Canadian hemlock

Tsuga canadensis (L.) Carri~re i

P. apterus

5th instar larvae

Topical

+++

83

Hemlock

T. heterophu

G. mellonella

pupae

Injection

-

86

Sarg.

'

Yew

Taxus brev/folia Nutt.

P. apterus

5th instar larvae

Topical

+++

83

American larch

Larix laricina (Du Roi) K. Koch

P. apterus

5th instar larvae

Topical

++

83

European larch

L. decidua Mill.

P. apterus

5th instar larvae

Topical

+

83

Southern pine

Pinus echinata Sarg.

P. apterus

5th instal" larvae

Topical

Pine

P. contorta Engelm.

pupae

Injection

G. mellonella I

83 86

i i

Red spruce

Picea rubra Sarg.

P. apterus

5th instar larvae

Topical

Spruce

Pc. sitchensis (Bonn.) Cam

G. mellonella

pupae

Injection

Juniperus procumbens

P. apterus

Juniper

P. apterus

Thuja plicata Donn ex D. ii

i

I

5th instar larvae i

J. virginiana L.

Red cedar

-

86

t

,,.

Juniper

83

..

5th instar larvae

G. mellonella

Topical

_.

l

Topical

pupae

Injection

Don

83 i

-

:

-

! 86

83

i ii

i

i

ii

i

The symbols represent the potency of the extract: +++ (high activity); ++ (moderate activity); + (slight activity); -(inactive).

!

JUVENILE HORMONES

COOCH 3

385

duvablone

Juvoclmene I

O

J u v o c i m e n e II HO

Bakuchiol

Echtnolone

Fig. (6). Structures of phytojuvenoids.

Juvabione, the compound responsible for this activity, was isolated from the balsam fir, Abies balsamea (L.) Miller, and identified as the methyl ester of todomamic acid, (+)-4(R)-[ l'(R)-5'-dimethyl-3'-oxohexyl]1-cyclohexene-1-carboxylic acid [87]. This compound is a sesquiterpenoid (Fig. (6)) with a cyclohexene group and an a,13-unsaturated methyl ester group; the chemical data for this compound are summarized in Appendix If, Table 1. The IR spectrum suggests that there is a carbonyl ester group present in conjunction with a double bond (1722 and 1645 cm -1) and also an isolated carbonyl group (1712 cm -l) [87]. Mass spectroscopy confirms

386

BEDE

and TOBE

the presence of the methyl ester and proton nuclear magnetic resonance identifies an isopropyl unit attatched to a non-asymmetric carbon atom and a methyl group attatched to a disubstituted carbon. Juvabione and juvabione-related compounds have been isolated from a number of gymnosperms (Appendix II, Table 2 and Table 3). There is a debate in the literature as to the proper stereochemical assignment of these compounds. This paper will follow the recommendations proposed by Manville [88]. The possible biosynthetic relationship between these compounds has also been proposed by Manville and coworkers [89, 90]. Application of juvabione to fifth larval instars of P. apterus produced the expected supernumerary sixth instars at the subsequent moult [87]. However, at higher doses, juvabione was also active against the box elder bug, Leptocoris trivittatus, and the mealworm beetle, Tenebrio molitor T a b l e 2.

Juvenoid Effects of Juvabione

!

Insect

Stage


t

ii

.,

Dose

|

Ref

k ....

Hemiptera

i

Pyrrhocoris apterus

5th larval instar

P. apterus

[

Topical

5 Ixg

87

5th larval instar

Topical

1.7 lag*

91

Leptocoris trivittatus

5th larval instar

Topical

100 p.g

87

Dysdercus cingulatus

5th larval instar

Topical

2.3 Ixg*

91

Graphosoma italicum

5th larval instar

Topical

- (500 btg)

91

Tenebrio molitor

pupae

Injection

500 p.g

87

T. molitor

pupae

Injection

+

92

T. molitor

pupae

Topical

- (500 I~g)

91

Dendroctonus pseudotsugae

adults

Topical

- (100 lag)

93

Manduca sexta

pupae

Injection

-

92

Antheraea polyphemus

pupae

Injection

-

92

Choristoneurafumiferana

eggs

Topical

Coleoptera

[

I

Lepidoptera

9

,

100 ~tg/egg mass .

94 ,,

.

.

A plus sign (+) represents an observed response where the effective concentration was not reported. A minus sign (-) indicates that the compound was inactive at the highest concentration tested (in brackets). Starred values represent the ED50; the dose at which 50% of the biological activity is observed.

JUVENILE HORMONES

387

(Table 2). Similar results were obtained following treatment of these insects with JH III. These results suggest that the JH-activity ofjuvabione is not specific to insects from the family Pyrrhocoridae, as was previously thought, but that these insects may be more sensitive to treatment. Pure juvabione was less active than the extract from balsam fir in the P. aperatus assay [84, 87]. Possible explanations for this observation are that there were differences in the strain of insect tested or, more likely, that the extract contained other compounds which either functioned as juvenoids or which acted as synergists to juvabione. In subsequent studies, the juvenoid effects ofjuvabione have been confirmed on fitch instars of P. apterus [91 ] and pupae of 7'. molitor [92] and observed on fifth instar nymphs of the red cotton stainer, Dysdercus cingulatus [91]. The variability in the sensitivity of these insects may reflect differences in the rate of penetration across the cuticle, metabolism and excretion etc. (Table 2). In subsequent studies, topical application of juvabione to T. molitor pupae was inactive [91]. This reflects differences in the mode of treatment; activity was observed if the pupae were injected with juvabione but absent if it was topically applied. Juvabione was inactive against pupae of the tobacco hornworm, Manduca sexta, and the polyphemus moth, ,4ntheraea polyphemus [92] and the last instar larvae of the pentamid bug, Graphosoma italicum [91 ]. Bioactivities of structually related juvabiones have also been investigated (Table 3). The isomer of juvabione, epijuvabione, and dehydroepijuvabione inhibited normal development of fifth instars of P. apterus [95]; epijuvabione was approximately ten times more effective than dehydroepijuvabione in the assay [96]. Both these compounds are also active on fifth instar nymphs of five Dysdercus species, which are also in the family Pyrrhocoridae" D. intermedius, D. superstitiosus, D. fluvoniger discolor, D. chaquensis and D. cingulatus, [96, 97]. These compounds are inactive against G. italicum, T. molitor, the Colorado potato beetle, Leptinotarsa decemlineata, the crickets, Gryllus domesticus and Acheta domesticus, the locust, Locusta migratoria, and the wax moth, Galleria mellonella [95, 96]. The juvenoid activity of dihydrojuvabione is similar to juvabione [92]. Bioactivity was observed against P. apterus fifth instars and T. molitor pupae, but this compound was inactive following injection into pupae of the Lepidopterans, M. sexta, and A. polyphemus. As expected, the proposed juvabione biosynthetic intermediates, dehydrojuvabione, juvabiol and its isomer, and dehydrojuvabiol and its isomer, were also active against fifth larval instars of P. apterus, and D. cingulams, but inactive against last instars of G. italicum and the pupae of T. molitor [91 ]. In addition to their effects on metamorphosis, these compounds also disrupt embryonic development. The viability of P. apterus eggs is severely reduced following treatment of adult females or freshly laid eggs with partially purified "paper factor" [98]; this activity is not observed

388

BEDE and TOBE

following application of the extract to eggs of O. fasciatus or R. prolixus. Potent ovicidal activity was observed following application of juvabione to eggs of the spruce budworm, Choristoneurafumiferana, a lepidopteran forest pest (Table 3) [94]. Interestingly, balsam fir, ,4. balsamea, the conifer from which juvabione was first isolated, is a host plant of the spruce budworm. Treatment of C. fumiferana eggs with dihydrojuvabione also reduced hatching [92]. Application of this compound to the adult female of the Douglas-fir beetle, Dendroctonus pseudotsugae, resulted in an increase in fecundity. This is significant because dihydrojuvabione has been isolated from the host plant of this beetle, the Douglas fir, Pseudotsuga menziesii (Beissn.) Franco. This suggests that coadaptation of the beetle and the host plant may have occurred such that dihydrojuvabione is not only non-toxic but increases the fecundity of the insect. T a b l e 3.

J u v e n o i d Activity of Structurally-Related J u v a b i o n e C o m p o u n d s i

m

Compound

Insect

Epijuvabione

Hemiptera

,

i

_

Stage

,

ii


Dose

Ref!

i

F i

l

.

,

Pyrrhorocis apterus

5th instar larvae

Topical

10 lag

96

Dysdercus intermedius

5th instar larvae

Topical

I0 lag

96

D. intermedius

5th instar larvae

Topical

5 lag

97

5th instar larvae

Topical

.

.

.

.

.

.

.

D. superstitiosus

. . . . . . . . .

i ; l

I lag .

.

.

97 .

.

.

.

D. discolor

5th instar larvae

Topical

0.5 lag

97

D. chaquensis

5th instar larvae

Topical

I lag

97

D. cingulatus

5th instar larvae

Topical

0.5 pg

97

Graphosoma italicum

5th instar larvae

Topical

- (100 pg)

97

Injection

- (1 mg)

95

Coleopteran

Tenebrio molitor

pupae i

T. molitor

pupae

Leptinotersa decemlineata

pupae

96 -

96

Orthoptera

Gryllus domesticus

last nymph stadium

Injection

- (I mg)

95

Acheta domesticus

last nymph stadium

Topical

- ( 100 lag)

96

! I

JUVENILE HORMONES

389

(Table 3) contd .....

i

Compound

i

Stage

Insect

i


l

|

,

. . . . . . .

F

,

,,

,

,

, ....

Dose

,,h

,

Ref ! i

I

last nymph stadium

Locusta migratoria

Lepidoptera

i Dehydroepi- i juvabione

Galleria mellonella

pupae

G. mellonella

pupae

Injection

- (1 mg) L -(100~tg)

95 !

96

Hemiptera

P. apterus

5th instar larvae

Topical

100 Ixg

96

D. intermedius

5th instar larvae

Topical

10 Ixg

96

D. intermedius

5th instar larvae

Topical

3 Ixg

97

D. superstitiosus

5th instar larvae

Topical

0.8 I,tg

97

D. discolor

5th instar larvae

Topical

0.1 Ixg

97

D. chaquensis

5th instar larvae

Topical

0.5 gg

97

D. cingulatus

5th instar larvae

Topical

0.5 l,tg

97

G. italicum

5th instar larvae

Topical

- (100 ~tg)

97

pupae

Injection

- (I mg)

95

Coleoptera T. molitor

,

T. molitor

pupae

- (100 g,g)

95

L. decemlineata

pupae

-

(lO0 p,g)

96

[

I

Orthoptera G. domesticus

last nymph stadium

Injection

- (1 mg)

95

Ac. domesticus

last nymph stadium

Topical

- ( i o o ~tg)

96

L. migratoria

last nymph stadium

Topical

- ( 1 0 o ~tg)

96

G. mellonella

pupae

Injection

- (1 mg)

95

G. mellonella

pupae

- (100 g,g)

96

Lepidoptera

390

BEDE and TOBE

(Table 3) contd.....

Compound

Insect

Stage


i

Dihydrojuvabione

Hemiptera P. apterus

Dose

Ref

i

I

i 9,_

5th instar larvae

Topical

T. molitor

pupae

Injection

Dendroctonus pseudotsugae

adult females

Topical

Manduca sexta

pupae

Injection

92

Antheraea polyphemus

pupae

Injection

92

Choristoneura fumiferana

eggs

Topical

reduction in hatching

92

P. apterus

5th larval instar

Topical

5 Ixg*

91

D. cingulatus

5th larval instar

Topical

2 Ixg*

91

G. italicum

5th larval instar

Topical

- (500 ~tg)

91

pupae

Topical

- (500 . g )

91

5th larval instar

Topical

5.6 ~tg*

91

Coleoptera 92 increase in fecundity

92

Lepidoptera

Dehydrojuvabione

Hemiptera


Juvabiol and isomer

Hemiptera J

P. apterus

,,,

D. cingulatus

5th larval instar

Topical

4.7 ~tg*

91

G. italicum

5th larval instar

Topical

- (500 ~ g )

91

Topical

- (500 ~ g )

....

,,


pupae

91

JUVENILE HORMONES

391

(Table 3) eontd ..... :,

Compound

Insect

Stage


Dose

Ref

!

b,,, ,,,

Dehydrojuva-biol and isomer

Hemiptera

,,,

|

P. apterus

5th larval instar

Topical

6.0 lag*

D. cingulatus

5th larval instar

Topical

0.7 lag*

G. italicum

5th larval instar

Topical

- (500lag)

91

- (500lag)

91

91

....

Coleoptera i

T. molitor

pupae

Topical i

A plus sign (+) represents an observed response where the effective concentration was not reported. A minus sign (-) indicates that the compound was inactive at the highest concentration tested (in brackets). Starred values represent the

ED50; the dose at which 50% of the biological activity is observed.

In balsam fir, juvabione is found predominantly in the wood; little activity is associated with the foliage or bark [85]. Interestingly, the free acids of juvabione and dehydrojuvabione, todomatuic acid and dehydrotodomatuic acid, were not detected in healthy balsam trees, Abies grandis and A. amabilis [99]. However, significant amounts of these compounds were present in the wood adjacent to sites infected by the balsam wooly aphid, Adelges piceae, suggesting that the synthesis of (+)todomatuic acid and its derivatives or transport of these compounds to the site of infection may be induced upon insect infestation. There are minor discrepancies regarding the biological activity of these compounds. For example, in one assay, juvabione is effective against pupae of T. molitor [87, 92] whereas in subsequent assays, it is inactive [91]. This difference is probably due to the method of delivery (injection vs topical application) which may reflect the ability of juvabione to cross the pupal cuticle or to differences in the metabolism of this compound depending on the route of entry. There also was a difference in the potency of some compounds, for example, epijuvabione and dehydrojuvabione [95, 96]. This could be related to the sensitivity of different strains of insects to the juvenoid or to differences in the physiological state of the insect. For example, in these bioassays, the test compound was applied to fifth larval instars of P. apterus but the age of the insects was not always specified. The activity of the juvenoid could be altered by the physiological stage of the insect and, in particular, the concentration of metabolic enzymes in the haemolymph which change dramatically during this period [57, 76]. This, along with the contradictory reports of the structural stereoisomers ofjuvabione and its derivatives [98, 100, 101, 102], presents a very complicated story.

392

BEDE and TOBE

Other Phytojuvenoids There are many examples of such JH mimics isolated from plants [42, 103]. In the following section, the activities of four of the better characterized JH mimics, juvocimene I and II, bakuchiol and echinolone, are reviewed to illustrate the diversity of the compounds which exhibit JH activity (Fig. (6)) and the range ofbioactivity (Table 4). Juvocimenes I and II are monoterpenoids isolated from the oil of sweet basil, Ocimum basilicum L. (Fig. (6)) (Table 4) [104]. These compounds demonstrate potent JH activity following application to last instar nymphs of the milkweed bug, O. fasciatus. Juvocimene II is approximately 10 times more active than juvocimene I, with biological activities (ED50) in the range of 5 pg and 50 pg, respectively [104]. Therefore, these compounds are far more potent then juvabione when applied to Oncopeltus [92]. The chemical data for these compounds are summarized in Appendix III, Table 1 and 2 and protocols for their chemical synthesis can be found in the references 105 and 106. Table 4.

Comparison of Phytojuvenoids

Compound ,,,

Plant

Plant part

Plant species

,,

Juv~imene

|, !

,

L.

Juvocimene Ii

Sweet basil

O. basilicum

Stage

Method of app.

Dose

Ref

Oncopeltus fasciatus

5th instar larvae

Topical

50 pg*

105

O. fasciatus

5th instar larvae

5 pg*

105

,,

Ocimum basilicum

Sweet basil

lmect

L.

.

b. . . . . . . . . .

seeds

Bakuchiol Echinolone

American coneflower

root

Psoralea cornifolia L .

Dysdercus koenigii

. . . .

,.

Tenebrio

Echinacea augustifolla D e .

molitor

.

Topical .

.

.

.

.

.

.

.

5th instar larvae

Topical

! 0 pg

108

pupae

Injection

0.97 ttg

109

5th instar larvae

Topical

30 ptg

110

......

Juvadecene

Pepper-tree

roots

Macropiper excelsum M i q .

O.

fasclatus

..,

Thujic acid

Red cedar

wood

Thuja plicata

.

.

.

.

T.

monitor

pupae

Injection

51 ! p g

I!I

D.

koenigii

5th instar larvae

Topical

blot reported

112

Donn .....

Tagetone

Marigold

whole plants

Tagetes minuta L. .

Starred values represent the EC50; this is the dose where 50% of the biological response is observed.

Bakuchiol is a phenolic monoterpene (meroterpenoid) isolated from the seeds of Psoralea corylifolia L. (Fig. (6)) [ 107]. Topical application of 10 lag of bakuchiol to fifth instar nymphs of D. koenigii, results in the metamorphosis to nymph-adult intermediates [108]. This juvenoid activity is comparable to juvabione where application of 0.6 lag to fifth instars ofP. apterus or 10 lag to last instar larvae of O. fasciatus, produced morphological abnormalities in 50% of the adults [91, 92]. The chemical data for bakuchiol is compiled in Appendix III, Table 11 and synthesis of the racemate methyl ether from geraniol has been reported [ 113].

JUVENILE HORMONES

393

Root extracts of the American coneflower, Echinacea augustifolia DC., also exhibited juvenoid activity [114]; ether extracts showed high morphogenic activity on T. molitor pupae but none was observed on fifth instar nymphs of O. fasciatus (500 p,g of extract). The active principle in the oil was identified as (E)- 10-hydroxy-4,10-dimethyl-4,11-dodeeadien2-one or eehinolone (Fig. (6)) [109]. However, chemically synthesized eehinolone was not active in the standard T. molitor pupal bioassay [ 115]. The authors of this report acknowledge that this may be due to a failure to synthesize the proper compound. However, the spectral data is consistent with that reported. They were also unable to isolate eehinolone from the roots of E. augustifolia. The chemical data for eehinolone are summarized in Appendix III, Table 12 and the synthesis of raeemie eehinolone has been reported [ 115, 116]. Other phytoehemieals which exhibit JH activity include juvadeeene (1(3,4-methylenedioxyphenyl)-trans-3-decene) isolated from roots of the pepper-tree, Macropiper excelsum Miq. [ 110], thujie acid (5,5-dimethyl1,3,6-eyeloheptatrien-l-carboxylie acid) extracted from the heartwood of western red cedar, Thuja plicata [111] and tagetone ((E)-2,6-dimethyl-5,7oetadien-4-one) from the marigold, Tagetes minuta L. [ 112]. There have been numerous reports of juvenoid activity of plant extracts (Table 5); however, to our knowledge, the compound(s) responsible for this activity have not been isolated and characterized. Plant secondary metabolites which mimic JH activity appear to be active on a narrow range of host species. What account(s) for this effect ? The majority of bioassays used last larval instars of P. apterus, O. faseiatus and pupae of T. molitor to test for activity of the juvenoids. Are these the most sensitive insects ? Six JHs have been identified to date; different homologs have been isolated from specific insect orders. Juvenile hormone III appears to be ubiquitous [ 12, 13] and, in most species, is the only JH present. Juvenile hormone I and II are important in the regulation of metamorphosis and ovarian maturation in Lepidoptera [5] and the bisepoxide appears to be the principle JH in higher Diptera [20]. Therefore, the nature of the JH in the test insect and the role that it plays in development must be considered in the selection of the bioassay; a compound which mimics the action of JH in P. apterus (Hemiptera) is unlikely to be active in a Lepidopteran insect. Originally, it was thought that these compounds could provide the structural basis for the design of pesticides because it is unlikely that insects would develop resistance to their own or closely related hormones [ 120]. However, this has proven not to be the ease. Most insects possess enzymes such as mixed function oxidases and esterases in their alimentary tract and haemolymph which are, among other functions, important for the metabolism of plant toxins [121, 122]. For example, insects which have developed resistance to insecticides may also show resistance to JH

BEDE and TOBE

394

Table 5.

Juvenoid Activity of Plant Extracts

,

Plant

,

Plant pa•

-

i r ~

..,

.

,

.,

,


Response

Ref

5th larval instars

Topical

+4+

117

5th larval instars

Topical

++

fasciatus

114

Tenebrio molitor

pupae

Topical

-

!!4

0/ascialas

5th larval instars

Topical

4+

114

pupae . . . 5th larval instars

Topical

Insect ,

,

Stage

Plant species |

,

Roots

Iris

Iris endata Thamb.

Dysdercus koenigii

Roots, stem, fruits

Iris

Iris douglasiana Herb.

Oncopeltus

Stem, leaves, fruits

Sweet pepperbush

Clethra aiternifolia L.

Root, root bark

White sassafras

Sassafras aibidum (Nutt.) Nees

O. fasciatus T. molitor

pupae

Topical

-

! 14

Stem, bark

Murray redgum

Eucalyptus camaldulensis

O. fasciatus

5th larval instars

Topical

4+

! 14

T. molitor

pupae

Topical

Twigs, leaves

Pitch pine

Pinus rigida Mill.

O. fasciatus

5th larval insists

Topical

+

I !4

T. molitor

pupae

Topical

Seeds

Lawson cypress

Chamaecyparis lawso-niana

O. fasctatus

5th larval instars

Topical

T. molitor .

.

.

Dehnh.

(Andr. Murray) Pari

ii i I

Topical

"

.

4+

._

114 i 14

! 14

! 14 -

I 14

7". molitor

pupae

Topical

.H-

I 14

Anthocephalus cadamba

D. cingulatus

5th larval instars

Topical

4-+

I 18

Lantana camara L.

D. cingulatus

5th larval instals

Topical

4+

I 18

Stem

Calophyllum sp. L.

D. cingulatus

5th larval instars i

Topical

~

118

Stem

Phyllanthus emblica

D. cingulatus

5th larval i insists i

Topical

-t-t+

!!8

5th larval instars

Topical

Stem Stem

Yellow sage

.

i

Stem

D. cingulalus

Erythrina indica Lam.

[

119

Stem

i i [

Auracaria excelsa R. Br

D. cingulatus

5th larval instars

Topical

Stem

j

Annona reticulata L.

D. cingulatus

5th larval , instara

Topical

Peltoforum inerme Benth.

D. cingulatus

5th larval instars

Topical

-14+

119

Manihot esculenta Pohl.

D. cingulatus

5th larval instars

Topical

+4-+

119

1 i

5th larval instals

Topical

+4+

119

9

Stem Stem

Cassava

"'

[ [

-4-++

119 119

Stem

Phyllanthas emblica L.

D. cingulalus

Stem

Tabernaemontana dichotoma

D. ctngulatus

5th larval instars

Topical

D. koenigii

5th larval instars

Topical

Aedes aegpti

larvae

Contact in water

112

Aedes aegpti

pupae

Contact in water

112

Roxb. Whole plant

Marigold

Tagetes minuta L.

i Muses domestica ,

pupae ,

I19 +++

Topical ,

.,

,

The symbols represent the potency of the extract: +++ (high activity); ++ (moderate activity); + (slight activity); - (inactive).

,

!i2

L 112

JUVENILE HORMONES

395

analogues [ 123, 124, 125]. This cross-resistance appears to be related to an increase in metabolic enzymes, particularly mixed function oxidases, including cytochrome P450 enzymes. Also, esterases are present at high levels in the insect haemolymph in the final instar and are thought to be partially responsible for the low JH III titers observed at this stage [57, 76]. Differences in such enzyme systems may account for the observed differences in the sensitivities of insect species to juvenoids. Further difficulty with the use of these analogues as a means of control of pest species is that the window of sensitivity to these compounds is short, e.g. to eggs, at metamorphosis or during reproduction. Treatment with these compounds may also result in an arrest of the insect in the feeding period. These compounds also have a relatively broad specificity and would not act exclusively on pest species. However, in certain circumstances, the synthetic JH analogues methoprene and hydroprene have been used successfully in insect control. Much of the interest in phytojuvenoids has focused on the isolation of compounds to use as models for the development of stable, potent pesticides. As a result, there is little information regarding the biological nature of these compounds. In most bioassays, the juvenoid was applied topically, biasing the screen for lipophilic compounds which are able to penetrate the insect cuticle and underlying epidermis. In nature, these compounds can be absorbed, ingested or inhaled depending on the plant and the life history of the insect on the plant. Little information is available regarding juvenoids which elicit these effects by oral administration. Similarly, there have been few reports regarding the amount and distribution of these compounds in the plant. To our knowledge, there is presently no information on whether the levels of these compounds in plant tissues are sufficiently high to affect insect herbivores or whether these compounds play another role(s) in vivo. Consequently, these compounds which have been defined in the literature as insect JH mimics do not meet our criteria: the present evidence suggests that they may function as phytojuvenoids but further studies must be performed. PLANT JUVENILE HORMONE ANTAGONISTS Phytochemicals, such as the precocenes, isolated from A g e r a t u m houstonianum [ 126], interfere with JH biosynthesis. In sensitive insect species, application of these dichromenes to larval instars results in precocious metamorphosis to sterile adults or sterility in adult females following treatment. Pesticides based on these compounds would be useful in the control of insects which are primarily destructive in their immature stages. Other phytochemicals which potentially function as "antijuvenile hormones" include dimethyl sciadinonate, isolated from the leaves of avocado, Persea americana Mill [127, 128]. Ingestion of this

396

BEDEand TOBE

compound results in the direct pupation of fourth instar larvae of the silkworm, Bombyx mori, bypassing the fourth moult. Precocenes

The bioactivities of precocene I, 7-methoxy-2,2-dimethyl chromene, and precocene II, 6,7-dimethoxy-2,2-dimethyl chromene, are well established (Fig. (7)). These two compounds have been isolated from plants throughout the family Asteraceae [ 129-136, 154]. The chemical data for these compounds and references for their synthesis are compiled in Appendix IV, Tables 13 and 14. There is a wealth of information on the effects of these compounds on various insect species [42, 137]. For simplicity, we will focus on the effects of precocene II on the susceptible Hemipteran bug, Oncopeltusfasciatus.

MeO M M e e ~

Precocene I

Precocene II

Fig. (7). Plant insectjuvenile hormone antagonists, the ageratochromenes.

Application of sublethal doses of precocene to the first, second and third instar nymphs of O. fasciatus results in premature metamorphosis to adultoids at the third, fourth and fifth stadium moult, respectively [126]. Interestingly, there is an "in-between" instar following the stage when the chromene is applied and when the effects are observed. These effects can be reversed by the topical application of JH; a decrease in both mortality and the number of insects which undergo precocious metamorphosis is observed following application of JH I to second instars which have been pretreated with precocene II [138]. Fourth instar larvae of O. faseiatus which were treated with precocene developed either into precocious adults or underwent apparently normal development through a fifth instar into an adult, with an increase in the preoviposition period and reduced fecundity observed in females [138, 139]. This stage of Oneopeltus was less sensitive to precocene; a hundred-fold increase in concentration was required to produce these effects [138]. Treatment of these precocious adult females with JH resulted in mating, although oviposition did not

JUVENILE HORMONES

397

ensue [ 126]. Application of precocene to the last nymphal stages has no effect and these insects develop into normal, reproductive adults [ 140]. Treatment of adult female O. fasciatus with precocene results in sterilization [ 126, 138, 139]. Examination of the ovaries revealed a marked difference between control and treated insects [126, 141]. In normal insects, oocyte development begins two to three days after adult moult and continues until day six, at which time oviposition normally begins. Treatment of gravid insects with precocene on day 5 after eclosion resulted in resorption of most oocytes. These eggs hatched and developed normally to third instar juveniles which then moulted into precocious adults [ 126]. Following application of precocene to newly emerged adult females, oocyte development is completely inhibited. Application of JH III to these insects resulted in a rapid increase in oocyte length, demonstrating that precocenes were not acting directly on the ovary. Transplantation experiments in which precocene II was added to CA maintained in vitro from mated adult female O. fasciatus, followed by implantation into fifth instar juveniles, demonstrated that the precocenes directly inactivate the CA and their effects are not the result of signals originating in the brain or other tissues [142]. In complementary experiments on the cockroach, Periplaneta americana, Pratt and Bowers demonstrated that incubation of the CA with precocene II directly inhibits JH III biosynthesis and release, even though in vivo, P. americana is relatively insensitive to these compounds [ 143]. The effects of precocenes on larval and adult O. fasciatus and their reversal by application of JH suggests that these compounds may be affecting JH biosynthesis by the CA. As with the ovary, profound differences are observed in CA volume between normal and precocenetreated animals [141]. After eclosion, the CA of mated adult females normally undergo a five-fold increase in volume over the next nine days. Treatment of these animals with precocene on day 5 results in a reduction in size. If the insect is treated with precocene immediately following the adult moult, the CA size does not change; even if JH III is added on day 5 to these animals, no further development occurs. However, in this species, there is no definitive evidence to suggest that CA volume is correlated with the rescue of JH. Morphological and ultrastructural studies of the CA of precocenetreated adult females of O. fasciatus demonstrate progressive necrotic degeneration, as compared to controls [ 144, 145, 146]. These studies have shown that precocene II not only inhibits the CA but actually stimulates its atrophy. Its specific cytotoxicity is attributed to epoxidation of the precocenes by the epoxidase which catalyzes the final step in JH III biosynthesis, generating extremely reactive 3,4-epoxy intermediates which alkylate cellular proteins [ 147, 148, 149]. This results in the atrophy of the CA and the resultant inhibition of JH biosynthesis [150]. Therefore, the destruction of the CA by precocene is responsible for its biological

398

BEDE and TOBE

activity. This occurs through its epoxidation by an enzyme which is only active at times of JH biosynthesis. Therefore, precocenes have no effect on inactive CA such as in those of last larval instars of O. fasciatus [ 140]. Generally, it is thought that these compounds are only effective in vivo against insects in the orders of Hemiptera, Homoptera, Dictyoptera and Orthoptera, with exceptions [42, 137]. Holometamolous insects are believed to be insensitive; high doses of precocenes may affect development in these and other insect species but it is thought that this action does not occur through inhibition of JH biosynthesis. Rescue experiments with reversal of precocene-induced effects counteracted with JH or JH analogues will allow the differentiation between endocrinespecific and non-specific effects. In vitro inhibition of JH biosynthesis in insect species which are insensitive to precocenes in vivo has also been demonstrated [ 151 ]. It is believed that the difference insusceptibility of different insect species to precocenes may, in part, be related to their metabolism by the insect. The metabolism of precocene II to the diol was demonstrated in nine insect species [ 147] and, presumably, occurs by mixed function oxidases present in the gut and fat body. The wax moth, G. mellonella, metabolized 77% of this compound as compared to 47% by O. fasciatus in the same time period. This difference in metabolic rate may account, at least partially, for differences in the sensitivity of these insect species. Precocene II has also shown antifeedant activity in a non-choice assay with the Mexican bean beetle, Epilachna varivestis [152]. It also has antifungal activity against Pyricuaria oryzae, whereas precocene I was nontoxic in this assay [ 153]. Neither compound demonstrated antibiotic activity against the yeasts, Saccharomyces cerevisiae and Candida albicans, the gram-negative bacteria, Pseudomonas fluorescens and Escherichia coli, and the gram-positive bacteria, Bacillus subtilis and Staphlococcus albus [130]. The distribution of precocene I and II in flowering plants of Ageratum houstonianum has been determined [134]. The highest amounts of precocene II is found in the leaves (1.4 lamol/g fresh weight (FW) to 6.7 ~mol/g FW in older leaves) and the flowerheads (21.5 I.tmol/g FW). It is invalid to compare a dose which would be topically applied versus ingested. However, with information on the amounts in the plant, it would be useful to determine if precocene II levels in the plant would be sufficient to either deter feeding or interfere with development of a susceptible insect species. Considering that application of 0.5 mg of precocene II is ovicidal to O. fasciatus eggs treated by fumigation [ 126], 10 ktg of precocene II resulted in precocious metamorphosis in O. fasciatus [139] and that 0.05 btmol/leaf disc deterred the feeding of E. verivestis [152], it is possible that these compounds are present in sufficient concentrations to play a role in protecting the plant against insect herbivory.

JUVENILE HORMONES

399

INSECT JUVENILE HORMONE III IN PLANTS To date, there has been only one report of the identification of an insect JH in a plant; in 1988, JH III and MF were isolated from the sedges Cyperus iria L. and C. aromaticus (Ridley) Mattf and Kiik [155]. Although JH III has only been detected in these two plant species, structurally similar sesquiterpenoids have been isolated from the roots of C. iria and a number of related Cyperus species (Fig. (8)) [ 156-161 ]. For C iria

Methyl- 1OR, 1 I-epoxy-3,7, I I-trimethyl2E, 6E-dodecadienoate

C monophyllus

C pilosus

C microiria

Methyl-3,7,11 -trimethyl- I I -hydroxy-2E, 6E, 9Z-dodecadienoate

"~-

+

Methyl-3,7, i l-trimethyl- I I-hydroxy-2E, 6E, 9E-dodecadienoate

-!-

+

Methyl-3,7,1 I-trimethyl-2E, 6E, 9Z, I ! Z-dodecatraenoate

"[-

+

Methyl-3,7,1 i-trirnethyl-2E, 6E, 9E, I ! E-dodecatraenoate

~

"]"

Farnesol Methyl farnesoate

C polystachyos

C serotinus

+

+

+

+

+

+

+

+

+

Juvenile Hormone HI: methyl-10R. 11-epoxy-3.7,11-trimethyl-2 E,6Edodecadienoate (CtoH2eOs}

~ methyl 3,7,11-trlmethyl-11-hydroxy trans-2, trans-6, c/s-9-dodeeadfenoate (r methyl 3,7,11-trlrnethyl- 11-hydroxy tmns-2, tmns-6, tr~Ls-9-dodecadlenoate

1

o"

OH methyl 3,7',11-trlmethyl tr~s-2, tn~ls-6, ds-9,l 1-dodeeadlenoate (C16He4021 methyl 3,7',11-trlmethyl trots-2, tr~s-6, trans-9,11-dodeeadlenoate

oi" Methyl F~-nesoate: methyl a,7,11-trlrnethyl trans-2, t r ~ s 6,10-dodeeatrlenoate (e~6H26021

oi" Farnesol: 12N, 6~1-3,7,11-trlmethyl-2,6,10-dodeeatrlen- 1-ol (r 15H2601

Fig. (8). Linear sesquiterpenoids isolated from the roots of Cyperus species [ 155-161 ].

400

BEDE and TOBE

example, MF, the immediate biosynthetic precursor of JH Ill in insects, has been identified in extracts from C iria and three other Cyperus species, C monophyllus, C pilosus and C serotinus [155, 158, 160, 161], as well as from grape skins [ 162] and the bark of Polyalthia viridis Craib [ 163 ]. The linear sesquiterpenoid methyl (E,E)- 10,11 -dihydroxy-3,7,11 trimethyl-2,6-dodecadienoate has been isolated from dried roots of a canopy tree Hortia regia Sandwith., and the root bark of an African rainforest tree, Cleistopholis patens (Benth.) Engl. and Diels, and the stem bark of C1. staudtii (Engl. & Pierre) [ 164, 165, 166]. This compound is structurally identical to JH III except that the epoxide has been hydrated to a vicinal diol, a reaction which readily occurs in the presence of acid or base. These reports suggest that JH III, MF and structurally similar compounds are present in diverse plant species and are more prevalent than previously thought. Distribution of Juvenile Hormone III in Cyperus iria

The distribution of JH III was monitored in C. iria throughout plant development using a radioimmunoassay [ 167]. In seven-month old plants, the amount of JH III in root tissue is 27.2 + 3.3 ~g/g FW [ 168]. This is approximately thirty-five times the whole body concentration of day 5 adult females ofD. punctata [57]. The total level of JH III in the roots was approximately 600 times and 300 times the amount found in either the inflorescence or leaf tissue, respectively, at this stage of development. This profile was maintained over the course of development, with the highest concentrations of JH III observed in the root tissue and substantial quantities also in the leaf tissue. The overall amount of JH III per gram plant tissue was less than that originally reported [ 155]; this discrepancy probably reflects the different cultivars and environmental growth conditions. Possible Biological Role(s) of Juvenile Hormone III

The isolation of JH lII from C. iria and C. aromaticus, the high concentrations found in C iria throughout development and the extraction of structurally similar compounds from closely related species as well as other plant species suggests that this compound may play an important biological function(s) in these plants. At this point, the nature of this role is speculative; however, it is possible that JH III may be involved in plantinsect, plant-plant, plant-nematode or plant-fungal interactions. At present, there are few reports on the ecology of C. iria and associated insects and the information available is vague and contradictory. In the initial report on the isolation of JH III from C. iria, third stadium grasshopper nymphs, Melanoplus sanguinipes, were raised on either

JUVENILE HORMONES

401

wheat seedlings or C. iria; no difference was observed in growth. However, upon moulting to adults, those reared on C. iria displayed deformed wings and colour changes indicative of development under conditions of excess JH [ 155]. Adult female grasshoppers fed on C. iria were infertile and their ovaries contained only immature oocytes [ 155]. In field studies, eggs of the Dipteran leafminer, Hydrellia sp., which were laid on C. iria leaves did not hatch [ 169]. However, in some reports, C. iria had no effect on insect development. For example, nymphs of the planthoppers, Nisia strovenosa and N. nervosa were reared successfully on C. iria and C. rotundus L., although more insects reached maturity on C. rotundus [ 170]. Also, in a feeding preference study, the rice stink bug, Oebalus pugnax, did feed on C. iria, although the mean number of feedings was less than that observed on other plants such as the Vasey grass, Paspalum urvillei Steud. [ 171 ]. It is also possible that these compounds may reduce plant competition by inhibiting the germination and growth of plants in the immediate vicinity. In the search for allelopathic agents in invasive weeds, methyl farnesoate and farnesol were isolated from tubers of C. serotinus Rottb. [ 161 ]. At concentrations of 1 mM, these compounds inhibited the growth of lettuce and rice seedlings. In other studies, treatment of seeds with famesol (83.3 IxM) stimulated root elongation in barley but had a slight inhibitory effect on mustard, tomato, spinach and wheat [ 172]; no effect was observed on lettuce, carrot or cabbage. At higher concentrations (516.6 ~M), famesol inhibited root growth in barley. The high levels of JH III in the plant coupled with the above evidence of allelopathic activity of two of its biosynthetic precursors, farnesol and MF [161], raises the possibility that these compounds may be involved in the inhibition of germination and growth of surrounding plants. Allelochemicals may also be released into the environment as a defence against phytoparasitic nematodes [173]. Juvenile hormones have been shown to affect the development of nematodes [ 174]. Juvenile hormone I and MF inhibited hatching of the eggs ofHaemonchus contortus [175] and application of JH III (3.4 mM) to third-stage larval females of the rodent hookworm, Nippostrongylus brasiliensis, resulted in a 50% reduction in egg production [176]. The biosynthetic precursor of JH III, farnesol, inhibits development of larvae of the nematode Trichinella spiralis [ 177]. However, in the few studies done, these compounds do not appear to have an effect on plant parasitic nematodes [ 175, 178]. CONCLUSION It is apparent from these examples that plants produce compounds which are able to interfere with the endocrine system of insects. It is tempting to speculate that they may be responsible, or at least partially responsible, for the protection of the plant against insect attack. Unfortunately, most

402

BEDE and TOBE

work has focussed on the effects of these compounds on the insect and the development of synthetic pesticides based on their structure. Little is known about the distribution of the compounds in the plant, whether their concentration is sufficient to inhibit insect attack or whether they are induced upon attack to protect the plant tissues. Also, in most studies, the effects of topically applied compounds were observed whereas the dietary effects, which is more likely the mode of entry, on these insects is not known. The mechanisms of insect metabolism of these compounds have also not been extensively studied. There are many examples of plants which produce insect hormones, for example, the insect moulting hormone, 20-hydroxyecdysone [179]. However, to date there had only been one report of the identification of an insect JH from a plant [ 155]. It is obvious from the previous section, that we are in the preliminary stages of determining the role of JH III in C. iria. In an attempt to understand the role of this compound in the plant, we have characterized the distribution of JH III developmentally. At present, we can only speculate as to its possible biological activity(ies). ACKNOWLEDGEMENTS We thank C. Garside and K. Yagi for thoughtful comments on the manuscript and P. Bowser for help with the manuscript. Funding of this research was provided by a National Sciences and Engineering Research Council of Canada Operating Grant (S.S.T).

JUVENILE HORMONES

403

APPENDIX I. JUVENILE HORMONE DATA Table 1.

Chemical Data for Juvenile Hormone II1, Methyl-10R,ll-epoxy-3,7,11trimethyl 2E,6E-dodecadieneoate [21, 38, 180-183]

Beilstein Registry Number: 1316317 MW 266.38 C!6H2603 Boiling point: 125-126~ (0.08 mm3) 30~ (0.02 mm 3) Ultraviolet spectroscopy (ethanol): ~, = 221 rim, ~ = 14 350 Infrared spectroscopy (film): 2950, 1720, 1215, !130 cm"1. 1720, 1650 cm" 1.

Table 2.

Mass Spectral Data for Juvenile Hormone III [23] ,

Mass (m/z) ,

i

,

,

,,

,,

M+ (M-H20)

C16H260 3 C16H2402

234

(M-CH3OH) +

C15H2202

206

(M-CH3OH + CO) +

C14H220

i 95 163 153 135

C 12H 1902 C 11HI5 O C 10H 170 CIOHI5

114 81 71 43 ,

(M-C4H70) + (195-CH3OH) (M-C6HgO2) (153-H20) (] 95-CO + CH3OH ) (M-C I OH 16O) (M-C I OH 1703) (C12H1902) (C13H1903)

C6H 1002 C6H9 C4H70 C3H7 ....

266 248

M+ (M-H20)

C!6H2603 C16H2402

234

(M-CH3OH) +

C 15H2202

195 163 135

(M-C4H70) + (195-CH3OH) (153-H20) (195-CO + CH3OH) (M-CIoHI60) (M-C I OH 1703) (C 12H 1902) (C13H1903)

C 14H ! 902 C 11HI5 O CIOHI5

i,

i

,,i

i

266 248

114 81 * 71 43

,

Ion Fragment 15 eV

i

70 eV

C6HI002 C6H9 C4H70 C3H 7 i

i

||l

m

Bolded masses represents ions unique to the low energy spectra. An asterix denotes the base peak. Other references: 180, 183, 184.

BEDE and TOBE

404

T a b l e 3.

Proton Nuclear Magnetic Spectra of Juvenile Hormone III i

i

i

ill

i

. . . .L$:~ i s )

.

i

.

.

x.v~

-,_, tul

t

' "

218(d,J-15Hz} "

9

,,

ii

" ..

-

o:;

.

2 . 7 4 (t, J = 6 H z ) 8 1.2 1.63

Assign men t double peak=two methyl groups attached to epoxide s, 3H, C-7

5 1.27 & 1.32 1.65

Assign ment s, 2 x 3H, CH3 at C-I I

8 1.26 1.3 1.62

s, 3H, CH 3 at C-7

1.7

2.1 2.5

epoxide proton

2.18 2.74

3.61

carboxyl methyl vinyl protons C-6 vinyl protons C-2

3.72 5.22 5.75

Assignment s, 3H, CH3 at C-I 1 s, 3H, H-12 s, 3H, CH3 at C-7 m, 4H, H-8, H-9 m, 4H, H-4, H-5

d, sH, J--I.5 Hz, CH 3 at C-3 t, J=6 Hz, IHz, IH, H-10 2.70t, J=6 Hz, IH, H-

10 5.12 5.59

s, 3H, OCH3 m, IH, H-6 m, IH, H-2

CCI 3

Solvent:

[271

Reference: ,

,

_

3.69 5.14 5.67

s, 3H, OCH3 t, J=6 Hz, IH, H-6 br. s, IH, H-2

CDC! 3

CDC! 3

[381

[1831

,,,

_

Chemical shifts are reported at 8 values in ppm. Tetramethylsilane was the internal standard. Other references: 180, 182

T a b l e 4. l,

,,,,,

13C Nuclear Magnetic Spectra of Juvenile Hormone ,,

I I I ( C D C I 3 ) [183]

,,,

8 50.9 167.2 I 15.7 159.9 41.1 26.1 123.8 135.5 26.5 27.7 64.2 58.2 25 25 16.2 18.9

,,

Assignment OCH 3 C-l C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C- l 0 C-I l C-12 CH 3 at C-l l CH 3 at C-7 CH 3 at C-3

i

Chemical shifts are reported as 8 values in ppm. Tetramethylsilane was the internal standard. Numbering of juvenile hormone III as illustrated in Appendix I, Table 3.

i

,t

JUVENILE HORMONES

Table 5.

405

References for Juvenile H o r m o n e III

Extraction and Chromatography: Radioimmunoassay: Radiochemical assay: Chemical synthesis:

12, 15, 37, 185-197. 167, 198-201. 45, 46, 47, 49, 50. 38, 180-184, 202-211.

APPENDIX II. JUVABIONE AND ANALOGS Table 1.

C h e m i c a l Data for Juvabione, (+)-4(R)-[l'(R)-5'-Dimethyl-3'-oxohexyl]-lcyclohexene-l-carboxylic acid methyl ester [87, 90, 92, 101, 102, 212, 213]

Beilstein registry number: 2125665 MW 266.38 C16H260 3 Ultraviolet spectroscopy (ethanol): ~,= 222 nm, e = 13 600 Infrared spectroscopy (carbon disulfide):1722, 1712, 1645 cm "I. Mass spectroscopy (m/z): 266.2 (M+), 234, 206, 167, 166, 139, 135, 134 (base peak), 127, 107, 105, 91, 85, 79, 77, 69, 67, 57, 43, 41. H+-NMR (CDCI 3, I00 Hz): 8 (ppm) 6.96 IH, multiplet,H2 3.72 3H, singlet, methyl ester 0.90 6H, doublet, J = 6 Hz, C-5' methyls 0.86 3H, doublet, J 6 Hz, C-I' methyl 2.8-1.1 13H 13C NMR: 209.8, 167.3, 138.9, 129.8, 52.1, 51.0, 47.4, 32.3, 29.4, 24.9, 24.2, 22.3, 16.2. Chemical synthesis (references): 212, 213, 214. =

Table 2.

Chemical Data of Juvabione and Related Compounds

Compounds (+)-Todomatuic acid (+)-Juvabione (+)-Epijuvabione (+)-Dehydrojuvabione (+)-Dehydroepijuvabione (+)-Dihydrojuvabione Juvabiol Isojuvabiol Epijuvabiol

References 102, 215. 87, 89, 90, 92, I 01, 214. 95,214. 88, 89, 90. 89, 90, 95. 92. 89, 90, 91. 89, 90, 91. 89, 90.

406

BEDE and TOBE

Table 3.

Juvabione and Structurally Related Compounds Isolated from Conifers

(+)-Todomatuic acid

Japanese fir (Abies sachalinensis Schmidt )1 Douglas fir (Pseudotsuga menziesii (Beissn.) Franco) 10

(+)-Juvabione (4R, I'R)

Balsam fir (,4. balsamea (L.) Miller) 2 Douglas fir (P. menziesii) 3,9 Alpine fir (A. lasiocarpa (Hook.) Nutt.) 4 Silver fir (.4. alba Mill.) 5

(+)-Epijuvabione (4R, I'S)

Balsam fir (A. balsamea) (Czechoslovakian) 6

(+)-Dehydrojuvabione (4R,I'R)

d. balsamea 7 A. lasiocarpa 4 A. aiba 5

(+)-Dehydroepijuvabione (4R,I'S)

A. balsamea (Czechoslovakian) 6 A. lasiocarpa 4 ,4. alba 5

(+)-Dihydrojuvabione

P. menziesii 3

Juvabiol (4R, I'R,3'S)

A. balsamea 8 A. lasiocarpa 4 A. alba 5

Isojuvabiol (4R, I'R, 3'R)

A. balsamea 8 A. alba 5

Epijuvabiol (4R, I'S, YS)

A. lasiocarpa 4 A. alba 5

References: I. 215

6.

95

2.

87

7.

88

3.

92

8.

91

4.

90

9.

!01

5.

89

10.

102

JUVENILE HORMONES

407

APPENDIX HI. PLANT JUVENILE HORMONE MIMICS Table 1.

Chemical Data for Juvocimene I, 1-Methoxy-4-[6-methyl-4-(2-methylpropenyl)-octa-l,5,7-trienyl]-benzene [ 105, 106] ,,

MW

282.42

|

C20H260

Ultraviolet spectroscopy (ethanol):

~, = 237 nm, e = 23 000 ~. = 260 nm, e = 22 000

Infrared spectroscopy (cm'l):

3010, 2910, 1640, 1610, 1580, 1513, 1440, 1375, 1295, 1250, 1175, 1105, 1035, 985, 965, 890, 835.

Mass spectroscopy (m/z):

282 (M+), 147 (base peak), 135, 93.

H + N M R (CDCI 3, 100 Hz):

8 (ppm)

p-Methoxycinnamyl:

7.25

2H, doublet, aromatic proton

6.85

2H, doublet, aromatic proton

6.32

I H, doublet, alkenyl proton

6.00

I H, doublet, alkenyl proton

2-Methylbutadienyl:

Isobutenyl:

13C NMR:

3.81

3H, singlet, methoxy

2.42

2H, broad triplet, aikenyl proton, aromatic ring

6.38

I H, broad doublet, vinylic moiety

5.37

I H, broad doublet

5.11


4.96


1.78

3H, doublet, methyl group

5.04

I H, double septet

3.37

1H, broad quintet, methine proton

!.72

3H, doublet, allylic methyl group

! .66

3H, doublet, allylic methyl group

158.6 (C), 141.7 (CH), 136.1 (CH), 132.6 (C), 131.3 (C), 130.7 (C), 130.3 (CH), 127.0 (CH), 126.4 (CH), 113.9 (CH), 110.8 (C8), 55.3 (CH30), 39.8 (C3), 38.3 (C4), 25.8 (CH3), 18.2 (CH3), 12.1 (CH3)ppm.

408

Table 2.

BEDE and TOBE

Chemical Data for Juvocimene II, 3-[l-[3-(4-Methoxyphenyl)-2-propenyl]-3methyl-2,4-pentadienyl]-2,2-dimethyl oxirane [105] ,,,

MW

,

298


~, = 227 nm, e = 29 000 k = 262 nm, e = 25 000

298 (M+), 207, 147 (base peak), 91.

H+-NMR (CDCI 3, 100 Hz): p-Methoxycinnamyl:

8 (ppm) 7.26 6.85 6.4O 6.08 3.81 6.41 5.39 5.16

2-Methyl-trans- 1,3-bu ta dienyl:

2H, doublet, aromatic proton 2H, doublet, aromatic proton I H, doublet, alkenyl proton 1H, doublet, alkenyl proton 3H, singlet, methoxy I H, broad doublet, vinylic moiety I H, broad doublet I H, broad doublet, vinylic moiety 1H, double septet 3H, doublet, methyl group I H, doublet, epoxy proton 2 x 3H, singlet, methyl groups 3H, singlet

5.03 1.76 2.71 1.33 1.30

1,2-Epoxy-2-methylpropyl:

,l,llll

i

,

,

Chemical Data for (+)-Bakuchiol [107, 216] ,

MW

,

C20H260 2


Table 3.

,

256

,,,

,

,,

CI8H240

Ultraviolet spectroscopy (ethanol): ~. = 260 nm, e - 18400 Infrared spectroscopy (cm'l): 3350, 1530, 1245, 980, 822.


H+-NMR:

256 (M+), 213, 174, 173 (base peak), 158, 145, 107, 93, 83, 79, 77. See paper for fragmentation schematic.

5 (ppm) 6.70

4H, AA'BB' quartet, J = 8.5 Hz Aromatic H's

5.90

2H, quartet, J = 16 Hz, A7,8

4.60-5.93

3H, multiplet, AI 6,17

4.9 1.60 1.51 1.13

IH, olefinic, A2,3 3H, singlet, C-2 methyl 3H, singlet, C-2 methyl 3H, singlet, C-6 methyl i

i

ii,

,

JUVENILE HORMONES

Table 4.

MW

409

Chemical Data for Echinolone, ( E ) - 1 0 - H y d r o x y - 4 , 1 0 - d i m e t h y i - 4 , 1 1 dodecadien-2-one [109, 114, 115]

224.2

C14H240 2

Infrared spectroscopy (cm"I):

3440, 3060, 1710, 1632, 1440, 1360, 1230, 1160, 900, 715.


148, 133, 111. 8 (ppm) 5.95 5.32 5.20 5.00 3.05 2.07 1.62 1.22

H+-NMR (CDCI3):

i H, doublet, J = 10, 18 Hz, CH=CH2 I H, triplet, J - 7 Hz, vinylic proton IH, doublet, J = 1, 18 Hz, CH2=CH IH, doublet, J = I, 10 Hz, CH2=CH2 2H, singlet, CH2COCH 3 3H, singlet, CH3CO 3H, singlet, methyl group at double bond 3H, singlet, tertiary CH 3

APPENDIX IV. PLANT JUVENILE HORMONE ANTAGONISTS Table 1.

Chemical Data for Precocene I, 7-Methoxy-2,2-dimethyl-2H- chromene [129, 217-2211

Beilstein Registry Number: 133917 MW

190.24

C12H140 2


~. -- 279 nm, e = 5 670 ~. = 322 rim, e = 6 750 3050, 1630, 1600, 1450 cm "I. 1640, 1615, 1570, 1500, 1025 cm "I. 1610, 1470, 1380, 1200, 1050 cm "I.

Infrared spectroscopy

(neat): (neat): (CHCI3):


190 (M+), 175 (base peak).

H+-NMR (CDCI3):

13C NMR (CDCI3):

(ppm) 6.6-6.8 6.19 5.49 3.77 1.40

3H, multiplet, benzene ring H 1H, doublet, J - 10 Hz, 4-H IH, doublet, J - 10 Hz, 3-H 3H, singlet, OCH 3 6H, singlet, 2 x CH 3

76.15 (C-2), 127.67 (C-3), 121.81 (C-4), 126.80 (C-5), 106.47 (C-6), 160.58 (C-7), 101.95 (C-8), 154.08 (C-8a), 114.50 (C-4a), 27.89 (2CH3).

410

BEDE and TOBE

Table 2.

C h e m i c a l Data for Precocene II, 6,7-Methoxy-2,2-dimethyl-2H- c h r o m e n e

[129, 218, 220-224] ,,

, |

,,|

,

i

,,

i

,

Beilstein Registry Number: 160683 MW

220.27

CI3H160 3


= 278 nm, e = 3 580 ~. = 322 nm, e = 6 750

Infrared spectroscopy (neat): (CHCi3):

1640, 1613, 1575, 1502, 1350, 1360, 1010, 1610, 1470, 1370, 1260, 1210 cm "1.


220 (M +)

H+-NMR (CDCI3):

8 (ppm) 6.42, 6.54 6.25 5.48 3.83, 3.84 1.42

13C NMR (CDCI3):

750 cm "1.

2H, each singlet, benzene ring H I H, doublet, J = 10 Hz, 4-H I H, doublet, J = 10 Hz, 3-H 6H, singlet, 2 x OCH 3 6H, singlet, 2 x CH 3

75.94 (C-2), 128.19 (C-3), 121.94 (C-4), 110.09 (C-5), 149.84 (C-6), 147.35 (C-7), 101.18 (C-8), 143.20 (C-8a), 113.12 (C-4a), 27.67 (2CH3), 55.9 (OCH3), 56.5 (OCH3).

Synthesis of precocene !, precocene I1 and general chromenes (references): 147, 217, 219-240. Radiolabelling of precocene II (references): 147, 148, 230, 241.

ABBREVIATIONS:

BSA CA DMADP FDP FW GC IDP JH MS MF MVA NAD § NADPH

Bovine serum albumin Corpora allata Dimethyl allyl diphosphate Famesyl diphosphate Fresh weight Gas chromatography Isopentenyl diphosphate Juvenile hormone Mass spectroscopy Methyl famesoate Mevalonate Nicotinamide adenine dinucleotide (oxidized form) Nicotinamide adenine dinucleotide phosphate (reduced form)

JUVENILE HORMONES

411

REFERENCES [1]

Kafka, F. In The Metamorphosis, in the Penal Colony and Other Stories; W. Muir and E. Muir, Tranls.; Schlocken Books: New York, 1975; pp. 67-132. [2] Slfima, K. In Herbivores, Their Interactions with Secondary Plant Metabolites; G.A. Rosenthal and D.H. Janzen, Eds.; Academic Press: New York, 1979; pp. 683-700. Bowers, W.S. In Herbivores, Their Interactions with Secondary Plant [31 Metabolites. Vol I. The Chemical Participants; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 431-456. [4] Harboume, J.B. In Introduction to Ecological Biochemistry; Academic Press: New York, 1993; pp. 117-120. [5] Gilbert, L.I., Rybczynski, R., Tobe S.S. In Metamorphosis; L.I. Gilbert, J.R. Tata and B.G. Atkinson, Eds.; Academic Press: New York, 1996; pp. 59-107. [6] Rosenthal, G.A.; Janzen, D.H. In Herbivores, Their Interactions with Secondary Plant Metabolites, Academic Press: New York, 1979. [7] Rosenthal, G.A.; Berenbaum, M.R. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol I. The Chemical Participants, Academic Press: New York, 1991. [8] Wigglesworth, V.B. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 1-24. [9] Rhoades, D.F. In Herbivores, Their Interactions with Secondary Plant Metabolites; G.A. Rosenthal and D.H. Janzen, Eds.; Academic Press: New York, 1979; pp. 1-54. [lO] Futuyma D.J.; Keese, M.C. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol II. Ecological and Evolutionary Processes; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 439-475. [ll] Hay, M.E., Steinberg, P.D. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol II. Ecological and Evolutionary Processes; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 371-413. [12] Schooley, D.A. In Analytical Biochemistry of Insects; R.B. Turner, Ed.; Elsevier Science: Amsterdam, 1977; pp. 241-287. [13] Tobe S.S.; Feyereisen, R. In Endocrinology of Insects; R.G.H. Downer and H. Laufer, Eds.; A.R. Liss: New York, 1983; pp. 161-178. [14] Meyer, A.S.; Schneiderman, H.A.; Hanzmann, E. Ko, J.H. Proc. Natl. Acad. ScL U.S.A., 1968, 60, 853. [151 Judy, K.J.; Schooley, D.A.; Dunham, L.L. ; Hall, M.S.; Bergot, B.J.; Siddall, J.B. Proc. Natl. Acad. Sci. U.S.A., 1973, 70, 1509. [16] Sparagana, S.P.; Bhaskaran, G.; Dahm, K.H.; Riddle, V..I. Exp. Zool., 1984, 230, 309. [17] Bergot, B.J.; Jamieson, G.C.; Ratcliff, M.A.; Schooley, D.A. Science, 1980, 210, 336. [18] Bergot, B.J., Baker, F.C., Cerf, D.C., Jamieson, G., Schooley, D.A. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds., Elsevier Science: Amsterdam, 1981; pp. 33-45.

412

[19]

BEDE and TOBE

Schooley, D.A., Baker, F.C., Tsai, L.W., Miller, C.A., Jamieson, G. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones; J.A. Hoffmann and M. Porchet, Eds.; Springer-Verlag: Berlin, 1984; pp. 373-383. [20] Lefcvere, K.S.; Lacey, M.J.; Smith, P.H.; Roberts, B. Insect Biochem. Molec. Biol., 1993, 23, 713. [21] Bowers, W.S.; Thompson, M.J.; Uebel, E.C. Life Sciences, 1965, 4, 2323. [22] Trost, B.M. Accounts of Chemistry, 1970, 3, 120. [23] Liedtke, R.J.; Djerassi, C. J. Org. Chem., 1972, 37, 2111. [24l Dunham, L.L.; Henrick, C.A.; Smith, D.H.; Djerassi, C. Organic Mass Spect., 1976, 11, 1120. [25] Silverstein, R.M., Bassler, G.C., Morrill, T.C. In Spectrometric Identification of Organic Compounds 4th Ed., John Wiley and Sons: New York, 1991, pp. 15-16. [26] Thomas, A.F.; Willhalm, B.; Mfiller, R. Organic Mass Spect., 1969, 2, 223. [27] Meyer, A.S.; Hanzmann, E.; Schneiderman, H.A.; Gilbert, L.I.; Boyette, M. Arch. Bcem. Bphys., 1970, 13 7, 190. [28] Kuhnz, W.; Rembold, H. Org. Mag. Res., 1981, 16, 138. [29] Nakanishi, K.; Goto, T.; It6, S.; Natori; S. Nozoc. S. In Natural Products Chemistry, Vol. 1, Academic Press: New York, 1974, pp. 72-78. [30] Meyer, A.S.; Hanzmann, E.; Murphy, R.C. Proc. Natl. Acad. Sci. U.S.A., 1971, 68, 2312. [311 Nakanishi, K.; Schooley, D.A.; Koreeda, M.; Dillon, J. Chem. Comm., 1971, 1235. [32] Giese, Ch.; Spindler, K.D.; Emmerich, H. Z. Naturforsch., 1977, 32c, 158. [33] Kramer, K.J.; Dunn, P.E.; Peterson, R.C.; Law, J.H. In The Juvenile Hormones; L.I. Gilbert, Ed.; Springer-Verlag: Berlin, 1976; pp. 75-95. [34] van Tamelen, E.E. Ace. Chem. Res., 1968, 111. [35] Mumby, S.M.; Hammock, B.D.J. Agric Food Chem., 1979, 27, 1223. [36] Schooley, D.A.; Judy, K.J.; Bergot, B.J.; Hall, M.S.; Jennings, R.C. In The Juvenile Hormones; L.I. Gilbert, Ed.; Springer-Verlag: Berlin, 1976; pp. 101117. [37] Bergot, B.J.; Ra/cliff, M.; Schooley, D.A.J. of Chromatography, 1981, 204, 231. [38] Anderson, R. J.; Henrick, C.A.; Siddall, J.B.; Zurflilh, R. J. Am. Chem. Soc., 1972, 94, 5379. [39] Dunham, L.L.; Schooley, D.A.; Siddall, J.B.J. Chromat. Sci., 1975, 13, 334. [40] Croston, G.; Abdel-Aal, Y.A.I.; Gee, S.J.; Hammock, B.D. Insect Bcem., 1987, 17, 1017. [41] Pfiffner, A. In Aspects of Terpenoid Chemistry and Biochemistry; T.W. Goodwin, Ed.; Academic Press: New York, 1971; pp. 95-135. [42] Wakabayashi, N.; Waters, R.M. In CRC Handbook of Natural Pesticides, Vol III; E.D. Morgan and N.B. Mandava, Eds.; CRC Press: Boca Raton, 1985; pp. 87-151. [43] Szibbo, C.M.; Rotin, D.; Feyereisen, R.; Tobe, S.S. Gen and Comp. Endocrin., 1982, 48, 25. [44] Engelmann, F. Advances in Insect Physio., 1979, 14, 49. [451 Tobe, S.S.; Stay, B. Adv. in Insect Physio., 1985, 18, 305. [46] Tobe, S.S.; Pratt, G.E. Biochem. J., 1974, 144, 107. [47] Pratt, G.E.; Tobe, S.S. Life Sci., 1974, 14, 575. [48] Smith, A.F.; Yagi, K.; Tobe, S.S.; Schal, C. J. Insect Physiol., 1989, 35, 781.

JUVENILE HORMONES

[49]

413

Feyereisen, R.; Tobe, S.S. Anal. Biochem., 1981, 111, 372.

[50] Tobe, S.S.; Clarke, N. Insect Biochem., 1985, 15, 175. [5~1 Roth, L.M. Ann. Rev. Ent., 1970, 15, 75. [52]

[53] [54] [55] [56] [57]

[58] [59] [60] [61] [62] [63]

[64] [65]

[66] [67] [68] [69]

[70] [71] [72] [73] [74] [751 [76] [77] [78] [79]

[80]

Tobe, S.S. In Insect Biology of the Future; M. Locke and D.S. Smith, Eds.; Academic Press: New York, 1980; pp. 345-367. Weaver, R.J.; Pratt, G.E.; Finney, J.R. Experientia, 1975, 31, 597. Weaver, R.J.; Pratt, G.E. Phys. Ent., 1977, 2, 59. Tobe, S.S.; Stay, B. Gen. Comp. Endocrin., 1977, 31, 138. Gadot, M.; Chiang, A.-S.; Schal, C. J. Insect Phys., 1989, 35, 537. Tobe, S.S.; Ruegg, R.P.; Stay, B.A.; Baker, F.C.; Miller, C.A.; Schooley, D.A. Experientia, 1985, 41, 1028. Lanzrein, B.; Gentinetta, V.; Abegglen, H.; Baker, F.C.; Miller, C.A.; Schooley, D.A. Experientia, 1985, 41, 913. Monger, D.J. In Methods in Enzymology, Vol. 110; J.H, Law and H.C. Rilling, Eds.; Academic Press: New York, 1985; pp. 51-58. Sutherland, T.D.; Feyereisen, R. Mol. and Cell. Endocrin., 1996, 120, 115. Svoboda, J.A.; Kaplanis, J.N.; Robbins, W.E.; Thompson, M.J. Ann. Rev. Ent., 1975, 20, 205. Feyereisen, R.; Koener, J.; Tobe, S.S. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds.; Elsevier Science: Amsterdam, 1981; pp. 81-92. Baker, F.C.; Mauchamp, B.; Tsai, L.W.; Schooley, D.A.J. Lipid Res., 1983, 24, 1586. Schooley, D.A.; Baker, F.C. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 363-389. Cusson, M.; Yagi, K.J.; Ding, Q.; Duve, H.; Thorpe, A.; McNeil, J.N.; Tobe, S.S. Insect Bcem., 1991, 21, 1. Feyereisen, R.; Pratt, G.E.; Hamnett, A.F. Eur. J. Bcem., 1981, 118, 231. Wang, Z.; Ding, Q.; Yagi, K.J.; Tobe, S.S.J. Insect Phys., 1994, 40, 217. Reibstein, D.; Law, J.H. Bcem. Bphys. Res. Comm., 1973, 55, 266 Hammock, B.D. Life Sciences, 1975, 17, 323. Hammock, B.D.; Mumby, S.M. Pest. Biochem. Physiol., 1978, 9, 39. Law, J.H. In Biosynthesis of Isoprenoid Compounds, Vol. 2; J.W. Porter and S.L. Spurgeon, Eds.; John Wiley and Sons: New York, 1981; pp. 507-534. Brindle, P.A.; Baker, F.C.; Tsai, L.W.; Reuter, C.C.; Schooley, D.A. Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 7906. Brindle, P.A.; Baker, F.C.; Tsai, L.W.; Schooley, D.A. Arch. Insect Bcem. Physiol., 1992, 19, 1. Peter, M.G.; Shirk, P.D.; Dahm, K.H.; R/Slier, H. Z. Naturforsch., 1981, 36c, 579. Sparagana, S.P.; Bhaskaran, G.; Barrera, P. Arch. Insect Bcem. Phys., 1985, 2, 191. Hammock, B.D. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 431-472. Rotin, D.; Feyereisen, R.; Koener, J.; Tobe, S.S. Insect Bcem., 1982, 12, 263. Mundall, E.C.; Tobe, S.S.; Stay, B. J. Insect Phys., 1981, 27, 821. Riddiford, L.M. In Insect Juvenile Hormone: Chemistry and Action; J.J. Menn and M. Beroza, Eds.; Academic Press: New York, 1972; pp. 95-l 11. Masner, P.; Shima, K.; Landa, V. Nature, 1968, 219, 395.

414

[81]

[82] [83] [84]

[85] [86] [87]

[88] [89]

[90] [911 [92] [93] [94] [95] [96] [97]

[98] [99] [lO0] [lO1] [102] [103] [104]

[1o5] [106] [107] [108] [109]

[llO] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120]

BEDE and TOBE

Saxena, B.P.; Koul, O.; Tikku, K. Indian J. Exp. Biol., 1978, 16, 703. Steele, C.L.; Lewinsohn, E.; Croteau, R. Proc. Natl. Acad. ScL U.S.A., 1995, 92, 4164. Slfima, K.; Williams, C.M. Proc. Natl. Acad. Sci. U.S.A., 1965, 54, 411. Slfima, K.; Williams, C.M. Biol. Bull., 1966, 130, 235. Slfima, K.. Ent. Exp. Appl., 1969, 12, 721. Mansingh, A.; Sahota, T.S.; Shaw, D.A. Can. Ent., 1970, 102, 49. Bowers, W.S.; Fales, H.M.; Thompson, M.J.; Uebel, E.C. Science, 1966, 154, 1020. Manville, J.F. Can. J. Chem., 1975, 53, 1579. Manville, J.F.; Book, K.; von Rudloff, E. Phytochem., 1977, 16, 1967. Manville, J.F.; Kriz, C.D. Can. J. Chem., 1977, 55, 2547. Manville, J.F. Can. J. Chem., 1976, 54, 2365. Rogers, I.H.; Manville, J.F.; Sahota, T. Can. J. Chem., 1974, 52, 1192. Ibaraki, A.; Sahota, T.S. Can. Forestry Bi-Monthly Res. Notes., 1976, 32, 3. Retnakaran, A. Can. Ent., 1970, 102, 1592. v CereS', V.; DoleJ~. L.; Lfibler, L.; Sorm. F.; Slfima, K. Coil Czech. Chem. Comm., 1967, 32, 3926. v Slfima, K.; Such~', M.; $orm, F. Biol. Bull., 1968, 134, 154. $uch~'. M; Slfima, K.; Sorm. F. Science, 1968, 162, 582. Slfima, K.; Williams, C.M. Nature, 1966, 210, 329. Puritch, G.S.; Nijholt, W.W. Can. J. Bot., 1974, 52, 585. Blount, J.F., Pawson, B.A., Saucy, G. Chem. Comm., 1969, 715, 1016. Sakai, T.; Hirose, Y. Chem. Letters, 1973, 491. Sakai, T.; Hirose, Y. Chem. Letters, 1973, 825. McDonough, L.M. In Isopentenoids in Plants: Biochemistry and Function; W.D. Nes, G. Fuller and L.-S. Tsai, Eds.; Marcel Dekker: New York, 1984; pp. 81101. Bowers, W.S.; Nishida, R. Science, 1980, 209, 1030. Nishida, R.; Bowers, W.S.;Evans, P.H.J. Chem. Ecol., 1984, 10, 1435. Mestres, R.; Mufloz, E. Syn. Comm., 1996, 26, 1309. Mehta, G.; Nayak, U.R.; Dev, S. Tetrahedron, 1973, 29, 1119. Bhan, P.; Soman, R.; Dev, S. Agric. Biol. Chem., 1980, 44, 1483. Jacobson, M., Redfem, R.E.; Mills Jr., G.D. Lloydia, 1975, 38, 473. Nishida, R.; Bowers, W.S.; Evans, P.H. Arch. lnsect Bcem. Physiol., 1983, 1, 17. Barton, G.M., MacDonald, B.F., Sahota, T.S. Can. For. Serv. Bi-monthly Res. Notes, 1972, 28, 22. Saxena, B.P.; Saravastava, J.B. Ind. J. Exp. Biol., 1973, 11, 56. Damodaran, N.P.; Dev, S. Tetrahedron, 1973, 29, 1209. Jacobson, M.; Redfem, R.E.; Mills Jr., G.D. Lloydia, 1975, 38, 455. Cooke Jr., M.P. Jr. Org. Chem., 1979, 44, 2461. Orsini, F.; Peizzoni, F. J. Org. Chem., 1980, 45, 4726. Saxena, B.P.; Srivastava, J.B. Experientia, 1972, 28, 112. Prabhu, V.K.K.; John, M. Ent. Exp. Appl., 1975, 18, 87. Prabhu, V.K.K.; John, M. Experientia, 1975, 31, 913. Williams, C.M. Sci. American, 1967, 217, 13.

JUVENILE HORMONES

415

[121] Sparks, T.C.; Hammock, B.D. In Pest Resistance to Pesticides; G.P. Georghiou and T. Saito, Eds.; Plenum Press: New York, 1983; pp. 615-668. [122] Dowd, P.F.; Smith, C.M.; Sparks, T.C. Insect Bcem., 1983, 13, 453. [123] Dyte, C.E. Nature, 1972, 238, 48. [124] Cerf, D.C.; Georghiou, G.P. Nature, 1972, 239, 401. [125] Vinson, S.B.; Plapp Jr., F.W. ,I. Agr. Food Chem., 1974, 22, 356. [126] Bowers, W.S.; Ohta, T.; Cleere, J.S.; Marsella, P.A. Science, 1976, 193, 542. [127] Kaneko, C.; Tsuchiya, T.; Ishikawai, M. Chem. Pharm. Bull., 1963, 11,271. [128] Chang, C.-F.; Isogai, A.; Kamikado, T.; Murakoshi, S.; Sakurai, A.; Tamura, S. Agr. Biol. Chem., 1975, 39, 1167. [129] Suga, T.; Shishibora, T.; Kosela, S.; Sood, V.K. Phytochem., 1975, 14, 308. [130] Proksch, P.; Proksch, M.; Towers, G.H.N.; Rodriguez, E. J. Natl. Products, 1983, 46, 331-334. [131] Tamayo-Castillo, G.; Jakupovic, J.; Bohlmann, F.; Castro, V.; King, R.M. Phytochem., 1989, 28, 139. [132] Ferracini, V.L.; Roewer, I.; Gao, F.; Mabry, T.J. Phytochem., 1989, 28, 1463. [133] Castro, V.; Tamayo-Castillo, G.; Jakupovic, J. Phytochem., 1989, 28, 2415. [134] Siebertz, R.; Proksch, P.; Witte, L. Phytochem., 1990, 29, 2135. [135] Zdero, C.; Bohlmann, F., Niemeyer, H.M. Phytochem., 1991, 30, 693. [1361 Gonz~ilez, A.G.; Aguiar, Z.E.; Grillo, T.A.; Luis, J.G.; Rivera, A.; Calle, J. Phytochem., 1991, 30, 1137. [137] Staal, G.B. Ann. Rev. Ent., 1986, 31,391. [138] Masner, P.; Bowers, W.S.; K~ilin, M.; Miihle, T. Gen. Comp. Endocrin., 1979, 37, 156. [139] Zamorano, G.; Carrau, T.M.; Enguix, A.L.; Martinez-Pardo, R.; Cachaza, A.N. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds.; Elsevier Science: Amsterdam, 1981; pp. 311-314. [140] Unnithan, G.C.; Nair, K.K. Ann. Ent. Soc. Amer., 1979, 72, 38. [141] Bowers, W.S.; Martinez-Pardo, R. Science, 1977, 197, 1369. [142] Milller, P.J.; Masner, P.; K~ilin, M.; Bowers, W.S. Experientia, 1979, 35, 704. [143] Pratt, G.E; Bowers, W.S. Nature, 1977, 265, 548. [144] Unnithan, G.C.; Nair, K.K.; Bowers, W.S. Insect Phys., 1977, 23, 1081. [145] Liechty, L.; Sedlak, B.J. Gen. Comp. Endocrin., 1978, 36, 433. [146] Sedlak, B.J. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 25-60. [147] Ohta, T.; Kuhr, R.J.; Bowers, W.S.J. Agric. Food Chem., 1977, 25, 478. [148] Soderlund, D.M.; Messeguer, A.; Bowers, W.S.J. Agric. Food Chem., 1980, 28, 724. [149] Soderlund, D.M.; Feldlaufer, M.F.; Takahashi, S.Y.; Bowers, W.S. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds., Elsevier Science: Amsterdam, 1981; pp. 353-362. [150] Bowers, W.S.; Evans, P.H.; Marsella, P.A.; Soderlund, D.M.; Bettarini, F. Science, 1982, 217, 647 [~51] Feldlaufer, M.F., Bowers, W.S. Gen. Comp. Endocrin., 1982, 46, 377. [152] Srivastava, R.P.; Proksch, P. Naturwissenschaften, 1990, 77, 438. [153] Satoh, A.; Utamura, H.; Ishizuka, M.; Endo, N.; Tsuju, M.; Nishimura, H. Biosci. Biotech. Biochem., 1996, 60, 664. [154] Proksch, P.; Rodriguez, E. Phytochem., 1983, 22, 2335. [155] Toong, Y.C.; Schooley, D.A.; Baker, F.C. Nature, 1988, 333, 170.

416

BEDE and TOBE

[156] Iwamura, J.; Komaki, K.; Komai, K.; Hirao, N. Nippon Nogeikagaku Kaishi, 1978, 52, 379. [157] Iwamura, J.; Kameda, M.; Komai, K.; ttirao, N. Nippon Kagaku Kaishi, 1978, 11, 1552 (Chem. Abstr. 90: 69086h). [158] Iwamura, J.; Komaki, K.; Komai, K., Hirao, N. Nippon Nogei Kagaku Kaishi, 1978, 52, 561 (Chem. Abstr. 90: 135072d). [159] Iwamura, J., Komaki, K., Komai, K.; Hirao, N. Nippon Kagaku Kaishi, 1978, 53, 255 (Chem. Abstr. 90: 164723x). [160] Iwamura, J. Nippon Nogei Kagaku Kaishi, 1979, 53, 343 (Chem. Abstr. 92: 1162321). [161] Komai, K.; Sugiquaka, Y.; Sato, S. Kinki Daigaku Nogakubu Kiyo, 1981, 14, 57 (Chem. Abstr. 95: 162961c). [162] Versini, G., Rapp, A.; Dalla Serra, A.; Pithier, U., Ramponi, M. Vitis, 1994, 33, 139. [163] Kijjoa, A.; Pinto, M.M.M.; Pinho, P.M.M., Tantisewie, B., Herz, W. Phytochem., 1990, 29, 653. [z64] Jaeobs, H.; Ramadayal, F.; McLean, S.; Perpiek-Dumont, M.; Puzzuoli, F.; Reynolds, W.F..I. Natl. Products, 1987, 50, 507. [165] Waterman, P.G.; Muhammad, I. Phytochem., 1985, 24, 523. [166] Tane, P.; Ayafor, J.F.; Sondengam, B.L. Phytochem., 1988, 27, 3986. [167] Goodman, W.G.; Orth, A.P.; Toong, Y.C., Ebersohl, R.; Hiruma, K.; Granger, N.A. Arch. Insect Bcem. Bphys., 1995, 30, 295. [168] Bede, J.C.; Goodman, W.G.; Tobe, S.S. Pure Appl. Chem., 1998, in press. [169] Meneses, C.R.; Garcifi de la Osa, Centro Agrieola, 1988, 15, 90 (CABS 07910772). [170] Dela Cruz, C.G. International Rice Res. Newsletter, 1986, 11, 26 (CABS 07404038). [171] Naresh, J.S.; Smith, C.M. Ent. Exp. Appl., 1984, 35, 89. [172] Wardle, K.; Short, K.C. Bcem. Phys. Pflanzen, 1982, 177, 210. [173] Hasan, A. In Allelopathy: Basic and Applied Aspects; S.J.H. Rizvi and V. Rizvi, Eds.; Chapman and Hall: London, 1992; pp. 413-441. [~74] Davey, K.G. In Endocrinology of Selected Invertebrate Types; H. Laufer and R.G.H. Downer, Eds.; Alan R. Liss" New York, 1988; pp. 63-86. [175] Rogers, W.P. Comp. Bcem. Physiol., 1978, 61A, 187. [176] Glassburg, G.H.; Teffl, P.M.; Bone, L.W. Proc. Helminthol. Soc. Wash., 1983, 50, 62. [177] Meerovitch, E. Can. J. Zool., 1965, 43, 81. [178] Johnson, R.N., Viglierehio, D.R. Experimental Parasitology, 1970, 27, 301. [179] Adler, J.H.; Grebenok, R.J. Lipids, 1995, 30, 257. [18o] McCormick, J.P.; Schafer, T.R.J. Org. Chem., 1977, 42, 387. [181] Ichinose, I.; Hogogai, T., Kato, T. Synthesis, 1978, 605. [182] Kleijn, H., Westmijze, H.; Meijer, J.; Vermeer, P. J. Roy. Neth. Chem. Soc., 1981, 100, 249. [~83] Rodriguez, J.B.; Gros, E.G.Z. Naturforsch., 1990, 45b, 93. [184] Meyer, W.; Spiteller, G. Liebigs Ann. Chem., 1993, 1253. [185] Trautmann, K.H.; Schuler, A.; Such ~, , M.; Wipf, H.-K. Z. Naturforsch, 1974, 29c, 161. [186] van Broekhoven, L.W.; van der Kerk-van Hoof, A.C.; Salemink, C.A.Z. Naturforsch, 1975, 30c, 726.

JUVENILE H O R M O N E S

417

[187] Lanzrein, B.; Hashimoto, M.; Parmakovich, V.; Nakanishi, K. Life Sciences., 1975, 16, 1271. [188] Peter, M.G.; Dahm, K.H.; R611er, H. Z. Naturforsch., 1976, 31c, 129. [1891 Bergot, B.J.; Sehooley, D.A.; Chippendale, G.M.; Yin, C.-M. Life Sciences, 1976, 18, 811. [190] Hamnctt, A.F.; Pratt, G.E.J. Chromatography, 1978, 158, 387. [191] Hagenguth, H.; Reinhold, H. Z. Naturforsch., 1978, 33c, 847. [192] Hagenguth, H.; Rembold, H. J. Chromatography, 1979, 170, 175. [193] Mauchamp, B.; Lafont, R.; Jourdain, D. J. Insect Phys., 1979, 25, 545. [194] Huibregtse-Minderhout, L.; van tier Kerk-van Hoof, A.C.; Wijkens, P.; Biessels, H.W.A.; Salemink, C.A.J. Chromatography, 1980, 196, 425. [195] Rembold, H.; Hagenguth, H.; Rascher, J. Anal. Bcem., 1980, 101, 356. [196] KSrtv~lyessy, G.; Sz6r,'tdi, S.; Sztruhar, I.; Lad,-tnyi, L. J. Chromatography, 1984, 303, 370. [197] Rembold, H.; Lackner, B. J. Chromatography, 1985, 323, 355. [198] Strambi, C.; Strambi, A.; De Reggi, M.L.; Hrin, M.H.; Delaage, M.A. Fur. J. Bcem., 1981, 118, 401. [199] Granger, N.A.; Goodman, W.G. In Immunological Techniques in Insect Biology; L.I. Gilbert and T.A. Miller, Eds.; Springer-Vcrlag: New York, 1988; pp. 215251. [200] Goodman, W.G.; Coy, D.C.; Baker, F.C.; Xu, L.; Toong, Y.C. Insect Bcem., 1990, 20, 357. [2011 Goodman, W.G.; Huang, Z.-H.; Robinson, G.E.; Strambi, C.; Strambi, A. Arch. Insect Bcem. Phys., 1993, 23, 147. [202] Johnson, W.S.; Li, T.-T.; Faulkner, D.J.; Campbell, S.F.J. Am. Chem. Soc., 1968, 90, 6225. [203] Loew, P.; Siddall, J.B.; Spain, V.L.; Werthemann, L. Proc. Natl. Acad. Sci. U.S.A., 1970, 67, 1462. [204] Locw, P.; Siddall, J.B.; Spain, V.L.; Werthemann, L. Proc. Natl. Acad. Sci. U.S.A., 1970, 67, 1824. [205] van Tamelen, E.E.; McCormick, J.P.J. Am. Chem. Soc., 1970, 92, 737. [206] Loew, P.; Johnson, W.S.J. Am. Chem. Soc., 1971, 93, 3765. [207] Hcnrick, C.A.; Schaub, F.; Siddall, J.B.J. Am. Chem. Soc., 1972, 94, 5374 [208] Ohki, M.; Mori, K.; Matsui, M. ,4gric. Biol. Chem., 1974, 38, 175. [209] Sehooley, D.A.; Bergot, B.J.; Goodman, W.G.; Gilbert, L.I. Bcem. Bphys. Res. Comm., 1978, 81, 743. [210] Adams, P.H.J. Labelled Cmpds. Radiopharm., 1988, 25, 395. [211] Messeguer, A.; S,~nehez-Baeza, F.; Casas, J.; Hammock, B.D. Tetrahedron, 1991, 47, 1291. [212] Pawson, B.A.; Cheung, H.-C.; Gurbaxani, S.; Saucy, G. Chem. Comm., 1968, 1057. [2131 Trost, B.M.; Tamaru, Y. Tetrahedron Letters, 1975, 44, 3797. [214] Pawson, B.A.; Cheung, H.-C.; Gurbaxani, S.; Saucy, G. J. Am. Chem. Soc., 1970, 92, 336. [215] Tutihasi, R.; Hanazawa, T. J. Chem. Soc. Japan, 1940, 61, 1041 (CA (1943) 37: 258). [216] Prakasa Rao, A.S.C.; Bhalla, V.K.; Nayak, U.R.; Dev, S. Tetrahedron, 1973, 29, 1127. [2171 Mann, J.; Kane, P.D. Tetrahedron Letters, 1985, 26, 4677.

418

BEDE and TOBE

[2~8] T'm~ir, T.; Seb6k, P.; KSv6r, K.; J~tszber6nyi, J.C. dcta Chim. Hung., 1988, 125, 303. [219] Miranda, M.A.; Primo, J.; Tormos, R. Heterocycles, 1988, 27, 673. [220] Iyer, M.; Trivedi, G.K. Syn. Comm., 1990, 20, 1347. [221] Bissada, S.; Lau, C.K.; Bemstein, M.A.; Dufresne, C. Can. J. Chem., 1994, 72, 1866. [222] Uchiyama, M.; Overeem, J.C.J. Roy. Neth. Chem. Soc., 1981, 100, 408. [223] Miranda, M.A.; Primo, J.; Tormos, R. Heterocycles, 1991, 32, 1159. [224] Solladi6, G.; Boeffel, D.; Maignan, J. Tetrahedron, 1996, 52, 2065. [225] Smith, L.I.; Ruoff, P.M.J. Am. Chem. Soc., 1940, 62, 145. [2261 Livingstone, R.; Watson, R.B. J'. Chem. Soc., 1957,1509. [227] Sehweizer, E.E., Shaffer, E.T.; Hughes, C.T.; Berninger, C.J.J. Org. Chem., 1966, 31, 2907 [228] Mechoulam, R.; Yagnitinsky, B., Gaoni, Y. Jr. Am. Chem. Soc., 1968, 90, 2418. [229] Anderson, W.K.; LaVoie, E.J.; Whitkop, P.G.J. Org. Chem., 1974, 39, 881. [230] Ohta, T.; Bowers, W.S. Chem. Pharm. Bull., 1977, 25, 2788. [23~] Bongini, A.; Cardillo, G.; Orena, M.; Porzi, G.; Sandri, S. Tetrahedron Letters, 1979, 2 7, 2545. [232] Sartori, G.; Casiraghi, G.; Bolzoni, L.; Casnati, G. J. Org. Chem., 1979, 44, 803. [2331 Tsukayama, M.; Sakamoto, T.; Horie, T.; Masumura, M.; Nakayama, M. Heterocycles, 1981, 16, 955. [234] Kawase, Y.; Yamaguchi, S.; Horita, H.; Takeno, J.; Kameyama, H. Bull. Chem. Soc. Jpn., 1982, 55, 1153. [235] Strunz, G.M.; Brillon, D.; Gigu6re. Can. J. Chem., 1983, 61, 1963 [236] Cort6s, M.J.; Haddad, G.R.; Valderrama, J.A. Heterocycles, 1984, 22, 1951. [237] Pandey, G., Krishna, A. 3. Org. Chem., 1988, 53, 2364. [238] Tiabi, M.; Zamarlik, H. Tetrahedron Letters, 1991, 32, 7251. [2391 Kulkami, S.A.; Paradkar, M.V. Syn. Comm., 1992, 22, 1555. [240] Aukrust, I.R.; Noushabadi, M.; Skatteb~l, L. Polish J. Chem., 1994, 68, 2167. [241] Hsai, M.T.S.; Grossman, S.; Schrankel, K.R. Chem.-Biol. Interactions, 1981, 3 7, 265.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, VoL 22

419


ANTIULCER AND GASTROPROTECTIVE ACTIVITY OF FLAVONIC COMPOUNDS: MECHANISMS INVOLVED M.J. M A R T I N * ; C. A L A R C O N DE LA LASTRA; V. MOTILVA; C. LA CASA

and

Dept. of Pharmacology, Faculty of Pharmacy. University of Seville, Prf Garcia Gonz6lez s/n, 41012-Seville, Spain A B S T R A C T : The flavonoids comprise a large group of unique compounds that are widely distributed in the plant kingdom. In recent years, they have been reviewed for their wide range of biological activities, focusing in particular on the potential therapeutic use of this class of molecules as antiinflammatory, antiallergic, antiviral, anticancer or immunostimulant drugs. This pharmacological potential is probably due to the capability of flavonoids to interact with important cellular processes in which key enzymes such as cyclooxygenase, lipooxygenase, phospholipase A2, NADH-oxidase or glutathion reductase are involved. Other interesting studies also reported the capacity of some flavonoids to interact with oxygen activated species since they are strong scavengers of lipid radicals. These properties, including the antiinflammatory and antioxidant mechanisms and the capacity to inhibit cellular apoptosis, could also be related with an important antiulcerogenic and protective effect on gastric mucosa. Numerous authors have demonstrated the ulcer-protecting properties of these substances against different experimental models, such as restraint-stress, absolute ethanol, reserpine, acetic acid and pyloric occlusion. This review deals with the gastroprotective effects of flavonic compounds, the mechanisms involved, and the possible structure-activity relationships.

INTRODUCTION O f the substances identified in plants, the flavonoids represent one of the most important and interesting classes of biologically active compounds. They are present in a wide variety of plants, and are especially common in leaves, flowering tissues, pollens and fruits. These compounds are also abundant in w o o d y parts such as stems and bark, and they are an important part of human nourishment. Flavonoids have low molecular weight and occur naturally as aglycons, glycosides and methylated derivatives. The aglycons generally consist of a benzo-(-pyrone which in the position 2 or 3 is substituted by a phenyl ring (Fig. 1).

420

MART|N et aL

1

Flavans

0 X Y D A

0 Dihydroxhalcones

oHZ) Flavan-3-ols (Catechtns)

T I

0 N

0

0 Chalcones

Flavanones

.OAD OH Flavan-3-dlols (Leucocyanidins)

Flavylium salts

~ CH-~ Aurones

0 Flavones

421

ANTIULCER AND GASTROPROTECTIVE ACTIVITY

{Fig. I). contd.....

0 0 X Y D A T I 0 N

%

0

Flavanolols {Dihydroflavonols}

Anthocyanldlns

L E V

0

L

0

Flavonols Fig. (1). Principal structural groups of natural flavonoids (from Bombardelli & Morazzoni) [33].

Hydroxylation occurs naturally in position 3, 5, 7, 3', 4' and 5'. Glycosidation with L-rhamnose, D-glucose, glucorhamnose, galactose or arabinose in position 3 or 7 is frequent in nature. Methyl ethers and acetyl esthers of the alcohol groups are known to occur naturally. The pharmacological profile of these agents presents a wide range of activities affecting different biological systems. The empirical use of flavonoids as drugs has acquired scientific confirmation in the last few years. Numerous studies describe its capability to interact with important cellular processes in which keyenzymes are involved such as cyclooxygenase [ 1-5] lipoxygenase [2, 4-10], xanthine oxidase [ 11-14], phospholipase A2 [15, 16], cyclic nucleotide phosphodiesterases [17], proteinkinase C [18], hyaluronidase [19], reverse transcriptase [20, 21 ], mitochondrial succinoxidase [22], NADH-oxidase [23, 24], glutathione reductase [25], glutathione S-transferases [26]. A considerable body of research work deals with the action of flavonoids upon cell membrane permeability [27], biosynthesis of prostaglandins [28-31], their ability to

422

MARTIN et aL

capture free oxygen radicals [32-34], and to modify the synthesis and liberation of histamine [35], or the neutrophil function [36]. The bibliographic research shows that flavonoids possess widespread biological activities, including antimierobial [37-39], antihelmintie [40], mutagenie [41-42], carcinogenic [40, 43, 44], antieareinogenie [42, 45, 46], antioxidant [34, 38, 47, 48], antiinflammatory [49-53], antiallergie [54], antiviral [55, 56], endocrine [57], antihyperlipidemie [58], and antidiarrheie [59-61 ] properties. In the last few years, many papers have been published on the antiuleer effects of numerous flavonoids. Several of these compounds prevent gastric mueosal lesions produced by different methods of experimental ulcers, and they are able to reduce the number and intensity of the lesions [65-69]. Peptic ulcer disease is one of the most common pathological processes of the gastrointestinal tract. It is associated with chronic inflammation of the gastric mueosa and, in the ease of duodenal ulceration, with the duodenal mueosa. It is characterized by frequent recurrence and high incidence. Approximately 10% of the Western population develops this disease, which is associated with high management costs and a substantial reduction in the quality of life of patients. Numerous pharmacological strategies have been used for the treatment and prevention of this pathological process, including antiaeid, antieholinergie or H2antihistaminergie drugs, and more recently proton pump inhibitors. However the gastroduodenal phytotherapy has not developed in a similar way. In the past century, opium extract was frequently used in the treatment of severe pain associated with peptic ulcer disease. Other sedative remedies prescribed included belladonna, hyoseiamus, eonnium, and cannabis, drugs with analgesic or antieholinergie properties. Over the years, numerous plants have been used in folk medicine against gastroduodenal disorders. The development of phytoehemieal techniques has allowed the identification and isolation of numerous principles from some of them, such as alkaloids [70], sesquiterpen laetones [71, 72], or polyphenolie compounds [66-69]. These antiuleer properties have also been related to flavonie extracts of different species, in which phytoehemieal study has demonstrated that these polyphenols are the prevailing compounds (Fig. 2). FLAVONOIDS WITH ANTIULCER ACTIVITY A traditional remedy frequently used in gastrointestinal phytomedicine has been licorice root, Glizyrrhiza glabra. Its gastroprotective effects are attributable to its flavonoids, the flavone liquiritoside and chalcone isoliquiritoside (1-1.5%), which induce defensive mechanisms of the gastric mucosa such as the stimulation of mucous secretion and also

ANTIULCERAND GASTROPROTECTIVEACTIVITY

Rhamnoglucosyl-

423

0

OH

~OCH

3

O

Neohesperidin

Hesperidin OH

O

.,yOH

,~OH

0 Llqulritoslde

OH

0 Narlngenln

OH Rhanmoglucosyl-

~

OH

H

O Narlngin

OH

O Apigenln

OH

OH

OH

OH H

H

I OH OH

O Luteollne

Quercetin

MARTINet aL

424

(Fig. 2). contd .....

OH OH H

H

I OH

O ~ O H Genlsteln

~=

O-Rhanmoglue~yl

Rutln

/OH

CH-~~~--OH

~

C

HO~ ~ oH Sulphuretln

H__~O -"0

H0 H

l--z

MarltlmeUn (3',4',6,7-Tetrahydroxyaurone) OH

H

H

(2',4'-Dlhydroxychalcone)

lsollqulritoslde

0 Sofalcone


425

(Fig. 2). contd.....

Y OH

O !

Sophoradin

Fig. (2). Flavonoids with antiulcer activity.

enhance the healing of the lesions [73-75]. More recently, glycyrrhetic acid has also been implicated and its structure has been used as the starting compound for the synthesis of carbenoxolone, an antiulcer substance with cytoprotective properties (Fig. 3). Precisely the steroid structure of both compounds is also responsible for the side effects, edema, hypertension and electrolytic imbalance. COOH

COOH

)H-CH 2

Glyclrretlc acid

Carbenoxolone

Fig. (3). Structural relation between glycirretic acid and carbenoxolone.

Another isoprenyl chalcone, sophoradin, isolated from the root of

Sophora subprostata [76], exhibits an antiulcer effect in both Shay's

pylorus ligated rats [77], and water-immersed and restraint-stressed rats [78]. In addition to sophoradin, isoliquiretin and isoliquiritigenin have been reported chalcones as possessing antiuleerogenic action, and some reports show that an isoprenyl unit enhances this activity [79]. Kyogoku et al. [80] have synthesized thirty new chalcones which are sophoradin analogues. Several of theses having one or two isoprenyl groups, with or

426

MART|N et al.

without a carboxymethyl group, were found to possess antiulcer activity in both experimental models, and their potencies were equal to that of sophoradin (Table 1). In the course of its screening, they succeeded in finding a new amiulccr compound, 2'-carboxymethoxy-4,4'-bis(3-methyl2-butenyloxy)chalcone (Su-88), sofalcone, which has been used clinically in Japan since 1984 with satisfactory results. The main metabolites of this agent had been detected in human plasma and urine [81 ]. Thus, Hatayama et al. [82] undertook the synthesis and the study of the antiulcer effect of these metabolites. This activity was examined in ulcers provoked by Shay's pyloric ligature, water immersion restraint stress, and histamine induction. Two of the main metabolites in human plasma showed a considerable protective effect, and the compound excreted in human urine showed a somewhat weaker activity. Table 1.

Isoprenyl Chalcones with Antiulcer Activity (Kyogoku et al., 1979) 3

4'

6' 6

3'

0

C-2'

C-Y

C-4'

C-5'

C-2

H

H

i

Pr

OH H

I

OH

Pr

OH

H

OH

C-3 |

C-4

C-5

OPr

H

Inhibitory ratio Shay's rats Stress rats i

/ill

H

ill

+++

+++

,,,

OPr

H

++

+++

OPr

H

+++

++

OPr

H

+++

+++

OPr

H

++

+++

H

H

+++

++

i

OH

Pr

OPr

OH

H

..

H

OPr

H

OPr

H

!

i !

OR H

Soforadin

OH

OPr

i

P

H

OPr

H

H

OPr

H

Pr

OH

H

H

OPt

H

+++

+++

OR

H

H

H

+++

+

H

Pr

OH

Pr

+++

+++

I

!

!

|

,

/ Pr:

/ - ~ ~

R: C H 2 C O H

+: 1 1 - 4 0 %

++: 4 1 - 7 0 %

+++: 7 1 - 1 0 0 %

i


427

Another synthetic derivative of sophoradine, solon, was studied by Konturek et al. [83] in order to verify the antisecreting and gastroprotective effects on different experimental models: stress and water immersion, acidified aspirin, and absolute ethanol. They found an important cytoprotective effect which was maximum between 60 and 90 minutes after administration. Other authors have shown the antiulcerigenic effect of 2 ' , 4 ' - d i h y d r o x y e h a l e o n e , 2',4'-dihydroxy-3'methoxyehaleone and neohesperidin dihydroehaleone [84]. Bidens aurea is a herbal plant commonly used for its digestive and sedative properties. The antiulcer efficiency of a flavonic fraction obtained from the flowering tops of this species on gastric damage induced by restraint stress, acetic acid, and absolute ethanol has been demonstrated [85-87]. Oral treatment with the ether fraction of the flavonic extract gave a high level of gastric protection, and its effectiveness was comparable to that of ranitidine and omeprazol. The phytochemical analysis of this fraction showed the presence of polyphenolic compounds, mainly aurones and chalcones, and the genins maritimetin and sulfuretin were identified [88, 89]. Thus, it is possible to attribute the gastroprotective effect to auronic and chalconic compounds. Apart from chalconic derivatives, other flavonic compounds, such as flavones, flavanones, bioflavonoids and anthocyanidins, also exhibit antiulcer activity. C i n n a m o m u m cassia (Chinese cinnamon), a species containing numerous flavonic derivatives (epieateehol, epieateehol-O-glueoside and dieyelie-O-glueosides), has been used in traditional Chinese medicine for its analgesic, antipyretic and tonic properties [90]. The aqueous extract showed an effect comparable to cimetidine, a potent antisecretory agent, preventing the ulceration induced by stress and cold, and contrarily to cimetidine, it inhibited the ulceration induced by serotonin. It also reduced the secretion of acid and pepsin and increased the mucosal blood flow. Similar gastroprotective results were obtained in other experimental models, such as in the lesions induced by phenylbutazone and oral administration of ethanol. The species Chamomilla recutita (chamomile) is rich in phenolic compounds, cumarines, phenolic acids and, especially, flavonoids such as 13-D-glyeosyl-7-apigenine and their acetylated derivatives, luteolin glucosides, quereetin heterosides and their methylated genines [90]. Apart from its known antispasmolytic activity, it also protects against ulceration mediated by indomethacin, stress and absolute alcohol. The leaves of Catha edulis also have a high content of polyphenolic compounds, tannins and flavonoids. The isolated flavonic fraction showed a significant antiulcer activity against the lesions induced by phenylbutazone and pyloric ligature in rats [91]. It is proven that the compounds responsible for this activity are the major constituents of the

428

MARTIN et aL

ether extract: kaempferol, quercetin, myricetin and dihydromyricetinrhamnoside. A study on the antiulcer activity of genistein, a flavonoid appearing in several species of the genus Genista, showed its effectiveness in preventing ulcer caused by reserpine and phenylbutazone [92]. This result is in agreement with those obtained by Rainova et al. [93], who found that the mixture of flavonoids extracted from Genisto rumelico (luteolin, luteolin-7-glucoside and genistein) exhibited dose-dependent protection in rats. In a study of five models of classic ulcer (pyloric ligature, stress, reserpine, phenylbutazone and 5-hydroxytryptamine), genistein was the most active. The studied compounds did not show antisecretory effect nor did they induce changes in pH or in the pepsine concentration of the gastric juice. The floral sumity of Dittrichia viscosa, a species abounding in the Mediterranean region, has been frequently used in folk medicine over the years for the treatment of gastroduodenal symptoms. Some studies have demonstrated the antiulcerogenic effect of this species against different experimental models of acute or chronic gastric lesions [94]. Ether extraction showed that the major constituents were quereetin, 3-0methylquereetin, naringenin, and 7-O-methylaromadendrine [95, 96], and a further study was undertaken to determine its gastroprotective effect. The gastric lesions induced by the oral administration of necrotizing agents (100% ethanol, HC1 0.6 N and 30% NaCI) were reduced sharply following an oral pretreatment with the flavonic extract and this protective effect could be related to an increase in gastric mucus as well as to the extract's glycoprotein contents [97]. Reyes et al. [98] have investigated the antiulcerogenicity of the flavonoid fraction (ethylacetate extract) of Erica andevalensis, a species endemic to southern of Spain. Oral treatment with the purified extract of the major flavonoid, myrieetin-3-O-D-galaetoside, was found to be effective against gastric ulceration induced by cold-restraint stress, pyloric ligature, and absolute ethanol in rats. The complex flavonoid-lignan silymarin, a hepatoprotective agent from Sylibum marianum, was effective in preventing gastric ulcer induced by experimental stress (restraint and cold), pyloric ligature and ischemiareperfusion in rats [99, 100].The animals treated with silymarin, showed a significant reduction in both number and severity of the lesions; however, the volume of the gastric secretion was not altered, although the concentration of histamine decreased remarkably. Nevertheless, in ethanolinduced ulcers, pretreatment with s i l y m a r i n did not prevent the formation of lesions. Quereetin, a flavone which has numerous pharmacological effects, including antioxidant [101 ], antiinflammatory [ 102], antithrombotic [ 103], antitumoural [ 104, 105], and antibacterial [ 106, 107] ones, has also been tried in the prevention of ulcers induced by some experimental models


429

[108, 109]. The authors showed the cytoprotective properties of this flavonoid and the participation of its antioxidant properties in the prevention of gastric lesions induced by absolute ethanol [110]. Some studies revealed the protective properties of naringenin against the different types of experimental lesions [108, 111, 112]. A time-effect study showed the maximum effectiveness of this flavone to be after 4 hours of oral administration. Rutin, quercetin-3-rhammnosylglucoside, is known for its antiinflammatory and vasoactive properties, diminishing capillary permeability and exerting a vasoconstrictive effect on the peripheral blood vessels [38]. Seeing that gastroduodenal pathology is accompanied by edema and vascular alterations, a new experiment was designed to verify the possible influence of the carbohydrate side chain on the gastroprotective effect [113]. The authors showed that administering rutin before the necrotizing agent, ulceration was essentially prevented, as with quereetin. By contrast, in the same experimental model, ulceration was also prevented by naringin, the glucoside of naringenin, but the effective doses were significantly higher than those of the genine, showing that the inclusion of the sugar side chain in the molecule decreases the gastroprotective activity [ 114]. Another flavonoid, hypolaetin-8-glueoside, found in many species of the genus Sideritis, is traditionally used in Spain for its antiinflammatory and digestive properties [115, 116]. It was isolated from Sideritis leucantha and its antiulcerogenic properties studied by Alcaraz and Tordera [117]. The authors found that this compound significantly reduces the gastric lesions induced by absolute ethanol, acetyl salicylic acid and, Shay's pyloric ligature. Many other flavonic compounds have been reported to exhibit an interesting antiulcer effect: mecyadanol, (+)-cyanidanol-3, (+)-catechin, 3O-methyl-(+)-catechin, 3-methoxy-5,7,3',4'-tetrahydroxyflavan, apigenin7,4'-diorthomethylether, hesperidin, and amentoflavone [ 118-121 ]. MECHANISMS INVOLVED IN THE ANTIULCEROGENIC EFFECT OF FLAVONOIDS Recently there have been extraordinary advances in the understanding of the pathophysiology and treatment of gastrointestinal disorders. However, many questions regarding acid-related diseases remain to be answered. Traditionally, ulceration of the gastric mucosa was regarded as being caused by excessive gastric acid secretion. The significance of HCI in peptic ulcer disease had already been recognized in 1910, by Schwartz, "without acid gastric juice, no peptic ulcer" [122], and became the basis upon which ulcer therapy was designed: anticholinergic drugs, histamine H2 receptor antagonists, antiacids, and more recently, proton pump inhibitors. But also it was admitted that the enhancement of acid secretion

430

MARTIN et aL

alone is unlikely to produce lesions, because many patients with ulcers secrete acid at normal rates. The discovery, in 1979, by Robert [123] of the remarkable ability of prostaglandins (PG) to increase the resistance of the gastric mucosa to damage started research on the role of defensive factors, such as the mucus-bicarbonate barrier, mucosal blood flow, neutralization of free radicals, stabilization of lisosomal membranes or nitric oxide on the gastric mucosal integrity. Numerous causes can contribute to the genesis of peptic ulcer, so the imbalance between the aggressive and defensive factors plays an important role. Aggressive factors include both endogenous (acid, pepsin, gastrin, leukotrienes, free radicals or Helicobacter pylori) and exogenous (food, NSAID, nicotine or stress) mediators (Table 2). Table 2.

Aggessive and Defensive Factors of Gastric Mucosa i

Aggessive factors i

i

,,

i

,

,

Defensive factors i|

9 Acid secretion

9 Mucus-bicarbonate barrier

9 Pepsine

9 Prostaglandins

9 Helicobacter pylori

9 Endogenous antioxidants

9 Electrolytic balance

9 Mucosai blood flow

9 Leukotrienes

9 Stabilization of the mastocyte membrane

9 Oxygen free radicals

9 Regulation of gastrointestinal motility

9 Neutrophil activation and adherence

9 Nicotine

9NSAID

9 Nitric oxide

9 Stress

9 Epithelial restitution i

A series of papers has been published in the last few years about the mechanisms involved in the antiuleerogenicity of flavonoids. These compounds can modify both aggressive and defensive factors.

Aggressive Factors

Acid Secretion Stimulation of parietal cells for gastric acid secretion is a complex process mediated both centrally by vagal parasympathic fibers and peripherically by release of histamine from the fundic mucosal enterochromaffin-like


431

cells. Other mediators include at least acetylcholine from the vagus and gastrin from the G cells of the antrum. H2 receptor antagonists are extremely effective inhibitors of acid secretion during pentagastrin stimulation and are able to inhibit partially the secretion due to vagal stimulation. Since neither gastrin nor acetylcholine binds to H2 receptor, it is apparent that histamine must therefore operate on the pathway of stimulation initiated by these mediators. Thus it seems likely that histamine rather than gastrin is the final mediator of acid secretion. Histamine has been involved in the pathogenesis of gastric ulcers produced by restraint stress, pyloric ligature and other methods because it increases vascular permeability and acidity [124, 125]. Senay and Levine [ 126], and more recently Marazova et al [ 127], related the ulcerogenesis induced by immobilization and cold to an increase of histamine synthesis, resulting from stimulation of histidin-decarboxylase activity. This enzyme was found at high concentrations in the glandular mucosa of the rat. In other experimental models, the mechanisms involved in mucosal lesion production are, among others, an enhancement of vagal activity leading to hyperacidity, and an increase of mast cell degradation with a high liberation of histamine. However, the role of this amine against ulcerogenic agents such as ethanol and other necrotizing agents is not clear. Some authors [128] consider the possibility of a gastroprotective effect of histamine, mediated by stimulation of PG synthesis, because it prevents the appearance of the hemorrhagic bands caused by these agents. Palitzsch et al. [129] suggest that these gastroprotective processes might be induced through H3 receptor. Some flavonoids have been reported to possess antisecretory activity in vivo [ 130]. Yamahara et al. [ 131 ] also found that vexibinol, a flavonol obtained from Sphora significantlyn, inhibits basal and 2-deoxy-D-glucosestimulated acid secretion in rats but possesses no or a much weaker effect on acid secretion stimulated by histamine, carbachol or pentagastrin. Saziki et al. [132] reported that sofaleone significantly reduced the volume and acidity of gastric juice in pylorus-ligated rats. Similar results were obtained by Alcaraz and Tordera [117] with hypolaetin-8-glueoside. In the same experimental model, the flavonic extract of Stachytarpheta cayennensis also inhibited the basal acid secretion as well as that induced by histamine and bethanecol, and the authors suggest that this inhibition could be due to the level of histamine release and H2 receptor interaction [ 133]. Other agents such as quereetin, naringenin, (+)-eyanidanol-3 and m e e y a d a n o l are able to inhibit the enzyme histidin-decarboxylase, decreasing the histamine levels in the gastric juice [134-136]. Konturek et al. [83] and later Martin et al. [108] found that this inhibition was not accompanied by a parallel reduction of acidity. Similar results were obtained by Alarc6n et al. [99] when they assayed the antiulcerogenic

432

MARTI[N et aL

effect of silymarin on pylorus-ligated gastric secretion and lesions. The authors suggest that mechanisms other than antisecretory ones are involved in the preventive effect of these agents. The last mediator of gastric secretion in the parietal cell is an H§ +ATPase (proton or acid pump) which is a member of the phosphorylating class of ion transport ATPases. Hydrolysis of ATP results in ion transport. This chemical reaction induces a conformational change in the protein that allows an electroneutral exchange of cytoplasmic H § for K § The pump is activated when associated with a potassium chloride pathway in the canalicular membrane which allows potassium chloride efflux into the extracytoplasmic space, and thus results in secretion of hydrochloric acid at the expense of ATP breakdown. The activity of the pump is determined by the access of K § on this surface on the pump. In the absence of K § the cycle stops at the level of the phosphoenzyme [137]. The high efficacy of proton pump inhibitors (PPI) in the abrogation of acid secretion is based on the fact that their inhibition is independent of the pathway of stimulation. In fact, these agents block acid production in response to all stimulants. It is this specific blockade of the final stage in the complex chain of acid production that enables PPI to avoid many of the clinical problems noted with other antisecretory drugs. Murakami et al. [138], showed that some flavonie compounds, such as the chalcone derivatives xanthoangelol and 4 - h y d r o x y d e r r i c i n from the root of Angelica keiskei are potent inhibitors of the proton pump in vitro. The same authors [139] studied the antisecretory mechanisms of sophoradin, its synthetic chalconic derivative sofalcone, and chalcone. The order of potency in inhibiting the enzyme was sophoradin>sofalcone>ehaleone. This is compatible with the antisecretory activity shown in pylorusligated rats in vivo, in which sophoradin was more effective than sofalcone in reducing the acid output, while chalcone had little effect on acid output [132]. These compounds were also shown to be effective in the in vitro proton transport mediated by H+,K+-ATPase. The ATP hydrolytic sites for the proton pump are located at eytosolic sites and the high affinity K + sites are on luminal face across the membrane [140]. The enzyme is phosphorylated at cytosolic sites by ATP in the presence of Mg 2+. Then the enzyme-phosphate complex is dephosphorylated by luminal K § The kinetic studies carried out by Murakami et al. [ 139, 141 ] demonstrated that the inhibition of the gastric pump by sofalcone, chalcone and quercetin was competitive with respect to ATP and noncompetitive with K § In this way, Beil et al. [142] showed that quercetin, flavone and flavanone locked acid formation in parietal cells in response to histamine and cAMP stimulation, flavanone being the most potent inhibitor. H § K+-ATPase was inhibited by all of them, and this inhibition increased with lowering ATP concentration. The steady-state phosphorylation level of the enzyme was also dose-


433

dependently reduced by quereetin. The authors suggest that these flavonoids may bind to ATP sites competitively with ATP and inhibit the formation of intermediate phosphoenzymes, thereby inhibing H+,K+ATPase activity. Although it is unlikely that flavonoids cause inhibition of acid secretion in vivo by blocking the proton pump activity, these results suggest that this mechanism could be involved at least partly.

Helicobacter pylori Since Warren and Marshall, in 1983, isolated Helicobacterpylori from the human stomach, this microorganism began to be considered an important factor in gastric disorders [143]. Now it is accepted to be the cause of chronic active type B gastritis [ 144], and strong evidence suggests that H. pylori infection is a major causative factor for peptic ulcer disease [146, 147]. In fact, H. pylori is present in over 90% of patients with duodenal ulceration. The prevalence of infection in gastric ulcer patients is somewhat lower and more variable, 70-90% [148, 149]. However, probably the most compelling evidence for a causal relationship between H. pylori and peptic ulcer disease is the prolonged remission from reulceration that follows successful eradication of the organism [150]. The exact mechanism by which H. pylori causes peptic ulceration remains unclear, although it is thought to involve mucosal damage mediated by the virulence factors of the organism, mucosal inflammatory response to tissue damage and H. pylori antigens, disturbances in gastrin, somatostatin and acid secretion, and the development of duodenal gastric metaplasia [ 149, 150]. The acceptance that H. pylori infection has a causal role in peptic ulcer pathogenesis has already had a considerable impact in the management of this disease. Today, a combination of antibacterial (amoxicillin, clarithromycin, tetracyclin, metronidazole, tinidazole or bismuth compounds) and antisecretory agents (H2-receptor antagonists or PPI) constitutes the most important pharmacological strategy in the treatment of this disease. The use of these drugs combined in a regime of triple or quadruple therapy achieves eradication rates of around 90% [151, 152]. A recent study carried out by Beil et al. (1995) [142] shows that flavone, flavanone and quereetin inhibited H. pylori growth in a concentration-dependent manner. The most potent compound was flavone, with an antibacterial activity similar to that reported by colloidal bismuth subcitrate. Therefore, from the known digestive properties of flavonie agents, antisecretory action and inhibition of H.pylori growth, it appears that these agents could have a therapeutic potential, ideal for treatment of gastrointestinal disorders associated with H.pylori infection, e.g. type B gastritis and duodenal ulcer.

434

MARTIN et as

Leukotrienes

Leukotrienes (LT) are a class of eicosanoids which derive from the arachidonic acid cascade, via 5-1ipoxygenase (5-LOX), and they are involved as possible mediators of gastric damage and ulceration [53, 154]. Recent investigations have indicated that these agents play an important role as mucosal aggressive factors. In rats, LT have been shown to be released in response to necrotizing agents such as absolute ethanol [ 155( and hydrochloric acid [156]. The exogenous LT have been reported to exert strong action on mucosal microcirculation, they cause potent vasoconstriction, vascular stasis and an increase in vascular permeability [157], and may contribute to the maintenance of the chronic injury, especially if there is inflammatory infiltration. Furthermore, LTC4 and LTB4, although not ulcerogenic themselves, aggravated mucosal injury induced by various noxious agents or non steroidal anti inflammatory drugs, NSAID [ 154, 158, 159]. A number of reports have suggested that LT synthesis inhibitors, LTB4 receptor antagonists, and LTD4 receptor antagonists might protect against the formation of gastric lesions, and therefore they may be considered as possible clinical agents [158,160, 161 ]. Several flavonoids have been shown to inhibit 15-LOX activity from soybean, 12-LOX from blood platelets [2] or 5-LOX from leukocytes [1, 162]. Some authors have established that the 3',4'-diol substitution in the B ring (catechol) is the most important requirement for the inhibition by flavonoids of arachidonate lypoxygenases [163, 164]. These conclusions were supported by the results obtained by Moroney et al. [1], and they confirm that an additional hydroxyl substituent in the body of the molecule confers the most potent and selective inhibitory effect of 5-LOX. By contrast, the glycosylation considerably reduces potency. In this study, quereetin (3',4',5,7-tetrahydroxyflavonol), rutin (quercetin-3glucoside), n a r i n g e n i n (4',5,7-trihydroxyflavanone), h y p o l a e t i n (5,7,8,3',4'-pentahydroxyflavone), and hypolaetin-8-glueoside were found to be selective 5-LOX inhibitors, and the order of potency was quereetin>hypolaetin>naringenin>rutin>hypolaetin-8-glueoside. All of them exhibit antiulcerogenic effect. Thus, it is possible that this property could be due, at least in part, to inhibition of LT synthesis. However this supposition requires further investigation. Oxygen Free Radicals

Oxygen-generated free radicals have been shown to be implicated in many pathophysiological conditions and in the toxicity of xenobiotics.They provoke severe changes at cellular level leading to cell death, because owing to their extreme reactivity, they attack the essential cell constituents, such as nucleic acids, proteins or lipids. They also induce peroxidation of the


435

membrane lipids and this action lead to the formation of a series of toxic compounds, such as epoxides, aldehydes, and new free radicals. An important intermediate in biological actions is the superoxide anion radical, which is formed in vivo during the reduction of molecular oxygen. This species can produce hydrogen peroxide (H202) a highly toxic product, which in turn gives rise to the hydroxyl radical (.OH) by reaction with transition metal ions in the body. The (.OH) radical is very reactive and one of the strongest oxidizing agents [165]. On the other hand, gastric mucosal injury has also been associated with periods of ischemia in several different clinical settings, including trauma [166], ethanol exposure [167], acetic acid [168], NSAID [169], or ischemia-reperfusion [170]. When a tissue is subjected to ischemia, a sequence of chemical reactions is initiated which may ultimately lead to cellular dysfunction and necrosis [ 171]. Although no single process can be identified as the critical event in ischemia-induced damage, most studies indicate that depletion of cellular energy stores and accumulation of toxic metabolites may contribute to cell death. Sources of reactive oxygen metabolites include the xanthine oxidase system, which is modified during ischemia, since it produces the superoxide anion radical and H202 during reperfusion. These oxygen radicals may then be converted to the highly cytotoxic hydroxyl radical by the iron-catalyzed Haber-Weiss reaction. This initiates the process of lipid peroxidation which, in turn, results in the production and release of substances that recruit and activate polymorphonuclear leukocytes [172]. The substances which are able to hinder their formation or with capacity to capture the formed free oxygen radicals are thus potential antiulcerogenic agents. It has been known for a long time that most of the flavonoids and their derivatives are principles capable of capturing free oxygen radicals, peroxides of fatty acids as well as hydroxyl groups. The capture of superoxide free radicals has been evidenced by the formation of trans-trans hydroperoxides of linoleic acid by flavonoids [173, 174]. The capture of hydroxyl radicals responsible of numerous cell degradations is what indicates its relation with factors of cellular protection. Letan [175] proposed a series of structural requirements that flavonoids had to fulfil in order to have antioxidative action: one free OH group in position 3 one double bond between carbons 2 and 3 one keto group in position 4 of the pyrone ring free OH groups in positions 5 and 7 free OH groups in positions 3' and 4' In this context, the sugar moiety masks the antioxidant activity of flavonoids. A possible explanation for this could be that the lower

436

MARTIN et aL

lipophilicity of the glycoside prevents its access to the lipid membranes, the site of lipid peroxidation. This was evident when Rekka and Kourounakis investigated the effect of 7-mono-,7,4'-di-,7,3',4'-tri and 5,7,3',4'-tetra-hydroxiethyl rutosides and 7,3',4'-tri-hydroxiethyl quercetin on non-enzymatic lipid peroxidation in vitro. In this study quereetin was found to be the most antioxidant [ 176]. Interactions between a number of flavonoids, and superoxide anion have been described by many authors [177, 178, 179]. In contrast, only a few data are available relating to the hydroxyl radicals scavenging activity of flavonoids. Cillard and Cillard in 1988 [180] reported the percentage of scavenging of the hydroxyl radical produced by the decomposition of hydrogen peroxide or peroxides of fatty acids such as linoleic acid identified by HPLC. In that study, some antiulcer flavonoids as quereetin and naringenin, exerted a scavenging of about 48% and 36% respectively. More recently, interesting studies also reported the strong hydroxyl radical scavenging capacity of rutin [181 ] and silymarin [48]. There are many studies about the relation between the free oxygen radical scavenging activities of many flavonoids and their effects on the enzymes which take part in the metabolism of arachidonic acid, cyclooxygenase (COX) and lipoxygenase (LOX). Duneic [182] shows that the antiradical action affects the COX activity in several ways. In most of the cases, at high substrate concentrations the enzymatic activity was intensified and at low concentrations it was inhibited. Apparently, the influence of the antiradical properties on the activity of enzymatic metabolism of the arachid0nic acid in vitro might also be due to the effect of these agents on the active center of the enzymes. Robak et al. [ 183, 184] studied a good amount of flavonoids, analyzing their influence on COX and LOX activity, and evaluating their antioxidant properties. They showed that most of them stimulated COX. Rutin and quercetin exerted a strong stimulating action, while naringenin, naringin and hesperidin did not stimulate COX. In addition, quereetin, naringenin and silymarin were found to be relatively selective inhibitors of 5-LOX [ 1, 185-187] inhibiting therefore the biosynthesis of LT. A number of papers show that among flavonoids there are strong scavengers of lipidic radicals [33, 34]. For example, quercetin and Gingko biloba extract show an important antilipoperoxidant action or a weak effect (rutin and kaempferol), moreover hesperidin increases the rate of autoxidation. The discrepancy between the activity of these flavonoids seems to be important in explain the different effects of, for example, quercetin (inhibitor) and rutin (stimulator) on the cell COX activity. It is, in fact, important to remember that COX activity includes lipoperoxidative steps and thus it is inhibited by antilipoperoxidant agents. Chelating activity gives another possibility to flavonoids to interact with the oxidation of membrane lipids, and it is considered important for


437

their inhibitory action on microsomal lipid peroxidation induced by an iron dependent system [33]. On the other hand, there is growing evidence for the role of the nitric oxide radical (.NO) in vascular endothelial damage. It is speculated that (.NO) scavenging activity may contribute to the therapeutic effects of the flavonoids. On the basis of this hypothesis,recently the (.NO) scavenging capacity of certain flavonoids, as hydroxyethylrutosides, has been investigated by Van Acker et al. [ 188]. Neutrophil Activation and Adhesion Free oxygen radicals produced in gastric pathology not only come from the affected tissue, but they can also be originated in several biological reactions, pointing up the activation of neutrophils in the extracellular space. Recently, the degree of neutrophil infiltration in the gastric mucosa has been related to the genesis of the lesions [189, 190]. Neutrophils contain an NADPH oxidase which reduces molecular oxygen to the superoxide anion radical and these leukocytes are the primary mediators of reperfusion-induced increases in microvascular permeability. The leukocytes are attracted by the chemotactic factors liberated in the states of ischemia. These chemotactic factors favor the gastrodamaging process and the way they release phagosomes full with oxygen free radicals into the environment. These endogenous agressors stimulate the mucosal parenchyma and many types of cell to produce the inflammatory damage: Mastocytes are stimulated and release: histamine, leukotrienes (LT), interleukin-1 (IL-1), platelet activating factor (PAF), proteases and peroxidases. The endothelial cells of the capillaries liberate LT, nitric oxide (NO) and endothelin- 1. The neutrophils adhere to the endothelium, block the capillaries and damage the endothelial integrity. Activated neutrophils produce reactive oxygen metabolites and release a variety of cytotoxic proteins, e.g. proteases, lactoferrin, and they also secrete the enzyme myeloperoxidase (MPO) which catalyzes the formation of potent cytotoxic oxidants such as hypochlorous acid (HOCI) from H202 and chloride ions and N- chloramines [172]. The inflammatory mediators cause mucosal ischemia and an increase of the capillary permeability affecting the fluid which is extravasated to the interstices, causing mucosal edema. Neutrophils and erythrocytes are moved to the extravascular compartment. The epithelial surface appears hemorrhagic. The mucosal ischemia extends, microcirculation gets

438

MARTIN et aL

congested with stagnant blood, the tissue becomes hypoxic and necrotic cells appear even in the deepest layers of the mucosa. It is evident that agents capable of inhibiting the activation of these granulocytes could be also evaluated as potentially antiulcerogenic. Recent investigations seem to confirm this hypothesis. As shown by Alarc6n de la Lastra et al. [100] using a model of gastric injury induced by ischemia-reperfusion in rats, pretreatment with silymarin, the hepatoprotective principle of Silybum marianum L. prevented post-ischemic injury. These protective effects were specifically related to reduction of MPO activity as index of polymorphonuclear leukocyte infiltration after injury. These findings indicated that inhibitory effects of silymarin on neutrophil function seem to contribute significantly to its gastroprotective actions. By contrast, the inhibition of neutrophil infiltration did not appear to be involved in the antiulcer effect of quercetin in gastric mucosal injury induced by 50% ethanol, an experimental model in which there is a considerable leukocyte influx into the gastric mucosa [ 110]. Treatment of human endothelial cells with cytokines such as interleukin-1, tumoral necrosis factor-alpha (TNF-alpha), or interferongamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation may support a role in maintaining gastric mucosal integrity. A recent experimental study indicated that one of the most potent flavones, apigenin, exhibited a dose and time-dependent, reversible effect on adhesion protein expression, as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited TNF-alpha- induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression [ 191]. By the same token, a flavonoid, 2-(3-amino-phenyl)-8-methoxychromene-4-one, markedly inhibited TNF-alpha induced vascular cell adhesion molecule-1 (VCAM-1) in a concentration-dependent fashion of human aortic endothelial cells, but had no effect on intercellular adhesion molecule- 1 (ICAM- 1) [ 192]. A number of experimental studies have been performed looking at the gastroduodenal damaging effects of non-steroidal antiinflammatory drugs (NSAID) such as indomethacin [193]. In these studies, orally administered 5-methoxyflavone inhibited indomethacin-induced leukocyte adherence to mesenteric venules, suggesting a role of inhibition of leukocyte adherence in gastroprotective activity of this flavonic compound.

Defensive mechanisms In addition to antisecretory mechanisms, the enhancement of the defensive mucosal factors seems to play an important role in the antiulcer effects of


439

the flavonoids. The term "mucosal defense" refers to the factors that permit the mucosa to withstand frequent exposure to substances with a wide range of pH, osmolarity and temperature, and to noxious agents and bacterial products capable of causing local and systemic inflammatory reactions. However, the mucosa can repair such injury quickly, thereby limiting it to the most superficial layer of cells and preventing entry into the systemic circulation of substances detrimental to the organism. Their resistance can also be enhanced when irritants are present in the stomach. Thus, the ability of the mucosa to resist significant injury is attributable to a dynamic process rather than to a static barrier. Although numerous flavonic substances present gastroprotective properties, the mechanisms involved in mucosal defense, are not sufficiently explained and, naturally, they are not the same for each flavonoid. Mucus-bicarbonate Barrier

Morris et al. [194] demonstrated that a primary effect of many cytoprotective agents may be stimulation of mucosal secretion, resulting in the formation of a barrier that may attenuate the damaging effects of necrotizing agents. The role of the so-called mucus-bicarbonate barrier in protecting the mucosa from injury induced by acid and pepsin is one of the most controversial aspects of mucosal defense. The mucous gel is proposed to form a continuous "blanket" over the mucosal surface, which traps bicarbonate secreted by the epithelium and therefore acts as a layer in which luminal acid that diffuses toward the epithelium is neutralized. In addition, the mucus plays an important role in restricting the bacterial movement to the surface epithelium; bacteria become trapped in the gel and are eventually excreted in feces. It is also capable of acting as an antioxidant, and so can reduce mucosal damage mediated by these factors produced by bacteria or immunocytes [ 195, 196]. An additional element in mucosal defense is the coupling of defensive and aggressive factors, so that mucosal protective mechanisms are enhanced when acid secretion is occurring. In this way, some authors [ 197, 198] attributed mucus hypersecretion in cases of severe damage to an autodefense mechanism of increased synthesis in the intact glandular zones, to aid restitution of the lesioned areas. The viscous and gel-forming properties of mucus secretions are derived from mucin glycoprotein constituents (5%); they also contains protein, lipid and nucleic acid, much of it derived from dislodged epithelial cells and bacteria [ 199]. These constituents have been shown to enhance the viscous properties of mucins in vitro [200, 201], and such interactions may be significant towards determining mucus gel properties in vivo. Considering the recorded data, we can assert that the role played by gastric mucus in the cytoprotection mediated by flavonic substances is

440

MART|N et at

variable. Quercetin, naringenin, hypolaetin-8-glucoside, solon and sofalcone [202, 203] provoke a clear stimulation of mucus synthesis and secretion, also enhancing the glycoprotein content with an increase of total proteins, hexosamines, neutral glycoproteins and sulfated macromolecules [109, 112, 117]. The same results were obtained from the flavonic fraction of Bidens aurea [86, 89] and Dittrichia viscosa [97]. A rheological study showed that preparations of adherent gastric mucus gel scraped gently from the mucosal surface of rats treated with naringin had significantly increased viscoelastic properties [114]. These findings seem to indicate that the enhancement of mucus characteristics is related to the gastroprotective effects of these compounds. Concerning ulcers induced by absolute ethanol, pretreatment with silymarin stimulated mucus secretion, but did not modify the concentration of total proteins and hexosamines [99]. By contrast, in the same experimental model, Reyes et al. [98] found that mucus amount was not modified by the ethylacetate extract of Erica andevalensis, although there was an increase in the concentration of its components. P6rezGuerrero et al. [113] showed that rutin, the glycoside of quercetin, has a protective effect, although it did not induce any changes in the amount of mucus or in its glycoprotein content. These results confirm that the mucus layer alone is incapable of protecting the underlying mucosa [204]. It is necessary that other defensive mechanisms, such as bicarbonate secretion by the non-parietal cells [205], the restoration of vascular factors [206], or the inhibition of leukocyte adherence [198], come into play. Prostaglandins

In comrast to LT, many natural prostaglandins (PG), particularly of the E series, inhibit gastric acid secretion. However, over and above antisecretory activity, these agents also share the property of "cytoprotection". This term was proposed by Robert in 1979 [123] to account for the ulcer-reducing effect of prostaglandins by a mechanism other than inhibition of gastric secretion. Gastric secretory inhibition is in itself somewhat protective, so separation of these two effects is important. Numerous studies evidence the role and the influence on the gastric defensive mechanisms of these mediators [92, 207-209]. There is evidence that PG may have a physiological role in regulating mucosal mucus; moreover they increase gastric non-parietal cell secretion and duodenal bicarbonate secretion, and improve the lysosomal and vascular integrity [210]. The stabilizing effects of PG on the microvasculature may be particularly important, since it appears that one of the first abnormalities in the pathogenesis of mucosal injury is an increase in microvasculature permeability. Finally, it has been shown that PG limit the depth of mucosal destruction in injury induced by absolute ethanol,

ANTIULCER AND GASTROPROTECTIVE ACTIVITY'

441

permitting more rapid restitution of the damaged superficial epithelium, and they normalize the gastric secretion which was inhibited after administering the necrotizing agents. These findings, described by Robert et al. [211 ], show that prostaglandins maintain the morphologic integrity of parietal cells even in the presence of this class of agents. The antiulcer effect of some flavonoids might be related to their ability to increase the mucosal levels of these prostanoids. This is the case of solon, which inhibits the metabolizing enzyme 15-OH-PG-DH 15hydroxy-prostaglandindehydrogenase [83]. The cytoprotective mechanism would be similar to the one of carbenoxolone, a drug frequently used in experimental models because of its ability to stimulate the mucosal defenses. Some flavonoids have been shown both to inhibit and to stimulate production of prostaglandins in vitro [28, 212]. Flavone and flavanone induced PGE2 production in isolated gastric mucosal cells, but since these compounds did not stimulate the prostanoid production in gastric cells exposed to arachidonic acid, it is likely that both flavonoids enhance prostaglandin formation by acting as cofactors of cyclooxygenase [ 142]. Other flavonoids that are able to enhance the luminal release of PG in vivo are the glucoside of hypolaetin [ 117], the anthocyanidin pigment IdB 1027 [213], and the flavonic extract of Bidens aurea [214]; therefore their antiulcer effect could be related to an enhancement of PGE2 levels in gastric mucosa and also to a generalized stimulation of the defensive mucosal mechanisms. Alarc6n et al. [ 109] designed an assay to verify the possible role of the mucosal prostaglandins in the antiulcerogenic effect of quereetin on ethanol-induced gastric lesions in rat. The animals were pretreated orally with this flavonoid before administering the ulcerogenic agent, and the degree of alcohol-ulceration was notably reduced. The total amount of mucus as well as its content in glycoproteins increased.When indomethacin was administered subcutaneously to the animals pretreated with quereetin, the protective effect reverted partially, the intensity of the lesions increasing and the synthesis of mucus and concentration of hexosamines decreasing. This may point to a possible participation of prostaglandins in this effect. However, Beil et al. [142] found that quereetin was not effective in stimulating PGE2 production in vitro. These data are in agreement with those of Alcaraz and Hoult [6], who showed that this flavonoid has no effect on prostaglandin formation in fragments of rat caecum. Similar results were obtained with naringenin on mucosal lesions induced by necrotizing agents [112]. Therefore, the antiulcerogenic effect does not seem to be mediated through the cyclooxygenase pathway, because in this experimental model, this flavonoid does not increase the synthesis of mucosal PGE2. Rather, the gastroprotection induced by quereetin and narigenin might be related to

442

MARTIN et aL

an enhancement of mucus secretion and enrichment of its glycoprotein content. Endogenous Sulfh ydryls

Sulfhydryl compounds (SH), like PG, are endogenous cytoprotective agents. SH-containing compounds, and also agents that modify SH groups, oxide SH groups or bind SH groups, prevent the acute hemorrhagic erosions caused by ethanol, nonsteroidal antiinflammatory drugs or stress in animal models [215, 216]. Depletion of GSH results in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption. In contrast, a rise in gastric SH levels limits the production of oxygen-derived free radicals, and could be related with cellular protection [217]. In addition to free-radical scavenging activity, SH compounds, as well as PG, may maintain a good blood flow that allows the energydepen-dent rapid restitution to cover initial epithelial surface damage [218, 219]. Some experimental studies have demonstrated that flavonoids, such as quereetin [ 110] and the flavonic extract from Bidens aurea (Aiton) Sherff [89] prevented the gastric necrosis induced by 50% v/v ethanol, and produced a significant enhancement of gastric mucosal SH content. Therefore, it was assumed that endogenous SH contributed to the functional mechanisms of protection by flavonoids in this experimental model. Blood Flow and Nitric Oxide

Gastric mucosal flow plays a central role in preventing damage to the gastric lining.The remarkable effectiveness of this vascular defensive system occurs at all levels of gastric tissue organization, from the gross arterial anastomotic network to the ultrastructure of mucosal capillaries and collecting venules. This is because it supports the defensive mechanisms by supplying oxygen and fuel to the mucosal cells involved in maintaining the transepithelial barrier against HCI. Blood flow also removes metabolic waste and CO2 resulting from mucosal metabolism and from neutralization of influxing acid. In addition, mucosal blood flow is critically important in helping control intratissue pH, because it is the vehicle by which bicarbonate is transported into the superficial mucosa and by which excess protons are removed from the tissue [220]. Besides protecting mucosal tissue from infiltrating gastric acid, mucosal blood flow constitutes an important aspect of tissue resistance to nonspecific chemical assaults [221]. Toxic agents directly injure the mucosal cells and also stimulate the contraction of smooth vascular muscle, slowing the mucosal blood flow and congesting the


443

microcirculation at this level. The available data show that stasis of blood flow and increased vascular permeability reflect early pathogenic factors in the development of toxic agents such as ethanol on gastric hemorrhagic erosions [167, 222]. In less than 3 minutes after a necrotizing insult, two circulatory responses become evident. These responses are (a) vasospasm of venules and dilation of arterioles reflecting vascular congestion and slowing or stasis of blood flow, and (b) increased capillary permeability to the macromolecules of, plasma, which causes a net flux of fluid out of the plasma into the interstitium [223]. It is obvious that agents capable of preventing an increased microvascular permeability and of restoring an adequate blood flow in the affected areas, might contribute to regeneration of the lesions. This is the case of the flavonoids quereetin and naringenin which, in a model of chronic ulcer induced by 5% acetic acid in rats [ 111 ], induced an important proliferation of blood vessels in the internal area of the ulcerative formation, revealing that the enhancement of angiogenesis could restore the damaged microcirculation. Recently, Blank et al. [193], using laser doppler flowmetry have demonstrated the vascular mechanisms of 5-methoxyflavone-induced protection against acute gastric damage induced by a nonsteroidal antiinflammatory drug (indomethacin) in rats. The finding that this flavonoid significantly increased gastric vascular perfusion suggested that these effects could contribute to the flavonoid's gastroprotective activity. In this regard, several investigators have proven that some flavonoids show interesting effects upon the wall of the smallest blood vessels and particularly on the perivascular tissue. This is the case of Erica andevalensis, whose ethyl acetate extract showed a marked dosedependent effect on the mucosal vascular permeability against the proinflammatory mediator histamine [98]. Furthermore, pretreatment with the flavonic extract of Oxyris quadripartita significantly diminished the increase in the mucosal permeability induced by bradykinin in rats, evidencing a marked antiedema and vasoprotective action of this compound. These results confirmed that Oxyris quadripartita exerted protective mucosal activity through vascular mechanisms [224]. In addition, recent experimental studies indicated that the flavonic extract obtained from the flowers of Bidens aurea exerted antiulcer activity on ethanol-induced mucosal damage through a complex mechanism involving a significant inhibition of the enhanced vascular permeability in the gastric mucosa [89]. Thus, data are available which explain the critically important role played by gastric blood flow in the mechanisms of flavonoid-induced gastroprotection in experimental gastric ulcer. Furthermore, it is abundantly clear that significant advances have been made in the knowledge of the physiology of nitric oxide over the last few years. There

444

MARTIN et aL

are numerous experimental studies revealing the important role of this endogenous vascular-active substance in the mechanisms of gastroprotection [225, 226]. Nitric oxide itself, as well as known releasers of nitric oxide, protects significantly against ethanol- and HCl-induced gastric damage, and this effect is not altered by indomethacin pretreatment, suggesting mechanisms independent of those involving prostaglandins which are also known vasodilators [223]. Nevertheless the role of nitric oxide in flavonoids antiulcer activity has not yet been investigated, and further studies are required in this line. FLAVONOIDS AND APOPTOSlS Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as in toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as in acute or chronic toxicities. Examples of apogens are numerous, and include endogenous regulatory proteins and hormones as well as xenobiotic chemicals, oxidative stress, anoxia, and radiation. This can lead to inappropriate survival and pathological accumulation of aberrant cells [227]. Alternately, suppression of apoptosis through, for example, interference in cell signaling, can result in pathological accumulation of aberrant cells and diseases such as tumors [228]. The process of controlled cell death during development has been recognised for over a century [229], and is often described as "gene-directed cell death" because it is an integral component of development involving gene-directed steps [230]. Researchers originally distinguished apoptosis as a mode of cell death distinct from necrosis based on morphological criteria [231 ]. Apoptosis was recognised to occur under certain pathological conditions e.g., viral hepatitis as well as under physiological ones e.g., atrophy of the postlactational breast. In contrast, necrosis never occurs under physiological conditions and is a common consequence of the severe insults frequently studied by toxicologists. Although apoptosis was defined two decades ago, necrosis is the mode of cell death most familiar to biological scientists, except perhaps embryologist. Diseases characterized by the accumulation of cells include cancer, autoimmune diseases, and certain viral illnesses. Cell accumulation can result from either increased proliferation or the failure of cells to undergo apoptosis in response to appropriate stimuli. Although much attention has been focused on the potential role of cell proliferation in these so-


445

called proliferative disorders, increasing evidence suggests that alterations in the control of cell survival are important in their pathogenesis [232]. It is well known that many diet products, including flavonoids, reduce the risk of cancer, and recently some authors have tested the effect of flavonoids on various cell lines. Quereetin is one of the most abundant natural flavonoids, being present in various common vegetables and fruits [232]. As a semi-essential metabolite in plant life, quercetin was considered to be an inert, nonessential component of the human diet. It was assumed that quercetin had no specific effect on human health. However, following the discovery in 1977 that quercetin was mutagenic in the Ames and other short-term in vitro tests, interest in this agent increased. Growing concern for human safety regarding dietary quercetin was prompted a few years ago by reports that the flavonoid caused an increase in the incidence of different tumors. A number of long-term, well controlled feeding experiments with rats, mice and hamsters followed. These studies did not confirm the reported carcinogenity of quercetin. Instead, they indicated that quercetin might exert a protective effect in reducing the number of spontaneous tumors or the incidence of chemically induced ones [234]. Meanwhile, quercetin, together with other flavonoids, became the subject of intensive investigations not only by toxicologists and nutritionists, but also by immunologists, enzymologists, pharmacologists, and other medical scientists. In several in vitro experiments various flavonoids showed growth-inhibitory effects on cells from various human cancers: colon, breast, ovarian, gastrointestinal, and leukemic cells [104, 235-239]. It appears that a number of the biological effects of quercetin and other flavonoids may be explained by their antioxidative activity and ability to scavenge free radicals. Some flavonoids may act extracellularly, others intracellularly, and still others by both mechanisms and simultaneously at various sites. More than a dozen different mechanisms for the protective effect of flavonoids as anticarcinogens have been suggested. Because they are antioxidants, it is likely that their antioxidative property is, at least in part, responsible for their reported anticarcinogenic potential. Other mechanisms include their capacity to scavenge free radicals, to chelate, to block or trap ultimate carcinogen electrophiles by forming innocuous products in a nucleophilic chemical reaction, to inhibit the promotion phase of carcinogenesis, and to modulate the balance between activation and inactivation processes of specific enzymes in the liver [42]. Other authors have demonstrated that flavonoids inhibit the growth of malignant cells through mechanisms including inhibition ofglycolysis, macromolecule synthesis and enzymes, freezing the cell cycle, and interaction with estrogen type II binding sites synthesis [240, 241 ]. Wei et al. [242] have recently indicated that quercetin displays antitumor activity by triggering apoptosis and the agent was found to

446

MARTIN et aL

increase the amount of cells at G~ and S without affecting total cell quantity. Other studies have shown that flavonoids specifically inhibit the synthesis of heat shock proteins at the level of mRNA accumulation and transcription without affecting the other synthetic processes [241 ]. Heat shock proteins are known to play an important role in the protein metabolism and survival of cells [243]. The data indicated that quercetin induces apoptosis in tumor cells through inhibition of heat shock proteins and expression. Several reports have indicated that topoisomerase-II inhibitors induce apoptosis in thymocytes and other cell types [244]. Many agents have been reported to trigger the apoptotic program in thymocytes. Schneider et al. [234] found that the isoflavonoid compound genistein induces apoptosis in a distinctive human subpopulation through the inhibition of topoisomerase-II. At the gastrointestinal level, detailed studies by Potten and colleagues of epithelial cell kinetics in the mouse gastrointestinal tract have shown that apoptosis is rare in normal epithelium one apoptotic body being seen every 5 th crypt [245]. The paucity of apoptotic cells on histological section reflects the rapid kinetic of apoptosis and the removal of apoptotic cells by phagocytosis. This, and an absence of inflammatory response, probably explain why apoptosis went unnoticed for so long. Apoptosis has been shown in a number of diseases of the gastrointestinal tract 9 Excessive apoptosis 9Melanosis coli, Shigella flexneri dysentery, graft versus host disease, AIDS, inflammatory bowel disease. Defective apoptosis 9carcinogenesis. Neither is it known whether apoptosis plays an important part in disease pathogenesis nor whether apoptosis is merely part of the normal clearance of damaged cells. However, the main importance of apoptosis in intestinal disease concerns carcinogenesis, and hence the potential of treatment for cancer. It is now realised that, in general, anticancer agents do not kill by necrosis but rather by causing sensitive cancer cells to commit suicide by the induction of apoptosis [246]. Recently the effect of different flavonoid compounds (bioehaninA,

daidzein, genistein, genistin, pruneetin, puerarin and pseudobaptigenin) on cell proliferation of various cancer cell lines derived from the gastrointestinal tract has been demonstrated [247]" Stomach cancer: HSC-39K6, HSC-40A, HSC-41E6, HSC-42H, HSC-43C1, HSC-45M2, SH101-P4. Esophageal cancer : HEC-46R1. Colon cancer: HCC-48B2, Hcc-50D3. Fibroblast 9ST-Fib, ST-Fib2.


447

The results suggest that two of them, bioehanin A and genistein, inhibit the growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis. The authors hypothesized about the mechanisms involved. It was shown that tyrosine kinase inhibitors, such as erbstatin and tyrophostins, had an antineoplasic effect against human tumors of mammary, oesophageal, and maxillary origins in xenograph systems [248, 249]. In the study, they examined the antineoplasic effect of biochanin A or genistein upon athymic nude mice bearing human stomach tumors. Biochanin A significantly inhibited HSC45M2 and SH10 l-P4 tumor growth. Data suggested that biochanin A may serve as useful anticancer drug. However, growth of HSC-41E6 tumor was not suppressed by the flavonoid, suggesting that action of this compound is cell-type specific. Much more needs to be learnt about cell death in the gastrointestinal tract. A major challenge is to understand fully the factors that regulate apoptosis and to develop therapies that can manipulate apoptosis for the treatment of cancer. CONCLUSIONS The flavonoids comprise a large group of naturally occurring low molecular weight substances found in essentially all plant parts. They have been extensivel studied and reported to possess widespread biological activities. They possess a high capability to interact with important cellular processes mediated by different classes of enzymes. Other interesting studies also reported the capacity of some flavonoids to interact with active-oxygen species since they are strong scavengers of free radicals. These properties, together with the anti-inflammatory activity, could be involved in the antiulcerogenic effect of flavonoids. Some of them are inhibitors of aggressive mechanisms of the gastric mucosa, such as acid secretion, H. pylori, leukotrienes or neutrophil infiltration, and a parallel enhancement of defensive factors, such as enrichment of mucus gel, release of prostaglandins and endogenous sulfhydryls, restoring the mucosal blood flow or preventing the increase of microvascular permeability. The data reported in this review show that the inclusion of the sugar side chain in the molecule reduces both antioxidant and gastroprotective activities. Therefore, from the known digestive properties of flavonic agents, it appears that they could have a therapeutic potential for treatment of gastrointestinal disorders. At the same time, it has been reported recently that some flavonoids inhibit the synthesis of heat shock protein, which plays an important role in the protein metabolism and survival of cells. Quercetin, genistein and other flavonic agents induce apoptosis in different tumor cells through inhibition of heat shock protein and expression. Although these findings could suggest a possible anticancer therapy, it is necessary to learn much

448

MARTIN et aL

more about the effect of flavonoids on cell death in the gastrointestinal tract. ACKNOWLEDGEMENTS The autors are grateful to B.Berenguer for helpful assistance in the elaboration of the manuscript. RE~REN~ [1]

Moroney, M.A.; Alcaraz, M.J.; Forder, R.A.; Carey, F.; Hoult. R.S.d. Pharm. Pharmacol., 1988, 40, 787. [2] Robak, J.; Shridi, F.; Wolbis, M.; Kr61ikowska, M. Pol. J. Pharmacol. Pharm., 1988, 40, 451. [3] Laughton, M.J.; Evans P.J.; Moroney, M.A.; Hoult, J.R., Halliwell, B. Biochem. Pharmacol., 1991, 42, 1673. [41 Grupe, R. Agents Act., 1991, 32, 79. [5] Hoult, J.R., Moroney, M.A.; Pay~t, M. Methods Enzymol. 1994, 234, 443. [61 Alcaraz, M.J.; Hoult, J.R.S. Arch. Int. Pharmacodyn., 1985, 278, 4. [71 Ban, M.; Tonal, T.; Kohno, T.; Matsumoto, K.; Horie, T.; Yamamoto, S.; Moskowitz, M.A.; Levine, L. Stroke, 1989, 20, 248. [8] Ferrandiz, M.L.; Ramachandran Nair, A.G.; Alcaraz, M.J. Pharmazie, 1990, 45, 206. [91 Kalkbrenner, F.; Wurm, G.; Bruchhausen, F. Pharmacology, 1992, 44, 1. [lo] Abad, M.J.; Bermejo, P.; Villar, A. Gen. Pharmacol., 1995, 26, 815. [11] Ito, M.; Moriyama, A.; Matsumoto, Y.; Takaki, N.; Fukumoto, M. Agric. Biol. Chem., 1985, 49, 2173. [12] Ito, M.; Ono, Y.; Kai, S.; Fukumoto, M. J. Nutr. Sci. Vitaminol., 1986, 32, 635. [13] Constantini, L.; Albasini, A.; Rastelli, G.; Benvenuti, S. Planta Med., 1991, 58, 342. [141 Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165. [151 Gil, B.; Sanz, M.J.; Tereneio, M.C.; Ferrandiz, M.L.; Bustos, G.; Payfi, M.; Gunasegaran, R.; Aicaraz, M.J. Life Sci., 1994, 50, 333. [16] Gil, B.; Sanz, M.J.; Terencio, M.C.; Gunasegaran, R.; Paya, M.; Alcaraz, M.J. Biochem. Pharmacol., 1997, 53, 733. [17] Cazenave, J.P.; Beretz, A.; Anton, R. In Flavonoids and Bioflavonoids; L. Farkas; M. Gabor; F. Kallay, Ed.; Elsevier : Amsterdam, 1986; pp. 373 -380. [18] Ferriola, P.C.; Cody, V.; Middleton, E. Jr. Biochem. Pharmacol., 1989, 38, 1617. [19] Kuppusamy, V.R.; Khoo, H.E.; Das, N.P. Biochem. Pharmacol., 1990, 40, 397. [20] Vlietink, A.J.; Vanden Berghe, D.A.; Haemers, A. In Plant Flavonoids in Biology and Medicine II. Biochemical, Cellular and Medicinal Properties; V. Cody; E. Midldleton Jr.; J.B. Harbome; A. Beretz, Ed.; Alan R.Liss: Now York, 1988; pp.283 -299.


[21] [221 [23] [24]

[25] [26] [27]

[28] [29] [30] [311 [32] [33]

[34] [351 [36] [37]

[38] [39]

[40] [41] [42] [43] [44]

[45] [46] [47]

[48] [49]

[50] [52]

[53] [541

449

Ono, K.; Nakane, I.; Fukushima, M.; Chermann, J.C.; Barr(~-Sinoussi, F. Eur. J. Biochem., 1990, 190, 469. Hodnik, W.F.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 35, 2345. Tauber, A.L.; Fay, J.R.; Marietta, M.A. Biochem. Pharmacol. 1984, 33, 1367. Hodnik, W.F.; Bohmont, W.J.; Capps, R.S.; Pardini, R.S. Biochem. Pharmacol., 1987, 36, 2873. Elliot, A.J.; Scheiber, S.A.; Thomas, C.: Pardini, R.S. Biochem. Pharmacol., 1992, 44, 1603. Zhang, K.; Das, N.P. Biochem. Pharmacol., 1994, 47, 2063. Gabor, M. Flavonoids and Bioflavonoids, Farkas, I.; Gabor, M.; Kallay, F. Ed. Szeged, Budapest, 1985. Bauman, J.; Bruchhausen, F.; Wurm,G.; Nauyn -Schmiedeberg, S. Arch. Pharmacol., 1979, 307, 73. Slater, T.F.; Eakins, M.N. In New trends in the therapie of liver disease. Bertr A. (Ed.), Karger: Basel, 1984, pp. 84 -88. Elia, G.; Santoro, M.G. Ann. N.Y. ,4cad. Sci., 1994, 744, 323. Hodnick, F.W.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 14, 2345. AIcaraz, M.J.; Mas, J.A.; Ubeda, A.; M,~fiez. Planta Med. 1991, 57 (Suppl. 2), All0. Bombardr E.; Morazonni, P. Chimicaoggi, 1993, 11, 25. Wolf, B.; Michel, C.; Saran, M. Meth. Enzymol., 9194, 234, 420. Hayes, N.A.; Foreman, J.C.J. Pharm. Pharmacol., 1987, 39, 466. Midleton, E.; Drzewiecki, G.; Tatum, J. Planta Med., 1987, 325. Havsteen, B. Biochem. Pharmac., 1983, 32, 1141. Robak, J.; Gryglewski, R.J. Pol. J. Pharmacol., 1996, 48, 555. Middleton Jr., E. Trends Pharmacol. Sci., 1984, 5, 335. McClurr J.W. In The Flavonoids; J.B. Harbome; T.J. Mabry; H. Mabry, Ed.; Academic Press: New York, 1975; Vol. 2, pp.1011 -32. Czeczot, H. ,4cta Biochim. Pol., 1994, 41, 144. Stavric, B. Clin. Biochem., 1994, 27, 245. Brown, J.P. Mutation Res., 1980, 75, 243. Gaspar, J.; Rodrigues, A.; Laires, A.; Silva, F.; Costa, S.; Monteiro, M.J.; Monteiro, C.; Rueff, J. Mutagenesis, 1994, 9, 445. De Crosse, J.J. Cancer N.Y., 1982, 50, 1982. Yamamoto, S.; Aizu, E.; Jiang, H.; Nakadate, T.; Kiyoto, Y., Wang, J.C.; Kato, R. Carcinogenesis, 1991, 12, 317. Stadler, R.H.; Markovic, J.; Turesky, R.J. Biol. Trace Elem. Res., 1995, 47, 299. De Groot, H.; Dehmlow, C.; Raven, U. Meth. Find. Exp. Clin. Pharmacol. 1996, 18 (Suppl. B), 25. Alcaraz, M.J.; Hoult, J.R.S. Biochem. Pharmacol., 1985, 34, 2477. Gabor, M.; Razga, Z. Acta Physiol. Hung., 1991, 77, 197. Borissova, P.; Valcheva, S.; Belcheva, A. ,4cta Physiol. Pharmacol. Bulg., 1994, 20, 25. Tordera, M.; Ferrandiz, M.L.; Alcaraz, M.J.Z. Naturforsch., 1994, 49, 235. Da Silva, J.A.; Oliveira, A.B.; Lapa, A.J.J. Pharm. PharmacoL, 1994, 46, 118. Nassis, C.Z.; Haebisch, E.M.; Giesbrecht, A.M. Braz. J. Med. Biol. Res., 1992, 25, 929.

450

MARTIN et aL

[55] Brinkwoth, R.I.; Stoermer, M.J.; Fairlie, D.P. Biochem. Biophys. Res. Comm., [56] [57]

[581 [59]

[601 [611 [62] [63]

[641 [65] [66]

[67] [681 [691

[701 [71] [72] [73] [74] [75] [76] [77] [781 [79]

[80] [81]

[821 [83] [84]

1992, 188, 631. Ohnishi, E.; Bannai, H. Antiviral Res., 1993, 22, 327. Baker, M.E. Proc. Soc. Exp. Biol. Med., 1995, 208, 131. Farboodniay, M.A.; Ray, A.B.J. Nat. Prod., 1993, 56, 989. G~ilvez, J.; Crespo, M.E.; Jim6nez, J.; Su,Srez, A.; Zarzuelo, A. J. Pharm. Pharmacol., 1993, 45, 157. G61vez, J.; S,-tnchez de Medina, F.; Jim6nez, J.; Torres, M.I.; Fern6ndez, M.I.; Nuflez, M.C.; Rlos, A.; Gil, A.; Zarzuelo, A. Planta Meal, 1995, 61, 302. Sanchez de Medina, F., Gfilvez, J.; Romero, J.A.; Zarzuelo, A. J. Pharmacol. Exp. Ther., 1996, 278, 771. Morazonni, P.; Bombardelli, E. Fitoterapia, 1995, 66, 3. Ferrandiz, M.L.; Bustos, G.; Pay6, M.; Gunasegaran, R.; Alcaraz, M.J. Life Sci., 1994, 55, 145. Closa, D.; Torres,M.; Hotter, G.; Bioque, G.; Leon, O.S.; Gelpi, E.; Rosell6, C.; Catafau, J. Prostaglandins Leukot. Essent. Fatty Acids, 1997, 56, 331. Maschenko, N.; Lipkan, G.N.; Voitenko, G.N. Pad. Fiziol. Eksp., 1981, 6, 47. Barnaulov, O.D.; Manicheva, O.A.; Kamissarenko, N.F. Khim.Farm. Zh., 1983, 17, 945. Manicheva, O.A.; Barnaulov, O.D. Rastit. Resur., 1984, 20, 256. Barnaulov, O.D.; Manicheva, O.A.; Yasinov, R.K.; Yakoulev, G.P. Rastit. Resur., 1985, 21, 85. Khrenova, D.H.; Dargaeva, T.D.; Nikolaev, S.M.; Fedotovskika, N.N.; Brutko, L.I.; Farmatsiya, 1986, 35, 46. Martin, M.J.; Alarc6n, C.; Motilva, V.; La Casa, C.; Villegas, I.; Keijjoa, A. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 164. Guardia, T.; Guzm~n, J.A.; Pestchanker, M.J.; Guerreiro, E.; Giordano, O.S.J. Nat. Prod., 1994, 57, 507. Maria, A.O.; Wendel, G.H.; Guardia, T.; Guzmfin, J.A.; Pestchanker, M.J.; Guerreiro, E.; Giordano, O.S. Biol. Pharm. Bull., 1995, 18, 1784. Nomura, H. Fukuoka IshL 1959, 50, 354. Takagi, K.; Ishii, Y. Arzneim. Forsch., 1967, 17, 1544. Bruneton, J. Elementos de Fitoqulmica y Farmacognosia, Acribia: Zaragoza, 1991. Komatsu, M.; Tomomori, K.; Hatayama, K.; Mikuriya, N. Chem. Phar. BulL, 1970, 18, 602. Sasajima, M.; Nakane, S.; Saziki, R.; Saotome, H.; Hatayama, K.; Kyogoku, K.; Tanaka, I. Folia Pharmacol. Jpn., 1978, 74, 897. Takagi, K.; Okabe, S. dpn. s PharmacoL, 1968, 18, 9. Adami, E.; Marrazzi-Ubert, E.; Turba, C. Arch. Int. Pharmacodyn., 1964, 147, 113. Kyogoku, K.; Hatayama, K.; Yokomori, S.; Saziki, R.; Nakane, S.; Sasajima, M.; Sawada, J.; Ohzeki, M.; Tanaka, I. Chem. Pharm. Bull., ! 979, 27, 2943. Hayashi, Y.; Umehara, T. Yakuri to Chiryo, 1982, 10, 2697. Hatayama, K.; Yokomori, S.; Kawashima, Y.; Saziki, R.; Kyogoku, K. Chem. Pharm. Bull., 1985, 33, 1327. Konturek, S.J.; Radecki, T.; Brozowski, T.; Drodowiitz, D. Eur. J. PharmacoL, 1986, 125, 185. Su~.rez, J.; Herrera, M.D.; Marhuenda, E. Phytoter. Res., 1996, 10, 616.

ANTIULCERANDGASTROPROTECTIVEACTIVITY

[85] [86] [87]

[88] [89]

[901 [91] [921 [93]

[94] [95] [96] [971 [98] [99]

[~00] [101] [102] [103] [104]

[io5] [106] [107]

[~o8] [109] ill0] [lll] [112] [113] ill4]

451

Ayuso, M.J.; Marhuenda, E.; Martin, M.J. Plant. Mdd. Phytothdr., 1986, 20, 255. Alarc6n de la Lastra, C.; Martin, M.J.; La Casa, C.; Motilva, V. J Ethnopharmacol., 1994, 42, 16 I . Martin Calero, M.J.; La Casa, C.; Motilva, V.; L6pez, A.; Alarc6n de la Lastra, C. Z. Naturforsch., 1996, 51c, 570. Pardo, M.P.; Fernandez, C.I. Pharm. Mediterranea, 1978, 12, 259. La Casa, C.; Martin, M.J.; Alarc6n de la Lastra, C.; Motilva, V.; Ayuso, M.J.; Martin, C.; L6pez, A. Z. Naturforsch., 1996, 50c, 85. Rombi, M. 100 Plantes Medicinales. Composition, mode d'action et interet therapeutique, Romart: Nice, 199 I. Ageel, A.M.; Parmar, N.S.; AI -Meshal, I.A., Tariq, M. FIP Abstract, 1984, 43. llarionov, I.; Rainova, L.; Nakov, N. Farmatsiya, 1979, 26, 39. Rainova, L.; Nakov, S. Phytother. Res., 1988, 2, 137. Martin, M.J.; Alarc6n de la Lastra, C.; Jim6nez, B.; Marhuenda, E.; Moreno, J. Pharm. Mediterranea, 1986, 16, 573. Grande, M.; Piera, F.; Cuenca, A.; Tones, P.; Bellido, S.I. Planta Med., 1985, 35, 414. Martin, M.J.; Alarc6n de |a Lastra, C.; Marhuenda, E.; Delgado, F.; Torreblanca, J. Phytother. Res., 1988, 2, 183. Alarc6n de la Lastra, C.; L6pez, A.; Motilva, V. Planta Med., 1993, 59, 497. Reyes Rutz, M.; Martin-Cordero; Ayuso, M.J.; Toro, M.V.; Alarc6n de la Lastra, C. Phytother. Res., 1996, I 0, 303. Alarc6n de la Lastra, C.; Martin, M.J.; Marhuenda, E. J. Pharm. Pharmacol., 1992, 44, 929. Alarc6n de la Lastra, C.; Martin, M.J.; Motilva, V.; Jimenez, M.; La Casa, C.; L6pez, A. Planta Med., 1995, 61, I 16. Bors, W.; Michel, C.; Saran, M. Meth. Enzymol., 1994, 234, 420. Mascolo, N.; Pinto, A.; Capasso, F. J. Pharm. Pharmacol., 1987, 40, 293. Landolfi, R.; Mower, R.L., Steiner, M. Biochem. Pharmacol., 1984, 33, 1525. Scambia, G.; Ranevett, F.; Panici, B.; Piantelli, M.; Bonniammo, G.; De Vizenzo, R.; Ferrandina, G. Br. J. Cancer, 1991, 62, 942. Argullo, G.; Garnet, L.; Besson, C.; Demige, C.; Remesy, C. Cancer Lett., 1994, 87, 55. Waage, S.; Hedin, P. Phytochemistry, 1985, 24, 243. Panasiak, W.; Wleklik, M.; Oraczewska, A.; Luozak, M. Acta Microbiol. PoL, 1989, 38, 185. Martin, M.J.; Motilva, V.; Alarc6n de la Lastra, C. Phytother. Res., 1993, 7, 147. Alarc6n de la Lastra, C.; Martin, M.J.; Motilva, V. Pharmacology, 1994, 48, 56. Martin, M.J.; La Casa, C.; Alarc6n de la Lastra, C.; Cabeza, J.; Villegas, I.; Motilva, V. Z. Naturforsch., 1997, 53C, 82. Motilva, V.; Alarc6n de la Lastra, C.; Martin, M.J. Phytother. Res., 1992, 6, 168. Motilva, V.; Alarc6n de la Lastra, C.; Martin, M.J.J. Pharm. Pharmacol., 1993, 45, 89. P6rez-Guerrero, C.; Martin, M.J.; Marhuenda, E. Gen. Pharmacol., 1994, 25, 575. Martin, M.J.; Marhuenda, E.; P6rez-Guerrero, C.; Franco, J.M. Pharmacology, 1994, 49, 144.

452

MARTIN et aL

[1~5] [ll6] [117] [118]

Villar, A.; Gasc6, M.A.; Alcaraz, M.J.J. Pharm. Pharmacol., 1984, 36, 820. Villar, A.; Gasc6, M.A.; Alcaraz, M.J.J. Pharm. Pharmacol., 1987, 39, 502. Alcaraz, M.J.; Tordera, M. Phytother. Res., 1988, 2, 85. Wendi,P.; Reimann, H.J.; Swobwda, K.; Hennings, G.; Blumet, G. Ach. PharmacoL, 1980, 313 (SuppL), 238. [1191 Albertani -Borowski, N. Drug of the Future, 1983, 8, 209. [120] Parmar, N.S. Int. J. Tiss. Res., 1983, 4, 415. [121] Gambhir, S.S.; Goel, R.K.; Das Gupta, G. Indian. J. Med. Res., 1987, 85, 689. [122] Schwartz, K. Beitr. Klin. Chir., 1910, 67, 96. [123] Robert, A. Gastroenterology, 1979, 77, 761. [124] Takeuchi, K.; Furukawa, O.; Nishiwaqui, H.; Okave, S. Gastroenterology, 1987, 93, 1276. [125] Takeuchi, K.; Matsumoto, J.; Ueshima, K.; Ohuchi, T.; Okabe, S. Digestion, 1992, 51, 203. [126] Senay, E.C.; Levine, R.J. Proc. Soc. Exper. Biol. Med., 1967, 125, 122 I. [127] Marazova, K.; Lozeva, V.; Belcheva, A. Agents Actions, 1993, 38, C301. [128] Arakawa, T.; Satoh, H.; Fukuda, T. Hepato -Gastroenterol., 1990, 13, 166. [129] Palitzsch, K.D.; Horales, R.E.; Kronauge, J.F.; Szabo, S. Gastroenterology, 1989, 26, 381. [130] Albinus, N.; Friseh, G.; Henningns, G. Agents and Actions, 1983, 13, 249. [131] Yamahara, J.; Moehizuki, M.; Chisaka, T.; Fujimura, H.; Tamai, Y. Chem. Pharm. Bull., 1990, 38, 1039. [132] Saziki, R.; Arai, I.; Kobayashi, N.; Mutoh, K.; Sasajima, M.; Ohzeki, M. Pharmacometrics, 1976, 18, 7690. [1331 Vela, S.M.; Souccar, C.; Lima-Landman, M.T.; Lapa. A.J. Planta Med., 1997, 63, 36. [134] Parmar, N.S.; Ghosh, M.N. Eur. J. Pharmacol., 1981, 68, 1981. [135] Parmar, N.S. Int.J.Reac., 1983, 5, 415. [136] Hayes, N.A.; Foreman, 1L Jr. Pharm. Pharmacol., 1987, 39, 466. [137] Modlin,I.M. From Prout to the Proton Pump, Schnetztor -Verlag GmbH" Konstanz, 1995. [138] Murakami, S.; Isobe, Y.; Kijima, H.; Muramatsu, M.; Aihara, H.; Otomo, S.; Baba, K.; Kozawa, M. J. Pharm. Pharmacol., 1990, 42, 723. [139] Murakami, S.; Muramatsu, M.; Aihara, H.; Otomo, S. Biochem. PharmacoL, 1991, 42, 1447.

[1401 [141] [142] [143] [1441 [145] [146] [147] [148]

[149] [150]

Ray, T.K.; Nandi, J. Biochem. J., 1986, 233, 231. Murakami, S.; Muramatsu, M.; Otomo, S. J. Enzyme Inhib., 1992, 5, 293. Beii, W.; Birkholz, C.; Sewing, K.-Fr. Arzneim. Forsch., 1995, 45, 697. Warren, J.R.; Marshall, B.M. Lancet, 1983, i, 1273. Sipponen, P.; Sepp/tl/l, K.; Aarynen, M.; Helaske, T.; Kettunen, P. Gut, 1989, 30, 922. Figura, N.; Guglielmetti, A.; Rossolini, A.; Barberi, A.; Cusi, G.; Musnanno, R.A.J. Clin. Microbiol., 1989, 27, 225. Sipponen, P.; Hyv/trinen, H. Scand. J. Gastroenterol., 1993, 28 (suppl 196), 3. M~graud, F. Eur. J. Gastroenterol. Hepatol., 1993, 5 (suppl 1), S17. Hunt, R.H.; Malfertheiner, P.; Yeomans, N.D.; Hawkey, C.J.; Howden, C.W. Eur. J. Gastroenterol. Hepatol., 1995, 7, 658. Tytgat, G.N.J. Aliment. Pharmacol. Ther., 1994, 8, 359. Malfertheiner, P.; Haiter~ F. In The year in Helicobacter pylori, Current Science: London, 1994.


453

[151] Lamou|iatte, H.; Cayla, R.; Zerbib, F.; Talbi, P.; M6graud, F. Gut, 1995, 37 (Suppl 2), A 151.

[1521 Goddar, A.F.; Logan, R. Eur. J. Gastroenterol., 1995, 7, 1. [153] Wallace, J.L.; McKnijht, G.W.; Keenan, C.M.; Byles, N.I.A.; McNaughton, W.K. Gastroenterology, 1990, 98, 1178. [1541 Asako, H.; Kubes, P.; Wallace, J.L.; Gaginella, T.; Wolf, R.E.; Granger, D.N. Am. J. Physiol., 1992, 262, G903. [155] Boughton -Smith, N.K. Meth. Find. Exp. Clin. Pharmacol., 1989, 11, 53. [156] Osada, T.; Goto, H.; Tsukamoto, Y.; Nakazawa, S.; Sugiyama, S.; Ozawa, T. Dig. Dis. Sci., 1990, 35, 186. [157] Wittle, B.; Oren -Wolman, N.; Guth, P.H. Am. J. Physiol., 1985, 248, G580. [158] Konturek, S.J.; Brzozowski, T.; Drozdowicz, D.; Beck, G. Dig. Dis. Sci., 1988, 33, 806. [159] Pihan, G.; Rogers, C.; Szabo, S. Dig. Dis. Sci., 1988, 33, 625. [160] Vaananen, P.M.; Keenan, C.M.; Grisham, M.B.; Wallace, J.L. Inflammation, 1992, 16, 227. [161] Miyata, K.; Kamato, T.; Nishida, A.; Takizawa, K.; Takeda, M. Eur. J. Pharmacol., 1995, 276, 165. [162] Vob, C.; Sept~lveda-Boza, S.; Zilliken, F.W. Biochem. Pharmacol., 1992, 44, 157. [163] Yamamoto, S.; Yoshimoto, T.; Furukawa, M.; Horie, T.; Watanabe-Kohno, S. J. Allergy Clin. Immunol., 1984, 74, 349. [~64] Welton, A.F.; Tobias, L.D., Fiedler-Nagy, C.; Anderson, W., Hope, W., Meyers, K.; Coffey, J.W. Prog. Clin. Biol. Res., 1986, 7, 33. [165] Byung, P.Y. Physiol. Rev., 1994, 74, 139. [166] Lucas, C.E.; Sugawa, C.; Riddle, J.; Rector, F.; Rosenberg, B.; Walt, A.J. ,4cta Surg., 1971, 102, 266. [167] Whittle, J.R. Br. J. Pharmacol., 1993, 110, 3. [168] Motilva, V.; Martin, Ma J.; Luque, I.; Alarc6n de la Lastra, C. G en. PharmacoL,1995, 27, 545. [169] Avila, J.R.; Alarc6n de la Lastra,C.; Martin, M.J.; Motilva, V.; Luque, I.; Delgado, D.; Esteban, J.; Herrerias, J.M. Inflamm. Res., 1996, 45, 83. [170] Alarc6n De La Lastra,C.; Motilva, V.; Cabeza, J.; Martin, M.J.J. Pineal Res., 1997, 23, 47. [171] Granger, D. N.; J. Kourthuis Ann. Rev. Physiol., 1995, 57, 31 I. [172] Zimmerman, B.J.; Granger, D.N. Hepato-Gastroenterology, 1994, 41, 337. [173] Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25, 383 [174] Husain, R.S.; Cillar J.; Cillard, P. Phytochemistry, 1987, 26, 2489. [1751 Letan, A. J. Food Sci, 1986, 31, 518. [176] Rekka, E.; Kourounakis, P.N.J. Pharm. Pharmacol., 1991, 43,486. [1771 Robak, J.; Gryglewski, R.J. Biochem. Pharmacol. 1988, 37, 837. [178] Sichel, G.; Corsaro, G., Scalia, M, Di Bilio, A.J.; Bonomo, R.P. Free Radical Biol. Med. 1991, 11, 1. [179] Capatano,A.L. Angiology, 1997, 48, 39. [180] Cillard, J.; Cillard, P. S.T.P. Pharma, 1988,4, 594. [181] Negre -Saivayrr A.; Affany, A.; Hariton, C.R. Pharmacology, 1991,42, 262. [182] Duneic, Z. Folia Med. Cracov, 1987, 2813, 283. [183] Robak, J.; Duneic, Z.; Rzadkowska -Bodalska, H. Pol. J. Pharm. Pharmacol., 1986, 38, 483. [184] Robak, J.; Ryszard, J.; Gryglewski, R.J. Biochem. Pharmacol., 1988, 37, 837.

454

MARTIN et al.

[185] Yoshimoto, T.; Furukawa, M.; Yamamoto, S.; Horie, T.; Watanabe-Kohno, S. Biochem. Biophys. Res. Comm., 1983, 116, 612.

[1861 Koch, H.P., LOftier, E. Meth. Find Exp. Clin. Pharmacol. 1985, 7, 13. [187] Cholbi, M.R., Experientia, 1991, 47, 195. [~88] Van -Ackcr, S.A.; Tromp, M.N.; Haenen, G.R.; Van der Vijgh, W.J.; Bast, A. Biochem. Biophys. Res. Commun,1995, 25, 755. [189] Andrews, F.J., Malcontcnti,C.; O'Brien, P.E. Dig. Dis. Sci., 1994, 37, 1356. [1901 Granger, D. N.;. Kourthuis, J. Ann. Rev. Physiol., 1995, 57, 31. [1911 Gerritsen, M.E.; Carley, W.W.; Ranges, G.E.; Shcn, C.P.; Phan, S.A.; Ligon, G.F.; Perry, C.A. Am. J. Pathol. 1995, 147, 278. [192] Wolle, J.; Hill, R.R.; Ferguson, E.; Devall, L.J.; Trivedi, B.D.;Newton, R.S.; Saxena, U. Arterioscler. Thromb. Vasc. Biol. 1996, 16, 1501. [193] Blank,M.A.; Ems, B.L.; O'Brien, L.M.; Weisshaar, P.S.; Ares, J.J.; Abel, P.W., McCafferty, D.M.; Wallace, J.L. Digestion, 1997, 58, 147. [194] Morris, G.P.; Basu, B.K.; Holitzner, C.A.; Williamson, T.E.J. Clin. Gastroenterol., 1993, 17 (Suppl. 1), $5. [1951 Grisham, M.B.; Von Ritter, C.; Smith, B.F.; La Mont, J.T.; Granger, D.N. Am. J. Physiol., 1987, 253, G93. [196] Allen, A.; Cunliff% W.J.; Pearson, J.P.; Venablcs, G.W.J. Int. Med., 1990, 228 (S upp l 732), 83. [197] Allen, A.; FlemstrSm, G.; Garner, A.; Kivilaakso, A.E. Physiol. Rev., 1993, 73, 823. [1981 Wallace, J.L.; Granger, D.N. FASEB J., 1996, I0, 731. [1991 Bell, A.E.; Sellers, L.A.; Allen, A.; Cunliffe, W.J.; Morris, E.R.; Ross -Murphy, S.B. Gastroenterology, 1985, 88, 269. [200] Murty, V.L.; Sarosiek, J.; Slomiany, A.; Slomiany, B.L. Biochem. Biophys.Res. Commun., 1984, 1221,521. [2011 Mall, A.S.; Hutton, D.A.; Coan, R.M.; Sellers, L.A.; Allen, A. Biochem. Soc. Trans., 1988, 16, 585. [202] Suwa, T.; Fukushima, K.; Kyogoku, K.; Edo, H.; Mori, Y. Japan J. Pharmacol., 1984, 34, 89. [203] Slomiany, B.L.; Liau, Y.H.; Mizuta, K.; Slomiany, A. Biochem. Pharmacol., 1987, 36, 3273. [204] Brooks, F.P.; Cohen, M.D.; Soloway, L.D. Peptic Ulcer, Churchill -Livingstone: New York; 1985. [205] Bahari, H.M.N.; Ross, I.N.; Tumberg, L.A. Gut, 1982, 23, 513. [2o61 Whittle, B. Meth. Find. Exp. Clin. Pharmacol., 1989, I 1, 35. [2071 Walt, R.P. Scand. J. Gastroenterol., 1990, 25 (Suppl 174), 29. [2081 Holtz, J. Z. Gastroenterol. Verh., 1991, 26, 162. [2091 Robert, A.; Leung, F.W.; Guht, P.H. Gut, 1992, 33, 444. [210] Wilson, D.E. In Gastric Cytoprotection a Clinician's Guide; Hollander, D.; Tamawski, A.S., Ed.; Plenum Publishing Co.: New York, 1989; pp 169-183. [211] Robert, A.; Nezamis, J.E.; Lancaster, C. Am. J. Physiol., 1983, 245, G I 13. [212] Baumann, J.; Wurm, G.; Bruchhausen, F. Arch. Pharm. Weinhein., 1980, 20, 627. [2~3] Mertz-Nielsen, A.; Munck, L.K.;Bukhave, K.; Rask -Madsen, J. Ital. d. Gastroenterol., 1990, 22, 288. [214] Alarc6n de la Lastra, C.; La Casa, C.; Motilva, V.; L6pez, A.; Martin, M.J. Phytomedicine, 1996/97, 3, 327. [215] Itoh, M.; Guth, P.H. Gastroenterology, 1985, 88, 1162.


455

[216] Takeuchi, K.; Megumu, O.; Hiromichi, N,: Okabem S. ,1. Pharmacol. Exp. Ther., 1988, 248, 836. [217] Konturek, P.K.; Brzozwski, T.; Konturek, S.; Dembinski, A. Gastroenterology, 1990, 98, 909. [218] Szabo, S.; Pihan, G.; Dupuy, D. In New Pharmacology of Ulcer Disease. Szabo, S.; Moszik, G. Eds. Elsevier: New York, 1987, pp. 424 -446. [219] Szabo, S.; Vattay, P.1990 Gastroenterol. Clin. North Am. 19, 67. [220] Oates, P.J. In Gastric Cytoprotection, a Clinician's Guide. Hollander, D. ; Tarnawski, A.S. Eds., Plenum Publishing Corporation: New York, 1989, pp. 125 -164. [221] Szabo, S.; Goldberg, I. Scand d. GastroenteroL,1990, 25 (suppl 174), 1. [222] Giavin, G.; Szabo, S. F,4SEB 3., 1992, 6, 825. [223] Jacobson, E.D. Gastroenterology, 1992, 102, 1788. [224] Gomez, M.E.; Ayuso, M.J.; Toro, M~ V.; Alarc6n de la Lastra, C. Meth. Find. Exp. Clin. Pharmacol., 1994, 16 (suppl 1), 131. [225] MacNaugton, W.; Cirino, G.; Wallace, J.L. Life Sci. 1989, 54, 1869. [226] Konturek, S.J.; Brzozowski, T.; Majka, H.; Pykto, P.J. Eur. 3.Pharmacol., 1993, 239, 215. [227] Williams, G.T. Biochem. Cell. Biol., 1994, 72, 450. [228] Zakeri, Z.; Lockshin, R.A. Ann. N. Y. Acad. Sci., 1994, 71, 9229. [229] Lockshin, R.A.; Zakeri, Z.F. Ann. N. Y. Acad. Sci., 1992, 663, 249. [230] Zakeri, Z.F.; Quaglino, D.; Latham, T.; Lockshin, R.A. FASEB d., 1993, 74, 78. [231] Bellamy, C.O.; Malcomson, R.D.; Harrison, D.J.; Wyllie, A.H. Semin. Cancer. Biol., 1995, 616, 285. [232] Guillouf, C.; Grana, X.; Selvakumaran, M.; De Luca, A.; Giordano, A.; Hoffma N,; B.Liebermann,; D.A. Blood, 1995, 85, 2698. [233] Larocca, L.M.; Piantelli, M.; Teofili, L.; Leone, G.; Ranelletti, F.O. Exp. Hematol., 1996, 24, 496. [234] McCabe, M.J.; Orenius, S.; Biochem. Biophys. Res. Com., 1993, 194, 945. [235] De Vincenzo, R.; Scambia, G.; Benedetti Panici, P.; Ranelletti, F.O.; Bonanno, G.; Ercoli, A.; Delle Monache, F.; Ferrari, F.; Piantelli, M.; Mancuso, S.; Anticancer Drug Res., 1995, 10, 490. [236] Scambia, G.; Ranelletti, F.O.; Panici, P.B.; De Vincenzo, R.; Bonanno, G.; Fer randina, G.; Piantelli, M.; Bussa, S.; Rumi, C.; Cianfriglia, M. Cancer Chemother. Pharmacol., 1994, 344, 64. [237] Larocca, L.M.; Giustacchini, M.; Maggiano, N.; Ranelletti, F.O.; Piantelli, M.; Alcini, E.; Capelli, A.J; Urology, 1994, 152, 1033. [238] Scambia, G.; Ranelletti, F.O.; Panici, P.B.; Piantelli, M.; De Vincenzo, R.; F errandina, G.; Bonanno, G.; Capelli, A.; Mancuso, S. Int. J. Cancer, 1993, 54, 466. [239] Yoshida, M.; Yamamoto, M.; Nikaido, T. Cancer Res., 1992, 52, 6681. [240] Hosokawa, N.; Hirayoshi, K.; Kudo, H.; Takechi, H.; Aoike, A.; Kawai, K.; Nagata, K. Mol. Cell Biol., 1992, 123, 498. [241] Koishi, M.; Hosokawa, N.; Sato, M.; Nakai, A.; Hirayoshi, K.; Hiraoka, M.; Abe, M.; Nagata, K.; Jpnan J. Cancer Res., 1992, 831,222. [2421 Wei, Y.Q.; Zhao, X.; Kariya, Y.; Fukata, H.; Teshigawara, K.; Uchida, A. Cancer Res., 1994, 54, 4957. [2431 Welch, W.J. Physiol. Rev., 1992, 722, 36. [2441 Onishi, Y.; Azuma, Y.; Kizaki, H. Biochem. Mol. Biol. Int., 1994, 32, 112.

456

[245] [246] [247] [248]

MARTf[Net as

Potten, C.S. Cancer Metastasis Rev., 1992, 11, 179. Dive, C.; Evans, C.A.; Whetton, A.D. Semin. Cancer Biol., 1992, 34, 27. Yanagihara, K.; Ito, A.; Toge, T.; Numoto, M. Cancer Res., 1993, 535, 821. Toi, M.; Mukaida, H.; Wada, T.; Hirabayashi, N.; Toge, T.; Hori, T.; Umezawa, K. Eur. J. Cancer, 1990, 267, 24. [249] Yoneda, T.; Lyall, R.M.; Alsina, M.M.; Persons, P.E.; Spada, A.P.; Levitzki, A.; Ziberstein, A.; Mundy, G.R. Cancer Res., 1991, 51, 4435.

Atta-ur-Rahman(Ed.)Studies in Natural Products Chemistly, Vol. 22 9 2000 ElsevierScienceB.V. All rightsreserved

457

SIMPLE FLAVONES POSSESSING COMPLEX BIOLOGICAL ACTIVITY S. TAHARA* and J. L. INGHAMw

Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan and w of Food Science and Technology, University of Reading, Whiteknights, P.O. Box 226, Reading RG6 2AP, England, U. K. ABSTRACT: Two simple flavones, each of which exhibits distinct biological activity despite their closely related structures, have been recognized by detailed bioassays, and bioassay-orientated isolation procedures. The identity of both flavones has been confirmed by synthesis. One of these compounds, 5-methoxy-6,7-methylenedioxyflavone has been found in an extract of Polygonum lapathifolium L. subsp, n o d o s u m (Polygonaceae) using a screening test devised to detect antidotes against the benzimidazole fungicide, benomyl (or its active principle MBC, l H-benzimidazol-2ylcarbamic acid methyl ester). The other compound, 5-hydroxy-6,7methylenedioxyflavone, is a host-specific signalling substance that exudes from spinach roots and attracts zoospores of the phytopathogenic fungus ,4phanomyces cochlioides the cause of spinach root rot. This review describes the bioassay, isolation and identification of these active compounds, and compares their activity with that of various other related, and unrelated, chemicals of either plant or synthetic origin. The possible ecochemical role and mode of action of flavone and non-flavone antidotes and attractants is briefly discussed.

GENERAL INTRODUCTION The flavonoids, and their close chemical relatives, the isoflavonoids, easily comprise the largest and most widespread group of naturally-occurring secondary compounds. Since the isolation, in the mid-1840's, of pure apiin (now known to be the flavone apigenin-7-O-apiosylglucoside) from seeds of parsley (Petroselinum sativum, syn..4pium petroselinum; Umbelliferae), many thousands of flavonoids and isoflavonoids have been chemically identified. Most of the flavonoids found in green plants are oxygen-ring heterocyclics, with the vast majority being chalcones/dihydrochalcones (non-heterocyclic), flavones (including the 3hydroxyflavones, or flavonols), flavanones (dihydroflavones), aurones and anthocyanidins/anthocyanins. Of these, the flavones, which are based on 2-phenylchromone (1), are the most commonly encountered, being essentially ubiquitous within the families and genera of the higher plants. Most flavones occur in the Plant Kingdom as simple aglycones, with hydroxyl, methoxyl or methylenedioxy substituents, or as O- or C-linked

458

TAHARA and INGHAM

glycosides, but more complex molecules are also regularly, and increasingly, described in the phytoehemical literature.

8

"~

HO.

5'

6' i

7

o.

6

114 O

5

2

.o

o.

luteone

1 2-phenylchromone

~f' ) OH

H

I

OH

2 ima

:Z 11.

Allium ascalonicum L.

Liliaceae

AfL I.nd

plant

12.

Allium cepa L.

Liliaceae

Ind, Pak, M.E., E.S. Afr, S.E. Asi, costa

bulb

36,47

epilepsy & infantile convulsions +PTZ

,,

13.

Liliaceae

Ailium sativum L.

70% ethanolic ext.

-STN

I 4 I

C. Asi, Ind, Pak, Sri, i E.N.S. Aft', costa, Bri, N. Eur, Egy, Mex, U S A

bulb

32

....

complex preparation

convulsive affections +PTZ

32 ,

I 37

70% ethanolic ext.

32

14.

Ailophylus africanus Beouv.

Sapindaceae

trop Aft"

leaves

convulsions

decoction with leaves of Ocimum basilicum L.

48

15.

Aloe vera L.

Liliaeeae

pant

sap ofleaves

epilepsy & convulsions in children

-

35

A. vera var chinensis Hain. Berger. ,

Tai, Indchi, Chi

....

> Z ,H >

,,q m

16.

i Alstonia boonei De Wilid.

Apocynaceae

Afr

17.

Alstonia venenata R.Br.

Apocynaceae

W.Pen

18.

Alstonia scholaris R.Br.

Apocynaceae

W. trop Aft', trop Aus, Sri, S. Chi, Ind, Ids, Indochi, Pak, Phil, Mal, Bur, Jav

! 9.

Ampelopsis japonica Thunb. Mak.

Vitacese

Kor, Chi, Jap

Anagallis arvensis L.

Primulaceae

Ind, Him, Pak

20.

.

i21. )

.

.

.

.

.

Commelinaceae ....

Annona muricata L.

Annonaceae

W. Pen, temp and trop Him, Ind Gha, Ind

!_

23.

Annonidium monni (Oliv.) Eng]. et. Diels

70% ethanolic ext.

32

leaves

+PTZ +EST

aqueous ext.

49

epilepsy.

,5o,51

epilepsy

35

roots

anticonvulsive

35

herb (whole plant)

epilepsy & hysteria

34, 50, 52

root bark

infantile convulsions

o

epilepsy

leaves

convulsive seizures

bark

epilepsy

ripe fruit

.

.

Aneilema scapiflorum Wight.

+PTZ

,l

L i

22.

.

plant

Annonaceae

C a m

.

.

.

.

34, 50 53 +PTZ

ethanolic ext.

54 55

l

24.

Apium graveolens L.

Umbelliferae

Chi, Ind, N.W. Him

seeds +IVIES -PTZ -STN

25.

Aralia continentalis L.

Araliaeeae

Chi

NM

26.

Arisaema consanguineum Schott.

Aracear

S.W. Chi

tuber

27.

A. amurense var serratum Maxim Nak.

Araceae

Kor

28.

A. heterophyllum.B.

Araceae

W.C. Chi

,

+

3-n-butylphthalide (6) 3-nbutyl 4-5-dihydrophthalide (7) alkaloid fraction

56 57

volatile oil

58

convulsions in children

35

c o m a

anticonvulsant

35

tuber

epilepsy

J,

i

35

29.

A. amurense Maxim

Araceae

N. Chi

tuber


30.

A. japonicum Maxim.

Aracear

Chi, Tai, Jap

tuber

sedative for convulsions

Armillarta mellea Vahl. ex Fr. Quei.

31.

Tricholomataceae

Chi

dried mycelia

Arrabidaea platyphylla Bur.

Bignoniaceae

Bra

roots & leaves

33.

Artemisia verlotorum

Compositae

Bra, Spa

whole plant

34.

Artocarpus heterophyllus Lame

Momcear

Bur, Chi, Phil, Mal, Indchi, Ind, Bng

wood

Asarum macranthum Hook.

Aristolochiaceae

Tai

36.

Asparagus officlnalis L.

Liliaceae

Bulg, Ind

37.

Asparagus racemosus Willd.

Liliaceae

Ind, Him, Ksh

35 35

..

+IVIES +PTZ +STN

..

32.

I

epilepsy +MES +PTZ +3-mercapta -propionic acid +pilocarpine

aqueous ext.

59

decoction

60

95% ethanolic ext.

61

35

sedative for convulsions

. .

35.

Bacopa monnieri L.

38.

Scrophulariaceae

Ind, Pak, Mad, Phi, Sri

Bramia monnieri L. Herpestis monniera H. B. et K.

+EST -PTZ -STN

NM

Balsamodendron sp. vat. Commiphora myrrha (Nees) ..... E n s l .

Burseraceae

J Aft, Asi, Ara

ethanolic ext.

62

epilepsy

36

anticonvulsant

63

whole plant

epilepsy

50, 52, 64

root stalk & leaves

epilepsy & nocturnal

34, 36, 37

epilepsy

whole plant

39.

35

hysteria

gum resin from stem

epilepsy

+AUD +PTZ -IVIES

fraction aqueous ext. & 95% ethanolic ext.

65 66

37

Ot lint r

epilepsy

Barringtonia racemosa L. Roxb

Lecythidaceae

Ind, Sub Him

Basilicum polystachyon L. Moench

Labiatae

Jav, Aus

herb

sedative in convulsions & epilepsy

Benincasa hisplda (Thunb.) Cong.

Cucurbitaceae

lnd, Pak, Jay, trop, Chi, Mal, Ids

fruit

epilepsy & hysteroepilepsy

juice/decoction

fresh fruit

epilepsy

juice

B. cerifera Savi Bersama abyss!nica Fresen.

Melianthaceae

trop .S..Afr

roots

epilepsy

Boerhavia diffusa L.

Nyctogynaceae

Aft', lnd, Pak

root bark

anticonvulsive

B. repens L.

roots

Bombax malabaricum DC.

Malvaceae

Ind

seeds

Brassica nigra L. Koch

Cruciferae

Pak, Ksh, Ind

~

Buchnera cruciata Ham.

Scrophulariaceae

Butea monosperma (Lam.) Kuntze

33 +PTZ +PTZ +PTZ -EST

epilepsy

methanolic ext. 70% ethanolic ext. aqueous ext.

53 32 49

-

34

epilepsy

47

Chi

epilepsy

35

Papilionoideae

Pak, Ind

epitepsy

47

Caesaipinia puicherrima L. Sw.

Leguminosae

pant

flowers

convulsions m children

C bonduc L. Roxb

Caesalpiniaceae

Pak, Ind, S. Afr Nig

seeds plant

convulsions

+PTZ +STN

in a mixture

35

oil

53, 67 32

70% ethanolic ext.

i

51.

Mimosaceae

Calliandra portoricensis Jacq.

Afr

roots

32

anticonvulsant +PTZ +STN

70% ethanolic ext.

32

roots

+PTZ +EST

aqueous ext.

49, 68

stem

+PTZ +EST

aqueous ext.

68

-PTZ -EST

alkaloidal ext.

68

A8 and A9 tetrahydrocannabinols (8 and 9)

69, 70

plant

roots & stem ......

-6AtrO Cannabinaceae

Cannabis sativa L.

52.

N1d

Ind, Pak, Sub Him

+EST

-PTZ

SP-175 (I0)

71

i

2-q

cannabidiol (11) 72, 73, 74

2

-q

alcoholic ext.

34, 36, 47, 50, 64 75

epitep.sy

juice

48


complex preparation

48

alkaloidal ext.

76

+PTZ +dexamphetami ne +EST 53.

Gentianaceae

i Canscora decussata Sehult. Roem. Roxb.

fresh plant

Ind, Sri, Bur, trop Aft

+EST

whole plant i

Rubiaceae

Canthium guein:ii Sond.

54.

Rubiaceae

Canthium bibracteatum Baker. Hiern.

55.

.

.

.

.

.

.

.

trop Aft"

.....

,

i

trop Aft

leaves

. . . . . . . .

roots

...........

.

56.

Capparis baducca

Capparidaceae

Jap

57.

Cardamine pratensis L.

Cruciferae

Ksh, W. Tib

flowering t o p s .

epilepsy

Umbeilifereae

Ksh, S. Ara, N. Him

seeds

anticon vuisant

Rutaceae

Mex

58.

59. ~,,

.

Carum carvi L. .

.

.

.

,,

Casimiroa edulis Llave et Lex. ~

i

,,

leaves .....

leaves

juice

epilepsy

u

,..

-6

50

+IVIES +PTZ

.

77

aqueous ext.

78

I !

i

. .

60.

Cassia accidentalis L.

Leguminosae

trop

seeds


complex preparation

37

61.

Cassia sophera L.

Leguminosae

trop, Ind

seeds


complex preparation

37

62.

Catunaregam nilotica Stapf. Tirvengadum

Rubiaceae

trop Aft

roots


decoction

48

stem bark

convulsions

infusion

wood

epilepsy

,,,

63.

Cedeus libani Barrel var. Deodara Hook. f.

Coniferae

N.W. Him, Ksh

64.

Celastrus paniculta Willd.

Celastraceae

Sub Him, lnd

65.

Celtis cinnamomea Lindi.

Ulmaeear

Thi, Ids, Sik, Him, Bng, Ind

wood scrappings

66.

Centeila asiatica L.

Umbeiliferae

lnd

leaves plant

.

67.

Centipeda orbicularis Lour.

Compositae

Pak, Ind

.

.

.

a,nll,iepi/eptic

68.

Cerbera odollam Gaertn.

Apocynaceae

S.C. Vie, Cam, Ind

Cimicifuga dahurica

Ranunculaceae

USSR

70.

Cinchona spp.

Rubiaceae

S. Ame, Ind, Jay, Sri

bark

71.

Cinnamomum camphora L. F. Nces Eberm.

Lauraceae

Pak, Chi, Tai, Jap, Ind, Chi, Kor, lnd, Sri, Bra, Jam, Tai, Mau, Med regions

stearoptene from trunk root and branch camphor obtained by sublimation of chipped wood.

Cissampelos pareira L.

mucronata)

(C.

Menispermaceae

Aft, Ind

,,J

convulsions +PTZ +MES

leaves

plant

63 35

epilepsy

69.

72.

48

37

aqueous ext. 50% ethanolic ext.

79 80

.

47

+PTZ

95% ethanolic ext.

81

+STN +CAM

70% ethanolie tincture

82

+E

quinine (12)

anticonvulsant

83

35

epilepsy & hysteria

+PTZ +STN

camphor with olive oil

64

70% ethanolic ext.

32

73.

Cissus integrifolia (Bak) Planch.

Vitaceae

Tan

roots

epilepsy

74.

Citrullus colocynthis Schrad.

Cucurbitaceae

Mor

fruit

antiepileptic

75.

Citrus aurantium L.

Rutaceae

76.

Citrus maxima (Burro.) C. grandis L. C. decumana L.

Rutaceae

Ciausena anisata (Willd) Oiiv.

Rutaceae

77.

Aft, Ind, Chi, Ecu, Egy, S. Fra, W. Ind, Ira, Isr, Med, Mor, Pak, Por, .Spa, Tur, USA lnd, Pal
2 m,,l

r :Z ,
2 ,q ;>.

35, 36

N

epilepsy

47

roots

epilepsy

47, 50, 86

leaves

epileptic & convulsive af.fections

37, 64

epilepsy

87

,.q ,< ,.q

85.

Convolvulus arvensis L.

Convolvulaceae

Bulg, Ind, Him

NM

ethanolic ext. ,

Convolvulus pluricaulis

Convolvulaceae

Ind

dried plant

+EST +tremo-rine

95% ethanolic ext.

88

87.

Corydalis sp.

Fumariaccae

Chi

tuber

+PIC +PTZ

chloroform A ext. chloroform B ext.

89

88.

Crocus sativus L.

Iridaceae

Pak, Ksh, Irn, Spa, F.E., Chi

dried stigma

sedative in convulsions

89.

Crossostephium chinese L.

Compositae

Jap, Chi, Tai, Phi

leaves

infantile convulsions

90.

Cucumis sp. L.

Cucurbitaceae

Pak, Kor, Chi, Indchi, Ids, trop Aft, Med, W. Asi

stalks of unripe fruit

epilepsy

,

35 decoction

seeds

epileFsy

rhizome

convulsions

35

/~ts

37

Cucumis co!ocynthis L.

[92.

Curcuma aromatica Salisb.

Zingiberaceae ,

Ind, Chi, Indchi

Cuscuta europea L.

Convolvulaceae

Eur, W.C. Asi

.94.

Cynanchum saccatum

Asclepiadaceae

roots

+AUD

95.

Cynanchum decipiens

Asclepiadaceae

roots

+KND

96.

Cynanchum otophyllum

Asclepiadaceae

roots

-EST +AUD

97.

Cynodon dactylon L. Pers.

Graminae (Poaceae)

93.

Ind, Pak, Indigenous to tropics and subtropics of both hemispheres

35 35

91.

_

62

86.

,,

i

+EST -PTZ -STN

plant

oil

epilepsy & hysteria

3 alkaloids (names NM) . , 90

otophylloside A and B (14 and 15)

91

juice

34, 50, 51, 52,

leaves

+PTZ +EST

aqueous & alcoholic ext.

grass

-PTZ +IVIES

aqueous ethanolic ext.

98.

Cyperus esculentus L.

Gramineae

Mex, Ind

rhizome

99.

Cyperus rotundus L.

Cyperaceae

pant

rhizome roots

epilepsy epilepsy

+PTZ +STN

64

64

49 92

infusion

93

complex preparation 70% ethanolic ext.

35 32

100.

Cystoseira unsenoides L. Roberts

Cystoseiraceae

Spa

whole plant (alga)

101.

Datura fastuosa L.

Solanaceae

Ind, Pak

seeds

102.

Datura metel L.

Solanacear

.

103.

Daucus carota W.

Apiaceae (Umbelliferae)

Asi, Eur, costa, Ind, Pak

104. Deinbollia borbonica Scheff.

105.

106. 107.

Sopindaceae

Delphinium consolida var consolida

Ranunculaceae

Delphinium denudatum Wall.

Ranunculaceae

Desmodium adscendens Sw.

trap E. Afr

Bulg

+PTZ

34

epilepsy

47

red flowers in the centre of umbel roots

epilepsy

67

anticonvulsant

95

roots

convulsions

decoction

leaves

epilepsy & convulsions

concoction

NM

roots

Papillionaceae

Ghana

plant leaves

epilepsy

+F.ST

ethanolic

+PTZ

95% ethanolic ext.

+PTZ +KA

Desmodium polycarpum DC.

Leguminosae

Ind

plant

convulsions

109.

Desmodium tr~orum DC.

Leguminosae

Ind, Him, Pak, Chi, Phi, Med, Ids, Phi

leaves

convulsions & convulsions m children ..

110.

Desmodium pulchellum Benth.ex Baker

Leguminosae

Chi, Mal, Ids, Phi

1 1 1.

Dichrostachys cmerea L. Wight & Am.

Mimosaceae (Leguminosae)

Afr, Pak, Ind, Sri, N. Aus, Mal

Dictamnus albus L.

48

ext.

-PTZ -STN Ksh, W. temp Him

Rutaceae

Ind, Ksh, W. Him, Indochin Mal

94

epilepsy

108.

112.

methanolic ext.

decoction ethanolic ext.

root bark

epilepsy

47 96 97

50

complex preparation

37, 50, 51, 52 35

convulsions in infants undefined parts plant

62

decoction

98

epilepsy

64

hysteria

34, 50

lilt

113.

Diospyros usmabarensis F. White

114.

115.

.

.

.

.

E. & S.E.Aft"

Ebenaceae

epilepsy

roots

.

decoction

99

ethanolic ext.

62

,

Echium vulgare

Boraginaceae

Bulg

NM

Ekebergia senegalensis A.

Meliaceae

Sen

bark

+EST -PTZ -S'IN

epilepsy

53

Juss. .......

Elaeocarpus ganitrus Roxb.

116.

Elaeocarpaceae

Ind, Nep

....

epileptic fits

fruit .....

1 ! 7. [ Elaeocarpus sphaericus K.

Tiliaceae

Aft, lnd, Nep

fruit

Elaeocarpaceae

Ind

nuts

.

+IVIES

I

90% ethanolic ext.

epilepsy

53

epilepsy

5O

Schum.

118,

Elaeocarpus tuberculantus Roxb.

50, 85 I00

.....

119.

Emilia coccmea Don.

Nig

Compositae

.

.

.

.

+p'rz +EST

leaves .

.

.

.

120.

Erythrina stricta Roxb.

Fabaceae (Leguminosae)

Ind, Nep

bark

! 2 I.

Erythrina variegata L.

Leguminosae

Ind

bark

122.

Erythroxylum spp.

Erythroxylaceae

epilepsy

+STN +PTZ

~

,

123. ! 24.

Euphorbia hirta L.

Euphorbiaceae ......

Aus, Ind, Pak, pant

plant NM

Euphorbia fisheriana

Euphorbiaceae

Tai

125.

Euphorbia nyikae Pax.

Euphorbiaceae

Ken, Tan, S. Aft

126.

Euphorbta tirucalli L.

Euphorbiaceae

i

,,,

i Aft, Ind, B.ng

convulsions +E

,

roots

epilepsy

fresh milky latex

epilepsy +PTZ

aerial parts

aqueous ext.

49

.......

powder

50, 51

alkaloidal fraction

I01

cocaine (16)

102, 103

decoction

35

alkaline ext.

I04

decoction

33

with other ingredients

33

50% ethanolic ext.

I05

decoction

35, 50, 64

J~

I

127.

Excoecaria agallocha L.

Euphorbiaceae

! Ind, N. Aus, New Cal, ...... from S.E. Asi to poly

leaves

epilepsy

= ,

128.

Ferula alliacea Boiss.

Umbelliferae

Ira, Afg, Pak,

129.

Ferula foetida Re•el.

Umbelliferae

USSR

130.

Ferula galbaniflua Boiss. et.

Umbeiliferae

Per

gum resin

epilepsy

Moraceae

trop & S. Afr

root bark

covulsions

gum resin

epilepsy, hysteria & infantile

36, 37, 50, 52, 85

convulsions 47

Buhse 131.

Ficus capensis Thunb.

,,

132.

Flemingia strobilifera R.Br.

Leguminosae

31

Ind, Pak, Bng

roots

epilepsy, hysteria & nocturnal epilepsy

-

50, 87

trop S. Afr, trop & subtrop Asi, Aus, Mas

roots

epilepsy

decoction

33

......

,,

133.

Flueggea virosa Willd. Voight

Euphorbiaceae

134.

Galeopsis ladanum L.

Labiatae

Poi

overground parts

+PTZ -EST

aqueous ext. (lyophilized)

106

2-q

135.

Galium sylvaticum

Rubiaceae

Bulg

NM

+EST -~Z -STN

ethanolic ext.

62

2 ,< C

136.

Galium verum L.

Rubiaceae

W. Him, Ksh

plant

epilepsy & hysteria

juice/decoction

50

137.

Galphimia glauca Cav.

Malpighiaceae

Mex

2 ,q

shrub aerial parts

epilepsy

.,

....

decoction with Strychnos madagascariensis

.,

+PTZ +STN

107 methanolic ext.

138.

Gentiana compestris

Gentianacear

Ind, temp N.W. Him, Ksh

NM

139.

G. crassicaulis Duthie

Gentianaceae

Chi

roots

convulsions

35

140.

G. dahurica Fisch.

Gentianaceae

Chi

roots

convulsions

35

141.

G. decumbens L. f.

Gentianaceae

Pak

roots

convulsions

35

142.

G. fetisowii Reget Wink.

Gentianaceae

Chi

roots

convulsions

35

,

. . . . . .

-q ,< ,q N

N

swertianoline (45)

108

143.

Gentiana macrophylla Pallas

Gentianaceae

Mon

roots

convulsions

35

144.

G. tibetica King

Gentianaceae

Chi

roots

convulsions

35

145.

G. wutaiensis Marqund

Gentianaceae

146.

Gossypium herbaceum L.

Malvaceae

Gynandropsis pentaphylla Wiild.

roots

convulsions

35

Ind, Pak, Bang, E.S.

seeds

epilepsy

34, 47

Pak, Ind

seeds

convulsions

67

F i)

leaves

epilepsy

Afr, Egy

,

147.

Tib

Capparidaceae , ,

148.

Gyrocarpus americanus Jacq.

Gyrocarpaceae

149.

Haplophyllum dubium

Rutaceae

150.

Haplophyllum perforatum

151.

H. ~labrinu m

152.

Hedeoma pulegioides L. Pets.

+EST +PTZ

dubinine (17)

109

Rutaceae

seeds

+PTZ +CAM

haplophylidin (18)

110

Rutaceae

roots

infusion

! 11

decoction

99

Ame

~ ....

+STN L

convulsions & spasms

whole plant

Helichrysum setosum Ha rv.

Composi,tae

Tan

leaves

154.

Hemidesmus mdicus R. Br.

Asclepiadaceae

Bng, Ind

~

,

, epilepsy

155.

Heracleum sibiricum

Umbelliferae

156.

Heracleum verticillatum

Umbelliferae

157.

Hesperethusa crenulata Roxb. Roem.

Rutaceae

158.

Hibiscus abelmoschus L.

Malvaceae

Ind

seeds

159.

Himanthalia elongata L.S.F. Gray

Himanthaliaceae

Spa

whole plant (alga)

160.

Hippeastrum v i t t a t u m .

Amaryllidaceae

USSR

NM

+EST +PTZ +NIC -STN

roots

Bur ,

furocoumadns (sphondin

112, 113

pimpinellin, bergapten, isopimpineilin, angelicin & isoberl~apten) (19 to 24)

epilepsy

leaves

35

. . . . .

,

,

Malpighiaceae

47 36

epilepsy & nocturnal epilepsy

,,

Hiptage benghalensis Kurz.

63

leaves

~

153.

161.

decoction

,,

Mau, Ksh, Rod, Ind, Nep, Bng

tincture

34

+PTZ

chloroform ext.

114

+PTZ -STN

hippeastrine (25)

115

decoction

116

hysteria

epileptic fits

plant .

162.

Holarrhena floribunda 9 (G.Don) Dur et Schinz.

,,

Hoslundia opposita Vahl.

163.

Nig

Apocynaceac .....

Labiatar (Lamiaccae)

,

trop & S. Afr, Mad, Nig

aqueous ext.

49


decoction with roots of Cassia petersiana Bolle.

33

convulsions

decoction with Grewia stuhlmannii Schum K. aqueous ext.

+EST +PTZ

leaves ,,

roots whole plant leaves

33 49

+PTZ +EST 164., Hoya australis R. Br.

, Asclepiadaceae

F~

leaves

convulsions

165., Humboldtia vahliana Wight

, Legumin0sae

Ind

bark

epilepsy

50

166.

Hyoscyamus niger L.

Solanaceae

Ksh, lnd

epilepsy & nocturnal epilepsy

47 36

167.

Hyptis suaveolens Poit.

Labiatae

Nig, Bra, Ind, Bng

leaves

+PTZ +EST

volatile oil

49

Icacinaceae

Nig

tuber

+PTZ -STN

50% mcthanolir ext.

117

1 6 9 . llex aqu!folium L.

Aquifoliaceae

Gre, Eur, Ind, temp

epilepsy

170. [ Impatiens repens Moon

Balsaminaceae

Sir

epilepsy

Leguminoseae (Papilionaceae)

Ind, Sri, Sen, W. trop Afr, Phi, Egy, Ids, Ame, Chi, Bra, Mal, Bur

lcacina trichantha Pflamzenfam

168. ,

,

63

decoction

)

87 64

i

171.

Indigofera tinctoria L.

roots

epilepsy

leaves

epilepsy

unspecified parts 172., Ipomoea hederacea L. Jacq 173.

lpomoea hispida Roem & Schult

,

Convolvulaceae

I Convolvulaceae

I !

epilepsy

Ind, Him Ind

powdered ext. with other ingredients juice juice with honey decoction

plant

epilepsy


34, 67 64 98 ! 98 I !47 50

~

-l

r,

74

Jatropha curcas L.

Aft', Ind, tropAme

Euphorbiaceae

i

I roots & fresh leaves

convulsions & fits +PTZ -STN

roots

17 5 .

datropha gossypifolia L.

Euphorbiaceae ,,,

Air, lnd, Bra

roots & leaves

anticonvulsant

176.

J. multi~ida L.

Euphorbiaceae

S. Ame, Ind, (cultivated)

roots & leaves

anticon vulsant

177.

Juniperus macropoda Boiss.

Cupressaceae

Ind, Nep, Him, Pak

berries

178.

Kochia prostrata

Chenopodiaae

...

infusion

,

~

,

+PTZ

32

70% ethanolic ext. 70% ethanolic ext.

32

.

32

+EST

essential oil

118

+STN -PTZ

70% ethanolic ext.

119

(tincture)

Khaya ivorensis A. Chem.

Meliaceae

Aft"

stem bark

febrile convulsions in children

+PTZ

70% ethanolic ext.

120

180.

Khaya senegalensis A. Juss

Meliaeeae

Afr

stem bark

febrile convulsions in children

+PTZ

70% ethanolic ext.

120

181.

Lagochilus sp.

Labiatae

ext.

121

182.

Lammaria ochroleuca de la Pylaie

Laminariaceae

methanolic ext.

122

183.

Lantana camara L.

Verbenaceae

infusion 70% ethanolic ext.

53

decoction and concoction

99

i 179.

NM

+STN +CAF +PIC +CAM

deep seas

whole plant (alga)

-PTZ

Air, Gui, Ind, trop Ame

roots leaves

trop Aft

leaves

convulsions in children epilepsy

i i

.

.

.

.

.

.

.

.

.

.

anticonvulsant

,,

.

184.

Launaea cornuta Oliv. & Hiern. C. Jeffrey

185.

Lavandula stoechas L.

Labiatae

Por, Can, Med, As. min

-

186.

Lavandula sp.

Labiatae

Eur, Med, Asi

-

187.

Ledebouriella seseloides Wolff.

Umbelliferae

Chi, Man

roots

Compositae

+PTZ +STN

47 +PTZ +NIC +EST

convulsions & spasms .

oil vapour

123

~

35

188.

Leonurus cardiaca L.

Labiatae

Gre, Pak

189.

Leucas lavandulifolia J.Sm.

Labiatae

Irop Asi & Aft"

epilepsy leaves

Leucas zeylanica R.Br.

87

convulsions, epileptic seizures & coughing spasms

35

decoction or infusion

190.

191.

Licaria puchury major.

Lauraceae

192.

Limnophila chinensis Osb.

Scrophulariaceae

lndchi

branchlets & leaves

antispasmadic in convulsions

Rutaceae

Ind, Pak, Nep, Him, Bnl~,

leaves

epilepsy

seeds

Men'. 193.

Limonia crenulata Roxb.

+E~T

essential oil fraction

124

decoction with other ingredients

35 50

,

194,

Limonia acidissima L.

Rutaceae

Ind, Ids

i ! leaves

epilepsy

195.

Linum usitatissimum L.

Lineae

Ind

I seeds & flowers

hysteria

196.

Lobelia inflata L.

Campanulaceae

Ame, Ind

whole plant

epilepsy, hysteria & convulsions

197.

Maerua angolensis DC

Capparidaceae

Aft"

roots & leaves

epilepsy

34, 37 seed oil

111 decoction with Ricinus

epilepsy Magnolia obovata Thunberg

Magnoliaceae

Jap, Chi

Wilson

Magnoliaceae

Jap, Chi

199.

Mangifera odorata L.

Anacardiaceae

200.

Maprounea africana Muell.

Euphorbiaceae

201.

Marsilea sp.

bark

bark

Marsileaceae

Con Ind

plant leaves NM

mm

2

84

decoction +STN +Pie +PTZ +intra-cerebrovent-ricular inj of penicillin G-K

Magnolia officinalis Rehder et

....

Z ,.q

communis L.

roots 198.

34

hysteroepilepsy +PTZ

99

;r,,

ether ext.

125

,.q

magnolol (26)

126

compound mixture

35

ethanolic ext.

127

N

,.q

marsiline (27)

i

128

tirol

,'

,

,J

Marsilea minuta

202.

Marsileaceae

. . . .

Ind ,,

_ 203.

M. rajasthanensis

Marsileacear

204.

Martynia annua L.

Pedaliaceae (Martyniaeeae)

205.

M. diandra Glox.

206.

Matricaria aurea L.

207.

,,

+PTZ -EST

leaves ,,

Ind

,,

129

marsiline (27)

.....

129

leaves

50, 51, 86, 130

Mex, Ind, Nep, Pak

leaves

epitepsy

decoction

i Martyniaceae

Ind, Pak

fruit

epileptic attacks

decoction

I Coml:msitae

Pak

plant & flowers

hysteria

52

M. chamomilla L.

Compositae

lnd

dried flower heads

hysteria

34

208.

Melia azedarach L.

Meliacear

lnd, Pak, Bng, Sub Him

plant

209.

Melissa oflqcinalis L.

Labiatae

N.W. Gre, Pak

210

Meilittia usaramensis Taub.

Pa ilionaceae

Ken, Tan, Moz

Rutaceae

Phi

"

211.

~,,

Micromelum compressum Blco. ! Men'.

,

P

+PTZ +STN

,,

70% ethanolic ext.

131

32

epilepsy &. hysteria

~

roots

convulsions

decoction

31

young shoots

infantile con vulsions


35

132

o

,

212.

Moghania strobilifera L. St. Hil. ex Jacks.

Leguminosae

Pak, Ind, Mal, Phi, Chi, Bur, from Ind to Phi

roots

epilepsy

35

Moringa oleifera Lamk.

Moringaceae

trop, Ind, PaL Him, E. Air, Sri, Mal, Bur, Phi

roots, root bark, leaves, gum, flowers & seeds

epilepsy & hysteria

roots

nocturnal epilepsy

34, 37, 50, 64,

.....

213.

70% ethanolic ext. ....

Moringa concunensis Nimmo

Moringaceae

used as a substitute ! f o r M. oleifera

.

.

.

.

.

215. [ Muntingia .calabura L. 216. ! Musa paradisiaca L. var sapientum Kumtzc Musa sapientum L. ,

,

Elaeoearpacee Musaceae

trop Ame, Indchi

flowers

Pak, lnd, Chi, Phi, trop

stem sap

antihysteric i epilepsy & hysteria

32 67

4

.

35, 47, 52, 67

36

+PTZ +STN 214.

~t

i

L

,,,

I

:infusion .

sap

.

.

.

.

.

.

35

35, 50, 52

217.

50

epilepsy

Ind, Chi

Mylitta lapidescens Horan. (Fungus)

I i

,

,

decoction

31

218.

Myrica salicifolia Hochst. ex. A.Rieh.

Moraceae

trop Afr

roots

convulsions

219.

Myrtus communis L.

Myrtaceae

Ind, from Med to Him

leaves

epitepsy

34, 50

220.

Nardostachys jatamansi DC.

Valerianaceae

Alp Him, Bhu, Nep, Sik

roots & rhizome

epilepsy, hysteria, convulsive affections & hysteroepilepsy

34, 37, 50, 67, +MES

221.

Nerium oleander L.

222.

223.

Apocynaceae

Newbouldia leavis (Beauv.) Seem. ex Bureau

Bignoniaceae

Nicotiana tabacum L.

Solanaceae

As min, Med

Lag, Gha, Nig

leaves & bark leaves bark & root bark leaves

epilepsy + PIC + BIC


convulsions

Ind, Pak

jatamansone (isolated from essential oil) (28) fraction B-I fraction B-3

36, 47, 64, 86

133 64 134

infusion

53

+PTZ +EST

aqueous ext.

49

+PTZ

70% ethanolic ext.

32

+PTZ

70% ethanolic ext.

32

+~Z

juice 70% ethanolic ext.

33 32

+~Z -STN

70% ethanolic ext.

32

+EST +~Z

70% ethanolic ext.

135

decoction

33

oil

136

+STN

!

224. ....

Ocimum amencanum L.

Labiatae

Ind

225.

Ocimum basilicum L.

Labiatae

Asi, trop

226.

leaves

Ocimum gratissimum L.

Labiatae

Ind, (cultivated)

Ocimum sanctum L.

Labiatae

Ind, Pak, Phi

leaves

Ocimum suave Willd.

Labiatae

E.W. Aft, trop Asi

roots & leaves

Oleam miilefolii

Oleaceae

Pol

plant

epilepsy

i i 227. t

228. 229.

,

epilepsy & convulsions +PTZ

"..dl

230.

Origanum vulgare L.

Labiatae

Pak, Chi, Ksh, Indchi, Mal

-

epilepsy

23 !.

Ormocarpum kirkii S. Moore

Papilonaceae

E. & S. Aft"

roots

epilepsy

232.

paeonia albiflora (Pallas)

Paeoniaceae

Chi, Jap

233.

Paeonia emodi Wall.

Ranunculaceae

Him, Ksh, Pak

tubers

234.

Paeonia lactiflora Pall. vat trichocarpa Bunge.

Paeoniaceae

Jap

roots

.

35

-PTZ

roots

decoction

31

paeoniflorin (29)

137 34, 36, 47, 50, 52

epilepsy, convulsions, hysteria & hysteroepilepsy . pentagalloyiglucose (30)

+PTZ EEG

138

paeonifiorin (29)

-PTZ EEG albiflorin (31) methanolic ext. aqueous/acetone ext. aqueous ext.

+PTZ EEG +PTZ EEG +PTZ EEG +PTZ EEG 235.

Paeonia officmalis L.

Paeoniaceae

S. Eur, W. Asia

tubers

epilepsy, hysteria & convulsions

236.

Paeon!a ' suffruticosa Andr.

Paeoniaceae

Chi, Jap

dried.root bark

convulsions

237.

Palisota ambigua CB. Clarke.

Commelinaceae

Con, C. Air

leaves

epilepsy

238.

Panax ginseng C. A. Meyer

Araliaeear

Ame, E. & S.E. Asi, Bulg, Cana, Chi, F.E., Jap, Kor, Tib

roots

.

.

.

.

.

37, 50

~

35

99.9% ethanolic ext.

139

neutral saponins

140

~

111

.

-IviES +PTZ +NIC +STN ,,

,,

239.

Panax quinquefolius L.

Araliaeeae

Ame

roots

convulsions

240.

Paris polyphylla Sin.

Liliaeeae

Pak, Chi, Tai, lndchi

roots

epilep.~y

Passiflora incarnata

Passifloraceae

Jap

NM

241.

+PTZ -MES -PIC

~

.

+PTZ

_

.

concoction .

.

.

35

.

maltol and ethyl maltol (32 & 33). ,

141

acidic and neutral triterpene glycosides ..

142

.....

242.

Patrinia intermedia .

.

.

.

.

.

Valerianaceae

USSR

roots

+STN

243.

Rutaceae

Peganum harmala L. .

.

.

.

.

.

.

.

.

,

.

,

Ind, Pak, N. Aft, Eur, Mon, Rus, Tib, Tur

seeds

,

34

hysteria

i

decoction with Helichrysum setosum Harv.

31

244

Phoenix reclinata Jacq.

Orchidaceae

E. & S. Aft

roots

epilepsy

245.

Phyllanthus emblica L. (Syn. Emblica officinalis Gaertn ).

Euphorbiaceae

Ind, Ira, N. Ame, Nep, Cub

fruit


246.

Phyllostachys nigra Lodd. var henonis Mitford

Graminae

Kor, Chi

stem


complex preparation with raw ginger

35

247.

Phyllanthus urinaria L.

Euphorbiaceae

Nig, Moz, N. Ame, S. Ame, Ind, Sri, Nep, Chi, Mal, S. Kor, Indchi, Ids, Aus, Fij, Fra, Neth

roots

epilepsy & convulsions in children

used with dog's ear

144

used with Marrubium album

144

blossoms

+

alkaloids

145

dried unripe fruit

+AUD +MES -PTZ -PIC

75% ethanolic ext.

146

,

248.

Picnomon acarna L. Cass.

Compositae

Gre

249.

Piper iongum L.

Piperaceae

Gre, Chi, Jav, lnd, Sum, Tai, Bor, Sri, MaLl

250.

,

,

Piper methysticum

Piperaceae

143

leaves

,

,

,

NM

+EST

+PTZ +EST

2 tam

r-, kawain (34) dihydrokawain (35) methysticin (36) I dihydromethysticin (37) demethoxy yangonin (38) yangonin (39)

147 r

m

am

251.

Piper nigrum L.

Piperaceae

Piper retrofractum

Ind, Pak, Sri, Bra, Jav, Indochi, Ids, trop Asi, Bor, E.S. Aft, Phi, Mad, Nig, Chi

+STN +PTZ +IVIES -IVIES

fruit

seeds

piperine (40)

150 151 152 150

+

+PIC +AUD +I/C Fe + 1/V tub +l/V g!u 252.

Pithecolobium saman Benth.

253.

Plumbago :eylanica L.

254.

Polygonum japonicum Meisn. P. bistorta Sensu Chinese auct. Non L.

255.

Pongamia glabra Vent.

Leguminosae

: Plumbaginaceae Polygonaceae I

Leguminosae

Jam, trop, Ame

leaves

Ind, W. Pen, Bng

~

Chi, Tai, Jap, lndchi, Kor, Pak, S.E. Asi

rhizome

lnd, Pak

+PIC +NIC -PTZ -STN

alkaloidal fraction

153

+PTZ

70% ethanolic ext.

32

convulsions

epilepsy

,. . . . . . .

148, 149, 150

35

several complex prescript!ons

37

256.

Pothos scandens L.

Araceae

Chi, Mal, Ind

leaves

convulsions

257.

Psidium guyava L. P. guajava L.

Myrtaceae

Ame, Ind, Pak, subtrop, trop, Chi, Phi, S.E. Afr, Egy

leaves

ext. & tincture

37, 64

fresh leaves

epilepsy & convulsions in children convulsions

concoction

31

.

.

.

.

.

35

258.

Psychotria curviflora Wall.

Rubiaceae

Indchi, Mal

leaves

convulsions

poultice

35

259.

Punica granatum L.

Punicaceae

Ksh, Him, Ind, Pak

~

epilepsy.

~

47

260.

Quercus infectoria Oily.

Fagaceae

Randia esculenta Lour. Men'.

Rubiaceae

....

,,

261.

Gre, Syr, As.min, Ind, . . . . Ira,' Irq . . . . Indchi

47

epilepsy wood

convulsions

decoction with other plant drugs

35

1

Rauwolfia serpentina L. Benth.

262.

Rauwolfia vomitoria L.

263. 9

Apocynaceae

Apocynaceae

|

I

, Rosaeeae

,.266. ' Ruta chalespensis L.

, Rutaceae

267.

t

Ruta graveolens L. (Ruta graveolens var augustifoliea Pers W.)

....

f

epilepsy

stem bark 1

.

.

.

.

.

.

,,

!

leaves

i

,,,

|

Rutaceae

..,

S. Eur, Ind, Sri, (cultivated)

herb leaves seeds

35 51 130 154

+PTZ +STN

70% ethanolic ext.

32

+PTZ

70% ethanolic ext.

32

alcoholic ext.

155

~

Air, N. Ame, Arg, C.Asi, Bre, Chi, S. Eur, Sub Him, Ind, Ira, Mex, Pak, Parag lnd

with rose water decoction reserpine (41 )

.

+PTZ

.

epilepsy 1

Euphorbiaceae

.265:,, Rubus ellipticus

epilepsy

herb

Aft"

i

Ricinus communis L.

264.

=

Pak, Ind, trop Afr, C. Ame, Bur, Chi, Sri, Indchi, Thi, Sum, N. Bor, W. Lao, trop Asi

i

infantile conyu.!sions

+EST

L

Ruta tuberculata Forsk. Boiss.

269.

,,

|

Salvia nemorosa sub sp. i amplexicanlis

.

epilepsy hysteria convulsions & fits in children

juice used with other ingredients

and Z!ngiber purpuream

:

.

.

.

.

,

270. ] Salvia sclarea

I Sapindus emarginatus L. Roxb.

+EST -PTZ -S'IN

ethanolic ext.

62

Bulg

NM

+EST -PTZ -S'IN

ethanolic ext.

62

Pak, Ind

Sapindaceae t

i 272. ~ Sapindus . . . mukorossi . . Gaertn.

seed capsule .

i Sapindaceae

. . . . . . . . . .

Ind, Pak, Nep, Him, Bng, Chi

epileptic fits

Sapindus trifoliatus L. b

Ind, Bng, Sri

Sapindacear ,,

274. j Saussurea. lappa C.B. Clarke

|

.

.

.

Ksh

fruits & seeds

epilepsy

fruit & pulp of fruit

epilepsy, hysteria & hysteroepilepsy

.

'roots 9

.

9

|

2

9

epilepsy

r :Z ,
physcion-8-O-13-D- glucoside > trans-piceid > physcion > emodin-8-O-~-D-glucoside > chysophanic acid. Wang [63] has shown that the anthraquinone, emodin, inhibits the cytotoxic effect of trichomonads in astrocytic cell cultures in a dose dependent manner. Such inhibition suggests the in vitro production of an anti-trichromonal agent. This action may be due to the generation of hydrogen peroxide extracellularly [64] and intracellularly [65]. The quinoid structure of emodin can be activated to the semiquinone radical intermediate, which may react with oxygen to produce a superoxide anion radical, hydrogen peroxide and hydroxyl radical [66]. The hydrogen peroxide could form the hydroxyl radical which may cause intracellular DNA damage [67]. T a b l e 2.

C y t o t o x i c i t y o f S i x C o n s t i t u e n t s from the R o o t s o f Polygonum cuspidatum a

Cpd. name

Cpd. No. ,i

ED50 ,

,,

mb

rb

i ii

Chysophanic acid

2093-+257

0.518+0.281

0.965+0.031

Emodin

18.7:1:4.7

1.938+0.082

0.985+0.010

.

6

Physcion

7

8

.

.

....

.

990+617

1.007+0.587

0.96x'-0.075

Emodin-8-O-l]-D-glucoside

2180

0.794

0.975

Physcion-8-O-[~-D-glucoside

286

. . . .

,

,,,

14

Piceid

519

0.714 ,=,

.

0.858 .

1.552

.

.

.

.

0.824 .

aFrom Yeh et al. [9] b where m is the slope of median effect; r = linear correlation coefficient of the plot. Compounds 1-5 are anthraquinones. Compounds I and 4 are structurally similar to emodin. Structures 3 and 5 are glucoside derivatives of 2 and 4.

Deoxyribonucleic Acid and Ribonucleic Acid Precursor Activity The effect of the compounds isolated from Polygonum cuspidatum on precursor incorporation into DNA and RNA was investigated by Yeh et

618

OGWURU a n d ADAMCZESK!

al. [10]. The inhibition of DNA precursor incorporation at 100 ~tM (40~tg/mL) concentration was emodin > physcion-8-O-13-D-glucoside > physcion > emodin-8-O-13-D-glucoside > chysophanic acid > piceid (table 3). The inhibition of RNA precursor incorporation at the same 100 ktM (40~g/mL) was slightly different from the DNA incorporation results. The order of potency was emodin > physcion > physcion-8-O-13-D-glucoside > chrysophanic acid > piceid > emodin-8-O-13-D-glucoside. With regard to the inhibition of RNA precursors incorporation, the glucoside derivatives of the anthraquinones, emodin and physcion, have reduced biological activity as compared to the parent compounds. Table 3.

Effect of Isolated C o m p o u n d s from Polygonum Precursor Incorporation into DNA and R N A

cuspidatum on the

% inhibition of precursor incorporation

into Cpd. No.

Cpd. Name

conc(lxM)

i

5

RNA

Chysophanic acid

10 100

30.15:12.3 38.25:14.1

8.6:1:4.0 19.7+8.7

Emodin

10 100

25.3-l-8.6 56.4+5.2

0.2 53.7+!4.0

Physcion

10 100

31.6"!"8.9 46.85:0.8

7.0:1:3.0 49.6:!:14.0

10 I00

15.05:3.1 41.3+5.6

trans-piceid ( 1 6 ) = trans-resveratrol-O4-~-glc (17) > cis-piceid (19) > cis-resveratrol-O4-~-glc (20), while the order of PKC activity was trans-resveratrol-O4-~5-glc (17) > cis-resveratrol-O4-~5-glc (20) > cis-resveratrol (18)> trans-resveratrol (15) > trans-piceid (16) --- cis-piceid (19). Table 4.

Kinase Inhibitory Activity of the Isolated Stilbenes

Cpd.No.

Compound Name

PTK

PKC

15

trans-Resveratrol

6 x 101

4 x 101

Piceid

2 x 102

2 x 102

ResveratroI-O4q3-glc

2 x 102

0.3 x 101

18

cis-Resveratrol

5 x 101

3xi01

19

cis-Piceid

5 x l0 2

2 x 102

2O

cis-Resveratroi-O4-~-glc

>8 x 102

0.6 x 101

16 17

trans-

trans-

From Jayatilak et al. [33]

The cis-isomer of resveratrol possessed the highest PTK inhibitory activity followed by trans-resveratrol. This implies that the presence of free phenolic groups is associated with PTK activity. Trans-resveratrolO4-[3-glc inhibited PKC with a significant potency. It's activity against PKC was better than trans-resveratrol, thus impling that there is no requirement for the free phenolic substituent on the trans structure for PKC inhibition to occur. On the other hand, the near absence of PKC activity in both the trans- and cis-piceids indicates that the phenol group at the Rl position is important. The mechanism of inhibition of protein tyrosine kinase could be the binding to a tyrosine-containing peptide or substrate that is essential for enzyme activity. This might mean that these stilbenes are competitive inhibitors with respect to the protein peptide or substrate [68]. The action of three anthraquinones (emodin, physcion and emodin-O[3-D-glucoside), isolated from the root of Polygonum cuspidatum, on protein tyrosine kinase was investigated by Jayasuriya et al. [ l l ] (see Table 5). As revealed in Table 5, emodin posseses the strongest inhibitory activity against PTK in p56 Ick cells of the three anthraquinones. The substitution of the C6-OH group with -OMe in physcion, [69] or C8-OH

620

OGWURU a n d ADAMCZESKI

with glucose in emodin-O-l]-D-glucoside [12] (7) completely abolishes the inhibitory activity against protein tyrosine kinase. This indicates that the presence of free hydroxyl groups at the C-8 and C-6 positions is necessary for the inhibition of protein tyrosine kinase. The mechanism of action proposed by Jayasuriya et al. [10] suggested that emodin was a competitive inhibitor of PTK in p56 Ick cell with respect to ATP (Ki = 15 ~M) and a non-competitive inhibitor with respect to the tyrosinecontaining substrate [ 10]. Table 5.

Protein Tyrosine Activity Inhibitory Activity of Emodin, Physcion and Emodin-O8-[~-D-glucoside Against PTK in p56 lck Cells Compound No.

Compound

5

Emodin

IC50 (Ixg/mL)

Physcion 7

>800

Emodin-O8-~-D-glucoside

>800

i

FromJayasuriya et al. [ !0]

Zimmerman and Sneden [ 13] investigated the effects on protein kinase C of two vanacosides, namely, vanacoside A (28)and B (29), isolated from Polygonum. The PKC inhibitory activity (IC50 values) was 44 I.tg/ml and 31 Ixg/ml for vanacosides A and B, respectively. The octacetate derivative (30) of these compounds was inactive in PKC inhibition assays. This indicates that the presence of a free hydroxyl or phenolic group is essential for the inhibitory activity that is characteristic of the vanacosides.

Phytotoxic Activity Inoue et al. [70] examined the acetone extracts ofPolygonum sachalinense and found that the neutral acidic fraction from TLC agar plate was responsible for the phytotoxic properties of the plant. The compounds identified from the solvent extracts include emodin and physcion and their glucoside derivatives. The glucoside derivatives showed very little phytotoxtic activity. The activity of the compounds on root and hypocotyl or coat sheath growth in lettuce was also examined. It was found that emodin caused growth inhibition of mature lettuce plants at a concentration of over 100 ppm (3.7 x 10-4 M), and inhibition was observed at a concentration of 50 ppm (1.85 x 10-4 M) in lettuce seedlings. No phytotoxicity was observed for emodin or physcion glucosides at 200 ppm against lettuce seedlings, in plant growth bioassays.

BIOACTIVE NATURAL PRODUCTS FROM POLYGONUM SPECIES

621

Oncogene Signal Inhibitory Activity If a cell replicates while DNA damage is present, permanent alterations to the genome can be produced. This may modify proteins that regulate gene expression. The critical genetic alterations which underlie the process of tumorigenesis involve at least two types of genes" the oncogenes and oncosuppressor or tumor suppressor genes. The cellular oncogenes code for oncoproteins that are involved in signal transduction and the proliferation of cells. Signal transduction is believed to be altered by cellular oncogenes or tumor suppressor genes during the transformation of normal cells into malignant cells. Protein kinases encoded or modulated by oncogenes were used to prescreen potential antitumor activity of extract from Polygonum. The results show that emodin, displayed a highly selective inhibitory activity against Src-Her-2/neu and ras oncogenes.

Antitumor Activity The antitumor activity of some phenolic components of Polygonum cuspidatum was investigated by Ryu et al. [ 12]. The compounds identified in the acetic acid fraction included trans-resveratrol (3,5,4'trihydroxystilbene, 15) and cis-piceid (3,4',5-trihydroxystilbene-3-O-[3-Dglcoside, 19). These compounds were tested in several tumor cell lines which included A-549 (non small cell lung), SK-OV-3 (ovarian), SK-MEL2 (skin), XF498 (CNS) and HCT15 (colon). In agreement with other published bioactivity results, the activity of the glucoside derivative was less than that of trans-resveratrol [71]which was active against all the tumor cell lines tested) and is shown in Table 6. Table 6

In vitro A n t i t u m o r Activity of t r a n s - R e s v e r a t r o l and cis-Piceid. T h e ED$0

Value of Each C o m p o u n d was Defined as the Concentration (~g/ml) that Caused 50% Inhibition of Cell Growth In vitro EDs0 Values for Cell Lines ,

i

trans-Resveratrol (15) i ,,,=,

c/s-Piceid (19)

'%

A549

3.5

50.4

SK-OV-3

3.7

>50

SK-MEL-2

2.4

42.8

XT498

3.8

>50

HCTI5

3.5

>50

OGWURU a n d ADAMCZESKI

622

The difference in the chemical structure of trans-resveratrol and cispiceid is that the latter has a glucose at the Rn position while the former has a hydroxyl group. This implies that the presence of the hydroxyl substituent is necessary for antitumor activity of the stilbene. The difference in activity may also be due to geometric stereochemical differences between the cis and trans double bonds and/or the poor solubility of the glucoside derivative in water which inhibits the interaction of this compound with cellular components. Effects of Condensed Tannins on Digestive Enzymes Tannins are another class of compounds extracted from P o l y g o n u m species of plants. They have been shown to have adverse effects on the growth of chicks and rats [ 16-20]. Tannins are classified into hydrolyzable and condensed types. The hydrolyzable tannins contain ester or glucoside bonds and are readily decomposed by acids, while the condensed tannins contain the benzene nuclei. The inhibitory effect on trypsin activity is more marked with condensed tannins than with hydrolyzable tannins. [72] Horigome et al. [73] tested the effects of tannin administration on the digestive enzymes in the intestine of rats. They found that the activities of the enzymes trypsin and cx-amylase in the three segments of the intestine (lower, middle and upper) was significantly depressed in rats fed on a test diet containing 10g/kg of tannin when compared with the rats fed a basal diet (Table 7). On the other hand, lipase activity in the upper and lower T a b l e 7.

Effects of T a n n i n s on the Activities of the Digestive E n z y m e s in the Small Intestine of Rats

Segment of small intestine Tannin in diet trypsin

absent present

Upper

Middle

5.3 +0.6

Lower

15.7 + 2.1

17.0+2.1

7.8"*:!: 1.1

13.4"-t"0.6

11.9:1:0.9

17.2 +2.1

13.4"*:1:0.6

4.5**+0.4

14.2" *:1:1.2

16.6"*:t: 1.0

2.0

+ 0.5 ......

a-amylase

absent present

,

lipase

absent present

24.7 + 3.0 21.4 + 0.4

,

,

39.0+2.8 47.4"* + 4.8

39.2 :t: 9.3 38.8:1:6.2

*p 50 Ixg/ml) [109, 110] may be due to the use of only low amounts of these plant parts in the production process. As is evident from these investigations date of harvesting, plant parts collected and selection of solvent may substantially influence the pattern of active constituents in plant extracts. Thus, it is not astonishing that variable biological effects are observed for different crude extracts, underlining the need for standardization to achieve reproducible pharmacological and clinical activity. Unfortunately, however, also for

S T U D I E S ON H Y P E R I C U M P E R F O R A T U M - ST. J O H N ' S W O R T

667

isolated, pure constituents of Hypericum no clear receptor binding profile has yet emerged. Thus, in a recent investigation no inhibition of ligand binding to receptors and related binding sites of the two excitatory amino acids GABA and glutamate could be demonstrated for three characteristic compounds (hypericin 1, kaempferol 43 and hyperforin 1 0 ) a t concentrations between 10 and 100 ~tM, while a methanol total extract was found to inhibit the agonist binding site of the NMDA receptor halfmaximally at a concentration of 7 lag/ml [ 109, 110]. In contrast, in another study hypericin 1 was found to bind to the NMDA receptor (Ki about 1 laM), although no affinity of a crude extract to this receptor was detected at a concentration of 5 lag/ml [ 103]. With the exception of NMDA-antagonistic activity, no biologically relevant interaction of hypericin 1 with an extensive battery of receptors and other binding sites has been described [103, 106, 107, 109]. Since NMDA antagonists prevent HIV-1 gpl20-induced neurotoxicity, it has been suggested that the interaction of Hypericum extracts and constituents with NMDA binding sites may mainly play a role in their antiviral activity [103]. However, a methanolic Hypericum extract was devoid of protective effects against NMDA- and gpl20-induced cytotoxicity and did not influence the gpl20-stimulated release of arachidonic acid in primary rat neurons [112]. Functional NMDA antagonists are active in animal models used for the evaluation of antidepressants, and chronic administration of NMDA antagonists to rodents results in downregulation of cortical 13-adrenoceptors, a phenomenon well known for many antidepressants, indicating that interaction with the NMDA receptor complex may well be related to the antidepressant activity of Hypericum [ 109]. Hyperforin 10, which was recently identified to be a major biological active constituent of Hypericum [ 113], was examined for receptor affinity in a assay panel analog to the screening program of the NIH [114]. Significant inhibition of ligand binding was observed for the following binding sites (Ki values are given in parenthesis): adenosine (86 lxM), CCKA (0.4 laM), CCKB (17 ~tM), and chloride channel (6.7 ~tM). Kaempferol 43, a characteristic flavonoid constituent of Hypericum, was not found to influence ligand binding in seven selected receptor assays at concentrations up to 100 ~tM [ 107]. A general mechanism which appears to be of major importance with regard to the long-term effects of psychotropic drugs on neurotransmission involves receptor adaptation, e.g., changes in receptor density and/or signal transduction. For example, it has been shown that all classes of antidepressants reduce the functional sensitivity and the number of postsynaptic B-adrenoceptors in the frontal cortex of the rat brain and presumably in the brain of depressed patients following 2-3 weeks' administration, even though there is no evidence that these drugs directly interact with these binding sites. The time of onset of receptor

668

ERDELMEIER et aL

desensitization or down-regulation approximately parallels the time it takes for the therapeutic effect of the antidepressants to become evident. Other adaptive changes in response to chronic treatment with antidepressant drugs include a down-regulation of 5-HT2 receptors and an up-regulation of t~l-adrenoceptors [96, 107]. Mtiller et al. [104] investigated if Hypericum extracts induce similar adaptive changes. Wistar rats were subchronically (14 days) treated with imipramine (20 mg/kg p.o.) or a methanolie Hypericum extract (240 mg/kg p.o.) and then the number and affinity of 13-adrenoceptors and 5-HT2 receptors in the frontal cortex was examined. In agreement with most findings in the literature imipramine treatment led to a significant decrease in the density of both receptors. Likewise, application of Hypericum extract induced a reduction of 13-adrenoceptors by about 16 %, while the same treatment caused a significant increase in the number of 5-HT2 receptors by approximately 15 %. The affinity of the radioligands used (3H-DHA and 3H-ketanserine, respectively) was not affected by either treatment. An increase of both 5HTIA and 5-HT2 receptors was also observed in a second study after oral administration of the same extract at a rather high dose of 2700 mg/kg for 26 weeks [115] . Interestingly, when rats are treated with electroconvulsive shock therapy a similar pattern of down-regulation of 13adrenoceptors and elevation of 5-HT2 receptors is usually observed. These findings may indicate that different clinically effective treatments may have divergent effects on 5-HT2 receptor density [ 104]. In contrast to the above mentioned in vivo studies, in preliminary investigations it was found that a methanolic extract from St. John's Wort (5 - 500 ktM) significantly reduced the expression of serotonin receptors at the plasma membrane of rat pheochromacytoma cells (PC-12) [ 116]. Effects on Neurotransmitter Uptake _

Besides an inhibition of MAOs, an antagonism on a2- or an agonism on 5HTIA receptors it is now generally believed that most antidepressant drugs operate initially by suppression of synaptosomal uptake of neurotransmitters like serotonin and/or noradrenalin [104]. In a preliminary study, a Hypericum extract was shown to cause a 50 % inhibition of serotonin uptake by rat synaptosomes at a concentration of 6.2 lag/ml [112]. This observation has now been confirmed by other investigators [ 104, 106]. Surprisingly, these authors found that in contrast to all other known antidepressants a methanolic extract from St. John's Wort in addition to serotonin (IC50 2.4 Ixg/ml) inhibited also the synaptosomal uptake of noradrenalin (IC50 4.5 Ixg/ml) as well as dopamine (IC50 0.9 ~tg/ml). Recently, these investigations were extended to include the evaluation of a standardized extract as well as those of some ingredients on the uptake of glutamate and GABA [ 109, 110]. Hypericum extract was found

STUDIES ON HYPERICUM PERFORATUM - ST. JOHN'S WORT

669

to potently inhibit the synaptosomal uptake of both amino acid neurotransmitters (IC50 for L-glutamate: 21 lag/ml; IC50 for GABA: 1.1 ~tg/ml). Neither the naphthodianthrone hypericin 1 nor the flavonol aglycone kaempferol 43 expressed any effect on these uptake systems at concentrations of 10 laM. A comparative study of the above mentioned methanolic extract (containing 1.5 % hyperforin 10) with an extract prepared by extraction with supercritical carbon dioxide (containing 38.8 % hyperforin 10), indicated, that hyperforin 10 is a major active inhibitor of neurotransmitter uptake in different Hypericum preparations [ 117]. As is evident from Table 2, pure hyperforin 10 like the two extracts exerted a strong inhibitory action on the synaptosomal uptake of all five neurotransmitters examined. Although the IC50 values for L-glutamate uptake were generally higher than the corresponding values for the other neurotransmitters, the rank order of potencies was neither comparable for both extracts nor for pure hyperforin 10. In addition, the IC50 values of the two extracts did not strictly correlate with their hyperforin content. These observations suggest that, although hyperforin 10 is a potent inhibitor of neurotransmitter uptake, it is either not the only active constituent of the extracts or other compounds in the extracts modulate its efficacy [ 117]. T a b l e 2.

Inhibition of Synaptosomal Uptake of Different Neurotransmitters By pure Hyperforin 10 as well as a C O 2 a n d a Methanol Extract f r o m Hypericum perforatum. Given are the Mean IC50 Values (+ SD) in ttg/ml. The Numbers in Parenthesis Represent the Corresponding Hyperforin 10 Concentration in nM, Which for the Extracts was Calculated from Their Hyperforin 10 Content (CO2-Extract : 38.8 %; Methanol Extract: 1.5%) |ill

Neu rot ransm itter

Hyperforin 10

CO2-Extract

Methanol-Extract

Serotonin (5-HT)

0.110 • 0.024

0.26 • 0.06

2.43 • 0.40

(205)

(188)

(67)

0.043 • 0.013

0.25 • 0.08

4.47 • 2.05

(80)

(181)

(123)

0.055 + 0.010

0.056 + 0.025

0.85 + 0.14

(102)

(41)

(24)

0.099 • 0.022

0.12 • 0.04

1.11 :!:0.06

(184)

(87)

(31)

0.445 + 0.369

2.83 + 1.80

21.25 + 10.47

(830)

(2045)

(586)

Noradrenalin

Dopamine

Gamma-Aminobutyrie acid (GABA)

Glutamate

Data adapted from [ ! 17]

670

ERDELMEIER et aL

Studies on Psychotropic Activity in Experimental Animals A comprehensive examination of an alcoholic Hypericum extract, standardized for its hypericin 1 content (0.3 mg/ml fluid extract; 7 mg/g lyophilized extract), in several animal models considered to indicate psychotropic and in particular antidepressant activity of test substances was first performed by Okpanyi and Weischer [118]. The extract dosedependently (2 - 10 mg/kg hypericin 1 p.o.) enhanced the exploratory activity of mice in a foreign environment, significantly prolonged the ethanol-induced sleeping time (2.4 and 6 mg/kg hypericin 1 p.o.), and within a narrow dose range (strongest effect at 2 mg/kg p.o. hypericin 1) exhibited reserpine antagonism with respect to hypothermia but not with respect to catalepsy or ptosis. Similar to imipramine, Hypericum extract significantly increased the activity of mice in the water wheel (12 mg/kg hypericin 1 p.o.). This later effect was also observed after intraperitoneal injection of pure hypericin I (20 mg/kg). While a single treatment with the extract (6 and 12 mg/kg hypericin 1 p.o.) did not influence the aggressive behavior of socially isolated male mice, a significant decrease was observed after oral application for three weeks. In contrast to imipramine, administration of the extract for five days (2 mg/kg hypericin 1 p.o.) did not counteract the clonidine-induced depression of exploratory activity in male gerbils. Winterhoff and colleagues [119] investigated the activity of a lyophilized St. John's Wort total extract (containing 0.015% hypericin 1) and a Hypericum extract without hypericin 1 in three short-term experimental models in mice and rats. The extracts were orally applied in solution or as suspension. Hypericum extract without hypericin 1 (500 mg/kg) given as solution or suspension caused a distinct increase of dopamine concentration in the hypothalamus of rats 4 h after gavage. The suspension of the same preparation additionally caused a pronounced increase of the 5-hydroxyindoleacetic acid content, a major product of monoamine oxidase catalyzed metabolism of serotonin. No significant effect was observed for the hypericin 1 containing total extract, indicating that this constituent antagonizes the activity of other compounds of the extract and thus is not responsible for this action. The authors suggest that the reduced activity of the solution may be due to interference with the vehicle used (20 % 1,2-propanediol, 20 % ethanol 96 %, 40 % glycerol, 15 % H20 5% Hypericum extract). Reserpine antagonism was observed in mice independent from the application of Hypericum extract before or after injection of reserpine. This observation in combination with the increased hypothalamic content of 5-hydroxyindoleacetic acid does not support the suggested inhibitory effect of St. John's Wort extract on MAO in vivo (see above). In contrast to the study of Okpanyi and Weischer [ 118] in this investigation the narcotic effect of ketamine was


671

clearly reduced by Hypericum total extract indicating a central stimulating effect of this preparation. The above described experiments were recently confirmed and extended by the same group of authors [ 120, 121. For these studies a methanolic extract containing between 0.24 and 0.32 % hypericin was employed. Significant and dose-dependent effects on the central nervous system were observed which included a decrease in ketamine-induced sleeping time (50 - 500 mg/kg p.o.) and an increase in body temperature (250 and 500 mg/kg p.o.). Two behavioral assays frequently employed to evaluate the potential efficacy of prospective antidepressant drugs in mice or rats were used. In the tail suspension test (500 mg/kg p.o.) and the forced swimming test (optimal effect at 250 - 500 mg/kg p.o. each 24, 19 and 1 h before the evaluation), a significant decrease of immobility was observed. The activity of the extract in the forced swimming test did not diminish during daily treatment for three weeks, indicating that non-specific effects can be excluded as the cause for decreased immobility. No effect on spontaneous motility was observed in the open field test. The two dopamine receptor antagonists sulpiride (antagonizes mainly D2 receptors) and haloperidol (unspecific dopamine antagonist) reversed most of the effects of Hypericum extract, pointing to a common, dopamine-mediated action in the different behavior test models. The authors also report that DL-13butyrolactone, which according to these investigators is supposed to reduce the firing rate of dopaminergic neurons, and ct-methyltyrosine, which inhibits synthesis of dopamine and noradrenalin, completely abolished the reduction of immobilization in the tail suspension test after treatment with Hypericum. Furthermore, apomorphine-induced decrease of body temperature was enhanced in mice previously treated with St. John's Wort. A profound effect of Hypericum extract on the dopaminergic system in rats was also indicated by an enhanced hypothalamic quotient of homovanillic acid to dopamine after acute treatment (500 mg/kg p.o.) and a reduced serum concentration of prolactine following treatment for three weeks (125 mg/kg p.o.t.i.d.). Evaluation of six fractions from a methanolic Hypericum extract in the forced swimming test revealed significant activity for two fractions at oral doses comparable to the whole crude extract. These fractions contained mainly flavonoids and hypericin l/pseudohypercin 2, respectively. Indeed, the activity of the hypericin 1/pseudohypericin 2 containing fractions exceeded those of the original extract as significant effects were still observed at a dose corresponding to only 18 mg/kg of the native preparation [121]. Significant effects of pure hypericin 1 (1.5 mg/kg p.o.) were observed on the ketamine-induced sleeping time, and in the forced swimming test, while no change of spontaneous motility in the open field assay and the ratio of homovanillic acid/dopamine in the rat hypothalamus was observed [ 120]. However, the activity of pure hypericin in these tests was usually lower as suggested by its content in crude extracts. Recently,

672

ERDELMEIER et aL

it has been reported that native extracts and fractions obviously contain at least one compound which improves the solubility of naphthodianthrones. This may explain why pure hypericin and pseudohypericin produced only borderline effects in the forced swimming test while extract fractions enriched for naphthodianthrones caused a remarkable decrease of duration of immobility [ 122]. For a hydroalcoholic extract of Hypericum perforatum, produced by successive extraction of dried aerial parts with petroleum ether, 1,2dichlorethane and ethanol (50 % v/v), a sedative effect in mice has been reported [ 123]. Theauthors observed a bell-shaped dose-response effect on spontaneous motility with maximal activity at an oral dose of 26.5 mg/kg p.o, while pentobarbital-induced sleeping time was most significantly prolonged at the lowest dose applied (13.25 mg/kg p.o.). No effect on neuromuscular transmission was observed in three different test models (chimney test, traction test and rota-rod test). After separation of the crude extract in fractions containing mainly flavones, naphthodianthrones or amino acids, it was not possible to clearly attribute the effect of the native extract to a particular group of constituents. Thus, the authors conclude that activity of the hydroalcoholic extract may results form the cumulative effects of different compound, but they do not offer any explanation for the lower activity of the extract at higher doses. In a comparative study, the effects of aqueous ethanolic extracts from Hypericum perforatum and Hypericum calycinum on the central nervous system were investigated in several behavioral models in mice [ 124]. Like the both antidepressant drugs desipramine and trimipramine the two extracts (250 mg/kg i.p.) reduced swimming performance, decreased the rota-rod locomotory activity and displayed an analgetic effect in the tailflick test. In addition, the extract from H. perforatum diminished the exploratory behavior in the hole-board test, while no such effect was observed for H. calycinum. Whereas the decreased swimming time appeared to be resistant to blockade by opioid antagonist, the analgetic effect may involve 8-opioid receptors as this activity of the extract could be reversed by the 0pioid antagonist naltrindol but not naloxone [ 125]. Results from this study provide evidence against the involvement of hypericin 1 in the CNS activity of St. John's Wort extracts as Hypericum calycinum does not contain this naphthodianthrone derivative. For a similar extract from Hypericum hyssopifolium, whose chemical composition has not yet been determined, only a weak analgetic effect was observed [ 125]. Unfortunately, the significance of the activities observed is limited by the fact, that reference drugs and extracts were applied at doses which caused sedation of the experimental animals. Learned helpnessless in rats is a validated animal model of depression and can be prevented by long-term treatment (7 - 21 days) with classical antidepressants (e.g., imipramine, clomipramine, fluoxetine), whereas acute treatment is usually ineffective. Single oral administration of a

STUDIES ON H Y P E R I C U M P E R F O R A T U M - ST. J O H N ' S W O R T

673

hydroalcoholic Hypericum extract (equivalent to 3 mg/kg hypericin) 1 h before induction of helpnessless significantly increased the escape reaction when tested 24 h later (mean number of escapes :!: s.e.m.: 14.3 _+ 2.3 versus 1.3 + 0.9 in control animals). This protective effect was retained and even enhanced in rats treated for 10 days b.i.d, with the same dose. The antidepressant activity after acute as well as chronic treatment was antagonized by administration of the dopamine D~ antagonist SCH 23390 (0.03 mg/kg s.c.) and the 13-adrenoceptor/serotonin 5-HTIA receptor antagonist pindolol (5 mg/kg i.p.) [126]. Exploitation of the antidepressant action of an ethanolic and a carbon dioxide extract from St. John's Wort in two animal paradigms of depression, learned helpnessless and forced swimming test in rats, did not reveal any activity after acute treatment. However, repeated oral administration of these extracts for three consecutive days dosedependently reduced the immobility time in the forced swimming test and antagonized the escape failures induced by prior exposure to inescapable shocks [ 117]. Generally, the ethanol extract caused significant effects at a dose range between 50 and 300 mg/kg p.o., whereas the CO2-extracts displayed an equieffective activity at ten times lower doses. Chemical analysis demonstrated that the ethanolic extract contained all currently known constituents of these preparations. In contrast, the only quantifiable constituent of the CO2-extract was hyperforin 10 (38.8 %). The dose effect curves of both extracts were almost superimposible when calculated on the basis of their hyperforin 10 content. Thus, these results suggest that hypericin I and other compounds which can not be extracted from St. John's Wort by supercritical CO2 are not essential for the antidepressant action of therapeutically used preparations. Rather the phloroglucinol derivative hyperforin 10 appears to be an important antidepressant constituent of Hypericum extracts. Indeed, activity of pure hyperforin 10 could now be established in both above mentioned animal models of depression [ 114]. Activity of preparations form St. John's Wort on the central nerveous system has also been indicated by their action on in vivo monitored field potentials from four brain areas of freely moving rats. Quantitative analysis after oral application of two different extracts (containing 0.29 or 0.103 % napthodianthrones, respectively) at doses equivalent to 0.5, 1 or 2 mg/kg naphtodianthrones revealed a late onset (3 to 4 h after application) of effects which were mainly related to the frontal cortex. Decreases of electrical power throughout all frequency ranges is suggested by the authors to be similar to the activity pattern of antidepressive and/or analgetic drugs. Since alpha l and alpha2 frequencies were particularly affected, the involvement of serotonergic and dopaminergic transmitter systems is considered as possible mode of action. Almost identical results were obtained after treating the animals for 8 consecutive days [127]. Using exactly idential methodology, different results were recently

674

ERDELMEIER et aL

obtained in the same laboratory with another Hypericum extract [128]. Oral application at a dose equivalent to 1 mg/kg total hypericins resulted in an increase of delta power densitiy, followed by an increase in theta and alpha2 frequencies. These changes started during the second hour after administration and were most intense during the third and forth hour. Brain areas mainly affected were the frontal cortex and hippocampus. Hypotheses developed from earlier trails under identical experimental conditions would indicate that this extract mainly acts at cholinergic and dopaminergic transmitter systems. Although these contradictory results might be caused by differences in the composition of extracts, inherent methodical and technical insufficiencies of the test system cannot be excluded.

Antiviral Activity Antiviral activity of extracts prepared from Hypericum perforatum has first been reported by Russian scientists [ 129,130]. The acetone fraction and the water-soluble part of an ethylacetate extract, which contained catechins and flavonoid aglycones, were the most active preparations. In addition, inhibitory action on influenza virus was observed with a fraction prepared by extraction with hot water which also contained some catechins. The in vitro antiviral activity of natural products and in particularly of flavonoids and hydrolyzable or condensed tannins is well established [131]. However, the discovery that the two aromatic polycyclic diones hypericin 1 and pseudohypericin 2, originally isolated from Hypericum triquetrifolium, inhibit the replication and the infection cycle of retroviruses has caused considerable interest [ 132]. Meruelo et al. [132] examined the effects of both naphthodianthrones on two murine leukemia viruses. When either compound was injected intravenously (50 ~tg) in mice concomitantly with or 1 day after inoculation with Freund leukemia virus, the animals were completely protected. Similarly, antiviral activity was observed in vivo against infection with radiation leukemia virus. Although not all results could be reproduced by other investigators these findings have basically been confirmed by further work on many different viruses from various species including HIV-1 (for review see 134]. In spite of a great number of published studies on the antiviral activity of hypericin 1 and pseudohypericin 2, the mode and site of action is still not satisfactory solved. Tang et al. [135] found that the antiviral activity of hypericin 1 is dependent on the presence of a viral lipid membrane, while it is ineffective against non-enveloped viruses, e.g. adeno- or poliovirus. In a number of studies it has been observed that the antiviral activity of hypericin 1 is greatly enhanced or entirely dependent on its photoactivation by visible light possibly explaining discrepancies in the findings among different groups [136,137]. Thus, the concentration needed to induce a significant anti-HIV effect in vitro decreased from about

S T U D I E S O N H Y P E R I C U M P E R F O R A T U M - ST. J O H N ' S W O R T

675

1 lag/ml in the dark to a concentration of 0.01 ~tg/ml in the presence of light [ 138]. Variously, the antiviral activity of hypericin 1 has been suggested to be caused by an interference with viral replication (e.g., assembly, budding, or shedding of newly synthesized viruses) or by immediate virucidal effects [132, 133, 135, 136]. In addition, an inhibitory effect on HIV-1 reverse transcriptase has been reported [139]. As purified reverse transciptase from both avian myeloblastosis virus and murine leukemia virus were entirely unaffected by hypericin 1, the observed inhibition appears to result form a photodynamically induced cross-linking of reverse transcriptase with other viral proteins [140]. Since hypericin 1 has also been shown to directly interact with DNA [ 141] and to inhibit protein kinase C [142] mitochondrial succinoxidase [143] and tyrosine kinase activity of epidermal growth factor receptor [144], it has been suggested that these effects might as well be involved in its antiviral action [134]. A comprehensive discussion on the antiviral activity of hypericin 1 is included in three recent reviews on the chemical and biological properties of this compound [134, 145, 146]. The effective virucidal activity of hypericin 1 has now triggered its evaluation in human clinical trials for the treatment of HIV and chronic hepatitis C infection [ 147].

Antibacterial Activity The antibacterial effects of Hypericum extracts are well established. Already in 1951 a Hypericum extract with antibiotic activity was patented in the USA for the purpose of food preservation [148]. Based on the traditional external use of St. John's Wort oil for the treatment of injuries, bruises, myalgies, swellings and bums (see below), ethanolic and aqueous extracts have also been recommended as wound disinfectant and for the therapy of paradontosis [149]. However, Neuwald and Hagenstr6m [ 149] were unable to detect any antibacterial effects of St. John's Wort oil, while a strong activity against Staphylococcus aureus was observed for acetonic extracts particularly of those prepared from inflorescences. This observation could not be accounted for by known constituents of Hypericum like hypericin 1, tannins or essential oils. Extensive studies on the antibacterial action of Hypericum have been performed by Russian scientists. As a result, the enriched extracts Imanin and Novo-lmanin were developed which have been used clinically for the treatment of purulent wounds [1]. In 1971, hyperforin 10 was isolated as the active principle from these preparations [33]. Hyperforin 10 was found to be active against methicillin resistant Staphylococcus aureus (minimal inhibitory concentrations 0.1 Ixg/ml) and a range of other Gram-positive bacteria, while no or only weak activity was observed for Gram-negative bacteria and fungi. Barbagallo and Chisari [ 150] compared the activity of lipophilic extracts from three different Hypericum species (H. perforatum, H. perfoliatum and H. hircinum). H. perforatum was found to be the most

676

ERDELMEIER et aL

active with a stronger activity against Gram-positive then Gram-negative bacteria. Likewise, antibacterial, antifungal and antimalarial effects have repeatedly been reported for phloroglucinols and other compounds from different species of the genus Hypericum [e.g., 51, 55, 56, 58, 60, 151 ].

Antitumor Activity In a survey on the use of plants for the treatment of cancer there are 14 entries referring to Hypericum as a folk remedy for various neoplastic conditions [152]. Seegers demonstrated a normalization of the disturbed respiratory activity of mouse ascites carcinoma cells and discussed the antitumor activity of St. John's Wort on theoretical considerations [ 153]. An alcoholic Hypericum extract was found to inhibit the aerobic glycolysis of human brain tumor slices, while the glucose metabolism of normal rabbit brain slices was not affected. This is an interesting finding, as aerobic glycolysis, i.e. production of lactic acid despite the presence of oxygen, is typically enhanced in tumor cells. Flavonoids were identified as active constituents. Highest activity was observed for hyperoside 42, whereas the naphthodianthrone hypericin 1 did not inhibit the production of lactic acid [154]. However, tests by the National Cancer Institute (USA) indicated little promise of Hypericumperforatum against cancer [ 155]. Following detection of the antiviral activity of hypericin I (see above), a number of observations have suggested that this compound may as well represent a potential anti-cancer therapy. These biological properties include the light-dependent inhibition of protein kinase C [142], the photosensitized inhibition of mitochondrial succinoxidase [ 143], and the photoinduced inhibition of epidermal growth factor receptor (EGF-R) tyrosine kinase activity [ 144]. Inhibition of EGF-R tyrosine kinase by hypericin 1 was shown to be irreversible, non-competitive and time as well as temperature dependent. The IC50 increased from 0.75 ~tM in the dark to 44 nM with light illumination for 30 min. This effect was presumably due to a type I photosensitization mechanism since exclusion of oxygen did not alter the inhibition curve. Some Ser/Thr protein kinases (e.g., protein kinase A, casein kinase 1 and 2) and the enzyme 5'-nucleotidase were not inhibited even at concentrations > 100 ~tM [144]. However, the same authors recently reported that hypericin 1 in addition to protein kinase C also caused the light-dependent inhibition of certain other Ser/Thr kinases (e.g. protein kinase CK-2, mitogen-activated kinase) and the insulin receptor tyrosine kinase, while it was ineffective towards the cytosolic tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. These results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin 1 [ 156]. In contrast to the above mentioned studies, Richter and Davies [ 157] observed no inhibition of EGF-induced tyrosine phosphorylation of the EGF-R in HN5 squamous carcinoma


677

cells, but hypericin 1 caused a dose- and time-dependent reduction of EGF receptor number and affinity. In addition, hypericin 1 was found to inhibit the mitogenic effects of EGF, acidic fibroblast growth factor (aFGF) and platelet-derived growth factor (PDGF) in NR6/HER cells. Thus, although hypericin 1 inhibits EGF signaling it obviously does not act specifically on the EGF-R pathway. These conflicting results may reflect differences in the assay systems used. While previous investigations were performed with membrane preparations, Richter and Davies exposed intact cells to hypericin 1, which may have limited access of the compound to the intracellularly located active site of the EGF-R tyrosine kinase. Studies on EMT6 mouse mammary carcinoma cells demonstrated that hypericin 1 is a highly effective inhibitor of cell growth in a concentration range of 1 - 50 lxM. The effect appeared to completely depend on illumination and oxygen suggesting a type II photosensitizing reaction. Interestingly, cellular uptake of hypericin 1 occurred under both hypoxic and aerobic conditions [158]. A 1000-fold photopotentiation has been observed with a normal rat epithelial cell line (FRTL-5) and neoplastic rat MPTK-6 cells using hypericin 1 as photosensitizer [159]. The considerable potential of hypericin 1 as a sensitizer for the photodynamic therapy of cancer was also supported by investigations with the human fibroblast cell line MRC5. The results obtained indicate that type I as well as type II photosensitization mechanisms are involved in the cytotoxic effect of hypericin 1 [ 160]. Analysis of the growth inhibiting effect of hypericin 1 in a number of different cell lines revealed that this compound induces apoptosis [e.g., 161, 162, 163, 164]. A dramatic difference in the sensitivity of several human and mouse cell lines towards photoactivated hypericin 1 has been noticed. The differential cytotoxic effect did not correlate with the expression of the EGF-R or the P 170 glycoprotein in the cells, but phototoxicity was shown to depend on the cellular uptake of hypericin 1 [165]. In comparison to pigmented melanoma cells amelanotic tumor cells were found to be more sensitive to the phototoxic activity of hypericin 1, an effect which is obviously imposed by the protective action of melanin against free radical induced damages [166]. Conversely, treatment of human malignant glioma cells with hypericin 1 prior to ionizing radiation has been found to synergistically enhance cell killing, and it has been suggested that this effect is caused by a hypericin-induced depletion of intracellular stores of free radical scavengers [ 146]. Subcutaneous tumors that developed after implantation of human mammary carcinoma cells (MX-1) in athymic mice regressed following local injection of hypericin 1 and exposure to visible light [ 167]. Tissue uptake and distribution of hypericin 1 was measured in rabbits and in nude mice xenografted with P3 human squamous cell carcinoma to assess the value of this naphthodianthrone as in vivo sensitizer for laser photoactivation of solid tumors. Maximum Hypericum levels were seen in

678

ERDELMEIER et al.

both species 4 h after intravenous injection with the following rank order of concentrations: lung > spleen > liver > blood > kidney > heart > gut > tumor > stomach > skin > muscle > brain. Elimination of hypericin 1 was rapid in most murine organs with residual levels below 10 % of maximal concentrations by 7 days compared with a retention of 25 - 30 % in the tumor tissue and several organs. These results suggest that hypericin 1 may be a useful photosensitizer for laser interstitial therapy of human cancer [ 168]. The in vivo antitumor activity of hypericin 1 was evaluated in athymic nude mice xenografted with A431 cells. The substance was intraperitoneally administered at different doses and the tumors were locally irradiated 2 h later with white light (180 J/cm 2) using a cold light source. If treatment was started one day after tumor inoculation, a dosedependent antitumor effect was observed. Complete inhibition of the tumor growth was achieved with 2.5 mg/kg hypericin 1. When the efficacy of a single hypericin I dose (5 mg/kg) followed by a single light treatment was investigated on established tumors (60 mm3), an 80% reduction of tumor mass was seen. Furthermore, an accumulation of hypericin 1 in A431 xenogratts was observed alter local light irradiation [ 169]. Recently, the first experimental clinical investigation on the local use of hypericin 1 as photosensitizer for photodynamic therapy in a patient with recurrent malignant mesothelioma has been reported [170]. A phase I clinical trail utilizing orally administrated hypericin 1 in patients harboring recurrent malignant gliomas is ongoing [146]. A review on the use of hypericin 1 in adjuvant brain tumor therapy has lately been published [146]. Other Activities

Various preparations from St. John's Wort have been used in the management of wounds and injuries at least since the times of Dioscorides (lst century A.D.) and Plinius (23 - 79 A.D.) [4, 171]. Oleum Hyperici, a still utilized crude product prepared by infusing fresh blooms from St. John's Wort in vegetable oil and subsequent maceration for several weeks, is applied externally to dispel traumas, bruises, bums, scalds, ulcers, sores, swellings and myalgies [46]. Used internally Oil of St. John's Wort is recommended for the treatment of dyspeptic conditions [3]. It has been suggested that the therapeutic action of Oleum Hyperici is due to its content of the phloroglucinol derivative hyperforin 10 [172], which possesses antibacterial activity (see above). Unfortunately, hyperforin 10 and its homologues show low stability in the oil, possibly explaining the failure of Neuwald and Hagenstrrm [ 149] to demonstrate an antibacterial effects of Oil of St. John's Wort. Hypericin 1, for which antiinflammatory activities have been described (see below), could not be identified in Oleum Hyperici [46]. Whether lipophilic breakdown products of hypericin 1 and hyperforin 10 are involved in the clinical effects of the oil has apparently


679

not been investigated. It has been speculated that essential oils may be responsible for the wound healing potential of Hypericum extracts [ 1]. Hypericum extracts are extensively used in industry for the manufacturing of cosmetics and dermatological products, such as sun creams, antiphlogistie ointments or shampoos [ 173]. Investigations on the anti-irritant potential of several substances commonly employed in cosmetic formulations unraveled a protective activity of an oily Hypericum extract against croton oil induced skin irritation in the rabbit [174]. A phase II clinical study has now been initiated to evaluate synthetic hypericin 1 as a topically applied, light-activated therapy for specific skin diseases including psoriasis, cutaneous T-cell lymphoma, warts, and Kaposi's sarcoma [175]. Despite the extensive traditional and current use of Hypericum in skin care and skin disorders targeted pharmacological studies related to these applications have rarely been performed. In a preliminary experimental study in human volunteers, equal quantities of hydroglycolic extracts from

Calendula officinalis, Matricaria chamomilla, Anthemis nobilis, Tilia chordata, Centaurea cyanus and Hypericum perforatum were added at a 5 % level into the aqueous phase of 0.5 % hydrocortisone cream. When this preparation was applied to artificially induced skin abrasions, it was found that the plant extracts containing cream accelerated the healing time on an average of 16 % (3.4 days) versus the control I173). However, from this study no conclusions on the active ingredients and the mode of action can be drawn. Accelerated wound healing and reduced inflammation after topical application of Hypericum could possibly depend on the inhibition of protein kinase C by hypericin 1 [ 142]. In this context, it has been reported that hypericin 1 suppresses O2 generation and respiratory burst of neutrophils after stimulation via both protein kinase C-dependent as well as -independent pathways. In addition, NADPH oxidase activity and tumor necrosis factor t~-induced tyrosyl phosphorylation of neutrophil proteins were inhibited by hypericin 1 in a light- and concentrationdependent manner. For the light-dependent inhibition oxygen was required. Thus, the results suggest that the light-dependent suppression of O2 generation by hypericin 1 is caused by inhibition of tyrosine kinase, protein kinase C, and NADPH oxidase through an oxygen-dependent mechanism, possibly involving both type I and II photosensitization mechanisms [ 176]. In accordance with these observations, it has been shown that hypericin 1 reduces the release of arachidonic acid and the production of leukotriene (LT) B4 by human neutrophils. An even stronger effect was observed for a total extract of Hypericum perforatum. Hypericin 1 also inhibited the production of interleukin l t~ in lipopolysaceharide-stimulated and non-stimulated human monocytes. Interestingly, a similar effect was observed for a Hypericum extract made free of hypericin I. This finding and the observed effect of the crude

680

ERDELMEIER et al.

extract on LTB4 synthesis might be explained by the presence of other active compounds in the extract, for example flavonoids, which are known to inhibit 5-1ipoxygenase. While hypericin 1 had no effect on NO production in LPS-stimulated monocytes, it increased NO syntheses in non-stimulated cells, an effect which is probably caused by a nonenzymatic, light-induced action of the compound [ 177]. Analgetic activity in mice has been described for a total flavonoid extract produced from the shoots of H. perforatum [ 178]. It appears that the active flavonoids belong to the quercetin group. In vivo the effects of water extracts from St. John's Wort, mainly containing carbohydrates and polyphenols, were investigated on the graftversus-host reaction and the production of anti-sheep red blood cell antibodies in mice. The extracts displayed a variable pattern of immunomodulating activity, whose intensity and direction largely depended on the mouse strain used, on the quantity of antigen applied and on the dose of extract administered [179]. Similarly, in another study immunostimulating as well as immunosuppressive effects were observed. Thus, a polyphenol fraction from Hypericum perforatum enhanced the activity of mononuclear phagocytes and improved cellular and humoral immunity, while a lipophilic fraction was found to elicit an immunosuppressive action [ 180]. A growing number of basic and clinical studies indicate a linkage between the nervous and the immune system and particular attention has been paid to the possible role immunological dysregulations may play on neurological disorders [181]. Therefore, the above described antiinflammatory and immunomodulatory effects of Itypericum may also be relevant for its antidepressant activity. In a preliminary study the effect of a methanolic extract on the cytokine synthesis in diluted blood samples from five healthy volunteers and four depressive patients was examined after stimulation of cells by phytohemagg|utinin and lipopo|ysaccharide. At a rather high concentration of the extract (10 mg/ml) the release of interleukin-6 was strongly suppressed, whereas a weaker reduction of synthesis of interleukin-l~ and tumor necrosis factor-t~ was observed [182]. Investigations on rabbit ileum preparations revealed a spasmolytic activity of Hypericum constituents. The strongest effect was observed for a flavonoid-rich ethyl acetate fraction, which possessed about 0.7 % of the potency of papaverine hydrochloride [ 1]. A relaxing activity on porcine isolated coronary arteries after contraction with histamine, prostaglandin F2ct and KCl-depolarization has also been reported for procyanidin containing fractions from St. John's Wort. Vasoactive properties appeared to positively correlate with the molecular mass of the procyanidins. An inhibition of phosphodiesterase and/or antagonism of angiotensine converting enzyme has been suggested by the authors as possible mode of action [ 183].


681

Investigations in our laboratories have demonstrated that the phloroglucinol hyperforin 10 dose-dependently inhibits contractions of guinea pig ileum preparations induced by several neurotransmitters and also to desensitize this organ against the action of acetylcholine. Further studies indicated that hyperforin 10 most potently antagonizes 5-HT3 receptor mediated responses both in vitro and in vivo. Thus, the IC50 value of the compound for inhibiting 5-HT3 receptor induced contractions of the guinea pig ileum in vitro was 180 nM and amounted to 10 mg/kg after oral administration. Oral treatment of rats with a hyperforin-rich carbon dioxide extract reduced serotonin induced bradycardias in anesthetized rats, a reaction which is also supposed to be mediated via 5-HT3 receptors [ 113, 114]. As Itypericum is traditionally used for the treatment of gastrointestinal as well as hepatic and biliary disorders [184], there is strong evidence that this therapeutic effect may be due to this action of hyperforin 10. In relation to these clinical indications, it is worth mentioning that hyperforin 10 interacts with ligand binding to cholecystokinin (CCK) A and B receptors [114]. The choleretic and hepatoprotective activity of an alcoholic extract from St. John's Wort has also been confirmed experimentally. Pretreatment with Hypericum extract increased bile flow in cannulated rats (500 mg/kg intraduodenally) and significantly reduced barbiturate sleeping time in mice (500 mg/kg i.p.) following CC14-induced hepatic injury. Hepatic excretion of hypericin 1 was confirmed by the detection of the compound in the bile of treated animals [ 184]. Other pharmacological activities described for Hypericum extracts include diuretic [ 185]. antioedematous [ 186] and anthelminthic properties [187]. In yeast cells highly diluted hypericin 1 has been reported to stimulate respiratory activity [ 1]. Moreover, general roborant and tonic effects of small doses of hypericin l, reflected in an increased vitality and growth rate of farm and laboratory animals, have repeatedly been described [ 1].

Toxicological Studies A comprehensive evaluation of the toxicological profile of a methanolic extract from St. John's Wort was recently presented at the 2nd International Congress of Phytomedicine in Munich [188]. Without mentioning the animal species, the no-effect level of this preparation following single oral application has been reported to be above 5000 mg/kg. After treatment of rats and dogs for 28 days unspecific toxic symptoms (e.g., reduced body weight, changes in hematological and clinical-chemical parameter) which indicate a slight damage to liver and kidney were observed at oral doses of 900 and 2700 mg/kg. These doses correspond to about 70 and 200 times the recommended daily therapeutic dose in humans. No effect of treatment was observed at a dose of 300

682

ERDELMEIER et aL

mg/kg. Histopathological investigations revealed a mild hypertrophy of the zona glomerulosa of the adrenals. Reproductive functions (fertility, embryonic development, pre- and postnatal development) were obviously not influenced by treatment with Hypericum extract. However, it is not possible to adequately assess these toxicological studies and in particularly the investigations on reproductive toxicology as important experimental details were not provided and these evaluations are not yet published as full papers. Contradictory results of mutagenicity studies have caused a controversial discussion on the genotoxic potential of Hypericum extracts [ 189, 190, 191 ]. Thus, it has repeatedly been shown that extracts from St. John's Wort possess a mutagenic activity in the reverse mutation test with different strains of Salmonella typhimurium. As indicated by the pattern of activity (e.g., a stronger effect in strain TA98 than in TA100 and a significant increase of revertants in the presence of rat liver microsomes) there was strong evidence that the flavonol quercetin 38 is the major or sole mutagenic principle in Hypericum extracts [ 192, 193, 194]. The mutagenicity of quercetin 38 in the Ames test and in Drosophila, as well as chromosomal aberrations and sister chromatid exchanges in mammalian cells are now well established [194, 195]. Interestingly, the natural occurring 3-flavonol glycosides were found to be non-mutagenic, but could be activated by a variety of mixed glycosidases [ 196]. This activation may also take place by enzymatic hydrolysis during drying, storage, processing or ingestion of the plant material [194]. Hypericin 1, another constituent of Hypericum extracts, was found to be negative in the Salmonella mutagenicity assay [192]. Since the Ames test is a short time in vitro test in prokaryotes its significance for mammalian cells has sometimes be challenged [ 197]. Thus, the genotoxicity of an aqueous-ethanolic Hypericum extract was investigated in different in vivo and in vitro test systems with mammalian cells. All the in vitro assays (hypoxanthine guanidine phosphoribosyl transferase (HGPRT)-test in V79 cells [0.3 - 4 ~l/ml], unscheduled DNA synthesis (UDS) in primary rat hepatocytes [0.014 - 1.37 ktl/ml] and cell transformation test with Syrian hamster embryo cells [0.75 - 10 ~tl/ml]) as well as the in vivo tests (mouse fur spot test [1 - 10 ml/kg p.o.] and chromosome aberration test with Chinese hamster bone marrow cells [ 10 ml/kg p.o.]) were found to be negative [ 197]. In contrast, a weak but dosedependent increase in unscheduled DNA synthesis in rat hepatocytes has been reported for an ethanolic extract by another group [ 192]. Carcinogenicity studies with Hypericum extracts have not yet been reported. However, a number of such studies have been performed with quercetin 38 which is contained in these preparations and has been shown to be positive in a variety of genotoxicity tests as mentioned above. Although many studies in rats and mice did not show evidence of


683

carcinogenicity [e.g., 198, 199, 200, 201], other reports indicated an increased incidence of tumors [ 195, 202]. At present the health risk of quercetin 38 for humans can not be estimated with confidence. The total amount of quercetin 38 ingested with the different therapeutically used Hypericum extracts is typically around 3 mg. Compared with the daily food intake, which is calculated to be about 50 mg [203, 204], the medical use of these preparations appears to represent a minimal additional hazard to human health. Moreover, there is even evidence that quercetin 38 possesses antimutagenic and anticarcinogenic activity [ 193,205]. The genotoxicity of St. John's Wort oil has been investigated in the Ames test. Similarly to alcoholic extracts, the preparation was found to dose-dependently increase the number of revertants and this effect was markedly enhanced in the presence of a metabolic activation system [ 192]. The ingestion of St. John's wort by grazing animals has long been known to be a,;sociated with the development of photosensitization [206]. The animals develop skin erythema, edema, and blisters particularly at the white areas of skin which become subsequently dry and necrotic. In addition, psyc,homotoric excitement (e.g., restlessness, scratching of affected parts) is frequently observed. This condition is commonly known as hypericism [207]. Horsely [208] was able to reproduce this condition in animals by the oral administration of the red pigment extracted from leaves. St. John's Wort poisoning has subsequently been classified as a primary photosensitivity on the basis that all the pathological changes are the result of the photodynamic action of the absorption of hypericin 1 from the ingested plant, without interfering with the function of any other organ than the skin [206]. As mentioned before, under anaerobic conditions hypericin acts as a type I photosensitizer forming semiquinone and superoxide radicals. In the presence of oxygen the photodynamic action of naphthodianthrones is caused by the production of singlet oxygen (type II mechanism) [ 134, 209, 210]. These highly reactive oxygen species are able to interact with lipids, proteins or carbon hydrates resulting in damage to nucleic acids, inactivation of enzymes or membrane dysfunction [211]. As a very lipophilic molecule, hypericin 1 has also been shown to associate with biological membranes [210]. When isolated hypericin 1 was administrated orally to rats tbllowed by exposing the animals to sunlight, it was found that 1-2 mg/rat resulted in death of the animals within 1 - 2 h. Mice treated with 0.25 - 0.5 mg of hypericin 1 and exposed to a 2,000 W lamp for 30 min died within 24 h. In contrast, mice injected with 3 - 4 mg and kept in the dark survived [212]. In quantitative studies in calves, toxic symptoms were observed after feeding dried aerial parts of St. John's Wort at doses of 3 g/kg and above by stomach tube. Chemical analysis revealed that this dose contained about 370 ~tg/kg hypericin 1 [206]. Again, toxic effects were only observed after exposure of animals to light. Chronic hypericin

684

ERDELMEIER et aL

ingestion in farm animals causes weight loss, failure to gain weight, reduced milk yield and wool production as well as diminished reproductive performance [213]. Sheep given different dosages and frequencies of Hypericumperforatum had decreased hemoglobin, red blood cell counts, packed cell volume, total protein, glucose, cholesterol, triglycerides, and serum alkaline phosphatase activities. Blood urea nitrogen, sodium, potassium, bilirubin and the activities of lactate dehydrogenase as well as various aminotransferases were increased [214]. The photosensitizing activity of hypericin 1 is sometimes referred to as an immunotoxic response [e.g., 3]. However, there is no evidence that immunological mechanisms are involved in this adverse reaction. The first case of a phototoxic skin reaction in a female elderly patient after taking the recommended daily dose of Hypericum extract for 3 years has recently been described by Golsch and colleagues [215]. The patient presented itching and edematous erythemas at skin regions exposed to light. UV-B sensitivity was found to be increased (expressed as a decreased minimal erythema dose) while no allergic reaction to Hypericum could be provoked in a prick test. This case, however, was quite unusual in that the phototoxic reaction was provoked by UV-B light, while hypericin 1 is known to absorb UV-A wave-lengths, and the morphology of the skin reaction was rather atypical. No other case of a phototoxic skin reaction on antidepressant doses of Hypericum extracts has been reported in the literature so far. Several investigations were then initiated in order to elucidate the relationship between Hypericumextract, sunlight and the skin. S iegers and coworkers [216] incubated cultured human keratocytes with photosensitizing substances (psoralene, chlorpromazine, hypericin) and three Hypericum extracts. After 24 hours of incubation, the cells were irradiated with UV-A light of defined energy and the substance concentrations lethal for 50 % of cells (LD50) were determined. The lowest L D50 values were found with 5-methoxypsoralene and hypericin 1, whereas Hypericum extracts had only a weak effect on UV-A sensitivity. The lowest LD50 found with a Hypericum extract after 700 mJ/cm 2 UV-A irradiation was more than 1000fold the steady state maximum plasma concentration (Cmax) after intake of 3 x 600 mg/day, which is twice the recommended dose, of a standardized methanolic extract. Based on animal toxicity studies [206], the researchers came to the conclusion that the minimum phototoxic dose is 30 to 50 times the amount of hypericin 1 ingested with the recommended daily dose of Hypericum extracts in affective disorders [206]. Brockm611er and colleagues [217] included 13 healthy volunteers in a fourfold cross-over trial. Single doses of 900 mg, 1800 mg and 3600 mg of the methanolic Hypericumextract LI 160 and placebo were administered to the subjects in a randomized sequence. Before and 4 hours after drug intake, when the plasma concentrations of hypericin 1 and


685

pseudohypericin 2 were at maximum, small areas on the back were irradiated with increasing doses of solar simulated irradiation (SSI) which consisted of ultraviolet UV-A and UV-B light, while separate areas were irradiated with increasing doses of UV-A light. The minimal erythema dose (MED) of SSI and the minimal tanning dose of UV-A were determined 20 hours after irradiation. The minimal erythema dose was not altered at any dose of Hypericum extract suggesting that there is no pathological photosensitivity of single doses. The mean minimal tanning dose was slightly reduced to 7.6 J/cm 2 after 3600 mg of Hypericum extract while it was 9.2 J/cm 2 after placebo. A further 50 volunteers took 1800 mg/day of Hypericum extract LI 160 for 15 days. Sensitivity to SSI and UV-A light was measured before the first and 4 hours after the last drug intake. A marginal decrease in mean MED from 0.17 to 0.16 and a slight drop in mean MTD were observed. There was, however, no correlation between hypericin 1 and pseudohypericin 2 on the one hand and MED or MTD on the other hand. In conclusion, even under steady state treatment with twice the dose recommended for depressive disorders, only a minimal increase in daylight sensitivity was found along with a mildly enhanced tanning reaction. This ties in with the results of in vitro and animal studies and the fact that only one case of phototoxicity in a patient taking the antidepressive dose has been documented thus far. And even this case is of questionable relevance because of atypical features. Pharmacokinetic Studies

Due to their complex composition the pharmacokinetic assessment of herbal medications generally imposes serious technical and regulatory problems. As the active principle(s) of plant extracts are otten not known it is difficult to decide which constituent(s) should actually be studied [218]. In the absence of a well defined therapeutically relevant chemical entity, characteristic constituents of herbal preparations are frequently employed for the purpose of standardization. Correspondingly, pharmacokinetic evaluations of Hypericum extracts have almost exclusively been based on the analysis of the naphthodianthrones hypericin 1 and pseudohypericin 2 which represent typical products of members of the genus Hypericum and are considered to be involved in some of their clinical effects. Pharmacokinetic evaluations of hypericin 1 and pseudohypericin 2 in experimental animals are restricted to studies in mice. Following intravenous injection of 17.5 mg/kg of synthetically prepared hypericin peak concentrations of 27.8 ILtg/mlwere measured at 10 min and decreasing values could be followed for a period of 240 h (10 ng/ml). The data were well adjusted to a two-compartment model with a distribution phase (tl/2tx) of 2 h and an elimination half life (tl/2~) of 38.5 h. The volume of

686

ERDELMEIER et aL

distribution amounted to 12.6 ml [219]. In a second study in mice, hypericin 1 and pseudohypericin 2 isolated from plants after labeling in vivo with 14C were administered orally to 8 female animals. Distribution of radioactivity in various organs and blood was followed for up to 6 h. Blood levels of both substances were maximal after 6 h. Significant amounts of radioactivity could also be detected in liver, kidney, brain and especially in muscle. The same investigators determined the concentration of hypericin 1 in serum of a single test person following oral application of an aqueous-ethanolic Hypericum extract (containing 1.0 mg hypericin). Hypericum could first be detected after 3.5 h (0.45 ng/ml) and continuously raised, with the exception of a small decrease after 6 h, for the whole of the observation period of 8 h (4.21 ng/ml) [220]. A preliminary investigation in human volunteers [221] has recently been extended by the same authors [222]. Single-dose pharmacokinetics of hypericin 1 and pseudohypericin 2 were studied in 12 male subjects after oral administration of 300, 900 and 1800 mg of a methanolic extract from aerial parts of St. John's Wort containing 0.083 % hypericin 1 and 0.175% pseudohypericin 2. Characteristic pharmacokinetic parameters for the medium dose are summarized in Table 3. Although hypericin 1 and pseudohypericin 2 are closely related chemically and were released from the same galenical preparation they displayed a quite distinct pharmacokinetic behavior. Most remarkable was a lag time of almost 2 h before hypericin 1 could be measured in plasma, whereas pseudohypericin 2 appeared much earlier in the systemic circulation (0.4 h). Disproportionally lower values for Cmax and AUC at the lower dose for both naphthodianthrones might be caused by an incomplete saturation of various binding sites. Two week treatment with 300 mg extract three times a day resulted in a median steady-state trough level of 7.9 ng/rnl hypericin 1 and 4.8 ng/ml pseudohypericin 2, respectively. The corresponding peak plasma concentrations were 8.8 and 8.5 ng/ml for hypericin 1 and pseudohypericin 2, respectively. Following intravenous injection of 3.6 ml Pharmacokinetic Parameters of Hypericin 1 and Pseudohypericin 2 After Oral Administration of 900 mg of a Methanolic Hyperic..um extract to Human Volunteers

Table 3.

ii

,,

,

,,%,,

,,,

,

,

,,,,,,,,,

,

9

L

i

,,,-"

i

Lag time of absorption (tlag) (h)

1.9

Maximum plasma concentration (Cmax) (ng/ml)

7.2

12.1

Time at Cmax (tmax) (h)

6.0

3.0

Area under the curve (AUCo_oo) .(h.pg.l-I)

198

140

Elimination half life (tl/2f5) (h)

43.1

24.8

,

_ _

Data adapted from [222]

,

,,

...

ii

Pseudohypericin 2

Hypericin 1

Parameter

0.4 , - - ,

i

STUDIES ON H Y P E R I C U M P E R F O R A T U M - ST. J O H N ' S W O R T

687

of a diluted extract (containing a total of 115 I.tg hypericin and 38 I.tg pseudohypericin) in two volunteers, peak concentrations of about 27 ng/ml hypericin 1 and 6.7 ng/ml pseudohypericin 2 were measured. Hence, both compounds were initially distributed into a volume of approximately 4 to 5 liters which roughly correlates with the blood volume. Kinetic parameters ai'ter intravenous administration corresponded to those estimated after oral application. The systemic bioavailability of hypericin 1 and pseudohypericin 2 was calculated to be about 14 and 2 1 % , respectively. Neither of both naphthodianthrones could be detected in free or conjugated form in the urine. The results of the above cited study by Kerb et al. [222] are at variance for a number of pharmacokinetic parameters with an earlier evaluation in which the same extract was given to two human volunteers [223]. Although plasma levels of hypericin 1 were similar in both trials, in the previous investigation maximal concentrations were already observed after about 2.5 h, the concentration response was linear in the range from 200 to 1200 mg and the elimination half life was only 6 h. Since hypericin 1 has been demonstrated to nonspecifically bind with high affinity to proteins, detergents, and lipids [224], food intake may interfere with its absorption after oral treatment. However, as the volunteers in both studies fasted overnight before drug application in the morning this aspect seems not to account for the observed differences. Pharmacokinetic experiments conducted in rats and man demonstrate that hyperforin 10 is absorbed after oral application [225]. After administration of 300 mg/kg of an ethanolic Itypericum extract containing 4.93% hyperforin l0 to rats, maximum plasma concentration was found to be 370 ng/ml reached after 3 h. Elimination half-life was 3 h, clearance (Cl) 70 ml/min/kg. Table 4.

Pharmaeokinetic Parameters of Hyperforin 10 After Administration of 300, 600 and 1200 mg Hypericum Extract (Containing 4.93~ Hyperforin 10) to Human Volunteers. (Mean + sem, n=6, * p21 improved significantly more in HAMD and CGI than those with HAMD

Studies in Natural Product Chemistry, Volume 22: Bioactive Natural Products, Part C

Studies in Natural Products Chemistry: Bioactive Natural Products Part L

Studies in Natural Product Chemistry, Volume 27: Bioactive Natural Products, Part H (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry, Volume 29: Bioactive Natural Products (Part J) (Studies in Natural Product Chemistry Series)

Studies in Natural Products Chemistry, Bioactive Natural Products

Studies in Natural Product Chemistry, Volume 25: Bioactive Natural Products, Part F

Studies in Natural Product Chemistry, Volume 18: Stereoselective Synthesis, Part K (Studies in Natural Products Chemistry)

Bioactive Natural Products (Part I)

Bioactive Natural Products (Part G), Volume 26 (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry, Volume 33: Bioactive Natural Products (Part M)

Studies in Natural Product Chemistry, Volume 15: Structure and Chemistry, Part C (Studies in Natural Products Chemistry)

Bioactive Marine Natural Products

Studies in Natural Products Chemistry, Volume 35

Bioactive Natural Products

Studies in Natural Products Chemistry, Volume 31: Indices Part A

Medicinal chemistry of bioactive natural products

Medicinal Chemistry of Bioactive Natural Products

Natural Product Chemistry at glance

Studies in Natural Product Chemistry, Volume 20: Structure and Chemistry, Part F, (Including Cumulative Index Volumes 1-20) (Studies in Natural Products Chemistry)

Comprehensive Bioactive Natural Products, Volume 8: Quality Control & Standardization

Comprehensive Bioactive Natural Products, Volume 4: Antioxidants & Naturaceuticals

Comprehensive Bioactive Natural Products, Volume 6: Extraction, Isolation & Characterization

Comprehensive Bioactive Natural Products, Volume 1: Potential & Challenges

Progress in the Chemistry of Organic Natural Products, Volume 93

Progress in the Chemistry of Organic Natural Products, Volume 93

Progress in the Chemistry of Organic Natural Products, Volume 89

Progress in the Chemistry of Organic Natural Products, Volume 92

Progress in the Chemistry of Organic Natural Products, Volume 94

Cp2TiCl in Natural Product Synthesis

Natural Product Chemistry at a Glance

Saponins (Chemistry and Pharmacology of Natural Products)

Studies in Natural Product Chemistry, Volume 22: Bioactive Natural Products, Part C

Studies in Natural Products Chemistry: Bioactive Natural Products Part L

Studies in Natural Product Chemistry, Volume 27: Bioactive Natural Products, Part H (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry, Volume 29: Bioactive Natural Products (Part J) (Studies in Natural Product Chemistry Series)

Studies in Natural Products Chemistry, Bioactive Natural Products

Studies in Natural Product Chemistry, Volume 25: Bioactive Natural Products, Part F

Studies in Natural Product Chemistry, Volume 18: Stereoselective Synthesis, Part K (Studies in Natural Products Chemistry)

Bioactive Natural Products (Part I)

Bioactive Natural Products (Part G), Volume 26 (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry, Volume 33: Bioactive Natural Products (Part M)

Studies in Natural Product Chemistry, Volume 15: Structure and Chemistry, Part C (Studies in Natural Products Chemistry)

Bioactive Marine Natural Products

Studies in Natural Products Chemistry, Volume 35

Bioactive Natural Products

Studies in Natural Products Chemistry, Volume 31: Indices Part A

Medicinal chemistry of bioactive natural products

Medicinal Chemistry of Bioactive Natural Products

Natural Product Chemistry at glance

Studies in Natural Product Chemistry, Volume 20: Structure and Chemistry, Part F, (Including Cumulative Index Volumes 1-20) (Studies in Natural Products Chemistry)

Comprehensive Bioactive Natural Products, Volume 8: Quality Control & Standardization

Comprehensive Bioactive Natural Products, Volume 4: Antioxidants & Naturaceuticals

Comprehensive Bioactive Natural Products, Volume 6: Extraction, Isolation & Characterization

Comprehensive Bioactive Natural Products, Volume 1: Potential & Challenges

Progress in the Chemistry of Organic Natural Products, Volume 93

Progress in the Chemistry of Organic Natural Products, Volume 93

Progress in the Chemistry of Organic Natural Products, Volume 89

Progress in the Chemistry of Organic Natural Products, Volume 92

Progress in the Chemistry of Organic Natural Products, Volume 94

Cp2TiCl in Natural Product Synthesis

Natural Product Chemistry at a Glance

Saponins (Chemistry and Pharmacology of Natural Products)

Recommend Documents