Studies in Natural Product Chemistry, Volume 25: Bioactive Natural Products, Part F

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Studies in Natural Products Chemistry Volume 25 Bioactive Natural Products (Part F)

studies in Natural Products Chemistry edited by Atta-ur-Raliman

Vol. 1 Vol. 2 Vol. 3 Vol. 4 Vol. 5 Vol. 6 Vol. 7 Vol. 8 Vol. 9 Vol. 10 Vol. 11 Vol. 12 Vol. 13 Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. 19 Vol. 20 Vol. 21 Vol. 22 Vol. 23 Vol. 24 Vol. 25

Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidaton (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bloactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bloactive Natural Products (Part B) Bloactive Natural Products (Part C) Bloactive Natural Products (Part D) Bloactive Natural Products (Part E) Bloactive Natural Products (Part F)

Studies in Natural Products Chemistry Volume 25 Bioactive Natural Products (Part F)

Edited by

Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

2001 ELSEVIER Amsterdam - London - New York - Oxford - Paris - Shannon - Tokyo

ELSEVIER SCIENCE Ltd The Boulevard, Langford Lane Kidlington, Oxford OX5 1GB, UK

© 2001 Elsevier Science Ltd. 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 IDX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also contact Global Rights directly through Elscvier's home page (http://www.elsevier.com), 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: (+1) (978) 7508400, fax: (+1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (+44) 207 631 5555; fax: (+44) 207 631 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 2001 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for.

ISBN:

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FOREWORD The present volume of Studies in Natural Product Chemistry covers many important classes of bioactive natural products. The article on bloactive indole alkaloids by Reija Jokela et. al., describes the acid-catalysed epimerization of these compounds and resulting pharmacological activities. Toshihiro Akihisa & Ken Yasukawa present the antitumor-promoting and anti-inflammatory activities of triterpenoids and sterols from plants and fungi, many of which are of medicinally importance. A number of articles on bioactive terpenes Include oleanene glucuronides (James Kinjo et. al.,) biotransformation of terpenoids by microorganisms (Jan C.R. Demyttenaere), cycloartane and oleanane saponins (Luisella Yerotta et, al.,) and labdane-type diterpenes (Cost as Demetzoo et. al.,) as well as metabolism of the tomato saponin alpha-tomatine by phytopathogenic fungi (Manuel Ruiz-Rubio et. al.,). An interesting article has been written by James Zlegler, et. al.,) on heme aggregation inhibitors. Intercellular communication in higher plants continues to be of interest and in addition to the known phytoharmones i.e. auxins, cytokinins, gibberellins, abscisic acid and ethylene, certain bioactive peptides have been identified in signal transductions involved In plant defence, growth and development which is discussed by Andreas Schaller. The articles on the biosynthesis of brassinosteroids by Jochen Winter, immunopotentlating effects of a glycoprotein from Chlorella vulgaris by Kuniaki Tanaka et. al. and hepatoprotective plants components by Shiegeoshi Kadota et. al., should also be of considerable interest. The induction and regulation of biosynthetic activity of phytoalexins in carrot cells is reviewed by Fumiya Kurosaki. The plant inhibition of eukaryote signal transduction induced by secondary metabolites is another important area which is comprehensively described by Gideon M. Polya. Two articles written by Juan Duarte et. al., & J. Galvez et. al., cover the important effects offlavonoidson cardiovascular and gastrointestinal disorders respectively, while the bioactivity of phenolic componds in higher plants is described by Juan M. Ruiz et. al. Marine substances continue to be important sources of bioactive natural products. Recent advances in marine natural products are covered by M.J. Abad et. al., in detail. Biologically active halogen compounds described by Gerhard Laus and marine sulfurcontaining natural products reviewed by Carolos Jimenez and biological activities chlorogenic acids discussed by Motoyo Ohnishi et. al., should also be of interest to the readers. Ethanolic extracts of Crocus sativus and Its components in learning behaviour and long term potentiation of memory processes is another recent area of interest which has been reviewed by Y. Shoyama et. al.,). It is hoped that Volume-25 of "Studies in Natural Products Chemistry" would prove to be of considerable interest to the readers and represent important another additions this growing series books natural product chemistry. I would like to express my thanks to Dr. Shakeel Ahmad for his assistance in the preparation of the index. I am also grateful to Mr. Muhammad Asif and Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.

Atta-ur-Rahman

Ph.D. (Cantab), SaD. (Cantab)

April, 2001

This Page Intentionally Left Blank

PREFACE When Professor Atta-ur-Rahman informed me, many years ago, of his desire to launch a new series which would become 'Studies in Natural Products Chemistry', I enthusiastically encouraged him to do so but pointed out what a difficult task it would be. First, the expert authors able to write the very numerous chapters of such a work would have to be found. Then, a logical sequence to the series would have to be determined. Finally, because a large number of books, reviews etc. already exist which dealt with particular areas of the chemistry, biology and therapeutic applications of natural products (in particular, Sir Derek Barton's posthumous 'Comprehensive Natural Products Chemistry'), something new would have to be offered. So we have now arrived at Volume 25 and I am very pleased to see that our colleague Atta-ur-Rahman has once more realised a Herculean feat. Taken together, this still uncompleted work Is colossal In breadth and, especially, extremely useful for ail scientists, not only those working in the natural products field. At a time when much (too much?) is being said about combinatorial chemistry, here we are face-to-face with part (and only a small part at that) of the products of the Creator's own combinatorial chemistry, which, it is believed, took 4.8 billion years to produce. Thus, sincere thanks are due to our colleague Atta-ur-Rahman who has since become Minister of Science and Technology in his mother country, Pakistan. And heartfelt thanks also to the authors of this Volume 25 as well as of ail preceding volumes for the considerable contribution they have made to our entire scientific community. And we now impatiently await the coming volumes. P.Potier April. 2001


ix

CONTENTS Foreword

v

Preface

vii

Contributors

xi

Acid-catalysed epimerization of bioactive indole alkaloids and their derivatives

M. BERNER, A. TOLVANEN AND R. JOKELA

3

Antitumor-promoting and anti-inflammatory activities of triterpenoids and sterols fi'om plants and fungi T. AKIHISA AND K. YASUKAWA

43

Bioactive oleanene glucuronides obtained from fabaceous plants J. KINJO AND T. NOHARA

89

Biotransformation of terpenoids by microorganisms JAN C.R. DEMYTTENAERE

125

Cycloartane and oleanane saponins from Astragalus sp. L. VEROTTA AND N.A. EL-SEBAKHY

179

Labdane-type diterpenes: Chemistry and biological activity C. DEMETZOS AND K.S. DIMAS

235

Metabolism of the tomato saponin a-tomatine by phytopathogenic fungi M. RUIZ-RUBIO, A. PEREZ-ESPINOSA, K. LAIRINl, T. ROLDAN-ARJONA, A. DIPIETRO AND N. ANAYA

293

Heme aggregation inhibitors: Antimalarial drugs targeting an essential biomineralization process J. ZIEGLER, R. LINCK AND D.W. WRIGHT

327

Bioactive peptides as signal molecules in plant defense, growth and development A. SCHALLER

367

Enzymes involved in the biosynthesis of brassinosteroids J. WINTER

413

Immunopotentiating effects of a glycoprotein from Chlorella vulgaris strain CK and its characteristics K. TANAKA, Y. SHOYAMA, A. YAMADA, K. NODA, F. KONISHI AND K. NOMOTO

429

Hepatoprotective effect of plant components: Inhibition of tumor necrosis Factor-ot-dependent inflammatory liver injury K. HASH, Q. XIONG AND S. KADOTA

459

Induction and regulation of biosynthetic activity of phytoalexin in carrot cells F. KUROSAKl

483

Inhibition of Eukaryote Signal Transduction Components by Plant Defensive Secondary Metabolites G.M. POLYA

513

Flavonoids and cardiovascular diseases J. DUARTE, F. PEREZ-VIZCAINO, J. JIMENEZ, J. TAMARGO AND A. ZARZUELO

565

Effects of flavonoids on gastrointestinal disorders J. G A L V E Z , F. S A N C H E Z D E MEDINA, J. JIMENEZ AND A. ZARZUELO

607

Bioactivity of the phenolic compounds in higher plants J.M. RUIZ AND L. ROMERO

651

Bioactive natural products from marine sources M.J. ABAD AND P. BERMEJO

683

Biological activities of natural halogen compounds G. LAUS

757

Marine sulfur-containing natural products C.JIMENEZ

811

Absorption, metabolism and biological activities of chlorogenic acids and related compounds H. MORISHITA AND M. OHNISHI

919

Effects of ethanol extract of Crocus sativus L. and its components on learning behavior and long-term potentiation H. SAITO, M. SUGIURA, K. ABE, H. TANAKA, S. MORIMOTO, F. TAURA AND Y. SHOYAMA

955

Subject Index

971

CONTRIBUTORS MJ. Abad

Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

K.Abe

Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033,Japan

Toshihiro Akihisa

College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan

Nuria Anaya

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

P. Bermejo

Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

Mathias Berner

Laboratory of Organic Chemistry, Helsinki University of Technoloy, Espoo, Finland

Costas Demetzos

School of Pharmacy, Department of Pharmacognosy, Panepistimiopolis Zografou 15771, University of Athens, Athens, Greece

Jan C.R. Demyttenaere

Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

Konstantinos S. Dimas

International Institute of Anticancer Res., Kapandriti, 19014, Attiki, Greece

Antonio Dipietro


Juan Duarte

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

Nadia A. El-Sebakhy

Faculty of Pharamcy, Alexandria, Egypt

J. Galvez


Koji Hase

Institute for Consumer Healthcare, Yamanouchi Pharmaceutical Co. Let., Tokyo 174-8612, Japan

University

of

Alexandria,

Carlos Jimenez

Departamento de Quimica Fundamental, Universidade de A Corunia, 15071 A Coruiia, Spain

Jose Jimenez


Reija Jokela


Shigetoshi Kadota

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

Junei Kinjo

Faculty of Pharmaceutical Sciences, University, Kumamoto 862-0973, Japan

Fumiko Konishi

Research Laboratories, Chlorella Industry Co. Ltd., 1343 Hisatomi, Chikugo City, Fukuoka 833-0056, Japan

Fumiya Kurosaki

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan

Khalid Lairini


Gerhard Laus

Immodal Pharmaka GmbH, Bundestrasse 44, A-6111 Volders, Austria

Rachel Linck

Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282-1530, USA

F. Sanchez de Medina


S. Morimoto

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Hideko Morishita

Faculty of Education, Wakayama University, Sakaedani Wakayama 640-8510, Japan

Kiyoshi Noda


Toshihiro Nohara

Faculty of Pharmaceutical Sciences, University, Kumamoto 862-0973, Japan

Kumamoto

930

Kumamoto

Kikuo Nomoto

Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0054, Japan

Motoyo Ohnishi

Department of Pharmaceutical Science, Institute of Medical Science, Kansai Shinkyu Medical College, 1-11-2 Wakaba Kumatori Sennan Osaka 590-0482, Japan

Alonso Perez-Espinosa

Departamento de Genetica, Facuhad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

Francisco PerezVizcaino

University Complutense of Madrid, Department Pharmacology, School of Medicine, Madrid, Spain

Gideon M. Polya

Department of Biochemistry, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia

Teresa Roldan-Arjona


Luis Romero

Departamento de Biologia Vegetal, Facultad de Ciencias, Universidad de Granada, 18071-Granada, Espafia

Juan M. Ruiz

Departamento de Biologia Vegetal, Facuhad de Ciencias, Universidad de Granada, 18071-Granada, Espafia

Manuel Ruiz-Rubio


H. Saito


Andreas Schaller

Institute of Plant Sciences, Federal Institute of Technology Zurich, CH-8092 Zurich, Switzerland

Y. Shoyama


Yikihiro Shoyama

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0054, Japan

M. Sugiura


of

Juan Tamargo

University Complutense of Madrid, Department Pharmacology, School of Medicine, Madrid, Spain

H. Tanaka


Kuniaki Tanaka


F. Taura


Arto Tolvanen


Luisella Verotta

Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via Venezian 21, 20133 Milano, Italy

Jochen Winter

Max-Planck-Institut fur Zuchtungsforschung, Carl-vonLinne-Weg 10, D-50829, Koln, Germany

David W. Wright


Quanbo Xiong

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

Akira Yamada

Kurume University Research Center for Innovative Cancer Therapy, and Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume City 830-0011, Japan

Ken Yasukawa

College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan

Antonio Zarzuelo


James Ziegler


of

Bioactive Natural Products


Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 25 © 2001 Elsevier Science B.V. All rights reserved.

ACID-CATALYSED EPIMERIZATION OF BIOACTIVE INDOLE ALKALOIDS AND THEIR DERIVATIVES MATHIAS BERNER, ARTO TOLVANEN AND REIJA JOKELA* Laboratory of Organic Chemistry, Helsinki University of Technology, Espoo, Finland ABSTRACT: The acid-catalysed epimerization reaction of bioactive indole alkaloids and their derivatives is reviewed. The three mechanisms, which have been proposed for the P-carboline-type indole alkaloids, are discussed. Through recent developments, evidence for all three mechanisms has been obtained, which shows the complexity of the epimerization reaction. The epimerization seems to depend on structural features and reaction conditions making it difficult to define one universal mechanism. On the other hand, the isomerization mechanism of oxindole alkaloids has been widely accepted. The acid-catalysed epimerization reaction provides a powerful tool in selectively manipulating the stereochemistry at the epimeric centre and it can also have a marked effect on the pharmacology of any epimerizable compound. Therefore, examples of this reaction in the total synthesis of indole alkaloids are given and pharmacological activities of some C-3 epimeric diastereomers are compared. Finally, literature examples of acid-catalysed epimerization reactions are presented.

INTRODUCTION It was suggested back in the early 1950s that yohimbine-type alkaloids epimerize at the centre attached to the 2-position of indole [1], which corresponds to C-3 in the biogenetic numbering [2]. Interest in the acidcatalysed epimerization reaction was aroused during the structure elucidation of the pharmacologically important indole alkaloid reserpine (1) in the 1950s [3]. The equilibration of reserpine (1) and its derivatives in acid provided important stereochemical information on the reserpine skeleton. The epimerization of reserpine (1) resulted in the more stable C3 epimer, isoreserpine (2). When 1 was, for example, refluxed in acetic acid (AcOH) for 3 days, isoreserpine (2) was obtained in 60% yield [4], Scheme (1). Due to inversion of the configuration at C-3, reserpine (1) and isoreserpine (2) differ in sterical shape, and the pharmacological properties of the two compounds are dissimilar: isoreserpine (2) is completely inactive [5]. Thus, acid-catalysed epimerization can have a marked effect on the pharmacology of any epimerizable compound.

H CH30

CH3O H' C H 3 0 0 C ^ ^ 6 \ x ^ ^ OTMB J

6CH3

CHaOOC*^ " Y " ÔTMB ^

6CH3

TMB = 3,4,5-trimethoxybenzoyl Scheme (1). Epimerization of reserpine (1).

The epimerization reaction has been surveyed earUer by three different groups [6-8]. Kametani and Ihara [6] dealt with the epimerization and racemization of isoquinohne and indole alkaloids. In a review of the Pictet-Spengler condensation, Cook and Cox [7] included a short paragraph on the epimerization of natural products (indole alkaloids), and, more recently, Lounasmaa and co-workers [8] published a review of the epimerization reaction of reserpine (1) and other indoloquinolizidines. Our purpose here is to give in-depth information on the C-3 epimerization reaction in the synthesis of bioactive indole alkaloids and their derivatives, including oxindole alkaloids and 2-substituted indoles, and serve as a guideline for predicting the epimerization behaviour of indole alkaloids in general. We also compare the pharmacological activities of some C-3 epimeric diastereomers. Articles dealing with the epimerization reaction are not easy to find. The term epimerization is often not mentioned in the abstract or title of an article and hence the discovery of a specific publication is sometimes pure coincidence. Furthermore, some authors use the term isomerization instead of epimerization, which naturally makes the search even more complicated. By definition, epimerization is the alteration of one asymmetric centre (the given compound has more than one asymmetric centre) but isomerization is the process whereby a compound is converted into an isomer [9]. Isomerization is therefore a more general term, resulting in an abundance of references and making it virtually impossible to track down all the publications of interest. We therefore apologize if we have omitted any crucial publications from this review.

EPIMERIZATION OF p-CARBOLINE-TYPE INDOLE ALKALOIDS AND THEIR DERIVATIVES Mechanistic Discussion In 1955, Wenkert and Liu [10] proposed a mechanism for the acidcatalysed C-3 equihbration of alio- and epialloyohimbane (Mechanism 3). Three years later, in 1958, two other mechanisms (Mechanisms 1 and 2) were introduced by Woodward and co-workers in connection with the first total synthesis of reserpine (1) [11]. In Mechanism 1, Scheme (2), protonation takes place at C-7 (the 6position of indole) and is then followed by enamine formation via proton abstraction at C-3. The change of configuration at C-3 is completed by enamine protonation and subsequent proton cleavage at C-7.

Scheme (2). Presentation of Mechanism 1.

Mechanism 2, Scheme (3), involves initial protonation at C-2 and the formation of an intermediate enabling cleavage of the C-2 - C-3 bond to give an iminium ion. Acid-induced recyclization of the iminium species gives rise to the epimerized product. In Mechanisms 1 and 2 the reactions are considered to proceed via an equilibrium concentration of the free base. Mechanism 3, Scheme (4), which was first proposed by Wenkert and Liu [10], starts with protonation at Nb, the most basic site and so the most likely to be protonated first. After protonation, the C-3 - Nb bond is cleaved due to participation of the indole nitrogen lone pair, giving a carbocation intermediate. Ring reclosure is assisted by the Nb lone pair to effect the inversion at C-3.


S

1 H ,

f^^N

ii

n

f-^

i»H


A feature common to all mechanisms is the necessity of the Na nitrogen lone pair and aromatic n electrons. Recently, Lounasmaa and coworkers [12] proved the vital role of the Na nitrogen lone pair in the epimerization reaction by chosing c/^-deethylebumamonine (3) as a target molecule. Compound 3 possesses a lactam moiety, which blocks the lone pair of Na. Deethylebumamonine (3) was not therefore expected to epimerize; nor did it, as was experimentally proven. When, however, the amide system was removed, c/.y-deethyldihydroebumamenine (4) epimerized readily when treated with refluxing trifluoroacetic acid (TFA) ovemight, resulting in an equilibrium ratio of 20:80 (4:5), Scheme (5). By analogy, it has been reported that Na-sulfonamido p-carbolines also failed to epimerize under acidic conditions [13].

— ^

no epimerization

20h

ii refl* 16h

Scheme (5). Epimerization experiments with compounds 3 and 4.

To test the necessity of aromatic n electrons in the epimerization reaction, indolo[2,3-a]quinoUzidines with different substituents at C-10 were prepared [14], Subjecting these compounds to epimerization conditions (TFA, 90°C, 60 min) gave the following results, Table 1. Table 1. Epimerization experiments with 10-substituted indolo[2,3-a]quinolizidines.

: l r RXJCJU^N

+ ^XXJC^N

H CHsOOC^

Starting ester 6

R H

Transicis ratio 74:26 (6:7) No epimerization

8

NO2

9

NH2

No epimerization

10

NHAc

Traces of cw-epimer

11

OH

32:68(11:12)

13

Br

Traces of c/5-epimer

CHaOOC^

Deactivating groups such as the nitro or bromo group hinder or slow down epimerization whereas activating groups such as the hydroxy group accelerate it. The amino group, originally electron donating and strongly activating, protonates immediately in acidic solution and becomes electron withdrawing and strongly deactivating; this hinders the epimerization reaction. An increase or decrease in the electron density of the aromatic A ring is therefore crucial for the epimerization reaction, thus underlining the vital role of the aromatic n electrons. Further proof was provided by MacPhillamy and co-workers [4] who in the 1950s had already demonstrated that reserpine (1) epimerizes with greater ease than deserpidine (11-demethoxyreserpine). Proton Abstraction at C-3 (Mechanism 1) In general. Mechanism 1 has not attracted much support. Lately, however, evidence in its favour has started to accumulate. Enamine formation by hydrogen abstraction in Mechanism 1 provides a basis for deuterium incorporation/abstraction experiments. In their pioneering work, Gaskell and Joule [15] prepared 3-deuteroisoreserpine and subjected it to epimerization conditions (AcOH, IIS'^C). Samples withdrawn from the reaction mixture showed that epimerization had occurred without loss of label. Only more vigorous conditions (AcOH, 140°C, 3 d) resulted in totally unlabelled reserpine (1) and isoreserpine (2). On the other hand, when 3-deuteroisodeserpidine was treated with AcOH (118°C), deuterium was lost more rapidly than the epimerization advanced. Hence, Gaskell and Joule concluded that deuterium abstraction occurs via Mechanism 1 and that reprotonation of the enamine intermediate takes place stereospecifically, leading to the original base and not to epimerization. Reports have recently been published of protonation at C-7 (a prerequisite for Mechanism 1) under strongly acidic conditions [16,17]. While investigating the protonation site of Rauwolfia alkaloids, Balon and co-workers [16] showed through ^^C NMR studies that protonation takes place at C-7 in 18M H2SO4. Experiments by Royer et al. [17] indicated that reserpine (1) and isoreserpine (2) are transformed into the corresponding 2,7-dihydro compounds (14) (55%) and (15) (87%) by NaBH3CN in TFA at room temperature. Scheme (6). This is only possible through protonation at C-7. Further evidence was provided by deuterium incorporation at C-7 when TFA-d was used.

NaBHgCN CH30

TFA, rt

CH3O

OTMB

CH3OOC

CH300C^ ^ V ^ ÔTMB 6CH3

OCH3

14 (H-2P, H-3p, H-7P) 15 (H-2a, H-3a, H-7a)

1 (H-3p) 2 (H-3a)

Scheme (6). Reduction of reserpine (1) and isoreserpine (2) with NaBH3CN.

Deuterium incorporation into reserpine (1) with a strong deuterated acid (TFA'd) was investigated by Lounasmaa et al. [18]. Treating reserpine (1) with refluxing TFA-J overnight yielded hexadeuterated reserpine (16) and isoreserpine (17) (ratio 15:85), Fig. (1). When the reaction was repeated with a shorter reaction time (5 min), only 10,12dideuterated reserpine and isoreserpine were obtained, proving that, even with a strong acid, epimerization and deuteration at the epimeric centre occur at different rates. This finding imphes, at the least, that Mechanism 1 is not primarily responsible for the epimerization reaction of reserpine (1). Interestingly, in a review of Woodward's [11] reserpine synthesis, Nicolaou and Sorensen recently suggested that the acid-catalysed epimerization reaction might occur via Mechanism 1 [19].

CH30

0CH3 0CH3

16(p.D) 17 (a-D) Fig. (1). Hexadeuterated reserpine (16) and isoreserpine (17).

0CH3

10

Other examples of deuterium incorporation at the epimeric centre have also been reported. When treated with a deuterated acid (DCl/MeOD and TFA-J, respectively), both P-carboline derivative 18 [20] and vinylogous urethane 19 [21] resulted in deuterium incorporation at C-1 and C-12b (both correspond to C-3), respectively. Fig. (2). Mechanisms similar to that of Mechanism 1 were suggested to explain the epimerization. It should be noted, however, that, as pointed out above, mere deuterium incorporation is not sufficient evidence for Mechanism 1. Were Mechanism 1 alone responsible for the epimerization, both deuterium incorporation and epimerization would have to happen at the same rate.

COOH

CHaCHzOOCr H^v; "COOCH2CH3 H

18

19

I

H

''

Fig. (2). P-Carboline derivative 18 and vinylogous urethane 19.

Another way to test the operation of Mechanism 1 is to subject C-12b alkyl substituted indolo[2,3-a]quinolizidines to epimerization conditions. Should Mechanism 1 be operative, these compounds could not epimerize. Thus, C-12b methyl substituted indoloquinolizidines 20 - 25 with different structural features were prepared by Lounasmaa and co-workers [22], Fig. (3). As well as testing Mechanism 1, they investigated the effect of different structural features on the epimerization reaction in general.

cc

][CH3

COOCH3

COOCH3

20 (a-CHs)

22 (a-CH3)

24 (a-CH3)

21 (P-CH3)

23(P-CH3)

25(p-CH3)

Fig. (3). Indolo[2,3-«]quinolizidine derivatives 20 and 21, lactams 22 and 23 and vinylogous urethanes 24 and 25.

11

The "normal" indoloquinolizidines 20 and 21 resemble compounds such as reserpine (1). Considering the results of the deuterium experiments (see above), one could expect compounds 20 and 21 to epimerize under acidic conditions. And indeed, refluxing 20 or 21 in TFA ovemight yielded an equilibrium ratio of 55:45 (21:20). Lactams 22 and 23 were also expected to epimerize, since prior results (see below) suggested that Mechanism 3 is active in the epimerization reaction of these compounds. Compounds 22 and 23 epimerized in deaired TFA resulting in an equilibrium ratio of 65:35 (23:22). Winterfeldt and co-workers [23] have reported epimerization of similar compounds under acidic conditions. Not only were vinylogous urethanes known to epimerize with great ease but deuterium was also known to be incorporated at room temperature at the epimeric centre (see above) [21]. Furthermore, Wenkert and co-workers [24,25] have suggested an epimerization mechanism for vinylogous urethanes analogous to Mechanism 1, Scheme (7). Epimerization of 24 and 25 was therefore expected to produce interesting results. When different mixtures of vinylogous urethanes 24 and 25 were treated with TFA, the ratios remained unchanged. Thus, the methyl group at C-12b had hindered epimerization, which meant that vinylogous urethanes epimerize via Mechanism 1. Systematic acidcatalysed study of compounds 20 - 25 proved the marked effect of structural features on the epimerization reaction.

C00CH3

C00CH3

C00CH3

C00CH3

Scheme (7). Epimerization mechanism for vinylogous urethanes.

C-2 - C'3 Bond Cleavage (Mechanism 2) Mechanism 2, which constitutes a retro Pictet-Spengler type process, has long been held responsible for the acid-catalysed epimerization reaction

12

of reserpine (1) [15,26]. The main reason for this is the outcome of the trapping experiments of Gaskell and Joule [15], who obtained two bond scission products 26 and 27 when reserpine (1) was refluxed in Zn/AcOH for 24 h, Fig. (4). 2,3-Secoreserpine (26) suggests Mechanism 2 but 3,4secoreserpine (27) points to Mechanism 3.

CH30

OTMB

OTMB 0CH3

26

0CH3

27

Fig. (4). 2,3-Secoreserpine (26) and 3,4-secoreserpine (27).

Ahhough compound 27 was obtained in a much higher yield than was 26, Gaskell and Joule concluded that Mechanism 2 is active in the epimerization reaction of reserpine (1). They discredited Mechanism 3 because of the incapability of the metho salts 28 and 29 to epimerize. Instead, treatment of 28 and 29 with AcOH (140°C, 3 d) resulted in inversion of Nb to yield 30 and 31, respectively. Fig. (5). It was concluded that the inversion probably occurs via C-3 - Nb bond scission. 91 ^CH3 CH30

CH3O H' CHsOOC^ > ^

C H 3 0 0 C ' ' ^ ^ V ^ ' ^ OTM B

°^"^

28

CH3O

"OTMB

^^"'

29

CH3O H""|

H'

CHsGOC" "^Y'^^ÔTMB

CH300C^ ^ V ^

6CH3

30 Fig. (5). Metho salts 28-31.

ÔTMB

6CH3

31

13

Moreover, in the case of reserpine (1), the methoxy group at C-11 increases the electron density at C-2 and hence supports Mechanism 2, Fig. (6) [27].

Fig. (6). Resonance stabilization of the methoxy group.

In conjunction with their studies on electrophiUc substitution in indoles, Jackson and co-workers [28] suggest that the initial protonation occurs at C-7 as in Mechanism 1, followed by hydride rearrangement of the indole P-hydrogen to the a-position. The epimerization reaction would then take place as depicted in Mechanism 2. Hence, Gaskell and Joule and Jackson et al. differ only in the matter of initial protonation. In 1989, Cook and co-workers [29] reinvestigated the epimerization reaction in connection with reserpine (1). One of their key observations, based on the results of Martin et al. [30] and of Sakai and Ogawa [31], is that Mechanism 2 cannot be primarily responsible for the epimerization reaction of reserpine (1). Both Martin and co-workers and Sakai and Ogawa report that the iminium species 32, Fig. (7), cyclizes under acidic conditions mainly to reserpine (1) and not to isoreserpine (2). If Mechanism 2 alone were responsible for epimerization, then isoreseipine (2), not 1 should be the main product. Mechanism 2 was accordingly discredited.

CH30

£00H

H" CHaOGC^ " Y ^ ÔTMB 22 Fig. (7). Iminium species 32.

6CH3

14

C-5 - A^^ Bond Cleavage (Mechanism 3) If only three mechanisms are considered and two of them are discredited, then the third must be active. And indeed, a substantial body of evidence has built up in favour of Mechanism 3. Cook and co-workers have thoroughly investigated the acid-catalysed epimerization reaction with P-carboline derivatives [13,32,33]. Their conclusive work has provided strong evidence for Mechanism 3. When compound 33 was treated with TFA-J (2.9 equiv.), the more stable C-1 epimer 34 was obtained in high yield. Moreover, no deuterium had incorporated at C-1, thus ruling out Mechanism 1. A reduction experiment with NaBH4 on 33 yielded the C-1 epimer 34 and a ring cleavage product 35. Compound 35 provides strong evidence for Mechanism 3. In a control experiment, compound 34 was subjected to reductive conditions similar to those used for 33. The experiment resulted only in starting material, proving that intermediate 35 truly arises from the epimerization reaction. Scheme (8) [13]. Cook and co-workers had earlier suggested that reserpine (1) epimerizes analogously to p-carboline derivatives and hence via Mechanism 3 [29]. H

COOCH3

kjCI^^

H ^."v. I^COOCHa

^.vTs.

NaBH4 KJ^J CH2CI2, rt

H

35

KJ COOCH3 Ph

TFA NaBH4

+ 34

O

no reaction

CH2CI2, rt

Scheme (8). Epimerization experiments conducted by Cook and co-workers.

15

Lounasmaa et al also investigated the epimerization behaviour of lactams [12]. They found that lactams such as 36 epimerize with great ease. Epimerization of 36 with TFA at room temperature resulted in an equilibrium ratio of 70:30 (36:37) within a reaction time as short as 2 h, Scheme (9).

CH3

Scheme (9). Epimerization of lactam 36.

Being protonated at the carbonyl oxygen in strongly acidic media, lactams enable delocahzation of the Nb lone pair, Fig. (8).

Fig. (8). Delocalization of the Nb lone pair in a protonated lactam.

The canonical forms displayed above show a similarity with Mechanism 3, namely, the positive charge at Nb. Proof of the involvement of Mechanism 3 was therefore sought. When 36 was refluxed in a Zn/TFA solution for 3.5 h the secolactam 38, Fig. (9), was obtained in 22% yield. Compound 38 indicates that Mechanism 3 is active in the acid-catalysed epimerization of such lactams, a finding in accordance with the previously reported result that C-12b methyl substituted lactams epimerized under acidic conditions (see above).

16

Fig. (9). Secolactam 38.

Racemization of P-Carbolines In connection with the preparation of (+)- and (-)-tetrahydroharmine (compounds 39 and 40 in Fig. (10), respectively) the racemization of these compounds was studied by Chrisey and Brossi [34]. They demonstrated that, under acidic conditions, the pure enantiomers racemized with relative ease, and suggested that the racemization resembled epimerization of reserpine (1) and 1,3-disubstituted tetrahydrop-carbolines. Therefore, the mechanism responsible for the racemization would be one of those mentioned above. CH3O CH30 H CH3

39

u

40

NH -> H CH3

NH

CH3O

H CH3

41

Fig. (10). (+)-Tetrahydroharmine (39), (-)-tetrahydroharmine (40), and (-)-tetrahydroroeharmine (41).

Cook and co-workers suggested that partial racemization had occurred in the acid/base mediated isolation of (-)-tetrahydroroeharmine (41), Fig. (10) [35]. Proof was obtained by treating 41 with TFA/CH2CI2 at room temperature, which resulted in racemization of 41. In an experiment with TFA'd, deuterium was incorporated only at C-5 and C-8, not at the epimeric centre, indicating that a mechanism analogous to Mechanism 1 was not active. Reddy and Cook, in contrast, proposed that the mechanism depicted in Scheme (10) was responsible for the racemization of (-)-tetrahydroroeharmine (41). This mechanism is analogous to Mechanism 3. Interestingly, compounds with more than one asymmetric

17

centre, e.g. reserpine (1) and 1,3-disubstituted tetrahydro-P-carbolines, do not racemize under epimerization conditions [29,33].

CH

"V^^JI ^Q A ^

i l l ^ A s ^ NH

bond rota and and reclosure redo ^, CH36

Scheme (10). Racemization mechanism for (-)-tetrahydroroeharmine (41).

Synthesis of Bioactive Indole Alkaloids and Their Derivatives by Utilizing the Acid-Catalysed Epimerization Reaction Obtaining the correct stereochemistry for a given compound is of vital importance for the pharmaceutical industry because pharmacological properties often depend on it. For indole alkaloids and their derivatives, the acid-catalysed epimerization reaction provides, contrary to the more traditional oxidation/reduction procedure, a convenient tool to influence selectively the configuration at C-3 (biogenetic numbering). The following examples demonstrate the high utility of the epimerization reaction. Woodward's Total Synthesis of Reserpine Reserpine (1) with its remarkable pharmacological properties remains an attractive synthetic target. The first total synthesis of reserpine (1), by

18

Woodward and co-workers [11] in the 1950s, constitutes one of the most classical acid-catalysed epimerizations in indole alkaloid chemistry. After the key intermediate 43 had been prepared, it was reacted with 6methoxytryptamine (42) in benzene, Scheme (11).

.OrTi 42 CH3OOC

I.POCI3 •

2. NaBH4, MeOH

L " ^,,'T ^

CHaOGC^ ^Y"^ ^ OAc

CHsGOC^^^Y^ ^ OAc

43 ^^"^

44 . KOH,

OCH3

f ^

MegCCOOH

MeOH ^ „ ^ J k . J k

•"

2. NaBH4, MeOH

H H'

2.DCC, pyridine

46 . NaOMe,

'•

xylene, A

OCH3

\ \ i

MeOH ^ „ ^ J k s J k

[..•"

47

2.TMBC1, pyridine

OCH3 ^^"'

H H H"' CHaOOC^ ^ S ^ "*OTMB 6CH3 (±)-l

Scheme (11). The final stages of Woodward's reserpine synthesis.

Immediate sodium borohydride (NaBH4) reduction gave lactam 44. Bischler-Napieralski cycHzation of 44 followed by NaBH4 reduction yielded (±)-methyl-0-acetyl-isoreserpate (45). The correct stereochemistry at C-3 was obtained by first lactonizing compound 45; epimerization with pivalic acid then resulted in (±)-reserpic acid lactone (47). Treatment with base followed by acylation with TMBCl yielded racemic reserpine. The stereochemical considerations involved in the epimerization reaction will be discussed later.

19

Synthesis of Deethyleburnamonines by Lounasmaa and Co-workers An efficient route to both cis- and ^ra«5-deethylebumamonines is a further example of the efficacy of the epimerization reaction [36], These unnatural compounds are close derivatives of the well-known, pharmacologically important indole alkaloid, ebumamonine. For the synthesis of c/^-deethylebumamonine (3), Scheme (12), trans-QSi^x 6 was epimerized in refluxing TFA to give a readily separable mixture of starting material and m-ester 7 (ratio 22:78).

^

OgCii

THF*

H CHaOGC

7

N

pyridine P^"^^"'

k^N^si^N

DMF*

H TsO-

49

k A H^ ^

Mô" kJsAl^''

NC-

50 Scheme (12). Lounasmaa et al synthesis of deethylebumamonine (3).

After the correct stereochemistry at C-12b had been obtained, the methyl ester group was reduced with lithium aluminium hydride to yield alcohol 48. Tosylation of the alcohol and subsequent replacement of the tosylate with cyanide resulted in nitrile 50. Finally, acid treatment of 50 resulted in the target compound, deethylebumamonine (3).

20

Synthesis of the Tangutorine Skeleton by Jokela and Co-workers Tangutorine (51), Fig. (11), an indole alkaloid recently isolated from the leaves of Nitraria tangutorum [37] constitutes an interesting synthetic target for pharmacological evaluation. The carbon framework of 51 was therefore prepared to investigate, for the first time, the conformational and stereochemical features of this ring system [38]. The epimerization reaction served as a tool in the studies.

OH

Fig. (11). Tangutorine (51).

Preparation of the dodecahydro benz[/]indolo[2,3-a]quinolizidine ring structure relied on the Fry reaction [39] of salt 52, which, after subsequent acid-induced cyclization, yielded tlu*ee compounds, 53 - 55, Scheme (13).

OTX. „ e

l.KCN,6NHCl, NaBH. 2. 50% AcOH

52

Scheme (13). Preparation of compounds 53-55.

The unexpected a-amino nitrile 53 could easily be converted via AgBF4 treatment followed by reduction to the tangutorine model 56, Scheme (14).

21

l_AgBF4 2. NaBH.

Scheme (14). Conversion of a-amino nitrile 53 to the tangutorine skeleton 56.

After the Fry reaction, the tangutorine skeleton 56 could also be obtained via an epimerization sequence, Scheme (15). Since the catalytic hydrogenation of 54 yielded predominantly the wrong isomer (H-3, H-19 and H-20, all p), an alternative approach was sought to exploit compounds 54 and 55. Refluxing 54 in TFA overnight resulted in a ratio of 40:60 for compounds 55 and 54. Compound 57 was obtained by catalytic hydrogenation of 55, after which the tangutorine skeleton was formed via acid-catalysed epimerization of 57 (ratio 60:40, 56:57). Hence, by an epimerization sequence, compounds 54 and 55 can be converted into the desired target molecule 56.

+ 54

55 refl.*

Scheme (15). Epimerization sequence to obtain tangutorine model 56.

54

140:601

+

57

22

Stability and Conformational Analysis of Epimers The different conformations of indolo[2,3-^]quinolizidines play an important role in predicting the thermodynamically more stable epimer (see below). The indolo[2,3-a]quinolizidine system can exist in three main conformations: one C/D trans ring juncture (conformation a) and two C/D cis ring junctures (conformations b and c). These are in equilibrium by nitrogen inversion and c/^-decalin type ring interconversion. Ring C is considered to be in a half chair conformation and ring D in a chair conformation, Scheme (16) [40]. c/5-decalin type ring interconversion

a

C Scheme (16). The three conformations of indolo[2,3- promotion^progression [1-3] [Fig. (1)]. Among these stages, in contrast to both the initiation and progression stage, animal studies indicate that the promotion stage takes a long time to occur and may be reversible, at least early on. Therefore, the inhibition of tumor promotion in the multiple stages is expected to be an efficient approach to cancer control [4-7]. Cancer chemoprevention is defined as the use of specific natural and synthetic chemical agents to reverse or suppress carcinogenesis and prevent the development of invasive cancer. There is a growing awareness in recent years that dietary non-nutrient compounds can have important effects as chemopreventive agents, and there has been considerable work

44

on the cancer chemopreventive effects of such compounds in animal models. Several classes of natural products are now known as chemopreventive agents which included carotenoids, curcumin (turmeric yellow), limonene, quercetin (4), retinoids, tea tannins, and terpenoids [3,6,8-14].

Initiation epidermis

Promotion

' MMWBw.«MuwmaMU4Uwiiiwiiiiiwiwaiitti]yi)iiiiyiii»i^^

Death

^cxX3r)P3P"'o*^3Sc^::v?5rd^^ carcinomas

"• apoptosis no threshold irreversible short-lerm

I Progression

- threshold - reversible - long-term

- invasion - metastasis no threshold irreversible

Fig. (1). Critical steps and characteristics in multistage carcinogenesis [6]

In the course of our research on potential antitumor-promoters (cancer chemopreventive agents) from edible plants and fungi, and from crude herbal drugs, we have found that various triterpene alcohols and sterols and their oxygenated derivatives showed inhibitory effects on inflammation in mouse ear induced by 12-(9-tetradecanoylphorbol-13-acetate (TPA; 1), an in vivo primary screening assay for antitumor-promoters, and on tumor promotion in two-stage carcinogenesis in mouse skin initiated by 7,12-dimethylbenz[a]anthracene (DMBA, 2) and promoted by TPA. In this review, we summarize the inhibitory activities of triterpenoids and sterols (mostly from plants) on TPA-induced inflammation and on tumor promotion during carcinogenesis with DMBA and TPA, as well as in several other assays for antitumor-promoters. In addition, we have summarized anti-inflammatory and anti-allergic activities of these compounds evaluated using various assay systems described in the literature. We will not refer to activities of triterpenoidal and steroidal saponins because they have been subject to recent reviews [15-18]. The

45

Strategy of chemoprevention is different from that of cancer chemotherapy where the agents are directed against cancerous or fully promoted cells and seek to selectively kill the cell based on some aspect of its aberrant biochemical equilibrium [19-21]. BIO ASSAY FOR ANTITUMOR-PROMOTERS Bioassay systems used for screening antitumor-promoting compounds are listed in Table 1. They are classified into in vitro assays using subcellular structures, in vivo anti-inflammation assays induced by the tumor promoters, i.e,, croton oil, 12-0-hexadecanoyl-16-hydroxyphorbol-13aceate (HHPA), and TPA (1), and in vivo experimental carcinogenesis assays. The following in vitro assays are used usually as primary screening assays: Epstein-Barr virus early antigen activation inhibition (EBV) [22-27], TPA-stimulated *^^Pi (radioactive inorganic phosphate) incorporation in HeLa cells (human cervical cancer cells) inhibition (HeLa) [28,29], TPA-induced ornithine decarboxylase inhibition (GDC) [30,31], and protein kinase C (Ca^"*^- and phospholipid-dependent protein kinase) inhibition (PKC) [25,32]. Many compounds that active in EBV and HeLa assays have been proved to be inhibitors of tumor promotion on two-stage carcinogenesis tests in vivo. Assays for the inhibition of croton oil induced edema (CRO) [33,34], HHPA induced edema (HPA) [35], and TPA induced edema (TPA) [31,36,37] are used as rapid in vivo preliminary tests for screening antitumor-promoting substances. Among the existing models for experimental carcinogenesis studies, two-stage carcinogenesis in mouse skin with DMBA and TPA is being performed most frequently [8,37-42] since skin proves to be a unique target to differentiate between the effect of a test agent as either an anti-initiator or an antipromoter under appropriate experimental conditions [8]. Further studies using animal models have been undertaken for carcinogenesis of skin [28,38,41-43], colon [44], liver [45], lung [46], and mammary [42,47] as shown in Table 1. Assay for linhibition of TPA-induced Inflammatory Ear Edema in Mice For screening antitumor-promoting agents, we used TPA-induced inflammatory ear edema in mice [Fig.(2)] [36,37,48]. TPA (l|Lig/ear) dissolved in acetone (20 |LI1) was applied to the right ear of ICR mice by means of a micropipette. A volume of 10 |al was delivered to both the inner and outer surfaces of the ear. The samples or their vehicles, methanol-chloroform-water (2:1:1,20 |Lil) or chloroform (20 |a,l), as control, were applied topically about 30 min before TPA treatment. For ear

46

Table 1. Bioassay Systems for Screening of Compounds with Antitumor-Promotion, Anti-Inflammatory and Anti-Allergic Activities Described in This Review Code Bioassay system Assay for Antitumor Promoters In vivo primary screening assay CRO Croton oil induced edema inhibition HP A 11-0 -Hexadecanoyl-16-hydroxyphorboI-13-acetate (HHPA) induced edema inhibition TPA 12-0 -Tetradecanoylphorbol-13-acetate (TPA; 1) induced edema inhibition In vitro primary screening assay Inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced EBV by TPA TPA-Stimulated ^^Pi incorporation in HeLa cell inhibition HeLa TPA induced ornithine decarboxylase inhibition ODC Protein kinase C inhibition PKC In vivo assay Colon Inhibition of A^-methyl-A^-nitrosourea (MNU) induced colon tumors Liver Inhibition of A^-diethylnitrosamine/phenobarbital induced hepatic tumors Inhibition of 4-(methylnitrosoamino)-l-(3-pyridyl)-l-butanone (NNK) Lung induced lung tumors Mammary I Inhibition of MNU induced mammary tumors Mammary 11 Inhibition of spontaneous mammary tumors Skin I Inhibition of 7,12-dimethylbenz[fl ]anthracene (DMBA; 2)/TPA induced skin tumors (Topical application) Skin II Inhibition of DMBA/TPA induced skin tumors (Oral application)

References"

Skin III application) Assay for Anti-Inflammatory and Anti-Allergic Compounds In vivo assay AA Arachidonic acid induced edema inhibition AIP Adjuvant induced polyarthritis inhibition BRA Bradykinin induced edema inhibition CAR Carrageenan induced edema inhibition CPS Capsaicin induced edema inhibition CTN Cotton pellet granuloma inhibition DEX Dextran induced edema inhibition Delayed-type allergy suppressant activity DTA Ethyl phenylpropiolate (EPP) induced edema inhibition EPP FAA Formaldehyde induced arthritis inhibition HSI Histamine induced edema inhibition ITA Immediate-type allergy suppressant activity PAF Platelet aggregation factor (PAF) acether induced edema inhibition SER Serotonin induced edema inhibition In vitro assay ACM Anticomplementary activity cAK Cyclic AMP-dependent protein kinase inhibition

[28,38]

" Only some representative references were given.

[33,34] [35] [31,36,37,48] [22-27] [28,29,39] [30,71] [25,32] [44] [45] [46] [47] [42] [8,37,39-42] [43]

[56,89,93,104] [105,106] [107] [64,89,108,109] [110] [89] [108] [111,112] [62] [105,108] [107] [112] [107] [64] [113,114] [115,116]

47

Table 1. Continued. Code

COX COX2

HLE HPT HSR HYA iNOS 5L0X 12L0X 15L0X PLA2

SOF

Bioassay system Cyclooxygenase inhibition Inducible cyclooxygenase inhibition Human leucocyte elastase inhibition Hepatoprotective activity Histamine release inhibition Hyaluronidase inhibition Inducible nitric oxide synthase inhibition 5-Lipoxygenase inhibition 12-Lipoxygenase inhibition 15-Lipoxygenase inhibition Phosphoslipase A2 inhibition Inhibition of A^ -formylmethionyl-leucyl-phenylalanine (FMLP) induced superoxide formation from rat neutrophils

Sample Img/ear

TPA l^g/ear

1

Ear Edema Measurement

i 30 min

References'* [93,102,117] [118] [93,119] [115,120] [91,121-123] [124] [97,99-101] [93,102,117] [117] [102,125] [126,127] [122]

1

^~-"—

m

6hr

Fig. (2). Assay of TPA-induced inflammation in mice [37]

thickness determinations, a pocket thickness gauge with a range of 0-9 mm, graduated at 0.01 mm intervals and modified so that the contact surface area was increased to reduce the tension, was applied to the tip of the ear. The ear thickness was measured before treatment (a), and 6 h after TPA treatment (b =TPA alone; b' =TPA plus sample). The following values were then calculated: Edema A is induced by TPA alone (b - a). Edema B is induced by TPA plus sample (b' - a). Edema A ~ Edema B Inhibition ratio (%) = x 100 Edema A Each value was the mean of individual determinations from 4-5 mice. The 50% inhibitory dose (ID50) values were determined by the method of probit-graphic interpolation for four dose levels. This assay is

48

advantageous since it requires only few milligrams of sample, and it can be performed within one day. The activities of inhibitors on TPA-induced inflammation almost parallel their inhibitory activities on tumor promotion [49]. Assay for linhibition of DMBA/TPA Induced Skin Tumors in Mice For the highly inhibitive compounds in the TPA-induced inflammatory assay, we evaluated their antitumor-promoting activity by a standard initiation-promotion protocol [Fig. (3)] [37,40]. Initiation was achieved by a single application of 50 |ig of DMBA (2), a well known tumor initiator, to the skin of backs of 8-week-old female ICR mice. From one week after initiation, 1 |Lig of TPA (1) was applied twice a week until week 20. The test compound (2.0 or 0.2 |iM) dissolved in acetone-dimethyl sulfoxide (DMSO) (9:1, 100 |il) was applied 30 min before each TPA treatment. Groups of 15 mice were used. The number of tumors was counted weekly. Initiation

Promotion

i{

DMBA DMSO-acetone (195 nM) (,.9)

DMSO-acetone TPA TPA (1.7 nM) (1.7 nM) (1:9) in acetone in apetone

DMSO-acetone (1:9)

t/ 30 min

30 min . Twice a week

1 week

1

1-

DMSO-acetone TPA TPA (1:9) (1.7 nM) (1.7 nM) in Acetone in êtone

30 min

{ Twice a week

y30 min , 1

20 weeks

i+

DMBA Test compound TPA Test compound TPA Test compound TPA Test compound TPA (195 nM) (2.0 or 0.2 nM) (1.7 nM) (2.0 or 0.2 ^M) (1.7 nM) (2.0 or 0.2 îM) (1.7 nM) (2.0or0.2^M) (1.7 nM) in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone

1 week

•J-/4 30 min

1

30 min . Twice a week

1

30 min

20 weeks Fig. (3). Method of two-stage carcinogenesis assay in mouse skin [37]

•1: Twice a week

y. 30 min .

49 C13H27COQ

IV. Lanostane

II. Ergostane

(17) 3P-OH. 5:6. 7:8. 9:11. 22:23Etetraene (18) 3P-0H. 5:6, 7:8, 22:23E-triene (19) 3P-0H. 6:7, 22:23E-diene, 5a,8a-epidioxy (20) 3P-0-FER, 5:6-ene, 24R+S Stigmastane

(42) 3a-0Me. 7:8-ene, 23-oxo; 26-COOMe (43)3p-OH. 7:8. 9:11, 24:25triene, 21-COOH (44) 3P-0H, 8:9-ene (45) 7p,15a-diOH, 3, 11, 23-trioxo. 8:9-ene, 26-COOH (46)3p,7p-diOH. 11.15-dioxo, 8:9-ene. 26-COOH (47)3p,7p,15a-triOH. 11.23dioxo, 8:9-ene. 26-COOH (48)7p,12p.diOH. 3. 11,15, 23tetraoxo, 8:9-ene, 26-COOH (49)3p.16a-diOH, 7:8, 9:11, 24:25-triene, 21-COOH (50) 3p,16a-diOH, 8:9, 24:25diene, 21-COOH (51) 3p-0Ac. 16a-0H, 8:9, 24:25-diene. 21-COOH (52) 3P-0H. 8:9. 24:25-diene

(21) 3p,7a-diOAc, 5:6-ene (22) 3p,7p-diOAc, 5:6-ene (23)3p-OH, 5:6, 7:8.9:11. MeO ^ ^ ^^ (5) O ^ 22:23E-tetraene. 24p(R) (24) 3P-0H. 4a-Me. HO,^>S1AAOH 7:8,24:24^Z-diene (25) 3P-0H, 5:6. 7:8. 22:23Etriene.24p(R) (26) 3P-0H. 6:7, 22:23E-diene, O^^v^^^v^ (6) 5a,8a-epidioxy, 24p(R) I. Cholestane (27) 3P-OH, 5:6. 24:24Ê-diene (53) 3-0X0, 7:8.9:11-diene. 21-COOH (28) 3p,4p-diOH. 5:6-ene (54)3p-OAc,16a-OH,7:8.9:11-diene. (29) 3p.7a-diOH, 5:6-ene 21-COOH (30) 3p.7p-diOH, 5:6-ene (55) 3p,16a-diOH. 7:8, 9:11-diene, (31) 3P-0H, 5:6-ene, 7-oxo (7) 3P-0H, 5:6-ene 21-COOH (8) 3P-0H, 4.4-diMe, 5:6-ene (32) 3p-0H. 7:8-ene (56) 3a.16a-diOH. 7:8. 9:11-diene. (9) 3-0X0, 4,4-diMe, 5:6-ene (33) 3B.0H, 5:6-ene 21-COOH (10)3p-OH,4,4-diMe, (34) 3P-0-FER. 5:6-ene (57) 3p-0-Bz-p.0H. 16a-0H.

rrr

5a,6a-epoxy

(35) 3P-0H. 7:8, 22:23E-diene (11) 3-0X0. 4,4-diMe. (36) 3p.0H. 5:6. 22:23E-diene 5a,6a-epoxy )^J. 00 ô L.^L. ^ r (12) 3a,5a-diOH. 4.4-diMe (37) 3p.6p-diOH. 4:5-ene (13) 3P-0H. 4.4-diMe, 7:8-ene(38) 3p,6a-diOH (14) 3p.0H, 4a-Me, 7:8-ene (39) 33 ep-diOH (15) 3-OH, 4-Me. 3:4-ene ) ' J^' ^.^ 16 3p.0H, 5:6-ene, 7-0X0 ^ ^ J ' ^ ' f ^° ^ ^ (41)3,6-dioxo, 4:5-ene

Fig. (4). Structures of compounds described in this review

7:8. 9:11-diene. 21.C00H (^^l f'^^^ ^(11)^ne (59) 3p-0Ac. 16a-0H. 8:9-ene. 21-CO6H (60)3-0X0. 16a,25-diOH, 7:8. 9:11-diene. 21-COOH (61) 3p.16a-diOH.8:9-ene.21-COOH

50

(81) 24:25-ene (82) 24,25-epoxy (24R+S) (83) 20:21-dlhydro(20R). 13:17, 24:25-dJene (84) 20:21-dihydro(20S). 13:17. 24:25-dlene (85) 24-methylene

HOOC/,

ROOC

(62) (63) (64) (65)

R=H, 24-methylene R=Me, 24-methylene R=H, 24:25-ene R=H, 24-methylene, 25-OH

VIII. Euphane

7C^^^ 'h

(99) 8:9, 24:25-dlene (100)7:8, 24:25-diene (101) 7:8-ene, 24,25-epoxy (24R+S)

V. Cycloartane

IX. Tirucallane

21//,.

HOOC

VII. Cucurbitane (66) 3 P - 0 H . 24:25-ene (102) R=oxo. 7:8, 24:25-diene, (67) 3P-0-FER, 24:25-ene 27-COOH (68) 3-0X0, 21-COOH, 24,25(103) R = a - O H . 7:8, 24,25-diene, epoxy (24R+S) 27-COOH (69) 3 P - 0 H . 21-CHO, 24:25-ene (104) R=p-OH. 7:8. 24:25-diene (70) 3 P - 0 H , 21-COOH. 24:25-ene (105) R=p-OH, 8:9, 24,25-diene (71) 3-0X0, 21-COOMe, 24,25epoxy (24R+S) RO^ (72) 3 P - 0 H . 24-methylene X. Quasslne QH (88) R=H (73) 3P-0-FER, 24-methylene H04^x%,N\C00Me (89) R=H, 24:25-dihydro (74) 3 P - 0 H . 21-COOMe, (90) R=H, 7-0X0 24:25-ene (75) 3-0X0, 21-COOMe, 24:25-ene (91) R=Ac, 7-0X0 (92) R=H, 7-0X0, 24:25-dihydro (76) 3-0X0, 21-CHO, 24:25-ene

xffix: H

(77) 3-0X0, 2 1 - C O O H , 24:25-ene (78) 3-0X0, 2 I - C H 2 O H , 24:25-ene

(106)3:4-ene. R=Bz ^R^107)3:4-ene,R=Q

2V/,.

CHO

(93) R = a - O H , R^=H,

R2=AC

(94) R=oxo R^=H, 23:24.ene, R2=AC

(95) R=a-OH, R^=H,23:24-ene, R2=H

(96) R=a-OH,

=H. R^=H

(97) R=a-OH.

=H, 15-0X0,

2=L 23:24-ene. R''=H

( 9 8 ) R = a - O H , R ^ = H , 15-0X0, R2=H

Fig. (4). Continued-1

(108)3:4-ene,R=^

"^

(109)3:4-ene, R=^

"^

(IIO)ip-OH,

"^

(111)ip-0H, (112)3-OH. 3:4-ene

-•or

(113)R=Ac,3:4-ene

OAc

51 (137) 3p,16p-diOH, 28-CH2OH (138)3|3-OH, 16P-0H (139) 3P.O-OCC13H27. 16P-0H (140)3p-O-OCCi5H3i, 16P-0H (141) 2a.3p-diOH. 28-COOH (142)3p-OH,28-COOH (143)3-0X0, 28-COOH (144)3p.16p-diOH (145) 3P-0H, 28-COOH, 3O-CH2OH (160)R=H,R^=P-OH, R2= Me '2=1Me (161)R=H,R^=oxo, R' 2=r (162) R=H, R'=oxo, R^=CH20H 3l=, (163) R=H, R'=:p-OH, R^=CH20H

COOR (146) R=H, 2a-0H. R^=COOH (116)3p-OAc,27-COOH R^=Me (117)3p-OAc,28-CH20H (118) 3P-0AC. 28-COOH (147) R=H, R^=Me. R 2 = C 0 0 H 164) R=H,2-CN. 12:13-ene (119)3p-OH 165) R=H.2-CN, 9:11-ene, (148) R=0-CCi7H35, R^=Me. (120)3p-OAc 12-0X0 R2=COOH (121)3p-OH,30.CH2OH 166)R=H. 11-0X0.12:13-ene (149) R=PHT-Na. R^=Me, (122)3p-0-PHT-Na, 167)R=H. 11-0X0.13:18-ene R2=COOH 30-CH2O.PHT-Na 168) R=H, 12-0X0 (123)3p-0-SUC-Na, (150)R=PHT-Na,R^=Me, 169) R=H, 9:11-ene, 12-0X0 30-CH2O-SUC-Na R2=C00Na 170)R=H.9:11-epoxy (124)3p-0-P03Na2, 171) R=H,2-0H. 12:13-ene (161)R=H, R^=Me, 30-CH2OPO3Na2 172)R=H,2-OMe, 12:13-ene (125)3p-0-PHT-Na, 173) R=Me, 12:13-ene 3O-CH2OH 174) R=H (126) 2a,3p-diOAc,28-COOH 175)R=H.9:11-ene (127)1,3-dloxo 176)R=H, 12:13-ene (128) 3p,16p-diOH. 28-COOH 177) R=H,2-CHO. 12:13-ene (129)3p,15a,16a-triOH. ;i78)R=H, 11:12, 13:18-diejjie 28-COOH (130)3p-OH,28-CH2OH (131) 3-oxo,22p-OH.28-COOH (152)R=H,2a-OH, R'=CH20H. (132) 3-0X0. 22P-0-TIG. R2=C00H 28-COOH (153)R=H. 2a-0H. R^=CH20H. (133)3p-OH.22p-0-TIG. R2=COOMe 28-COOH (154)R=Ac,2a-OAc, R^=CH20Ac. R 2 = C 0 0 H (179) R=H. Me, 13:18-ene >1. (155)R=Ac.2a-OAc, (180) R=H, R'=Me, 18:19-ene (134) 3-0X0.22pR^=CH20Ac, R2=C00Me (181) R=H. R^=CH20H. 28-COOH (156) R = H , R ^ = C H 2 0 H . R 2 = C O O H 9:11. 12:13-diene (135)3p-OH.22p-i (157)R=H. R ^ = R 2 = C H 2 0 H (182) R=PHT-Na, R^=CH2028-COOH (158)R=PHT-Na, R^=R2=: PHT-Na. 9:11.12:13-diene CH20-PHT-Na (136) 3-0X0.22p-CL^^S^ (183) R=H, R^=CH20H, 28-COOH ^ (159)R=H,2a-OH, R^= 11:12,13:18-dlene CH2OH. R2=Me (184) R=PHT-Na,R^=CH20PHT-Na,11:12.13:18-diene Fig. (4). Continued-2

rr

'TV

52 (137) 3p,16MiOH. 28-CH2OH (138)3p-OH, 16P-0H (139) 3P-O-OCC13H27. lep-OH (140)3p-O-OCCi5H3i, 16P-0H (141) 2a.3p-diOH. 28-COOH (142) 3P-0H. 28-COOH (143) 3-0X0, 28-COOH (144)3p.16p-diOH (145) 3P-OH. 28-COOH. 3O-CH2OH (160) R=H, R^=p-OH. R2=Me (161)R=H, R^=oxo, R^=Me (162) R=H. R^=oxo. R 2 = C H 2 0 H (163) R=H. R^=p-OH. R 2 = C H 2 0 H

COOR (146) R=H. 2a-0H. R^=COOH. (116)3p-OAc.27-COOH R2=Me (117)3p-OAc.28-CH20H (118) 3p-0Ac, 28-COOH (147) R=H, R^=Me, R 2 = C 0 0 H 164)R=H.2-CN, 12:13-ene (119)3p-OH 165) R=H.2-CN, 9:11-ene. (148) R=0-CCi7H35. R'=Me (120)3p-OAc 12-0X0 R^=COOH (121)3p-OH.30-CH2OH 166)R=H, 11-0X0.12:13-ene 1=11 (149) R=PHT-Na, R'=Me (122)3p-0-PHT-Na, 167)R=H. 11-0X0,13:18-ene R2=COOH 30-CH2O-PHT-Na 168) R=H, 12-0X0 (150)R=PHT-Na,R'=Me (123)3p-0-SUC-Na, 169) R=H, 9:11-ene, 12-0X0 30-CH2O-SUC-Na R2=C00Na 170)R=H.9:11-epoxy (124)3p-0-P03Na2. 171) R=H.2-0H, 12:13-ene (151)R=H.R^=Me, 30-CH2OPO3Na2 172)R=H,2-OMe. 12:13-ene R2=COO-CCI7H35 (125)3p-0-PHT-Na. 173) R=Me. 12:13-ene 3O-CH2OH 174) R=H (126) 2a.3p-diOAc.28-COOH 175) R=H. 9:11-ene (127)1.3-dioxo 176) R=H. 12:13-ene (128) 3p.16p-diOH, 28-COOH 177) R=H,2-CH0.12:13-ene (129)3p,15a,16a-triOH. 178) R=H. 11:12. 13:18-diejjie 28-COOH (130)3p-OH,28-CH2OH (131)3-oxo,22p-OH, (152) R=H. 2a-0H, R^=CH20H, 28-COOH R2=COOH (132)3-0X0. 22P-0-TIG. (153) R=H. 2a-0H. R^=CH20H. 28-COOH R2=C00Me (133)3p-OH.22p-0-TIG. (154)R=Ac,2a-OAc. 28-COOH 32^, R'=CH20Ac. R^=COOH (179) R = H 13:18-ene (155)R=Ac,2a-OAc. 18:19-ene (134) 3-0X0,22p. R'=CH20Ac. R^=COOMe ( I 8 I ) R=H. R^=CH20H 28-COOH 32=. (156) R=H.R'=CH20H.R^=C00H 9:11.12:13-diene (157)R=H, R ^ = R 2 = C H 2 0 H (135)3p-OH,22p-< (182) R=PHT-Na. R^=CH2028-COOH (158)R=PHT.Na. R^=R2= PHT-Na, 9:11, 12:13-diene CH20-PHT-Na (136) 3-0X0,22p-OLx'*v^/ (183) R=H, R^=CH20H, 28-COOH ^ (159)R=H,2a-OH, R^= 11:12.13:18-diene CH2OH, R2=Me (184) R=PHT-Na,R^=CH20PHT-Na.11:12.13:18-diene Fig. (4). Continued-2

Rcrys

'XT

^CY

53

COOH

(207) 3-0X0, 1:2-ene, 28-COOH XIV. Taraxastane (208) 2a.3p,19a-triOH, 28-COOH (209)3a.16p-OH (210) 3P-OH. 28-COOH (211)3p-OH,28-CH20H

(185)2a,3p-diOAc, 18P-0H, 5:6,12:13-diene (186) 2a,3p-diOAc, 5:6.12:13-diene (187)2p.3a-diOAc. 5:6, 12:13-diene (188) 2a,3a-diOH, 5:6,12:13-diene (189) 3-0X0, 11:12,13:18-diene (190)3a-OH, 11:12,13:18-diene

Multiflorane p (230) 3p.16p-diOH, 20:30-ene (231) 3P-O-OCC15H31, 16P-0H. 20:30-ene (232) 3p,16p-diOH. 20:21-ene (233) 3P-O-OCC13H25.16P-0H. 20:21-ene (234) 3P-O-OC15H31.16P-0H. 20:21-ene (235) 3p,16p-diOH, 28-CH2OH, 20:21-ene (236)3p,16p,22a-triOH, 20:21-ene (237)20:21-ene (238) 3P-0H, 20:21-ene (239) 3P-0H, 20:30-ene (240) 3P-0AC. 20:30-ene

(212)3p-OH, R=CH20H (213)3p-0-SUC-K, R=CH20H (214) 3P-0-SUC-K, R=CH20-SUC-K (215)3p-0-PHT-K, R=CH20-SUC-K (216)3p-OH, R=COOH (217) 3P-0-SUK-K, (191) R=H,R^=R2=Me,R^=CH20H R=COOK (192) R=PHT-Na. R^=R2=Me, (218)3p-0-PHT-K, R^=CH20-PHT-Na R=COOK (219)3a-OH. R=CH20H (193)R=H, R^=R2=Me, R^= XV. Taraxerane (220) R=Me COOH. 11-0X0 (221)3p-OH,R=Me. 7-0X0 (194) R=H,R^=R^=Me,R2=CH20H (222) 3a-0H. R=CH20H. 7- 0x0 (195) R=H,R^=R2=CH20H,R2=Me -. R^

XII. Ursane

~ (241) (242) 3P-0H

2^ 24

(196) 3p- OAc. 11-0X0, 24-COOH (197) 3p- OH (198)3p- OAc (199) 3p- O-OCC15H31 (200) 3p- O-OCC17H31 (201) 3a OH, 24-COOH (202) 3p, 16p-diOH (203) 3p- O-OCC13H27,16P-0H (204) 3p- O-OCCisHai.iep-OH (205) 3,1 1-dioxo, 1:2-ene, 28-COOH (206) 3 a-OH, 28-COOH


XVI. Glutlnane (223)3p-OH, 7:8.9:11-diene, R=CH20H (224) 3P-0-SUC-K, 7:8, 9:11diene, R=COOK (225) 3a-0H, 7:8. 9:11-diene. R=CH20H (226) 3a-0Bz. 7:8. 9:11dlene, R=CH20H (227) 3P-0H, 7:8-ene. R=Me (228)9:11-ene. R=Me (229) 3a-0Ac. 7-oxo. 9:11-ene, R=CH20Ac

(243) 3P-0H. 5:6-ene. 10a-H (244)5:10-ene

54

XVII. Friedelane ROOq..

(259)3p-0-OCi5H3i. XXi. Arborinane 16P-0H (260) 3a-0H (261)3p,16p-dlOH, 28-CH2OH (262) 3P-0H. 3O-CH2OH (263) 3P-0H, 30-CHO (281) R=H (264) 3P-0H (282) R=Ac (265) 3p.0Ac (266) 3P.O-OCC15H31 XXII. Other Triterpenes (267) 3P.O-OCC17H31 (268) 3-0X0 (269) 3P-0-CAF, 28-COOH \^ (270) 3P.O-SO3K, 28-COOH (271) 3P-0-SUC, 28-COOH HO

(247) 3P-0H (248) 3-0X0 HOOa.

(272) 28-COOH (273) (274)12:13-ene, 18P-H

(249) R=H (250) R=Ac

HOO9.,

XIX. Hopane , H '

H I

OH

(251)

XVIII. Lupane

(275)17:21-ene, R=H OH OH

(276)R=^X^

OH OH

3 0 v ^

(277)13:18-ene (278)12:13-ene (252) 3p-0Ac, 30-CHO (253) 3p-0Ac. 28-COOH (254) 3P-0BZ, 28-COOH (255) 3P-0H, 28-CH2OH (256) 3-0X0, 28-CHO (257) 3P-0H, 28-COOH (258)3p, 16p-diOH

XX. Moretenane

H nr

^\H

(279) R = H (280)


R=AC

(288)

^^

55 XXIII. Spirostane

XXV. Phytoecdysone

*

^

0

H OMe

FER (feruroyi) (289) 3P-0H, 12-0X0 (290) 3,12-dioxo, 4:5-ene (291)3|3-OH

(295) R=H.

Ritf^^_

ZQH\—0

XXIV. Cardiac steroid

(296) R=H, Ri="^^p^'^'YÔ (297)R=OH. R^=^1f^^^'^^H Structures of abbreviated moieties

o

P

PHT (pthalyl)

.OH O s u e (succinyl)

(292) R=H, R^=H, R2=Me (293) R=H, R^=OH, R 2 = C H 2 0 H ,

^

11a-0H (294) R=H, R^=OH. R^=CHO

Ac (acetyl) Bz (benzoyl)

OH CAP (caffeoyi)

. TIG (tigulyl)


INHIBITORY EFFECTS OF TRITERPENOIDS AND STEROLS ON PRIMARY SCREENING ASSAYS FOR ANTITUMORPROMOTERS Table 2 lists all compounds discussed in this paper. Their structures are shown in Fig. (4). They are oxygenated compounds having the following skeletons: regular steroids [cholestane (I), ergostane (II), and stigmastane (III)], tetracyclic triterpenoids [lanostane (IV), cycloartane (V), dammarane (VI), cucurbitane (VII), euphane (VIII), tirucallane (IX), and quassine (X)], pentacyclic triterpenoids [oleanane (XI), ursane (XII), multiflorane (XIII), taraxastane (XIV), taraxerane (XV), glutinane (XVI), friedelane (XVII), lupane (XVIII), hopane (XIX), moretenane (XX), and arborinane (XXI)], other plant steroids [spirostane (XXIII), cardiac steroid (XXIV), and phytoecdysone (XXV)]. Most of the semi-synthetic compounds listed were preparedfromnatural products by simple chemical modification. The bioassay systems in which the compounds exhibited inhibitory effects, together with the major sources of the compounds, are included in the Table. Inhibitory Effects on TPA, HHPA, and Croton Oil-Induced Inflammatory Edema

56

The inhibitory effects of the sterols and triterpenoids on TPA-induced inflammatory ear edema in mice are shown in Table 2. The inhibitory effects of three reference compounds, quercetin (4), a known inhibitor of TPA-induced inflammation in mice, and of two commercially available anti-inflammatory drugs, indomethacin (5) and hydrocortisone (6), were included for comparison. As is evident from Table 2, most of the compounds examined exhibited activity almost equivalent to or higher than quercetin (4). Inhibitory effects on the other experimental models were also included in Table 2. Sterols and Their Derivatives Cholesterol (7), a representative animal-sterol, did not show an appreciable inhibitory effect, whereas its 24-ethyl homologues, sitosterol (33) and stigmasterol (36) [50], the most ubiquitous phytosterols, exhibited activity almost equivalent to that of 4. A^-Unsaturated sterols, schottenol (32) and spinasterol (35), were more active than their A^-isomers, 33 and 36, respectively [50]. Ergosterol (18), a typical fungal sterol possessing a A^* -conjugated-diene system in the nucleus, exhibited higher activity than those of the above animal- and phytosterols [36,51]. Oxygenation of the nucleus of sterol usually enhanced the activity. Thus, all of the oxygenated sterols assayed, with the exception of 7a-hydroxysitosterol (29) and its diacetate (21), markedly inhibited inflammation induced by TPA [51,52]. Acetylation of the hydroxyl group at C-3 exerted almost no influence on the activity of sterols (29/21 and 30/22) whereas esterification with ferulic acid enhanced the activity (34/33). Methylation at C-4 of sterol nucleus was one of the other factors affecting activity enhancement. Thus, in general, 4-methylsterols (14,15) and 4,4dimethylsterols (8,13) exhibited higher activity than 4-desmethylsterols. A similar structure-activity relationship was observed also in the HHPA-induced inflammation on mouse ear [35]. Whereas cholesterol (7) did not show inhibitory activity, several 4,4-dimethylcholestane derivatives, 8-12, exhibited activity. 4,4-Dimethylcholestane-3a,5a-diol (12) was the most potent inhibitor: its activity was comparable to that of ursolic acid (210) [35]. Compound 12 reduced also the inflammation induced by teleocidin B (3), one of the indole alkaloid-type of tumor promoters [53]. Triterpenoids Most of the tetracyclic and pentacyclic 3-monohydroxy triterpenoids

57

examined exhibited higher activity than 4-desmethylsterols. This is consistent with the above observation that C-4 methylation of a steroid or triterpenoid nucleus enhances the activity. Further oxygenation, e.g., hydroxylation, carbonylation, or carboxylation, in addition to at C-3 enhanced the inhibitory effect of triterpene alcohols as has been observed for lanostane-, cucurbitane-, oleanane-, ursane-, taraxastane-, and lupane-type compounds. Several lanostane-type compounds, viz., dehydroeburiconic acid (53), dehydropachymic acid (54), dehydrotrametenolic acid (43), 16a-hydroxytrametenolic acid 3-O-acetate (51), poricoic acid A (62), and poricoic acid B (64) [37,54-56] showed a strong inhibitory activity which was at the same level as hydrocortisone (6). Twenty-five triterpenoids from Compositae flowers, viz., twelve monohydroxy, seven dihydroxy, and four trihydroxy triterpenoids, were evaluated with respect to their anti-inflammatory activity induced by TPA [57-59]. It was observed that these triterpenoids examined markedly inhibited the inflammation with 0.03-0.8 mg/ear of the 50% inhibitory dose. There was a close relationship between the hydroxylation of triterpenoids and the inhibitory effects. Di- and trihydroxy triterpenoids always showed higher activity than their corresponding 3 p-monohydroxy compounds. In the case of A -taraxastenes, faradiol (232; 0.2 mg/ear), showed a higher inhibitory effect than its 3p-monohydroxy homolog, v|/-taraxasterol (238; 0.4 mg/ear). Further hydroxylation of 232 at C-22a to give heliantriol C (236; 0.03 mg/ear) enhanced the effect markedly. Heliantriol C (236), brein (202; ursane deriv.), and heliantriol B2 (261; lupane deriv.) showed a fairly strong inhibitory effect which was almost comparable with that of hydrocortisone (6). Esterification with a fatty acid or with ferulic acid at C-3 of triterpene alcohols exerted almost no influence on the activity as has been observed with lupane- (264/265) and cycloartane- (66/67, 72/73) type compounds and a C-3 monohydroxy triterpenoid (264/266). However, esterification at C-3 with a fatty acid reduced the inhibitory activity of some dihydroxy triterpenoids (138/139,140; 202/203, 204; 230/231; 232/233, 234). Three lanostane carboxylic acids, 16a-hydroxydehydrotrametenolic acid (49) [60], 16a-hydroxytrametenolic acid (50) [60], and dehydrotumulosic acid (55) [60,61], and dehydropachymic acid (54) [60] and pachymic acid (59) [61], have been reported to be active against topical anti-inflammatory activities induced by TPA. Ten pentacyclic triterpenoids, viz., six oleananes (119,130,142, 147,156, and 193), three ursanes (197,208, and 211), and one lupane (264) were considerably active against TPA-induced edema. Of those compounds, the triterpenoid acids (142,147,193, and 208) were the most active. However, erythrodiol (130) surpassed all others [62]. Three triterpenoids 210 [63], 255, and 257 have been evaluated by the TPA-induced inflammatory ear edema assay [64]. The anti-inflammatory effects of glycyrrhetic acid (147), and its

58

derivatives on TPA-induced mouse ear edema were studied [65]. Among the derivatives of 147 tested, six dihemiphthalate derivatives, i.e., di-O-hemiphthalates of olean-12-ene-3p,30-diol (deoxoglycyrrhetol; 120), oleana-9(ll),12-diene-3p,30-diol (181), and oleana-11,13(18)-diene3p,30-diol (183), and their disodium derivatives, 122, 182, and 184, respectively, inhibited most strongly ear edema on both topical (ID50, 1.6 mg/ear for 120,2.0 mg/ear for 181, and 1.6 mg/ear for 183) and oral (ID50, 88 mg/kg for 122, 130 mg/kg for 182, and 92 mg/kg for 184) administration. Compound 147 and deoxoglycyrrhetol (121), the parent compounds, produced little inhibition by oral administration at less than 200 mg/kg. In this study, the mechanisms of TPA-induced ear edema were investigated with respect to the chemical mediators. The formation of ear edema reached a maximum 5 h after TPA application (2 |ag/ear) and the prostaglandin E2 (PGE2) production of mouse ear increased with the edema formation. TPA-induced ear edema was prevented by actinomycin D and cycloheximide (0.1 mg/ear, respectively) when applied during 60 min after TPA treatment. It has been suggested that the dihemiphthalate derivatives of triterpenes derivedfiôm147 by chemical modification are usefiil for the treatment of skin inflammation by both topical and oral application [65]. Among five triterpenoids isolated from Calendula officinalis flowers, P-amyrin (119), faradiol (232), v|/-taraxasterol (238), taraxasterol (239), and lupeol (238), the diol 232 was the most active. It showed a dose-dependent effect with a potency that equals that of indomethacin (5) in the topical anti-inflammatory assay with croton oil [33]. Esterification at C-3 of 232 with a fatty acid reduced the activity by more than 50% [33] consistent with our observation in the TPA-induced assay described above. The anti-inflammatory properties, as determined by croton oil-induced edema of mouse ear, of faradiol-3-O-myristate (233) and its 3-0-palmitate (234), the main components of lipophilic extracts of C officinalis flowers, were shown to be contribute significantly to the pronounced antiphlogistic activity of the lipophilic extracts of C. officinalisflowers[34]. Inhibitory Effects on TPA-induced EBV Activation The in vitro EBV-EA activation inhibition assay uses EBV genome-carrying lymphoblastoid cells (Raji cells derived from Burkitt's lymphoma). Many compounds which inhibit EBV-EA induction by tumor promoters have been demonstrated to act as inhibitors of tumor promotion in vivo [16,22,42,66-68]. This assay has an advantage since it obtaines the information on tiie cytotoxicity from the viability of Raji cells. High viability of these cells is an important factor in developing a compound for the chemoprevention of cancer [16]. So far, a number of triterpenoids and steroids from plants and their derivatives have been shown to possess

59

inhibitory effects on EBV-EA activation induced by TPA. They are derivatives of cycloartane [24], cucurbitane [26,69,70], quassine [71], oleanane [22,23,27,42,72-74], ursane [22,42], multiflorane [67], taraxastane [42,67], taraxerane [42,67], glutinane [67], friedelane [75], lupane [42,72], hopane [67], cardiac steroid [76], and phytoecdysone [68], as shown in Table 2. Glycyrrhetic acid (147) and retinoic acid are known in vivo antitumor-promoters which inhibit EBV-EA induction by tumor promoters [77]. Oleanolic acid (142) and ursolic acid (210) significantly inhibit the activation induced by TPA and teleocidin B (3) as do 147 and retinoic acid [22]. Enhancement of the inhibitory activity was found in 3-oxo derivatives of 142 and 210, while either loss of oxygen functionality at C-3 of 210 or oxidation at C-3 of 147 led to the reduction of the activity [22]. Two 3-0-acetyl oleananes, 3-0-acetylerythrodiol (117; 68% inhibition at 1x10^ mol ratio compound/TPA; 80% cell viability) and acetyloleanolic acid (119; 64% inhibition; 80% viability), showed remarkable inhibitory effects with preserved higher viabilities of Raji cells than their free alcohols, erythrodiol (130; 81% inhibition; 50% viability) and 142 (70% inhibition; 60% viability) [72]. Under the same assay conditions, betulinic acid (257) exhibited complete inhibition of activation with 80% cell viability [72]. Arjunolic acid triacetate (154; 66% inhibition at 1x10^ mol ratio compound/TPA) and arjunolic acid triacetate methylester (155; 73%) [23], and lantadene B (134; 41%) and lantadene C (136; 45%), and the hydroxylated derivative of ketone 134, i.e., 135 (59%) [27], with always >80% cell viability at the assay conditions, appear to have valuable potency as antitumor-promoters. Three cucurbitanes [25-acetyl-23,24-dihydrocucurbitacin F (93; 42% inhibition), cucurbitacin F (95; 37%), and 23,24-dihydrocucurbitacin F (96; 45%)] [69], ten quassines (106 - 115; 100%) [71] (in which enhancement of the activity by a methylenoxy bridge and side chain was observed), a taraxastane (taraxasterol, 239; 64%), and a taraxerane (taraxerol, 242; 57%) [42], two friedelanes (2,3-dihydroxy-24norfriedela-l,3,5(10),7-tetraen-29-oic acid, 249; 57%; and its diacetoxy derivative, 250; 64%) [75], three phytoecdysones: decumbesterone A (296; 34%), cyasterone (297; 24%), and polypodine B (298; 34%) [68], all showed strong inhibitory effects on EBV-EA induction at 1x10 mol ratio compound/TPA with the preservation of high viability of Raji cells. Twenty-three triterpenoid hydrocarbons isolated from ferns were screened [67]. The following eight exhibited strong inhibitory effects at 1x10^ mol ratio compound/TPA with >80% viability of Raji cells [67]. They are: multiflor-8-ene (220; 45%), multiflor-9(ll)-ene (228; 41%), taraxastane (237; 43%), taraxerane (241; 37%), glutin-5(10)-ene (244; 52%), hop-17(21)-ene (275; 51%), neohop-13(18)-ene (277; 37%), and neohop-12-ene (278; 37%). It should be mentioned that the inhibitory

60

effects of these eight compounds were stronger than those of glycyrrhetic acid (147; 46% inhibition at 5x10^ mol ratio compound/TPA and >80% viability of Raji cells) and retinoic acid, which are known to be strong antitumor-promoters [68]. Inhibitory Effects on TPA Stimulated ''^Pi Incorporation in HeLa Cells The inhibitory effect on in vitro TPA-stimulated ^^Pi-incorporation into phospholipids of HeLa cells as the primary screening test is known to correlate well with antitumor-promoter effects in vivo [28,39,78.]. Seventeen triterpenoids isolated from cacti and ten derivatives have been examined for the inhibition of TPA stimulated "^^Pi-incorporation into phospholipids of HeLa cells [79]. Echinocystic acid (cochalic acid; 128; 50% inhibition at 50 |ig/ml of compound with 50 nM of TPA), erythrodiol (130; 42%), queretaroic acid (145; 41%), oleanolic acid (142; 48%), and betulinic acid (257; 100%) showed significant inhibitory activities with 257 being the most active. The conclusion was that, in the case of lupane and oleanane type triterpenoids, the presence of the free carboxyl, formyl, or hydroxymethyl group at C-28 is important for the inhibitory effect [79]. Several other triterpenoids have been reported to be active in this assay, viz., seven oleananes (entagenic acid, 129 [43]; 18a-olean-12-ene3p,28-diol, 194 [39,43]; glycyrrhetic acid, 147 [43]; hederagenin, 156 [43]; olean-12-ene-3p,23,28-triol, 159 [39,43]; and its 18a-epimer, 195 [39,80]; and 18a-deoxoglycyrrhetol, 191 [39,43]); and two other compounds, viz., abiesenonic acid methyl ester (42) [29] and ursolic acid (210) [43]. Inhibitory Effects on TPA-induced ODC Accumulation Induction of epidermal ODC is a characteristic biochemical alteration elicited by TPA and may be representative of the effects of phorbol esters with strong tumor promoting activity [81]. A single application of TPA (5 |ig) resulted in a substantial and transient increase of epidermal ODC activity in mice with a peak at about 4 h after TPA treatment, and the induction was potently inhibited by treatment (5 |LIM) of the mouse skin with sitosterol (33, 65% inhibition) and three lupane type triterpenoids: betulin (255; 79%), betulinic acid (257; 89%), and lupeol (264,96%) [30]. The inhibitory effect on TPA-induced ODC activity was further reported for ursolic acid (210; 45% inhibition at 2.0 |aM/5nM of TPA) [31]. Inhibitory Effects on Protein Kinase C

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TPA-type tumor promoters can activate both phospholipid and Ca^^-dependent protein kinase C (PKC), an enzyme activated by endogeneous diacylglyceroi released by an activation of phospholipase C. PKC actually constitutes a family of several isozymes and is widely accepted as one of the major intracellular targets of TPA-type tumor promoters. Activated PKC undergoes phosphorylation of proteins regulating cellular differentiations and/or proliferation [6]. Glycyrrhetic acid (147) has been demonstrated to inhibit PKC activity (90% inhibition at 1 mM) [25]. The potent antagonism of tumor promotion in mouse skin by 147 may be a consequence of both its binding interactions with steroid receptors and its inhibition of PKC [25]. Nine different PKC isozymes have been identified recently by cDNA coding [6]. Several lupane type triterpenoids have been examined on the inhibitory effect against isozymes of PKC, such as piI-H, y-H, 5-H, and s-H. Dihydrobetulinic acid (272) and succinyl betulinic acid (271) showed inhibitory effect against 5-H [IC50: 272, 46|aM and 271, 49|iM] and y-H PKC [IC50 : 272, 74^M and 271, 37fiM] [32]. Inhibitory activities against y-H PKC were also observed with compound 254 (a lupane 3-benzoate) and 270 (a lupane 3-sulfate). None of the triterpenoids examined showed inhibition against pII-H and 8-H PKC(IC5o>150^M)[32]. ANTITUMOR-PROMOTING ACTIVITIES OF TRITERPENOIDS AND STEROLS Inhibitory Effects on Skin Tumors Glycyrrhetic acid (147) was the first triterpenoid shown to inhibit the tumor promotion with DMBA and TPA in mouse skin [38]. The inhibitory effect was also demonstrated for oleanolic acid (142) and ursolic acid (210). The activities of 142 and 210 were comparable to that of retinoic acid, a known inhibitor of tumor promotion [66]. For some of the compounds with highly inhibitory activities in the TPA-induced inflammatory assay (Table 2), we have evaluated their antitumor-promoting activity on two-stage carcinogenesis by DMBA and TPA in mouse skin and have found that all of the compounds evaluated possessed remarkable activity. They are two ergostanes: ergosterol (18) [36] and ergosterol peroxide (19) [51], two stigmastanes (sitosterol, 33 [30] and stigmasterol, 36 [50]), three lanostanes (16a-hydroxytrametenolic acid 3-0-acetate, 51 [54], pachymic acid, 59 [54], and poricoic acid B, 64 [54]), a cycloartane (cycloartenol ferulate, 67) [48], an euphane (euphol, 101) [82], a multiflorane (karounidiol, 225) [83], three taraxastanes (faradiol, 232 [40,84,85], heliantriol C, 236 [40,84], and taraxasterol, 239 [84,85]), and four lupanes (betulin, 255 [86], betulinic acid, 257 [30,86],

62 100

U

1 g

o

S

5 10 15 Weeks of promotion


20

Fig. (5). Inhibitory effect of taraxasterol (239) and faradiol (232) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting 1 week after initiation by a single topical application of 50 ng of DMBA, 1 ^g of TPA was applied twice weekly. Topical application of 239 (2.0 fimol), 232 (2.0 nmol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). • = +TPA with vehicle alone; O = +TPA with 239; A = +TPA with 232 [85].

lupeol, 264 [86], and lupeol acetate, 265 [86]). Of the seventeen compounds evaluated, two 3p-acetoxy-16a-hydroxylanost-8-enes with a carboxyl group at C-21 (51 and 59) and a 3p,16p,22atrihydroxytaraxastene (236), exhibited the highest activity. Fig. (5) A shows the time dependence of skin tumor formation in the groups treated with DMBA plus TPA with or without 2.0 |iM each of taraxasterol (239) and faradiol (232). The first tumor appeared at week 11 in the group treated with DMBA/TPA. In the groups treated with DMBA/TPA and 239, the first tumor appeared at week 13. Of mice treated with DMBA/TPA, 73% were tumor-bearing at week 20, as compared 20% in the group treated with DMBA/TPA and 239. Fig. (5) B shows the average number of tumors per mouse. The group treated with DMBA/TPA produced 7.1 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and 239 had 1.0 tumor per mouse. The treatment with 232 caused an 80% reduction in the average number of tumors per mouse at week 20. In the group treated with DMBA/TPA and 232, no tumors had appeared at week 20 [85]. Fig. (6) A shows the time dependence of skin tumor formation in the group treated with DMBA/TPA, with or without 0.2 |aM each of 232 and heliantriol C (236). The first tumor appeared at week 8 in the group treated with DMBA/TPA. In the group treated with DMBA/TPA and 232 and 236, the first tumor appeared at weeks 10 and 13, respectively. The percentage of tumor-bearing mice treated with DMBA/TPA was 93% at week 20,

63

100

e

10 o E

50

S

i

*a

S

ON

O

10

15

Weeks of promotion

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20

Fig. (6). Inhibitory effect of faradiol (232) and heliantriol C (236) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting 1 week after initiation by a single topical application of 50 ^g of DMBA, 1 fig of TPA was applied twice weekly. Topical application of 232 (0.2 fimol), 236 (0.2 nmol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). • = +TPA with vehicle alone; A = +TPA with 232; O = +TPA with 236 [54].

whereas the percentages in the groups treated with DMBA/TPA and 232 and 236 were 40% and 20%, respectively. Fig. (6) B shows the average number of tumor per mouse. The group treated with DMBA/TPA produced 8.6 tumors per mouse at week 20, whereas the group treated with DMBA/TPA and 232 and 236 had 2.9 and 0.9 tumors per mouse, respectively. The treatment with 232 and 236 caused 66% and 90% reductions, respectively, in the average number of tumors per mouse at week 20 [40]. Of the three compounds tested, the trihydroxy compound 236 was the best anti-tumor promoter, followed by the dihydroxy compound 232 and monohydroxy compound 239 of which activity was paralleled with the inhibitory effect on TPA-induced inflammation (Table 2). Tests of variousflavonoidsdescribed in the literature [87] also showed a close relationship between the inhibitory activities on tumor promotion and on TPA-induced inflammation. Fig. (7) A and shows the time dependence of skin tumor formation in the groups treated with DMBA/TPA, with or without 0.2 îM each of three lanostanes, pachymic acid (59), 16a-hydroxytrametenolic acid 3-0-acetate (51), and poricoic acid B (64) [54]. Thefirsttumor appeared at week 11 in the group treated with DMBA/TPA. In the groups treated with DMBA/TPA and 59, 51, and 64, the first tumor appeared at week 13, 14, and 13, respectively. The percentage of tumor-bearing mice treated with DMBA/TPA was 73% at week 20, whereas the percentages in the groups

64 100

^

JB 08

o B 5 10 15 Weeks of promotion

20


20

Fig. (7). Inhibitory effect of pachymic acid (59), 16a-hydroxytrametenoIic acid 3-O-acetate (51) and poricoic acid B (64) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting I week after initiation by a single topical application of 50 fig of DMBA, 1 ^g of TPA was applied twice weekly. Topical application of 59 (0.2 ^mol), 51 (0.2 ^mol), 64 (0.2 ^mol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). # = +TPA with vehicle alone; O = +TPA with 59; A = +TPA with 51; A = +TPA with 64 [40].

treated with DMBA/TPA and 59, 51, and 64 were 27, 33, and 33%, respectively. Fig. (7) B shows the average number of tumors per mouse. The group treated with DMBA/TPA produced 7.1 tumors per mouse at week 20, whereas the groups treated with DMBA/TPA and 59, 51, and 64 had 1.2, 0.6, and 2.3 tumors/mouse, respectively. The treatments with 59, 51, and 64 caused a 83, 92, and 68% reduction, respectively, in the average number of tumors per mouse at week 20 [54]. The inhibitory effects of 0.2 |LiM of 51,59, and 64, as well as of the two taraxastanes (232 and 236) [40], corresponded to those of 2.0 |LIM of the other sterols (18,19,33, and 36) and triterpenoids (67,101, 225, 239, 255, 257, 264, and 265) described above, and, therefore, compounds 51, 59, and 64 had about ten times the activity of the other compounds on tumor promotion induced by TPA in mouse skin. The following compounds have further been revealed to possess activity on tumor promotion by TPA in two-stage carcinogenesis initiated with DMBA in mouse skin: a lanostane (abiesnonic acid methyl ester, 42) [29]; two cucurbitanes (cucurbitacin F, 95, and 23,24-dihydrocucurbitacin F, 96) [69]; nine oleananes (arjunolic acid triacetate, 154, and arjunolic acid triacetate methylester , 155 [74], erythrodiol, 130 [39], lantadene A, 132, and lantadene B, 134 [45], olean-12-ene-3p,23,28-triol, 157, and 18a-glycyrrhetinic acid, 193 [88], and 18a-olean-12-ene-3p,28-diol, 194, and 18a-olean-12-ene-3p,23,28-triol, 195 [39,43]); a taraxerane (taraxerol,

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242) [42]; two hopanes [hop-17(21)-ene, 275, and neohop-13(18)-ene, 277] [67]; and a phytoecdysone (cyasterone, 297) [68]. It must be pointed out that the 18a-oleanane triterpenoid 195, prepared from glycyrrhetic acid (147), was 100 times more active than 147 [39]. 01ean-12-ene-3p,23,28-triol tri-0-hemiphthalate sodium (158), on oral administration, has been proved to suppress carcinogenesis in mouse skin induced by DMBA and TPA [43,89]. This is the first report of an effective oral administration of triterpenoid suppressing skin tumor promotion in mice. Glycyrrhetic acid (147) has also been demonstrated to noticeably suppress the promoting effect of teleocidin B (3) on skin tumor formation in mice induced by DMBA [28,38]. Inhibitory Effects on Colon, Liver, Lung, and Mammary Tumors Several sterols and triterpenoids have been shown to possess preventive activities, both on skin tumors and on tumors of internal organs, v/z., colon, liver, lung, and mammary tumors. The compounds that inhibit nitrosamine formation in the assays described below have been suggested to be inhibitors of carcinogen formation and they inhibit 4-(methylnitrosamino)-1 -(3 -pyridyl)-1 -butanone (NNK) induced tumors by acting as blocking (anti-initiating) agents [3]. The inhibitory effect of sitosterol (33) on colon tumor formation in rats treated with the carcinogen A'^-methyl-A^-nitrosourea (MNU) has been studied, and it has been demonstrated that compound 33 nullified in part the effect of this direct-acting carcinogen on the colon [44]. This suggested that phytosterols may have a protective dietary action to retard colon tumor formation. Squalene (287) is an acyclic triterpenoid and a key biosynthetic intermediate for the other cyclic triterpenoids and sterols. Its chemopreventive efficacy on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) has been assessed [90]. Oral administration of 287 inhibited total ACF induction and crypt multiplicity by 46%, and at a level of 1% 287 did not show any significant effect on serum cholesterol (7) levels. Squalene (287) is a characteristic constituent of olive oil, and epidemiologic and laboratory studies have suggested a cancer protective effect and/or lack of a tumor promoting effect by dietary olive oil as compared with other types of non-marine oils. Compound 287 significantly suppressed ACF formation and crypt multiplicity in the colon. This strengthened the hypothesis that 287 has chemopreventive activity against colon carcinogenesis [90]. In addition, compound 287, as well as olive oil, by oral administration, has been revealed to possess inhibitory effect on NNK-induced lung tumorigenesis thus demonstrating that dietary olive oil and 287 can effectively inhibit NNK-induced lung tumorigenesis

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[46]. The inhibitory effect of olean-12-ene-3p,23,28-triol tri-0-hemiphthaiate sodium (158), ursoHc acid (210), and betulinic acid (257) on lung tumors have been reported [43]. Lantadene B (134), on oral administration, has an inhibitory effect on mouse hepatic tumors induced by A^-nitrosodiethylamine and phenobarbital [45]. The effect of dietary cholesterol (7) on mammary tumor development was examined in female Sprague-Dawley rats exposed to the carcinogen MNU [47]. Tumor incidence in the compound 7 group (67%) was significantly lower than in the control group (96%) which suggested that dietary 7 inhibits mammary tumor development in this model. Taraxasterol (239), on oral administration, showed remarkable inhibitory effects on mouse spontaneous mammary tumors using C3H/0iJ mouse [42]. THE MECHANISMS OF ANTITUMOR-PROMOTION The mechanisms by which antitumor-promoters suppress the tumor promotion are not Imown, but may be due to the following effects: (i) inhibition of polyamine metabolism; (ii) inhibition of arachidonic acid metabolism; (iii) protease inhibition; (iv) induction of differentiation; (v) inhibition of oncogene expression; (vi) inhibition of PKC; and (vii) inhibition of oxidative DNA damage [3,6,91]. The polyamine content of cells is correlated to their proliferative, and often, their neoplastic capabilities. A key enzyme in the polyamine biosynthetic pathway, ornithine decarboxylase (ODC), catalyzes the convertion of omithine to putrescine. Phorbol ester promoters such as TPA cause increased ODC activity and accumulation of polyamines in affected tissues. Diacylglycerol activated PKC, and the potent tumor promoter, TPA, binds to, and activates PKC, in competition with diacylglycerol. PKC stimulation results in phosphorylation of regulatory proteins that affect cell proliferation. Some chemopreventive agents have inhibitory activity towards PKC. Refer to recent review articles for further discussion [3,6,91]. A TRITERPENOID AS A TUMOR-FROMOTER As discussed in the previous sections, a number of triterpenoids with great structural diversity have antitumor-promoting activities. In contrast, 28-deacetylbelamcandal (288), a spiroiridal-type triterpenoid, has recently been reported to possess tumor promoting activity [92]. Compound 288, which stimulated differentiation of human promyelocytic leukemia (HL-60) cells (a fast method for screening TPA-type tumor promoters).

67

bound to and activated PKC, and induced tumor necrosis factor-a release from HL-60 cells in the same manner as TPA. In an in vivo study, groups treated with 100 jag DMBA plus 400 nM of 288 showed 64.3% tumor incidence by week 20. Compound 288 represents a new structural class of mouse skin-tumor promoters. Iridal-type triterpenoids are characteristic constituents of plants in the genera of Iris and Belamcanda, and it has been suggested that other congeners of 288 may be potential mouse skin-tumor promoters. ANTI-INFLAMMATORY AND ANTI-ALLERGIC ACTIVITIES OF TRITERPENOIDS AND STEROLS Inflammation is one of physiological responses of organisms when they suffer physically or chemically induced stress, and comprises complex processes influenced by chemical mediators [35]. The mediators belong to different chemical classes, such as biologically active amine (histamine, serotonin), proteins and peptides (hydrolytic enzymes, cytokines, growth factors, colony stimulating factors, complement factors, antibodies, kinines), activated oxygen species (superoxide anion, hydroperoxide, hydroxyl radicals), and lipids (PAF, prostanoids, leukotrienes) [93]. Anti-inflammatory compounds can suppress the inflammation by inhibiting activity or by interaction with one or several of the above cited chemical mediators. Many triterpenoids have been used as antiinflammatory remedies in folk medicines and have been reported to possess anti-inflammatory and anti-allergic activities in various experimental models. In Table 1 are listed selected fourteen each of in vivo and in vitro bioassay systems, among a number of assay systems reported in the literature, applied to triterpenoids and sterols for their anti-inflammatory and anti-allergic activities. Table 2 includes the bioassay systems in which individual triterpenoids and sterols exhibited significant activities. The concept that inflammation and carcinogenesis are related phenomena has been the subject of many studies that have attempted to link these two processes in a mechanistic fashion [94-99]. In connection with this, as is evident in some triterpenoids such as oleanolic acid (142), glycyrrhetic acid (147), and ursolic acid (210) in Table 2, those possessing anti-inflammatory activity in various experimental models exhibited antitumor-promoting activity as well. This suggests that most of the other anti-inflammatory triterpenoids and sterols listed in Table 2 might have antitumor-promoting properties which have not been evaluated, yet. Two inflammatory enzymes, inducible nitric acid oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) have critical roles in the response of tissues to injury or infectious agents. These inducible enzymes are

68

essential components of the inflammatory response, the ultimate repair of injury, and carcinogenesis [97,99]. More than eighty novel triterpenoids have been synthesized from 142 and 210, and have been tested for their ability to suppress the de novo formation of two enzymes, iNOS and COX-2 [97]. Two synthetic oleananes, 3,ll-dioxoolean-l,12-dien-28-oic acid (166) and 3,12-dioxoolean-l-en-28-oic acid (168), were found to be highly active. Then, new derivatives with electron-withdrawing substituents at the C-2 position of 3-oxoolean-l-en-28-oic acid were synthesized [97,100]. Among them, 2-cyano-3,12-dioxooleane-l,9dien-28-oic acid (164) was 400 times potent than previous compounds prepared as an inhibitor of iNOS (IC50, 0.4 nM). The potency of 164 was similar to that of dexamethasone, although 164 does not act through the glucocorticoid receptor [98,101]. CONCLUSION Even though the antitumor promoting activities of many of the naturally occurring triterpenoids and sterols from plants and fungi described in this review are not remarkably high, use of these compounds might be advantageous because they are considered to be non-toxic or less toxic and to show fewer side effects [91,102,103]. Thus steroids and triterpenoids are possible candidates for cancer chemopreventive agents. In the future, more mechanistic-oriented basic research is needed to elucidate the mechanisms of action. Studies of derivatives of these naturally occurring triterpenoids and sterols are also necessary to elucidate the structure-activity relationship and to guide the development of novel antitumor-promoters. ABBREVIATIONS Refer to Table 1 for abbreviations. ACKNOWLEDGEMENT We thank Dr. W. C. M. C. Kokke (ChiRex Cauldron, Malvern, Pennsylvania, U.S.A.) for his help during the preparation of this review.

Table 2. Triterpenoids, and Sterols and Their Oxygenated Derivatives from Plants and Fungi and the Bioassay Systems in which the Compounds Exhibited inhibitory Activities Compound Reference Compound Quercetin lndomethacin Hydrocortisone 1. Cholestane Cholesterol 4,4-Dimethylcholesterol 4,4-Dimethylcholest-5-en3-one 4,4-Dimethyl-5,6aepoxycholestan-3~-ol 4,4-Dimethyl-5,6a-epoxycholestan-3-one 4,4-Dimethylcholestane-3a,5a-diol 4,4-Dimethyllathosterol Lophenol 4-Methylcholest-4-en-3-01 7-Oxochoiesterol 11. Ergostane 9(11)-Dehydroergosterol Ergosterol Ergosterol peroxide 24-Methylcholesterol ferulate Ill. Stigmastane 7a-Acetoxysitosteryl acetate 7B-Acetoxysitosteryl acetate 7,9(1 I)-Bisdehydroporiferasterol Citrostadienol 'See Table 1 for the assay system. IDSO(mgtear).

Code Source and Occurrence 4 5 6

The most common flavonoid in higher plants Commercial Commercial

Animal fats; Minor sterols of many higher plants 8 Prepared from 7 9 Prepared from 7 10 Prepared from 7 11 Prepared from 7 12 Prepared from 7 13 Prepared from 7 14 Liliaceae; Solanaceae; Cactaceae 15 Prepared from 7 16 Woolwax 7

Bioassay Systema TPAD Other Assays 1.6

136,371

0.3 0.03

[36,371 [36,371

>2.0 Mammary I; HPA

[35,37,47]

0.3

[30,35,37] [351 I351 [351 [351 [30,371 [30,371 130,371 [37,5 11

>2.0 0.3

20 21 22 23 24

Prepared from 33 Prepared from 33 Chlorella vulgaris Widespread occurrence in higher plants

>2.0 0.5 >2.0

19

HPA HPA HPA HPA HPA

0.4 0.2 0.3 0.2

Chlorella vulgaris (Chlorella) Major sterol of fungi and yeast; Chlorella vulgaris Chlorella vulgaris Rice bran

17 18

References

0.2 0.3

Skin I Skin I

A CM

[37,851 [36,371 [36,37,5I] [481 [37,521 137,521 [37,851 [I 141 OI

\O

Table 2. Continued-1 Compound 7-Dehydroporiferasterol . . 7-Dehydroporiferasterol peroxide Fucosterol 4$-Hydroxysitosterol 7a-Hydroxysitosterol 7$-Hydroxysitosterol 7-(;brositosterol Schottenol Sitosterol Sitosterol ferulate Spinasterol Stigmasterol Stigmast-4-ene-3$,6$-diol Stigmastane-3$,6a-diol Stigmastane-3$,6$-diol Stigmastane-3,6-dione Stigmast-4-ene-3,6-dione N. Lanostane Abiesenonic acid methyl ester Dehydroeburiconicacid Dehydropachymic acid Dehydrotrametenolic acid Dehydrotumulosic acid 24,25-Dihydrolanosterol 3-Epidehydrotumulosicacid Ganodelic acid A Ganodelic acid B Ganodelic acid C Ganodelic acid D 30-u -Hydroxvbenzoyldehydrotumulosicacid

2 0

Code Source and Occurrence TPAD Other Assays 25 ChloreNa vulgaris 0.5 26 Chlorella vulgaris 0.7 27 Brown algae; Solanaceae; Olive; Rice bran >2.0 28 Trichosanrhes kirilowii (Cucurbitaceae) 0.8 seeds >2.0 29 Trichosantheskirilowii seeds 30 Trichosantheskirilowii seeds 0.6 31 Cucurbitaceae 1.0 1.0 32 Theaceae; Cucrubitaceae; Amaranthaceae 33 Major sterol of most higher plants 1.8 Colon, Skin I, CAR, ODC 0.2 34 Rice bran 1.1 35 Theaceae; Cucurbitaceae; Spinach 36 Major sterol of most higher plants 1.9 Skin I 0.6 37 Prepared from 33 38 Trichosantheskirilowii seeds 0.5 0.6 39 Prepared from 33 40 Polygonum chinensis (Polygonaceae) HSR, SOF HSR, SOF 41 Polygonum chinensis 42 53

54 43 55 44 56 45 46 47 48 57

Prepared from abieslactone isolated from Abies mariessi Fomes oficinalis (Polyporaceae) Poria COCOS Poria cocos Poria cocos Solanaceae seeds; Animal fats Poria cocos Ganodenna lucidum (Polyporaceae) Ganoderma lucidum Ganoderma lucidum Ganoderma lucidwn Poria cocos (Hoelen; Polyporaceae)

Skin I, HeLa 0.03 0.02 AA 0.03 AA TPA AA, CAR, PLA2 0.2 0.09 HSR HSR HSR HSR 0.3 AA

References 137.511 . [37,511 [30,371 137,521 [37,521 [37,521 137,521 [37,50 [20,30,44,50,128,129] [481 [20,501 [30,37,50] [37,52] [37,521 [37,521 [I221 [I221 [29,130] [37,551 [37,55,56,60] [37,55,56] [60,61,126] [30,371 [37,551 11231 [ 1231 [I231 [I231 [56]

Table 2. Continued-2 Compound

16a-Hydroxydehydrotrarnetenolicacid 16a-Hydroxytrametenolicacid 16a-Hydroxytrametenolicacid 3-0 -acetate Lanosterol 24-Methylenelanost-9(1 l)-en-3P-ol Pachymic acid Polyporenic acid C Poricoic acid A Poricoic acid AM Poricoic acid B Poricoic acid D Tumulosic acid V. Cycloartane Cycloartenol Cycloartenol ferulate 1-oic (24R ,S)-24,25-Epoxy-3-oxocycloartan-2 acid 3B-Hydroxycycloart-24sn-21-al 30-Hydroxycycloart-24en-21-oic acid Methyl (24R ,S)-24,25-epoxy-3-oxocycIoartan2 1+ate 24-Methylenecycloartanol 24-Methylenecycloartanol ferulate Methyl 25,26,27-Trisnor-3-oxocycIoartan-24-al21-oate Methyl 3B-hydroxycycloart-24-en-2I-oate Methyl 3-oxocycloart-24en-2l-oate 3-0xocycloart-24en-214 3-Oxocycloart-24-en-21sicacid 3-0xocycloart-24-en-21-01

Code 49 50 51 52 58 59 60 62 63 64 65 61

Source and Occurrence Poria COCOS Poria cocos Poria COCOS Solanaceae seeds; Yeast; Animal fats Theaceae seeds Poria cocos and other fungi Polyporus spp. and other fungi Poria COCOS Poria COCOS Poria COCOS Poria COCOS Poria COCOS

66 67 68

Widespread occurrence in higher plants Rice bran, y-Oryzanol Prepared from 77

69 70 71

Prepared from 77 Prepared 6om 77 Prepared from 77

72 73 79

Widespread occurrence in higher plants Rice bran Prepared from 77

74 Prepared from 77 75 Prepared from 77 76 Prepared from 77 77 Lansium domesticum (Meliaceae) 78 Prepared from 77 25,26,27-Trisnor-3-oxocyc10artane-21,24-dial 80 Prepared 6om 77 VI. Dammarane Dammaradienol 81 Dammar resin; Shea butter; Theaceae

TPAD Other Assays TPA TPA 0.02 Skin I 0.2 0.4 0.04 Skin 1, AA, CAR, PLA2 0.1 0.03 AA 0.08 0.02 Skin 1, AA 0.1 0.2 0.3 0.2

CAR Skin I EBV

[37,59,133] 1481 1241

EBV EBV EBV

1241 [241 1241

EBV

[20,37,59] [48] 1241

EBV EBV EBV EBV EBV EBV

1241 1241 [241 [24,134] [241 1241

0.2 0.2

0.8

References 1601 [601 [37,54,55] [20,37,55] [37,131] [37,54,55,61,126,132] 137,551 [37,55,56] [37,55,56] [37,54,55,56] 137,551 137,551

137,591

c .I

Table 2. Continued3 Compound (24R ,S)-24,25-Epoxydammaradienol Isoeuphol Isotimcallol Mansumbinone Mansumbinoic acid 24-Methylenedammarenol V11. Cucurbitane 25-Acetyl-23,24-dihydrocucurbitacinF Cucurbitacin B Cucurbitacin F I Oa-Cucurbitadienol 23,24-Dihydrocucurbitacin F 24-Dihydrel Oa-cucurbitadienol 7-0~0-1Oa-cucurbitadienol

74x0-10a-cucurbitadienol acetate 7-0~0-24-dihydro- 1Oa-cucurbitadienol 15-OxocucurbitacinF 15-0~0-23,24dihydrocucurbitacinF VIII. Euphane Butyrospermol (24R ,S)-24,25-Epoxybutyrospermol Euphol IX.Tirucallane Masticadienoic acid Shinol (3B-Masticadienolic acid) A'-~imcallol Timcallol

4

Code Source and Occurrence 82 Theaceae seeds 83 Theaceae seeds 84 Theaceae seeds 86 Commiphora incisa (Burseraceae) resin 87 Commiphora incisa resin 85 Shea butter Hernsleya carnosflora (Cucurbitaceae) rhizomes 94 Wilbrandia ebracteata (Cucurbitaceae) 95 Cowania mexicana (Rosaceae) leaves and branches 88 Cucurbitaceae seeds 96 Hemrleya carnosflora rhizomes 89 Prepared from 88 90 Trichosanthes kirilowii (Cucurbitaceae) seeds 91 Prepared from 88 92 Prepared from 88 97 Cmvania mexicana leaves and branches 98 Co~vaniamexicana leaves and branches

TPAD Other Assays 0.5 0.3 0.3 CAR CAR 0.5

93

99 Theaceae seeds 100 Theaceae seeds 101 Euphorbia spp. latex; Theaceae seeds

[691

CAR EBV

11351 [26,691

Skin I, EBV >2.0 0.7 0.7 0.4

102 Schinus terebinrhifolius (Anacardiacee)

berries 103 Schinus rerebinrhifolius berries 104 Theaceae seeds 105 Euphorbia spp. latex; Theaceae seeds

EBV

>2.0

0.6 0.5 0.2

0.8 0.4

N

References [37,131] [37,131] [37,131] [log] [ 1091 [37,131]

[37,136] [26,691 [37,136] [37,136]

EBV EBV

[37,136] [37,136] [261 [261

Skin l

[37,131] [37,131] [37,82,131]

PLAZ

11261

PLA2

[ 1261 [37,591 11311

Table 2. Continued-4 Compound X. Quassine Bruceanol-A Bruceanol-B Bruceanol-C Bruceanol-D Bruceanol-E Bruceanol-G Bruceantin Isobruceine-B Dehydrobruceantinol Dehydrobruceantin XI. Oleanane 3p-Acetoxylean-l2en-27-oicacid 3 - 0 -Acetylerythrodiol Acetyloleanolic acid B-Amyrin B-Amyrin acetate &Amyrin Arjunolic acid Arjunolic acid methylester Arjunolic acid triacetate Arjunolic acid triacetate methylester Crategolic acid

Code Source and Occurrence

TPA' Other Assays

References

106 107 108 109 110 111 112 113 114 115

Brucea antidysenterica (Simaroubaceae) Brucea antidysenterica Brucea antidysenrerica Brucea antidysenterica Brucea anridysenterica Brucea antidysenterica Brucea antidysenrerica Brucea antidysenterica Brucea anridysenierica Brucea antidysenterica

EBV EBV EBV EBV EBV EBV EBV EBV EBV EBV

1711 1711 1711 ~711 1711 [711 [711 [711 [711 [711

116 117 118 119 120 179 152

Vitex negundo (Verbenaceae) seeds Eupteleapolyandra bark Prepared from 142 by acetylation Widespread occurrence in higher plants Prepared from 119 by acetylation Theaceae seeds Cochlorspermumtinctorium (Cochlorspermaceae) rhizomes Prepared from 152 Prepared from 152 Prepared from 152 Boswellia serrata resin Prepared from 142 Prepared from 142

CAR EBV EBV CAR, CRO,EPP, 5LOX 5LOX EBV

[I371 [721 1721 [33,37,59,62,138] 11381 [37,131] 123,741

EBV Skin I, EBV Skin I, EBV ACM iNOS C O X , iNOS

123,741 [23,741 [23,741 11131 [lo31 [98,101]

2-Cyano-3-oxooleana-l,12-dien-28-oic acid 2-Cyano-3,12dioxoolana-1,9(11)-dien-28-oic

153 154 155 146 164 165

acid Deoxoglycyrrhetol Deoxoglycynhetol di-PHT 2Na

121 Prepared from 147 122 Prepared from 147

Deoxoglycyrrhetol-di-0-SUC 2Na

123 Prepared from 147

0.4 0.3

SLOX, l2LOX TPA AA.BRA,CAR,COX,CPS, DTA, HPT, HSI, 5LOX 12LOX, PAF COX, 5LOX l2LOX

[I171 [43,65,107,110,117, 1391 [I 171

Table 2. Continuedd Compound Deoxoglycyrrhetol-di-0-phosphate 4Na Dwxoglycyrrhetol3-0-PHT Na 18a-Deoxoglycyrrhetol 18a-Deoxoglycyrrhetol-di-0 -PHT 2Na

Code Source and Occurrence TPAD Other Assays References 124 Prepared from 147 I2LOX [I171 125 Prepared from 147 SLOX, I2LOX [I171 191 Prepared from 147 HeLa [431 192 Prepared from 147 AA [I391 2a,3~-Diacetoxy-l8-hydroxyoleana-5,12-dien-185 Vitex negundo (Verbenaceae) seeds CAR 11371 28-oic acid 2a,3B-Diacetoxyolean-5,12-dien-28-oicacid 186 Vitex negundo seeds CAR 11371 2~,3a-Diacetoxyolean-5,12-dien-28-oic acid 187 Vitex negundo seeds CAR 11371 126 Prepared from 141 by acetylation EBV 1721 2,3-Di-0 -acetylmaslinic acid 2~,3a-Dihydroxyoleana-5,12-dien-28-oic acid 188 Vitex negundo seeds CAR 11371 127 Eupteleapolycndra (Eupteleaceae)bark EBV 1721 1,3-Dioxoolean-12-ene acid 166 Prepared from 142 COX2, iNOS [97,100,101] 3,l I-Dioxoolean-1,12-dien-28-oic 3,11-Dixoxooleana-l,13(18)-dien-28-oicacid 167 Prepared from 142 iNOS 11001 3,12-Dioxoolean-1-en-28-oic acid 168 Prepared from 142 COX2, iNOS [97,100] 3,12-Dioxooleana-l,9(11)-dien-28-oicacid 169 Prepared from 142 iNOS [100,101] 128 Sapogenin from Echinocystis spp., 0.2 EBV, HeLa [16,30,37,43,140-1421 Echinocystic acid (Cochalic acid) Albizzia spp. Entagenic acid 129 Entada phaeseolides (Leguminosae) HeLa [431 acid 170 Prepared from 142 9 0 1K-Epoxy-3-oxoolean-l-en-28-oic iNOS 11001 Erythrodiol 130 Olive, grape, Compositae, and other plants TPA Skin l,CAR DTA, EPP, HeLa [39,43,62,79,11 I] 180 Theaceae seeds Germanicol 0.9 [37,131] 147 Sapogenin from GIycyrrhiza glabra and Glycyi-rhetic acid (Glycyrrhetinic acid) [16,25,28,35,38,43,62, 0.1 Skin I, 111, AA, ACM, cAK, C A R , EBV, EPP, HeLa, 67,68,86,88,89,93,107, some other plants HPA, HSI, PKC 113,115,1431 Glycynhetinyl stearate 148 Prepared from 147 CTN 1891 149 Prepared from 147 Glycyrrhetinic acid 3-PHT Na TPA 143,651 I2LOX 11171 150 Prepared from 147 Glycyrrhetinic acid 3-PHT 2Na 193 Commercial; Prepared from 147 TPA Skin I,cAK, CAR, EPP, HPT [62,88,115] 18a-Glycyrrhetinic aicd 156 Sapogenin from Clematis, Holboellia , 0.1 AA. ACM, CAR, CRO, EPP, [20,37,43,59,62,93,124 Hederagenin Hedera spp. HeL& HYA ,144, 1451 131 Prepared from 132 by alkaline hydrolysis EBV 1271 228-Hydroxyoleanonicacid 2-Hydroxy-3-oxooleana-1,12-dien-28-oic acid 171 Prepared from 142 iNOS fl00,lOll

2

Table 2. Continued-6 Com~ound Lantadene A "Lantadenol A" Lantadene B "Lantadenol B" Lantadene C Longispinogenin Maniladiol Maniladiol3-0 -myristate Maniladiol3-0 -palmitate Maslinic acid 2-Methoxy-3-oxooleana-1,12-dien-28-oicacid Methyl 3-oxooleana-1,12-dien-28-oate Oleanonic acid Olean-12ene-3a, 168-diol Oleanolic acid

Code Source and Occurrence TPAD Other Assavs 132 Lantana camara (Verbenaceae)leaves Liver, Skin I, EBV EBV 133 Prepared from 132 by NaBH4 reduction Liver, Skin I, EBV 134 Lantana camara leaves EBV 135 Prepared from 134 by NaBH, reduction 136 Lantana camara leaves EBV 137 Compositae flowers 138 Compositae flowers 139 Edible chrysanthemum flower 140 Edible chrysanthemum flower 141 Eupteleapolyandra bark EBV 172 Prepared from 142 iNOS iNOS 173 Prepared from 142 143 Commerical; Prepared from 142 EBV, iNOS HPT 144 Canarium album (Burseraceae) Skin 1, ACM, AIP, CAR, cAK, 142 Occurs as glycosides in olive leaves, sugar beet, panax rhizomes, etc. COX2,DEX,EBV,EPP, FAA, HeLa, HLE, HPT, HYA, iNOS Skin I, HeLa 157 Prepared from 156 Olean-12-ene-38,23,28-trio1 Lung, Skin 11 Olean-l2-ene-3P,23,28-trioltri-PHT Na 158 Prepared from 156 159 Commiphora merkeri (Burseraceae) roots CAR Olean-12-ene-2a,3~,23-triol TPA 181 Prepared from 147 Oleana-9( 1I), 12-diene-313.30-diol TPA AA,BRA,CAR,COX,CPS. Oleana-9(11),12-diene-3P,30-diol di-PHT 2Na 182 Prepared from 147 DTA, HPT, HSI, 5LOX I2LOX PAF TPA Oleana-11,13(18)-diene-3~,30-diol 183 Prepared from 147 TPA AA, BRA, CAR, COX, CPS, Oleana-1 1,13(18)-diene-3P,30-diol di-PHT 2Na 184 Prepared from 147 DTA, HPT, HSI, 5LOX, 12LOX PAF Skin I, HeLa 18a-Olean-l2-ene-3D,28-diol 194 Prepared from 142 Skin I, HeLa 18a-Olean-l2-ene-3(3,23,28-triol 195 Prepared from 156 3-Oxoolean-1-en-28-oic acid iNOS 174 Prepared from 142

References 127,451 1271 [27,451 [271 1271 137,581 [37,581 [ 1461 11461 1721 [100,101] [loo] [22,100] [ 1201 [22,30,31,37,43,62,66, 72,93,103,108,113,115, 118,124,147,148] [39,431 [89,130] 11491 [651 [43,65,80,89,107,110, 1171 1651 [43,65,80,89,107,110, 117,1391 [39,431 [39,431 [I 001

Table 2. Continued-7 Compound 3-Oxooleana-1,9(1 I)-dien-28-oic acid 3-Oxooleana-1,12-dien-28-oicacid 3-Oxooleana-1,12-dien-2-al-28-oic acid 3-Oxooieana-I, 11,13(18)-trien-28-oic acid Papyriogenin A

Papyriogenin C Queretaroic acid Soyasapogenol B Soyasapogenol E Stearyl glycyrrhetinate Wistariasapogenol A Wistariasapogenol B XII. Ursane Acetyl-l I-keto-S-boswellic acid (AKBA) a-Amyrin a-Amyrin acetate a-Amyrin palmitate a-Amyrin linoleate D-Boswellic acid Brein Brein 3 - 0 -myristate Brein 3-0-palmitate 3,ll-Dioxoursa-I, 12-dien-28-oic acid 3-Epiursolic acid 3-Oxoursa-I, 12-dien-28-oic acid Tormentic acid Urs-l2ene-3a, 16p-diol(3-Epibrein)

Code Source and Occurrence 175 Prepared from 142 176 Prepared from 142 177 Prepared from 142 178 Prepared from 142 189 Aglycone of papyrioside L-Ila from Tefrapannrpapyrifenrm(Araliaceae) leaves 190 Aglycone of papyrioside L-IIb 145 Sfenocereussfellatus (Cactaceae) 160 Wistaria brachybotrys (Leguminosae) knots 161 Sapogenin of wistariasaponin D 151 Prepared from 147 162 Wistaria brachyborrys knots 163 Wistaria brachybortys knots

TPAD Other Assays iNOS iNOS iNOS iNOS CAR

CAR HeLa EBV

EBV CTN EBV EBV

Boswellia serrafa resin 5LOX HLA Widespread occurrence in higher plants 0.2 cAK, CAR, EPP Prepared from 197 by acetylation 5LOX Synthesis; Alstonia boonei (Apocynaceae) AIP, cAK Prepared from 197 cAK, 5LOX Gum resin of BosweNia serrata ACM Compositae flowers; Canarium album 0.05 HPT fruits 0.2 Edible chrysanthemum flower 0.2 Edible chrysanthemum flower iNOS Prepared from 210 Prepared from 210 COX2 Prepared from 210 iNOS Poferiumancisfroides (Rosaceae) TPA CAR, EPP 209 Canarium album (Burseraceae) h i t and HPT 196 197 198 199 200

References [I 001

Table 2. Continued-8

Rhododendron spp. (Ericaceae)

Uvaol

211 Olive oil; Ilex latifolia (Aquifoliaceae);

0.1

CAR,COX2,DTAA,EBV 72,91,93,102,111,113, HeLa,HLE,HPA,HPT, 115,118,119,130,158HSR, 5LOX ISLOX, O X , 1601 SER CAR, EPP, ELE, HPT [37,58,62,93,115]

Compositae

X I I I . Multiflorane Bryonolol Bryonolol3-SUC K Bryonolol di-SUC 2K Bryonolol di-PHT 2K Bryonolic acid Bryonolic acid 3-SUC 2K Bryonolic acid 3-PHT 2K 3-Epibryonolol 3-Epikarounidiol 3P-Hydroxylmultiflora-7,9(1I)-dien-29-oic acid 3-SUC 2K Karounidiol Karounidiol3-benzoate Multiflor-8-ene Multiflorenol Multiflor-9(11)-ene 7-Oxoisomultiflorenol 7-Oxodihydrokarounidiol 7-0xo-8P-multiflor-9(I1)-ene-3a,29-diol diacetate XIV. Taraxastane Amidiol Amidiol3-0 -palmitate

212 213 214 215 216 217 218 219 223 224

Trichosanthes kirilowii seeds Prepared from 212 Prepared from 212 Prepared from 212 LuJa cylindrica cell suspension culture; Bryonia dioica roots Prepared from 216 Prepared from 216 Trichosanthes kirilowii seeds Trichosanthes kirilowii seeds Prepared from 216

>2.0 DTA, ITA DTA, ITA DTA, ITA DTA, ITA DTA, ITA

Trichosanthes kiriloivii seeds Trichosanthes krriloivii seeds Fern rhizomes Benincasa hispida (Cucurbitaceae)fruits Fern rhizomes Trichosanthes kirilowii seeds Trichosanthes kiriloivii seeds Trichosanthes kirilowii seeds

0.4 0.2

230 Compositae flowers 231 Edible chrysanthemum flower

DTA, ITA DTA, ITA 0.2 0.6 DTA, ITA Skin l EBV HSR EBV 0.2 0.3 0.8

Table 2. Continued-9 Compound Faradiol Faradiol3-0 -myristate Faradiol3-0 -palmitate Heliantriol Bo Heliantriol C Taraxastane v-Taraxasterol Taraxasterol Taraxasterol acetate XV. Taraxerane Taraxerane Taraxerol XVI. Glutinane Glutinol (Alnusenol) Glutin-5(lO)ene XVII. Friedelane Celastrol 2,3-Dihydroxy-24-norfriedera-1,3,5(10),7tetraen-29-oic acid 2,3-Diacetoxy-24-norfriedera-1,3,5(10),7tetraen-29-oic acid Epifriedelanol Friedelin Pristimerin Regenol B XVIII. Lupane

3~-Acetoxylup-20(29)-en-30-al Acetyl betulinic acid Benzoyl betulinic acid

w 4

Code Source and Occurrence 232 Compositae flowers 233 Edible chrysanthemum flower 234 Edible chrysanthemum flower 235 Compositae flowers 236 Compositae flowers 237 Fern rhizomes 238 Compositae 239 Compositae

TPA" 0.2 1.0 0.9 0.1 0.03 0.4 0.3

240 Echinops echinatus (Compositae) 241 Fern rhizomes 242 Compositae; Rhododendron spp.; Euphorbia spp.

Other Assays Skin I, CRO CRO CRO Skin I EBV CRO

Mammary 11, Skin I, CRO, EBV AIP, CAR. FAA

References [33,34,37,40,58,85] [33,146] [33,146] [37,581 [37,40,58] [671 [33,34,37,59] [33,37,42,59,85] [lo51

EBV Skin I, EBV

[671 [37,42,59]

243 Benincasa hispida fruits 244 Fern rhizome

HSI EBV

[I211 [671

245 Triptetygium wiflordii (Celastraceae) 249 Prepared from 245

EBV EBV

[751 1751

250 Prepared from 245

EBV

[751

CAR CAR

[I631 [20,30,37,163]

iNOS EBV

1991 [751

EBV PKC

[37,861 [72] [32]

247 Calyophyllumapetalum 248 Cork; Lingnania chungii ;Calyophyllum apetalum 246 Celastraceae; Hippocrateaceae 251 Tripterygium wilfordii (Celastraceae) 252 Prepared from 264 253 Prepared from 257 by acetylation 254 Prepared from 257 by benzoylation

0.4

0.9

0.8

Table 2. Continued-10 Compound Betulin (Betulinol) Betulonaldehyde Betulinic acid Calenduladiol Calenduladiol 3-0-palmitate Dihydrobetulinicacid 3-Epilupeol Heliantriol B2 30-Hydroxylupeol

3~-Hydroxylup-20(29)-en-30-al Lupeol

Code Source and Occurrence 255 Birch bark; Corylus avellana (Betulaceae); Vicia.faba 256 Prepared from 264 257 Cornusflorida (Cornaceae); Ziryphus vulgaris (Rhamnaceae) 258 Compositae flowers 259 Edible chrysanthemum flowers 272 Prepared from 257 260 Prepared from 264 261 Compositae flowers 262 Flourensia heteroleptis and other higher plants. 263 Prepared from 264 264 Widespread occurrence in higher plants

269 270 271

Vernonia cinerea and many other higher plants Acacia dealbata ;Stevia Prepared from 264 Acacia dealbata ; Cneorum tricoccon Prepared from 264 Pluchea lanceolata (Compositae) stem and leaves Pyracantha crenulata (Rosaceae) Prepared from 257 Prepared from 257

275 277 278 276

Fern rhizomes Fern rhizomes Fern rhizomes Bacterium Zymomonas mobilis

Lupeol acetate

265

Lupeol palmitate Lupeol linoleate Lupenone Lupanol Neolupenol (lup-12-en-3P-01)

266 267 268 273 274

Pyracrenic acid Sulfonyl betulinic acid Succinyl betulinic acid XIX.Hopane Hop-1 7(2 1)-ene Neohop-13(18)-ene Neohop-12-ene Tetrahydroxybacteriohopane

TPA" Other Assays References 0.2 Skin I , cAK, CAR, ODC,SER [30,37,64,86,115] 1.O 0.3

Lung, Skin I, CAR, EBV,HeLa, ODC, SER

0.2 0.3 PKC 0.4 0.05 0.6 0.6 0.6 0.6 0.6

137,861 [30,37,64,72,79,130, 164) 137,581 [I461 [321 137,861 137,581 [37,861

137,861 Skin I, AIP, cAK, CAR. CRO, [30,33,37,62,86,116, EPP ,ODC 165,1661 Skin 1 [30,37,86] cAK AIP, cAK

CAR

[30,37,116] [115,165,166] 137,861 [37,861 11671

CTN PKC PKC

1891 1321 1321

Skin I, EBV Skin I, EBV EBV AA, ISLOX

1671 [671 1671 1104,1261

1.7 0.2

I \O .

Table 2. Continued-11 Compound XX. Moretenane Moretenol Moretenol acetate XXI. Arborinane Sorghumol Sorghumol acetate XXII. Other Triterpene Bacchara-12,2 I-dien JS-ol Helianol Limonin Sasanquol Squalene 28-Deacetylbelamacandal XXIII. Spirostane Hecogenin 24R -Spirost-4-ene-3,12-dione Tigogenin XXIV. Cardiac Steroid Digitoxigenin Ouabagenin Strophanthidin Decumbesterone A Cyasterone Polypodine B

-

Code Source and Occurrence

TPAD Other Assays

279 Pluchea lanceolata stem and leaves 280 Pluchea lanceolafa stem and leaves

CAR CAR

281 Pluchea lanceolata (Compositae) roots 282 Pluchea lanceolata roots

CAR CAR

Theaceae seeds Compositae flowers; Theaceae seeds Evodia rutaecarpa (Rutaceae) fruits Camellia sasanqua (Theaceae) seeds Fish liver oils; Yeast lipids and higher ~lants 288 Iris tectorum (Iridacee) rhizomes 283 284 285 286 287

289 Polygonurn chinensis ;Commercial 290 Polygonurn chinensis (Polygonacae) 291 Commercial 292 293 294 295 296 297

Commercial Commercial Commercial Ajuga decumbens (Labiatae) Ajuga decumbens Ajuga decumbens

0.8 0.1 AA, BRA, CAR 0.4 >2.0 Colon, Lung

Skin tumor promoter CAR, HSR, SOF HSR. SOF CAR

EBV EBV EBV EBV Skin I, EBV EBV

References

[37,13 I] [37,591 [20,169] [I701 [30,37,46,89]

81

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[146] Ukiya, M.; Akihisa, T.; Yasukawa, K.; Kimura, Y.; Koike, K.; Nikaido, T.; Kasahara, Y; Kumaki, K., Paper Abstracts for the 42nd Ann. Meeting of Coll. Sci. TechnoL, Nihon Univ., 1998, N-20, p. 1160. [147] Kim, D. S.; Oh, S. R.; Lee, I. S.; Jung, K. Y; Park, J. D.; Kim, S. I.; Lee. H.-K. Phytochemistry, 1998, 47, 397-399. [148] Kapil, A.; Sharma, S. J. Pharm. Pharmacol, 1995, 47, 585-587. [149] Fourie, T. G.; Snyckers, F. O. J. Nat, Prod, 1989, 52, 1129-1131. [150] Sugishita, E.; Amagaya, S.; Ogihara, Y J. Pharm.Dyn., 1983, 6, 287-294. [151] Sugishita, E.; Amagaya, S.; Ogihara, Y J. Pharm. Dyn, 1982, 5, 379-387. [152] Konoshima, T.; Kozuka, M.; Haruna, M.; Ito, K., J, Nat. Prod. 1991, 54, 830-836. [153] Safayhi, H.; Mack, T.; Sabieraj, J.; Anazodo, M. L; Subramanian, L. R.; Ammon, H. R J. Pharmacol Exp. Then, 1992,261, 1143-1146. [154] Safayhi, H.; Sailer, E. R.; Ammon, H. R Mol Pharmacol, 1995,47,1212-1214. [155] Sailer, E. F; Subrammanian, L. R.; Rail, B.; Hoemlein, R. F; Ammon, A. P.; Safayhi, H. Br J. Pharmacol, 1996, 117, 615-618. [156] Kweifio-Okai, G.; Bird, D.; Eu, R; Carroll, A. R.; Ambrose, R.; Field, B. Drugs, Exp. Clin. Res., 1994,20, 1-5. [157] Kweifio-Okai, G.; De Munk, F; Rumble, B. A.; Macrides, T. A.; Cropley, M. Res. Commun. Mol Pathol Pharmacol, 1994, 85, 4 -55. [158] Hsu, H.-Y; Yang, J. J.; Lin, C. C. Cancer Lett., 1997, 111, 7-13. [159] Shukla, B.; Visen, R K. S.; Patnaik, G. K.; Tripathi, S. C; Srimal, R. C. Phytotherapy Res., 1992,6,74-79. [160] Simon, A.; Najid, A.; Chulia, A. J.; Delage, C; Rigaud, M. Biochim. Biophys. Acta, 1992,1125,68-72. [161] Akihisa, T.; Yasukawa, K.; Kimura, Y; Takido, M.; Kokke, W. C. M. C; Tamura, T. Chem. Pharm. Bull, 1994,42, 1101-1105. [162] Tabata M.; Tanaka, S.; Cho, H. J.; Uno, C; Shimakura, J.; Ito, M. J. Nat. Prod., 1993, 56, 165-174. [163] Chaturvedi, A. K.; Parmar, S. S.; Bhatngar, S. C; Misra, G.; Nigam, S. K. Res. Commun. Chem. Pathol Pharmacol, 1974, 9, 11-12. [164] Mukherjee, R K.; Saha, K.; Das, J.; Pal, M.; Saha, B. R Planta Med.1997, 63, 367-369. [165] Geetha, T.; Varalakshmi, R; Latha, R. M. Pharmacol Res., 1998, 37, 191-195. [166] Geetha, T.; Varalakshmi, R Gen. Pharmacol, 1999,32,495-497. [167] Chawla, A. S.; Kaith, B. S.; Handa, S. S.; Kulshreshtha, D. K.; Srimal, R. C. Fitoterapia, 1991, 62,441-444. [168] Chawla, A. S.; Kaith, B. S.; Handa, S. S. Indian! Chem., 1990,29B, 918-922. [169] Matsuda, H.; Yoshikawa, M.; linuma, M.; Kubo, M. Planta Med.l99S, 64, 339-342. [170] Akihisa, T.; Yasukawa, K.; Kimura, Y; Yamanouchi, S.; Tamura, T. Phytochemistry 1998,^5,301-305. [171] Peana, A. T.; Moretti, M. D. L.; Manconi, V.; Desole, G.; Pippia, P. Planta Med., 1997,63,199-202.


Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B .V. All rights reserved.

89

BIOACTIVE OLEANENE GLUCURONIDES OBTABVED FROM FABACEOUS PLANTS

JUNEI KINJO and TOSHfflIRO NOHARA Faculty of Pharmaceutical ScienceSy Kumamoto University, Kumamoto 862-0973, Japan ABSTRACT: Oleanene-glucuronide (OG) is defined as an clean-12-ene type triterpene with a C-28 methyl group and a glucuronic acid moiety linked at C-3 of the triterpene. Soyasaponin I is a representative OG. It has been revealed that OGs are widely distributed in fabaceous plants and show several biological activities. As a part of our study on the chemical constituents of fabaceous plants, we have obtained over 191 OGs from 40 fabaceous plants. Furthermore, in the course of our study on hepatoprotective drugs, we devised the conditions for an in vitro assay method using immunologically induced liver injury on primary cultured rat hepatocytes and confirmed hepatoprotective actions of more than 40 OGs and the related compounds. Structure-activity relationships for the sapogenol moiety suggested that the p-hydroxy group at C-21 would enhance hepatoprotective activity; on the contrary, the hydroxy group at C-23, 24, 29 and 30 could reduce the activity. On the other hand, the free carboxylic acid group at C-28 may mediate cytotoxicity toward liver cells. The structure-hepatoprotective relationships of the sugar moiety suggested that the skeleton with glucuronic acid linked at C-3 was a crucial unit in mediating hepatoprotective activity. In the case of a disaccharide chain bound at C-3, an oxygen-bearing group at C-5" seems to enhance the hepatoprotective activity. The terminal rhamnopyranosyl group of fabatrioside seems not to be necessary for the activity. Since antiviral activities of OGs against herpes simplex virus type 1 were reported, we also examined antiherpetic activity of some OGs and related compounds. A trisaccharide group shows greater action than a disaccharide group. Monoglucuronide did not show any antiherpetic activity. Further, the sapogenols showed more potent antiherpetic activity than those of their saponins. This structure-activity relationship was completely different from that obtained from hepatoprotective and antiherpetic activities. The mechanism of antiherpetic activitiy of sapogenols might be different from that of saponins. Furthermore, since OGs were known to have not only anti-complementary but also anti-nephritic activities, we tested some OGs toward the classical pathway. Monoglucuronides and diglycosides were most potent then followed by triglycosides, whereas the aglycones exhibited increase of hemolysis. These results indicate that the glucuronic acid moiety is important for expression of anti-complementary activity. The anti-complementary activity of the OGs with a free acid form of glucuronic acid was more potent than that of sodium salt or methylester forms. Furthermore, reduction of the glucuronic acid moiety decreased significantly their activity. The free acid form of the glucuronic acid moiety seemed to contribute to the potency. The hydroxy group at C-24 did not affect the anti-complementary activity except for the methylester forms.

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INTRODUCTION Saponins are glycosidic compounds present in many edible and inedible plants. Structurally, they are composed of a lipid-soluble aglycone consisting of either a sterol or, more commonly, a triterpenoid and watersoluble sugar residues differing in type and amount of sugars [1]. Because of their amphiphilic nature, they are highly surface-active. Their biological activity is closely related to the chemical structures that determine the polarity, hydrophobicity and acidity of compounds [1]. A recent review by Safayhi and Sailer shows that pentacyclic triterpenes, including the oleanane skeleton, might be a rich natural resource of lead compounds for anti-inflammatory drug development [2]. Some oleanane-type triterpene saponins are known to exhibit anti-hepatic (hepatoprotective) action. Among them, glycyrrhizin [3-7] and saikosaponins [8, 9] are the most well-known. Oleanene-glucuronide (OG) would be defined as an olean-12ene type triterpene with a C-28 methyl group and a glucuronic acid moiety linked at the C-3 of the triterpene. Soyasaponin I (1) [10, 11] is a representative OG, and glycyrrhizin also belongs to the OG species. It was been revealed that OGs are widely distributed in leguminous plants and show several biological activities. For example, anti-hepatitis [12, 13], antihypercholesteremia[14], anti-urolithiasis [15], anti-inflammatory [16] and anti-nephritic [17] activities were confirmed in experimental in vivo models. Furthermore, antiviral

[18], anti-complementary

[17] and calcium-

dependent potassium channel-opening [19] activities were examined using in vitro models. In view of the fact that leguminous plants are widely distributed and used as foodstuff and folk medicines, we have focused on these plants and are trying to develop natural medicines after proving the effectiveness of these crude drugs and to find the lead compounds among these natural sources. Herein, we describe the structures of OGs and some of their biological activities, discussing the structure-activity relationships.

91

EXTRACTION, ISOLATION AND DISTRIBUTION Puerariae Flos (the flowers of Pueraria lobata) is a crude drug used for counteraction to alcohol intoxication in traditional Japanese and Chinese therapeutic systems [20]. We have found that total OGs were effective for alcohol intoxication [21]. It was also effective for an experimental in vivo model of hepatic injury [22]. That is, the total OG decreased the alanine aminotransferase (ALT) level in high fatty food-induced and CCI4-induced experimental liver injury. After repeating some chromatographic technique, we obtained three OGs together with several isoflavones [20]. On the other hand, Abri Herba, the whole plant of Abrus cantoniensis, is used as a folk medicine for infectious hepatitis in China. Its efficacy has been substantiated clinically [23]. Chiang et d. confirmed the efficacy of the total saponin fraction in a pharmacological experimental model [24]. We also reported that the total saponin fractions of this plant were effective for experimental liver injuries in an in vivo model induced by CCI4, Fig. (1) [25]. 4000^ (IU/1) AST

3000 J

ALT

2000 J 1000 J

control

CCI4

Soya I

OG-I

OG-II

Rg. (1). Effects of Extract of Abri Herba in Mice Treated with CCI4 Effects of Soyasaponin I (Soya I), total OG-l (CXJ-I) and total CXJ-D (OG-H) on CCI4 induced liver injury by oral administration. These doses were each 500mg/kg. Each column represents mean of 10 mice. *p -OH]

HO

Fig. ( 1 4 ) . Structure-Antiherpetic Activity Relationships of Oleanene Glucuronides

ANTICOMPLEMENTARY ACTIVITY OF OG The complement system is a humoral effector of inflammation which is activated by a cascade mechanism through the classical and/or alternative pathway [62]. Activation of the system is nomially beneficial for the host. However, excessive activation may evoke pathological reaction in a variety of immunological and degenerative diseases and hyperacute rejection in transplantation. Therefore, the modulation of complement activity should be useful in the therapy of inflammatory diseases. As described previously, the mechanism of our immunological liver injury is regarded as being caused by complement-mediated cell damage [50, 51]. In connection with this view, Shinohara et cd, reported anticomplement actions of soyasaponin I analogs [17]. The order of potency was soyasaponins III (l-'2 |Llg/ml) > IV (3-^5 |Xg/ml) > SBMG (5 |ig/ml) » I (125 |Lig/ml) > II (100-150 |J.g/ml). The order was very similiar to the order for hepatoprotective activity obtained from our experiments. Shinohara et al also revealed anti-nephritis action of those analogs on an experimental in vivo model. Since the antibody-complement system plays an important role in humoral immunity, we were interested in the structure-hepatoprotective relationships of the individual OGs. At first, we tested sophoradiol

118

glucuronides together with soyasapogenol B glucuronides (Table 6). Table 6. Anti-complementary Activity of Soyasaponin I, Kaikasaponin III and Related Compounds Substance

Classical Pathway (CP) IC50

Alternative Pathway (AP) % inhibition (32^lg/ml)

Soyasaponin I (1)

67 ^M

14%

Soyasaponin HI (6)

32^lM

15%

3|XM

-19 %* 1% -18 %* -7%* -2%* 15%

SBMG(12) Soyasapogenol B Kaikasaponin EQ (21) Kaikasaponin I (2 6) SoMG Sophoradiol Azukisaponin V (2) Dehydrosoyasaponin I (7 2)

-50% (32|Lig/ml)* 62|iM 13 ^M 13 ^M -47% (32|Lig/ml)* 14 ^M 50 îM

-

Soyasaponin 11(3)

26 ^M

-

Wistariasaponin D (7 4)

15 ^M

-

Astragaloside VIII (4) Lupinoside PA4 (16) Azukisaponin II (7)

21 ÎM 8^M 3^M

-

Kudzusaponin A3 (3 0) Lupinoside PA, (4 0) Lupinoside PA3 (3 6) Dt-E(94)

120|ilM

-

29|IM 61 |LlM

-

*

-

Dt-C (91)

niiM

-

Dt-B(92)

28 ÎM

-

-

-

* hemolytic

Among them, diglycosidic glucuronides (6 and 21) showed the most potent anti-complementary activity, followed by monoglucuronides (12 and SoMG) and triglycosidic glucuronides (1 and 21). Sophoradiol and soyasapogenol B did not show the inhibition of hemolysis, but rather promoted it under the presence of serum on the classical pathway, whereas

119

all of them showed very weak or no hemolytic effects on the alternative pathway of the complement system (Table 6). The anti-complementary activity of each saponin was influenced by the nature of glucuronic acid (Table 7), where the free acid (COOH) showed much more potent activity than the sodium salt (COO"Na"*") or methylester (COOCH3). Reduction of the acid (glucuronic acid) to the alcohol (glucose) of saponins decreased significantly their activity. The free acid form of the glucuronic acid moiety seemed to contribute to the potency. These results indicate that the glucuronic acid moiety is important for expression of the anti-complementary activity. In contrast to hepatoprotective action, the hydroxy group at C-24 did not affect the anticomplementary activity. However, in the case of the methyl ester of sophoradiol glucuroides (Table 7), the anti-complementary activity was significantly depressed. Therefore, the hydroxy group at C-24 of soyasapogenol B glucuronides might enhance anti-complementary activity instead of the methoxy carbonyl moiety at C-6 of glucuronic acid. Table 7. Anti-complementary Activity of Various Analogs for the Glucuronic Acid Moiety of Oleanene Glucuronides on CP Substance

IC50

Free acid (-COOH) Soyasaponin I Soyasaponin lU SBMG Kaikasaponin III Kaikasaponin I SoMG

10 JIM 6.8 iiM 7.9 jlM 44|iM 2.6 JIM 9.9 JIM

Sodium salt (-COONa^ -11%(35|LIM)* 42|LlM 15|IM 50 ^M 14 ^M 30|LtM

% inhibition Methyl ester (-COOCH3) 30|XM 40 ^M 35|XM 180 jlM 210 |IM 660|LlM

Reduced (-CH2OH) -5%(100^M)* -2%(100^M/

4%(100|IM) 29%(100|iM) 12%(100jiM) -20%(100|IM)*

hemolytic

Various other oleanene glucuronides having a characteristic functional group were also tested and the structure-anti-complementary activity

120

relationships were discussed (Table 6). Within the trisaccharide group of soyasapogenol B, the order of anticomplementary activity was azukisaponin V (2) > astragaloside Vni (4) > soyasaponin II (3) > soyasaponin I (1). Therefore, the potency of the sugar moieties was in the order of Rha-(l-^2)-Glc-(l->2)-GlcA > Rha(l-^2)-Xyl-(l->2)-GlcA > Rha-(l-^2)-Ara-(l-^2)-GlcA > Rha-(1~>2)Gal-(l-»2)-GlcA. Similarly, within the disaccharide group of soyasapogenol B (6 and 7), the potency of the sugar moieties was also in the order of Glc-(l->2)-GlcA > Gal-(l-^2)-GlcA. The saponin having an a-hydroxy group at C-3" (Glc or Xyl) seemed to show greater action. When the anti-complementary activities of 1 and 7 2 which have the same trisaccharide group (S,) were compared, the order of potency was dehydrosoyasaponin I (72) > soyasaponin I (1). This meant that the potency of the sapogenol moieties was in the order of soyasapogenol E > soyasapogenol B. Similarly, in the case of another trisaccharide group (S4, 4 and 7 4), the potency of the sapogenol moieties was also in the order of soyasapogenol E > soyasapogenol B. The information that the hydroxy group at C-29 reduced activity was similar to that on the structure-hepatoprotective relationship. In contrast, the sugar moiety in the E-ring enhanced anti-complementary activity. Further, we tested some oleanolic acid-type glucuronic acids. Although bisdesmosidic saponins showed moderate anti-complementary activity, a monodesmosidic saponin (94) showed cyototoxicity. Since some inflammation, including hepatitis and nephritis, is caused by excessive immunoreaction, it might be possible that OGs in the edible fabaceous plants play an important role for suppression of some inflammation. In Fig. (15), the obtained structure-anticomplementary activity relationships are shown.

121 Hemolytic ? Essential

Not Important

COOH

Reduce Enhance ? COOH* > COONa* > COOMe » CH2OH

Reduce

Fig. ( 1 5 ) . Structure-Anticomplementary Activity Relationships of OGs

ABBREVIATIONS OG: ALT:

= Oleanene-Glucuronide = Alanine Aminotransferase

AST: SBMG: SoMG: HSV-1:

= Aspartate Aminotransferase = Soyasapogenol B Monoglucuronide = Sophoradiol Monoglucuronide = Herpes Simplex Virus Type 1

ACKNOWLEDGEMENTS I express my appreciation to Dr. H. K. Lee of Korea Research Institute of Bioscience and Biotechnology for measurement of anti-complementary activity. I am grateful to Dr. K. Yokomizo and Prof. M. Uyeda in this faculty for measurement of antiherpetic activity.

122

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Prod., 1995, 55, 1372-1375. [59] Ushio, Y.; Abe, H. Planta Medica, 1992, 58, 171-173. [60] Sindambiwe, J. B.; Calomme, M.; Geerts, S.; Pieters, L.; Vlietinck, A. J.; Vanden Berghe, D. A. J, Nat. Prod, 1998, 61, 585-590. [61] Kurokawa, M.; Basnet, P.; Ohsugi, M.; Hozumi, T.; Kadota, S.; Namba, T.; Kawana, T.; Shiraki, K. J. Pharmacol. Exp. Ther., 1999, 289, 12-1%. [62] Park, S.-H.; Oh, S.R.; Jung, K.Y.; Lee, I.S.; Ahn, K.S.; Kim, J.H.; Kim, Y.S.; Lee, J.J.; Lee, H.-K. Chem. Pharm. Bull, 1999, 47, 1484-1486. [63] Kinjo, J.; Matsumoto, K.; Inoue, M.; Takeshita, T.; Nohara, T. Chem. Pharm. Bull, 1991,39, 116-119. [64] Kinjo, J.; Hatakeyama, M.; Udayama, M.; Tsutanaga, Y.; Yamashita, M.; Nohara, T.; Yoshiki, Y.; Okubo, K. BioscL Biotech. Biochem., 1998, 62, 429-433. [65] Cui, B.; Sakai, Y.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 136-138. [66] Cui, B.; Inoue, J.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992, 40, 3330-3333. [67] Kinjo, J.; Yamashita, M.; Nohara, T. In Food Factors for Cancer Prevention; Ohigashi, H.; Osawa, T.; Terao, J.; Watanabe, S.; Yoshikawa, T., Eds.; SpringerVerlag: Tokyo, 1977, pp. 323-327. [68] Ding, Y.; Kinjo, J.; Yang, C ; Nohara, T. Chem. Pharm. Bull, 1991, 39, 496498. [69] Yahara, S.; Emura, S.; Feng, H.; Nohara, T. Chem. Pharm. Bull, 1989, 37, 2136-2138. [70] Kubo, T.; Hamada, S.; Nohara, T.; Wang, Z.-R.; Hirayama, H.; Ikegami, K.; Yasukawa, K.; Takido,M. Chem. Pharm. Bull, 1989, 37, 2229-2231. [71] Udayama, M.; Kinjo, J.; Yoshida,N.; Nohara, T. Chem. Pharm. Bull, 1998, 46, 526-527. [72] Hirakawa, T.; Okawa, M.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 2000, 48, 286-287. [73] Kinjo, J.; Kishida, F.; Watanabe, K.; Hashimoto, F.; Nohara, T. Chem. Pharm. Bull, 1994,42, 1874-1878. [74] Yahara, S.; Irino, N.; Takaoka, T.; Nohara, T. Natural Medicines, 1994, 48, 312313. [75] Arao, T.; Idzu, T.; Kinjo, J.; Nohara, T.; Isobe, R. Chem. Pharm. Bull, 1996, 44, 1970-1972. [76] Cui, B.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992, 40, 2995-2999. [77] Cui, B.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1993, 4], 553-556. [78] Takeshita, T.; Yokoyama, K.; Ding, Y.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1991,59, 1908-1910. [79] Ding, Y.; Takeshita, T.; Yokoyama, K.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 139-142. [80] Ding, Y.; Tian, R.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 1831-1834. [81] Ding, Y.; Tian, R.; Kinjo, J.; Nohara, T.; Kitagawa, I. Chem. Pharm. Bull, 1992,40,2990-2994. [82] Kinjo, J.; Fujishima, Y.; Saino, K.; Tian, R.; Nohara, T. Chem. Pharm. Bull, 1995, 43, 636-640. [83] Udayama, M.; Kinjo, J.; Nohara, T. Phytochemistry, 1998, 48, 1233-1235. [84] Kinjo, J.; Udayama, M.; Hatakeyama, M.; Ikeda, T.; Sohno, Y.; Yoshiki, Y.; Okubo, K.; Nohara, T. Natural Medicines, 1999, 5i, 141-144.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry', Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOTRANSFORMATION OF TERPENOIDS BY MICROORGANISMS JAN C.R. DEMYTTENAERE Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, 8-9000 Gent, Belgium; e-mail: Jan. Demvttenaere(^rug. ac. be ABSTRACT: In recent years, theflavourmarket has undergone a tremendous 'back-tonature' demand, which is illustrated by the consumers' preference for 'natural' flavourmg substances instead of synthetic 'artificial' compounds. These natural products can be obtained by extraction from plant material, but they can also be produced by biotechnological processes using micro-organisms. De novo synthesis of 'bioflavours', such as volatile esters, and biotransformation of monoterpenes are fields of investigation that gain a growing interest. Terpenes are obtained from the essential oil of many plants and are relatively cheap. They are usually isolated from the oils by rectification. This renders abundant monoterpenes, such as a-pinene and limonene, inexpensive starting materials for chemical and biochemical transformations. Thefiingalbiotransformation of these natural precursors to more valuable aroma compounds offers a very interesting alternative source of naturalflavours.This review article deals with the biotransformation of some monoterpenoids and sesquiterpenoids by both fiingi and bacteria. As substrates myrcene, ocimene, geraniol, nerol, citronellol, citral, citronellal, linalool, limonene (and related compounds), pinenes, menthol, camphor, pulegone, ionones, nerolidol, famesol, caryophyllene, valencene, patchoulol, etc... are discussed.

INTRODUCTION 1 Definition of 'Natural Flavours' As consumers want more and more 'natural flavours' instead of synthetic ones, there is a trend to focus on the production of these natural flavour substances [1,2]. In the United States of America the Code of Federal Regulations (CFR 101.22.a.3) defines the term 'natural flavour' to include not only animal or plant derivatives but also products obtained from enzymatic and fermentative processes. According to the regulations of the US FDA (Food and Drug Administration) guidelines (1958), a natural flavour must be produced from natural starting materials and the endproduct must be identical to a product already known to exist in nature. Thus, biocatalytic, but not chemical transformation of natural substances can be legally labelled as natural [3]. The European guidelines (88/388/EWG of June 22, 1988; 91/71/EWG and 91/2/EWG of January 16, 1991) define natural aroma compounds as isolated by 'physical, enzymatic, or microbiological processes or traditional food preparation

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processes solely or almost solely from the foodstuff or the flavouring source concerned' [4]. However, there is a difference between natural, nature-identical and artificial products. Natural products are directly isolated from plant or animal sources, by physical processes, such as extraction, maceration, distillation,... Microbial production of flavours is also a source of natural products. Nature-identical compounds are produced synthetically, but are chemically identical to their natural counterparts. Artificial flavour substances are compounds that have not yet been identified in plant or animal products for human consumption. They are made synthetically, have the same or similar smell and other properties as some natural flavours, but are chemically totally different. For example, vanillin can be obtained via at least five different ways: (i) by isolation from the orchid (Vanilla planifolia), which is a very expensive method; (ii) by tissue culture followed by extraction; (iii) by microbial transformation of eugenol, the main compound of clove; (iv) from lignine by synthesis, and (v) from guaiacol, a natural aroma compound, with comparable molecular structure. Only the vanillin obtained via the first three methods is natural. The other routes afford a nature-identical vanillin. Since not only the isolation from nature but also biotechnological processes (the use of microorganisms and enzymes) are a source of 'natural flavours' [5], the term 'bioflavours' will be used. 2 Bioflavours In 1987, the term 'bioflavours' was used for the first time as the title of an intemational symposium at Wiirzburg University (Bioflavour *87). In his introductionary lecture, Drawert defined 'bioflavours' as 'natural* and 'naturally produced' flavours [6]. Many important food aromas originate from biochemical pathways. These pathways comprise microbial reactions, endogenous and exogenous enzymatic action, and plant metabolism. In the past, flavour research concentrated on characterising the important chemicals in foods responsible for their specific aroma. Less information is therefore available on the biogeneration of flavours. At present, however, a renaissance of studies of natural flavours, including their biogeneration can be observed [1]. A number of factors appear to be responsible for the renewed interest in bioflavour research, e.g,: - consumers increasingly reject artificial flavours and demand natural ones;

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- bioflavour formation may provide possibilities for producing industrially important secondary metabolites which are not available by conventional procedures; - advances in genetic research and genetic engineering permit speculations to explore bioflavour production. Within the field of biotechnology, food technology is the oldest and economically most important part. Ten years ago, about 90% of the industrially produced flavours and fragrances were of synthetic origin, the rest was mainly derived from agricultural sources [6]. In contrast to repeated predictions, the trend to natural flavours is unbroken. Today, food flavours share about 2 billion US $ in the world-wide 5 billion US $ market for volatile flavours and fragrances [4]. Natural aromas account for 60% and 40%, respectively, of the total sales in Europe and in the United States of America. The respective figures for Japan and the remaining countries are 10% and 5%. In Germany however, about 70% of all food flavours used in 1990 were natural [7]. 3 Scope and Production of Bioflavours Some advantages of the biotechnological production of flavours are [6]: - the products may possess the legal status of a natural compound; - the high substrate and reaction specificity of enzymes guarantee a defined stereochemistry; - optimised reaction conditions lead to complex, uniform products and to constant productivity; - multiple-step reactions, which are not possible in aqueous solution by chemical means, proceed under mild conditions; - adverse extemal influences such as unfavourable climate, pest infestations, economical or ecological drawbacks can be ignored. There are many different ways to produce bioflavours: plant tissue and cell cultures can be used, microorganisms, such as bacteria, yeasts, fungi and algae can produce flavours de novo and through bioconversions, and enzymes can be applied. 3.1 Plant Tissue and Cell Culture

A large number of various fine chemicals is derived from plants, e.g., drugs, pigments, and other biologically active substances. In the past, their production by means of plant cell cultures has attracted the interest of many researchers. Although most plant cell cultures have been unable to

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produce an adequate yield of flavour substances, a few examples exist where tissue cultures exhibited an increased production compared to that of the plant. On the other hand, a variety of volatiles, different from those occurring in the plant, has been isolated from cell cultures [8]. As de novo synthesis has been proven unsuccessful in most cases, biotransformation of added precursors has been studied extensively. There is evidence that plant cell cultures retain an ability to transform specifically exogenous substrates administered to the cultured cells. Therefore, plant cell cultures are considered to be useful for transforming cheap and plentiful substances into rare and expensive compounds by using the cell culture as a bioreactor. For instance, cofactor dependent specific conversions of terpenoids in suspension cultures of aromatic plants often proceed with high yields and negligible amounts of byproducts. In Fig. (1), three examples of biotransformations of terpenes by plant cell cultures are shown (after [6]). I Salvia officinalis

_ _

^

Borneol (1)

CHO

r v ^

1 ^

Conversion rate no 0/

78/.

Camphor (2)

Melissa officinalis • 2h

Citronellal (3)

I I Is^ CHjOH

99.5%

Citronellol (4)

66%

Valencene (5)

Nootkatone (6)

Fig. (1). Biotransformation of terpenes by plant cell cultures (after [6])

3.2 Use of Microorganisms

A very efficient way to obtain bioflavours is by biotechnological processing using microorganisms [5]. The microbial production of aromas offers many advantages: the circumstances under which the reactions occur are generally mild: e.g., neutral pH, ambient temperature. There are

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two important ways to produce microbial flavours: de novo production of aromas, also called biosynthesis and the transformation of natural precursors into interesting flavour compounds, called bioconversion [9]. An extensive overview on the biotranformation of terpenoids by microorganisms will be given in the next chapter. The capacity of microorganisms to produce pleasant odours has been known at least since the beginning of this century [10]. Terpenoids are natural compounds responsible for the characteristic smell of essential oils. Many of the terpenoid producing microorganisms are fungi growing on decaying fir-wood and belonging to the ascomycetes or basidiomycetes. One of the most important examples is the genus Ceratocystis, producing a variety of terpenoid alcohols with a very attractive smell [11]. Esters also constitute a group of important flavour compounds. They are the main aroma components found in fruits (apples, pears, ...). For example, bananas contain 12-18 ppm acetates. The price of the pure flavour compounds, when isolated from fruit, can range between 10,000 and 100,000 US $/kg! In the past, research has been carried out by our group about the microbial production of fruity esters by the yeast Hansenula mrakii and the fimgus Geotrichum penicillatum [10]. A fermentation was developed whereby fusel oil was continuously converted into a mixture of 3-methylbutyl acetate (isoamyl acetate) and 2-methylbutyl acetate, the 'character impact compounds' of banana flavour. A very well known dairy product is Roquefort cheese, its flavour is generated by mould action. This so called 'Blue cheese flavour' is attributed to methyl ketones and is formed by the degradation of fatty acids by Penicillium roquefortii. The production of these bioflavours has also been investigated by our group [12,13] and will not be further discussed here.

BIOTRANFORMATION OF TERPENOIDS BY MICROORGANISMS Introduction Terpenoids (often referred to as isoprenoids) constitute the largest group of natural products. They belong to the most important flavour and fragrance compounds, and are found in the microbial, plant and animal kingdoms [14]. They offer a very wide variety of pleasant and floral scents. Living organisms synthesize a remarkable diversity of isoprenoids [15]. More than 23,000 different compounds have been isolated and all contain one or more isoprene unit, the building block of these compounds [16]. Thus, the

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Structure of terpenoids is built by combining these isoprene (7) units, usually joined head to tail. Terpenes are classified according to the number of isopentenyl units (rule of Ruzicka, Fig. (2)), e,g,, 2: monoterpenes (C10H16), 3: sesquiterpenes (C15H24), 4: diterpenes (C20H32), 6: triterpenes (C30H48X and higher: polyterpenes.

or

Isoprene (7)

Myrcene (8)

Ocimene (9)

Fig. (2). Rule of Ruzicka: head to tail coupling of two isoprene units, giving myrcene

Until 1993, all terpenes were considered to be derived from the classical acetate/mevalonate pathway involving the condensation of three units of acetyl CoA to 3-hydroxy-3-methylglutaryl CoA, reduction of this intermediate to mevalonic acid and the conversion of the latter to the essential, biological isoprenoid unit, isopentenyl diphosphate (IPP) [17,18,15]. Recently, a totally different IPP biosynthesis was found to operate in certain eubacteria, green algae and higher plants. In this new pathway glyceradehyde-3-phosphate (GAP) and pyruvate are precursurs of isopentenyl diphosphate, but not acetyl-CoA and mevalonate [19,20]. So, an isoprene unit is derived from isopentenyl diphosphate, and can be formed via two alternative pathways, the mevalonate pathway (in eukaryotes) and the deoxyxylulose pathway in prokaryotes and plant plastids [16,19]. An extensive overview of the biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants is given by Chappell [17]. Abundant sources of terpenoids are the essential oils. They consist of a complex mixture of terpenes or sesquiterpenes, alcohols, aldehydes, ketones, acids and esters [21]. The monoterpenes are subdivided into three groups: acyclic, monocyclic and bicyclic (there is only one tricyclic terpene: tricyclene). Each group contains hydrocarbon terpenes, terpene alcohols, terpene aldehydes, ketones, oxides etc... The isolation of terpenes from plants entails several problems (e.g., very low concentrations). Therefore other sources of these flavour compounds are searched for: microorganisms for example (especially bacteria and fungi) are used for the production of terpenoids [22]. Since terpenoids are very important flavour and fragrance compounds, the biotransformation of terpenes offers a very interesting source of novel

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flavours, the so-called bioflavours. A recent review of the biotechnological production of flavours and fragrances is given by Krings and Berger [7]. This literature review will deal with the biotransformation of terpenoids by microorganisms, more specifically fungi and bacteria. 1 Monoterpenes

LI Acyclic Monoterpenes 1.1.1 Acyclic hydrocarbon monoterpenes

Myrcene, ocimene As can be seen from Fig. (2), myrcene (8) is the most simple terpene: in nature, it is formed directly by head to tail linking of two Cs-biological isoprene units, isopentenyl pyrophosphate [14]. Rearrangement of one double bond gives its isomer ocimene (9). The biotransformation of these two acyclic hydrocarbon terpenes however, is not very well documented. One of the earliest reports [23] describes the degradation of )ff-myrcene (8) by a strain of Pseudomonas putida, commencing with the oxidation of the terminal methyl group. In 1985, the bioconversion of acyclic terpenes and related structures with a terminal isoprenoid group was described [24]. The preferred microorganism for this reaction was Diplodia gossypina ATCC 10936. The main reactions were hydroxylation reactions, Fig. (3). On oxidation, myrcene (8) gave next to the diol (10) (yield up to 60%) also a side-product (11) that possesses one carbon atom less than the parent compound, in yields of 1-2%.

Diplodia gossypina

Myrcene (8)

I X^-OH

10

+

I ^,CH

11

I k,^

Myrcene (8)

G. applanatum Pleurotus sp.

Myrcenol(12)

Fig. (3). Hydroxylation of myrcene by Diplodia gossypina (after[24]) and by Ganoderma applanatum and Pleurotus sp. (after [25])

One of the most recent publications dealing with the bioconversion of myrcene [25] described its transformation to a variety of oxygenated metabolites, with Ganoderma applanatum, Pleurotus flabellatus and Pleurotus sajor-caju possessing the highest transformation activity. The extracted metabolites represented a complex mixture of numerous acyclic and monocyclic metabolites of which myrcenol (12) (2-methyl-6-

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methylene-7-octen-2-ol), with a fresh-flowery impression, dominated the yield and sensory impact of the mixture, Fig. (3). 1.1.2 Acyclic monoterpene alcohols and aldehydes

Geranioly nerol citronellol citral citronellal Geraniol (3,7-dimethyl-(£)-2,6-octadien-l-ol) (20) occurs in nearly all terpene-containing essential oils, frequently as an ester. Palmarosa oil contains 70-85% geraniol; geranium oils and rose oils also contain large quantities. Geraniol is a colourless liquid, with a flowery-rose-like odour [26]. Nerol (14) is the Z-enantiomer of geraniol and occurs in small quantities in many essential oils where it is always accompanied by geraniol. Its name originates from its occurrence in neroli oil. It has a fresh, sweet rose-like odour and a bitter flavour [27]. Citronellol (3,7dimethyl-6-octen-l-ol) (4) has been found in about 70 essential oils and in the oil of Rosa bourbonia. The Bulgarian rose oil contains more than 50% L-citronellol, whereas East African geranium contains more than 80% of the D-isomer [27]. L-Citronellol, which is also called rhodinol, has a sweet, peach-like flavour and is more delicate than D-citronellol which has a bitter taste. Citral (3,7-dimethyl-2,6-octadien-l-al) (26) occurs as Z(neral) and E- (geranial) isomers, analogous to the corresponding alcohols, nerol and geraniol. Natural citral is nearly always a mixture of the two isomers. It is found in lemongrass oil (Cymbopogon flexuosus (Nees.) Stapf.) in concentrations of up to 75% [27], in Lit sea cubeba oil (up to 75%) and in small amounts in many other essential oils. The citrals are colourless to slightly yellowish liquids, with an odour reminiscent of lemon [26]. (-f)-Citronellal (3,7-dimethyl-6-octen-l-al) (3) occurs in citronella oil at concentrations of up to 45%; Backhousia citriodora oil contains up to 80% (~)-citronellal. Racemic citronellal is found in a number of Eucalyptus citriodora oils at concentrations of up to 85%. It is a colourless liquid with a refreshing lemon-, rose-type odour, similar to balm mint [26]. The first detailed work on the microbial degradation of the acyclic monoterpene alcohols and aldehydes was reported in the early part of 1960 [28-31]. They studied the metabolism of citronellol, citronellal, geraniol and geranic acid by the soil bacterium, Pseudomonas citronellolis. They observed that the metabolism of these acyclic monoterpenes is initiated by the oxidation of the primary alcohol group to the carboxyl group, followed by carboxylation of the C-10 methyl group Off-methyl) by a biotindependent carboxylase [31]. The carboxymethyl group is eliminated at a later stage as acetic acid. Further degradation follows the jff-oxidation pattern. The details of the pathway are shown in Fig. (4) (after [32]).

133

CHO

Citronellol (4)

Citronellal (3)

Citronellic acid (13)

C02

10

CH-OOOH

CH2OH

Nerol (14)

CHjOH OOOH

Geraniol(20)

Geranial (21)

Geranic acid (22)

19

Fig. (4). Microbial degradation of citronellol, nerol and geraniol by Pseudomonas citronellolis (after [32])

The microbial transformation of the aldehydes citronellal (3) and citral (26) by Pseudomonas aeruginosa was also reported [33]. This bacterium, capable of utilising citronellal or citral as the sole carbon and energy source, has been isolated from soil by the enrichment culture technique. It metabolised citronellal (3) to citronellic acid (13) (65%), citronellol (4) (0.6%), dihydrocitronellol (23) (0.6%), 3,7-dimethyl-l,7-octanediol (24) (1.7%) and menthol (25) (0.75%), Fig. (5). The metabolites of citral (26) were geranic acid (22) (62%), l-hydroxy-3,7-dimethyl-6-octen-2-one (27) (0.75%), 6-methyl-5-heptenoic acid (28) (0.5%), and 3-methyl-2-butenoic acid (29) (1%). In a similar way, citral was converted to geranic acid by P. convexa [34]. The biotransformation of citronellol and geraniol by P. aeruginosa, P. citronellolis and P. mendocina was also reported by another group [35]. In 1977, a Pseudomonad was isolated from soil by enrichment culture technique with linalool as the sole source of carbon and energy [36]. The bacterial strain was later identified as Pseudomonas incognita and given the name 'linalool strain'. It was also capable of growing on geraniol, nerol and limonene. The biotransformation of geraniol by this

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microorganism was studied. Shake cultures were treated with geraniol (20). At the end of the incubation period (40 hr), the contents were pooled, acidified with 3 N HCl and extracted with ether and separated in their neutral and acid components. In the neutral fraction (1.4 g) the following metabolites were found: 3-(4-methyl-3-pentenyl)-3-butenolide (30) (25 mg), geranial (21) (130 mg), unreacted geraniol (20) (900 mg), a hydroxyketone (40 mg): l,3-dihydroxy-3,7-dimethyloct-6-ene-2-one (32), which upon treatment with sodium metaperiodate yielded the oxidation product 6-methyl-5-hepten-2-one (33), and a triol (20 mg): 3,7dimethyloct-6-ene-l,2,3-triol (31). The acidic fraction (2.0 g) yielded as major metabolite geranic acid (22) (1.75 g), and a keto acid (20 mg): 7methyl-3-oxo-6-octenoic acid (19), Fig. (6).

Citral(26)

22

27

28

29

Fig. (5). Degradation of citroncllal and citral by Pseudomonas aeruginosa (after [33])

An analogous experiment was run by the same group to study the bioconversion of nerol. It was degraded in essentially the same way as geraniol. Identical products as from geraniol were recovered as major metabolites, together with neral and neric acid [37]. Finally the biotransformation of limonene was carried out with the same microorganism in the same fashion as with geraniol and nerol. The details of this research will be discussed in the chapter of cyclic monoterpenoids (1.2.1). Based on these data, two pathways for the degradation of geraniol (20) were proposed by Madyastha [32], Fig. (7). Pathway A involves an oxidative attack on the 2,3-double bond resulting in the formation of an epoxide. Opening of the epoxide yields the triol (31) which upon oxidation forms a ketodiol (32). The ketodiol (32) is then converted to 6methyl-5-hepten-2-one (33) by an oxidative process. Pathway B is

135

initiated by the oxidation of the primary alcoholic group to geranic acid (22) and further metabolism follows the mechanism as proposed earlier for P, citronellolis [28,31]- In the case of nerol (the Z-isomer of geraniol) degradative pathways analogous to pathways A and B as in geraniol are observed. It was also noticed that P. incognita metabolises acetates of geraniol, nerol and citronellol much faster than their respective alcohols [38]. oo-o

CHjOH

CHO

CHjOH

Geraniol (20) Periodate OOCH

22

CDOH

19

Fig. (6). Degradation of geraniol by Pseudomonas incognita (after [36])

CO2 + H2O 9^ 7

Geraniol (20)

Triol (31)

Ketodiol (32)

6-Methyl-5-hepten-2-one (33)

B

GOGH

)S-Oxidation

Geranial (21)

Geranic acid (22)

y9-Keto acid (19)

Fig. (7). Pathways of degradation of geraniol by Pseudomonas incognita (after [32])

136

In the eighties, the bioconversion of monoterpene alcohols by fungi had not been studied intensively [32]. However, a strain of Aspergillus niger was isolated from garden soil, able to transform geraniol, citronellol and linalool to their respective 8-hydroxy derivatives. This reaction was called 'cy-hydroxylation' [39,40]. Fermentation of citronellyl acetate with A. niger resulted in the formation of a major metabolite, 8-hydroxycitronellol accounting for approx. 60% of the total transformation products, accompanied by 38% citronellol. Fermentation of geranyl acetate with A. niger gave geraniol and 8-hydroxygeraniol (50 resp. 40% of the total transformation products). However, in the case of linalyl acetate, besides linalool and 8hydroxylinalool which were formed to the extent of 25 and 45% of the total products resp., small amounts of geraniol and a-terpineol (together 25%) were also formed. Nearly 40% of the acetates were metabolised in 72 hr. One of the most important examples of fungal bioconversion of the monoterpene alcohols citronellol, geraniol and nerol and the terpene aldehyde citral is the biotransformation by Botrytis cinerea. Fig. (8). B. cinerea is a fungus of high interest in winemaking [41]. In an unripe state of maturation the infection of grapes by B, cinerea is very much feared, as the grapes become mouldy ('grey rot'). With fully ripe grapes however, the grovrth of 5. cinerea is desirable; then the fungus is called 'noble rot' and the infected grapes deliver famous sweet wines, such as, e.g., Sautemes of France, Tokay Aszu of Hungary or Trockenbeerenauslese wines of Germany [42]. Several key components among the volatiles of grapes and wines are terpenoid compounds. The last years, the influence of 5. cinerea on the monoterpene fraction of wine aroma has been studied intensively, especially the biotransformation of geraniol, nerol, citronellol, linalool and citral. One of the first reports in this area dealt with the biotransformation of citronellol (4) by B. cinerea [43], Fig. (8). The substrate was metabolised predominantly to the co-hydroxylation product (J?)-2,6-dimethyl-2-octene1,8-diol (34) and its reduction product 2,6-dimethyl-l,8-octanediol (35). Minor amounts of (Z)-2,6-dimethyl-2-octene-l,8-diol (36), 3,7-dimethyl1,7-octanediol (37),p-menthane-3,8-diol (38), isopulegol (39), 7-hydroxy6-methylhept-5-en-2-one-l-ol (40) and 2-methyl-y-butyrolactone (41) were found. It was also found that with grape must, citronellol (4) was completely metabolised, while using a synthetic medium an incomplete transformation was noticed, also yielding 6-methyl-5-hepten-2-one (33) and citronellic acid (13) as degradation products [42]. The same group also investigated the bioconversion of citral (26) [44], Fig. (8). A comparison was made between grape must and a synthetic medium. When using grape must, no volatile bioconversion products were found. With a synthetic medium, biotransformation of citral (26) was observed yielding predominantly nerol (14) and geraniol (20) as reduction products and as

137

.CH,OH

Fig. (8). Biotransformation of citroneliol, geraniol, nerol and citral by Botrytis cinerea (after [4245])

138

minor compounds the co-hydroxylation products (2£',6Z)-2,6dimethylocta-2,6-diene-1,8-diol (47), (2E, 6jE)-2,6-dimethylocta-2,6-diene1,8-diol (43), 6-methyl-5-hepten-2-one (33), 6-methyl-5-hepten-2-ol (52), 7-hydroxy-6-methylhept-5-en-2-one-l -ol (40) and 2-methyl-ybutyrolactone (41). Finally the bioconversion of geraniol (20) and nerol (14) was described by the same group [45], Fig. (8). When using grape must, a complete bioconversion of geraniol (20) was observed yielding as predominant product from co-hydroxylation (2jB,6£)-2,6-dimethyl-2,6octadiene-l,8-diol (43) and its reduction product (£)-3,7-dimethyl-2octene-l,8-diol (44). The following minor compounds were found: (2Z, 6^-2,6dimethylocta-2,6-diene-l,8-diol (42), 2,6-dimethyM,8-octanediol (35),/?menth-l-en-9-ol (45), 7-hydroxy-6-methylheptan-2-one (46), 7-hydroxy-6methylhept-5-en-2-one (40). (Z)-2,6-Dimethyl-2,7-octadiene-1,6-diol (48), (j5)-2,6-dimethyl-2,7-octadiene-l,6-diol (49), (Z)-3,7-dimethyl-2octene-l,8-diol (50) and 3,7-dimethyM-octene-3,8-diol (51) were bioconversion products formed after rearrangement of geraniol to nerol and linalool. With a synthetic medium, geraniol was not completely metabolised. Analogous results were obtained with nerol (14): complete transformation took place with grape must, yielding predominantly the cohydroxylation product (2£,6Z)-2,6-dimethylocta-2,6-diene-l,8-diol (47) and its reduction product (Z)-3,7-dimethyl-2-octene-l,8-diol (50). On synthetic medium, the substrate (14) was not completely metabolised, resulting in low yields of (2£',6Z)-2,6-dimethylocta-2,6-diene-l,8-diol (47) and /7-menth-l-en-9-ol (45). The most important metabolites from citronellol (4), geraniol (20), nerol (14) and citral (26) are displayed in Fig. (8). In the same year the biotransformation of these monoterpenes by B. cinerea in model solutions was described by another group [41]. Although the major metabolites found were co-hydroxylation compounds, it is important to note that these authors only identified the JJ-isomers in the extracts and that some new compounds were detected that were not described by the previous group, Fig. (9). Geraniol (20) was mainly transformed to (2£,5£r)-3,7-dimethyl-2,5-octadiene-l,7-diol (53), (£)-3,7dimethyl-2,7-octadiene-1,6-diol (54) and (2£:,6£)-2,6-dimethyl-2,6octadiene-l,8-diol (43), nerol (14) to (2Z,5£)-3,7-dimethyl-2,5-octadiene1,7-diol (55), (Z)-3,7-dimethyl-2,7-octadiene-l,6-diol (56), and (2£,6Z)2,6-dimethyl-2,6-octadiene-l,8-diol (47). Furthermore a cyclisation product (57) was formed which was not previously described. Finally citronellol (4) was converted to trans- (60) and cis-vosQ oxide (61) (a cyclisation product not identified by the other group), (£)-3,7-dimethyl-5octene-1,7-diol (58), 3,7-dimethyl-7-octene-1,6-diol (59) and (£0-2,6-dimethyl-2-octene-l ,8-diol (34). The preparation of menthol from citronellal, pulegol or isopulegol by the fungus Penicillium digitatum was patented as early as 1955 [46]. To a

139

culture of P. digitatum propagated for 48 hr at 22°C on 1.5% brewer's wort, 2% citronellal was added and the culture was cultivated for 28 days at 22°C. Menthol was then separated by steam distillation, freezing and centrifugation and obtained in 93% yield. Q\p\\

X Geraniol(20)

53

Nerol(14)

55

56

47

57

58

59

34

60

Citronellol(4)

54

43

61

Fig. (9). Biotransformation of geraniol, nerol and citronellol by Botrytis cinerea (after [41])

One of the latest reports in this area described the biotransformation of citronellol by the plant pathogenic fungus Glomerella cingulata to 3,7dimethyl-1,6,7-octanetriol [47]. In all those examples submerged liquid fungal cultures and mycelia were used. Indeed, fungal spores are generally considered as a dormant stage in the life-cycle of fungi. However, also fungal spores are well known for their biocatalytic activity, e.g. the degradation of fatty acids to methyl ketones has been known as early as the late fifties. In 1958, Gehrig and Knight [48] were the first to describe the transformation of organic compounds by fungal spores: the conversion of octanoic acid to 2heptanone by Penicillium roqueforti spores. Another very important transformation known to be carried out by fungal spores is the biotransformation of steroids (triterpenoids) [49-51]. An example of this conversion is the hydroxylation of progesterone by Aspergillus ochraceus spores [52-54].

140

The ability of fungal spores of Penicillium digitatum to biotransform monoteqêne alcohols, such as geraniol and nerol and the mixture of the aldehydes, i.e, citral by has only been discovered very recently by our group [55,56,13]. Spores of Penicillium digitatum were inoculated on solid media. After a short incubation period, the spores germinated and a mycelial mat was formed. After two weeks, the culture had completely sporulated and bioconversion reactions were started. Geraniol, nerol or citral were sprayed onto the sporulated surface culture. After one or two days, the period during which transformation took place, the cultures were extracted. Geraniol and nerol were transformed into 6-methyl-5-hepten-2one by sporulated surface cultures. Spores retained their activity for at least two months. An overall yield of up to 99% could be achieved. The bioconversion of geraniol and nerol was also performed with sporulated surface cultures oi Aspergillus niger, Geraniol was converted to linalool, a-terpineol and limonene, and nerol was converted mainly to linalool and a-terpineol [57]. Linalool and linalyl acetate Linalool (3,7-dimethyl-l,6-octadien-3-ol) (62) occurs as one of its enantiomers in many essential oils, where it is often the main component. (i?)-(~)-Linalool for example occurs at a concentration of 80-85% in Ho oils from Cinnamomum camphora; rosewood oil contains ca 80%. (iS)(+)-Linalool makes up 60-70% of coriander oil [26]. The first data on the biotransformation of linalool date back to the seventies. The bioconversion of linalool to camphor by a newly isolated Pseudomonas pseudomallei (strain A) was described [58]. Other products were (£)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid (8-carboxylinalool), 2-methyl-2-vinyltetrahydrofixran-5-one and (£)-4-methyl-3-hexenoic acid. As mentioned before, the group of Madyastha isolated a soil Pseudomonad, Pseudomonas incognita by enrichment culture technique with linalool as the sole carbon source [36]. This organism, the iinalool strain' as it was called, was also capable of utilising limonene, citronellol and geraniol but failed to grow on citral, citronellal and 1,8-cineole. Fermentations were carried out with shake cultures containing 1% linalool as the sole carbon source. Fig. (10). It was suggested by the authors that linalool was metabolised by at least three different pathways of biodegradation. One of the pathways appeared to be initiated by the specific oxygenation of the C-8 methyl group of linalool (62), leading to 8hydroxylinalool (49), which was fiirther oxidised to linalool-8-carboxylic acid (63), probably via the aldehyde. The presence of furanoid linalool oxide (65) and the unsaturated lactone, 2-methyl-2-vinyltetrahydrofuran-5one (66) in the fermentation medium suggested another mode of utilisation of linalool. The formation of these compounds was believed to proceed

141

through the epoxidation of the 6,7-double bond giving rise to 6,7epoxylinalool (64), which upon further oxidation yielded (65) and (66). The presence of oleuropeic acid (74) in the fermentation broth suggested a third pathway. Two possibilities were proposed: (3a) water elimination giving rise to a monocyclic kation (72), yielding a-terpineol (73), which upon oxidation gave oleuropeic acid (74); (3b) oxidation of the C-10 methyl group of linalool (62) before cyclisation, giving rise to oleuropeic acid (74). This last pathway was also called the 'prototropic cyclisation' [32].

VôX~~^ oôX~

CDOH

3a^

7^8 Linalool (62)

67

68

69 OOOH

-< >-

Fig. (10). Bioconversion of linalool by Pseudomonas incognita (after [36])

Later it was found that P. incognita accepted linalyl acetate better than linalool as the sole source of carbon [59]. A microbial degradation of linalyl acetate leaving the acetoxy group intact was suggested. Only few literature data are available about the fungal biotransformation of linalool and its acetates. As mentioned before, the biotransformation of linalyl acetate by Aspergillus niger isolated from garden soil was studied [39,40]. Part of the unmetabolised substrate was

142

recovered from the cultures, together with linalool, 8-hydroxylinalool, aterpineol, geraniol and some unidentified products in trace amounts. The biotransformation of linalool by Botrytis cinerea has also been described [60]. After addition of linalool to botrytised must, a series of transformation products was identified: (£)- (49) and (Z)-2,6-dimethyl-2,7octadiene-l,6-diol (48), trans- (76) and cw-fiiranoid linalool oxide (77), trans- (78) and c/^-pyranoid linalool oxide (79) and their acetates (80, 81), 3,9-epoxy-p-menth-l-ene (75) and 2-methyl-2-vinyltetrahydrofuran-5-one (66) (unsaturated lactone), Fig. (11). Quantitative analysis however, showed that linalool was predominantly (> 90%) metabolised to (jEr)-2,6-dimethyl-2,7-octadiene-l,6-diol (49) by B. cinerea. The other compounds were only found as by-products in minor concentrations. Another example of fimgal bioconversion of linalool was described in literature: the biotransformation by Diplodia gossypina ATCC 10936 [61]. A conversion scheme for the bioconversion of both (if)-(-)- and (S)-(+)linalool was proposed.

^XQX,

aô^ 66

78

79

80

81

Fig. (11). Biotransformation products of linalool by Botrytis cinerea (after [60])

The bioconversion of linalool was also investigated by our group [62]. Biotransformation of (±)-linalool with submerged shaking cultures of Aspergillus niger^ particularly A. niger ATCC 9142 yielded a mixture of cis- and /raw^-furanoid linalool oxide (yield 15-24%) and cis- and transpyranoid linalool oxide (yield 5-9%). Biotransformation of (/?)-(•-)linalool (62a) with the same strain yielded almost pure trans-fyxxdôid (76) and /rara-pyranoid (78) linalool oxide (ee > 95), Fig. (12). These conversions were purely biocatalytic, since in acidified water (pH < 3.5) almost 50% linalool was recovered unchanged, the rest was evaporated. The biotransformation was also carried out with growing surface cultures.

143 HO,

:^:K HO

A. niger |]ATCC9142 '^ O HO "

(R)-(-)-LinaloI (62a)

Linalool epoxide (64)

/rfl[W5-furanoid linalool oxide (76)

/ra/?5-pyranoid linalool oxide (78)

Fig. (12). Biotransformation of (/?)-(-)-iinaiool by Aspergillus niger ATCC 9142 (after [62]) 1.1.3 Acyclic monoterpene ketones

The literature about the bioconversion of acyclic monoterpene ketones is very limited, since only few monoterpene ketones are known, such as tagetone (82a,b) and ocimenone (83a,b), Fig. (13). The bioconversion of these examples will not be discussed here. However, 6-methyl-5-hepten2-one (33), although not a terpene, but a very important degradation product of geraniol, nerol and citral (see 1.1.2), is also a possible precursor for interesting chiral compounds. Indeed, microbial asymmetric reduction of the prochiral 6-methyl-5-hepten-2-one (33) yields 6-methyl-5-hepten-2ol, sulcatol (84), an important aggregation pheromone of the ambrosia beetle, Gnathotricus sp. [63]. The chirality of the molecule is involved in its biological activity: one species, Gnathotricus sulcatus, responds to a mixture of 65% of isomer (5)-(+)-sulcatol and 35% of isomer (i?)-(-")sulcatol and does not respond to either one of these isomers alone. Another species, G. retusus is sensitive only to the iS-isomer and its response seems to be inhibited by the i?-enantiomer. Baker's yeast {Saccharomyces cerevisiae) and the anaerobic bacterium Clostridium tyrobutyricum for example gave the iS-enantiomer from 6-methyl-5hepten-2-one, while the two fungi Geotrichum candidum and Aspergillus niger gave the i?-enantiomer of sulcatol from the same ketone. The enantioselective synthesis of (S)-(+)-6-methyl-5-hepten-2-ol by asymmetric reduction of 6-methyl-5-hepten-2-one mediated by baker's yeast in 64% yield with 90% enantiomeric excess (ee) was also reported by another group [64]. 6-Methyl-5-hepten-2-one is a valuable precursor for microbial epoxidations and hence the production of chiral ethers with high optical purities. The biotransformation of 6-methyl-5-hepten-2-one (33) by Botryodiplodia malorum CBS 13450 to (i?)-sulcatol (84) was described [61], which is then epoxidised to the (55)-epoxide (85) and opened intramolecularly to c/5'-(2i?,57?)-2-(2'-hydroxyisopropyl)-5-methyltetrahydrofuran (86) and c/5'-(3iS',6^)-3-hydroxy-2,2,6-trimethyltetrahydropyran (87). Reduction of 6-methyl-5-hepten-2-one (33) with baker's yeast to (5)-sulcatol (88) which was used as substrate for Kloeckera corticis

144

yielded the rraw^-tetrahydrofuran, derivative (91), Fig. (14).

Z-Tagetone (82a)

£-Tagetone (82b)

(2i?,55)-pityol (90) and -pyran

Z-Ocimenone (83a)

£-Ocimenone (83b)

Fig. (13). Acyclic monoterpene ketones: tagetone and ocimenone

HoV^ 86 Botryodiplodia ^ malorum 84

85

"KX 87

HOY^

yeast

90

'OH Kloeckera^ corticis

89

"

» 91

Fig. (14). Bioconversion of 6-methyl-5-hepten-2-one (after [61])

Comparable results were also reported by another group [65]. Controlled conversion of 6-methyl-5-hepten-2-one by Botrytis cinerea resulted in the formation of (5)-(+)-6-methyl-5-hepten-2-ol (sulcatol) of 90% ee. In addition, {2R,5Ry, (25,55)-, (2i?,5S)- and (25,5i?)-pityol and the four enantiomers of 3-hydroxy-2,2,6-trimethyltetrahydropyran were found as biotransformation products of 6-methyl-5-hepten-2-one. A complete synthesis of optically pure (2i?,55)-pityol (90), a pheromone of the bark beetle Pityophtorus pityographus using a

145

chemoenzymatic route was also described [66]. The conversion consisted of a baker's yeast asymmetric reduction of 6-methyl-5-hepten-2-one (33) to (S)-sulcatoi (88), which was then submitted to an epoxidation carried out by A. niger. The last step of this reaction was a cyclisation [67] yielding (2i?,55)-pityol (90). L2 Cyclic Monoterpenes 1.2.1 Monocyclic hydrocarbon monoterpenes

Limonene and other compounds with ap-l-menthene skeleton Limonene (92) is the most widely distributed terpene in nature after a-pinene [68]. The (+)-isomer is present in Citrus peel oils at a concentration of over 90%; a low concentration of the (-)-isomer is found in oils from the Mentha species and conifers [26]. The first data on the microbial transformation of limonene date back to the sixties. A soil Pseudomonad was isolated by enrichment culture technique on limonene as the sole source of carbon [69]. This Pseudomonad was also capable of growing on a-pinene, jff-pinene, 1-p-menthene and/^-cymene. The optimal level of limonene for growth was 0.3-0.6% (v/v) although no toxicity was observed at 2% levels. Fermentation of limonene by this bacterium in a mineral-salts medium resulted in the formation of a large number of neutral and acidic products. Dihydrocarvone, carvone, carveol, 8-pmenthene-1,2-cz5'-diol, 8-p-menthen-1 -ol-2-one5 8-p-menthene-1,2-transdiol and l-p-menthene-6,9-diol were among the neutral products isolated and identified. The acidic compounds isolated and identified were perillic acid, jff-isopropenyl pimelic acid, 2-hydroxy-8-/?-menthen-7-oic acid and 6,9-dihydroxy-l-p-menthen-7-oic acid. Based on these data three distinct pathways for the catabolism of limonene by the soil Pseudomonad were proposed by the same group [70], involving allylic oxygenation (pathway 1), oxygenation of the 1,2-double bond (pathway 2) and progressive oxidation of the 7-methyl group to perillic acid (pathway 3) (see Fig. (15), after [68]). A fourth pathway, yielding a-terpineol and carried out by fungi such as Penicillium digitatum, P. italicum and Cladosporium and several bacteria will be discussed later. Also a fifth (hydroxylation in C-3) and sixth pathway (hydroxylation in C-4) will be mentioned later. The first pathway gives c/5-carveol (93), D-carvone (94) (an important constituent of caraway seed and dill-seed oils [27,71]) and 1-p-menthene6,9-diol (95). (+)-(5)-Carvone is a natural potato sprout inhibiting, fungistatic and bacteristatic compound [72,73]. It is important to note that L-(~-)-carvone (the 'spearmint flavour') was not yet described in microbial transformation [68]. However, the biotransformation of limonene to L-carvone was patented by a Japanese group [74]: a Corynebacterium species grown on limonene was able to produce about 10 mg/L of 99%

146

Pathway

\/

Carveol (93)

95

Pathway 2 ^

Limonene (92)

Limonene epoxide (96)

Dihydrocarvone (97)

COOH

COOH HOOC

HO' Limonene-l,2-diol (98)

OH

103

106

Pathway 3 (main pathway)

CHjOH

CHO

COOH

^ss

^

Perillyl alcohol (100) Perillaldehyde (101)

Perillic acid (102)

COOH OH

104

Fig. (15). Pathways for the degradation of limonene by a soil Pseudomonad (after [68])

COOH

105

147

pure L-carvone in 24-48 hr. Pathway 2 yields (-f)-dihydrocarvone (97) via intermediate limonene epoxide (96) and 8-p-menthen-l-oi-2-one (99) as oxidation product of limonene-1,2-diol (98). The third and main pathway leads to perillyl alcohol (100), perillaldehyde (101), perillic acid (102), constituents of various essential oils and used in the flavour and fragrance industry [27], 2-hydroxy-8-/7-menthen-7-oic acid (104), 2-oxo-8-/7menthen-7-oic acid (105), )ff-isopropenyl pimelic acid (106) and 4,9dihydroxy-l-/7-menthen-7-oic acid (103). As mentioned before, a Pseudomonas incognita was isolated by enrichment technique on the monoterpene alcohol linalool that was also able to grow on geraniol, nerol and limonene [36]. The metabolism of limonene by this bacterium was also investigated [37]. After fermentation the medium yielded as main product a crystallic acid, perillic acid, together with unmetabolised limonene, and some oxygenated compounds: dihydrocarvone, carvone, carveol, />-menth-8-en-l-ol-2-one, /?-menth-8ene-l,2-diol or/>-menth-l-ene-6,9-diol (structure not fiilly elucidated) and finally jff-isopropenyl pimelic acid. The same group has also isolated a strain of Pseudomonas putidaarvilla (PL-strain) from limonene and (+)-a-pinene as the sole carbon source that was capable of growing on (+)-limonene, (-+-)-a-pinene, (~)-apinene, jff-pinene, 1-p-menthene, 3-p-menthene andp-cymene as substrates [75]. Limonene was degraded to perillyl alcohol, perillaldehyde and perillic acid. More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4if)-(-f)-limonene to (4i?)-(+)-a-terpineol or (4S)-(+)-limonene to (45)(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfimie chemicals [27]. The first data onfimgalbioconversion of limonene date back to the late sixties [79,80]. Three soil microorganisms were isolated on and grew rapidly in mineral salts media containing appropriate terpene substrates as sole carbon sources. The microorganisms belonged to the class Fungi Imperfecti, and two of them had been tentatively identified as Cladosporium species. A Cladosporium designated Ti was isolated fi*om terpene-soaked soil using l-menthene (107) as the sole carbon source.

148

The major catabolic product isolated from the growth medium of this organism was found to be a cyclic 1,2-diol, identified as trans-pmenthane-l,2-diol (108). A similar but biochemically distinct Cladosporium sp. designated as T7 was isolated on D-limonene (92). The growth medium of this strain contained 1.5 g/L of the analogous product, /ra«5'-limonene-l,2-diol (109), Fig. (16). Minor quantities of the corresponding c/5-l,2-diol were also isolated. The third organism, designated as laboratory culture Tg, was isolated on 3-menthene and yielded /ra«5'-/7-menthane-3,4-diol. The same group [81] isolated a fourth microorganism from a terpene-soaked soil on mineral salts media containing D-limonene as the sole C-source. The strain, Cladosporium, designated T12, was capable of converting D-limonene into an optically active isomer of a-terpineol in yields of approx. 1.0 g/L.

Cladosporium ^ p

1-Menthene(107)

108

y'^

I ^

Cladosporium

Limonene(92)

109

Fig. (16). Biotransformation of 1-menthene and limonene by Cladosporium (after [79,80])

The fungal bioconversion of limonene was further studied [82]. Penicillium sp. cultures were isolated from rotting orange rind that utilised limonene and converted it rapidly to a-terpineol. Bowen [83] isolated two common citrus moulds, Penicillium italicum and P. digitatum, responsible for the postharvest diseases of citrus fruits. Fermentation of P. italicum on limonene yielded cis- and trans-c^rvtoX (93) (26%) as main products, together with cis- and /ra«5-p-mentha-2,8-dien-l-ol (110) (18%), (+)carvone (94) (6%), p-mentha-l,8-dien-4-ol (111) (4%), perillyl alcohol (100) (3%),/7-menth-8-ene-l,2-diol (98) (3%), Fig. (17). Conversion by P. digitatum yielded the same products in lower yields. The two alcohols /7-mentha-2,8-dien-l-ol (110) andp-mentha-l,8-dien-4-ol (111) were not described in the transformation studies where soil Pseudomonads were used [69]. The biotransformation of limonene by Aspergillus niger is a very important example of fungal bioconversion. Screening for fungi capable of metabolising the bicyclic hydrocarbon terpene a-pinene (see 1.2.2) yielded a strain of ^4. niger NCÎM 612 that was also able to transform limonene [75]. This fungus was able to carry out three types of oxygenative rearrangements. Fig. (18). The conversion of limonene (92) to a-terpineol (73) is an example of pathway 4 (cfr. supra).

149

110

111

carveol (93)

carvone (94)

perillyl alcohol (100)

98

Fig. (17). Biotransformation products of limonene by Penicillium digitatum and P. italicum (after [83])

• niger ,

Limonene (92)

a-terpineol (73)

c/5-carveol (93)

110

Fig. (18). Oxygenative rearrangements carried out by Aspergillus niger NCIM 612 on limonene (after [75])

More recently, the production of glycols from limonene and other terpenes with a 1-menthene skeleton was reported [84]. An extensive screening of 1000 different microorganisms showed that limonene was attacked by a large number of strains (320 strains). Accumulation of glycols during fermentation with several fungi was observed. The most appropriate strains were Corynespora cassiicola DSM 62475 and Diplodia gossypina ATCC 10936. An extensive overview on the microbial transformations of terpenoids with a l-p-menthene skeleton was published by Abraham et aL [85]. In 1985, the same group [24] investigated the biotransformation of (i?)(+)-limonene by the fungus Penicillium digitatum, A complete transformation of the substrate to a-terpineol by P. digitatum DSM 62840 was obtained with a yield of 46% pure product. The bioconversion of (4i?)-(-)-limonene to (4i?)-(-)-a-terpineol by immobilised fungal mycelia of Penicillium digitatum was described more recently [86]. The fungi were immobilised in Calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4°C. a-Terpineol production by the fungus was 12.83 mg/g beads per day, producing a 45.81% bioconversion of substrate. The optimum conversion temperature was 28°C and the optimum pH was 4.5. The highest

150

concentration of product was formed with a contact time of between 1 and 2 days [87]. A Japanese group also studied the biotransformation of limonene and related compounds (1-methylcyclohexene and cyclohexene) by Aspergillus cellulosae M-77 [88]. It is important to note tiiat (+)-limonene (92) was mainly converted to (-f)-isopiperitenone (112) (19%), (+)-limonene-l,2trans-6xo\ (109) (21%), (+)-c/5-carveol (93) (5%) and (+)-perillyl alcohol (100) (12%), Fig. (19). Although these alcohols (93,100,109) have been found by other authors in the past, the production of isopiperitenone (112) from limonene (92) had not been published before. The conversion of limonene to isopiperitenol by hydroxylation in the C-3 position and further oxidation of isopiperitenol to isopiperitenone is an example of the fifth pathway of limonene biotransformation. Only very recently, it was published by another group [89] that two unclassified strains of the basidiomycetes, Trichosporon, transformed (+)-limonene to isopiperitenone (0.05 and 0.4 g[L\ yield 2% and 20%), and transA,!dihydroxylimonene (0.6 g/L; yield 30%). This group also reported the conversion of (+)-limonene by the yeasts Arxula adeninivorans and Yarrowinia lipolytica to perillic acid.

Aspergillus cellulosae

92

112

100

93

109

Fig. (19). Biotransformation of (+)-limonene hy A. cellulosae (after [88])

Very recently, the purification and characterisation of an epoxide hydrolase, catalysing the conversion of limonene-1,2-epoxide to limonene1,2-diol has been described [90]. The enzyme was isolated from Rhodococcus erythropolis DCL14 and is induced when the microorganism is grown on monoterpenes. The authors found evidence that the enzyme, limonene-1,2-epoxide hydrolase is the first member of a new class (the third class) of epoxide hydrolases [91]. In a recent extensive overview on the biotransformation of terpenoids by Aspergillus spp., Noma and Asakawa [92] also mentioned a sixth pathway of limonene bioconversion: the hydroxylation at the C-4 position to give /7-mentha-l,8-dien-4-ol (111), Fig. (20), a compound also identified earlier as one of the bioconversion metabolites of limonene with Penicillium italicum [83]. In this review, the fifth pathway, leading to isopiperitenol (113) which isfiirtheroxidised to isopiperitenone (112) and its rearrangement product, piperitenone (114) is also discussed.

151

Hydroxylation products of this fifth pathway are 5-hydroxyisopiperitenone (115), 10-hydroxyisopiperitenone (116), 4-hydroxyisopiperitenone (117) and 7-hydroxyisopiperitenone (118), Fig. (20).

116

117

118

Fig. (20). Metabolic pathways 5 and 6 of limonene by Aspergillus spp. (after [92]) 1.2.2 Bicyclic hydrocarbon monoterpenes

a-Pinene The most abundant terpene in nature is a-pinene (119) which is industrially obtained by fractional distillation of turpentine [68]. (+)-aPinene occurs, for example, in oil from Pinus palustris Mill, at concentrations of up to 65%; oil from Pinus pinaster Soland. and American oil from Pinus caribaea contain 70% and 70-80% resp. of the (-)-isomer [26]. One of the earliest publications described the biotransformation of apinene by A, niger [93,94] A 24-hr shake culture of this strain metabolised 0.5% a-pinene (119) in 4-8 hr. After the fermentation the

152

culture broth contained a ketone, verbenone (121) (2-3%), an alcohol, cisD-verbenol (120) (20-25%), a diol, D-(+)-rraAi^-sobrerol (122) (2-3%), and a hydroxyketone, hydroxycarvotanacetone (123), Fig. (21). A. niger NCIM612

a-Pinene(119)

cw-Verbenol (120)

Verbenone (121)

trans-Sohrerol (122)

Hydroxycarvotanacetone (123)

Fig. (21). Bioconversion of a-pinene by Aspergillus niger NCIM 612 (after [93])

The degradation of a-pinene and other A^-menthene skeletons by Pseudomonas (PL-strain) was first investigated by Hungund et al [95]. A terminal oxidation pattern was proposed, leading to the formation of organic acids through ring cleavage. Shukla et al [96] described the fermentation of a-pinene and )ff-pinene in shake cultures by a soil Pseudomonas sp. (PL-strain) able to grow on a-pinene as the sole carbon source. A complex metabolite mixture was obtained composed of neutral as well as acidic compounds. Fig. (22).

Pseudomonas (PL-strain)

a-Pinene(119)

OOOH

(Jr™ Borneol (1)

Myrtenol (124)

Myrtenic acid (125)

OOOH

Phellandric acid (126)

Fig. (22). Bioconversion of a-pinene by Pseudomonas (after [96])

More recently, the degradation of a-pinene by Pseudomonas fluorescens NCIMB 11671 was described [97,98]. A novel pathway for the microbial breakdown of a-pinene (119) was proposed. Fig. (23). The attack is initiated by enzymatic oxygenation of the 1,2-double bond to form the epoxide (127). This epoxide then undergoes rapid rearrangement to produce a novel diunsaturated aldehyde, occurring as two isomeric forms. The primary product of the reaction (Z)-2-methyl-5-isopropylhexa2,5-dien-l-al (trivial name isonovalal) (128) can undergo chemical isomerisation to the jE-form (novalal) (129). Isonovalal, the native form of

153

the aldehyde, possesses citrus, woody, spicy notes, whereas novaial has woody, aldehydic, cyclene notes. The same bioconversion was also carried out by another bacterial strain, Nocardia sp. strain PI8.3 [99,100]. Also the biotransformation of a-pinene derivatives and other pinane monoterpenoids by Cephalosporium aphidicola has been described [101]. The best conversion was the oxidation of verbenol to verbenone (yield 61%). CHO mono-oxygenase

a-Pinene (119)

fC/^

dccyclising enzyme

a-Pinene epoxide (127)

f^ Ci\0 L ^

chemical isomerisation

Isonovalal = Z-isomer (128)

Novaial = £-isomer (129)

Fig. (23). Bioconversion of a-pinene by Pseudomonas fluorescens NCIMB 11671 (after [97])

j^Pinene jff-Pinene (130) is found in many essential oils. Optically active and racemic j9-pinenes are present in turpentine oils, although in smaller quantities than a-pinene [26]. Only very little is known about microbial transformations of )ff-pinene, which is an abundantly occurring natural terpene [68]. Shukla et aL [96] obtained a similarly complex mixture of transformation products from /?-pinene as from a-pinene through degradation by a Pseudomonas sp. (PL-strain). On the other hand, Bhattacharyya and Ganapathy [102] indicated that fungi such as A, niger NCIM 612, act differently and more specifically on the pinenes by preferably oxidising /?-pinene (130) in the allylic position to form the interesting products pinocarveol (131) and pinocarvone (132), besides myrtenol (124), Fig. (24). The same group [75] also described the conversion of ^-pinene by Pseudomonas putida-arvilla (PL-strain): a degradation pathway for the conversion of j5-pinene to bomeol was proposed. By enrichment culture technique, a bacterium was isolated from local sewage sludge, utilising caryophyllene as the sole source of carbon and energy [103]. Fermentation of ^-pinene by this culture in a mineral salt medium (Seubert's medium) at 30°C with agitation and aeration for four days yielded a few neutral and acidic transformation products. The metabolites isolated and identified were camphor (2), bomeol (1),

154

isobomeol (133), a-terpineol (73) and jff-isopropyl pimelic acid (134), Fig. (25). The organism \vsis idQntifiQd SiS Pseudomonas pseudomallai. Using modified Czapek-Dox medium and keeping the other conditions the same, the pattem of the metabolic products was dramatically changed. The metabolites then recovered were trans-pinocarveol (131), myrtenol (124), a-fenchol (135), a-terpineol (73), myrtenic acid (125) and two unidentified products. CHjOH Aspergillus niger NCIM612

Pinocarveol (131)

P-Pinene (130)

Pinocarvone (132)

Myrtenol (124)

Fig. (24). Bioconversion of ^-pinene by ^. niger (after [102])

COOI f'u^^

P. pseudomallai ^ Seubert's medium

f^^^^ ^ i ^

(^X^ Âx^

Camphor (2)

Isobomeol (133)

p-Pinene(130)

.OH f r i ' îx^

Bomeol(l)

COOH

OH

a-Terpineol (73)

COOH

CH,OH P. pseudomallai

^

"°;l)

.OH

modified Czapek-Dox medium

P-Pinene(130)

/ra«5-Pinocarveol (131)

p-Isopropyl pimelic acid (134)

Myrtenol (124)

a-FenchoI (135)

a-Teipineol (73)

Fig. (25). Bioconversion of j8-pinene by Pseudomonas pseudomallai (after [103])

Myrtenic acid (125)

155

1-2.3 Cyclic monoterpene alcohols

Menthol Together with a-terpineol, menthol (25) is one of the few terpene alcohols occurring widely in nature that have physiological properties making them important fragrance or flavour compounds [26]. There are in fact 8 isomers with a menthol (p-menthan-3-ol) skeleton, (-)-menthol (138) is the most important one, because of its cooling and refreshing effect. It is the main component of peppermint and commint oils obtained from the Mentha piperita and Mentha arvensis species. Many attempts have been made to produce (-)-L-menthol from inexpensive terpenoid sources, but these sources unavoidably also yielded the (±)-isomers: isomenthol, neomenthol, and neoisomenthol [68]. Especially Japanese researchers have been active in this field, maybe because of the large demand for L-menthol in Japan itself: 500 t/year [104]. Indeed, most literature deals with the enantiomeric hydrolysis of (±)-menthol esters to optically pure (~)-menthol. The asymmetric hydrolysis of DL-menthyl chloroacetate by an esterase of Arginomonas non-fermentans FERM-P-1 924 has been patented by the Japanese Nippon Terpene Chemical Co. [105,106]. Moroe et al [107] from the Takasago Perfumery Co. Ltd. claim that certain selected species of Absidia, Penicillium, Rhizopus, Trichoderma, Bacillusy Pseudomonas and others asymmetrically hydrolyse esters of (±)-menthol isomers such as formiates, acetates, propanoates, caproates, and esters of higher fatty acids. Fig. (26). Besides the hydrolysis of menthyl esters, the biotransformation of menthol and its enantiomers has also been published. The microbial transformation of menthol was studied by Shukla et al [108]. More recently Asakawa et al, [109] described the fungal biotransformation of (-)- and (+)-menthols by Aspergillus niger and A. cellulosae. A, niger converted (-)-menthol to 1-, 2-, 6-, 7- and 9-hydroxymenthols and the mosquito repellent-active 8-hydroxymenthol, whereas (+)-menthol was smoothly biotransformed by the same fungus to give 7-hydroxymenthol. A. cellulosae on the other hand, biotransformed (-)-menthol specifically to 4-hydroxymenthol. The bioconversion of (+)- and (--)-neomenthol and (+)-isomenthol by A. niger was studied later by the same group [110], mainly giving a hydroxylation. For a very detailed schematic overview of these reactions, we refer to the review given by Noma and Asakawa [92]. More recently, the fungal transformation of (-)-menthol by Cephalosporium aphidicola was reported [111]. Incubation of (-)menthol with this fungus for 12 days yielded four new metabolites, 10acetoxymenthol, 4a-hydroxymenthol, 3a-hydroxymenthol, and 10hydroxymenthol and two known compounds identified as 7hydroxymenthol and 9-hydroxymenthol.

156

Microorganism ^ O-CDCHj

(-)-Menthyl acetate (136)

^Y^'0-CDCH3

(+)-Menthyl acetate (137)

\ ^ O H

(-)-Menthol (138)

^^''O-COCHj

(+)-Menthyl acetate (137)

Fig. (26). Asymmetric hydrolysis of (±)-menthyl acetate to obtain pure (~)-menthol 1.2.4 Cyclic monoterpene ketones and norterpenoids

Camphor Both optical isomers of camphor (2) are found widely in nature, (+)camphor being more abundant. It is the main component of oils obtained from the camphor tree Cinnamomum camphora [26]. The hydroxylation of D-(-H)-camphor by Pseudomonas putida C\ was described [112]. The substrate was hydroxylated exclusively in its 5-exo- and 6-exo-positions. The earliest investigation of the degradation of (•f)-camphor dates back to the late fifties [113 and references cited therein]. Although only limited success was achieved in understanding the catabolic pathways, key roles for methylene group hydroxylation and biological Baeyer-Villiger monooxygenases in ring cleavage strategies were established [113]. A degradation pathway of (+)-camphor by Pseudomonas putida ATCC 17453 and Mycobacterium rhodochorus Ti was proposed [113]. Pulegone (i?)-(+)-Pulegone (139), a mint-like odour monoterpene ketone, is the main component (up to 80-90%) of Mentha pulegium essential oil (Pennyroyal oil) which is sometimes used in beverages and food for human consumption and occasionally in herbal medicine as an abortifacient drug. The biotransformation of (i?)-(+)-pulegone by fungi was investigated [114]. Most fungal strains tested, grown in a usual liquid culture medium, were able to metabolise (i?)-(+)-pulegone to some extent in a concentration range of 0.1-0.5 g/L; higher concentrations were generally toxic, except for one of the strains {Aspergillus sp.) isolated from mint leaves inftision, which was able to survive to concentrations of up to 1.5 g/L. The predominant bioconversion product was generally 5hydroxypulegone (140) (20-30% yield). Other metabolites were present in lower amounts (5% or less). Fig. (27). The formation of 5hydroxypulegone (140) was explained by hydroxylation at a tertiary

157

position. Its dehydration to piperitenone (114), even under the incubation conditions, during isolation or derivatisation reactions precluded any tentative determination of its optical purity and absolute configuration. This metabolism of (i?)-(+)-pulegone is very similar to the bioconversion pathway that was very recently published by another group [115]. Using the fungal strain Mucor piriformis, eight metabolites were isolated from the fermentation medium after conversion of (i?)-(-f)-pulegone (139), namely 5-hydroxypulegone (140), piperitenone (114), 6-hydroxypulegone (144), 3-hydroxypulegone (141), 5-methyl-2-(l-hydroxy-l-methylethyl)-2cyclohexen-1-one (142), 3-hydroxyisopulegone (146), 7hydroxypiperitenone (145), and 7-hydroxypulegone (147), Fig. (28).

Piperitenone (114)

141

142

143

Fig. (27). Bioconversion of pulegone hy Aspergillus sp. (after [114])

The biotransformation of (i?)-(+)-pulegone was also studied by a Japanese group [116]. The major bioconversion metabolite of this substrate with Botrytis allii was (-)-(lif)-8-hydroxy-4-/7-menthen-3-one. The secondary major product from this biotransformation was isolated and its structure established as piperitenone [117]. It is interesting to note that the same group also investigated the bioconversion of piperitone, the dihydrogenation product of piperitenone: a strain of Rhizoctonia solani was found able to hydroxylate the substrate preferentially at the 6-position [118,119]. Recently, the biotransformation of (+)-pulegone and other monoterpenoid ketones, like (-)-piperitenone, (+)- and (-)-carvone, (-)menthone and (-)-verbenone by yeasts and yeast-like fungi was also

158

Studied [120]. Only one organism, a Hormonema isolate (UOFS Y-0067), quantitatively reduced (-)-menthone and (-f-)-pulegone to (+)-neomenthol.

hydroxylation

(i?)-(+)-Pulegone(139)

hydroxylation

dehydration

5-HydroxypuIegone (140)

shydroxylation

Piperitenone(114)

allylic methyl oxidation

CHoOH

^ 3-Hydroxypulegone (141)

allylic alcohol rearrangement

142

6-Hydroxypulegone (144)

7-Hydroxypiperitenone (145)

.isomerisation

reduction of double bond

3-Hydroxyisopulegone (146)

7-Hydroxypulegone(147)

Fig. (28). Transformation of (7?)-(+)-pulegone by Mucor piriformis (after [115])

Carvone Carvone (94) occurs as (+)-carvone, (~)-carvone or racemic carvone. (AS)-(+)-Carvone is the main component of caraway oil (ca 60%) and dill oil and has a herbaceous odour reminiscent of caraway and dill seeds. (R)(-)-Carvone occurs in spearmint oil at a concentration of 70-80% and has a herbaceous odour similar to spearmint [26]. (S)-(H-)-Carvone (94) was used as substrate for bioconversions by selected microorganisms: five

159

bacteria and one fungus [121]. The substrate was reduced predominantly to both dihydrocarvones (97a, 97b) and to neo-isodihydrocarveol (148), Fig. (29). Sensitivity of the microorganisms to (5)-(+)-carvone and some of the products prevented yields exceeding 0.35 g/L in batch cultures. The fungus Trychoderma pseudokoningii gave the highest yield of neoisodihydrocarveol. O

(5)-(+)-Carvone (94)

97a

97b

OH

148

Fig. (29). Bioconversion of carvone (after [121])

(5)-(+)-Carvone is known to inhibit fungal growth of Fusarium sulphureum when the substrate was administered via the gas phase [73]. Under the same conditions, the related fungus, F. solani var. coeruleum was not inhibited. In liquid medium, both fungi were found to convert (5)(+)-carvone with the same rate, mainly to isodihydrocarvone, isodihydrocarveol and neo-isodihydrocarveol. As mentioned before, the biotransformation of (-f)- and (-)-carvone and other monoterpenoid ketones by yeasts and yeast-like fungi has been reported recently [120]. ^lonone j?-Ionone (149) and its derivatives are widely distributed in nature and represent important constituents of many essential oils [68]. The hydroxylation of jff-ionone by Aspergillus niger JTS 191 was reported [122]. Two major metabolites were isolated and the structures proposed as (iS)-2-hydroxy-j5-ionone (150) and (jf?)-4-hydroxy-^-ionone (151), Fig. (30). The complex was found to be very effective for tobacco flavouring at ppm level. For this research, more than 1000 microorganisms from various culture collections were tested for their abilities to convert ionones (a- and )5-ionones) to other aroma compounds. These microorganisms comprised over 150 fungi, 28 yeasts and more than 800 terpene utilising bacteria which were isolated from plants and soils [123]. Many of the bacterial isolates consumed the substrate without accumulation of interesting flavouring substances. Some fungi however {Aspergillus, Phialophora, or Rhizopus) successfully converted a- and ^-ionones to other aroma compounds. A, niger JTS 191 was selected as the most

160

suitable strain for the production of new aromatic metabolites from jiionone and ^-methylionone. O

0

0

6H P-Ionone (149)

O

a-Ionone(152)

(5)-2-Hydroxy-p-ionone (150)

O

3-Hydroxy-a-ionone (153)

(/?)-4-Hydroxy-p-ionone (151)

O

3-Oxo-a-ionone (154)

Fig. (30). Bioconversion of j5-ionone and a-ionone by Aspergillus niger JTS 191 (after [122,129])

A considerably different type of ^-ionone transformation was reported by Krasnobajew and Helmlinger [124]. They described the biotransformation of )ff-ionone by pregrown mycelia of Lasiodiplodia theobromae ATCC 28570. The fermentation of )ff-ionone had to be performed with pregrown cultures because the substrate inhibited the growth and led to lysis of the cells. Therefore mycelia cultures at the end of their logarithmic growth phase (after 20 to 50 hr) were employed. After 3 to 4 days the transformation capacity of the mycelia decreased considerably, and the fermentation broth was extracted. Under optimal conditions mycelia of L theobromae ATCC 28570 transformed \h g Pionone per litre of culture fluid. An essential-oil-type product with tobacco flavour could be obtained from the culture fluid. The biotransformation of j9-ionone by the same microorganism, L theobromae IFO 6469 was later patented by the Japan Tobacco, Inc. [125]: the metabolites were used as cigarette flavour improvers. More recently, the fed-batch biotransformation of )8-ionone by Aspergillus niger was described [126]. A commercially available strain, A. niger IFO 8541 was selected and was found to be an efficient biocatalyst for the biotransformation of ^-ionone into 2- and 4-hydroxy-)ffionone and 2-oxo-^-ionone. 4-Hydroxy-^-ionone was the main product with a mass yield close to 90%. It is interesting to note that the metabolism of )9-ionone involves a lag phase, which is a function of the

161

biomass concentration. Therefore, substrate addition was carried out after 100 hr of preliminary fungal growth in conical flasks. In a fed-batch operation, product formation was noticed about 40 hr after the first ^8ionone addition. 2- And 4-hydroxy-j5-ionone were the first compounds detected, 4-oxo-^-ionone appeared far later, after 320 hr cultivation. a-Ionone In 1978 Givaudan patented a transformation of a-ionone [127]: a fermentation of Botryodiplodia theobromae IFO 6469 with a-ionone (152) yielded a mixture of compounds (155 -157) with honeysuckle aroma. Fig. (31). The same fungi which efficiently biotransformed )ff-ionone also transformed a-ionone [128]. As observed for jS-ionone, a-ionone (152) transformations with Lasiodiplodia theobromae ATCC 28570 also suggested an oxygenase-type enzyme system to be responsible for the degradation of the molecule by loss of one C2-unit. The bioconversion of a-ionone, a-methylionone and a-isomethylionone by Aspergillus niger JTS 191 was described [129]. The major products from a-ionone (152) were cis- and rra«5-3-hydroxy-a-ionone (153) and 3oxo-a-ionone (154), Fig. (30). The biotransformation of a- and jff-ionones by ten kinds of Aspergillus spp., one of which was A. niger JTS 191, and other microorganisms was also studied later by another group [130]. The results with A. niger JTS 191 were essentially the same as the ones obtained by the previous group [129]. A complete schematic overview of all metabolites produced from a-ionone and ^-ionone is given by Noma and Asakawa [92]. o PK^'^^::^^ \^SJ^

Botryodiplodia (^^''^^T^^^^ ^ P'S''"^^^^" ^. (^^ 1 eo romae ^^X^^^^ O^^^X^^ G^^^Ô^

a-Ionone(152)

155

156

157

Fig. (31). Bioconversion of a-ionone by Botryodiplodia theobromae IFO 6469 (after [127])

2 Sesquiterpenes Sesquiterpenes and their derivatives are found together with monoterpenes in many essential oils. Many of them are important flavour and perfume compounds, some are of considerable importance for pharmaceutical applications [131]. As sesquiterpenoids contain one more isoprene unit than monoterpenes, a greater variety of structures is possible which is

162

manifested in nature by a tremendous diversity of this group of compounds [68]. Only around 30 different sesquiterpenes were known 20 years ago and these had nearly 15 different carbon skeletons. There are now almost 1000 known sesquiterpenoid compounds belonging to about 200 different skeletal families [132]. 2.1 Acyclic Sesquiterpenes 2.2.1 Acyclic sesquiterpene alcohols

Nerolidol Nerolidol (3,7,ll-trimethyM,6,10-dodecatrien-3-ol) (158, 162) is the sesquiterpene analogue of linalool (62). Because of the double bond at the 6-position, it exists as E- (158) and Z- (162) isomers. (+)-£^-Nerolidol occurs in cabreuva oil; (~)-nerolidol has been isolated from Dalbergia parviflora wood oils [26]. Arfmann et aL [133] investigated the hydroxylation of acyclic terpenes and analogues such as Z- and jE-nerolidol, famesol, nerylacetone and geranylacetone. Aspergillus niger ATCC 9142 and Rhodococcus rubropertinctus DSM 43197 were found to be the best suited strains. The bacteria exclusively oxidised the terminal methyl group to the primary alcohol (159) and further to the carboxylic acid (161), Fig. (32). The fungus on the other hand attacked parallel the double bond of the terminal isoprenyl group resulting in glycols (160) analogous to the transformation of 1-menthenes (see 1.2.1). The best yields with nearly 25% of primary alcohol (a)-hydroxy-£-nerolidol) (159) were obtained with A, niger ATCC 9142 and the substrate ^-nerolidol (158). Apart from the terminal oxidation and co-hydroxylation, hydroxylations in the chain of acyclic terpenes were also reported [112]. In a limited screen some microorganisms were found capable of introducing a hydroxy group in 8- or 9-position of the substrate JS-nerolidol. The epoxidation of the isoprenyl double bond of J?-nerolidol by Nocardia alba DSM 43130 was reported by the same group [112]. Terminal oxidations yielding both the co-hydroxylated product and the 10,11-diol were observed as parallel reactions. The same group also studied the bioconversion of Z- and £-nerolidol with three more fungal species: Diplodia gossypina, Corynespora cassiicola and Gibberella cyanea [112]. It was found that all strains hydroxylated the substrates to their respective vicinal diols (glycols). The highest yield was obtained with the strain G. cyanea (79.5%) and the substrate jE-nerolidol. Also hydroxyketones were found in lower yields (0.5-5%) and in some cases traces of epoxides were produced. More recently a very interesting report describing a completely different degradation pathway of nerolidol (158) by Alcaligenes eutrophus was

163

published [134]. This bacterial strain was isolated from soil by enrichment culture technique with nerolidol as the sole source of carbon and energy. Instead of the usual epoxidation of the 10,11-double bond, an epoxidation of the 1,2-double bond, followed by reduction of 166 to a triol (167) which was cleaved to a C2-ketol (glycolaldehyde) (168) and geranylacetone (169) was noticed, Fig. (33). This pathway is quite similar to the degradation of nerol and geraniol to 6-methyl-5-hepten-2-one (see Fig. (7)). To the best of our knowledge an analogous pathway for the degradation of linalool to 6-methyl-5-hepten-2-one has not been published before. The authors as well [134] claim that their proposed pathway for the biotransformation of nerolidol is hitherto unknown. Geranylacetone (169) was further reduced to (5)-(+)-geranylacetol (170), the "norsesquiterpene"-analogue of sulcatol. p

I

A. Niger

£-NerolidoI(158)

^y^^^::^^^^^.^^

+ "''ilT''^^^

159 (20%)

160

+ "^v>^

159 (16%)

161 (16%)

163 (1.5%)

164

163 (0%)

165 (25%)

A. niger

Z-Nerolidol(162) R. rubropertinctus

Fig. (32). a>-Hydroxylation of £- and Z-nerolidol (after [133])

These findings were also in contrast to the results they obtained carrying out the bioconversion of nerolidol with Aspergillus niger [135,136]. In this case, the two main metabolites identified were 12hydroxynerolidol (159) and 10,11-dihydroxynerolidol (160). The same reactions were noticed when dihydronerolidol was used as substrate: comethyl hydroxylation and dihydroxylation of the remote double bond.

164

H20 + C02 (5)-(+)-Geranylacetol (170)

Geranylacetone (169)

Glycolaldehyde (168)

Fig. (33). Proposed pathway for the degradation of nerolidol by Alcaligenes eutrophus (after [134])

Finally, a Japanese group also studied the biotransformation of Z- and £-nerolidol [137,138] with the plant pathogenic fungus Glomerella cingulata. Both Z- and £-nerolidol were mainly oxidised at the remote double bond. Z-Nerolidol (162) was transformed into (Z)-3,7,lltrimethyl-l,6-dodecadiene-3,10,ll-triol (164) while ^-nerolidol (158) was mainly oxidised to (£)-3,7,ll-trimethyl-l,6-dodecadiene-3,ll-diol (171); only small amounts of (£)-3,7,11-trimethyl-l,6-dodecadiene-3,10,11-triol (160) were obtained. Fig. (34).

Z-NerolidoI (162)

164

Fig. (34). Hydroxylation of £-nerolidol and Z-nerolidol by Glomerella cingulata (after [137,138])

165

Farnesol Farnesol (3,7,11 -trimethyl-2,6,10-dodecatrien-1 -ol) (181) is the sesquiterpene analogue of geraniol (20) and nerol (14), depending on its 2E' resp. 2Z-configuration. It is a component of many blossom oils. It is a colourless liquid with a linden blossom odour, which becomes more intense when evaporated, possibly due to oxidation [26]. The levels found in essential oils are generally low (0.5-1.0%) with the exception of cabreuva, which contains up to 2.5% famesol, and the distillate from flowers of Oxystigma buccholtzii Harms., which contains up to 18% famesol [27]. The first data about the bioconversion of famesol date back to the sixties: its degradation pathway is similar to that of geraniol and nerol. Seubert [139] showed that the degradation of famesol by Pseudomonas citronellolis proceeds through the oxidation of C-1 to give famesic acid, followed by carboxylation of the ^-methyl group. Subsequently, the 2,3double bond of the dicarboxylic acid is hydrated to a 3-hydroxy acid which is then acted upon by a lyase resulting in the formation of a j9-keto acid and acetic acid. The jff-keto acid readily enters the fatty acid oxidation pathway [29]. When famesol (181), as a mixture of the four isomers, was incubated with Rhodococcus rubropertinctus DSM 43197 for 23 hr, two products were obtained: a low yield (3%) of an 11-hydroxylated compound, 11hydroxygeranylacetone (182) and a higher yield (12%) of nerylacetone (174), Fig. (35) [133,140]. A common feature of both was that the famesol skeleton was shortened by a C-2 unit. It is interesting to remark that the transformation of famesol (181) to nerylacetone (174) is analogous to the conversion of geraniol or nerol to 6-methyl-5-hepten-2one. Aspergillus niger ATCC 9142 did not hydroxylate famesol, but reaction of the substrate with A. niger DSM 63263 yielded, after 48 hr, 4.5% 12-hydroxyfamesol (183), Fig. (35). HO.

J^^Z JL^^ Rhodococcus ^ ^ ^ ^^^ ^''^^'^"'^ÔH rubropertinctu^ ^^^^^ ^"^^ ^^'^^^ ^ DSM 43197 Famesol (181) 182 Mixt. of isomers Aspergillus niger 183 Fig. (35). Microbial transformation products of famesol (after [140])

174

166

The bioconversion of famesol by A. niger was investigated by Madyastha and Gururaja [135]: analogous transformation products as in the case of nerolidol were identified: 12-hydroxyfamesol and (5)-(-)10,11 -dihydroxyfamesol. More recently, the biotransformation of (2jE',6^-famesol (181a) was also carried out by a Japanese group [141] using the fungus Glomerella cingulata. At the first step, oxidation proceeded at the remote double bond to give (2£,6jEr)-3,7,ll-trimethyl-2,6-dodecadien-l,ll-diol (184) and (2£,6£)-3,7,ll-trimethyl-2,6-dodecadien-l,10,l 1-triol (185). In the second step, the diol (184) was further hydroxylated to (2£',6£:)-3,7,lltrimethyl-2,6-dodecadien-l,5,ll-triol (186), which was further isomerised to its (2Z,6£)-isomer (187), Fig. (36). In the course of this work, the same group [142] also investigated the biotransformation of (2Z,6Z)-famesol (181b) by this fungus, Glomerella cingulata. Oxidation of the remote double bond and isomerisation of the 2,3-double bond gave (2Z,6Z)-3,7,11-trimethyl-2,6-dodecadiene-l, 10,11triol (188) and (2J5:,6Z)-3,7,ll-trimethyl-2,6-dodecadiene-l,10,ll-triol (189) as major metabolites. Further degradation to (Z)-9,10-dihydroxy6,10-dimethyl-5-undecen-"2-one (177) was also observed, Fig. (36). 2.1.2 Acyclic sesquiterpene ketones

As in the discussion of the acyclic monoterpene ketones (see 1.1.3) instead of the sesquiterpene ketones, the structurally related compounds neryl(176) and geranylacetone (169) will be reviewed. Indeed, as mentioned before, neryl- and geranylacetone are the "norsesquiterpene"-analogues of 6-methyl-5-hepten-2-one, which is the j(?-oxidation product of nerol and geraniol, just like neryl- and geranylacetone are the bioconversion products of famesol (see 2.1.1). As mentioned before (2.1.1) Arfmann et aL [133] studied the cohydroxylation of the sesquiterpenes nerolidol and famesol and the related compounds neryl- and geranylacetone. This was done because cohydroxylated sesquiterpenes are important intermediates in the synthesis of industrially used fragrances and flavours. Aspergillus niger ATCC 9142 and Rhodococcus rubropertinctus DSM 43197 were unable to hydroxylate nerylacetone or its ^-isomer geranylacetone, at the co-position of the molecule. Incubation of nerylacetone with Mucor circinelloides CBS 27749 for 25 hr resulted in seven transformation products, including one co-hydroxylation compound, in 3% yield [140]. The group of Abraham et aL [112] expanded their studies of the hydroxylation of the sesquiterpenes nerolidol and famesol to the ketones neryl- and geranylacetone. Again similar results were obtained: Diplodia gossypina, Corynespora cassiicola and Gibberella cyanea were able to hydroxylate the substrates to the corresponding glycols (9,10-diols) with

I-

Fig. (36). Possible pathways for the metabolism of (2E,6E)-farnesol and (2562)-farnesol by Glomerella cingulata (after [141,142])

?i

168

high yields {ca 50-60%). In some cases smaller amounts of the epoxide and hydroxyketones were also noticed. The bioconversion of geranylacetone WiXh Aspergillus niger yielded the co-hydroxylation product 11-hydroxygeranylacetone and the vicinal diol (S)-(-)-9,10-dihydroxygeranylacetone as in the case of nerolidol and famesol [135]. The same reactions were noticed in the bioconversion of geranylacetol. More recently the group of Miyazawa et al. [137,138] investigated the biotransformation of neryl- and geranylacetone by the plant pathogenic fungus Glomerella cingulata. This research was undertaken in parallel with their studies of the sesquiterpene alcohol nerolidol (see 2.1.1). The results obtained with nerylacetone (176) were similar to those with Znerolidol (162): formation of a ketodiol (Z)-9,10-dihydroxy-6,10-dimethyl5-undecen-2-one (177). From geranylacetone (169) however, the main reaction obtained was a monohydroxylation yielding the hydroxyketone (£)-10-hydroxy-6,10-dimethyl-5-undecen-2-one (173), comparable with the transformation of ^-nerolidol (158), Fig. (37), resp. Fig. (34). 2.2 Cyclic Sesquiterpenes 2.2.1 Cyclic hydrocarbon sesquiterpenes

Caryophyllene and humulene Caryophyllene (190) is the main constituent (> 50%) of Copaiba (balsam) oils, which are obtained by steam distillation of the exudate (balsam) from the trunk of several species of Copaifera L. {Fabaceae), a genus of trees growing in the Amazon basin [26]. Copaiba balsam oils and balsams are used mainly as fixatives in soap perfumes. In 1979, Rama Devi [143] isolated a strain of Pseudomonas cruciviae by enrichment culture on caryophyllene. The major metabolite of this bioconversion was a product hydroxylated at the bridgehead and at the allylic position. The yield, however, was very low. Abraham et al [144-146] studied the biotransformation of caryophyllene (190) and humulene (196) by Diplodia gossypina (ATCC 10936) and two strains of Chaetomium cochliodes (DSM 63353 and ATCC 10195), Fig. (38) and Fig. (39). Sixty three products, including 49 that had never been described previously, were obtained and tested for their biological activity [147]. More recently, the bioconversion of (-)caryophyllene by Chaetomium cochliodes IFO 30576 was also studied by another group [148]. The substrate was first epoxidized at the C-C double bond, producing (-)-caryophyllene-4,5-oxide (191), which was then hydroxylated at the gem-dimethyl group and C-7 position giving 193.

Geranylacetone (169)

Nerylacetone (176)

178

Fig. (37). Hydroxylation of geranylacetone and nerylacetone by Glomerella cingulata (after [137,138])

170

Caryophyllene (190) D.

gossypina

CO(M

HO*"*'

194 (5%)

195

193 (12%)

Fig. (38). Main biotransformation products of caryophyllene by Diplodia gossypina and Chaetomium cochliodes (after [144,145])

OH HO

j3 Chaetomium cochliodes

Humulene(196)

HO'"

197

II i

^^ 198

'^

OH

199

Fig. (39). Hydroxylation of humulene by Chaetomium cochliodes (after [145])

Valencene Valencene (5), a sesquiterpene hydrocarbon isolated from orange oils is used as starting material for the synthesis of nootkatone (6), which is used for flavouring beverages [26] and which is a much sought-after aromatic substance [131]. Two bacterial strains, onefi-omsoil and the other from infected local beer, which utilised calarene as the sole source of carbon and energy have been isolated by enrichment culture techniques [149]. Both these bacteria were adapted to grow on valencene as the sole carbon source. Fermentations of valencene (5) by these bacteria of the genus Enterobacter in a mineral salts medium yielded several neutral metabolic products: dihydro alpha-agarofiiran (200) (7.5%), nootkatone (6) (12%), another ketone (201) (18%) and a-cyperone (202) (8%), Fig. (40).

171

Enterobacter

Valencene (5)

CO

Dihydro alpha-agarofuran (200) 7.5%

Ketone C14H22O (201) 18%

Nootkatone (6) 12%

a-Cyperone (202) 8%

Fig. (40). Biotransformation of valencene by Enterobacter sp. (after [149])

Very recently, the chemoenzymatic preparation of nootkatone from valencene was described [150]. Nootkatone was prepared from valencene by copper(I) iodide catalysed oxidation with tertAmtyX hydroperoxide and hydroxylated at C-9 by Mucor plumbeus and Cephalosporium aphidicola, 2.2.2 Cyclic sesquiterpene alcohols and ketones Patchoulol

Patchouli alcohol or patchoulol (203) is a major constituent (30-45%) of the patchouli essential oil which is extensively used in perfumery [68]. The essential oil is obtained by steam distillation of the dried leaves of Pogostemom cablin (Blanco) Benth. (Lamiaceae), Although it is the main component of the patchouli oil, this compound contributes less to the characteristic odour of the oil than nor-pachoulenol (206), present at a concentration of only 0.3-0.4%. A process for the production of the latter compound, via the microbial 10-hydroxylation of patchoulol (203) and subsequent oxidation of the 10hydroxypatchoulol (204) was published [151]. From the 350 microorganisms screened, four strains of Pithomyces species carried out regio-selective hydroxylation of patchoulol to 10-hydroxypatchoulol: Pithomyces sp. NRJ201 and P. chartarum NRJ210, isolated from soil situated in the neighbourhood of Kamakura, Japan, the most important ones. A method was developed by which 10-hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-litre fermentation jars at 2 to 4 g of patchoulol per litre and isolated as pure material in 30% yields. The

172

obtained hydroxylated product can easily be converted chemically to the industrially more important nor-patchoulenol (206), Fig. (41). An overview was given by Seitz [152]. ^^^^^2^^^>^

^^\.A^^^^V^

/yC>J

/K^J-

LL

1 Pifhomyces s p ^

Patchoulol (203)

02.Pt02^

^^î^ pT ^

\1

\ CHjCH

10-Hydroxypatchoulol (204)

Pb(0Ac)4^

yT^-^P^ OOOH 4-Carbohydroxypatchoulol (205)

"°C^C^ ^ i^KP Nor-patchoulenol (206)

Fig. (41). Regioselective lO-hydroxylation of patchoulol by Pithomyces sp. and subsequent chemical conversion to nor-patchoulenol (after [151,152])

Germacrone Hikino et aL [153] have investigated by enzymatic means the stereospecific epoxidation reactions of olefinic double bonds in the plant Curcuma zedoaria Roscoe. They studied the bioconversion of germacrone (207), a constituent of C zedoaria, by microorganisms in the hope of obtaining stereoselective epoxidation as in the case of the plant. Cunninghamella blakesleeana yielded three major products (208 - 210) from germacrone, Fig. (42). More recently, Asakawa et aL [154] described the biotransformation of germacrone by Aspergillus niger. A very unstable allylic alcohol (211) was obtained from the metabolite of germacrone along with (213), Fig. (42).

173

R = H(211) R = Ac(212) Fig. (42). Biotransformation of germacrone by Cunninghamella blakesleeana (after [153]) and by Aspergillus niger (after [154])

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

179

CYCLOARTANE AND OLEANANE SAPONINS FROM ASTRAGALUS SP. LUISELLA VEROTTA,*^ NADIA A. EL-SEBAKHY^ Dipartimento di Chimica Organica e Industriale, Universitd degli Studi di Milano, via Venezian 21, 20133 Milano-Italy; ^Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt ABSTRACT: Astragalus spp. (Leguminosae) represent a valid rural crop of ecological importance in many countries, and some species serve as foodstuffs and pharmaceutical emulsifiers. Astragalus species are used in Chinese traditional medicine as antiperspirant, antihypertensive, antidiabetic, diuretic and tonic. The pharmacologically active constituents of these Astragalus are of two different types, polysaccharides and saponins, and the most interesting pharmacological properties are hepatoprotective, inmiunostimulant and antiviral. Astragalus species are a source of cycloartane type saponins, derived from cycloartenol by oxidation at C-6, C-16, C-20, C-23, C-24, followed by possible ring closures with the formation of a 20,24-epoxide (cycloastragenol or cyclogalegenin) or a 20,25epoxide, or a 16,24- 20,24-diepoxide (cycloalpigenin), or a 16,23- 16,24-diepoxide (cycloorbigenin B). Approximatively 100 saponins mainly derivatives of the 20(R\24(S) form of cycloastragenol [3p,6a,16p,25-tetrahydroxy-20(/?),24(5)-epoxy9,19-cyclolanostane], (astragalosides or astraversianins), and more rarely of the 20{S), 24(R) form (cyclogalegenin), have been isolated. Some species also contain trihydroxyolean-12-ene saponins. Details on the isolation, purification and structural elucidation, along with recent results on the pharmacological properties of Astragalus cycloartane and oleanane saponins, will be discussed.

INTRODUCTION Saponins are widely distributed in the plant kingdom, and occur in many food plants (soybeans, chick peas, peanuts, mung beans, broad beans, kidney beans, lentils, garden peas, spinach, oats, aubergines, asparagus, fenugreek, garlic, sugar beet, potatoes, green peppers, tomatoes, onions, tea, cassava, yams), as well as in forage species (alfaalfa). Saponins are also contained in numerous herbal remedies. The useful biological applications of saponins, generally based on theii* membrane-disrupting properties, range from fish and snail poisons [1, 2], to potentially interesting anti-cancer agents [3] and ion channel-blockers [4, 5]. Other interesting biological applications for various specific

180

saponins include their uses as antiinflammatory, hypocholesterolemic, immune-stimulating, andflavor-modifyingsubstances [4, 6]. Saponins consist of a terpenoid core (the aglycone), having oxygenated positions bound to sugar moieties (up to ten monosaccharidic units). In water they form colloidal solutions which foam on shaking and precipitate cholesterol. When saponins are near cell membranes, their interaction with cholesterol may create pore-like structures that eventually cause the membrane to burst. Hemolysis is an example of this phenomenon (i.e. the distruction of erythocyte membranes, but not hemoglobin). Occasionally, they cause hypersecretion, which could explain their expectorant activities and also their toxicity to fish. Table 1. Plants containing cycloartane saponins.

Plant (family) Ranunculaceae

Genus

Distribution

Actaea

Asia

nd

Beesia

Asia

anti-inflammatory, analgesic

[71

Cimicifuga

Asia

antipyretic, analgesic, anti-inflammatory

[7]

Souliea

Asia

anti-inflammatory, analgesic

[7]

Thalictrum

fevers, hypertension, diarrhea, irritant, stomach ache

[81

anti-inflammatory, antip>Tetic, contraceptive sedative

[91

chest coughs, sypliilis, aphrodisiac, diarrhoea, bilharziasis, fever, anthelmintic, headaches, dysentery, dressing wounds, snake bites, vaginal orifice reduction digestive disorders, sweetener

[8, 10-131

antiperspirant, diuretic, tonic, anticancer, antidiabetic nd

[151

analeptic for the treatment of decline

[171

1 Rubiaceae

Mussaenda

Southern Africa, Europe, Asia, North America Asia

1 Passifloraceae

Passiflora

South America

1 Combretaceae

Combretum

Southern Africa

1 Leguminosae

Abrus

Oxytropis

Tropical Africa, South-East Asia Northern Africa, Asia, Europe Asia

Curculigo

Asia

Astragalus

Hypoxidaceae

nd. No details

Ref

Uses

[81

[141

[161

p lupenyl cation

Fig (1). Biogenelic pathway to cycloartane and oleanane saponins

I@ ti

oleanyl cation

182

Biogenetic pathway to cycloartane and oleanane saponins Tran^-squalene oxide, suitably positioned and folded on the enzyme surface (i.e. in a chair-boat-cliair-boat conformation), gives a polycyclic triterpene structure through a concerted carbocation-mediated cyclization. The resulting protosteryl cation then undergoes a series of Wagner-Meerwein 1,2 shifts. In plants, the final triterpene alcohol is cycloartenol. Should rran^-squalene oxide be folded in a chair-chairchair-boat conformation, then an identical carbocation mechanism ensues, and the transient dammarenyl cation (precursor of dammarane "ginseng" saponins) can evolve to oleanyl systems through ring expansion. Fig. (1)

"COOH

R'

R^

H

R^ mollic acid

a-L-arabinosyl

p-D-glucosyl

moilic acid glycoside

H

p-D-xylosyl

moliic acid xyloside

a-L-arabinosyl

P-D-4ACxylosyl

mollic acid 4Acxyloslde

p-D-xyloside

R^ mollic acid arabinoside

CH3 CH2

Jessie acid

CH2

Jessie acid arabinoside CH2

Vyis^CH3 0

a-Larabinosyl

CH3

moilic acid arabinoside

Fig. (2). Cycloartane saponins isolated in Combretum sp.

CH3

Jessie acid xyloside

183

Cycloartane saponins are relatively rare when compared to the many oleanane saponins (Table 1) and their occurrence is limited to herbs, shrubs and twinning plants (Astragalus, Abrus, Mussaenda, Passiflora), growing in dry, moderate climates. An exception is represented by Combretum which are trees diffused in tropical regions. Combretum genus, as well as Astragalus, contain both oleanane and cycloartane saponins (mollic acid and imberbic acid derivatives), thus possessing both the enzymatic pathways [10,11], Fig. (2).

R^

R^

R'

R*

R*

R«

R^

OH

H

OH

CH3

CH3

H

COOH

OH

H

OH

CH2-0-4AcRha

CHa

H

COOH

OH

H

O-Rha

CH2-0-4AcRha

CH3

H

COOH

OH

H

0-Rha

CH2-0-Rha

CH3

H

COOH

H

O-Rha

CH2-0-Rha

CH3

H

CH2OH

OH

H

O-Rha

CH2-0-4AcRha

CH3

H

CH2OH

OH

H

OH

CH2-0-Rha

CH3

H

COOH

OAc

H

OH

CH2-0-Rha

CH3

H

COOH

H

OH

OH

CH3

COOGIc

OH

Fig. (2). Oleanane saponins isolated in Combretum sp.

OH

CH3

Table 2. Astragalus species studied for their cycloartane and oleanane saponins Source

adsurgens alexandrinus Boiss. alopecurus amarus Pall babaagi basineri Trautv. brachyprerus cephalores Banks & Sol. var brevicalyx Eig. chrysopterus coluteocarpur cornplanatus R. Br. dissecrur dasyanrhus Pall.

ernesrii Comb exilis L. galegiformis L. glycyphyllos L hamosus L. kuhirangi (Nevski) Boriss. kulabensis illiensis L nlelanophrurius Boiss. rnembranaceus Bunge microcepllalus Willd.

Aglycone

China Mediterranean Coastal strip Russia Russia Russia Russia Turkey Turkey

cycloastragenol; 6a,25 dihydroxy, 3,164ioxo-9P,19cyclolanostane cycloastragenol: 3P,6a,16P,24(R),25 pentahydroxy 9~,19cyclolanostane

China Russia China Russia Russia

cycloastragenol, soyasapogenol B cycloastragenol cycloastragenol; soyasapogenol B; complogenin cycloastragenol, cyclocanthogenin cycloastragenol, 3P.7422 hydroxy-16,23epoxy-24.25.26.27tetranor-9p, 19cyclolanostane cycloalpigenin A cycloastragenol cycloastragenol cyclogalegenin cycloastragenol, soyasapogenol B, 19-keto-soyasapogenol B soyasapogenol B cycloastragenol cycloastragenol cycloastragenol 9~,19cyclolanostane cycloastragenol; 3P,6a,16P,24a,25-pentahydroxy, cycloastragenol, soyasapogenol B, huangqiyenin, cyclocanthogenin cycloastragenol, 3P,6cr, 16P,24a-tetrahydroxy-20,25-epoxy 9P,19-cyclolanostane

Russia Japan Russia Russia Russia Russia Russia Russia China Turkey China Turkey

cycloalpigenins cycloastragenol cycloastragenol cycloastragenol cycloastragenol, cyclocanthogenin cyclocanthogenin

Ref

nwngholicus Bunge oleijolius DC. orbiculatus Ledeb. pamirensis Ovcz. tk Rassulova peregrinus Vohl.

Turkey Russia Russia

pterocephalus Bunge pycnanthus

Russia Russia

quisqualis Bunge schachirudensis Bunge sieberi DC sieversianus Pall. sinicus L spinosus Vahl. taschkendicus Bunge tornentosus Lam.

Russia Russia Egypt China China Egypt Russia Egypt

tragacantha Habl. trigonus DC

Russia Egypt

trojantcs Stev. itninodus verrucosus Moris villosissinlrrs

Turkey Russia Italy Russia

cycloastragenol; la,3P,16P,27-tetrahydroxy9P, 19 cyclolanost-24Eene. cyclocanthogenin 1a,7P,24&25 tetrahydroxy-9P,19cyclolanostane cycloorbigenin B cycloastragenol cycloastragenol cycloastragenol 6dehydroxycycloastragenol, 24Rcycloartan-3P,6a,16P,24,25-pentaol, cycloastragenol

L. Verotta et al., unpublished [181 [921

cycloastragenol cyclogalegenin cycloastragenol soyasapogenol B; complogenin cycloastragenol cycloastragenol, cycloasgenin C 38 hydroxy[6a-acetoxy,23-ethoxy,16~,23(R)-epoxy-24,25,26,27-tetranor]-9~,19cyclolanostane cycloastragenol; cyclocanthogenin 25ene; cycloastragenol; 3~,6~,16~-trihydroxy-9~,19~yclolanost-24x0, 3 P . 6 ~16P-trihydroxy-9.19-cyclolanost-24ene, soyasapogenol B cycloastragenol; cyclocanthogenin, soyasapogenol B cycloastragenol cycloastragenol cycloastragenol

186

Astragalus genus is one of the richest source of cycloartane saponins. Oleanane type saponins are also found in Astragalus sp., but their occurrence is limited to structures common to Leguminosae. Fig (3) shows the naturally occurring cycloartanes and oleananes which have been isolated from different Astragalus species. The largest body of work on Astragalus saponins is found in the Russian literature [18]. The Tashkent group has been involved for years in the study of saponins from native plants. Unfortunately, most of their publications in the Russian literature were not available until recently as an English translation. In addition, confusion arose regarding the configuration at the centres involved in the formation of the tetrahydrofiiran ring (cycloastragenol and the less frequent cyclogalegenin are the peculiar skeletons of Astragalus sapogenins). A paper [19] eventually shed light on this misunderstanding, describing the X-ray analysis of the two diastereoisomers and solving the diatriba. Since this publication appeared in Russian, many authors remained unawai'e of these data for years. R = GH2CH3I H

soyasapogenol B

complogenin

Fig. (3). Tetranor-cycloartane and oleanane triterpenoids isolated from Astragalus sp.

No rational and corrected review on the so far described Astragalus saponins is presently available. Recently, a paper describing the pharmacological properties of Astragalus species has been published [20]. It reports an overview of the biological properties of the different metabolites present in the genus.

187

Fig.(3). Structures of the aglycones common to Astragalus sp.

29

28

OH

cycloastragenol

OH cyclogaiegenin

R = O; a-OH. H; ^-OH, H cycloalplgenlns 2^.0H

OH cycloorbigenin B

"

OH

cyciocanthogenin

ChfeOH

R = H. H; OH, H

188

The word Astragalus is derived from two Greek words "Astron" means a star and "Gala" means milk, for the belief that the presence of Astragalus plants in grass-land improved milk yield of livestock [21]. Genus Astragalus (Leguminosae) is one of the largest and most widely distributed genera of flowering plants [22, 23], it includes 2000 species, grouped into more than 100 subdivisions [24, 25]. The North American species (384) are chiefly perennial herbs or subshrubs while Old World species are more often shrubby. Many species in western and central Asia are spiny and develop phylloides or modified leaves in xerophytic habitats. Astragalus is the largest genus of the Russian flora with 889 species. It is represented in the mediterranean area by over 70 species, varied from small herbs to spiny shrubs [26]. Astragalus spinosus and A, trigonus are the most common species of the genus Astragalus in the western Mediterranean coastal region. While few of these species are native to Tropical Africa, many, if not most, still grow today in Egypt and Israel, on both sides of the Suez Canal. In fact, the Biblical World, bridging Africa via Egypt through Israel and Palestine to Europe and the Middle East, is at the crossroads of the African and the European Continent. Singular are these citations from ancient texts: "A little balm and a little honey, gum {Astragalus bethlehemiticus Boiss. ''Bethlehem)

Astragalus sieberi DC

Astragalus membranaceus Bunge

Tragacanth" "Gum"), myrrh, pistachio nuts, and almonds." Genesis 43:11. " I have gathered my myrrh with my spice..." (Astragalus gummifer Labill "Tragacanth", "Spice"). Song of Solomon 5. Reported ancient uses are for cancer, burns, cough, diarrhea. The medicinal properties oi Astragalus species are also described in the Chinese Pharmacopoeia [7]. Astragalus species are used in Chinese

189

Astragalus alexandrinus Boiss.

Astragalus bethlehemiticus Boiss.

Astragalus spinosus (Forssk.) Muschl..

Astragalus kahiricus DC.

190

traditional medicine as antiperspirant, antihypertensive, antidiabetic, diuretic and tonic [15]. In China, many pharmaceutical preparations containing Astragalus extracts or isolated compounds have been used. Water decoctions of the roots of A. membranaceus and A. sinicus were incorporated into pharmaceutical preparations for treatment of toothache or for oral hygiene. These extracts were found to remove tobacco stains, plaque and food residues from teeth. Many Astragalus species have attracted interest because of their apparent cytotoxic constituents and have been used for treatment of patients with leukemia and uterine cancers [27]. The most common use oi Astragalus is as forage for livestock and wild animals, although 32 sp have been recognized as of use in foods, medicines, cosmetics, as substitutes for tea or coffee, or as sources of vegetable gums [28]. However, a number of species are toxic for livestock and in many cases the toxins could be transferred to humans through meat or milk. Thus, there are two groups of Astragalus species that are closely related to health: the toxic species and the medicinal plants. The genus Astragalus appears highly uniform from a chemical point of view, with two kinds of pharmacologically active principles and three different kinds of toxic compounds. In the former group the polysaccharides and the saponins stand out, and, in the second, the indolizidine alkaloids (swansonine and its N-oxide derivative, and lentiginosine) [29-31], the nitrocompounds endecaphyllins (nitropropionic acid-glucose derivatives) and 3-nitropropyl-glucosides [32-34], and the seleniferous derivatives (seleno-cysteine, -cystathionine, -cystine, and methionine), are found [29, 35]. The triterpenes and saponins are the most widely studied secondary metabolites. Astragalus species are a source of cycloartane type saponins. They are characterized by a 9j8, 19 cyclolanost-24-ene-3jôl skeleton, whose oxygenated positions can be linked through acetal bondings to sugar moieties. All derive from the parent cycloartenol which undergoes oxidations (typical are at C-6, C-16, C-20, C-23, C-24), followed by possible ring closures with formation of a 20,24-epoxide (cycloastragenol or cyclogalegenin) or a 20,25-epoxide, or a 16,24-, 20,24-diepoxide (cycloalpigenin), or a 16,23-, 16,24-diepoxide (cycloorbigenin B). The 1oxidation is very rare. Biogenetically, the configuration at C20 is /?, although, rarely, a 20 (5) configuration has also occasionally been found. Fig. (4).

191

Fig. (4). 9.19 CYCLOLANOST-24-EN-3p-OL

Cycloartanes are found in the plants in free states as well as in the glycosidic form. Over than 100 saponins, mainly derivatives of the 20(R),24(S) form of 9p, 19 cyclolanost-24-.ene-3p-ol (cycloastragenol), named astragalosides or astraversianins, and more rarely the 20(5^, 24(R) form (cyclogalegenin), named cyclogaleginosides and sieberosides have been so far isolated (Table 3). Some species also contain trihydroxyolean12-ene saponins (Table 4). Isolation and purification Oleanane and cycloartane saponins can be isolated and structurally elucidated using similar techniques. A single chromatographic step is rarely sufficient to isolate a pure saponin from an extract. As a general rule, several preparative techniques are required to obtain the pure product. Saponins are generally extracted from plants through an alcoholic extraction of the defatted vegetable material. Due to the possible contemporary presence of acidic components (phenols and their acids, flavonoids, etc.) care should be taken about the pH of the alcoholic solution, which, if too low, can produce undesidered chemical modifications. Acidic methanol can hydrolize glycosidic bonds or produce transesterification. A subsequent useful step is the partition of the total dried alcoholic extract between n-butanol and water. This operation is important to eliminate mono- and disaccharides which complicate further separations.

rable 3. Cycloartane saponins found in Astragalus species

Rz

Et

Name

Astragalus sp. [Rel]

H

H

cycloastragenol

H

H

H

j3-D-xyl

H

H

trigonoside I

brachypterus [54], dissectus [60], kuhitangi [18], membranaceus [77], microcephalus [50], mongholicus [18], pamirensis [18], pterocephalus [18], sieversianus [18, 951, tragacantha [I 81, lminodus [ l 091 dissectus [60], rnembranaceus [79], pomirensis [I 81, tragacanrha [I 81, uninodus [I091 trigonus [I041

j3-D-glc

H H

brachyoside B

brachypterus [54], spinoslcs [98]

COCH3

H H

COCH3

I1

H

huangqiyenin D

membranaceus [80]

j3-D-xyl

kI

H

astraversianin X, cyclosieversioside E

alexandrinus [39], tnelanophrurius [71,72], pterocephalus [18] sieversianus [18, 961 , schachirudensis [18,93], uninodus [I091

boeticus [* *]

cyclosieversioside C astraversianin VI astraversianin V

schachirudensis [18, 931, sieversianus [18, 391, uninodus [109] sieversianus [96]

cyclosieversioside A, astraversianin I1 astraversianin III

melanophrurius [72], schachirudensis [18, 931 , sieversianus [18, 961, uninodus [I091 sieversianus [96] sieversianus [96]

astragaloside IV, cyclosieversioside F, astraversianin XTV, astramembrannin I astragaloside lI, astraversianin W isoastragalosideIt, astraversianin VIII cyclocephaloside I1 astragaloside I, astraversianin IV

alexandrinus [39], dissectus [60], kuhitangi [18], melanophrurius [71.72], membranaceus [76,79, 821, brachypterus [54], pterocephalus [18], p y c m t h u s [92], shachirudensis [18], sieversianus [95], spinosus [98], tragacantha [18], uninodus [log] brachypterus [54], melanophrurius [71,721, membranaceus [76, 791, mongholicus* [85], sieversianus [96], spinosus [98] membranaceus [76], sieversianus [96] microcephalus [54]

isoastragaloside I

brachypterus [54], membranaceus [76], melanophrurius [71.721. mongholicus* [85], spinosus [98], sieversianus [95], trigonus [lo41 membranacerrs [76, 821, spinosus [98]

acetylastragaloside I

membranaceus [76, 821

trojanoside A

rrojanus [ 1081

cyclocarposide

colureocarpus [581

cyclocarposide B

cycloaraloside A, astraverrucin I astraverrucin I1

amarus [45], membranaceus , peregrinus [u], verrucosus [1 lOa] verrucosus [1 lOa]

astraverrucin III

verrucosus [1 lOa]

cyclosieversioside D

basineri [53], schachirudensis [18, 931

cyclosieversioside B

basineri [53], schachirudensis [93]

cycloaraloside E

amarus [49]

trojanoside B

trojmus [log]

astragaloside W

kuhitangi [18], membranaceus [74],

astragaloside III

membranaceus [75,82], mongholicus* [85]

astrachysoside A

chrysopterus [56]

cyclosieversioside G , astraversianin XV astraversianin I X

sieversianus, [95, 961 chrysoprerus [56]

astraversianin XJ

sieversianus [95]

trigonoside II, askendoside D trigonoside Ill

lashkendicus [ l o l l , trigonus [104]

askendoside B

astragaloside VI

kulabensis [60],sieversianus [95]

C

P

astraversianin W

sieversianus [96]

astraversianin Xm

sieversianus [96]

cycloaraloside C, astrailienin A cycloaralosideD

illiensis [70],amarus [50],villosissimus [51] m r u s [48],peregrinus [u]

cycloaraloside B

amarus [46]

astraverrucin IV

peregrinus [u],verrucosus [llob]

astraverrucin V

verrucosus [l lob]

astraverrucin VI

verrucosus [ 1 lob]

cycloaraloside F

amarus [511, villosissimus [511,

astragaloside V

membra~ceus[75]

asernestioside A

ernesrii [61]

asernestioside B

ernestii [61]

asernestioside C

ernestii [61]

huangqiyegenin I

membranaceous [82]

huangqiyegenin A

membranaceous [811

R2

Name

Astragalus sp. [Ref]

H H H H H

cyclogalegenin

galegifonnis 1641. sieberi [94]

cyclogaleginosideB

galegifonnis [65]

cyclogaleginoside A

falcatus, galegifomus [65]

sieberoside I

sieberi [94]

sieberoside IJ

sieberi [94]

Rz

H

R3

H

Name

Asfragalus sp. [Ref]

cyclocanthogenin

tragacantha [ I 81

cyclocanthoside A

cephalotes [55]

cyclocanthoside E agroastragaloside I1

dissect us [60],cephalotes[55],microcephalus [54],melanophurius [71,72],tragacantha [I031 nronbranaceus* [82b]

agroastragaloside I

membranaceus* [82a]

cyclocanthoside D brachyoside C

cephalotes [55],kuhitangi [18],tragacantha [I81 brachypterus [54]

agroastragaloside N

membranaceus* [82c]

agroastragaloside V

membranaceus* [82c]

cephalotoside A

cephalotes [55]

brachyoside A

brachypterus [54]

cyclocanthoside G

rragacantha [103]melanophrurius [71,72]

5

74

75 76 77

a-L-rha(1--2)P-D-xyl PD-glc a-L-rha(1--2)PD-xyl a-L-ara(1--2)PD-xyl

H

H

P-D-glc

trojanoside C

trojanus [I081

P-D-glc

H

PD-glc

trojanoside D

trojanus [I081

P-D-glc

H

PD-glc

trojanoside E

trojanus [I081

P-D-glc

H

PD-glc

trojanoside F

trojanus [I081

Rz

R3

Name

Astragalus sp. [Refl

H H H

H H H

cycloasgenin

raschkendicus [I81

alexandroside I

alexandrinus [39]

askendoside C

taschkendicus [18]

H

H

askendoside A

taschkendicus [18]

P-D-glc

H

cyclopycnanthoside I pycnanrhus [92]

H

PD-&

askendoside G

taschkendicus [loo]

RI

Rt

Name

Asfragalus sp., [Refl

P-D-xyl

H H

macrophyllosaponin B

okifoiius [86]

macrophyllosaponin A

oleifolius [86]

P-D-xyl(4Ac) H

P-D-xyl(l--2)P-D-xyl

PD-glc

macrophyllosaponin C

oleifolius [86]

H

macrophyllosaponin D

olcifolitrs [86]

Astrngalus sp., [Refl

tomentoside I

H

CH2CH3

CH3

OAc

H

tomentosidc IJ

OH

H H

CH3

H

H

dayanthopenin

OAc

200

R

Name

Astragalus sp., [Ref]

6-oxo-cycloartan 3,16 diglucoside

trigonus [ 107]

91

j3-D-glc

O

92

p-D-g\c

a-OH

trigonus [106]

93

j8-D-glc

P-On

synthetic [106]

RO

R

Ri

94

^-D-glc

O

95

p-D-g\c

OH

Name

Astragalus sp,, [Ref] trigonus [105] trigonus [u]

!\.OH

Astragalus sp,, [Ref]

R 96

j8-D-xyl

)3-D-glc

cyclocephaloside I

microcephalus [83]

201

#

R

97

Ri

Name

Astragalus sp.^ [Ref]

H

0

cycloalpigenin A

alopecurus [41]

98

/3-D-xyl

0

cycloalpioside A

alopecurus [41]

99

H

a-OH, H

cycloalpigenin B

alopecurus [42]

100

P-D-xy\

a-OH, H

cycloalpioside B

alopecurus [42]

101

H

i3-OH, H

cycloalpigenin C

alopecurus [43]

102

j3-D-xyl

i3-0H, H

cycloalpioside C

alopecurus [43]

R

Name

Astragalus sp.^ [Ref]

103

H

cycloalpigenin

alopecurus [44]

104

/3-D-xyl

cycloalpioside

alopecurus [44]

202

Name

Ri

Astragalus sp.^ fRcf)

105

II

H

huangqiycgcnin 11

membranaceous (81)

106

)3-D.glc

H

huangqiycgcnin B

fnettibranaceous (81 ]

CH2OR2

Ri

Name

Astragalus sp.^ [Rcf]

107

H

H

)3-D-glc

mongholicosidc I

nionghoUcus (84)

108

CH3CO

OH

/J-D-glc

mongholicoside I

nwngholicus (84)

R 109

H

OH

R2

Name

Astragalus sp.^ (Ref)

H

cycloorbigenin B

orbiculatus [^9]

110

/3-D-xyl

H

H

cycloorbicoside A

orbiciilatus [SI]

111

/3-D-xyI

OH

H

cycloorbicoside B

orbiculatus [SS]

112

j3-D-xyl

OH

/3-D-gic

cycloorbicoside G

^rf7icw/arM5 (90)

* hairy roots; ** Assad, AM. PhD Pharm. Sci. Thesis, Faculty of Pharmacy, University of Alexandria (1984); (u): Verotta, L unpublished.

203

Table 4. Oleananane saponins found in Astragalus

23

24

species

CH2OH

R

Ri

R2

Name


113

H

H

H

soyasapogenol B

glycyphyllos [66]

114

i3-D-glc-UA

H

H

115

/3-D-gal(l—2)-)3-Dglc-UA Me ester j3-D-ara(l-~2)i3-D-glc )3-D-glc(l—2)-j3-Dglc-UA Me ester a-L-rha(l-2)^-D-gal(l-2)-/3-Dglc-UA and Me ester

H

H

H

116 117 118

119

120

121

122

123

124

a-L-rha(l-2)/3-D-glc(l—2)-i3-Dglc-UA Me ester a-L-rha(l--2)a-L-ara(l—2)-i3-Dglc-UA Me ester a-L-rha(l"2). j3-D-xyl(l—2)-j3-Dglc-UA and Me ester a-L-rha(l-2)^-D-xyl(l—2)-j3-Dglc-UA Me ester a-L-rha(l-2)j3-D-gal(l-~2)-)3-D. glc-UA Me ester a-L-rha(l-2)i3-D-xyl(l—2)-i3-Dglc-UA

sinicus [97] sinicus [97]

H

soyasaponin III Me ester soyasaponin FV

H

H

azukisaponin 11

H

H

soyasaponin I

H

H

azukisaponin V

trigonus [105], tribuloides [u] membranaceus [75], chrysopterus [56], sinicus [97], complanatus [59] trigonus [105]

H

H

soyasaponin n Me ester

sinicus [97]

H

H

astragaloside

complanatus [59], membranaceous [74]

P-D-g\c

H

j3-D-glc

H

H

0-/3-D-glc

vm

sinicus [97]

complanatus [59]

comploside 11

complanatus [59]

astrojanoside A

trojanus [iOS]

204

R

Name

125

H

complogenin

126

a-L-rha( 1 --2)-/3-D-xyl( 1—2)-i3D-glc-UAand Me ester a-L-rha( 1 -2)-j3-D-gal( 1—2)-j3D-glc-UA and Me ester

127


siniciis [97], complancuus [59] sinicus [97], complanatus [59]

[u]: Verotta et. Al., unpublished

A typical isolation strategy is the preliminary purification of the nbutanol extract over dextran supports like Sephadex LH20 or Fractogel TSK, followed by further fractionation of the crude saponin mixtures [111]. A new generation of polymers has been exploited for the initial purification steps. They are highly porous polymers (Daion HP-20, MCI gel CHP-20P (both from Mitsubishi Chemical Industries, Tokyo), Amberlite XAD-2) [112]. Methanol-water or acetone-water solvent gradients are used. The polar characteristics of saponins suggest to avoid unmodified silica gel stationary phases, which, if used, require water containing mobile phases to desorb the glycosides. Nevertheless, silica gel chromatography with chloroform-methanol-water as eluent is still the most popular and inexpensive method and is used in most of separations [51, 94]. A limited number of applications of centrifugal thin-layer chromatography for the separation of saponins have also been reported. The technique is a planar method earned out on a centrifugally accelerated inclinated plate, coated with a suitable sorbent. Solvent elution produces concentric bands across the plate which are spun off at the edges of the plate together with separated solutes and collected for subsequent analysis. Examples of

205

separations of cycloartane and oleanane saponins have been reported. Astragalus membranaceus saponins have been isolated through CTLC eluted with chloroform-methanol-water (100:30:3) [75]. The solvent system ethyl acetate-ethanol-water (8:2:1 or 16:3:2) was used on a starchbound plate to isolate cycloartane saponins from Passiflora quadrangularis [11, 113], Fig. (5). Chemically derivatized silica packings have obtained increasing popularity due to their chemical stability and good separation efficiency. RP8 and RP18 sorbents are the most frequent packings used. The commercially available particle sizes from 3 microns to 60 microns allow the use of vacuum chromatography (VLC) or medium to high pressure (MPLC or HPLC) chromatography depending on the desidered load/resolution result. Thus, analytical or preparative separations can be performed, the analytical method being easily tranferred onto a preparative separation, if the chemistry of the sorbents is similar. The solvents of choice are mixtures of methanol-water or acetonitrile-water using gradient conditions [55, 111, 114, 115]. Liquid-liquid partition methods have proved ideal for application to the field of saponins. Very polar saponins are amenable to counter-cunent chromatographic separation, especially as there is no loss of material by irreversible adsorption to packing materials. Counter-current chromatography (CCC) describes liquid-liquid chromatography without a sorbent, requiring two immiscible solvent phases. In most variants of CCC, one phase remains stationary while the second phase is passed through the stationary solvent component. The principle of separation involves the partition of a solute between the two immiscible solvents, the relative proportions of solute passing into each of the two phases being determined by the respective partition coefficients. Initially, the techniques profited of a gravitational stationary phase (Droplet Counter Current Chromatography, DCCC) where droplets of a mobile phase flows through an immiscible stationary liquid phase present in a series of vertical glass columns. Ternary or quaternary solvents are seldom used to produce two unmiscible phases, conveniently chosen through the profile of separation obtained by running a silica gel TLC plate with the water-saturated organic phase of the two phase aqueous solvent system [116]. The system chloroform-methanol-water in different ratios has been involved in the greatest number of applications [113, 117, 118]. The resolution is not high and separations require days, nevertheless the lack of irreversible adsorption and the consistences of loadings (up to 5 g) increased exponentially the study of bioactive saponins in the eighties.

206

"o C CO

E

o o o a

o CM

X

f

9. X X

a. c 5 Tt

O)

E

E CO CO CO

CO CO

|± CCS

£

8-

CM CO CD

X

I

I-

I i3

x:

O

I 2

t O

s O

&?

207

Centrifugal partition chromatography (CPC) relies on centrifugal force rather than gravitation for the retention of the stationary phase and solvents can be pumped at higher speeds through the instruments. In addition, no need for droplets formation is required. This allov^s shorter separation times, without loss of resolution, and an infinite choice of solvents with the only requirement of forming two immiscible phases, stable with the time. Chloroform-based systems have been mostly used for the separation of saponins due to their favourable partition coefficient towards such solvents. [116, 119,120]. Structure elucidation Saponins are constituted of a triterpenoid core to which, in one or more positions, a number of sugar unities are bound. Due to their biological activity their structures have been extensively studied and, at present, several hundreds structures have been elucidated. Such structural investigations often turned out to be very time consuming and tiresome, mainly due to the presence of the sugar moieties for which the site(s) of binding to the triterpenoid, the inter-glycosidic bonds and the conformation of them had to be assessed. This was generally achieved by chemical degradation leading to partial or total cleavage of the sugar moieties, which could be identified by classical methods. The NMR analysis of the intact molecules has been often bumpered by the extensive overlapping of most sugar signals. A relatively easier task was the identification of the terpenoidic portion of the saponins, for which a plenty of NMR information are reported in the literature. The conventional methods include hydrolytic studies followed by the characterization of the aglycone (sapogenin) and oligosaccharide moieties. The drawback of this procedure is the loss of information about the glycosilation site and sometimes about the anomeric configuration owing to anomerization of the reducing monosaccharide. The hydrolytic studies are usually performed under acidic conditions, the major disadvantage with cycloartane saponins being cyclopropane ring opening and rearrangement. In most oleanane-type saponins no degradation occurs during the acidic treatments, and the sapogenins can be extracted from the reaction mixture. Partial hydrolyses are of crucial interest, leading to the isolation of partially glycosidated saponins, for which comparison of the ^^C NMR spectra allow to determine interglycosidic linkages. Mixture of prosapogenins are usually obtained during controlled hydrolytic

208

3

cr

0O ^f-C

CO £ V> w © O

O EZ

O£co

cr

^

cc

o

I X O X I

o

o)X X X o)

209

210

conditions, causing accumulation of problems in products separations. Alkaline hydrolyses with methanolic sodium hydroxide are also performed when ester linked olisaccharidic chains are hypothesized to be present in the saponin [121], Fig. (6). The enzymatic hydrolysis is the method of choice when undesired reactions are to be avoided. j8-glucosidases (or /^-glucuronidases) from Helix pomatia digestive juice are now commercially available. They usually show a good hydro lytic performance, even if sometimes need long time reactions (days). Other enzymes are reported to have been used to specifically hydrolize saccharidic bonds like hesperidinase, j5-xylosidase, /?-galactosidase, and mixed crude enzymes like cellulase. Sugar identification and interglycosidic linkages require two steps: first the acid hydrolysis, and subsequent analysis (GC) of the silanized monosaccharidic units allow to determine number and type of sugars (if compared with appropriate standards) [117]; permethylation followed by methanolysis of the saponins identifies interglycosidic linkages if one is in possess of the partially methylated monosaccharides [122]. Sugar sequences can be interpreted through the NMR and mass of the permethylated alditol acetate [123], Fig. (7). This combination of chemical reactions and NMR analysis, anyway, requested some hundreds milligrams of pure saponin which often are not available. The numerous biological activities of saponins, as well as their widespread occurrence in the plants and the hundreds structures described so far, prompted the development of a strategy for their rapid, highly sensitive and, if possible, non degradative, structure elucidation. Mass spectrometry, obtained through the use of soft techniques (FAB, ESP MS) gives information about molecular weight (pseudomolecular ions [M+H]^ or [M-H]") and some very limited sequence and branching confirmation. [124-127]. Recourse to innovations in NMR spectroscopy is essential for further advances in the investigation of complex saponins. The use of a 600 MHz spectrometer gives the required sensitivity, consistent with the low amounts available by modern isolation techniques, while the use of sequential ID and inverse-detected 2D NMR techniques, couples the short time necessary to perform the experiments with the selfconsistency of the obtained results and, thus, the unambiguous structure assignment [128]. An essential prerequisite for deducing the structures of saponins by NMR spectroscopic studies is the unambiguous assignment of ^H and ^^C

211

o

•a '-3

c o •a o

^

c

s

•

212

resonances, which, in principle, involves three main steps: homonuclear correlations, heteronuclear correlations, tridimensional relationships. Recording of the broad-band decoupled ^^C NMR spectrum and DEPT experiments allows to obtain the number of carbon resonances (usually, even a non-quantitative spectrum gives good agreement between line intensities and number of carbon atoms) and carbon multiplicities. Recording of the ^H-^^C COSY spectrum allows to determine protons chemical shifts. F2 slices also allow to read coupling constants. Due to complexity of the spin systems, the determination of both proton chemical shifts and coupling constants of the aglycone can be achieved with "exclusive correlation spectroscopy" (E-COSY) [129]. This experiment favors the diagonal peaks in the diagonal multiplets, allowing the analysis of cross-peak multiplets even closer to the diagonal than for 2D-filtered COSY spectra. The advantage of the E-COSY experiment is a reduction of multiplets lines in the cross peak. It allows easy measurement of active constants and a very accurate measurement of passive coupling constants, thus it falls among the most appropriate experiments in order to obtain the largest number of couplings with the greatest accuracy possible [130]. It becomes the experiment of choice when overlapped signals occure endowed with multiple couplings, but it has been rarely used for the assignment of triterpene protons [104,131]. Selective excitation experiments (HOHAHA and ID-, 2D-TOCSY) determine spin-spin connectivities in isolated spin systems. Complete analysis of COSY spectra of saponins is often difficult because of peak overlap. Therefore it is very useful to be able to relay coupling information from an isolated proton (like the anomeric hydrogen of saccharides), which, properly excited, transfers the relayed coherence to coupled protons. This tranfer is blocked by a quaternary carbon or an heteronucleus, thus, in a TOCSY experiment, each network of mutually coupled protons can be detected by tracing the cross peaks from certain specific protons, or, by reading each ID subspectrum as an isolated spin system, through a series of ID TOCSY experiments, as shown in Fig. (8). The experiments allow to extract: 1) ^H-NMR subspectra of the monosaccharides; 2) ^H-NMR subspectra of isolated spin systems in the aglycone. Moreover, the correct choice of the mixing times, together with the peculiar in-phase multiplet structure (which contrasts with the antiphase structure in COSY spectra) permits the use of ID TOCSY experiments to correctly read vicinal couplings, even the low values undetermined by the less resolved COSY experiment. TOCSY experiments are frequently used for the determination of the aminoacid

Fig. (8). 1D-TOCSY experiments [128]

Alexandroside 1 [39] (Astragalus alexandrinus Boiss.)

Fig. (9). 1D-TOCSY e.uperirnents.

^«

CO' U>

I 6

I' .9

I

I

'H' I

I

'ii'

O

r-r-n-T-TT-i

I

M it )«

V

J"

i

I

I

t

215

216

constituents of peptides in which the aminoacid units are separated by amide carbonyl atoms. [39, 132-135]. The application to oligosaccharides is rarely reported [104, 131, 134, 135], Fig. (9). The subtraction from the total ^^C decoupled spectrum of the sugar ^^C resonances allows to determine the aglycone contribution. Through the ^H-^^C COSY (HMQC) spectrum, aglycone protons are identified and ^H-^H COSY (better E-COSY) or 2D-T0CSY experiments allow to assign all protons in the molecule (except for methyl groups). ^H-^^C long range COSY (HMBC, COLOC) allows to assign quaternary carbons, sugar interconnections and their linkages to the aglycone. At the end, 2D ROESY experiment assigns methyl groups, stereochemistry of the molecule and spatial arrangements. It also confirms sugar interconnections. Fig. (10). Thus, the rapid and unambiguous structure elucidation of complex saponins and their NMR full assignments can be performed with a combination of ID and 2D NMR techniques [136]. Since all the experiments can be run in an automated sequence, all the necessary information can be obtained during a "week end" acquisition. Moreover, the use of a 600 MHz spectrometer gives the necessary sensitivity to work with very low amounts of samples (few milligrams are enough), that, in natural product chemistry, often is a Umiting factor. 3.11, ddd, 11.2,9.0,12.3 3.80, tdd. 9.5.7.4,6.1 0.2r0.6,2d,4

/

H CH3 H 4.80, ddd. 7.9,8.0.6.9 3.55, dd, 11.2,5.0

2.55, d, 7.9

Fig (11). Typical *H NMR resonances of cycloastragenol.

217

31.2

H O '"•*' y^< ^^ 16.5

29X)

=68.3 O H

13C NMR signals of cycloastragenol l20(fl),24(S)-epoxy-9P.19 cyclolanostan-3p-16P,25-tetrolJ

For the identification of the skeletal type, the chemical shifts of cyclopropane protons are diagnostic of a cycloartane type saponin. They resonate between 0.2 and 0.6 ppm as doublets with ^J= 4 Hz. Characteristic skeletal protons are the protons on oxygen bearing carbons and olefinic protons (which are seldom undistinguishable from the anomeric protons, except for their multiplicities). A characteristic of the cycloastragenol skeleton is the deshielded C-22a proton which resonates at 3.11 ppm, a relatively *'free" spectral zone. Fig. (11). The ^^C resonances of cycloastragenol are also shown. They have been assigned through the strategy discussed before and are characteristic and diagnostic for the conformation described. As a comparison the resonances of the diastereoisomeric cyclogalegenin [20(5j,24(/?)] are reported in Fig. (12). Oleanane-type saponins present in Astragalus species are based on soyasapogenol B as the aglycone. The name clearly identifies its origin: soyasapogenol B is one of the aglycones found in soyabean saponins. Even the sugar chains have common characteristics: usually a /J-Dglucuronic acid is directly linked to the aglycone (at C-3) and carries (always bonded to C-2') a a-L-rhamno(l—2)-j8-D-gluco- or galacto- or xylopyranoside moiety, (see table IV). For the identification of the oleanane skeleton, diagnostic protons are at 5 5.51 (t, J= 4.5 Hz, H-12), and ca 5 3.30 H-18j3 (dd), which gives an NOE with H-12 and Me-30, and the protons of oxygenated carbons. A very recent review accurately describes the ^^C NMR spectra of glucuronide oleanane-type triterpenoid saponins [137].

218

I

o

q\

„ o

•.•- u> CO

^ / ^ o N

\

/

/

^ 0.25 mg/ml. Saponins isolated from soybean seeds inhibited HIV-1 replication in MT-4 cells at 0.5 |ig/ml (Nakamura et al. 1992) [159]. These saponins had a narrow therapeutic index and did not inhibit HIV-1 RT. One of them was found to inhibit HIV-induced cell fusion in MOLT-4 cells. Soyasaponin I and II were studied in vitro against herpes simplex virus type I (HSV-1). Soyasaponin II was more potent than soyasaponin I in the reduction of HSV-1 production. Soyasaponin II was also found to inhibit the replication of human cytomegalovirus, influenza virus, and human immunodeficiency virus type 1. This activity was not due to the inhibition of virus penetration and protein synthesis, but might involve a virucidal effect. When acyclovir and soyasaponin II were evaluated in combination for anti-HSV-1 activity, additive antiviral effects were observed for this virus [160]. Astragaloside II afforded almost 100% protection of Tlymphocytes in vitro against the cytophatic effects of HIV infection. However, the EC50 of ca. 2.5 x 10"^ molar was difficult to achieve in vivo [98].

Fig (13). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 pg/rnl Astragalus saponlns 6-0-fi-D-xyloplranosldes

Astragalus saponlns 6-0-p-D-glucopyranosldes

R = H Trigomside l (A. trigonus Dc) + 136 H HO

O OH

R = H cycloastragenol-6-0-glwoside (A. spinosus Vahl.) 64

+

~

(+ 202 at 1 pglml)

HO OH

+ 104

-E H

OH astraversianinX (A. alexandrinus Boiss.)

+ 105 0Ac

astraversianin VI

y w +48 OAc astraversianln II

1 OAc

astraversianin XIV

or Astragaloside IV

(A. alexandrinus biss.)

+a2

OH

(A. alexandrinus Boiss A. spinosus Vahl.)

OAc

Astragaloside I1 (A. trigonus DC A. spinosus Vahl.)

Hoa HO

Astragaloside I Trigonoside II

Trigonoside Ill

astraversianin XV

(A. trigonus DC)

(A. trigonus DC)

(A. alexandrinus Boiss.)

(A. trigonus DC A. spinosus Vahl.)

OH

Astixgaloside

Fig (14). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 pglml

Astragalus peregrinus

R = cydoastragenol

+ 161 (-19)

HOCH 2

Sieberoside 1

- 51

OH Sieberoside ll

+ 246

Astragalus trigonus DC OH

Glc 0

226

A modification of the immune response could be responsible for the antiviral and anticancer activity of saponins. The mode of action of immunostimulants involves an increased phagocytosis by granulocytes and macrophages, an activation of T-helper cells and a stimulation of cell division and transformation in lymphocytes. Astragalus saponin I (astragaloside IV) isolated from A. membranaceus, when given subcutaneously to mice, increased phagocytosis, bactericidal activity, and acid phosphatase activity by peritoneal macrophages [161]. The natural killer (NK) activity of human peripheral blood lymphocytes was suppressed by Astragalus saponins, especially Astragalus saponin I (astragaloside IV) and astraversianin XI at high concentrations (250 îg/ml), but was stimulated at concentrations 0.05-5 M.g/ml. [162]. 115 cases of leucopenia were treated with pure Astragalus preparation (PAP) at different doses. One group was treated with 15g of PAP twice a day and the other group with 5 g of PAP twice a day, for 8 weeks. A dose dependent significant increase in the count of white blood cells was observed [163]. The effect of Astragalus membranaceus on lymphocytes was studied. It stimulated the proliferation of murine spleen cells in vitro. [164]. The efficacy of Astragalus membranaceus oral liquor combined with routine therapy on T-lymphocyte subset of peripheral blood in viral myocarditis patients has been reported [165].

CH300C O ^ HO'

Ri = CHzCHa Ri = H

- 22

^

^

./•..CH.OH

HO OR

+22 H

(Astragalus tomentosus Lam) R=

Li^ÎÎ^/ Ô-'^^'^Z^^ "° 6H Fig. (15). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 ^g/ml.

Azukisaponin li -2 {Astragalus trlgor)u$DC) AzuWsaponln V +3 {Astragalus trigonus DC)

227

The immunological function of Astragalus membranaceus hairy roots was found to be comparable to that of the dry roots. The content of crude saponins and astragaloside IV in the hairy roots were 5.81 and 0.14%, respectively [166]. Recently, lymphocyte stimulation tests were performed in order to evaluate the immunomodulatory properties of Astragalus saponins. Lymphocyte proliferation or transformation is a process whereby de novo DNA synthesis takes place in response to a mitogen or any other appreciable stimulator (concanavallin A). Astraversianins II, and X, astragalosides I, II, IV and VI and cyclocantosides E and G isolated from A. melanophrurius were able to stimulate mouse lymphocyte proliferation in the concentration range of 0.01-10 îg/ml. At higher concentration, inhibition of thymidine incorporation was observed [72]. In the same assay, a number of cycloartane and oleanane saponins isolated from Egyptian Astragalus were tested. As shown in the Figures (13-15), 20(/?),24(5) epoxycycloartane (cycloastragenol), 20(5),24(/?)epoxycycloartane (cyclogalegenin) and A^"* cycloartane saponins gave higher stimulation (at the same concentration) than tetranorcycloartane and oleanane saponins. (Verotta, L. et al unpublished results). Cycloartane saponins seemingly contribute to the immunomodulatory properties of Astragalus^ in fact, curculigosaponin G from Curculigo orchioides was reported to significantly promote the proliferation of spleen lymphocytes in mice compared to controls, without a marked influence on antibody formation [167]. HOCHz R=

HO HO Ho\-.---^-\/

cH-jrÔ''

HO^^

O"

OH

Curculigosaponin G

ACKNOWLEDGEMENTS Work supported by Ministero deir University e della Ricerca Scientifica e Tecnologica of Italy.

228

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. Allrightsreserved.

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LABDANE -TYPE DITERPENES: CHEMISTRY AND BIOLOGICAL ACTIVITY COSTAS DEMETZOS^ and KONSTANTINOS S. DIMAS' ^School of Pharmacy, Department of Pharmacognosy, Panepistimiopolis Zografou 15771, University of Athens, Athens Greece Înternational Institute of Anticancer Res,, Kapandriti, 19014, Attiki, Greece ABSTRACT: The terpenoids are a class of natural products with biological activity. The number of isoprene units from which they are biogenetically composed is used for their classification. The diterpenes are another group from which several compounds with high biological activity have been produced. The most studied plant families are Asteraceae and Labiatae, as well as Conifers, which are the main sources of diterpenes. The lack of, or insufficient, chromatographic data (RI, Rt) as well as of reference compounds for most labdanes is the main reason for their absence from the chemical fingerprints of plant extracts. Also their use as chemotaxonomic markers within the species in which they occur is limited. The configuration at the C-13 carbon atom has been investigated, especially in the case of manoyl oxide, as well as the various isomers, the ratio of which determines antimicrobial activity. A variety of biological activities have been encountered in labdane diterpenes such as antibacterial, antifungal, antiprotozoal, enzyme inducing, anti-inflammatory activities and modulation of immune cell functions. More recent studies have shown that labdane exhibits significant cytotoxic and cytostatic effects against leukemic cell lines of human origin and interferes with the biochemical pathways of apoptosis and the cell cycle phases, as well as with the expression of several protooncogenes such as c-myc and bcl-l. This report underlines the role of these compounds, not only as tools for the study of the biochemistry and regulation of biochemical and metabolic pathways of mammalian cellular systems, but also as potential pharmacological agents in the fight against diseases such as cancer and heart disorders.

1. SECONDARY METABOLITES OF PLANTS The processes generating plant compounds have been separated into primary and secondary metabolism. Primary metabolism produces the basic products for the life of the plant like carbohydrates, amino acids, fatty acids, polysaccharides, proteins, lipids, RNA and DNA. The primary metabolites are produced in relatively large quantities and their distribution is universal. On the contrary, the secondary metabolites are

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not as common as they may exist only in a single plant species or be characteristic of specific groups of plants (i.e, genus) [1]. However, they are recognised as vital for the life of the plant even if their production is limited. This view is supported by the fact that secondary metabolites create a defence mechanism against bacterial, viral and fungal attacks analogous to the immune system of animals [1, 2, 3]. Additionally, they are linked to hormones that are responsible for the growth of the plants or the healing of their wounds. Secondary metabolites may also be found in animals, but 80% of all such metabolites known are of plant origin. An explanation for this wealth of plant-originated secondary metabolites may be the fact that plants are rooted and immobile. Hence they do not have the ability, vital to animals, to move away from danger. Plants do not respond to the environment in the same way as animals do. Nevertheless, they are exposed to weather conditions, soil factors, gradual environmental pollution, herbivorous animals or symbiotic organisms and other competing plants [3]. Plants are significant to the diet of humans and animals since they provide most of the essential nutrients and vitamins. Vitamins C (ascorbic acid), E (a-tocopherol) and K (phylloquinone) are biosynthesized by plants, while P-carotene, the precursor of vitamin A and ergosterol, the precursor of vitamin D, are also secondary plant metabolites. These metabolites are used in folk medicine and for industrial purposes, as raw materials for pharmaceutical and other products [3]. On the other hand, plants may produce substances, which are toxic and/or irritant to man. The classification of plants is primarily based on the similarities and differences that are displayed by their morphological and anatomical characteristics. In some instances this does not suffice since the morphological differences may not be genetically defined but have been caused by local bio-climatic factors. Nevertheless is apparent that secondary metabolites can contribute to the taxonomy of plants and their systematic evolution. There are many examples of cases where the morphological features are not clear and secondary metabolites serve to clarify the morphological classification {e.g. classification of the tribes of the family Asteraceae). It has also been proved to be significant to use all the secondary metabolites for the above purpose and not only one of their chemical groups [4].

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2 TERPENOIDS The terpenoids are secondary metabolites that are found in essential oils, resins, tissues of higher plants and micro-organisms, whilst recently some have also been located in liverworts [5,6]. The terpenoids are formed from linear arrangements of isoprene units. Fig. (1), which are derived from acetate metabolism through mevalonic acid (MVA). This pathway was found to be common to the whole range of natural terpenoid derivatives

/v Isoprene unit

Isoprene

Fig. (1). Isoprene units

[7]. It is useful to divide terpenoids into classes according to the number of C5 isoprene units they contain, each class being derived from a primary metabolite precursor. They are classified as hemiterpenes (C-5); monoterpenes (C-10); sesquiterpenes (C-15); diterpenes (C-20); sesteterpenes (C-25); triterpenes (C-30); and tetraterpenes (C-40). Higher polymers are found in materials such as rubber. Geranyl pyrophosphate is the primary metabolite from which monoterpenes are derived. Farnesyl pyrophosphate gives rise to the sesquiterpenes and through its conversion to squalene, to the triterpenes and steroids (C 18-30), while geranylgeranyl pyrophosphate is the primary metabolite precursor of the diterpenes and carotenoids (C-40) [4], Fig. (2). The terpenoids usually play a role in the growth or the defence of the organism that contains them. That is why some terpenoids are toxic, irritant or allergenic and some are repellent smelling (small terpenes)

238 Irregular monoterpenes

Hemiterpenes

Diterpenes

t

Monoterpenes

Carotenoids

Phytosterols

Steroids

Fig. (2). Primary metabolites precursor in the biogenesis of various terpenoids.

whilst some taste bitter. Nevertheless, they also play a primary role in the growth of the plants, e.g the growth hormone giberelline belongs to the terpenoid group [3]. Their significance in ecological biochemistry and pharmacology has been proven by a number of studies conceming their pharmaceutical or healing qualities in vitro and in vivo. Examples of these qualities are anaesthetic, analgesic, anthelmintic, antiepileptic, antiinflammatory, antirheumatic, antitumour, diuretic, expectorant, hypotensive, insecticidal, organoleptic (odour, taste), spasmolytic, toxic and purgative [8]. The terpenoids may also be used for the identification of the various taxa [9]. They were initially used extensively as taxonomic markers of gymnosperms, mainly due to their abundance in the leaves of conifers. In contrast, the terpenoids of angiosperms have been poorly researched, possibly due to their irregular distribution in such families e.g, in a survey of 34 species of Plectranthus (Labiatae) [10], 18 were found to have excellent oil profiles, with up to 32 components, but the remainder of the species failed to give any leaf volatiles. Compared with other secondary

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constituents, terpenoids tend to show more intraspecific variation. Different classes of terpenoids have been detected in individual species of almost every plant genus that has been studied extensively. Some variations occur at population level. Such differences in pattern are often correlated with geographical or ecological factors, so that the results of such surveys have been important in extending our knowledge of plant population structure. From the taxonomic point of view, the terpenoids have been mainly useful as an aid in: 1. Defining the species; 2. Detecting hybridisation in natural populations, which is a universal phenomenon in conifers; 3. Confirming the presence of geographical races; 4. Confirming generic and tribal limits. The taxonomy of species is important, because it puts the ecosystem in an order, and it also provides information about species and life evolution [3]. Generally terpenoids exhibit a variety of chemical structure and complexity. The elaboration of Gas Liquid Chromatography (GLC) method has made the detection of plant volatiles such as terpenoids easier, offering both qualitative and quantitative data. 3. DITERPENES The diterpenes (C-20) are derived from geranyllinalyl pyrophosphate or its C-13 allylic isomer, geranyl-geranyl pyrophosphate (GGPP). After protonation of GGPP, a cyclization process can be initiated, yielding copalyl PP. An alternative pathway leads to labdadienyl PP, which is the enantiomeric product. Fig. (3),[11]. These are usually found in plants as mixtures with other related compounds. They occur in nature as both normal and antipodal stereochemical series. Fig. (4) shows the general precursor of cyclic diterpenes, le geranyl geraniol (1), as well as the main diterpene skeletons classified by Rowe et al [12]. Ponsinet [13] has noted that the transformation of the water soluble geranyllinalyl pyrophosphate. Fig. (3), requires a set of enzymes different from those, which bring about the cyclizations of the water insoluble hydrocarbon, squalene, into the various triterpenes. This may explain why diterpenes and triterpenes rarely occur together in the same plant tissues.

240

a

CH3

—^''

f*7« C

5

Geranyllinalyl pyrophosphate R=pyrophosphate

t'•T; OPP

OPP Copalyl PP

GGPP

OPP

OPP

OPP H ^

GGPP

labdadienyl PP

OPP

Fig. (3). Biosynthetic pathways to the bicyclic diterpenes.

Usually diterpenes are found in plant resins and latexes where they are involved in their sticky texture. Resins often exude from the wounds of a

241

plant, playing an antimicrobial role. Nevertheless, some leaves, stems or woods of plants are covered with resin and latex despite the absence of

gibberellane

kaurane

Fig. (4). Structures of important classes of diterpenes.

wounds and are hostile to animal and predators; e.g. leaf-cutting ants avoid attacking such plants [10].

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The resin produced by conifers is rich in diterpenes. There is growing evidence that non-volatile diterpenes (e.g. kaurenic acid) inhibit the feeding of insects and hinder the growth of their larvae. This is achieved through the disturbance of the hormonal processes, especially of ecdysone synthesis [3]. On the other hand an adapted sawfly {Neodiprion seltifer) stores the chemical compound of the resin whilst still at the larval stage and uses it for its own chemical defence against birds. This diet of resin reduces the growth rate but the alternative of not feeding it from the tree would leave the insect without any chemical defence. Most other insects, which utilise diterpenes for defence, appear to make their own toxins from simple starting materials [3]. Toxicity to animals is especially associated with certain diterpene types, like grayanotoxin I, which occurs in some genera of the family Ericaceae [10]. The leaves and flowers oi Rhododendron sp. contain toxic diterpenes, able to contaminate the honey produced by bees [3,10]. Many Euphorbiaceae and Thymelaceae also contain toxic diterpenes, which have the additional property of being highly irritant and hence cocarcinogenic. The diterpene phorbol (1), which belongs to the tigliane skeleton. Fig. (5), was isolated after hydrolysis of the seed oil (croton oil) of Croton tiglium in 1931. Later it was also found in other plant species of the Euphorbiaceae family. The oil is still used in experimental cancer biology. Compound 2, Fig. (5), has been characterized as phorbol -12myristate-13-acetate (PMA) (2) and is the most potent tumor-promoting constituent of croton oil [14]. From the Euphorbiaceae family, various phorbol esters have been isolated from individual species of four genera: Croton, Sapium, Euphorbia and Ostodes. The natural occurring esters can be divided into two general types: the C-13 monoesters and the C-13, 20 diesters. The length of the C-13 ester chain varies from two - to sixteen carbons. Of interest is prostatin (3), Fig. (5), which has shown significant cytoprotective properties in human lymphocytic cells infected with the HIV - I virus [15]. A related family of compounds is the daphnane diterpenes. Resiniferatoxin (4), Fig. (5), is known for its potency as a proinflammatory agent [16]. Derivatives of compound 4 isolated from both the Euphorbiaceae and Thymelaceae families are well known for their antileukemic activity [17]. Ingenanes - Fig. (5) - are tetracyclic polyol esters isolated from the Euphorbia species. The parent compound is the polyol ingenol (5), derivatives of which have shown selective cytotoxic

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activities against several human cancer cell lines derived from leukemia, non-small-cell lung cancer, colon cancer, melanoma and renal cancer [18]. The phorbol esters have proved to be most potent tumor promoters for two-stage mouse skin carcinogenesis [19]. The major phorbol ester receptor has been identified as protein kinase C [20]. Computer modeling of phorbol esters and other classes of tumor promoters has shown an interesting similarity in the relative positions of certain heteroatoms and hydrophobic groups [21].

daphnane

tigliane

ORi 0R2

O

OH

1: 2:

R,=R2=H Ri=CO(CH2)l2CH3 R2= COCH 3

PhCH2

O2CCH2 -

Fig. (5). Diterpenes from Euphorbiaceae and Thymelaceae families.

ingenane

244

Taxol (1), Fig (6), is another important diterpene with anticancer properties. It was isolated in 1971 from Tcaus brevifolia Nutt. (Taxaceae), which is a slow growing shrub/tree, found in the forests of N.W Canada and USA. Taxol is one of over one hundred taxanes, which have been

Fig. (6). Structures of Taxol and taxane derivatives

characterized from various Taxus species. It belongs to a group of compounds with a four-membered oxetane ring and an ester side-chain, both of which are essential for anticancer activity. Taxol was isolated

245

from the bark off. brevifolia in low amounts (c. 0.01-0.02 %), but a content of up to 0.033 % was found in samples from leaves and twigs [22]. Taxol is derived from GGPP via cyclization reactions, employing the same mechanistic principles as in mono- and sesquiterpenes. Baccatin III (2), another taxane derivative, has been found (up to 0.2%) in the bark. A number of other taxane derivatives were isolated and characterized as 10 - deacetyltaxol, 10 - deacetylbaccatin III (3), cephalomannine (4) and 10 - deacetylcephalomannine (5) while some of them have been microbiologically transformed into taxol, Fig. (6). Cell cultures of T. baccata as well as microorganisms and enzymes, also offer excellent potential for the production of structurally related compounds and thus improve the yields of e.g 10-deacetylbaccatin III [23] in crude extracts. Taxol (Paclitaxel) is an important new anticancer drug for the treatment of ovarian cancer and is currently in clinical trials against metastatic breast cancers. It is also a potential drug for lung, head and neck cancer. Docetaxel (Taxotere) (6), (Fig. (6), is an analogue of taxol, which has been prepared from 10-deacetylbaccatin III, by semisynthesis. This compound has better water solubility than taxol and is being tested clinically against ovarian and breast cancer. The mechanism of action of Taxol involves the stabilization of ordinary cytoplasmic microtubules and the formation of abnormal bundles of microtubules [24, 25]. Various other biologically active diterpenes have been isolated from plants and are used by man for the treatment of a variety of diseases. The bark of Pinus strobus (Pinaceae), rich in diterpenes, among them manoyl oxide, is used in anticough syrups, whilst traditionally it has been used for the treatment of coughs, colds, congestion, injury, rheumatism and swelling by Indian Americans [26]. The antiinflammatory and antimicrobial activity of various diterpenes has been reported [27], as well as anti-fungal activity [28] and remarkable cytotoxic activity for some of them [27,29]. The ability to synthesize diterpenes is universal to plants, since phytol, the acyclic parent compound of the series, is present in ester attachment in the chlorophyll molecule and hence occurs in all green plants. Gibberellic acid is also widespread in the plant kingdom as a growth hormone. Besides phytol and gibberellic acid, the remaining diterpenoids are very restricted in occurrence and usually occur within one or only a few plant

246

families. The families in which the diterpenoids are regular constituents include among the gymnosperms, Pinaceae, Cupressaceae and Podocarpaceae and among the angiospemis, Ericaceae, Euphorbiaceae, Labiatae, Leguminosae and Thymelaceae. There are also records of diterpenes in individual species in many other angiosperm families [10]. This may be the reason why taxonomists use diterpenes as chemotaxonomic markers [30]. The diterpenes have been extensively used as taxonomic tools for the classification of various taxa mostly among the gymnosperms. The study of their diterpene content promoted the taxonomic reassessment of Tetraclinis articulata and Chamaecyparis obtusa (Cupressaceae) [10]. However the absolute stereochemistry must be examined carefully because the same species have been found to contain one or other enantiomeric series of diterpene. For example, the leaves of Podocarpus macrophyllus contain (-) kaurene and those of P. ferrugineus contain its enantiomer [31]. The relative configuration of diterpenes at carbon atoms 5, 8, 9 and 10 is derived from a concerted trans-anti-trans addition to the double bonds of the "all trans" acyclic precursor, geranyllinalyl pyrophosphate, folded in the most stable all chair conformation, Fig. (3). The two decalin rings have a trans configuration because this is the most stable conformation since only 2.7 kcal/mole are required, as opposed to the cis configuration, which requires 6-8.8 kcal/mole. It has been established though, that the trans is the active configuration since phorbol esters, as well as ingenane and daphnane derivatives are biologically active as trans isomers, while the cis conformation is inactive. Within a given plant source, the configuration at C-13 in the diterpenes varies with skeleton type. In fact, diterpenes of the same skeleton may have a different configuration at C-13. Sclareol and 13-£?/7/-sclareol from Salvia sclarea are examples of this phenomenon [32]. The same phenomenon has been observed in manoyl oxide isolated from several parts of Cistus creticus. The configuration at C-13 has been found to be different in different parts of the plant. The antibacterial activity was also found to be related to the C-13 configuration [33]. 4. LABDANE DITERPENES A very large number of diterpenoids possessing a labdane skeleton (1), Fig. (7), occur in nature [34]. The interest in studing labdanes is

247

r;6?

c?5^ 7 R=OH 8 R=OAc

Fig. (7). Labdane diterpenes

heightened due to the wide range of biological activities of these compounds [35]. They comprise a decalin system and a C-6 ring, which

248

may be open or closed with an oxygen atom, as in manoyl oxide and its derivatives. In the ring system of labdanes the substituents below the plane of the ring are drawn with a dashed bond indicating a-configuration, while when the substituents are drawn above the ring with a wedge bond, the p-configuration is designated. Usually, a-hydrogen atoms found at ring junctions are omitted for clarity, if they have the natural configuration. The antipodal of the normal series is indicated by the prefix ent' before the complete name of the compound. Labdane diterpenes have five chiral carbon atoms and they occur in nature in the two enantiomeric series. It has been observed that, in a single plant species, both enantiomeric labdanes co-occur [36,37]. The normal and the enantiomeric carbocation are produced from the achiral geranylgeranyl pyrophosphate (1), Fig. (8). The co-occurence of normal labdadienoic acid and antipodal

OPP

H ^ ^

OPP OPP

Fig. (8). The biosynthetic precursors of normal and enantiomeric labdanes

dihydroeperutic acid in the same plant was reported in 1967 [38]. A plant biosynthesizes the normal as well as the enantiomeric labdane - type diterpenes via 2 or via 3 intermediates, respectively. Fig. (8). From the leaves of Mimosa hostilis (Leguminoseae) both normal and antipodal labdanes have been isolated and their absolute configuration confirmed by x-ray crystallographic analysis [39]. The optical rotation of labdanes has been correlated to their structures and a total of 143 labdanes have been examined. The conclusion of this study was that this correlationship could provide information about the structure, as well as the stereochemistry at C-13. This study can also be used as a tool in order to correlate the optical

249

rotation and the structure for monoterpenoids, sesquiterpenoids and triterpenoids, which have comparable structures [40]. 4.1 THE ORIGIN OF LABDANES Labdanes have been isolated from several plant families, such as Asteraceae, Labiateae, Cistaceae, Pinaceae, Cupressaceae, Taxodiaceae, Acanthaceae, Annonaceae, Caprifoliaceae, Solanaceae, Apocynaceae, Verbenaceae and Zingiberaceae. In addition they have been isolated from marine algae of the genus Laurence, from Taonia atomaria and from the red alga Chondria tenuissima, from which has been isolated the bromoditerpene entA3-epi-concirmdiol, the structure of which was determined by x-ray crystallography [41]. The conifers are an important source of diterpenoids. Several labdanes have been detected in the neutral fraction of the oleoresin of Araucaria excelsa, including manool as well as nor-labdanes [42]. Some neutral diterpenoids, such as eA7^1abd-8, 13(E)-dien-15-ol and its acetate, were obtained after extraction of the resin of Araucaria bidwillii [43], while the normal series of the above labdanes have been isolated from Cistus creticus subsp. creticus [44]. From Pinus sylvestris, 3P-hydroxybiformene has been obtained, while from the needles of the American red wood pine {Pinus resinosa), 8,13-epoxy-labd-14-en-19-oic acid, was isolated. In Pinus nigra, labdane acids have been found to be the major acid components of its needles [45,46]. The occurrence of diterpenoids including labdanes in conifers has been published as a review of the chemistry of the order Pinales [47]. The genus Sideritis (Labiatae) has been an important source of novel diterpenoids, thus extensive studies have been done on this genus and a large number of labdanes have been isolated and identified in the past few years [48]. From Sideritis arborescens, andalusol was isolated and x-ray analysis was used to determine its absolute stereochemistry [49]. From another Sideritis species, i.e S. gomerae, gomeraldehyde (ent-S, 13,epoxylabd-15-al) and gomeric acid as well as their 13-epimers were isolated [50]. The chemical investigation of the aerial parts of S. nutans afforded, in addition to the known labdanes also isolated from S. gomeae, some new entAabdanQ oxides, such as gomerol, 13-epi gomerol, sidnutol and 3a-hydroxy-gomeric acid [51]. A derivative of manoyl oxide, namely

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borjatriol, has been isolated from S. mugronensis, while from S. arborescens, 6-deoxyandalusol and barbatol have been obtained. Borjatriol has been synthesized, using the diterpene larixol as the starting material [52]. The structures of borjatriol and of barbatol were determined considering their interrelationship with manoyl oxide [53, 54,55]. A series of andalusol derivatives have been obtained from S. foetens [56], while from the hexane extract of S, javalambrensis, eA7f-16-hydroxy-13-epimanoyl oxide was isolated [57]. From Sideritis canariensis and S. varoi (Labiateae), ribenol (^«f-3ahydroxy-13-e/7/-manoyl oxide) has been obtained and it was converted into 13-e/7/-manoyl oxide [58,59,60]. Ribenol was converted to its thiomidazolide (45% yield), by treatment with N, N'thiocarbonyldiimidazole [61]. This new ^-em/'-synthetic compound has been extensively studied against human leukemic cell lines [62]. Ribenol and its acetyl derivative have been isolated from the fruits of C. creticus subsp. creticus, in addition to other labdanes, and their percentage content in the extracts and in the essential oils of the leaves and fruits of the plant have been compared to that of C. creticus subsp. eriocephalus for chemotaxonomic purposes [33]. Ribenol and its acetyl derivative have first been identified as volatiles in the essential oil of the resin 'Ladano' and their chromatographic data using Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) were obtained [63]. In the same paper, a comparative study was carried out concerning the antistaphylococcal activity of these compounds and of manoyl oxide as a mixture of isomers. The results showed that ribenol had a better Minimum Inhibitory Concentration (MIC = 0.1 mg/ml) in contrast to the other compounds tested. From the plants Cistus ladaniferus, C. laurifolius, C. palinhae, C. clusii, C, symphytifolius and C. libanotis (Cistaceae) several labdanes have been obtained [64-71], while from C hirsutus (Cistaceae), 6p-acetoxylabd-8 (17) en-15-oic acid has been isolated and its structure determined considering its interrelationship with 6-oxocativic acid [43]. Isolation of several labdanes has also been reported from the genus Halimium of the Cistaceae family, their structures being determined by spectroscopic methods and by chemical transformations [72,73,74]. In the family of Asteraceae the ent- series of labdanes have been identified as the common components [75]. The following species of Asteraceae have yielded labdanes: Leyssera guaphaloides, Brickellia

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lemmonii, Baccharis petiolata, B. pedunculata, Aristeguetia buddleaefolia, Blepharisperum zanguebaricum, Stevia seleriana, Happlopappus pulchellus, H. arbutoides, H. parvifolius [76-82], Corymbium villosum [83], Chrysocephalum ambiguum [84] and Waitzia acuminata [85]. From Haplopappum parviflorus, twenty-one labdanes have been isolated which belong to seco-, nor-, and normal series. Sclareol (2), Fig.(7), however, was not isolated and as suggested in that report, it could be the common precursor of the isolated compounds [86]. Ent- labdanes have been isolated from Gutierrezia grandis, from Baccharis scoparia, from Oxylobus arbutifolius and from the Indian plant Phlogacanthus thyrsiflorus. The latter is used to treat bronchial conditions, while a similar action has also been reported for Andrographis paniculata (Acanthaceae) [87,88,89]. (-) Ozic acid has been isolated from the wild sunflower Helianthus occidentalis (Asteraceae) as cis- and transisomers [90]. In Austroeupatorium chaparense (Asteraceae) several furanoid 7p-acetoxylabdanes have been found [91], while other furan labdanes have been isolated from Galeopsis angustifolia (Labiatae) [92,93] and from Xanthocephalum linearifolium (Asteraceae) [94]. Dimeric labdanes have been obtained from the cones of Cunnighamia lanceolata [95] while dimeric labdanes, esters of malonic acid, have been previously obtained from the resin Ladano [96,97]. A number of labdane glycosides have been isolated and identified from the family of the Asteraceae. Gutierrezia sphaerocephala has given several glycosides, while from Aster spathulifolius a number of labdane 13-O-glycosides have been obtained [98,99]. Ent- labdanes of the same family have been isolated from Gutierrezia spathulata, from Haplopappus species and from Grindella species [98]. Glycosides derived from labdan-8(17), 13-dien3b,15,18-triol have been obtained from Rubus foliolosus which is used in Chinese traditional medicine [100]. From the root stalks of Gleichenia japonica some labdane glycosides have been obtained and they were found to be growth inhibitors to other plants [101]. From Viburnum suspensum (Caprifoliaceae) the gomojosides A-J have been obtained [102], while mitrariosides A-D have been obtained from Mitraria coccinea (Gesneriaceae) [103]. Gaudichaudioside F has been isolated as a bitter-tasting arabinoside of Baccharis gaudichaudiana. The phlomosides, a series of labdane glycosides isolated from the roots of the Tibetan plant Phlomis medicinalis (Labiateae), have also been used in folk medicine

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[101]. The aerial parts of Conyza steudellii (Asteraceae) have given new labdane xylosides, while from the leaves of Conyza trihecatactis, a xylopyranoside of 13-epi-sclareol has been isolated among other labdanes [104,105]. Havardic acids A-F (as methyl esters), have been isolated from another genus of the Asteraceae family, i.e Grindelia havardii [106]. 11oxo-manoyl oxide derivatives and coleosol, which is also a manoyl oxide derivative, have been obtained from Coleus forskohlii (Labiateae) [107,108] while from another plant of the Labiatae family, Roylea calycina a tumor inhibitory compound, namely precalyone, as well as calyone have been isolated [109]. From the plant Agathis robusta a group of labdane isomers at C-13 have been isolated and related to 13-e/?/-manool after interconvertion using lithium in diaminoethane [110]. The Hymenaea species, le H. ablongifolia and H. parvifolia, have been studied and enantiopinifolic acid as well as guamaic acid have been isolated, while enantio-lS-e/?/labdanolic acid has been isolated from Trachylobium verrucosum [111]. One of the well known labdanes is sclareol (syn. Labd-14-ene-8, 13diol) (2), Fig. (7), a ditertiary alcohol widely distributed in nature. It was first isolated from clary sage oil {Salvia sclarea, Labiatae) [112]. This oil is used in soaps, as a fragrance in cream and lotions, in food and beverages as a flavoring component [113] as well as in folk medicine [26,114]. Sclareol is an epimeric mixture at C-13 (ratio 9:1), where predominates the 13 R-epimer [115]. GC-MS analysis of the n-hexane extract of the liverwort Pleurozia acinosa (Jungermanniaceae), revealed a large peak of the compound which then was isolated and identified as 8,13-c//-ep/-sclareol. The spectral data of this compound was not shown to be close to those obtained from sclareol, from 13-ep/-sclareol, from ent-êpi -sclareol or from ent- 8,9 -di-epi- sclareol [116]. The percentage content of sclareol is up to 2% in clary sage oil, while in the concrete its concentration is up to 70% [114]. The Flavor and Extract Manufactures Association of the U.S.A [117] generally recognize Sclareol as a safe material. The use of clary sage oil in fragrances in the U.S.A amounts to about 10,000 lb/year. Sclareol is used on a commercial scale for preparing a series of Ambra odorants [118]. Cistus ladaniferus [119] is referred to as a plant having an amber odour and has been used as a perfumery raw material. Another plant which also belongs to the genus Cistus, namely Cistus creticus subsp. creticus (Cistaceae) contains sclareol in its leaves

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[44] and resin named Ladano [63]. Ambrox (3), Fig. (7), is reported as a key component of ambergris and it well known as an important ingredient for perfumery [119]. Ambrox is a degradation compound of sclareol and has been isolated from the oil of clary sage (Salvia sclarea), while it has also been shown to be present in Labdanum oil from C. ladaniferus (Cistaceae), as well as in cypress oil {Cupressus sempervirens) [118]. In the search for products for perfumery with ambergris odor, sclareol and manoyl oxide (4), Fig. (7), have been investigated and an oxidation process leading to the production of acetals has been examined and evaluated. The relationship between the structure of manoyl oxide and ambergris - type odour has also been examined and manoyl oxide was converted to its ketone or ethers [120]. 4.2, MICROBIAL TRANSFORMATION OF LABDANES Sclareol as well as several labdanes have been used as substrates of microbial cultures in order to produce compounds with higher potency and efficiency against various diseases. Microbial transformation of sclareol with Mucor plumbeus gave a mixture of triols from which the labd-14-en-3p, 8a, ISp-triol yielded in high amounts [121]. Sclareol has been referred to as a starting material to obtain interesting biologically active drimanes [122]. Sclareol possess antimicrobial properties against Gram positive and Gram negative bacteria [123,124]. Its \2-epi isomer has also shown an MIC of 250 |ig/ml, against Staphyloccoccus aureus, while sclareol was found to be more active against Staphylococci [63]. Manool (5), Fig. (7), which has also been obtained from Salvia sclarea as sclareol [125], is also a raw material for the commercial preparation of amber-type perfumes. In a biotransformation process that was carried out using Mucor plumbens, manoyl oxide, sclareolide and a 7a-hydroxy derivative of manool were obtained [126]. The oxidation of manool in order to produce ambergris-type perfumes has been examined and several known derivatives have been obtained [127]. The microbial transformation of entA3-epi'VCidinoy\ oxide by Rhizopus nigricans has been utilized to produce biologically active derivatives [128]. Incubation of eA7/-19-hydroxy 13-^j9/-manoyl oxide as well as e^/-3p-hydroxy IS-epimanoyl oxide with the fungus Gibberelafujikuroi rendered derivatives of

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manoyl oxide, the structure of which were determined using spectroscopic methods [129]. Ent -3p-hydroxy 13-£?p/-manoyl oxide (7) (ribenol), Fig. (7), has been used as starting material for a biotransformation process by Curvularia lunata. The in vitro micropropagation of the Sideritis foetens species has been described and several ^«/-manoyl oxide derivatives, as well as other labdanes, have been isolated from micropropagated plants. Recently, biotransformation by Curvularia lunata of some e«^13-e:/>/-manoyl oxides functionalized at C-3 or C-3 and C-12 produced derivatives, which inhibited the growth of the pathogenic protozoa, Leishmania donovani [130].

4.3. THE STEREOCHEMISTRY AT C-13 OF LABDANES The configuration at C-13 of the diterpenes has been a problem for many years. NMR spectroscopy using chiral shift reagents has been suggested as a method to differentiate manool from 13-ep/-manool [131]. Most of the diterpenes with a saturated side chain were present as mixtures of C13 epimers. Small differences in chemical shifts in the ^H and *^C - NMR spectra did not allow assignement of the stereochemistry at C-13 [132]. The absolute configurations of sclareol (2) and manool (5), Fig. (7), at C-13 have been determined [133]. As Hanson reported [134], the absolute stereochemistry assigned to some labdanes should be reexamined due to the enantiomers of labdanes. The biosynthetic pathways of sclareol and manool start from a geranyllinalool - type skeleton which cyclizes in a similar fashion as that described for cativic acid [135] and, via the intermediate 6, Fig. (7), sclareol is formed by hydration of 6, or manool by the loss of a proton [112,136]. Manoyl oxide (4), Fig. (7), has been isolated as a pure compound or identified via analytical techniques, in several plant species [33,44,63,137, 138,139]. Ohloff has shown that manoyl oxide can be prepared from sclareol [140]. The physical and chromatographic data of the synthetic and of the natural manoyl oxides have been compared and discussed [141]. Hodges and Reed have substantially contributed to the knowledge of the stereochemistry of manoyl oxide [141]. Manoyl oxide occurs in nature in both normal and antipodal configurations [58]. The 13-6?p/-manoyl oxide has been isolated after

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extraction of jack pine bark {Pinus banksiana Lamb.) and from Haplopappus parvifolius (Asteraceae) [142] as normal (a^ positive), while its antipode (a^ negative), also referred to as olearyl oxide, has been reported to occur in Olearia paniculata, the fungus Gibberella fujikuroi and the herbs Beyeria sp. [139]. The synthesis of 13-e/7/-manoyl oxide from sclareol has been reported [143]. Its derivatives have been isolated from natural sources, during microbial transformation [144-146] or from in vitro micropropagation of plants [147]. Synthetic derivatives of manoyl oxides have been synthesized for pharmacological purposes [148]. The circular dichroism curves of manoyl oxide and of its 13-epimers have been examined [149]. In studies related to antiinflammatory activity, [56] the \l>-epi isomer derivatives of manoyl oxide have been reported to be are active; however manoyl oxide and its isomers have not yet been studied. Recent studies on the two subspecies of Cistus creticus (Cistaceae) {i.e C.creticus subsp. creticus and C creticus subsp. eriocephalus) concerning manoyl oxides, have proven that the percentage content of the isomers of manoyl oxide varies, depending on the part of the plant and the polarity of the solvent which was used [33]. These results correlate with the antimicrobial activity and the percentage content of isomers of manoyl oxide in the mixture. No other pharmacological data are available up to now concerning the percentage content of the isomers of manoyl oxide except those for their antimicrobial activity. Hence, in order to elucidate the structures and the percentage content in the mixture of manoyl oxide isomers, an analytical approach like GC - MS was selected as a simple and rapid methodology [33]. The analysis by GC-MS of manoyl oxide as a mixture of isomers as well as the study of extracts and essential oils of the two C creticus subspecies, revealed the existence of more than one isomer which was difficult to separate and distinguish. The results showed different peaks which are indicative of the presence of isomers with different chromatographic data (RI: Retention Indices and Rt: Retention time). The fragmentation of manoyl oxide at the chiral center C-13 and the speed of the removal of CH3-I6 showed that there was different intensity for the peak with m/z 275 and 257 for the isomers [33]. Manoyl oxide is referred, to without any clarification, for the presence of isomers but never for their proportion [137,138]. This results in errors during the pharmacological evaluation of manoyl oxide and also in its use as a

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reference compound, irrespectively of its origin. In its ^H-NMR spectrum when manoyl oxide exists as a mixture of isomers, the aliphatic as well as the olefmic regions exhibited more than one initial signal [44]. The study of manoyl oxide derivatives i.e. 7 and 8 in. Fig (7), (i.e enthydroxy and ^w/-acetoxy-3p-manoyl oxides) isolated from Cistus creticus, by GC-MS resulted in only one peak indicative of the purity of the products [33]. From the ^H-NMR data it is clear that the 13-epz isomer was present in both derivatives [58,139]. The chromatographic data of the compounds 7 and 8 were recently published [33,63]. Hence, investigations have proven that, apart from the \l)-epi isomer, there are more isomers with varying intensities, which correspond to isomers that arise from the different configuration of C-8 chiral center [33]. This isomer showing a different configuration at C-8 has been isolated from the volatile leaf oil of Alaska (yellow) cedar and its structure has been confirmed using spectroscopic methods as well as chemical reactions [150]. The most important manoyl oxide derivative is forskolin (9), Fig. (7), (7p-acetoxy-8, 13-epoxy-la, 6p, 9a-trihydroxylabd-14-en-ll-one) [151153]. It belongs to the labdane series of diterpenes and was isolated from the Indian herb Coleus forskohlii fWilld.) Briq. (Labiatae). Since ancient times it has been used in Hindu and Ayurvedic traditional medicine [154]. The plant Coleus forskohlii (Willd.) Briq. has been extensively studied, and from its extracted roots a group of diterpenoids, with the basic skeleton of 11-oxo-manoyl oxide, have been isolated. The main compound, forskolin, presented remarkable chemical and biological properties [155]. Analogues of forskolin were then prepared by semisynthesis [156] or obtained by microbial transformations [157]. New analogues, more soluble than forskolin have shown activities comparable to and even higher than forskolin [158]. 4.4. LABDANES AS VOLATILE COMPOUNDS The lack of or the insufficience of chromatographic data (RI, Rt) and reference compounds for most of the labdanes [159], present basic problems in studies concerning the qualitative and quantitative analysis of plant extracts using GC or/and GC-MS analysis. Also their use as chemotaxonomic markers within species where they occur is limited. GC

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and GC-MS methods have been appUed to the detection and analysis of labdanes using capillary chromatographic columns, HP-5MS and CP-Wax [33]. Additionally, this analysis can be useful in finding out the fingerprints of extracts where labdanes exist, using GC or/and GC-MS, for standardizing plant extracts, which are used in phytotherapy. Anastasaki et aL, [33] have studied some labdanes isolated from Cistus creticus. The results obtained from GC and GC-MS analyses of these labdanes have contributed to a better understanting of the chemotaxonomic relationship between the two selected Cistus subspecies, le C. creticus subsp. creticus and C.creticus subsp. eriocephalus. [33]. 5. BIOLOGICAL ACTIVITIES OF LABDANES A variety of biological activities have been associated with labdane diterpenes including antibacterial, antifungal, antiprotozoal, enzyme induction, anti-inflammatory modulation of immune cell functions, as well as cytotoxic and cytostatic effects against human leukemic cell lines. The leaves of Cryptomeria japonica (Taxodiaceae) are traditionally used in Japan for the treatment of eczema. In Tibet the roots of Phlomis younghushbandii and of P, medicinalis (Labiateae) are used as an antifebrile and as a cough medicine. In East Africa the twigs of the shrub Premna oligotricha (Verbenaceae) are used as chewing sticks against the Gram positive bacteria responsible for dental caries {Streptococcus sp., Lactobacillus sp.), while the smoke formed by burning the plant is used to sterilise milk containers. On the island of Crete (Greece), Cistus creticus (Cistaceae) is used as a dermis malady, in arthritis and stomachache. Antifungal activity has also been shown by labdanes isolated from the seeds of Alpinia galanga and Aframomum daniellii (Zingiberaceae). Labdanes from Cistus creticus and from its resin 'Ladano', from Viburnum suspensum (Caprifoliaceae), Juniperus procera (Cupressaceae), Premna oligotricha (Verbenaceae) and from the sponge belonging to the genus Mycale, also exhibit strong antimicrobial activity. In 1998 the antiinflammatory activity of a product isolated from Cryptomeria japonica (Taxodiaceae) with labdane skeleton, was reported [35].

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5.1. ANTIBACTERIAL, ANTIFUNGAL AND ANTIPROTOZOAL EFFECTS Although an initial study by Soederberg et ai failed to show any activity against Staphylococcus aureus, subsequent studies have established significant antibacterial, antifungal and antiprotozoal activity for many labdane type diterpenes, especially for the labdanes of the manoyl oxide series [160]. In the study mentioned above, manoyl oxide acid and pinifolic acid, two resin acids isolated from Scots pine needles, were tested against 20 strains ofS. aureus using the disc diffusion method, but no significant activity was observed. Two other diterpenes of the manoyl oxide series, epigomeric acid and gomeric acid, tested by Darias et aL [27] against three Gram (+) bacteria {Bacilus subtilis. Micrococcus luteus and S. aureus) and two Gram (-) {Escherichia coli and Pseudomonas aeruginosa) exhibited a remarkable activity against the Gram (+) bacteria. As mentioned elsewhere in this text, the Cistacae is a family of plants from which a significant number of labdane diterpenes have been isolated. From the dried leaves of the Cistus incanus subsp creticus seven labdane type diterpenes have been isolated [44]. All the compounds were tested for antibacterial activity against S. aureus, S. epidermidis [Gram (+) bacteria] and P. aeruginosae, Enterobacter cloacae, Klebsiela pneumoniae and E, coli [Gram (-) bacteria] as well as for their antifungal activity against Candida albicans, Torulopsis glabrata and the opportunistic infectious fungus Saccharomyces cerevisiae [123]. These studies were carried out using the disc diffusion method, and with the exception of one diterpene, which was completely inactive, the other diterpenes showed significant activity against S. aureus, P. aeruginosae and K. pneumoniae. They exhibited zones of inhibition comparable to netilmicin, ceftazidine and cefriaxon, while they were more toxic than ampicillin. One of them {[(5R, 8R, 9R, 10R)-labdan-13 (E)-ene-8a, 15diol]} was also found to be active against C albicans [123]. Antibacterial activity against the Gram (+) bacteria S. aureus, S. epidermidis, B. subtilis, S. hominis and M luteus, and against C. albicans was also exhibited by the essential oil of the same subspecies, extracted either from the leaves or from the resin of the plant [63,161]. Analysis of the composition of these oils revealed that manoyl oxide isomers were the

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main compounds. However, no activity against the Gram (-) bacteria, (E. coli and P. aeruginosae), tested in the same studies was observed. In a subsequent study [162], it was reported that the essential oil obtained from the resin of another subspecies, Cistus creticus subsp. eriocephalus, where manoyl oxide isomers also predominated, exhibited significant antibacterial activity against four Gram (+) bacteria (S. aureus^ S. epidermidis, Str.faecilis, B. cereus and B, subtilis). Two Gram (-) bacteria {E. coli, P. aeruginosae) also tested in that study were found to be resistant. Furthermore the oil was active against C albicans. Manoyl oxide was also reported to be the main compound in the essential oil of Helichrysum rupestre [163], which exhibited remarkable antibacterial activity against S. aureus but moderate activity against the Gram (-) strains, P. aeruginosae, E. cloacae, K. pneumoniae and E. coli. Taking into account the above studies, it may be concluded that the manoyl oxide isomers exhibit major antibacterial activity, while a specificity of these labdane diterpenes against Gram (+) is also highlighted. Two other labdanes tested for antibacterial activity are sclareol and manool. They were reported by Ulubulen et aL to be active against S. aureus in a study performed using the disc diffusion method as well as the tube dilution test, exhibiting a minimum inhibiting concentration of 48.25 and 13.75 |ig/ml, respectively. These two compounds were, however completely inactive against C albicans and Proteus mirabilis [164]. Sclareol was tested and was found to exhibit antibacterial activity against S. aureus, P. aeruginosae and K, pneumoniae [123] while it also seemed to control well rust fungi on different kinds of bean [165]. A series of other labdane diterpenes isolated from various sources have also been reported to exhibit antimicrobial activity. Aulacocarpinolide and aulacocarpines A and B, labdane diterpenes isolated from the Cameroonian spice Aframonum aulacocarpos (Zingiberaceae), exhibited weak antibacterial activity against B. subtilis and fungus Mucur miehei [166]. Labdanes of the series of gomojosidae, isolated from the leaves of Viburnum suspensum (Caprifoliaceae) exhibited antibacterial activity against E.coli, Aeromonas salmonishida and B. subtillis [102]. Cryptotrienolic and isocupressic acid isolated from the bark of Juniperus procera were found to exhibit weak antibacterial activity when tested against B. subtillis, S. aureus, Str. durans, E. faecilis, and Mycobacterium

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itracellular [167]. Moderate antifungal activity was also attributed to labdane diterpenes from the seeds of Alpinia galanga (Zingiberaceae) [168] and the perennial herb Aframonum daniellii (Zingiberaceae) [169]. In addition labdane diterpenoids exhibiting antimicrobial activity have been isolated from aquatic environments. Mycaperoxides isolated from the Thai sponge [170] and a sulphated sesterterpene, hydroquinone halisulphate from the dark brown sponge (Halichondriidae) [171] were found to exhibit antibacterial and antifungal activity. Also a new furanoid labdane diterpene isolated from the ethanolic extract of Potomogeton nodosus (Potamogetonaceae) was reported to exhibit moderate inhibitory activity against Gram (-) and Gram (+) bacteria, such as B, subtillis, S. aureus, Str. faecilis, B. cereus, Shigella boydii, S. sonnei and S. shiga [172]. Some investigators have reported not only antibacterial and/or antifungal activity, but also a significant antiprotozoal activity. Ent 13epi'kQio manoyl oxides, as well as their derivatives produced through biotransformation from the fungus Curvularia lunata [144, 173], exhibited significant activity against the pathogenic protozoa Leishmania donovani. Biological activity against the same promastigote was also reported for another diterpene [(4S, 9R, lOR) methyl 18-carboxy-labda8, 13 (E)-diene-15-oate] isolated from the stem barks of Polyalthia macropoda (Annonaceae) [ 174]. 5.2. EFFECTS OF LABDANES ON MAMMALIAN

ENZYME

SYSTEMS 5.2.1 ADENYLATE CYCLASES The adenylate cyclases (AC) are a family of enzymes, which catalyze the synthesis of cyclic AMP (cAMP), from ATP. Cyclic AMP, a ubiquitous molecule in mammalian cells, plays a key role in controlling a vast number of biological processes, functioning as a major second messenger. The ACs are present in bacteria, where c-AMP plays a key role in the regulation of transcription in fungi, parasites and mammalian cells. The mammalians ACs (at least nine enzymes) are structurally unrelated to the bacterial ones consisting of 12 transmembrane helices and two cytoplasmic catalytic domains. They differ from each other in their

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activation or inhibition by Ca^V calmodulin, phosphorylation by protein kinases etc [175]. Among the labdane type diterpenes, forskolin, the manoyl oxide which is isolated from the roots of the herb Coleus forskohlii, exhibits a unique biochemical property. As Seamon and Daly [176,177] first demonstrated, this manoyl oxide can activate adenylate cyclase by interacting directly with the catalytic subunit or a closely associated protein of the adenylate cyclase system. The latest findings suggest that the forskolin regulatory site is located near the catalytic site at the dimer interface of the catalytic subunits, in a single deep cleft [175], Fig (9). Forskolin may act by promoting the catalytically optimum juxtaposition of the two domains, by forming a hydrogen bond between the oxygen of the first hydroxyl group and the first carbon atom of an aspartate. The most important difference between the catalytic and the forskolin binding sites, which otherwise are very similar, is the replacement of aspartate at the catalytic site and of serine at the forscolin-binding site. The forskolin-binding site is sterically closed and covers most of the solvent-accessible surface area of forskolin. The uniqueness of the mechanism of its action, marked out forskolin as an invaluable tool for the investigation of the role of c-AMP in biological processes. This manoyl oxide derivative is now used widely in most studies related to the role of c-AMP in cellular machinery as a powerful adenylate cyclase activator. Apart from forskolin, a number of other manoyl oxides have been shown to interact with the AC enzyme system. Biotransformation of certain e«r-13-£77/-manoyl oxides by Curvularia lunata resulted in compounds fiinctionalized in C-3 or in C-3 and C-12, which exhibited an AC stimulatory effect, although milder than that of forskolin (about 30 times less) [173]. The same activity was also ascribed to some synthetic derivatives of ^wr-8a-hydroxy-13 (16), 14 dien-18-oic acid methyl ester [178,179]. The biotransformation of ^^/-manoyl oxide-16-hydroxy 18-oic acid methyl ester with Rhizopus nigricans, however, resulted in carbomanoyl oxide which showed a selective inhibitory action on the activity of adenylate cyclase depending on the material initially used to stimulate the enzyme. This manoyl oxide inhibited the activity of the enzyme previously stimulated by forskolin but not by glucagon. A manoyl oxide {ent-^P, 6)ff-dihydroxy-13-e/7/-manoyl oxide) which also inhibited the activity of AC, was produced from the biotransformation of

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Forskolin

\«A^-Ay'

VwAÂy'

V-A.-AY'

Transmembrane helices CI domain

VWA^>V-/

es

. V«A-AY'

V«/yA«/

Membrane

C2 domain

Fig. (9). A) Schematical representation of the activation of intact adenylate cyclase (AC) b forskolin and Gsa (GTP- bound stimulatory G protein a subunit). Mammalian AGs consists o 12 transmembrane helices and two cytoplasmic catalytic domains (referred to as Ci and C2 represented as lightly shaded and black respectively), (a) Hypothetical basal state, (b) Th suggested forskolin-activated state, (c) Forskolin and GSa-activated state. B) Forskolin-binding site (f) in C1-C2 heterodimer. With S is represented the serine pai (Ser891-942), with which the Oi hydroxy 1 of forskolin makes hydrogen bond. (Modified from Ciirr. Opin. Struct. Biology, 1998, 8, 770. With the kind permission of J.H Harley).

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e«^3yff-hydroxy-13-e'p/-manoyl oxide, by Curvularia lunata [147]. The above studies show a clear structure-activity relationship, where the type and/or the position of substitutes may lead to compounds with either stimulatory or inhibitory activity, ahhough the pharmacophore part of the labdane diterpenes, concerning AC related activity, has not really been identified. S22. B-GLUCURONIDASE B-glucuronidase catalyzes the hydrolysis of various )ff-D-glucuronides, liberating free glucuronic acid. Extracts from Scoparia dulcis, a perennial herb of tropical and subtropical regions, have been reported to significantly inhibit the activity of y5-glucuronidase [180]. Further fractionation of the extract led to the isolation of three labdane type diterpenes, namely scoparic acids A, B and C.The most potent inhibitory diterpene was scoparic acid A showing an IC50 of 6.8x10"^ M (4 times less than that of glucosaccharo-1, 4-lactone, a well known yff-glucuronidase inhibitor) [181]. 5.2.3. PHOSPHOLIPASE A^ Phospholipase A2 (PL A2) catalyzes the hydrolysis of phospholipids esterified at the second carbon in the glycerol backbone. Arachidonic acid is commonly esterified in this position and the action of PLA2 releases arachidonic acid for subsequent metabolism via the cyclooxygenase and lipoxygenase pathways. Halisulphate isolated from the marine dark brown sponge (Halichondriidae) exhibits a 100% inhibition of PLA2at 16|j,g/ml [171]. Another natural labdane diterpene isolated from the Spanish herb Sideritis javalambrensis, ent-8 alpha-hydroxy-lambda-13 (16), 14-dien, was also found to inhibit non-pancreatic secretory phospholipase A2 [182] and human secretory synovial PLA2 at a concentration of 10'^ M [183]. 5.2.4. ALDOSE REDUCTASE Aldose reductase has been implicated in the pathogenesis of cataract in diabetic and galactosaemic animals. The enzyme catalyzes the reduction

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of glucose and galactose to their polyols, which then accumulate in large quantities in the lenses and ultimately lead to mature lens opacities. Preliminary investigations have shown that some metabolites isolated from the marine sponge Dysidea sp. could inhibit the activity of the enzyme aldose reductase [184].

5.2.5. PROTEIN KINASES Although originally discovered as an activator of phosphorylase kinase, it soon became apparent that protein kinase A (PKA) had a much wider role in eucaryotic metabolism. PKA is a c-AMP dependent enzyme, consisting of two regulatory (R) and two catalytic subunits (C), which dissociate when each of the R subunits binds two molecules of c-AMP. Agonistic c-AMP analogs, such as forskolin (and most probably other entmanoyl oxides, reported to affect c-AMP production), can activate PKA by increasing c-AMP levels and interfering in the modulation of the cAMP/PKA pathway [185]. Apart from PKA, some other protein-kinases were found to be controlled by forskolin, such as cytosolic sphingosine kinase in rat periosteal cells [186] and protein kinase B (PKB) [187]. The latter was found to be stimulated by the activation of PKA through a PI3 (phosphatidylinositol 3)-kinase-independent pathway. Furthermore, a distinct activation mechanism was suspected, other than that normally observed by growth factors such as insulin, since substitution of the serine at the S473 position of PKB with alanine could not prevent activation by forskolin. The JAK family of protein kinases in T lymphocytes can also be regulated by forskolin through the activation of PKA [188]. Thus it seems obvious that many other enzymes could be susceptible to control by forskolin. 5.3. ENDOCRINE EFFECTS OF LABDANES As mentioned earlier, the adenylate cyclase system is hormone sensitive and many hormones are capable of regulating the enzymes involved in either a stimulatory or an inhibitory manner, thus modulating

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the levels of intracellular c-AMP and eliciting the appropriate physiological responses. Forskolin has been shown to activate almost all hormone-sensitive adenylate cyclases in intact cells, tissues, or solubilized preparations of adenylate cyclase, with Effective Concentration (EC50) values between 5 and 15 |aM [175,189,190]. Low concentrations of forskolin ( 2 M

.C\Anal Biochem, 1985, 150, 76 - 85. [33] Lowry, O.H.; Rosebrough, N.J.; Fan, A.L.; Randall, R.J.; J. Biol Chem, 1951,193, 265 - 275. [34] Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, RA.; Smith, V:,Anal Chem, 1956, 28, 350 - 356. [35] Blumenkranz, N.; Asboe-Hansen, G.; Anal Biochem, 1973, 54, 484 - 489. [36] Dodgson, K.S.; Price, R.G.; Biochem. J, 1962, 84, 106 -110. [37] BarUett, G.R.; J. Biol Chem, 1959, 234, 466 - 468. [38] Doss, M.; Oette, K.; T. Klin Chem, 1965, 3, 125 -129. [39] Suzuki, J.; Kondo, A.; Kato, I.; Ikenaka, T.',Agric. Biol Chem, 1991,55,283 - 284. [40] Heinrikson, R.L.; Meredith, S.C.; Anal Biochem, 1984, 136, 65 - 74. [41] Hakomori, S.; J. Biochem, 1964, 55, 205 - 208. [42] Mort, A.; Parker, S., Mao, S.K.; Anal Biochem, 1983,133, 380 - 384. [43] Sauhiier, L.; Brillouet, J.M.; Moutounet, M.; Penhoat, C.H.; Michon, V; Carbohydr. Res,l991, 224,219'1?>5. [44] Tanaka, K.; Yamada, A.; Noda, K.; Hasegawa, T; Okuda, M.; Shoyama, Y.; Nomoto, K.; Cancer Immunol Immunother, 1998, ^5, 313 - 320. [45] Wexler, H.; J. Natl Cancer Inst, 1966, 36, 641 - 645. [46] Gomi, K.; Morimoto, M.; Nomoto, K,; Cancer Res, 1982, 42, 4197 - 4202. [47] Old, L.J.; Boyse, E.A.; Clarke, D.A.; Carswell, EA:,Ann NY Acad. Sci, 1962,101, 80-106. [48] Mitani, M.; Mori, K.; Himeno, K.; Nomoto, K.; J. Immunol, 1989,142,2148 - 2154. [49] Konishi, F; Mitsuyama, M.; Okuda, M.; Tanaka, K.; Hasegawa, T.; Nomoto, K.; Cancer Immunol Immunother, 1996, 42, 268 - 274. [50] SteflFen, EK.; Berg, R.D.; Infect Immun, 1983, 39, 1252 -1259. [51] Nomoto, K.; Yokokura, T.; Yoshikai, Y; Mitsuyama, M.; Nomoto, K.; Can. J. Microbiol, 1991, 37, 244 - 247. [52] Beuscher, N.; Scheit, K.H.; Bodinet, C; Kopanski, L.; PlantaMed, 1989, 55, 358 363. [53] Kawasaki, S.; Plant Cell Physiol, 1987, 28, 187 - 197. [54] Okamoto, H.; Minami, M.; Shoin, S.; Koshimura, S.; Shimizu, R.; Jpn. J. Exp. Med, 1966, 36, 175 - 186. [55] Tsukagoshi, S.; Hashimoto, Y; Fujii, G.; Kobayashi, H.; Nomoto, K.; Orita, K.; Cancer Treat. Rev, 1984,11, 131 - 155. [56] Chihara, G.; Hamuro, J.; Maeda, Y; Arai, Y; Fukuoka, F; Cancer Res, 1970, 30, 2776-2781. [57] Kikumoto, S.; Miyajima, T; Yoshizumi, S.; Fujimoto, S.; Kimura, K.; Nippon Nogeikagaku Kaishi, 1970, 44, 337 - 342. [58] Hirose, S.; Nagai, S.; Akatsu, T; Kobayashi, A.; Oohara, M.; Yakugaku-Zasshi, 1970, 96,419-424. [59] Kita, E.; Emoto, M.; Oku, D.; Hamuro, A.; Nishijima, F; Tanikawa, I.; Yasui, K.;

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Katsui, N.; Kashiba, S.; Nat. Immun. Cell Growth Regul, 1990, P, 387 - 396. [60] Kita, E.; Kamikaidou, N.; Oku, D.; Nakano, A.; Katsui, N.; Kashiba, S.; Nat. Immun. Cell Growth Regul, 1992,11, 46 - 55. [61] Wright, A.; Morrison, S.L.; J. Exp. Med, 1994,180, 1087 - 1096. [62] Nakamura, S.; Takasaki, H.; Kobayashi, K.; Kato, A.; J. Biol. Chem, 1993, 268, 12706-12712. [63] Ueda, T.; Iwashita, H.; Hashimoto, Y.; Imoto, T.; J. Biochem, 1996,119,157 -161. [64] Kieliszewski, M.J.; Lamport, D.T.; Plant J, 1994, 5, 157 -172. [65] Ryoyama, K.; Cancer Immunol. Immunother, 1992, 55, 7 - 13. [66] Hock, H.; Dorsch, M.; Kimzendorf, U.; Uberia, K.; Qin, Z.; Diamantstein, T.; Blankenstein, T.; Cancer Res, 1993, 53, 714 - 716. [67] Kuramoto, E.; Yano, O.; Shimada, S.; Cancer Immunol. Immunother, 1992,34,283 288. [68] Chai, J.G.; Bando, T; Kobashi, S.; Oka, M.; Nagasawa, H.; Nakai, S.; Maeda, K.; Himeno, K.; Sato, M.; Ohkubo, S.; Cancer Immunol. Immunother, 1992, 35, 181 185. [69] Matsumoto, T.; Himeno, K.; Mitani, M.; Mori, K.; Miake, S.; Nomoto, K.; J. Clin. Lab. Immunol, 1986,19, 83 - 89. [70] Mitani, M.; Mori, K.; Himeno, K.; Matsumoto, T.; Taniguchi, K.; Nomoto, K.; Cell. Immunol, 1985, 92, 22 - 30. [71] Kurata, S.; Tsuchiya, T.; Norimura, T; Yamashita, U.; J. Immunol, 1983,130, 496 500. [72] Nelson, M.; Nelson, D.S.; McKenzie, I.F.; Blanden, R.V.; Cell. Immunol, 1981, 60, 34 - 42. [73] Sinclair, N.R.; McFariane, D.L.; Low, J.M.; Cell. Inmiunol, 1981, 59, 330 - 344. [74] Tanida, S.; Uchida, H.; Taniguchi, K.; Nomoto, K.; Cancer Res, 1989,49, 284 - 288. [75] Dyall, R.; Nikolic-Zugic, J.; J. Exp. Med, 1995,181, 235 - 245. [76] Migita, K.; Eguchi, K.; Kawabe, Y; Tsukada, T.; Mizokami, A.; Nagataki, S.; J. Clin. Invest, 1995, 96, 727 - 732. [77] Bensinger, W.; Singer, J.; Appelbaum, R; Lilleby, K.; Longin, K.; Rowley, S.; Clarke, E.; Clift, R; Hansen, J.; Shields, T; Stoib, R; Weaver, C; Weiden, R; Buckner, CD.; Blood, 1993, 81, 3158 - 3163. [78] de Revel, T; Appelbaum, F.R; Stoib, R.; Schuening, R; Nash, R.; Deeg, J.; McNiece, I.; Andrews, R.; Graham, T.; Blood, 1994, 83, 3795 - 3799. [79] Hodgson, G.S.; Bradley, T.R.; Nature, 1979, 281, 381 -382.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 25 © 2001 Elsevier Science B.V. All rights reserved.

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HEPATOPROTECTIVE EFFECT OF PLANT COMPONENTS: INHIBITION OF TUMOR NECROSIS FACTOR-a-DEPENDENT INFLAMMATORY LIVER INJURY KOJI HASEj QUANBO XIONG,' SHIGETOSHI KADOTA *^ Înstitutefor Consumer Healthcare, Yamanouchi Pharmaceutical Co., Ltd, Tokyo 174-8612, Japan; andÎnstituteof Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan ABSTRACT: There is evidence to suggest that the upregulation of serum tumor necrosis factor (TNF-a) and the emergence of hepatocyte apoptosis are involved in the pathogenesis of human alcoholic and viral hepatitis. In experimental liver injuries induced by D-Galactosamine (D-GalN)/lipopolysaccharide (LPS), D-GalN/TNF-a or Propionibacterium acneslL?S, TNF-a induces hepatocyte apoptosis which triggers an inflammatory reaction and subsequent massive hepatocyte necrosis, playing a central role in the pathological process. These models provide a promising experimental basis not only for understanding the pathophysiological mechanisms of various hepatic disorders but also for evaluating the hepatoprotective efficacy of natural products. A diverse array of plant-derived compounds including saponins, polyphenols, iridoids and alkaloids have been reported to have a hepatoprotective effect in the TNF-adependent inflammatory liver injury models. Some of these compounds impede TNF-amediated hepatocyte apoptosis and consequently block the progression of liver injury, whereas others protect against hepatocyte necrosis occurring at the final stage. The protection against apoptosis by hepatoprotective compounds can be explained by the inhibition of TNF-a production from macrophages or by attenuation of the hepatotoxic fimction of TNF-a. This review discusses the recent progress in TNF-a-dependent liver injury models and the hepatoprotective action of plant constituents in such models.

INTRODUCTION A number of medicinal plants including Glycyrrhiza glabra, Silybum marianum, Picrorrhiza kurroa and Artemisia capillaris have been traditionally used for the treatment of hepatitis. These plants contain various compounds with unique structures such as alkaloids, terpenoids and polyphenols. To study the hepatoprotective phytoconstituents of medicinal plants is of value to provide a scientific basis for the traditional use. Additionally, the active constituents could be used as pharmacological tools for the study of the mechanisms underlying liver disease or as lead compounds for the development of new drugs for hepatitis. In fact, * To whom correspondance should be addressed. Fax: 81-76-434-5059. E-mail: [email protected]

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glycyrrhizin isolated from G. glabra, silymarin from S. marianum and picroliv from P. kurrooa have been developed as hepatoprotective drugs for viral chronic hepatitis and alcoholic hepatitis in Asia and Europe. Recent progress in the study of medicinal plants for treating liver diseases has resulted in the isolation of about 170 different phytoconstituents from 55 plant families [1]. In previous evaluations of hepatoprotective activity, direct hepatotoxins such as carbon tetrachloride (CCI4), bromobenzene, acetoaminophen or D-galactosamine (D-GalN) were used to induce e)q)erimental liver injury. These chemically induced liver injuries result from plasma membrane perturbation due to the generation of cellular radicals or the impairment of membrane component synthesis [2-4]. Consequently, too many phytoconstituents with antioxidant or membrane-stabilizing activity tended to exhibit hepatoprotective activity [5-10]. However, the majority of the cause of human hepatitis are triggered by immunological responses to viral infection, endotoxin or autoantigen [11-12]. Thus, liver injury models induced by direct hepatotoxins are considered to reflect only a limited aspect of human hepatitis. The evaluation for hepatoprotective activity of phytoconstituents in such models might produce e>q)erimental results inconsistent with those in the clinical situation. Therefore, new models related to immunological reactions have been recently proposed, namely, liver injuries induced by lip op oly saccharide (LPS), tumor necrosis factor-a (TNF-a) or concanavalin A (Con A) in intact or D-GalN-sensitized mice [13-14]. Recent progress in these models has revealed that TNF-a secreted from LPS-stimulated macrophages is a strong inducer of hepatocyte apoptosis, which triêrs an inflammatory reaction and massive hepatocyte necrosis [15-16]. The hepatic lesions induced in these models resemble those of human hepatitis, because upregulations of serum TNF- a concentration and hepatocyte apoptosis have been repeatedly reported as pathogenic symptoms in human hepatitis [17-22]. The TNF-a-dependent inflammatory liver injury models may therefore provide a more promising e)q)erimental basis for the evaluation of hepatoprotective agents. The first part of this review deals with the recent findings in the TNF-a-dependent liver injury models. The second part discusses the effect of plant-derived hepatoprotective agents on inflammatory liver injury and their pharmacological mechanisms. 1. TNF-a-dependent liver injury models TNF-a was originally recognized for its oncolytic effects on solid tumors, and subsequent investigations have demonstrated a pleiotropic effect mediating both acute and chronic inflammatory disorders. Several e>q)erimental liver injury models including D-GalN/LPS-, D-GalN/TNF-a-, Propionibacterium acnesILV?>', and concanavalin A (Con A) models are

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induced by host-immune responses including the activation of macrophages and T lymphocytes. A central role for TNF-a in these models has been reported in studies using neutralizing anti-TNF-a antibodies or knockout mice for TNF-a itself or receptors [23-27]. TNFa triggers hepatocyte apoptosis as well as necrosis, and induces chemoattractants which are essential for lymphocyte infiltration into liver [28-29]. This chapter deals with proceedings on TNF-a-dependent liver injury models. D-GaiN/LPS and D-GalN/TNF-a models: Administration of endotoxin (lipopolysaccharide; LPS) into different animals has been reported to cause multiple organ failure, resulting in lethal shock [30]. The liver is one of the main target organs for LPS toxicity. Numerous animal models have been developed to study the effects of LPS on liver injury. The simplest of these is bolus intravenous or intraperitonal injection of hi^-dose (ca. 1-40 mg/kg in rodents) of LPS [31-33]. However, this model may not be suitable for the evaluation of hepatoprotective efficacy, because LPS has a wide range of physiological activities and exerts unspecific deleterious effects on extrahepatic org^s such as the circulatory system, kidneys and lungs as well as liver. When other hepatotoxins are administered along with LPS, a synergic increase in liver injury occurs. For instance, co-injection of D-GalN (300-700 mglcg) and a subtoxic dose (1-100 /ig^g) of LPS into mice causes severe hepatitis [34]. In this model, liver injury is induced at 8 hr after intoxication without affecting other parts of the animal [35]. In contrast, a single injection of the above dose of D-GalN or LPS hardly affects mice. LPS is known as a strong stimulator that induces macrophages to secrete proinflammatory cytokines. Among the secretions, tumor necrosis factor-a (TNF-a) is thought to play predominant roles in liver injury and lethal shock induced by LPS, because administration of an anti-TNF-a antibody or a down-regulator of TNF-a production such as pentoxifylline abolish deleterious effects of LPS [23,24,30,36]. Although 10 | i ^ g of LPS is enough to induce TNF- a release, liver injury dose not occur in mice. This may be due to the fact that TNF-a triggers two distinct biochemical pathways; one that leads to apoptotic cell death in hepatocytes and another that leads to the induction of hepatoprotective proteins, probably nitric oxide synthase (NOS) and acute phase proteins [37-41]. D-GalN is converted to UDP-galactosamine (UDP-GalN) in hepatocytes, which depletes hepatocellular uridine phosphate such as UDP and UTP [42]. Depletion of uridine phosphate leads to inhibition of protein synthesis in hepatocytes, resulting in the suppression of the endogenous hepatoprotective proteins. Therefore, the susceptibility of animals to LPS hepatotoxicity increases more than 1000 times with the sensitization by D-GalN [34]. A similar sensitizing effect is seen when transcriptional inhibitors such as Actinomycin D (Act D) are administered with LPS or TNF-a [43]. The D-GalN/LPS-induced liver injury model has been

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frequently used to evaluate hepatoprotective agents [35,44,45]. Apoptotic symptoms such as DNA fragmentation and apoptotic bodies are observed in the livers of mice intoxicated with D-GalN/LPS or D-GalN/TNF-a [16]. A time course study on the pathological changes showed that hepatic fragmented DNA amount and serum ALT activity increase at 5 and 8 hr after intoxication, respectively. This means that hepatocyte apoptosis is induced at the early stage of the liver injury process and precedes hepatocyte necrosis [16]. Apoptosis has been recognized as a silent form of cell death under physiological conditions [46]. However, it appears that in some forms of liver disease, hepatocyte apoptosis actually triggers inflammation. Leist etal [16] postulated that in the D-GalN/LPS model, hepatocyte apoptosis may act as an initial step of liver damage, and subsequently, neutrophils are attracted by dying hepatocytes that are not removed swiftly enough under this pathological condition. Thus, neutrophils are thought to infiltrate liver tissue and to cause massive hepatocyte necrosis at the late stage. This concept was proved recently by a study with the caspase inhibitor Z-VAD, which allowed the selective blockage of apoptosis. Z-VAD treatment not only prevented caspase activation and apoptosis but also suppressed neutrophil transmigration and hepatocyte necrosis [15]. This indicates that a large number of hepatocytes undergoing apoptosis can represent a stimuli for primed neutrophils in sinusoids to transmigrate and activate, leading to massive hepatocyte necrosis. Therefore, prevention of apoptosis may be a new therapeutic approach to modulating inflammation and liver injury, although it remains to be elucidated how apoptosis signals inflammation [47,48]. Propionibacterium acnes/LPS model: Thehepatotoxicity of LPS and TNF-a can be enhanced by priming agents. It has been reported that severe hepatitis is induced by priming rodents with heat killed gramnegative bacteria namely Propionibacterium acnes or bacillus CalmetteGuerin (BCG) (ca. 1 mg^mouse), followed by injecting the mice with a small dose (ca. 1 /xg^mouse) of LPS after an interval of 7days [49,50]. The initial injection of BCG or P. acnes recruits mononuclear cells (MNCs) from the circulating system into the liver, which is prerequisite for the induction of LPS hepatotoxicity. Under this condition, the injection of a normally innocuous amount of LPS activates the infiltrating MNCs to release a huge amount of TNF-a, resulting in severe liver injury [27]. Plasma TNF-a activity rose sharply and reached a maximal level at 1 hr after LPS challenge, and then declined to near zero at 3 hr. Plasma ALT activity increased gradually from 4-24 hr, and more than 50% of mice died within 24 hr after LPS challenge. This liver lesion pathologically mimics fulminant hepatitis in humans, especially that associated with septic shock. This model has been one of the most popular animal models for the evaluation of hepatoprotective agents. The essential role of TNF-a in this model has been demonstrated by the observations that passive

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immunization against TNF-a or down-regulators of TNF-a such as PGE2 and dexamethasone protect mice from lethality and liver injury [27]. Liver damage in this model is histologically characterized by hepatocellular loss due to apoptosis and necrosis. Tsuji etal [51] recently reported that TNF receptor p55-deficient mice (TNFRp55"^") resist LPS-elicited hepatocyte apoptosis and liver injury as well as P. acwe^'-induced mononuclear cell infiltration, suggesting important roles forTNFRp55 in these pathological events. It should also be mentioned that Fas, which is a member of the nerve growth factor/TNF receptor superfamily, may contribute to induction of hepatocyte apoptosis in this model. The extent of P, acwe^'/LPS-induced hepatocyte apoptosis and liver injury is reported to be moderate in Fas-deficient Lpr/Lpr mice compared with wild mice. Fasseems to induce apoptosis together with TNFRp55 but through different pathways [51]. Alcohol and D-GalN models: It appears that TNF-a somewhat contributes to the pathogenesis of alcohol- and D-GalN-induced liver injury. In these two models, passive immunization of TNF-a markedly reduces liver injury [52]. Besides, ethanol or D-GalN-induced liver injury is significantly attenuated when Kupffer cells, a main source of TNF-a production, are destroyed with GdCla [53,54]. Ethanol-induced liver injury is diminished when gram negative bacteria in gut microflora are reduced by treatment with antibiotics or lactobacillus [55,56]. It is therefore hypothesized that chronic ethanol feeding or D-GalN administration increases the permeability of the gut to endogenous bacteria, resulting in the increased translocation of endotoxin into the blood, which stimulates Kupffer cells to produce TNF-a [57,58]. This hypothesis may be of clinical importance because patients with alcoholic liver disease and cirrhosis frequently have endotoxemia and hypercytokinemia [20,59,60]. In addition, serum TNF-a levels in patients with alcoholic hepatitis correlate inversely with patient survival ratio [18,19]. Pharmacological intervention with prostaglandins and glucocorticoids, which decrease TNF-a production, has been reported as an effective therapy in clinical liver diseases [61,62]. Con A model: As described above, LPS-related liver injury models are dependent on the activation of hepatic macrophages. On the other hand, a number of reports have suggested the potential involvement of T lymphocytes in chronic viral hepatitis and primary biliary cirrhosis [63]. In the 1990's, animal models of activated T cell-mediated liver injury were developed. Intravenous injection of mice with concanavalinA (Con A) at a dose of more than 1.5 mg^g leads to T-cell activation, resulting in liver injury within 8 hr of intoxication [64]. Con A is a T cell mitogenic plant lectin with high affinity for the hepatic sinus. Accumulation of Con A in the liver increases infiltration of circulating lymphocytes into the hepatic sinus and subsequent local proliferation. The activated lymphocytes in the liver release large amounts of cytokines that are essential for the pathology

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of liver injury in this model [13,65]. Pharmacological intervention with immunosuppresive drugs such as FK506 and dexamethasone abolishes the Con A-induced liver injury. Furthermore, mouse strains lacking T cells such as severe combined immunodeficient (SCID) mice and athymic nude mice show resistance to Con A-hepatotoxicity [64,65]. These results clearly revealed a central role for T cells in the development of liver injury. And, CD4'^ helper T cells are likely to be effector cells because pretreatment of mice with monoclonal antibody against CD4'^ but not CD8^ cells protected against Con A [64]. However, it is interesting to note that the activated lymphocytes start to infiltrate the liver tissue 8 hr after ConA injection, when liver damage has already begun. On the other hand, serum concentrations of most cytokines rise to a maximal level before the lymphocytes infiltrate, supporting that an early increase in cytokines is essential to induce liver injury [65,66]. Among these cytokines, interferon-y (IFN-y) is thou^t to predominantly correlate with the pathology, because passive immunization with anti-IFN-y but not with anti-IL-1 or anti-IL-6 antibody inhibits liver injury in this model [13,67, 68]. In contrast, IL-6 and IL-10 actually protect against liver injury by negatively regulating the production of IFN-y, suggesting a counter effect of some cytokines in this model [65,69]. The involvement of TNF-a in this model has been a subject of debate. Passive immunization with antiTNF-a abolished liver injury [13,65], whereas TNF-a gene-deficient mice are just as sensitive to Con A-induced liver injury as wild-type mice [70]. The reason for such a discrepancy in the involvement of TNF-a in this model is still unclear at present. Either the dose of Con A or genetic background of the mice used in each e^qperiment might affect the results. Additionally, mechanisms whereby INF-y and TNF-a induce liver injury also remain to be elucidated. Several authors suggested that similar to the D-GalN/LPS model, hepatocyte apoptosis is induced at the early stage of Con A-induced liver injury, via the TNF-receptor or Fas-lig^nd dependent pathway [13,16,70-72]. However, it is unclear why TNF-a could induce hepatocyte apoptosis without transcriptional arrest by D-GalN or Act D. Hepatocyte apoptosis may be caused by the synergistic action of TNF-a with IFN-y. On the other hand, other authors reported that the main pathological feature of the hepatic injury in the Con A model was hepatocyte necrosis, and that hepatocyte apoptosis is not found until the later stage of liver injury [73]. They reported that Con A administration caused a marked intrasinusoidal hemostasis, which consisted of erythrocyte agglutination, lymphocyte sticking to endothelial cells and platelet aggregation, resulting in congestion of the liver and consequently hepatocyte damage. Co-treatment with anti-TNF-a and anti-IFN-y monoclonal antibodies completely protected mice from hemostasis and liver injury. This suggested that IFN-y and TNF-a may be required for the induction of intrasinusoidal hemostasis [73]. Furthermore, TNF-a is well known as a strong inducer of adhesion molecules to recruit circulating

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lymphocyte in various cells including endothelial cells [31]. This effect is suggested to help the development of Con A-induced liver injury [68]. Thus, it is plausible that IFN-y and TNF- a are prerequisite for this model, and these cytokines may be related to the recruitment of lymphocytes to the hepatic sinus or the induction of intrasinusoidal hemostasis rather than direct cytotoxicity to hepatocytes. Tabid. Inflammatory liver injury models predominantly mediated byTNF-a Trigger Sensitiser Animal species D-GaIN (300-700 mg/kg, i.p.) Propionibacterium acnes (0.7-1 mg/mouse,/.v.) Pb acetate (15 mg/kg, /. v.) D-GalN (16 mg/mouse, i.p.) D-GalN (700 mg/kg, i.p.)

Effector cells M(l)

Ref

LPS(l-40mg/kg,/./?or/.v.) LPS(l-100^g/kg,/.p.) LPS(1-25 |ig/mouse, /.v.)

Mice, Rats, Others Mice, Rats, Rabbits M(j) M(}) Mice

[30,311 [34,351 [27,501

LPS(100ng/kg,/.v.)

Mice, Rats, Chicks, M(|) Baboons Mice M(t), T cells M(|), T cells Mice

[74,751

[761 SEB (50-100 pg/mouse, i.p.) [771 Anti-CD3 mAb (10 îg/mouse, /.v.) Md), T cells Con A (7.5-3Q__mg/kg, /.v.) Mice [Ml D-GalN, D-galactosamine; LPS, lipoplysaccharide; SEB, Staphylococcal enterotoxin B; Con A, concanavalin A; mAb, monoclonal antibody

2. Natural products with hepatoprotective activity against TNF-adependent liver injury Several crude drugs have traditionally been used for the treatment of liver diseases. Their pharmacological and biochemical actions have been evaluated scientifically using various e>q)erimental liver injury models. Following the experimental confirmation of a hepatoprotective property in some plants, numerous active compounds have been purified and identified. There are several reviews dealing with various aspects of hepatoprotective natural products [1,78,79]. We and other investigators have tried to find new hepatoprotective agents from natural resources using the inflammatory liver injury models mentioned in the first chapter. As a result, a series of plant-derived compounds including flavonoids, tetrahydroxanthone, caffeic acid derivatives, phenylethanoids, iridoids and alkaloids have been shown to have hepatoprotective activity. This chapter updates proceedings on the hepatoprotective effect and pharmacological mechanisms of phytoconstituents in TNF-a-dependent inflammatory liver injury. Glycyrrhizin (1) is a hepatoprotective saponin which consists of an aglycone glycyrrhizic acid and two molecules of glucuronic acid. Glycyrrhizin is derived from the root of licorice {Glycyrrhiza glabra L.) and allied plants (Leguminosae). There are many reports that suggested various bioactivities, namely, anti-inflammatory, anti-viral and hepatoprotective, of glycyrrhizin [80-84]. It has been used extensively as a therapeautic drug for chronic active hepatitis in Japan for decades. The

466

hepatoprotective effect of gjycyrrhizin on chemically-induced liver injuries is well established. For instance, pretreatment with glycyrrhizin (200 mg/kg,/.v. or i.p.) markedly inhibited the elevation of serum ALT and AST levels and the development of pericentral hepatocyte necrosis in CCI4- or allyl formate-induced liver injury in rats [85]. An in vitro study using primary cultured rat hepatocytes demonstrated that this compound exerts direct hepatoprotective activity against CCI4- or D-GalNcytotoxicity. This effect was much stronger in the aglycone glycyrrhizinic acid than glycyrrhizin. Additionally, we recently found that the oral or intraperitoneal administration of glycyrrhizin (100 or 200 mglcg) significantly inhibited the elevation of serum ALT activity after intoxication with D-GalN and LPS in mice [86]. Sibayama [85] reported that the mortality of rats after intoxication with LPS markedly decreased when glycyrrhizin (200 mg^g) was administered intravenously, althou^ no significant difference was seen in serum transaminase levels. These results prove the efficacy of glycyrrhizin in TNF-a-dependent inflammatory liver injury. In our data, glycyrrhizin provided 61% protection against elevation of the serum ALT activity at 8 hr after intoxication with D-GalN and LPS, whereas it inhibited neither hepatic DNA fragmentation nor apoptotic body formation, both of which are indicators of apoptosis, at the 5 hr time point [87]. This suggests that the hepatoprotective effect of glycyrrhizin in this model may be due to protection from secondary hepatocyte necrosis triggered at the fmal stage rather than inhibition of inflammation, although an anti-inflammatory effect through adrenocorticosteroid-like action of glycyrrhizin was proposed previously [88,89]. It is assumed that the membrane-stabilizing effect of glycyrrhizin is involved in the protection against necrosis [84]. COOH

GlcA-GlcA-0

,. -,

Fig. (1). Glycyrrhizin isolated from Glycyrrhiza glabra

It was recently examined whether glycyrrhizin affects T-cell- or TNFa-mediated cytotoxicity in vitro [90]. Glycyrrhizin suppressed T-cellmediated cytotoxicity against antigen-presenting cells; however, the effective concentration was as high as 200 [ig/mL. Since the serum concentration of glycyrrhizin after intravenous injection at the clinical dose is reported to be 30-60 |Lig^mL at the maximal level [91], this compound is unlikely to suppress T-cell-mediatedcytotoxicity in vivo. On the other hand, glycyrrhizin suppressed TNF-a-mediated cytotoxicity

467

against TNF-a-sensitive L929 cells. A significant inhibitory effect is observed even at a concentration of 2 jLtg^mL. However, it should be noted that the effect was very moderate: less than 50% protection even at 200 ILig^mL, the highest concentration tested. Although it was considered that such inhibition of immune-medicated cytotoxicity is one of the mechanisms whereby glycyrrhizin suppresses elevated serum transaminases levels in patients with chronic viral infection [90], there remains room for further investigation on this point. Tetrahydroswertianolin (THS; 2) is one of the main hepatoprotective constituents of SwertiajaponicaMakino, a very popular folk medicine in Japan. THS possesses an unique structure characterized by a partially-saturated xanthone frame (Fig. 1). Dreiding model analysis along with an analysis of the J-value from ^H-NMR andNOE experiments suggested that the cy clohexene ring in THS is likely to be in distorted halfchair conformation with an equatorial 5-OH and axial 8-O-glucose [92]. Additionally, using Mosher's method, its absolute configuration was determined to be 5-(i?) and 8-(iS) (Fig. 2). As to its hepatoprotective activity, pretreatment with THS (25 and 50 mglcg, s.c.) before intoxication with D-GalN and LPS in mice significantly attenuated the serum ALT elevation by 68 and 84%, respectively. A significant inhibition (80 and 91%) of the serum ALT elevation was also observed with the oral administration of THS (20 and 200 mg/kg). The hepatoprotective effect of THS was comparable to that of glycyrrhizin used as a positive reference. To find the active center of THS, the hepatoprotective activity of aglycone (tetrahydrobellidifolin; 3) and its derivative (l-hydroxy-3methoxyxanthone; 4) was investigated. The hepatoprotective activity was preserved on tetrahydrobellidifolin, whereas l-hydroxy-3metho)^^xanthone did not show any activity. These observations suggest that the cyclohexene-ring moiety may be important for the hepatoprotective activity of THS [86]. OCH3

O R O^ OH 2 : R = Glc 3 :R = H

r^j^^^NÔv^^-'^jîYÔCHa

O

OH

4

Fig. (2). Tetrahydroswertianolin and its derivatives

Further study demonstrated that THS significantly inhibited hepatic DNA fragmentation, the emergence of apoptotic bodies and chromatin condensation in D-GalN/LPS-induced liver injury in mice. The extent of the inhibitory effect of THS on hepatocyte apoptosis at 5 hr paralleled that on serum ALT elevation at 8 hr [87]. As mentioned above, in this model, a large number of hepatocytes undergoing apoptotic cell death can

468

represent a stimulus for primed neutrophils in sinusoids to transmigrate and activate, leading to hepatic inflammation and massive hepatocyte necrosis [15,30,48,93]. Therefore, prevention ofapoptosis will protect to some extent ag^nst hepatocyte necrosis. Our data clearly showed that THS inhibited hepatocyte apoptosis at the early stage of the development of liver injury, indicating that the suppression of hepatocyte apoptosis is one of the mechanisms by which THS protects mice from liver injury. Since it is established that the induction of hepatocyte apoptosis in the DGalN/LPS model is mediated by TNF-a, the interaction of THS with TNF-a might be related to their hepatoprotective activities. In a separate study, we found that THS significantly prevented the serum TNF-a elevation and hepatic mRNA induction that occurred as an early pathological event after D-GalN/LPS-intoxication. The inhibitory effect (65 to 78%) of THS on serum TNF-a elevation was observed at a dose range (10-200 mglcg) similar to that observed with the inhibitory effect (65% at a dose of 50 mg/kg)on hepatic apoptosis. On the other hand, pretreatment with THS did not attenuate mrTNF-a-induced hepatic apoptosis significantly in D-GalN-sensitized mice [87]. These results suggest that protection by THS against hepatic apoptosis induced by D-GalN and LPS may be due predominantly to the inhibition of TNF-a production. TNF-a is responsible for not only induction of apoptosis but also the initial inflammatory response such as e>q)ression of adherent molecules and sequestration of neutrophils in sinusoids [15,31]. The inhibition of TNF-a by THS, therefore, may contribute to the suppression of the initial inflammatory response as well. Acteoside (verbascoside; 5) is aphenylethanoid widely distributed in medical plants such as Cistanche deserticola Y. C. Ma. Phenylethanoids are a group of water-soluble natural products and were reported to have various bioactivities. Their antioxidant activity in particular is well documented [94]. There have been several reports on the hepatoprotective effect of acteoside and some other phenylethanoids on CCI4- or D-GalNinduced cytotoxicity in rat primary culture hepatocytes [95,96]. We recently found that pretreatment with acteoside (10 or 50 mg^kg, s.c.) significantly inhibited the elevation of serum ALT, fragmentation of hepatic DNA and formation of apoptotic bodies in the D-GalN/LPSinduced liver injury model. Also, this compound improved the mortality of mice after intoxication [97]. A similar effect was observed when acteoside was administered orally at a dose range of 20 to 100 mglcg. These results clearly showed that acteoside is effective against the hepatocyte apoptosis as well as necrotic liver damage. Interestingly, acteoside had no apparent effect on the elevation of serum TNF-a, but it partially prevented mouse TNF-a (100 ng^mL)-induced cytotoxicity in D-GalN (0.5 niM)-sensitized mouse hepatocytes at a concentration of 50 |LiM or more. Thus, the protective effect of acteoside on hepatocyte apoptosis and subsequent liver injury could be explained by attenuation of

469

the cytotoxic function rather than the production of TNF-a. It can be speculated that this compound may interfere with apoptotic signals such as the activation of caspase-3 protease. The fruits of Hovenia dulcis Thunb. (Rhamnaceae) is a traditional Chinese medicine used as a dotoxify ing agent for alcoholic poisoning [98]. The accelerating effect on alcoholic metabolism of the H2O extract of H, dulcis has been confirmed by e}q)eriments using rodents and human beings [99-101], although the mechanisms and active component(s) remain to be clarified. Recently, the MeOH extract of this plant was shown to possess hepatoprotective activity against CCI4- or D-GalN/LPS-induced liver injuries in rats and mice [102,103]. The MeOH extract has a good deal of dUiydroflavonoids such as (+)-ampelopsin (dihydromyricetin; 6) and its 0-methylated derivative hovenin I (7). Pretreatment with ampelopsin (100 mg/kg, p,o,) and hovenin I (25 or 50 mg1q)eriment, pretreatment with ampelopsin (100 mg/kg, p.o.) significantly reduced the extent of hepatic DNA fragmentation, and prevented the emergence of hepatocytes with chromatin condensation and apoptotic bodies at the early stage of D-GalN/LPS-induced liver injury in mice. The elevation of the serum TNF-a concentration was also suppressed by pretreatment with ampelopsin, which did not inhibit TNFa-induced hepatotoxicity in D-GalN-sensitized mouse hepatocytes (unpublished data of the author's group). The inhibitory effect of TNF-a production was commonly observed on other flavonoids, dismosin, hesperidin, naringin and rutin [104]. Particularly, intraperitoneal administration of naringin (8) (1 mg/mouse) was reported to inhibit LPS-induced TNF-a production by 94% in mice. Also, naringin pretreatment (1 or 3 mg/^mouse, ip.) markedly inhibited hepatocyte apoptosis after intoxication with D-GalN and LPS, and consequently protected mice from liver injury [104]. Based on these results, inhibition of hepatocyte apoptosis via suppression of TNF-a production may be of prime importance to the hepatoprotective mechanisms of flavonoids such as ampelopsin and naringin. Curcumin (9) is an ary Iheptanoid isolated from turmeric, the rhizome of Curcuma longa L. Turmeric has been used as a remedy for inflammatory in Asia for centuries. There are a number of reports supporting an anti-inflammatory effect of curcumin. Its hepatoprotective activity against inflammatory liver injury was also demonstrated in a recent study [105], where pretreatment with curcumin (50 mglcg, p.o.) significantly inhibited the elevation of serum transaminase activities after intoxication with D-GalN and LPS in mice. The hepatoprotective effect is presumably

470

mediated by inhibiting TNF-a production, because an in vitro study demonstrated that curcumin (5 |iM) markedly inhibited LPS-induced production of TNF-a and IL-1 in a human monocytic macrophage cell Hne, Mono Mac 6 [106]. A gel mobility assay indicated that the inhibition of these cytokines by curcumin is based on down-regulation of nuclear factor kappa B ( N F - K B ) , a key transcriptional factor which regulates the e>q)ression of cytokine genes through stimulation of LPS. Curcumin possesses two polyphenolic and one diketone functional group and is well known to have antioxidativeproperties. SinceNF-KB is an oxidative stress sensitive transcription factor [107], the antioxidant property of curcumin may help to inhibit N F - K B activation [106]. (-)-Epigallocatechiii gallate (EGCG; 10) is a main compound of green tea (Camellia sinensis) polyphenols and accounts for more than 40% of all polyphenols. EGCG has various bioactivities including scavenging of 0}q)ression [108,109]. Furthermore, it was recently reported that oral administration of green tea polyphenols (100 or 500 mg/kg) significantly suppressed the elevation of serum TNF-a concentration and markedly improved rat mortality after intoxication with LPS (40 mg1q)ression throu^ suppression of N F - K B activation [33]. Similar to curcumin, the antioxidant property of tea polyphenols is believed to play an important role in the inhibition of N F - K B . This mechanism may be consistent with the mode of action of some other polyphenolic compounds that inhibit TNF-a production, such as THS and ampelopsin. HO^^^

p

OHQ

^,0^"

Rha-Glc-O,

OH O 8

9

Fig. (3). Polyphenolic compounds with hepatoprotective acltlvity against TNF-a-dependent liver Injury

471

Gentiopicroside (11) and sweroside (12) are bitter secoiridoid glycosides widely distributed in Swertia spp, and Gentiana spp, (Gentianaceae). Thehepatoprotective effect of these compounds has been suggested in chemical liver injury models induced by CCI4 or Cd [110,111]. Besides, we found that pretreatment with gentiopicroside and sweroside (each 25 or 50 mg/kgj.p,) moderately inhibited the elevation of serum ALT activity in inflammatory liver injury induced by D-GalN and LPS in mice [86]. This finding was in line with that reported by Kondo et al. [110]; pretreatment with gentiopicroside (30 or 60 mglcg, Lp.) significantly inhibited the increase in the serum transaminase activities in a dosedependent manner after LPS challenge in BCG-primed mice. The hepatoprotective activity against inflammatory liver injury may be explained at least in part by the suppression of TNF-a production, because gentiopicroside significantly suppressed the acute increase in the serum TNF- a activity in a dose-dependent manner in the BCG/LPS model [110].

11

12

Fig. (4). Iridoids with hepatoprotective acitivity against TNF-a-dependent liver injury

The methanol extract (200 mglcg, ip,) of roots of Angelica Jurcijuga Kitagawa (Umbelliferae) was reported to protect mice from inflammatory liver injury induced by D-GalN (350 mg/kg, ip,) and LPS (10 lig^g, /./7.). By phytochemical analysis, coumarines and poly acetylenes were isolated as hepatoprote ctive compounds. Intraperitoneal administration (12.5 or 25 mg/kg) of isoepoxypteryxin (13), anomalin (14), isopteryxin (15) and falcarindiol (16) was found to significantly inhibit the increase in serum transaminase activities after intoxication with D-GalN and LPS [112]. These coumarines and poly acetylene were proposed to inhibit LPSinduced nitric oxide (NO) production by macrophages. However, it should be noted that the role of NO in the systemic inflammatory response is two-faced. On the one hand, overe)q)ression of NO is partly responsible for the vascular collapse and ensuing circulatory failure caused by LPS or TNF-a [113,114]. On the other hand, liver-derived NO is now considered an endogenous hepatoprotective factor [115]. Administration of NOS inhibitors (L-arginine-analogues) aggravates rather than inhibits hepatotoxicity and lethality induced by BCG/LPS, P. acnes/LPS, or TNFa [113,116,117]. Recent data indicated that NO prevents TNF-a-

472

induced hepatocyte apoptosis by inhibiting caspase-3 activation [118]. Inhibition of coagulation by NO is also proposed to contribute to the hepatoprotective effect [116,119]. Given these findings, it seems unreasonable to suppose that the inhibition of NO production is part of the hepatoprotective mechanism of coumarines and poly acetylene isolated from^. furcijuga. Instead, these compounds could impede the secretion of other deleterious factors like TNF-a from macrophages, resulting in hepatoprotection.

16 Fig. (5). Hepatoprotective coumarins and polyacetylene of Angelica furcijuga

Reports have suggested that some alkaloids protect against TNF-adependent liver injury. Pretreatment with an anti-malaria drug quinine (17) (230 mg/kg, up,) was shown to abolish plasma TNF-a elevation as well as subsequent hepatic DNA fragmentation and plasma transaminase and sorbitol dehydrogenase elevations in D-GalN/LPS-liver injury in mice. Also, it markedly improved mortality in mice after intoxication [120]. Activation of the K^ chaimel was reported to be important for LPSstimulated TNF-a production in human alveolar macrophages. Since quinine is a K^ channel blocker, it is plausible that this effect helps to inhibit TNF-a production both in vitro and in vivo [120,121]. Similar to quinine, intraperitoneal administration (30 or 100 m ^ g ) of sinomenine (18), an epimorphinan alkaloid from Sinomenium acutum Rehder et Wilson, was reported to significantly inhibit the elevation of serum transaminase activities in D-GalN/LPS-induced liver injury in mice in a dose-dependent manner [122]. Intraperitoneal administration (10 mg/kg)of bisbenzylisoquinoline (BBI) alkaloids, chondocurine (19), cycieanine (20), tetrandrine (21) and berbamine (22), also inhibited the elevation of serum ALT activity and improved mortality after LPS challenge in BCGprimed mice [123,124]. The hepatoprotective effect of sinomenine and BBI alkaloids is thought to result from suppression of TNF-a production.

473

because pretreatment with these alkaloids significantly reduced the increase in serum TNF-a activity after intoxication [122,124]. Colchicine (23), an alkaloid isolated from Colchicum autumnale, shows a very potent hep atop rotective effect against D-GalN/LPS-induced liver injury in mice; intravenous administration of only 0.5 mglcg colchicine suppressed the elevation of serum ALT activity to the normal level [125]. Interestingly, pretreatment with colchicine also markedly protected against liver injury and lethality induced by intravenous injection of TNF-a or LPS-treated macrophages into D-GalN-sensitized mice. On the other hand, in vitro treatment with colchicine (20 or 200 |iM) had no effect on LPS-stimulated TNF-a production in primary cultured bone marrow-derived macrophages. From these observations, the authors concluded that the hep atop rot ective effect of colchicine is due to antagonization of the hepatotoxic and lethal function of TNF-a [125]. This effect may be related to down-regulation of TNF-a receptors on target cells by colchicine as a consequence of microtubule depolymerization [126].

"N-CH3 H3C0.

23

H3CO..

H3C.,

Fig. (6). Alkaloids with hepatoprotective activity against TNF-a-dependent liver injury

474

The crude drug Saiko, the roots of Bupleurum falcatum, has been used in Oriental medicines for the treatment of hepatobiliary diseases. The effect of Sho-Saiko-To (Xiao-Chai-Hu-Tang), a preparation containing Bupleurum Radix, on chronic hepatitis is well defined by clinical trials in Japan and China [1,127]. Experimental data suggested that pretreatment with Sho-Saiko-To (500 mg^g, p.o.) blocks human TNF-a-induced lethality in D-GalN-sensitized mice. In this model, all of the control mice died within 24 hr after TNF-a challenge, whereas 80% of the Sho-SaikoTo-treated mice survived 72 hr. Decrease in rectal temperature seen at 1 hr after TNF-a challenge was also improved by Sho-Saiko-To pretreatment [128]. Based on phytochemical analysis of Bupleurum Radix, saikosaponins and other saponins have been proposed to have hepatoprotective effects. In a recent study, Bupleuroside i n (24), IV (25) and Xffl (26), and Scorzoneroside A (27), B (28) and C (29) were reported to show heap atop rot ective activity against D-GalN/LPS-induced liver injury at the dose range of 10 to 20 mg/kg[129,130].

Glc—Fuc—O^ ''CH2OH

Glc—Fuc—C ""CHgOH

24 : R = P-OH 25 : R = a-OH

26 OH OH OH

\

}

f

H

H

H

,vCOO—CHgi'-C—-C—C-'iCHgO—R2

CH2OH 'OH Ri—O' XHgOH

27: Ri = Fuc-GIc, R2 = Glc 28 : Ri = Fuc-GIc, R2 = H 29: Ri = Fuc, R2 = H

Fig. (7).Triterpene glycosides of Bupleurum scorzonerifolium

There is much interest in the pharmacological role of dietary nutrients in human health. Some nutrients have been recognized to have a hepatoprotective effect throu^ interaction with TNF-a. For instance, dietary supplementation (5%, at the e>q)ense of casein) with glycine (30), a non-essential amino acid, attenuated LPS-induced liver injury and

475

subsequent mortality in rats [131]. It has been shown that Kupffer cells have voltage-dependent Ca^^ channels, and increase of the intracellular Ca^"^ concentration ([Ca^^Jj) is essential for Kupffer cells to produce cytokines after LPS stimulation. An in vitro study using primary cultured Kupffer cells proved that a supplement of glycine (1 mM) to culture medium largely prevents the increase in [Ca^ ji due to LPS. This inhibitory effect by glycine appears to be mediated by an increase in chloride flux via activation of glycine-gated chloride channels [132]. Thus, the hepatoprotective effect of glycine is associated with a blunting of the LPS-induced elevation of [Ca ]i in Kupffer cells, thereby minimizing the production of proinflammatory cytokines includingTNF-a [131,132].

30

Fig. (8). Dietary neutrients reported to inhibit TNF-a production

Choline (31) has apparently similar biological activity to glycine. Previous studies have demonstrated that a choline-deficient diet significantly increases the mortality of rats due to endotoxin shock [133], whereas rats fed with a diet containing excess choline (0.025-0.4%) show resistance to LPS-induced liver and lung injuries, and subsequent mortality [134]. However, in contract to glycine, in vitro treatment with choline does not affect the LPS-induced [Ca^"^]i increase and TNF-a production in liver and alveolar macrophages, ahhough Ca^^ influx and TNF-a production in macrophages isolated from choline diet-fed rats is blunted by 40-60% compared with in control rats. In addition, glycine and choline synergistically protect against TNF-a production and endotoxin shock, suggesting that the protective mechanism of glycine against endotoxin shock is distinct from that of glycine [134]. It is hypothesized that choline alters the signaling cascade triggered by the binding of LPS to receptors on macrophages, possibly by increasing the ratio of membrane phosphatidylcholine to phosphatidylinositol whose turnover is essential for macrophage activation. Alternatively, choline may improve the LPSinduced decrease in membrane fluidity [134]. However, it remains to be elucidated whether the protective effect of choline against endotoxin shock is due only to inhibition of TNF-a production, because in the same report it was described that feeding choline had no effect on the increase in serum TNF-a after intoxication with LPS. Choline may affect also the susceptibility of hepatocytes and other target cells to TNF-a cytotoxicity.

476 Table 2. Natural products with hcpatoprotcctive activity against TNF-a-dependent liver injury Dose' Adm- Models Compounds Original plants Ref. (m^g) inist. Route Alkaloids: [123,1241 Berbamine Berberis vulgaris 10 X 3 i.p. BCG/LPS Colchicine Colchicum autumnal 0.5 X 2 i.v. D-GalN/LPS, 11251 D-GalN/TNF-a [123,1241 Chondocurine Chondodendron tomentosum 10 X 3 i.p. BCG/LPS Cycleanine Cissampelos imularis 10 X 3 i.p. BCG/LPS 1123,1241 Sinomenine Sinomenium acutum 30 i.p. D-GalN/LPS [1221 Tetrandrine Stephania tetrandra 10 X 3 i.p. BCG/LPS [1241 Quinine Cinchona officinalis 230 i.p. D-GalN/LPS [1201 Coumarlns: Isoepoxypterv'xin Anomalin Isoptety'xin

Angelica furcijuga Angelica furcijuga Angelica furcijuga

Iridoids: Gentiopicroside Sweroside Polyphenols (Flavonoids etc.): Acteoside (+)-Ampelopsin (Dihydromyricetin) Curcumin (-)-Epigallocatechin gallate Gomisin A Hovenin I Naringin Lithospermate B Tetrahydroswertianolin

12.5 12.5 12.5

i.p. D-GalN/LPS i.p. D-GalN/LPS i.p. D-GalN/LPS

Gentiana macrophylla

30 X 5

Swertiajaponica

25 X 2

i.p. BCG/LPS D-GalN/LPS, s.c. D-GalN/LPS

Cistanche deserticola Hovenia dulcis

10 X 2 100 X 2

P.O.

Curcuma longa 50 Camellia sinensis 100 Schizandra chinensis 100 Hovenia dulcis 25 Citrus aurantium var. daidai Umg/moiise) Salvia miltiorhiza 50 Swertiajaponica 50 X 2

Triterpenes: Bupleuroside III, IV and XlII Bupleurum scorzonerifolium Ginsenoside Re and Rgi Panax notoginseng Glycyrrhizin Glycyrrhiza glabra

D-GalN/LPS D-GalN/LPS

D-GalN/LPS LPS i.p. P. acneslLVS i.p. D-GalN/LPS i.p. D-GalN/LPS s.c. D-GalN/LPS P.O. D-GalN/LPS

P.O. P.O.

186,1101 [861 [971 [al [1051 [331 [91 [1031 [1041 [1351 [86,871

or s. c. 10 or 20 20 X 2 100 X 2 10

Scorzoneroside A, B and C

Bupleurum scorzonerifolium

Other phytoconstituents Falcarindiol Celosian

Angelica furcijuga Celosia argentea

Nutrients Choline

—

0.4%

—

5%

Glycine

s.c

[1121 [1121 [1121

12.5 10

i.p D-GalN/LPS i.p. D-GalN/LPS P.O. D-GalN/LPS or s. c. D-GalN/LPS i.p.

i.p. D-GalN/LPS s.c. D-GalN/LPS, P.acnesIL?^ Dietary LPS

[1291 [1361 [85,871 [1301 [1121 [44,1371 [1341

siippl.

Dietary LPS

[131,1321

suppl.

Minimally effective dose, [a] unpublished data of the author's group. D-GalN, D-galactosamine; LPS, lipoplysaccharide; BCG, bacillus Calmette-Guerin

CONCLUSION The TNF-a-dependent models are mostly characterized by the apoptotic cell death of hepatocyte at the early stage of liver injury. In these models, a large number of hepatocytes undergoing apoptosis can represent a Stimulus for primed neutrophils in sinusoids to transmigrate and activate, leading to hepatic inflammation and massive hepatocyte necrosis. This

477

means that prevention of apoptosis is an effective therapy for disrupting the progression of Uver injury. Indeed, some hep atop rot ective phytoconstituents protect against hepatocyte apoptosis and consequently block the progression of liver injury, althou^ others seem to prevent secondary hepatocyte necrosis triêred at the final stage without affecting apoptosis. Inhibition of hepatocyte apoptosis may be one important mechanism whereby phytoconstituents exert hep atop rot ective activity. Therefore, measuring apoptotic markers besides necrotic makers such as serum transaminase levels is useful to distinguish the mode of action of hepatoprotective compounds on inflammatory liver injuries. The protection against hepatocyte apoptosis and liver injury by natural products is presumably mediated by an interaction with TNF-a. The majority of the hepatoprotective compounds described here possess inhibitory activity against TNF-a production by macrophages. On the other hand, a few compounds attenuate the cytotoxic action of TNF-a. These observations clearly suggest that certain hepatoprotective phytoconstituents bear pharmacological potency that impedes the intracellular signaling pathway essential for TNF-a production in effector cells or TNF-a cytotoxicity in target cells. Elevation of the serum TNF-a level is frequently seen in patients suffering from alcoholic, viral or fulminant hepatitis, or cirrhosis, and there is an inverse correlation between the TNF-a elevation and the survival ratio of these patients. Continuing investigations on phytoconstituents with the TNF-adependent liver injury model are e>q3ected to provide new effective hepatoprotective agents. ABBREVIATIONS ActD ALT AST BCG Con A CCI4 D-GalN IFN-Y LPS MNCs NO NOS RT-PCR THS TNF-a TNF-R

=

actinomycin D alanine transaminase aspartate transaminase bacillus Calmette-Guerin concanavalin A carbon tetrachloride D-galactosamine interferon-y lipopolysaccharide mononuclear cells nitric oxide NO synthase reverse transcription-polymerase chain reaction tetrahydroswertianolin tumor necrosis factor-a TNF receptor

478

ACKNOWLEDGEMENT We would like to thank Emeritus Professor Tsuneo Namba and Dr Purusotam Basnet for their support and valuable comments on the manuscript. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11 [12 [13 [14 [15 [16: [17 [18 [19: [2o: [21 [22 [23 [24 [25 [26 [27 [28

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INDUCTION AND REGULATION OF BIOSYNTHETIC ACTIVITY OF PHT^TOALEXIN IN CARROT CELLS FUMIYAKUROSAKI Faculty ofPharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan ABSTRACT: The intracellular production of 6-methoxymellein, a phytoalexin of carrot, is induced by the addition of a wide variety of substances called elicitors. Active elicitor molecules appear to be fragments of pectic substances from carrot cell walls, and are liberated by partial hydrolysis of the walls with extracellular pectinase or proteases secreted by invading fimgi. The accumulation of 6-methoxymellein is controlled primarily by the rate of transcription of the genes which encode the biosynthetic enzymes. Transduction of elicitor signals in plant cells may involve a mechanism similar to that reported in odor-sensitive animal cells. It is likely that Ca^"^ acts as a second messenger, and plays a central regulatory role in expression of the genes that encode the enzymes for 6-methoxymellein biosynthesis. The increase in the cytoplasmic Ca^^ level is mediated by activation of the phosphatidylinositol cycle, liberating inositol trisphosphate and diacyl glycerol as messenger molecules. In addition, evidence has been gathered suggesting that cyclic AMP stimulates Ca^'^-influx by gating of cyclic AMP-sensitive cation channels with no accompanying cyclic AMP-dependent protein phosphorylation. Biosynthesis of 6-methoxymellein is catalyzed by two inducible enzymes, 6hydroxymellein synthase and 6-hydroxymellein-O-methyltransferase. 6-Hydroxymellein synthase is a multifunctional polyketide biosynthetic enzyme, and is an active catalyst only in the homodimeric form. Acetyl and malonyl moieties, which are the building units of 6-hydroxymellein, bind to the transacylase domain of the synthase, and are channeled to two SH-groups at the reaction center. The reaction catalyzed by 6hydroxymellein-0-methyltransferase proceeds by a bireactant sequential mechanism, and the activity of the enzyme is strictly controlled by its products.

INTRODUCTION It is widely recognized tliat plant cells are potentially rich sources of commercially important secondary metabolites. The production of secondary metabolites could be controlled by a mechanism by which

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enzymes are expressed or repressed. It is likely that the genes encoding specific enzymes in the biosynthetic pathway of desired products are sometimes repressed, or expressed very weakly. However, the mechanism involved in 'switch on' of the genetic information on secondary metabolism is at present very poorly understood. It is known that microbial invasion in plants often triggers the accumulation of antimicrobial substances called phytoalexins. Interaction between plants and microorganisms has been studied in several hostpathogen systems, and experimental results show that signal molecules which induce the synthesis of secondary products in plant cells are produced in the early stage of microbial infection. These molecules are called elicitors, and include peptides, polysaccharides and glycoproteins derived from microbial and plant cells. The response of plant cells to these elicitors was first studied from the phytopathological point of view to elucidate the regulation mechanism of phytoalexin production. Recent investigations have indicated that the treatment of plant cells with possible elicitors occasionally results in a rapid accumulation of secondary products. Here, several questions are addressed regarding the production of secondary metabolites by elicitors. 1) How is the signal of the elicitor recognized by plant cells? 2) How is the signal transduced in the cells? 3) How does the signal trigger the expression of genes? 4) How are enzyme activities controlled to produce secondary metabolites? Effective use of elicitors in producing usefiil metabolites in plant cells requires the elucidation of these biochemical mechanisms by which external stimuli regulate the genetic information. These questions are under active consideration, although at present very little is known about the molecular mechanisms underlying the recognition and transduction of elicitor signals. STIMULATION OF 6-METHOXYMELLEIN PRODUCTION IN CARROT CELLS Liberation of elicitors during host-pathogen interaction 6-Methoxymellein, an antifungal isocoumarin[Fig. (1)], was first isolated as the metabolite that is responsible for the bitter taste in cold-stored

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carrot roots [1]. Condon and Kuc [2] have shown that this compound accumulates in

Extracellular hydrolases

Cell wall fragments as elicitors

CH3O'

"^^

^ ^

'"'t

6-Methoxymellein production Fig. (1). Interaction between invading fungi and host plants in the elicitation of phytoalexin production

carrot roots after inoculation with Ceratocystis fimbriate, which causes black rot disease in sweet potato but is not pathogenic to carrot. The resulting production of 6-methoxymellein accounted for the resistance of carrot tissue to microbial infection. This compound inhibits the growth of various fungi in the concentration range 0.05 - 0.5 mM [3]. Preliminary studies indicate that heat-stable and water-soluble substances which show elicitor activity are released during interaction of carrot cells and the fungus [4]. The elicitor lost its activity after digestion with pectinase or proteases, suggesting that oligogalacturonides and/or peptides are jointly essential in inducing activity. Also, partial hydrolysates of pectic fractions of carrot cell walls prepared with these enzymes showed strong elicitor activity. These results suggest that extracellular hydrolases secreted by fungi, including pectinase and proteases, act to liberate oligosaccharides and peptides from carrot cell walls, and the fragments of the extracellular matrix of carrot bring about 6-methoxymellein production [Fig. (1)]. This was confirmed by an experiment in which filter-sterilized pectinase and trypsin were directly added to carrot cell

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culture. Biosynthetic activity of 6-methoxymellein was induced in the carrot cells, implying that eliciting substances are released from live carrot cells by the enzymatic action of these hydrolases. Stimulation of 6methoxymellein production was also observed on exposure of carrot cells to ethylene or metal ions [5]. Effect of esterification of oligogalacturoiiides on elicitor activity As with other secondary metabolites, phytoalexin production in in vitro culture of plant cells is influenced by culture age [6]. When 6methoxymellein production was triggered by adding partial hydrolysates of cell walls, its rate of production was very low in growing cells, but high in cells in the early stationary phase [7]. The release of active elicitor depended also on the growth stage of carrot cells. Partial hydrolysates obtained from carrot cells in the late logarithmic and early stationary phases yielded highly active elicitors, while those from the early logarithmic and late stationary phases showed very low activity. Asamizu et al. [8] reported that polygalacturonides in cell walls of cultured carrot are highly esterified, while those in aged cultures, which are a poor source of elicitor, consist mostly of non-esterified uronic acids. These results suggest that the elicitor activity of pectin fragments of carrot cell walls depends largely on the degree of esterification of uronide complexes. To verify this, partial hydrolysates of the pectic fraction of carrot cell walls were further treated with pectin esterase. After hydrolysis the elicitor activity had decreased to less than 20%, suggesting that some degree of esterification of polyuronides is required for the induction of 6-methoxymellein synthesis [9]. By contrast, Jin and West [10] reported that, in the induction of casbene synthesis in castor bean seedlings, the eliciting activity of tridecagalacturonides was decreased by methylesterification of carboxyl groups. The effect of chemical modification of oligogalacturonides therefore varies according to plant species. TRANSMEMBRANE SIGNALING MECHANISMS IN STIMULATION OF PHYTOALEXIN PRODUCTION Participation of Ca^^ as a second messenger

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When elicitor-active pectic fragments were analyzed by ion exchange and gel-filtration chromatography, the activity was found to be distributed in many fractions [11], suggesting that the elicitor consists not of a single molecule but a mixture of several active substances. This result led to an examination of whether these elicitors share a common signaling mechanism. Ca^"*" is an important second messenger in many physiological processes in both animal and higher plant cells, and calmodulin (CAM), a Ca^^-binding protein, plays a central role in many of these systems [12, 13]. 6-Methoxymellein production induced by oligogalacturonide was appreciably inhibited in the presence of the Ca^"^channel blocker verapamil [14]. Trifluoperazine and W-7 [N-(6aminohexyl)-5-chloro-l-naphthalenesulfonamide], a different class of inhibitors of CAM-dependent reactions, also caused marked inhibition. In addition, it was found that appreciable 6-methoxymellein biosynthesis was induced in carrot by treatment with Ca^"^-ionophore A23187. These observations strongly suggest that the increase in cytoplasmic Ca^^ level is an essential early event in eliciting 6-methoxymellein production. In potato and soybean, phytoalexin production is also a Ca^'^-dependent process, and the elicitor-induced responses were significantly inhibited by several Ca^'^-inhibitors [15, 16]. Activation of phosphatidylinositol cycle Further support for the hypothesis that Ca plays a central role in regulating phytoalexin accumulation is provided by experiments in which the tumover of phosphatidylinositol was measured in the plasma membrane of elicitor-treated carrot cells [17]. The carrot cells were first labelled with ['^H]myo-inositol and, after the addition of elicitors, acid extracts of the cells were analyzed chromatographically for the production of inositol trisphosphate (IP3). In cells treated with elicitor, the release of radioactive IP3 increased with time and attained a maximum at 3 - 5 min after treatment. Phospholipase activity responsible for the degradation of phosphorylated phosphatidylinositol increased correspondingly. Several reports have shown that IP3 induces rapid release of Ca^"^ from intracellular stores in animal cells [18, 19]. Studies on plant cells have also demonstrated that exogenous IP3 releases Ca^"^ from microsomal preparations at micromolar concentrations, although only limited

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information is available [20, 21]. Schumaker and Sze observed IPsinduced release of Ca^"^ from intact vacuoles of Avena seedlings [22]. Vacuoles are the most prominent organelles in plant cells, and normally contain 0.1 to 10 mM Ca^^; therefore they may serve as the principal Ca^^ store. Diacyl glycerol, another of the hydrolysates of phosphorylated phosphatidylinositol, is a known activator of protein kinase C in animal cells [23]. The present experimental results suggest that this protein kinase also participates in the expression of phytoalexin biosynthesis in carrot cells. We found that the synthetic diacylglycerol l-oleoyl-2-acetylrac-glycerol, which has been shown to be intercalated into cell membranes and to activate protein kinase C, induced 6-methoxymellein production in the absence of elicitor. A similar result was obtained for the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate, another activator of protein kinase C [17]. On the other hand the addition of H-7 [l-(5-iso-quinolinesulfonyl) -2-methyl-piperazine], a specific inhibitor for protein kinase C, resulted in suppression of phytoalexin production. These observations strongly suggest that a rapid breakdown of phosphatidylinositol in the plasma membrane of carrot cells takes place upon contact with elicitor molecules, resulting in the liberation of two types of second messengers, IP3 and diacyl glycerol. Role of cyclic AMP as a second messenger In contrast to Ca^^, the role of cyclic AMP (cAMP) as a second messenger in plant cells is still obscure, because there is no proof of the presence of cAMP-dependent protein kinase in plant cells. The existence of cAMP itself in plant cells has been confirmed [24, 25]; more recently, various works suggest that the cyclic nucleotide is involved in physiological events in plants [26]. We have found [14] that the addition of dibutyryl cAMP (Bt2cAMP) to carrot cell culture causes 6-methoxymellein production even in the absence of elicitor. Addition of several reagents which are known to change the intracellular level of cAMP, namely cholera toxin, which is an activator of adenylate cyclase, and theophylline, a phosphodiesterase (PDE) inhibitor, also led to production of 6methoxymellein, suggesting that elevation of the cAMP concentration in carrot triggers phytoalexin production in the cells. In fact, treatment of carrot cells with uronide elicitors led to a rapid but transient increase in the concentration of intracellular cAMP. Similar observations have been

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reported by Bolwell et al. [27], who tested the effect of various modulators of signal transduction processes on the induction of phenylalanine ammonia-lyase in Phaseolus vulgaris cell cultures. They found that cholera, petussis toxins and forskolin all stimulated synthesis of the enzyme. These reagents are known to activate adenylate cyclase, either through interaction with G-protein or directly. We examined changes in the activity of protein phosphorylation in carrot cells following treatment with either Bt2cAMP, forskolin or Ca^îonophore A23187 [28]. Addition of cAMP to cell extracts prepared from these treated cells did not cause any change in phosphorylation activity, indicating that cAMP-dependent kinase activity is absent or very low in carrot cells, as well as in most of the other plants. By contrast, the activities of Ca^^- and Ca^VCAM-dependent protein kinases increased markedly in both cytosolic and microsomal fractions after the treatment. Phosphorylation activity was stimulated not only by Ca^^-ionophore but also by Bt2cAMP and forskolin. Furthermore, although Bt2cAMP and forskolin can stimulate phytoalexin production in carrot cells, the effect was severely suppressed by diverse Ca^"^ channel blockers and CAM antagonists [28]. These observations suggest that cAMP acts as second messenger by stimulation of the Ca^'^-cascade, rather than by activating cAMP-dependent protein kinases. This view is supported by experimental results in which changes in the concentration of cytosolic Ca^^ in carrot cells were measured by a fluorescent Ca^"^-indicator (fluo-3) after treatment with the reagents [28]. The Ca^^ level in the cytoplasm of untreated carrot cells was found to be about 0.1 \iM, A marked increase in the intracellular concentration of Ca^"^ to 0.6 - 0.8 |iM was observed 3 6 min after the addition of BticAMP or forskolin. These results suggest that the increase in cytoplasmic cAMP level leads to the Ca^'^-influx into carrot cells. This conclusion was also drawn from an experiment in which the effect of cAMP on the Ca^^-flux was examined using '*^Ca^"^-loaded vesicles of plasma membrane [28]. Plasma membranes prepared by the two-phase partitioning method are generally composed of differently oriented sealed vesicles, some normal and some inside-out [29]. Incubation of these vesicles with "^^Ca^"^ in the presence of ATP results in selective placement of the radiolabelled ions into the inside-out vesicles by the plasma membrane-located Ca^^-ATPase [29]. When the ^^Ca^^loaded vesicles were incubated with cAMP a rapid release of "^^Ca "^ from the vesicles was observed; this discharge was specifically observed with

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cAMP among the nucleotides tested. These observations are consistent with the hypothesis that the cytoplasmic level of cAMP is raised by an appropriate stimulus, and the nucleotide triggers Ca^^-influx vîthout accompanying cAMP-dependent protein phosphorylation, probably through cAMP-sensitive ion channels. Synthesis and degradation of cyclic AMP Addition of forskolin to carrot cell culture caused an appreciable increase in adenylate cyclase activity. However, the increase was transient although the activator was present throughout the experiment [30]. In contrast to cyclase reported from other plant sources [31, 32] the forskolin-stimulated activity of the enzyme in carrot cell extracts was detected only when EGTA was included in the assay mixture, and the addition of exogenous Ca^"^ strongly inhibited the enzyme activity. The effect of various concentrations of Ca^"^ on adenylate cyclase activity was therefore studied using buffers with the concentration of free Ca^^ adjusted by the EGTA-Ca^"^ buffer system [33]. The activity of the cyclase was markedly affected by the free Ca^"^ concentration, and was maintained at a high level only when the Ca^"^ concentration was below 0.1 |LIM. This figure is close to the Ca concentration in cytoplasm in the resting state of various plant species. Constitutive activity of PDE was found in cultured carrot cells; this activity did not depend on either Ca^"^ or CAM. By contrast, a CAMdependent isoform of PDE (CAM-PDE) was induced in the cells by adding forskolin or Bt2cAMP to the culture [30]. Induction of CAMPDE activity in Bt2cAMP-treated carrot cells was markedly inhibited in the presence of verapamil, and addition of Ca^'*"-ionophore A23187 induced CAM-PDE [34]. These results suggest that increased Ca^"^, but not cAMP, in the stimulated carrot cells triggers induction of the PDE isoenzyme. Affinity of CAM-PDE to the substrate was low compared to constitutive PDE (Km values, 0.14 and 0.07 |uiM, respectively); however, V for the induced PDE was approximately 2.7 times higher than for the constitutive isoenzyme. These results suggest that synthesis and degradation of cAMP in cultured carrot cells are both controlled and switched on/off according to the concentration of Ca^^ in carrot cytoplasm. Adenylate cyclase activity is induced in the cells only in the resting state, and the enzyme activity is

491

automatically inhibited when the concentration of cytoplasmic Ca^^ increases and reaches the level of the excitatory state. The constitutive PDE, w^hich is insensitive to the cytoplasmic Ca^"*" level, is important in maintenance of the resting state of carrot cells, by keeping cellular cAMP and Ca^^ levels very low, while CAM-PDE induced in excited cells hydrolyzes the messenger nucleotide rapidly under conditions of high cAMP and Ca^"^, in vivo, as a response-decay mechanism. In animal cells, the cAMP-induced Ca'^'^'-influx through the nucleotidesensitive channels is terminated by the hydrolysis of cAMP, the ligand of the channels [35]. However, in cultured carrot cells, the cytoplasmic Ca^"^ concentration elevated by the stimulation of cAMP began decreasing even though the level of intracellular cAMP was high [28]. Furthermore, when a Ca^'^-influx was triggered by treating the cells with Bt2cAMP, the cytolasmic concentration of Ca^"^ returned to its base level after a few minutes, by which time the cAMP analogue was still present at a high concentration [28]. These results clearly indicate that, in contrast to animal cells, degradation of cAMP is not the immediate reason for the response decay of the cAMP-gated cation channel in carrot cells. We found [36] that the discharge of Ca^"^ from inside-out sealed vesicles of carrot plasma membrane was strongly inhibited when 9+

the suspension of the vesicles was supplemented with 1 îM free Ca , while Ca^"^ concentrations lower than 0.1 ^M did not affect Ca^'^-release. In addition, the inhibited Ca^^-flux across the plasma membrane was restored by the addition of CAM inhibitors and anti-CAM IgG [36]. These results suggest that the Ca^^-influx initiated by increases in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca^^ concentration reaches the threshold excitatory level in the cells. It is probable that CAM located in the plasma membrane plays an important role in the decay response of the cyclic nucleotide-gated cation channels. Ca^'^-dependent CAM binding to several target proteins in the plasma membrane has been reported in the pea [37]. However, this does not seem to be the case in closing the cAMP-sensitive cation channels of carrot cells, because Ca^^-loaded vesicles of plasma membrane which were repeatedly washed with EGTA-containing buffer showed similar resuks. CAM involved in this transmembrane signaling process should therefore be EGTA-stable, and probably partially embedded in the lipid bilayer as reported in the pea [37].

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Regulation of Ca^^-ATPase activity As with other eukaryotic cells [38], maintenance of low Ca^"^ concentration in the cytoplasm of non-stimulated higher plant cells is essential. The cytoplasmic Ca^"^ concentration of plant cells in the resting state, as described above, is generally maintained at approximately 0.1 ^M by the action of Ca^"^4ransporting systems [39] which sequester the ion into internal organelles, including endoplasmic reticulum, mitochondria, and vacuoles, or mediate its efflux to the cell exterior. It is known that Ca^^-pumping ATPase at the plasma membrane plays a key role in transporting Ca ^ to apoplastic spaces [39]. Characteristics of Ca^"^-translocating ATPase have been reported from a wide range of plants [39], although some are highly variable depending on the plant species. One of the most serious controversies over properties of ATPase is the role of CAM in regulation of the enzyme; inconsistent observations on the CAM-dependence of enzyme activity have been reported from several plants [40-43]. It is not yet clear whether this discrepancy represents genuine variation across species or is an experimental artifact. However, it seems that results depend partly on the fact that plasma membrane preparations obtained from higher plant cells sometimes contain the membranes of other organelles. A highly purified plasma membrane fraction from cultured carrot cells was prepared by the aqueous two phase-partition method [29], in order to reevaluate the role of CAM in regulating Ca^'^-ATPase at the plasma membrane of the cells. The Ca^'^-translocating activity of ATPase was considerably inhibited in the presence of different classes of CAM antagonists or anti-CAM IgG [44]. This Ca^"^-pumping activity decreased significantly when the plasma membrane preparation was washed with EGTA-containing buffer; however, it was restored to almost the control level upon adding exogenous CAM. These results suggest that Ca^^-ATPase at the plasma membrane of carrot cells is regulated by CAM, and the modulator protein associates with the enzyme in a manner dependent on the Ca^^ concentration [44]. The biochemical basis of CAM-induced stimulation of Ca^^-ATPase activity in carrot cells was studied further by determining the parameters of the Ca^"*"-translocating reaction of the enzyme in the presence and absence of exogenous CAM, using EGTA-treated plasma membrane [45]. The affinity of Ca^^-ATPase for Ca^^ was considerably increased by

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association with CAM, and Km values decreased from 11.4 |aM to 0.7 )LiM. These figures are close to those of CAM-dependent Ca^^-ATPase at the plasma membrane in animal cells [39]. Affinity of the enzyme for ATP was also increased in the presence of CAM, although the increase was low compared to that for Ca^"^ (Km values of 914 and 670 |LIM in the absence and presence of CAM). In contrast to the affinities for the substrates, the relative V values of the ATPase were similar or slightly decreased by the addition of CAM. It is well known that, in the excited plant cells having high Ca^^ concentration, CAM is activated by binding to the ion, and is able to associate with various CAM-dependent proteins [38]. The Ca^"^ concentration in resting plant cells, by contrast, is too low to activate CAM, resulting in the dissociation of the modulator from its target proteins, including Ca^"*"-ATPase [38, 39]. The Kca of the ATPase associated with CAM is similar to that of the cytoplasmic Ca^"^ level of excited plant cells (0.7 jaM), while the Kca of the ATPase without CAM increased markedly (11.4 |aM) though the cytoplasmic Ca^^ concentration in the resting cells is quite low. These observafions suggest that Ca^ÂTPase at the carrot plasma membrane plays an important role in the excited cells only as an 'acute' enzyme. However, Rasi-Caldogno et aL [43] pointed out that Kca decreased from about 10 |aM to about 0.1 |iM if the level of free Ca^"^ alone is considered. This low Km value of CAMdepleted Ca -ATPase for Ca is consistent with the transport protein involved in maintaining cytoplasmic Ca^"^ concentration at the submicromolar range being a 'house keeping' enzyme in resting cells. These results strongly suggest that, on binding of CAM, the affinity of the carrot Ca^^-ATPase for Ca^"*" is markedly increased, and this is the most important biochemical change behind the CAM-induced increase in pumping activity of the enzyme. Possible scheme for transduction mechanisms of elicitor signals These studies all support the hypothesis that external stimuli of the elicitor cause an increase in the cytoplasmic Ca^^ level via the phosphatidylinositol cycle and/or the adenylate cyclase system. Although an authoritative picture of this process cannot yet be given, possible signal transduction mechanisms are summarized in Fig. (2). At present the data

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are still fragmentary, so that it is important to leam more about the biochemical nature and function of the components involved in signal Elicitors

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-CX

both are tyrosine-phosphorylated by JAKs -^ tyrosinephosphorylated, activated STAT-a/STAT-b dimers translocate into the nucleus —> activated dimer binds to specific promoter elements —> specific gene expression [27, 28]. c. Ligand-activated protein tyrosine phosphatases (PTPases) couple ligand binding to phosphotyrosine protein dephosphorylation thus: ligand —> ligand-PTP -> ligand-PTP activated --> dephosphorylation of tyrosinephosphorylated-proteins [20]. d. Hormone-activated guanylate cyclase (GC) is activated by atrial natriuretic factor (ANF) to generate cGMP and thence promote vascular dilation and improved cardiac function. Thus, heart stress —> ANF -> ANF-GC -> ANF-GC activated -> increased cGMP -> increased natriuresis, diuresis, vascular dilation and cardiac function [29]. e. Hormone- or neurotransmitter-gated ion channels include such receptors for acetylcholine (AcCh) (nicotinic receptors), glutamate (Nmethyl-D-aspartate (NMDA) and non-NMDA receptors), glycine, serotonin and y-amino butyric acid (GABA). However it should be noted that some of these signalling molecules (e.g. AcCh, glutamate, serotonin and GABA) can also act through G-linked receptors). The signalling sequence involves the following: ligand (L) -> L-R ~> change in conformation of the L-R complex —> ion channel (for Na"*", K"*", CI" or Ca^^) opens -^ change in transmembrane potential (A\|/m) -> downstream

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effects (notably changes in cell excitability or cytosolic free Ca^"^ levels) [13-15]. f. Voltage-gated (V-gated) ion channels are protein pores specific for particular ions (e.g. Na"*" , K^ and Ca^^ channels) which open or close in response to changes in transmembrane potential (v|/m). Thus signallinginduced A\|/m —> V-gated channels open or close -4 change in permeability (AP) for specific ions —> A\|/m -> downstream effects as outlined in (e) above [13-15, 30]. g. Hormone-activated serine/threonine-specific receptor protein kinases (S/T-RPKs) include receptors for hormones of the transforming growth factor p (TGFp) family (that suppress cell proliferation), developmentally important activins (involved in mesoderm induction) and bone morphogenetic proteins (involved in bone formation). The pathway involved is as follows: H -> H-R -^ S/T-RPK activated -^ target protein serine/threonine phosphorylation -> downstream consequences (e.g. specific gene expression as a result of specific transcription factor phosphorylation and activation) [31]. h. Hormone receptors linked to heterotrimeric G proteins mediate signalling for ATP, adenosine, many peptide hormones, catecholamines (such as dopamine, epinephrine and norepinephrine), eicosanoids and histamine. In addition, glutamate, serotonin, GABA and acetylcholine (depolarizing and hence excitatory hormones/neurotransmitters that can act via ion channel receptors) can also act via G protein-linked receptors. The G proteins are heterotrimers composed of a, P and y subunits. Interaction with H-R complexes results in dissociation of a Ga subunit (as a complex with GTP) from a Gp-Gy complex. The Ga-GTP complex can interact with various effector proteins which are then activated and produce a variety of downstream effects. Reversibility of the system is achieved through the GTPase hydrolytic activity of the Ga subunit which generates Ga-GDP which then recombines to form the inactive Ga-GDPGp-Gy complex. Specificity in such pathways is achieved through specific hormone receptors and specific types of Ga subunits. This sequence can be summarized as follows: H ^ H-R -> H-R-Ga-GDP-Gp-Gy -^ H-R +

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Ga-GTP + Cp-Gy -> Ga-GTP activates effector proteins depending on what specific Ga subunit is involved -^ downstream effects [32-35]. A variety of Ga subunits interact with specific effector proteins and can be classified into various families, namely (for simplicity), Gas, Gaolf, Gai, Gao, Gaq and Gat. Gas-GTP (and Gaolf-GTP involved in olfaction) activate adenylate cyclase (AC) (thereby increasing cAMP levels and activating PKA and cAMP-gated Na"*" channels) and can also open Ca^"*" channels. Vibrio cholerae (cholera) toxin ADP ribosylates and inhibits the Gas and Gaolf GTPase, thus causing persistent activation of adenylate cyclase. In contrast, Gai-GTP inhibits adenylate cyclase, closes Ca^"^ channels and opens K^ channels. Gai is ADP-ribosylated and thus functionally inhibited by a Bordetella pertussis (hooping cough) toxin. Gao-GTP and Gaq-GTP are also variously ADP ribosylated by pertussis toxin and can activate phospholipase C (PLC) which catalyzes the formation from phosphatidylinositol-4,5-bisphosphate (PI45P2) of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). IP3 in turn increases cytosolic Ca^"*" by opening IPs-gated Ca^"^ channels in the endoplasmic reticulum (ER). Gat-GTP (transducin, the effector of lightactivated rhodopsin), activates cGMP phosphodiesterase, lowering cGMP levels in retinal rod cells and thereby closing cGMP-gated Na"^ channels as a critical step in visual perception. Gat is ADP ribosylated by both the cholera and pertussis toxins [32, 34]. EUKARYOTE SIGNAL-REGULATED PROTEIN KINASES All of the PM receptor-mediated signal transduction mechanisms described above ultimately act through the phosphorylationdephosphorylation of proteins through the action of signal-regulated protein kinases (PKs) [36-43] and phosphoprotein phosphatases (PPs) [18] as summarized in Fig. (1). Such reversible covalent modification is involved in metabolic regulation and in developmental regulation through control of cell division and specific gene expression. Indeed protein phosphorylation-dephosphorylation itself is regulated by the phosphorylation-dephosphorylation of protein kinases, PP inhibitors and PK- or PP-targeting proteins. Such phosphorylation-dephosphorylation processes can be the targets of particular plant-derived secondary metabolites. It has been estimated

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that there may be as many as 1000 PKs and hundreds of PPs encoded by a typical higher organism genome. Many signal-regulated PKs have been described and some of the better studied systems are briefly outlined below. Fig. (1) summarizes the signalling pathways leading to the activation of some of these major types of signal-regulated PKs. OUT

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Fig. (1). Signal transduction in eukaryotes involving plasma membrane-located receptors. PM, plasma membrane; OUT, outside the cell; IN, inside the cell; Ll-Lll, ligands 1-11; AC, adenylyl cyclase, cAMP, 3',5'-cyclic AMP; CaM, calmodulin; CaM PK, Ca^'^-CaM-dependent protein kinase; CDPK, Ca^'^-dependent protein kinase; cGMP, 3',5'-cyclic GMP; G, G protein; GC, guanylyl cyclase; IP3, inositol-1,4,5-triphosphate; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MLCK, myosin light chain kinase; P, permeability of PM to specific ions; PDK, phosphatidylinositolphosphate-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; PIP2, phosphatidylinositol-3,4-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PKA, cAMP-dependent protein kinase; PKB, insulin signalling-activated protein kinase; PKC, Ca^"^- and phospholipid-dependent protein kinase; PKG, cGMP-dependent protein kinase; PLC, phospholipase C; PLCy, phospholipase C-y; P-Pr, phosphoprotein; P-STAT, tyrosine phosphorylated STAT dimer (signal transducers and activators of transcription); PTPase, protein phosphotyrosine phosphatase; R, receptor; Ras, small G protein; Raf, MAPK kinase kinase; RSTK, receptor serine/threonine protein kinase; RTK, receptor tyrosine kinase; RPTP, receptor protein phosphotyrosine phosphatase; AP, change in permeability of PM to specific ions; A^', change in transmembrane potential.

a. Cyclic AMP-dependent protein kinase (PKA) is inactive as the holoenzyme (R2C2) which is activated by the binding of cAMP to the regulatory ( R ) subunits releasing the active catalytic ( C ) subunits: R2C2 (inactive) + 4 cAMP -> (R-cAMP2)2 + 2 C (active). PKA phosphorylates and activates phosphorylase b kinase, PP inhibitor-1 protein, site 1 on the

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glycogen targeting protein (thereby activating PPl) and triglyceride lipase. PKA phosphorylates and functionally inhibits CaM-dependent myosin light chain kinase (MLCK), glycogen synthase, site 2 on the glycogen targeting protein (thereby inhibiting PPl) and liver pyruvate kinase. The three dimensional structure of the PKA catalytic subunit has been determined and is very similar to the structure of the catalytic domains of a variety of other protein kinases [36]. Cyclic AMP levels are elevated by hormone receptors acting via Gas-GTP and are decreased by cAMP PDEs. A PKA inhibitor protein (the Walsh-Krebs inhibitor protein) inhibits free catalytic subunit, C. While cAMP is a "hunger" signal in eukaryotes and prokaryotes (signalling catabolite deficiency), fructose2,6-bisphosphate is a "plenty" signal in eukaryotes. Thus in animals cAMP promotes gluconeogenesis and inhibits glycolysis in the liver. PKA phosphorylates the liver dual fructose-6-phosphate-2-kinase / fructose-2, 6-bisphosphatase to inhibit the kinase and activate the phosphatase activity, thereby decreasing fructose-2,6-bisphosphate levels (a converse effect obtains with the muscle enzyme consonant with liver being a "glucose provider" and muscle a "glucose user") [43]. Finally, PKA phosphorylates CREB (cAMP response element binding) protein transcription factors that induce the synthesis of, for example, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase [44, 45]. Other targets of cAMP in eukaryotes are cAMP-gated Na^ channels (e.g. those involved in odor perception via Gaolf-GTP activation of adenylate cyclase, cell depolarization through the opening of cAMP-gated Na"^ channels and signal transmission to the central nervous system) [46], noting that Ca^^ and cGMP are also involved as second messengers in odor perception [47]. The Dictyostelium discoideum (slime mould) cAMP receptor is a 7-transmembrane a-helix-type receptor located on the PM and is involved in amoeboid form aggregation and differentiation to a spore-generating fruiting body) [48,49]. b. Cyclic GMP-dependent protein kinase (PKG) is a homodimer that is homologous to PKA and is activated by cGMP thus: E2 + 4 cGMP (E-CGMP2 )2 (active) [50, 51]. Cyclic GMP is generated by ANF-activated membrane-bound guanylate cyclase (GC) [29] or by soluble NO-activated GC, NO being generated by NO synthase that can be Ca^'^-CaM-activated [52, 53]. Cyclic GMP is hydrolyzed to 5'-GMP by cGMP PDEs. Activation of PKG leads to phosphorylation of specific proteins and, for

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example, to vascular dilation. However cGMP can also act via cGMPgated Na^ channels (e.g. this being critical in vision) [13-15]. c. The Ca^^- and phospholipid-dependent protein kinase (PKC) family consists of a variety of PKC isozymes that are variously activated by Ca^^, DAG and phospholipids [38, 50]. Cytosolic Ca^"^ is increased by hormones acting via RTKs (and subsequent PLC-y activation) or via Gao-GTP and Gaq-GTP (and subsequent PLC activation), both types of pathways yielding DAG and IP3, the latter mobilizing Ca^"*" from the ER. Cytosolic Ca^"^ is also increased by transmembrane potential depolarizing neurotransmitters and opening of voltage-gated Ca^^ channels, the latter events being directly communicated to the ER ryanodine receptors (RyRs) in skeletal muscle (or indirectly via cADPR synthase and Ca^'*"-CaMactivated cADPR interaction with the ryanodine receptor in other muscle cells). Ca^"*" is removed from the cytosol across the plasma membrane by the NaVCa^"*" antiporter (driven by a Na^ gradient generated by the Na"^, K^-ATPase) and into the ER and out of the cell by Ca^^-ATPases. PKC isozymes are variously involved in growth control and transcriptional activation by phosphorylating specific transcription factors that interact with TREs (tetradecanoylphorbolacetate (TPA) response elements) (TPA and other phorbol esters being plant-derived or semi-synthetic phorbolbased PKC activators). PKC also regulates growth via phosphorylation of key components such as histone HI, the epidermal growth factor receptor (a RTK) and phosphorylation and activation of Raf (a MAPKKK) [38, 42] (see (f) below). d. Ca^^-calmodulin (CaM)-dependent protein kinases are activated by the Ca^"*'-dependent regulator protein calmodulin (CaM) [55] and include CaM-dependent PKs I-IV [55] and myosin light chain kinase (MLCK) [56]. CaMKn is involved in specific gene expression, neuronal transmission and memory consolidation. MLCK has a key regulatory role in muscle contraction and in smooth muscle phosphorylates myosin light chains, thus permitting actin-myosin interaction and muscle contraction [13-15]. e. 5'-AMP-dependent protein kinase (AMPK) is a heterotrimer that is activated by 5'-AMP and requires activating phosphorylation by 5'-AMPactivated AMP kinase kinase (AMPKK). AMPK couples nutrient stress-

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or exercise-induced elevation of 5'-AMP to requisite metabolic and other responses. Activated phospho-AMPK phosphorylates and thus inhibits various proteins with the following effects: acetyl CoA carboxylase (decreasing malonylcoenzyme A and thereby inhibiting fatty acid synthesis and stimulating fatty acyl carnitine synthetase and fatty acid oxidation), creatine kinase (inhibiting the enzyme) and hydroxymethylglutarylcoenzyme A synthetase (thereby inhibiting cholesterol synthesis). AMPK phosphorylates and activates epithelial NO synthase (elevated NO activating soluble guanylate cyclase leading to cGMP-induced vascular dilation) and AMPK is also evidently involved in promoting glucose transport through mobilizing glucose transporters and in inhibiting apoptosis and transcription of particular genes [57]. f. PKB is a key protein kinase that is activated as result of insulin binding to the insulin receptor (a RTK) as summarized in the following scheme: insulin-insulin RTK -^ RTKs dimerise and are activated —> autophosphorylation on cytoplasmic tyrosines —> insulin R substrates IRS1 and IRS-2 are phosphorylated on tyrosine residues ~> phosphorylated RTKs and IRSs interact with effector proteins via phosphotyrosinebinding SH2 domains and, in particular, wortmannin-inhibited phosphatidylinositol-3-kinase (PI3K) is activated -^ PI45P2 to PI345P3 -^ PI34P2 (via PI345P3 5'-phosphohydrolase) -> the second messengers PI345P3 and PI34P2 activate PDKl (and a supposed second protein kinase, PDK2) by binding to pleckstrin homology (PH) domains on these protein kinases —> N-terminal region PH domains on PKB also bind PI34P2 and PI345P3 —> PKB is threonine- and serine-phosphorylated by PDKl (and PDK2) -^ PICB activated. Activated PKB phosphorylates various proteins including phospho-Bad (resulting in phosphoBad sequestration by 14.3.3 proteins and inhibition of apoptosis), p70S6K (causing ribsomal S6 protein phosphorylation and translational activation) and glycogen synthase kinase 3 (GSK3) (the inactivated phosphoGSK3 being unable to phosphorylate glycogen synthase, the active dephosphoglycogen synthase catalysing glycogen synthesis). PKB also contributes to GLUT4 activation (resulting in its translocation to the PM and glucose transport), activation by phosphorylation of the glycogen targeting subunit of glycogen-bound PPl (site 1 phosphorylation causing PPl activation and dephosphorylation and activation of phospho-glycogen

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synthase) -> glycogen synthase active -^ glycogen synthesis) and phosphorylation of eIF4E binding protein (thereby abolishing inhibition of eIF4E-dependent mRNA translation) [39, 58]. g. Mitogen-activated protein kinases (MAPKs) (or ERKs, extracellular signal regulated kinases) are activated by signalling cascades initiated by various signals including hormones such as insulin binding to specific RTKs. Subsequently a variety of effector proteins bind to the activated and tyrosine-phosphorylated RTKs or to each other by phosphotyrosine-binding SH2 domains or by SH3 domains that recognize proline-rich protein regions. This type of signalling pathway can be illustrated by the following example: insulin binds to its RTK receptor —> RTK aggregation, activation and autophosphorylation —> IRS-1 and IRS-2 tyrosine phosphorylation -> SHC (an adaptor protein) binds -> GRB2 (a further adaptor protein) binds -^ Sos (guanyl nucleotide exchange factor = GEF) binds and is activated —> Ras activation to yield active Ras-GTP ~> activation (by Ras-GTP) and phosphorylation (by PKC) of Raf (a MARK kinase kinase or MAPKKK) -> serine phosphorylation of MAPK kinase (yielding activated phosphoMAPKK) --> threonine and tyrosine phosphorylation of MAPK (involving a threonine-glutamate-tyrosine sequence phosphorylation) -> transcription factor phosphorylation -> specific gene expression) [59]. h. Cell division protein kinases (CDKs) of eukaryotes critically regulate passage of cells through the cell cycle that involves successive Gl, S (DNA synthesis), G2 and M (mitosis) stages. Cycle stage-specific CDKs are activated by specific threonine and tyrosine dephosphorylation, a particular threonine phosphorylation and by interaction with stage-specific cyclin proteins. The overall process is regulated exquisitely by hormoneinduced signalling, the synthesis and degradation of specific cyclins, the activation or de-activation of specific serine/threonine or tyrosine-specific protein kinases and PPs and by other regulatory components [60]. i. Histidine kinases (present in yeast, slime moulds and plants) autophosphorylate on histidine and are involved in subsequent aspartate phosphorylation on target proteins and protein kinase cascade regulation [42, 61].

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Of course plants also have signal transduction pathways but some major differences are apparent. Thus while possible elements of a cyclic nucleotide regulatory system have been found in plants including cAMP, cGMP, adenylyl cyclase, guanylyl cyclase, cyclic nucleotide-hydrolyzing phosphodiesterases and a number of cyclic nucleotide-binding proteins, functional equivalents of PKA or PKG have not been found in plants [6265]. Nevertheless there is evidence for the involvement of cGMP in stomatal opening [66] and in plant responses mediated by phytochrome proteins [64]. Further, proteins homologous to the cyclic nucleotide-gated ion channels of other eukaryotes are present in higher plants and an Arabidopsis gene (GCRl) encodes a putative seven transmembrane element receptor-like protein with similarity to the Dictyostelium cAMP receptor protein [64, 65]. Microinjection experiments have provided evidence for cADPR, NO and cGMP in induction of plant defense responses [67]. Finally, elements of Ca -mediated signalling, namely Ca^"^ pumps and channels, CaM, CaM-dependent protein kinases and Ca^'*'-dependent protein kinases (CaM-domain protein kinases) (CDPKs) have been resolved from higher plants [68, 69]. It is notable that a homologue of plant CDPK is found in the malaria-causing protozoan Plasmodium falciparum but not in other non-plant eukaryotes, suggesting that inhibitors of this CDPK may have therapeutic potential [70]. With this sketch of major eukaryote signalling pathways in mind, we can now consider the interaction of particular plant defensive secondary metabolites with particular signal transduction components of non-plant eukaryotes such as fungi and animals that consume plants. The reader is referred to some major compilations for the structures of most of the plant defensive compounds mentioned [1, 5, 6]. SIGNAL TRANSDUCTION TARGETS FOR PLANT DEFENSIVE SECONDARY METABOLITES I. Ligand-gated ion channel neurotransmitter receptors a. Acetylcholine receptors (nicotinic) bind the piperidine alkaloid nicotine and are excitatory (depolarizing), presynaptic and postsynaptic, acetylcholine-gated NaVK"*" channels involved in neurotransmission, including neuromuscular transmission [71-74]. Major plant-derived

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agonists of this kind of receptor include the quinolizidine alkaloid cytisine and the piperidine alkaloids nicotine, anabasine, nornicotine, (-)-lobeline and (+)-coniine [75, 76]. (+)-Coniine is from hemlock, Conium maculatum, (the agent of Socrates' judicial death) and related piperidine alkaloids from this source include pseudoconhydrine, y-coniceine and (+)N-methylconiine [1]. A variety of isoquinoline alkaloids are nicotinic receptor antagonists and consequently neuromuscular transmission blockers, notably the highly toxic bisbenzylisoquinoline alkaloid (+)tubocurarine, an important component of South American Indian curare. Other curare-like isoquinoline alkaloids include further bisbenzylisoquinoline alkaloids (berbamine, dauricine, isochondrodendrine (isobebeerine), macoline and rodiasine), Erythrina isoquinoline alkaloids (erysinine, erysotrine, erythratidine, a-erythroidine, p-erythroidine and dehydro-p-erythroidine), isococculidine, berberine and magnoflorine [1]. A variety of toxic diterpenoid alkaloids have curare-like properties including avadharidine, condelphine, delcoline, delcorine, elatine, karacoline, lappaconitine, N-desacetyllappaconitine and methyllycaconitine [1, 77]. A variety of toxic indole alkaloids also have curare-like activity including calebassine, caracurine, C-curarine, sapargine and toxiferine I [1]. It is notable that the snake 8 kDa polypeptide toxin a-bungarotoxin is also a nicotinic receptor antagonist that causes neuromuscular blockage and skeletal muscle paralysis [72]. b. lonotropic GABA (Y-aminobutyric acid) receptors (GABA class A and C receptors) are inhibitory (hyperpolarizing) GABA-gated CY channels that have sequence homology with nicotinic acetylcholine and glycine receptors. GABA is the main inhibitory neurotransmitter of the mammalian central nervous system (CNS) and GABA agonists have potential as anxiolytics and anticonvulsants [78-81]. The class A receptors are hetero-oligomeric, are modulated by steroids, barbiturates and benzodiazepines and are blocked by the phthalideisoquinoline alkaloids (+)-bicuculline and N-methylbicuculline and by the alkaloids (-)securinine and (+)-tubocurarine [82, 83]. GABA A receptor natural product agonists include GABA itself, the Amanita mushroom oxazole alkaloids muscimol and dihydromuscimol and the piperidine alkaloid isoguvacine [83]. The homo-oligomeric GABA C receptors are insensitive to bicuculline, steroids, barbiturates and benzodiazepines but are activated by GABA, and muscimol and bind isoguvacine [78, 79, 84].

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c. lonotropic glutamate receptors include N-methyl-D-aspartate (NMDA)-binding glutamate receptors (excitatory (depolarizing) NaVK"*" and Ca^"^ channels whose subunit composition can be regulated by the type of innervating neuron). Non-NMDA binding glutamate receptors are excitatory (depolarizing) receptors that are Na"*"/K^ channels, the opening of which and the consequent partial depolarization permitting NMDA receptors to open in response to glutamate. Activation of GABA A receptors can also facilitate NMDA receptor activation [85-90]. NMDA (as well as non-NMDA receptors) participate in long-term potentiation (LTP) of synaptic transmission that is involved in memory consolidation [90]. Non-NMDA glutamate receptors include those binding a-amino-3hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA) or kainic acid. The neurotoxins kainic acid and domoic acid are agonists of the non-NMDA glutamate receptors. The activity of the non-NMDA glutamate receptors is enhanced by PKA-catalyzed phosphorylation as a result of Dl dopamine receptor-mediated increase in cAMP concentration. Kynurenic acid is a non-selective ionotropic glutamate receptor antagonist and glycine is involved as a co-agonist in activation of NMDA receptors (Dserine also acting as a co-agonist of NMDA receptors). p-N-oxalylaminoL-alanine (L-BOAA) (from Lathyrus sativus), the causal agent of neurolathyrism in humans, has an excitatory effect on AMPA receptors and binds to NMDA receptors [91]. p-N-methylamino-L-alanine (LBMAA) (present in Cycas circinalis and implicated in a type of dementia in Guam) is also an NMDA agonist as well as a non-NMDA receptor agonist, albeit at much higher concentrations [91, 92]. The peptide derivative S-(4-hydroxybenzyl)glutathione from Gastrodia elata binds to kainic acid-binding glutamate receptors [93]. Bioactive polyamines such as spermine are present in animals and plants [1], have antifungal activity and can modulate NMDA receptors [94]. d. lonotropic serotonin (5-hydroxytryptamine) receptors (5HT3 class receptors) are excitatory (depolarizing) Na'**/K"^ channels that are involved in emesis and anxiety [95]. Plant compounds binding to the 5HT3 receptor include serotonin (5-hydroxytryptamine) itself and related bioactive and psychotomimetic indole alkaloids [1]. Ethanol and other alcohols may act via effects on the 5HT3 receptor [96].

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e. Glycine receptors are inhibitory (hyperpolarizing) glycine-gated CI" channels involved in neurotransmission and a well-known antagonist is the highly toxic indole alkaloid strychnine [1]. The benzylisoquinoline alkaloid (-)-laudanidine can produce strychnine-like effects of paralysis [1]. II. G-protein-linked receptors a. Acetylcholine receptors (muscarinic) are G protein-linked receptors (Ml, M2 and M3 variants), the cardiac receptor activating a G protein complex with resultant opening of a K"*" channel by the dissociated G protein py^^^pl^^' hyperpolarization and diminution of contraction [97]. A similar mechanism can be involved with some other G protein-linked receptors including somatostatin, serotonin, o2 adrenergic, dopamine (D2), opioid (|Li and 6) and GAB A (B) receptors [97]. Plant agonists include L(+)-muscarine, pilocarpine, pilosine, norarecoline and arecoline [1, 97]. Muscarinic receptor antagonists include the tropane alkaloids hyoscamine, atropine (the hyoscamine racemate) and hyoscine (scopolamine) [1, 97-99], the benzylisoquinoline liriodenine [100] and the steroidal alkalord ebeinone [101]. There is current interest in muscarinic agonists for treatment of some people with Alzheimer's Disease [102]. b. Adrenergic receptors for epinephrine and norepinephrine are G protein-linked receptors initiating various signalling pathways depending upon whether they are a l (elevating Ca^^ through phospholipase C activation through Gaq), a2 (mostly inhibiting adenylyl cyclase through Gai and depolarizing) and p adrenergic receptors (pl-AR, P2-AR and p3-AR) (elevating cAMP) [103-107]. The indole alkaloid yohimbine and the aporphine isoquinoline alkaloid xylopinine are a receptor blockers [108] and the aporphine isoquinoline alkaloid isocorydine and the bisbenzylisoquinoline alkaloid oxyacanthine are also adrenergic antagonists [1]. The legume-derived bioactive agmatine (l-amino-4guanidinobutane) binds to a2-adrenergic receptors and to the nonadrenergic imidazoline receptors that bind the hypotensive drug clonidine [109]. L-norepinephrine and dopamine are present in some plants and act as agonists for both a- and p-adrenergic receptors [1]. Further plant padrenergic receptor agonists include the phenylpropanoid alkaloids L-

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ephedrine, pseudoephedrine, D-cathinone and D-cathine [110]. The adrenergic receptors have a critical function in cardiac function, blood pressure [111], metabolism [13-15] and immune responsiveness [112]. c. Dopamine receptors are excitatory receptors involved in neurotransmission. Dopamine Dl receptors act via Gs, adenylyl cyclase activation, cAMP and PKA-phosphorylated, voltage-gated K"*" channel closure. D2 receptors act via Gao/Gai proteins causing adenylyl cyclase inhibition and ion channel modulation [113]. Dopamine is elaborated by some plants and the isoquinoline alkaloid (-)-salsolinol is a dopamine antagonist [1]. The Aconitum alkaloid sangorine is a D2 agonist [115]. Dopamine signalling and interacting elements are important targets for treatment of Parkinson's disease and schizophrenia [116]. d. Serotonin receptors (5HT1, 5HT2 and 5HT4 receptors) are excitatory (depolarizing) G protein-linked receptors acting via cAMP and PKA-phosphorylated, voltage-gated K"*" channel closure [117]. Plant agonists include serotonin (5-hydroxytryptamine) itself and various indole alkaloids including bufotenin (5-hydroxy-N,N-dimethyl tryptamine), Omethylbufotenine, gramine, hordenine, ergotamine, N,N-dimethyl tryptamine, mescaline, norharmane, harmine, ergine and harmaline [1, 118-120]. The indole hallucinogens psilocin (from the mushroom Psilocybe mexicana) and the synthetic lysergic acid diethylamide (LSD) also act as serotonin agonists [119]. The xanthone y-mî^gostin is a serotonin antagonist [121] and the alkaloids annonaine, nornuciferine and asimilobine bind to 5-HTl receptors [122]. Serotonin receptors and signalling are targets for treatment of depression and migraine. e. Metabotropic glutamate receptors (as opposed to the ionotropic glutamate receptors) are 7 transmembrane helix domain receptors that act via G proteins to modulate K"*" and Ca^"^ channels [123]. The mushroom compound ibotenate (the precursor of the hallucinogen muscimol) is an agonist of some metabotropic glutamate receptors [123]. f. GABA receptors (B-class receptors) are 7-TM helix, G protein-linked receptors that are like metabotropic glutamate receptors and activate Ca^"*" and K"*" channels [124]. GABA is present in a wide variety of Leguminosae seeds [1].

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g. Histamine receptors (HI, H2 and H3 receptors) are G protein-linked receptors. Allergic responses such as inflammation and bronchoconstriction involve HI receptors, heart rate increase and gastric secretion involve H2 receptors [125] and vascular H3 receptors are involved in hypertension [126]. Histamine itself occurs in some plants and the quinoline alkaloid vasicinal (7-hydroxypeganine) and the steroidal alkaloid tomatine (lycopericin) have some histamine antagonist activity [1]. h. Purine receptors bind adenosine and adenine nucleotides, with adenosine binding more tightly than ATP to PI (Al and A2) receptors and ATP binding more tightly than adenosine to P2 receptors [127-130]. Adenosine and dopamine receptors can interact. Thus adenosine Al receptors and dopamine D2 receptors act via Gai to inhibit adenylyl cyclase and adenosine A2A and dopamine Dl receptors act via Gas and Gaolf to activate adenylyl cyclase. Adenosine binding to Al and A2A receptors decreases the affinity of dopamine for Dl and D2 receptors, respectively. Thus adenosine binding to A2A receptors inhibits dopamineD2-mediated increase in Ca^"^ and inhibition of adenylyl cyclase. Adenosine binding to Al receptors inhibits dopamine-Dl-induced activation of adenylyl cyclase [130]. Thus adenosine can modulate dopamine responses in the CNS [130] and can have sedative and anticonvulsant effects in addition to having vasodilatory, bronchoconstricting and metabolic effects [127, 131]. The methylxanthines theophylline and caffeine are adenosine Al and A2 receptor antagonists but chronic administration can upregulate adenosine receptors [127]. The G protein-linked P2 receptors act via Gai or Gaq but it should be noted that P2x receptors are ionotropic and affect NaVCa^"^ channel opening [129]. i. Cannabinoid receptors include the CBl receptors (high in the CNS and coupled via G proteins to inhibit adenylyl cyclase, close Ca^^ channels and open K^ channels) and CB2 receptors (present in immune cells and acting via Gai proteins to inhibit adenylyl cyclase). CBl and CB2 receptors bind A^-tetrahydrocannabinol (from marijuana. Cannabis sativa) as well as the endogenous ligand anandamide (arachidonylethanolamide)

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[132]. However A^-tetrahydrocannabinol anatagonizes the peripheral CB2 receptor while acting as an agonist for the CNS CBl receptor [133]. Cannabinoid receptors mediate the appetite stimulant and psychoactive effects of cannabinoids which have therapeutic potential for relief from nausea and pain [132]. j . Opiate receptors mediate analgesic and anti-tussive effects and a number of well-known plant natural product narcotics and analgesics are agonists of such receptors, mimicking the effects of the endogenous peptide endorphin and enkephalin ligands [134-137]. Such plant products include the morphinan isoquinoline alkaloids morphine (the diacetate being heroin), codeine (3-0-methylmorphine), neopine (p-codeine) and thebaine [1, 138]. 0-demethylation of thebaine yields oripavine that can have both agonist and antagonist effects on opioid receptors [139]. Other isoquinoline alkaloids that have anti-tussive (but not analgesic) effects and which bind at receptors distinct from codeine and other opiates are the benzylisoquinoline papaverine, narceine and the phthalide isoquinoline alkaloids a-narcotine and narcotoline [1]. The isoquinolines (-)-salsolinol and tetrahydropapaveroline bind to an opiate receptor with affinities comparable to those of enkephalins and their analgesic effects are overcome by the opiate receptor antagonist naloxone [140]. The indole alkaloids ibogaine, ibogamine, coronaridine and tabernanthine bind to opiate receptors [141]. Plant-derived opiates and related compounds are of major importance medically because of their pain-relieving and narcotic effects [137]. k. Receptors for a variety of other peptide and non-peptide hormones acting via G proteins are potential targets for plant defensive compounds, including peptides. Thus a variety of plant secondary metabolites are uterotonic including the cyclic peptide kalata which has oxytocin-like activity in stimulating uterine contraction [142]. However these uterotonic agents are not necessarily acting as oxytocin receptor agonists and other uterotonic mechanisms are possible. IIL Ion channels and ion pumps a. Voltage-gated Na"*" channels are critical for neurotransmission and cell excitability [143]. Inactivation of the channel is blocked by the steroidal

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alkaloid veratridine [143] and by the highly toxic diterpenoid alkaloid aconitine from Aconitum [144]. A variety of related diterpenoid alkaloids having aconitine-like effects include aconifine, bikhaconitine, delphinine, falaconitine, indaconitine, jesaconitine, mesaconitine and pseudoaconitine [1]. The diterpenoid alkaloids lappaconitine, N-deacetyllappaconitine and ajacine block the Na"^ channel and thus can act as antagonists of aconitine [145]. b. K"*" channels are similarly critical to transmembrane potential- and Ca^'^-mediated signalling. Some K"*" channels are voltage-gated, others are modulated by G proteins (that are in turn regulated by particular hormones such as dopamine or adenosine) [146] and others are subject to Ca^"*"dependent activation [147]. A Ca^"^-dependent K"*" channel is opened by the lignan nordihydroguaiaretic acid (NDGA) [148]. Voltage-regulated K"*" channels involved in action potentials are blocked by the quinolizidine alkaloid sparteine (lupinidine) as well as by synthetic aminopyridine drugs [149]. ATP-sensitive K"*" channels (KATP channels) are blocked by ATP and have various roles including modulation of muscle, synaptic and endocrine functions [150, 151]. KATP channels control insulin secretion from pancreatic p-cells, are inhibited by synthetic sulphonylurea drugs used in treating non-insulin-dependent diabetes melittus (NIDDM) [150, 151] and are also inhibited by the legume-derived quinolizidine alkaloid sparteine [152]. c. Intracellular Ca^^ channels and PM voltage-gated Ca^"*^ channels are involved in signalling [153-159] and are targets for various plant defensive compounds. PM-located voltage-gated Ca^^ channels of various kinds (L, N, P, Q, R and T classes) have been resolved of which L-type Ca^^ channels are the best studied [153-156]. The L-type Ca^"*" channels are blocked by various synthetic drugs including phenylalkylamines (notably verapamil), benzazepines, benzthioazepines (notably diltiazem) and dihydropyridines [153]. Intracellular, ER-located, ligand-gated Ca^^ channels include the inositol-1,4,5-triphosphate (IP3) receptor, the ryanodine receptor (RyR), the NAADP receptor and the sphingolipid receptor [157-159]. The IP3 receptor Ca^^ channel is opened by IP3 generated as a result of G protein(or RTK)-mediated PLC activation [159].

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Ryanodine receptor Ca^* channels are opened by cADPR in a Ca^^CaM-dependent fashion and Ca^^, the diterpenoid alkaloid ryanodine and methylxanthines such as caffeine promote opening of the channel [157, 160]. Ryanodine can also negatively modulate the receptor [157]. Ryanodine receptors are involved in excitation-contraction coupling in skeletal muscle through direct interactions with V-gated Ca^^ channels [161, 162]. Both cADPR and NAADP are synthesized by ADP-ribosyl cyclase from NAD and NADP, respectively [163, 164] and cADPR may act as a second messenger (in addition to IP3) for chemokines such as interleukin-2 [165]. However there appears to be no direct involvement of the ADP-ribosyl cyclase activity of the ectoprotein CDS8 on Tlymphocytes and intracellular Ca^"*" signalling [166]. NAADP receptor Ca^^ channels are opened by very low NAADP concentrations but is blocked at higher NAADP concentrations [157]. Sphingolipid receptor Ca^^ channels may be opened by sphingosine-1phosphate or sphingosyl-phosphorylcholine [157]. d. The Na"*", K'*"-ATPase generates the Na"*" and K^ gradient across the PM that is required for cell excitability, action potential transmission and for Na^ gradient-dependent transport of metabolites and indeed of signalling molecules [14]. A variety of C23 and C24 triterpenoid-derived cardenolide and bufadienolide steroid glycosides are highly toxic but have cardiotonic activity and are potent inhibitors of the Na"^, K'^-ATPase, including the cardenolides digoxin (digoxigenin 3-O-tridigitoxoside) from Digitalis lanata and ouabain (ouabagenin 3-O-L-rhamnoside) from Strophanthus gratus and the bufadienolide scillaren A (scillarenin 3-0glucosylrhamnoside) from Scilla maritima. The diterpenoid alkaloid cassaine is also a potent inhibitor of the Na"**, K^-ATPase and the structurally related diterpenoid alkaloids cassaidine and erythrophleguine have similar digitalis-like effects [1, 167]. Digitalis, the dried leaves of foxglove {Digitalis purpurea), contains the Na"^, K^-ATPase inhibitor digitoxin and has been used as a cardiotonic for centuries [1]. Inhibition of the Na"*", K'*'-ATPase increases intracellular Na"*" and hence decreases Ca^'^/Na'*' antiport activity. Consequent elevated cytosolic Ca^"^ concentration increases heart contractility [14]. Various flavonoids inhibit the Na"", K^-ATPase [168, 169].

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e. Ca^"*" ATPases pump Ca^^ out of the cytosol across the PM or into the ER and can be Ca^'*'-CaM-activated. Thapsigargin is a sesquiterpenoid cell activator and secondary tumor promoter from Thapsia species. Thapsigargin is an inhibitor of the Ca^"*"-ATPase and consequently elevates cytosolic Ca^"^ concentration [170]. The prenylated xanthone a-mangostin is also an inhibitor of the Ca^'^-ATPase [171]. IV. Neurotransmitter converters and transporters A key element in any signalling is reversibility and neurotransmission across synaptic junctions requires neurotransmitter conversion to inactive entities or neuronal neurotransmitter uptake. Various plant defensive compounds interfere with these processes. Synaptic neurotransmitter transporters include glutamate transporters (that couple glutamate translocation to Na^ and K^ movement) [41] and transporters for GAB A, glycine, taurine, norepinephrine, dopamine and serotonin (that are coupled to Na^ and CI" movement) [42-44]. In addition there are vesicular monoamine transporters of the chromaffin granules of the adrenal medulla, stomach oxyntic cells and neurons that are variously involved in transport of epinephrine, norepinephrine, histamine, serotonin and dopamine into secretory vesicles, these transport processes being coupled to H^ movement [41, 45, 46]. A vesicle glutamate transporter in glutaminergic nerve endings similarly concentrates glutamate into vesicles in a process driven by a H^ gradient generated by a H^-ATPase [41]. a. Acetylcholinesterase (AcChE) hydrolyses acetylcholine to acetate and choline [178]. Plant derived acetylcholinesterase inhibitors include the indole alkaloids eseramine, eseridine, physostigmine (eserine) and physovenine, the sesquiterpenoid alkaloid N-(phydroxyphenthyl)actinidine, the steroidal alkaloid P-demissidine, the Amarillidaceae alkaloid galanthamine (galantamine) and the quinazoline alkaloid vasicinol (7-hydroxy peganine) [1, 179]. All of these alkaloids, like acetylcholine, have a critical protonatable N recognized by the enzyme. Physostigmine, galantamine and other AcChE inhibitors are of current interest for Alzheimer's Disease therapy [179]. b. Monoamine oxidases (MAO-A and MAO-B) catalyze the oxidative deamination of monoamine neurotransmitters, notably serotonin (5-

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hydroxytryptamine) [108]. The plant indole alkaloids harmaline and harmine [180], the chalcone isoliquiritogenin [1] and various isoquinoline alkaloids [108] are inhibitors of monoamine oxidase. c. Synaptic glutamate transporters are PM-located Na^-dependent transporters that remove glutamate (the major CNS excitatory neurotransmitter and a potential neurotoxin) from the extracellular space. In some cases K"*" is countertransported. A number of these transporters have been cloned [172, 173, 181-183] and inhibitors include analogues of glutamate and aspartate [173, 183]. d. NaVCr-linked monoamine neurotransmitter transporters are PMlocated and specific for various neurotransmitters including GABA, norepinephrine, serotonin, dopamine, glycine and taurine [172] and are targeted by some antidepressants, stimulants and antihypertensives [174]. GABA transporters (GAT1-GAT4) recover GABA after its synaptic release [172] and are variously inhibited by the phthalideisoquinoline alkaloids bicucuUine and norbicuculline and by the piperidine alkaloid guvacine, 3-aminopropionic acid (p-alanine) and the synthetic nipecotic acid (3-piperidinecarboxylic acid) [184, 185]. Some GABA transporter antagonists have anticonvulsant effects as a consequence of elevation of synaptic GABA [186]. Synaptic glycine transporters (GlyTl and GIyT2) are responsible for Na'^^/Cr -linked glycine transport and are the major means of removing synaptic glycine [187, 188]. GlyTl is inhibited by sarcosine (Nmethylglycine) [187]. Plant phorbol esters downregulate GlyTl through activation of PKC and oleoylacetylglycerol mimics this effect which is blocked by PKC inhibition [188]. Synaptic dopamine transporters are inhibited by various psychotropic alkaloids including the tropane alkaloid cocaine [189, 190], the indole alkaloid ibogaine [191] and by amphetamine (methylphenethylamine) and related compounds [192, 193]. Synaptic serotonin (5-hydroxytryptamine) transporters are inhibited by amphetamines, the tropane alkaloids cocaine and ecgonine [194] and by the indole alkaloid ibogaine (12-methoxyibogamine) and its demethylation product ibogamine [191, 195]. Hyperforin is a major antidepressant constituent of St. John's Wort (Hypericum perforatum) and inhibits serotonin uptake by elevating cytosolic Na"^ [196]. The additional

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major component hypericin does not appear to affect monoamine uptake but rather binds to apomorphine-binding sigma receptors [197]. Selective serotonin transport inhibitors are potential antidepressants [198]. e. Vesicular monoamine transporters (VMATl and VMAT2) of neuroendocrine cells transport various monoamine neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin and histamine) from the cytosol into vesicles prior to exocytotic release from the cell surface. VMATl is present in central, peripheral and enteric neurones and VMAT2 is present in chromaffin cells of the adrenals and in histaminestoring enterochromaffin-like cells of the stomach oxyntic mucosa [ITSIT?]. The VMATs are inhibited by the indole alkaloid reserpine (that is employed as a tranquillizer and antihypertensive agent) and also by phenylethylamine, amphetamine and amphetamine-related compounds [199, 200]. f. Vesicular glutamate transporters located in glutaminergic nerve endings accumulate glutamate into synaptic vesicles prior to release. This process is coupled to a H"*" gradient generated by a H"^-ATPase [201]. LGlutamate is ingested by herbivores and is neurotoxic in excess [1]. V. Cyclic nucleotide signalling targets of plant bioactive compounds Cyclic AMP acts as a hunger signal in non-plant eukaryotes that consume plants and its metabolic effects are mediated by PKA as outlined above [13-15]. This provides a rationale for the inhibition of PKA by representatives of a number of major classes of plant defensive secondary metabolites. PKA is required for pathogenicity towards plants of a variety of fungal pathogens [65, 202-206] and accordingly inhibition of PKA would be expected to interfere with fungal invasion. PKA is also involved in specific gene expression and development in higher organisms and is involved in cognitive processes of animals. Accordingly, inhibition of PKA might be expected to interfere with pathogenic fungal growth and perturb cognitive processes of herbivores such as insects [65]. Conversely, agents that elevate cAMP in animal cells can be toxic as evidenced by the toxic cAMP-elevating effect of cholera toxin in animal intestinal cells that results in excessive Na"*" and H2O loss [14, 207]. Similarly, Bordetella pertussis elaborates an invasive, Ca^^-CaM-activated adenylyl cyclase and

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hence elevates cAMP in the target animal cells [208]. This toxic effect of cAMP elevation provides a rationale for the defensive utility of adenylyl cyclase activation by the plant diterpenoid forskolin [209, 210] and of cAMP phosphodiesterase inhibition by plant-derived methyl xanthines such as theophylline and caffeine [211]. a. Adenylyl cyclase catalyzes the formation of cAMP from ATP and is indirectly modulated by a variety of plant natural products that interact with G protein-linked receptors as outlined above. The plant diterpene forskolin binds to the catalytic core of adenylyl cyclase and activates the enzyme [209, 210]. b. Cyclic AMP phosphodiesterases (cAMP PDEs) catalyze the hydrolysis of cAMP to 5'-AMP and cAMP PDE inhibition consequently elevates cellular cAMP concentration. There is a multiplicity of animal cyclic nucleotide PDEs [211-214] and these are variously inhibited by plant metabolites including methyl xanthines (such as caffeine, theophylline and theobromine) [211-217], a number of saponins [218], the chalcone isoliquiritigenin [219], the protopine alkaloid allocryptopine (phomochelidone or a-fagarine) [220], the benzylisoquinoline alkaloid papaverine [221], biflavones (amentoflavone, bilobetin, sequoiaflavone and ginkgetin) [222], the isoquinoline alkaloid atherosperminine [223], phenylpropanoid glycosides [224], dihydropyranocoumarins [225], dihydrofuranocoumarins [225] and a range of flavonoids [226]. c. Membrane-associated or soluble guanylyl cyclases catalyze the formation of cGMP from GTP [29, 227]. Atrial natriuretic factor (ANF) activates membrane-associated guanylyl cyclase (29, 227). A peptide having ANF-like activity in stimulating cGMP synthesis has been resolved from plants [228]. Soluble guanylyl cyclase is activated by NO which in turn is synthesized by NO synthase (NOS) [229-232]. Soluble guanylyl cyclase can be activated by CO generated by the heme oxygenase system [233]. NO is synthesized in plants [67] and the plant-derived vasorelaxant chalcone isoliquiritigenin activates soluble guanylyl cyclase [234]. Particular NOSs are activated by Ca^'^-CaM or regulated by phosphorylation by signal-regulated protein kinases [231] and plants elaborate a variety of secondary metabolites that are protein kinase inhibitors [235, 236].

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d. Cyclic GMP phosphodiesterases (cGMP PDEs) catalyze the hydrolysis of cGMP to 5'-GMP. A number of cGMP PDEs have been resolved from animal tissues and these enzymes are variously inhibited by some plant natural products including methyl xanthines and papaverine [211-217] . Cyclic GMP acting via PKG (or through cGMP-gated ion channels) has a vasodilatory effect of importance in relief of angina and of erectile disfunction, the synthetic specific cGMP PDE inhibitor viagra (sildenafil) being of importance for treatment of impotence [213]. e. Cyclic AMP-dependent protein kinase (PKA) mediates catabolite deficiency or hunger signalling resulting in specific gene transcription and metabolic alteration toward glycogenolysis, gluconeogenesis and fatty acid oxidation [13-15]. Representatives of some major classes of plant defensive secondary metabolites are inhibitors of PKA [235, 236]. Such plant-derived PKA inhibitors include flavonoids (3-hydroxyflavone, 5hydroxyflavone, 5,4'-dihydroxyflavone, 5,7-dihydroxyflavone, 3',4'dihydroxyflavone, 7,8-dihydroxyflavone, 3,3',4'-trihydroxyflavone, galangin, apigenin, 7-0-methyl-apigenin, 2,3-dihydroapigenin, naringin, luteolin, 2,3-dihydroluteolin, 3-0-methyl-2,3-dihydroluteolin, kaempferol, 4'-0-methyl-kaempferol, 7,8,3',4'-tetrahydroxyflavone, fisetin, 2,3dihydrofisetin, 2,3-dihydrofisetin, quercetin, 2,3-dihydroquercetin, 3-0rhamnosyl-quercetin, morin, tricetin, myricetin, quercetagenin, 3',4',5'-tri0-methyl-tricetin and hesperidin) [237], flavanols ((+)-catechin and (-)epicatechin) [237], a phenolic dimeric sesquiterpenoid (gossypol) [237], anthraquinones (alizarin, quinizarin, anthrarufin, chrysazine, anthraflavic acid, chrysophanic acid, emodin, purpurin and quinalizarin) [238], prenylated xanthones (y-mangostin and a-mangostin) [239], hydrolyzable tannins ( a range of such compounds with differing numbers of phenolic substitutents) [240], condensed tannins (a range of such catechin-based flavans with different degrees of complexity e.g. procyanidin dimer, trinmer and tetramer entities) [241, 242], amphiphilic triterpenoids (18-aglycyrrhetinic acid, 18-p-glycyrrhetinic acid, ursolic acid, oleanolic acid, betulin and asiatic acid) [243], a phenylpropanoid (curcumin) [244], prenylated isoflavones (warangolone, 8-y,Y-dimethylallylwighteone, 3'Y,Y-dimethylallylwighteone and nallanin) [245], an aporphine alkaloid (apomorphine) [246], a benzophenanthridine alkaloid (sanguinarine)

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[246], a stilbenoid (piceatannol) [247] and the phenolic ellagic acid [247]. A common feature of these compounds is a planar or quasi-planar hydrophobic nucleus linked to a hydrophilic element, a motif also found with a variety of potent PKA inhibitors that are synthetic [248] or microbe-derived [248, 249] as well as with PKA inhibitors that are synthetic phenanthrene-based [250], acridine-based [251, 252] or isoquinoline-based compounds [253], oxazine alkaloids (darrow red, nile blue A and oxazine 170) [246] and semi-synthetic prenylated xanthone derivatives [254]. This pattern suggests a possible binding site at the hydrophobic cleft of the PKA catalytic subunit that accomodates the purine ring of ATP [36]. However a variation from this pattern is found with some hydrophobic anti-inflammatory triterpenoid esters that are PKA inhibitors [256]. The naturally occurring PKA inhibitors described above are not necessarily specific for PKA. Thus various anti-inflammatory triterpenoid PKA inhibitors are also protease inhibitors [243, 255, 256]. Further, a variety of flavonoids that inhibit PKA also inhibit other protein kinases [235, 236] as well as inhibiting cyclic nucleotide PDE [226] and Na"',K''-ATPase[168, 169]. VI, Ca^^-mediated signalling targets The transient, signal-induced elevation of cytsolic free Ca^"*" concentration switches on a variety of Ca^^- and Ca^"*"-calmodulin (CaM)-dependent enzymes, notably protein kinases. Plants contain CaM and CaM-regulated enzymes [68] and accordingly elaboration of CaM antagonists would be damaging unless the CaM antagonists were stored in an inactive form or in a location remote from cytosolic CaM (e.g. in seeds or plant cell walls). Similarly CDPK rather than PKC represents a major means of mediating Ca^"*" signals in plants. As outlined below, plants elaborate a variety of PKC inhibitors and activators. a. The PKC family of protein kinases contains a variety of isozymes that are variously activated by Ca^"*", diacylglycerol (DAG) and phospholipids. PKC is activated by plant phorbol esters (tigliane diterpenoids) that act at the DAG-binding domain. The phorbol esters such as phorbol-12,13-dibutyrate (PDB) and 12-0-tetradecanoyl-phorbol13-acetate (TPA) are highly inflammatory compounds and secondary tumour promoters [38]. A variety of other PKC activators are elaborated

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by plants including other tigliane diterpenoid esters (e.g. those based on 4deoxyphorbol, 12-deoxyphorbol and 4,20-dideoxy-5-hydroxyphorbol nuclei), daphnane diterpenoids (daphnoretin, gnidimacrin) [257, 258], pyranocoumarins (decursin and decursin angelate) [259], a spiroiridal triterpenoid (28-deacetylbelamcandal) [260] and ingenol diterpenoids (17hydroxyingenol-20-hexadecanoate, ingenol-20-hexadecanoate, ent16a,17-dihydroxyatisan-3-one and ent-3p, 16a, 17-trihydroxyatisane) [261]. A glycosylated hydroxy fatty acid, namely glycosylated (IIS)hydroxyhexadecanoic acid (tricolorin A), displaces TPA from PKC [262]. The secondary tumour promoting (co-carcinogen) activity of phorbol esters can be rationalized in terms of an increase in levels of signalling pathway phosphoproteins phosphorylated by PKC and predisposing cells to transformation. The same rationalization can be applied to the secondary tumour promoting activity of thapsigargin (Ca^'^'-ATPase 9-4-

inhibition leading to increased Ca concentration and hence PKC activation) and of the PPl and PP2A inhibitors okadaic acid (from dinoflagellates) and the Microcystis microcystins (inhibition of dephosphorylation of the PKC-phosphorylated phosphoprotein) [18]. A variety of plant natural products inhibit PKC including flavonoids ( fisetin, quercetin, luteolin, hirsutenone, oregonin and hirsutanonol) [263], a sesquiterpenoid (gossypol) [264], anthraquinones (alizarin, quinizarin, anthrarufin, chrysazine, anthraflavic acid, chrysophanic acid, emodin, purpurin and quinalizarin) [238], hydrolyzable tannins [240], condensed tannins [241, 242], a phenylpropanoid (curcumin) [244], an aporphine isoquinoline alkaloid (apomorphine) [246], a stilbenoid (piceatannol) [247], the phenolic ellagic acid [247], diarylheptanoids (glycosylated (3R)-l,7-bis(3,4-dihydroxyphenyl)heptan-3-ol and the aglycone) [265], phenylethanoid glycosides (calceolareoside A, calceolsareoside B, forsythiaside, plantainoside D, leucosceptoside, acteoside and poliumoside) [266], corosolic acid [267], coumaryl glycosides (vanicoside A and vanicoside B) [268] and a xanthone (norathyriol) [269], noting that some semi-synthetic prenylated xanthones also inhibit PKC [254]. The stilbenoid magnolol inhibits phorbol ester activation of PKC [270]. PKC is involved in mitogenic signalling and inflammatory responses and some PKC inhibitors accordingly have anti-inflammatory effects [38]. b. The Ca^^-binding regulator protein calmodulin (CaM) activates a variety of enzymes including CaM-dependent kinases such as myosin

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light chain kinase (MLCK). Inhibition of CaM-dependent protein kinases could occur via inhibitor binding to CaM and affecting its function or through the inhibitor binding to the protein kinase. Thus the flavonoid quercetin and the sesquiterpenoid gosspol are inhibitors of CaMdependent MLCK and also interact with CaM as detected through 94-

inhibition of Ca -dependent enhancement of dansyl-CaM fluorescence [237, 271]. Further plant-derived MLCK inhibitors include other flavonoids (5,4'-dihydroxyflavone, 5,7-dihydroxyflavone, 3,3',4'trihydroxyflavone, galangin, apigenin, naringin, luteolin, kaempferol, 4'0-methyl-kaempferol, fisetin, morin, tricetin and myricetin) [271, 272], a phenolic dimeric sesquiterpenoid (gossypol) [271], anthraquinones (alizarin, quinizarin, chrysazine, anthraflavic acid, emodin, purpurin and quinalizarin) [238], condensed tannins [241, 242], an isoflavone (genistein) [245], aporphine alkaloids (apomorphine and boldine) [246] and a stilbenoid (piceatannol) [247]. c. Ca^^-dependent protein kinase or calmodulin domain-containing protein kinase (CDPK) is a major protein kinase in higher plants that is switched on by micromolar free Ca^"*" concentration [68, 69]. This type of protein kinase is not found in other eukaryotes except for Plasmodium, the protozoan causing malaria. Accordingly there is interest in Plasmodium CDPK as a potential target for selective chemotherapy [70]. Not surprisingly, plant CDPK is not inhibited by a wide range of plant-derived flavonoids (with the exception of 3,3',4'-trihydroxyflavone, 7,8,3',4'tetrahydroxyflavone, quercetin, tricetin and myricetin, noting that glycosylation of quercetin or methylation of tricetin abolishes inhibitory effectiveness) [271]. Of a wide range of anthraquinone-based PKA and PKC inhibitors tested, only purpurin is a good inhibitor of CDPK [238] and a variety of amphiphilic triterpenoid inhibitors of PKA do not inhibit plant CDPK [243]. The prenylated xanthones y-^ângostin and amangostin are good inhibitors of plant CDPK but these defensive components are located in the hull of the mangosteen fruit and hence away from the CDPK-containing cytosol [239]. Similarly, a variety of bark- and fruit-derived procyanidin and prodelphinidin-based condensed tannins are inhibitors of CDPK [241, 242]. CDPK is also inhibited by genistein [245] and the stilbenoid protein kinase inhibitor piceatannol [247].

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d. A large number of other serine/threonine protein kinases are encoded by higher organism genomes thus far examined [42, 50] and are likely to interact with plant defensive secondary metabolites. Serine/threonine-specific protein kinases that are involved in signalling cascades initiated by receptor tyrosine kinases (RTKs) are potential targets for the design of drugs against cancer and metabolic disease. Thus PKB is a serine/threonine protein kinase that is switched on by a pathway initiated by an insulin-insulin receptor tyrosine kinase interaction and PKB phosphorylates and reversibly inactivates a glycogen synthase kinase (GSK3). GSK3 is an enzyme that in turn phosphorylates and reversibly inactivates glycogen synthase. Accordingly, a synthetic compound or natural product that inhibits GSK3 might conceivably mimic insulinpromotion of glycogen synthesis [58]. However it must be appreciated that the catalytic domains of protein kinases are similar [36, 273-276] and, as is apparent from the above survey, there can be substantial overlaps of specificity of protein kinase inhibitors that interact with the catalytic domains of serine/threonine-specific (and indeed of tyrosine-specific) protein kinases. Thus casein kinase 11 is inhibited by the anthraquinone emodin [276] as are PKA and PKC [238]. Further examples of such protein kinase inhibition overlaps are given below. VII. Tyrosine kinase-mediated signalling Receptor tyrosine kinase-mediated signalling can be subject to interference by plant natural products at various levels. Thus plant inhibitory agents could act as agonists or antagonists with respect to particular receptors or inhibit downstream components such as RTK tyrosine kinase activity, Ras, Raf, MAPK, Src (a tyrosine kinase) or phospholipase C-y (PLCy). Thus PLCy is inhibited by the flavonoid amentoflavone [277] and a variety of flavonoids and other phenolic plant bioactives inhibit Src [278]. A further complexity is introduced if one considers "cross-talk" between pathways. Thus PKC can phosphorylate particular RTKs [22] and alter their function and is also involved in the activation by phosphorylation of the serine/threonine protein kinase Raf (a MAPKKK) [279, 280]. As outlined above, a variety of plant bioactives inhibit PKC. Genes encoding RTK-mediated pathway components (protooncogenes) can undergo mutation to oncogenes whose expression can contribute to oncogenic transformation. Accordingly there is much

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interest in synthetic or naturally-occurring substances that can inhibit the function of such oncogene products and hence inhibit or reverse cancerous transformation [281, 282]. RTKs bind the activating hormone and then phosphorylate target proteins or bind target adaptor and effector proteins through phosphotyrosine-SH2 domain interactions. A variety of plant natural products have been found which directly inhibit the tyrosine kinase activity of RTKs and in many cases also inhibit cell proliferation. Such plant-derived RTK inhibitors include polyphenols (purpurogallin [283], (-)-epigallocatechin gallate [284] and butein [285]), anthraquinones (emodin [286] and damnacanthal [287]), isoflavonoids (kievitone, genistein and genistin [288]), hydroxystilbenes (resveratol [289] and piceatannol [290]), flavonoids (desmal [291], kaempferol and quercetin [292]), the phenolic ellagic acid and synthetic compounds related to ellagic acid [293]. While piceatannol and genistein have been applied as selective tyrosine kinase inhibitors, piceatannol also inhibits PKA, PKC, MLCK and plant CDPK [247] and genistein also inhibits MLCK and (albeit poorly) PKA [245]. Ellagic acid is also an inhibitor of PKA and PKC [247], as is emodin [238]. VIII. Steroid hormone, taste and smell receptors Finally, brief mention must be made of plant compounds that mimic steroid hormones, notably the phytoestrogens (including particular isoflavones, coumestans and lignans) that bind to estrogen receptors [294], compounds that mimic insect developmental hormones such as juvenile hormone (including the sesquiterpenoids dehydrojuvabione, juvabione and juvenile hormone HI) [1] and compounds mimicking insect pheromones (such as the sesquiterpenoids a- and P-famesene) [1]. Taste receptors variously bind bitter and sweet entities, the signalling pathways involving changes in membrane conductance after ligand binding [295]. A variety of monosaccharides and disaccharides bind to sweet receptors and a notable bitter receptor ligand is quinine [1]. Smell signal transduction involves adenylyl cyclase, cAMP and cAMP-gated Na^ channels [46] and can also involve Ca^"^ and cGMP as second messengers [47]. Indoles such as skatole and indole itself are well known examples of

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"bad smelling" ligands that can act as insect attractants and "nice smelling" ligands include monoterpenes such as camphol [1]. FUTURE DIRECTIONS As indicated at the outset of this broad-based review, although of the order of ten thousand plant defensive compounds have been isolated and structurally characterized, only of the order of a thousand have so far been shown to interact with specific proteins and most of these biochemical sites of interaction are signal transduction components. The elucidation of such targets now permits rapid biochemical screening of complex ecosystems for plant-derived ligands for such proteins. Incisive technologies, including plasmon resonance-based analysis, are now available for rapidly quantitating such protein-ligand interactions. The recent major advances in genome sequencing and X-ray crystallographic structure determination of members of major protein families (most notably of protein kinases and hormone receptors, including receptor tyrosine kinases) will permit a more exacting progress towards filling in the plant defensive metabolite-eukaryote target protein matrix. ABBREVIATIONS AC AcCh AMPA AMPK AMPKK ANF cADPR cAMP CaM CaMPK CDPK CNS

= Adenylyl Cyclase = Acetylcholine = a-Amino-3-hydroxy-5-methyl-4-isoxazoleprionic Acid = 5'-AMP-dependent Protein Kinase = 5'-AMP-dependent Protein Kinase Kinase = Atrial Natriuretic Factor = Cyclic Adenosine-5'-diphosphateribose = 3',5'-Cyclic AMP = Calmodulin = Ca^^-CaM-dependent Protein Kinase = Ca^^'-dependent Protein ICinase = Central Nervous System

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CREB protein = cGMP = DAG = = ER = G = GABA = GC GEF = = GSK3 = H = IP3 = IP4 = JAK = L = L-BMAA = L-BOAA = LSD = MAPK = MAPKK = MAPKKK = MLCK = NAADP = NDGA = NIDDM = NMDA = NOS = NT = P = PDE = PDK = PI3K = PI34P2 = PI45P2 = PI345P3 = PK = PKA = PKB = PKC

cAMP Response Element Binding Protein 3',5'-Cyclic GMP Diacylglycerol Endoplasmic Reticulum G Protein y-Aminobutyric Acid Guanylyl Cyclase Guanyl Nucleotide Exchange Factor Glycogen Synthase Kinase 3 Hormone Inositol-1,4,5-triphosphate Inositol-1,3.4,5-triphosphate Janus Kinase Ligand P-N-Methylamino-L-alanine P-N-Oxazylamino-L-alanine Lysergic Acid Diethylamide Mitogen-activated Protein Kinase Mitogen-activated Protein Kinase Kinase Mitogen-activated Protein Kinase Kinase Kinase Myosin Light Chain Kinase Nicotinic Acid Adenine Dinucleotide Phosphate Nordihydroguaiaretic acid Non-Insulin-dependent Diabetes Melittus N-Methyl-D-aspartate Nitric Oxide Synthase Neurotransmitter Permeability of membrane to specific solutes Phosphodiesterase Phosphatidylinositol lipid-dependent Protein Kinase Phosphatidylinositol-3-kinase Phosphatidylinositol-3,4-bisphosphate Phosphatidylinositol-4,5-bisphosphate Phosphatidylinositol-3,4,5-triphosphate Protein Kinase cAMP-dependent Protein Kinase Insulin signalling-activated Protein Kinase Ca^^- and Phospholipid-dependent Protein Kinase

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PKG PLC PLCy PM PP P-Pr P-STAT PTPase R Ras Raf SM S/T-RPK RPTP RTK STAT TGFp TPA TRE AP A^

cGMP-dependent Protein Kinase Phospholipase C Phospholipase C-y Plasma Membrane Phosphoprotein Phosphatase Phosphoprotein Tyrosine Phosphorylated STAT Protein Phosphotyrosine Phosphatase Receptor Small G protein a MAPK Kinase Kinase Secondary Metabolite Receptor Serine/Threonine Receptor Protein Kinase Receptor Protein Phosphotyrosine Phosphatase Receptor Tyrosine Kinase Signal Transducer and Activator of Transcription Transforming Growth Factor p Tetradecanoylphorbolacetate Tetradecanoylphorbolacetate Response Element Change in Permeability of PM to specific solutes Change in Transmembrane Potential.

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FLAVONOIDS AND CARDIOVASCULAR DISEASES JUAN DUARTE; FRANCISCO PEREZ-VIZCAINO^ J0S6 JIMENEZ; JUAN TAMARGO^; ANTONIO ZARZUELO University of Granada, Department of Pharmacology, School of Pharmacy, Granada, Spain; and ^ University Complutense ofMadrid, Department of Pharmacology, School of Medicine, Madrid, Spain. ABSTRACT: Several epidemiological studies have found an inverse correlation between the dietary flavonoid intake and a reduced mortality from coronary heart disease and the incidence of stroke. We will focus our review on several mechanisms which have been suggested to explain these protective effects. a) Antiatherogenic eiffects. Flavonoids together with others antioxidants constitute two lines of defense in protecting cells against injury owing to oxidation of LDL: 1) at the LDL level, by inhibiting LDL oxidation due to their free radical scavenger activity, and 2) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidised LDL. Additionally, recent studies indicate that flavonoids can prevent the expression of adhesion and chemoattractant molecules. b) Antiaggregant effects. Flavonoids prevent platelet aggregation induced by several proaggregant stimuli although relatively high doses are required. Inhibition of platelet phosphodiesterases, inhibition of arachidonic acid metabolism and antioxidant effects have been suggested as possible mechanisms of action. c) Direct effects on vascular smooth muscle. The vasodilator effects of flavonoids in vitro is mainly endothelium-independent. The main mechanism of action seems to be related to their inhibitory effects on protein kinases. Some flavonoids, however, can produce endothelium-dependent contractile responses due to increased TXA2 production. d) Antihypertensive effects. Little information about the effects offlavonoidson blood pressure is available. However, recently, the chronic oral administration of quercetin has been shown to exert potent antihypertensive effects.

INTRODUCTION Flavonoids coiiq)rise a large group of secondary metabolites occurring widely throughout the plant kingdom. These low molecular weight polyphenolic compounds are foimd practically in all parts of the plants including fruit, vegetables, nuts, seed, flowers, and bark and are integral

566

part of the human diet. The term flavonoid as currently used refers to a large group of chemical compounds share the common skeleton of diphenylpyrans (C6-C3-C6). This basic structure allows a multitude of substitution patterns and variations leading several flavonoid classes, including flavonols, flavones, flavanones, catechins (or flavanols), anthocyanidins, isoflavones, dihydroflavonols, and chalcones Fig. (1). Over 4000 different flavonoids have been described, and the number is still increasing [1-3]. Until recently, the estimations most frequently cited pointed to a daily himianflavonoidintake of more than 500 mg/day (total flavonoids) [4,5]. However, more recent and accurate estimations gave an average of 23 mg/day ia of selected flavonols plus flavones [6] but this value varied among countries ranging from 6 mg/day in Finland to 64 mg/day in Japan [7]. The main sources offlavonoidsin the human diet are onions, apples, grapes, wine, tea, berries, herbs and spices. Among dietary flavonoids, quercetin is by far the most abimdant. A very wide range of biological actions offlavonoidshave been reported including antibacterial, antiviral, anti-inflammatory, antiallergic, antihepatotoxic, antiulcer, analgesic, hypoglycaemic, estrogenic, and antidiarrheic (for a review see [8-11]). In recent years, after the publication of several epidemiological studies showing an inverse association between the consumption of fruit and vegetables and cancer risk and mortality from coronary heart diseases, particular attention has been paid to their antitnutagenic, anticarcinogenic and cardioprotective activities (reviewed in ref [3,12]). In the present review, we analyse the cardiovascular effects of flavonoids in both in vitro and in vivo models and also the possible mechanisms inq)licated in the cardioprotective effects of these compounds. We will focus our review on the current status of research in these main topics: a) Their antiatherogenic effects and its relationship with their antioxidant properties (inhibition of low density lipoprotein [LDL] oxidation), b) the inhibition of platelet aggregation, c) the mechanisms involved in their vascular effects and their antihypertensive properties. EPIDEMIOLOGICAL STUDIES Cardiovascular diseases remain the main cause of death in developed countries. Studies relating the intake of dietary flavonoids to risk of cardiovascular disease (mortalityfromcoronary heart disease, incidence of

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FLAVANONES

CATECHINS

^^X ANTHOCYANIDINS

CHALCONES

Fig. (1). Generic structure of flavonoids

ISOFLAVONES

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myocardial inferction and stroke) have been observational in nature. Several studies have shown thatflavonoidintake was inversely related with mortality from coronary heart disease (table 1). Epidemiological studies in The Netherlands [6,13], Finland [14] and USA [15] as well as a crosscultural study performed in 16 cohorts from seven countries [7], have shown a significant inverse association between dietary flavonoids (mainly quercetin) and long term mortality from coronary heart disease and to a lesser extent with incidence of first myocardial inferction [6]. A study carried out in male USA health professionals found a modest non significant inverse association regarding long term mortality from coronary heart disease in men with previous history of ischaemic heart disease [16]. In contrast to the above studies, in the Caerphilly study (in which the major source of flavonoids was tea with milk) found a non significant increased mortality from ischaemic heart disease and a significant increase in total mortality in all quartiles of high flavonoid intake compared to the lowest quartile [17]. They hypothesised that this result may have been due to binding offlavonoidin tea with protein from milk, thus reducing flavonoid absorption. To summarise, a significant protective role for flavonoids in coronary heart disease was found in 3 out of 5 prospective studies, in addition to one cross-cultural study. One study showed a weak inverse and another a weak positive association between flavonol consumption and mortalityfromischaemic heart disease (Table 1). Additionally, men in the highest quartile of flavonol and flavone intake showed a reduced incidence of stroke in a Dutch cohort [18], while a prospective study in postmenopausal women foimd no association between totalflavonoidintake and stroke mortality [15]. The potential limitations, common in all epidemiological studies of diet and disease, are: a) mis-classification of dietary exposures, and b) lack of measure of changes in diet that occurred during the follow-up period, as their analyses were based on information from a single food frequency questionnaire at the beginning of the studies. The daily consumption of flavonoids is difficult to determine because the dietary supply is strongly dependent on feeding habits and, in this field, exhaustive tables on food composition are not always available. So, given the limitations of the diet assessment, the epidemiological evidence points to a protective effect of flavonols in cardiovascular disease but it is not conclusive.

Table 1. Summary of epidemiological prospective studies on flavonoid intake and coronary heart disease (CHD), myocardial infarction (MI) and stroke risk.

7

Zotpben Elderly Zotphen Finland (Tbe Netherlaods) (Tbe Netberlaoda)

Gender

Male

Male

Age (y) Outcome

65-84 (I) CHD mortality (2) MI incidence

50-69 Stroke incidence

Follow-up (y) Population size Relative risk (95% C.I.)

5 15 805 552 (I) 0.32 (0.15-0.71) 0.27 (0.1 14.70) (2) 0.52 (0.22-1 2.3)

CHD mortality

Scvcn Coootries

Female 49-59

CHD mortality

-

Japan) 2.6 (West

intake (mglday) (high vs. low) women Refaenw

1 113)

1 [IS]

[I 41

[I 61

[I 7l

[I 51

* Relative risk of highest versus lowest flavonoi 1 intake group, adjusted for agc diet and other risk factors for coronary heart disease. (C.I. = confidence interval)

i

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Furthermore, polyphenolics present in wine, of which flavonoids are important components, have been suggested to be responsible of the so called French paradox, that is, the unexpectedly low rate of mortality from coronary heart disease in French population despite an unfavourable e>qposure to known cardiovascular risk factors such as high saturated fat consumption [19-21]. Epidemiological studies in USA [22] and Denmark [23] reported that moderate red wine drinkers had a lower risk of coronary artery disease than participants with no alcoholic beverage preference. However, controversial results about the antioxidant capacity of human serum after red wine consuniption have been reported [24-27]. It is therefore uncertain whether wine constituents other than alcohol add to the cardioprotective effects of red wine. Tea is another important dietary source forflavonoidSpInfeet,about half of the flavonoid intake in western populations is derived from black tea. Tea was the major source of flavonoids in the Dutch [6,13] and Welsh studies [17]. Only a small number of studies investigated the association between tea consumption and cardiovascular disease risk. No association between tea consimiption and cardiovascular disease risk were reported in Scottish men and women [28] and in U.S. men in the Health Professionals foUow-up study [29]. However, in a Norwegian population an inverse association was reported between tea intake, serum cholesterol, and mortality from coronary heart disease [30]. Several studies reported that tea consumption did not affect plasma antioxidant activity [31] and hemostatic factors [32]. However, a recent prospective study (the Rotterdam study) of 3,454 men and women 55 years and older followed for 2 to 3 years, showed a significant, inverse association of tea intake with severe (> 5 cm the length of the calcified area) aortic atherosclerosis. Odds ratios decreased approximately 70 % for drinking more than 500 mL/day (4 cups per day). The associations were stronger in women than in men. However, the risk reductions for moderate and mild atherosclerosis were only weak or absent [33]. The protective role of dietaryflavonoidsagainst cardiovascular diseases in the above mentioned studies has been attributed to their inhibitory effects on LDL oxidation, their inhibitory effects on platelet aggregation. PHARMACOKINETICS A limitation for the understanding and relevance of these epidemiological

571

Studies was the scarce and conflicting data on the pharmacokinetics of flavonoids (for a review see [5,12]. Absorption from the diet is a prerequisite for a causal relation between flavonols and coronary heart disease prevention. In addition, the metabolism of flavonoids after absorption should not substantially inhibit their biological activities. Absorption of flavonoids from the diet was long considered to be negligible, as they are mostly bound to sugars as p-glycosides (with exception of catechins). Onlyfreeflavonoidswithout a sugar molecule, the so-called aglycones, were considered to be able to pass the gut wall, and no enzymes that can split these predominantly p-glycosidic bonds were thought to be secreted into the gut or present in the intestinal wall. The large intestine microflora, especially anaerobic species Bacteroides distasonis, B, uniformis and B. ovatus possess glycosidases liable to hydrolyse glycosides with a high degree of specificity in the distal ileum and in the caecum. Thus, only a marginal absorption of dietary flavonoids would be expected. However, several recent studies in rats and humans have revealed that flavonoids are absorbed in appreciable amounts, both as aglycones and as glycosides, in the small intestine [34]. Some but not all common dietary flavonoid glycosides can be deglycosilated by a hepatic beta-glucosidase [35]. In rats and humans, the absorption of orally administered quercetin aglycone was about 20 %, while isoflavone absorption was 10-20% [3637]. In ileostomy patients, absorption of quercetin glycosides from onions was 52%, 24% for quercetin aglycone and 20% for quercetin rutoside [38]. In rats after in situ perfiision of jejimum plus ileimi, quercetin (66,7% of perfiised) enters the enterocytes by a still unidentified mechanism. In these cells, quercetin is readily glucuronidated, methoxylated and sulfated [39]. The resulting metabolites can subsequently leave the intestinal cell possibly via a facilitated transport system, either across the apical pole (secretion of the conjugated forms in the lumen, 52,4%) or across the basolateral side for their fiirther transfer into the portal vein (14,3%). The liver fiuther metabolises the conjugated compoxmds arising from intestine, leading to an increase of their degree of glucuronidation, sulfation, and methoxylation [39]. The main plasma metabolites after quercetin administration are conjugated derivatives of quercetin and isorhamnetin, a 3'-0-methylated form of quercetin [40-42]. The conjugated metabolites show a long lasting

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presence in plasma (elimination half life of about 25 h) implying that repeated dietary intake would lead to an accumulation in plasma [43]. Excretion in bile of glucoronides and sulphates seems to be important. Bacteria in the colon hydrolyse conjugates and glycosides which is supposed to enable absorption of the liberated aglycones. Thus, conjugates can be reabsorbed and enter an enterohepatic cycle. However, these microorganisms also substantially degrade the flavonoid moiety by cleavage of the heterocyclic ring. Three main types of ring scission, depending on the ring structure, each leading to different phenolic acids or their lactones, have been postulated. These primarily produced phenolic acids are prone to secondary reactions such as p-oxidation, demethylation and dehydroxylation. The phenolic acids are absorbed and excreted with urine [5]. In conclusion, the effects of dietary flavonoids are expected to be dependent on the absolute content and the specificflavonoidspresent in the diet. Flavonoids are highly metabolised both at the intestinal level and in the liver. The pattem of glycosilation and the presence of other food components may also influence its bioavalability. Therefore, the results of epidemiological studies probably reflect the effects of long lasting metabolites whose pharmacology has been poorly analysed. At present, only the antioxidant capacities of the circulating metabolites of quercetin (glucurono-sufo conjugates of isorhamnetin and quercetin) were tested, showing that they also exhibit antioxidant properties [44]. ANTIATHEROGENIC EFFECTS Oxidised LDL are key factors ki the pathogenesis of atherosclerosis, the underlying cause of coronary heart disease, stroke, and peripheral arterial disease [45-47]. Endothelial cells and macrophages accelerate LDL oxidation, a process that is catalysed by heavy metals as copper and iron. Oxidised LDL are chemotactic for macrophages promoting their residence in the intima, cytotoxic to the endothelium, chemoattractant for monocytes, rapidly absorbed by macrophages leading to foam cell formation, appearance of fatty streak lesions and ultimately to growth of atherosclerotic plaques [48]. There is experimental evidence that certain antioxidants inhibit the formation of foam cells and slow the progression of atherosclerosis. Flavonoids can impair the generation of and neutralise

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superoxide anion [49], hydroxyl radical and lipid peroxide radicals [50]. These antioxidant properties of flavonoids, especially toward LDL, have attracted quite a lot of attention in recent years as a mechanism to explain the possible protective effect of these compounds against coronary heart disease. Antioxidant properties Flavonoids have the ability to act as antioxidants by a free radical scavenging mechanism with the formation of less reactive flavonoid phenoxyl radicals [Eq. (1) and (2)]. On the other hand, through their known chelating ability these compounds may inactivate transition metals ions (iron, copper), thereby suppressing the superoxide-driven Fenton Reaction, Eqs. (3) and (4), which is currently believed to be the most important route to activate oxygen species [51]. ROO- + Fl-OH -^ ROOH + Fl-OHO- -H Fl-OH -> H2O + Fl-0O2" + Fe (III) -> O2 -H Fe (II) Fe (II) + H2O2 -> Fe (III) + HO- + HO"

(1) (2) (3) (4)

There is much discussion in the literature regarding the relative contributions of these two mechanisms [52]. It is widely believed that the antioxidant ability of flavonoids reside mainly in their ability to donate hydrogen atoms and thereby scavenge the free radicals generated during lipid peroxidation. Despite the early realisation that the structures of these flavonoids allow them to form heavy metal complexes, metal chelation has generally been regarded to play a minor role in the antioxidant activity of these compounds and, hence, has not been intensively studied [53]. In fact, flavonoids inhibited LDL oxidation induced by ultraviolet light in the absence of iron and copper [54]. In general, optimal antioxidant activity offlavonoidis associated with the presence of multiple phenolic groups (hydroxyl groups increase the antioxidant activity, whereas methoxy groups suppress it), a carbonyl group at C-4, and free C3 and C5 hydroxyl groups. All these structural features are summarised in Fig. (2).

574

OH

O

B

o

P

^- H

Fig. (2). Structural criteria for effective free radical scavenger activity.

Substitution patterns on the B-rings appear to be the most important contributors. The 3*,4'-orthodihydroxy structure in the B rings confers the highest ability to the aryloxyl radical formed and participates in electron delocalization. A hydroxyl group at C-3 position is also beneficial to the

575

ability of flavonoids to inhibit lipid peroxidation [51]. The maximimi radical scavenging potential can be achieved by the combination of the 5-hydroxyl group in the A ring with the 3-OH group, the 2,3 double bond and the 40X0 function in the C ring. The 2,3-double bound (in conjunction with the 4-0X0 function) and the 3-hydroxyl group in the C ring are responsible for delocalization from B ring [50]. Therefore, quercetki, the most common flavonoid which has all these molecular characteristics, is one of the most potent antioxidant flavonoids. However, a number of studies have found prooxidant effects for many of these flavonoids. Because of their ubiquitous nature, the most widely studied have been quercetin and myricetin. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. The criteria for prooxidant activity of flavonoids remain unclear. Recently, it has been shown in a cell-free system, that prooxidant activities offlavonoidsincrease upon: a) the presence of easily autoxidizable catechol group in B-ring, b) an increase in the total number of hydroxy groups, and c) the conjugation between the A- and B-rings, i.e., the existence of flavone or flavonol structure [55,56]. However, the extracellular production of active oxygen species (superoxide, hydrogen peroxide, and hydroxyl radical) by dietary flavonols is not likely to occur in vivo [57]. Moreover, it has been found that naringenin, taxifoUn and kaempferol offer certain protection against quercetin toxicity, so that it is possible that the prooxidant effects can be minimised by the intake offlavonoidmixtures [58]. Mechanisms of inhibition of LDL oxidation Several mechanisms have been implicated in the protective effect of flavonoids in LDL oxidation. Fig. (3): A) Preservation of endogenous antioxidants. LDL particles contain antioxidants including tocopherols, p-carotene, lycopene, and retinyl stearate. LDL oxidation in vitro exhibits a lag phase corresponding to the time required for the endogenous antioxidants in LDL to be consumed [59]. The water soluble flavonoids (conjugated metabolites) located near the surface of phospholipid bilayers could prevent LDL oxidation by taking up the water soluble free radicals generated by copper through the Fenton reaction, decreasing the consumption of the LDL antioxidants contained ki

576

the lipid-water interface [44,60]. In fact, flavonoids decrease the consumption of endogenous a-tocopherol, thus delaying the increase in LDL uptake and oxidation by macrophages [60]. When flavonoids were added to the LDL solution, once the oxidation process was initiated, their antioxidant effect was related to the presence of vitamin E. When vitamin E was still present in the LDL,flavonoidsprolonged the lag phase for the formation of conjugated dienes, delaying the consumption of vitamin E and, therefore, the whole oxidation process. However, when flavonoids were added later on during the oxidation process, when vitamin E was already consumed, their addition neither regenerated the vitamin nor decreased or stopped the oxidation process. So, the flavonoids decrease LDL oxidation only if they are present in the early stages of the oxidation process, when endogenous antioxidants are still present [60]. B) Flavonoids reduce the formation or release of free radicals. LDL is oxidised by free radicals generated from endothelial cells, monocytederived macrophages, and smooth muscle cells [61]. Several flavonoids inhibit the release of reactive o>Q^gen species by stimulated human leukocytes or neutrophils [62,63] and attenuate this cell-mediated oxidation of LDL when added to the extracellular medium. Under these conditions, it is not clear whether the antioxidant effects of flavonoids were exerted within the lipoprotein particle, in the extracellular medium, or at the cellular level. Macrophage-mediated oxidation of LDL is considered to be of major importance during early atherogenesis, and it depends on the oxidative state of LDL and of the macrophages [61]. The LDL oxidative state is elevated when the ratio poly/mono unsaturated fatty acids is increased, and it is reduced by elevation of LDL-associated antioxidants. Flavonoids inhibit the oxidation of LDL by macrophages with concentrations producing 50% inhibition (IC50) ranging from 1-2 |aM for quercetin, fisetin, morin, and gossypetin to 15-20 |iM for galagin and chrysin [64]. Macrophage-mediated oxidation of LDL is affected by the balance between cellular oxygenases and antioxidants, such as the glutathione system. Flavonoids are known to inhibit cyclo-oxygenase and lipoxygenases. However, it appears that free radicals produced as a byproduct of these enzymes are not essential for LDL oxidation by macrophages [65]. In contrast, the activation of the macrophage NADPH oxidase, the enzyme responsible for the reduction of O2 to 02", can lead to a substantial LDL oxidation [66]. Flavonoids inhibit NADPH-oxidase activity in vitro, leading

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to a decrease of superoxide anion and hydrogen peroxide production [67], AntiQM 100 pM), can modify LDL increasing its uptake by macrophages by a non oxidative mechanism (non depletion of a-tocopherol in presence of myricetin was

580

observed) due to the aggregation of LDL particles caused by the covalent crosslinking of apo B-lOO molecules [81], The question arises as to whether or not theseflavonoidsmay modify LDL in vivo. This possibility seems unlikely as theseflavonoids,which are not the mainflavonoidsin the diet, probably do not reach plasma concentrations high enough to exert their effects. In addition, the modification of LDL by myricetin is prevented by serum, possibly because serum proteins, rather than the apo B-lOO of LDL, bind the bulk of myricetin. Thus, ifflavonoidsdo interact with LDL in vivo, they are likely to act by inhibiting its oxidation rather than modifying the LDL by themselves [64]. In conclusion, flavonoids together with others antioxidants constitute two lines of defense in protecting cells against injury owing to oxidation of LDL: a) at the LDL level, by inhibiting the LDL oxidation and the subsequent cytotoxicity, and b) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidised LDL. Effects on adhesion and chemoattractant molecules Recent evidence suggests that atherosclerosis is a chronic inflammatory process. The recruitment of mononuclear leukocytes and formation of intimal macrophage-rich lesions at specific sites of the arterial tree are key events in atherogenesis. Alterations of chemotactic and adhesive properties of the endothelium play an important role in this process [82]. Quercetin has been reported to inhibit the expression in glomerular cells of monocyte chemoattractant protein-1 (MCP-1) [83] a potent chemoattractant for circulating monocytes. Red wine reduced MCP-1 mRNA and protein expression in abdominal aorta of cholesterol fed rabbits after balloon injury and this effect was associated with a reduced neointimal hyperplasia [84]. The antioxidant-mediated inhibition of nuclear factor K B (NFKB) and the subsequent non selective reduction of cytokine transcription have been suggested to be responsible for these effects [85], Additionally, quercetin downregulated both phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis fector-a (TNFa)-induced intercellular adhesion molecule-1 (ICAM-l) expression in himian endothelial cells [86].

581

EFFECTS OF FLAVONOIDS ON PLATELET AGGREGATION Platelet-blood vessel interactions are implicated in the development of thrombosis and atherosclerosis [87]. Since Gryglewski and co-workers [88] showed that quercetin bound to platelets membranes, dispersed platelet thrombi adhering to rabbit aortic endothelium and prevented platelet adhesion and aggregation, several others researchers have established that particular flavonoids are effective inhibitors of platelet adhesion, aggregation and serotonin secretion (for a review see [2,5,8]). The degree of inhibition is dependent on the type of inducer and on the structure of the flavonoid. At 30 |LIM, fisetin, kaempferol or quercetin inhibit the platelet aggregation induced by arachidonic acid, whereas morin and myricetin are effective only at concentrations greater than 150 |nM [89]. Aggregations induced by adenosine diphosphate (ADP) and especially by platelet activating factor (PAF) are less affected by flavonoids, except by myricetin. Quercetin, fisetin and myricetin show a more pronounced inhibitory effect against collagen-induced aggregation. The flavonoid component of grape products, including red wine and purple grape juice inhibited collagen-mediated platelet aggregation [90]. This variety of flavonoids effects against different inducers influencing several pathways involved in platelet function, strongly suggests that the antiaggregatory effects of flavonoids cannot be attributed to a single biochemical mechanism [89]. Several mechanisms have been implicated on the inhibition of platelet aggregation induced by flavonoids: A) Inhibition of platelet phosphodiesterases (PDEs) [91], Quercetin and myricetin potentiated the anti-aggregatory action of prostacyclin (PGI2), a potent stimulator of platelet adenylate cyclase synthesised by the vascular endotheliimi, on ADP-induced platelet aggregation in washed himian platelets, and the elevation of platelet cyclic adenosine monophosphate (cAMP) elicited by PGI2 [89,92,93]. These effects are probably due to an inhibition of PDEs. As suggested by Ferrell and co-workers [92], this inhibition arisesfromthe similarity between the pyranone ring of flavonoids and the pyrimidine ring of adenine. B) Inhibition of arachidonic acid metabolisnL Most flavonoids tested (flavone, chrysin, phloretin,flavanone,apigenin and kaempferol) inhibited

582

cyclooxygenase activity although with marked differences ki potency. Myricetin and quercetin, however blocked both cyclooxygenase and lipoxygenase pathways at high concentrations (50 |LIM). At low concentrations (10 ^M) lipoxygenase was the primary target of inhibition [89]. Tzeng and co-workers [94], determined that flavonoids (iBsetin, kaempferol, morin and quercetin) were antiaggregant because they inhibited thromboxane B2 (TXB2) formation and also antagonised platelet aggregation induced by U46619, a thromboxane A2 (TXA2) mimetic receptor agonist. C) The antioxidant actions of flavonoids appear to participate in their antithrombotic action. Flavonoids bind to platelet membranes and scavenge platelet-generated lipid peroxides and free radicals, restoring the biosynthesis and the action of endothelial prostacyclin and nitric oxide (NO), respectively [49,88]. D) Other mechanisms. Genistein apparently can affect platelet function in ways possibly unrelated to protein tyrosine phosphorylation [95]. For example, platelet tyrosine phosphorylation stimulated by thrombin was only weakly êcted by genistein but it inhibited platelet aggregation and serotonin secretion. On the other hand, this isoflavone suppressed platelet aggregation, serotonin secretion and protein phosphorylation triggered by collagen and U46619. Moreover, daidzein, an inactive analogue, is capable. Eke genistein, of inhibiting the binding of U46619 to platelets with an associated reduction in collagen- or U46619-induced platelet responses. An inhibition of the intracellular mobilisation of Ca^"^ and of its influx across the plasma membrane could also play a role [96,97]. Inhibition of platelet aggregation by dietary flavonoid has been suggested to play a role in the protective effects of flavonoid-containing foods on ischaemic diseases [6]. As described above, the most abundant dietary flavonoid, quercetin, inhibited platelet aggregation in vitro (13-150 jiM) [94,98,99] and, in laboratory animals (5-50 mg kg"^) [100,101]. However, the effects of quercetin supplementation on human platelet aggregation have been poorly evaluated. Conquer and co-workers [102], investigated in a double-blind study with healthy volunteers, the influence of four capsules daily of a quercetin-containing supplement (1 g quercetin/day) or rice flour placebo for 28 days on plasma quercetin status and risk factors for heart disease. Quercetin intakes were - 50-fold greater than the dietary intakes associated with lower coronary heart disease

583

mortality on the basis of epidemiological studies. Subjects consuming quercetin-containing capsules had plasma quercetin concentrations ^ 23fold higher than those of subjects consimiing the control capsules. However, they did not observe any inhibition of either platelet aggregation or platelet release of TXA2. This may be due to the fact that the concentration of quercetin in the plasma of supplemented subjects (1.5 |j.M) did not reach high enough levels to exert an inhibitory effect on platelet activity. So, inhibition of platelet aggregation appears to require a minimum of 10 |LIM quercetia Similar results have been reported by Janssen and co-workers [103] in subjects consuming 220 g onions daily (containing 114 mg quercetin) or 5 g dried parsley daily (containing 84 mg of apigenin). These results suggests that the protective effect of food containing quercetin may be mediated via effects on risk factors other than inhibition of platelet aggregation and/or to fectors other than quercetin contained in those foods. VASCULAR EFFECTS OF FLAVONOIDS Another potential mechanism by which flavonoids may be protective in cardiovascular diseases is by their direct effects on vascular smooth muscle cells either as vasodilators or as inhibitors of proliferation. However, vasoconstrictor effects have also been reported for some flavonoids. Various natural, chemically modified and mixtures of flavonoids are widely used therapeutically as venous protective or venotonic drugs in chronic venous insufficiency and haemorrhoidal attacks. Flavonoids have been found to inhibit increased vessel wall permeability,fluidchanges in the capillary bed and diffusion of plasma proteins. In addition, they may exert a protective effect on the perivascular tissues due to their antihyaluronidase effect and the inhibition of lysine oxidase (producing crosslinks in collagen and elastin) and lysosomal hydrolases (degrade glycosamines). All these effects may account for the venotonic effects of these drugs [5]. However, the venous effects offlavonoidsare out of the scope of the present review. Vasodilator effects Several studies foimd that flavonoids and other polyphenolic compounds

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present in food exhibit vasodilator effects in different isolated vascular preparations [104-117]. The vasorelaxant response evoked byflavonoidsis considered mainly endothelinm-independent [108-111,115-117]. However, others investigators have found that some polyphenolic compounds [104106] produce endothelium-dependent vasorelaxation, Endothelium-dependent relaxation

The vascular endothelium lies at the interface between the circulating blood cells and the vascular smooth muscle cells and plays a crucial role in regulating blood flow and vascular tone [118]. In addition, vascular endothelium can synthesise and release different relaxant &ctors such as NO, prostanoid derivatives and the so called endothelium-derived hyperpolarizing fector [119] and also endothelium-derived contracting fectors (endothelins, vasoconstrictor prostanoids and superoxide anions) [120]. In many vascular pathologies, such as hypertension, diabetes and atherosclerosis, endothelium-dependent vasorelaxation to different vasodilator agonists is reduced. One of the mechanisms accounting for this endothelial dysfunction is a decreased release of NO [121]. Certain wines, grape juice, grape skin extracts [111], red wine polyphenol conqx)unds (RWPCs) [104-106] and defined polyphenols contained in wine, such as, leucocyanidol [104], delphinidin and oligomeric condensed tannins [105] cause endothelium-dependent vasorelaxation in vitro. The imderlying mechanisms involved in this vasodilator effect are far fi-om clear. Fitzpatrick and co-workers [111] foimd that certain wines, grape juice and grape skin extracts, which are known to contain polyphenols, can induce endothelixmi-dependent vasorelaxation of rat aorta probably via NO release, enhanced biological activity of NO or protection against breakdown by superoxide anions. As described above, flavonoids scavenge superoxide anions protecting the inactivation of NO induced by this fi'ee radical [50]. In feet, the synthetic flavonoid 6, 7-dimethoxy-8-methyl-3', 4', 5-trihydroxyflavone protects endothelium-dependent relaxation in rabbit ear and basilar arteries fi'om high levels of superoxide anion possibly by scavenging superoxide anion [113]. However, very recently, the group of Stoclet showed that a red wine extract enriched in RWPCs induced endothelium-dependent relaxation in rat aorta via an enhancement of endothelial NO synthesis, rather than enhanced biological activity of NO or

585

protection against breakdown by superoxide anions [104,106]. This effect was aboUshed after removal of extracellular calcium or in the presence of

endothelial cell

Flavonoid

NO

vascular smooth muscle cell

q)erimental colitis [154-159], since they are able to reduce colonic glutathione depletion or to decrease colonic malonyldialdehyde (MDA) production, two biochemical markers of induced lipoperoxidative insult. This effect can be considered of great value because free radicals, including reactive oxygen and/or nitrogen metabolites, have been proposed to play a key role in the pathophysiology of human IBD [160, 161]. Moreover, in the last few years special attention has been paid about the dual role of nitric oxide (NO) m the pathogenesis of IBD [162]. It has been shown that physiological levels of NO, generated by the constitutive isoforms of nitric oxide synthase (cNOS), exert a direct protective effect in the acute intestinal inflammatory states, mainly through inhibition of leukocyte-endothelium interaction and of the increase in epithelial permeability, two key events in the early stages of intestinal inflammation [163]. On the contrary, NO synthesis is markedly augmented in states of chronic intestinal inflammation, largely due to the upregulation in the inducible isoform of NOS (iNOS), which results in several indirect deleterious/proinflammatory effects via generation of reactive nitrogen oxide species derivedfromthe interaction of NO and O2", such as nitrogen dioxide, dinitrogen trioxide and peroxynitrite, that promote oxidative stress and tissue injury [164]. It is plausible that the antioxidant and/or scavenging properties of flavonoids can interfere with NO metabolism; first, flavonoids may preserve the beneficial effects of NO by directly scavenging superoxide anions [165], which are thought to inactivate the physiological NO that is generated within endothelial cells and protects the gut from inflammatory insult [163]; second, it has been shown thatflavonoidsinhibit iNOS [166] and act as potent scavengers of peroxynitrite [167], and thus they could ako prevent the indirect deleterious effects of NO on the intestinal system. However, the antioxidant activity offlavonoidscannot be considered the only mechanism involved in their beneficial effects. Another mechanism that can participate is their ability to inhibit lipoxygenase activity, decreasing leukotriene B4 (LTB4) production [24]. LTB4 has been long thought to be a key proinflammatory mediator in IBD. In fact, blockade of LTB4 synthesis [168] or of the LTB4 receptor [169] has proven beneficial in experimental colitis. Diflferent flavonoids have been shown to decrease colonic LTB4 production in colitic rats [154-159], but a cause-effect relationship has not been consistently found [154, 158]. It is plausible that both mechanisms, antioxidative activity and inhibition of LTB4 synthesis, may cooperate in their beneficial effects, since LTB4 has been shown to potently promote

627

neutrophil chemotaxis, adherence and degranulation in the TNBS treated colon [169], whereas free radical overproduction results in a multitude of deleterious effects in the gastrointestinal tract, including direct cytotoxicity, which promotes further release of proinflammatory mediators [161, 170]. In fact, flavonoid treatment resulted in most of the studies in a significant reduction of colonic myeloperoxidase activity, an enzyme predominantly found in the azurophilic granules of neutrophils which is considered as a sensitive marker of neutrophil infiltration, and is extensively used for this purpose [171], Moreover, it has been demonstrated that flavonoids, mainly as aglycones, are inhibitors of neutrophil fimction and MPO activity [24], which can also contribute to the intestinal antiinflamatory activity. In addition to the above mentioned mechanisms, a preservation of colonic absorptive function, which is profoundly altered in intestinal inflammation, can account for the beneficial effects of flavonoids in these intestinal conditions. In fact, it has been proposed that abnormal intestinal permeability is an underlying factor in the pathogenesis of human IBD [172], because a leaky epithelium may allow the entry into the lamina propria of bacterial o dietary antigens that are poorly cleared by the mucosal immune system or of a bacterial product that initiates an uncontrolled inflammatory response [173], It has been shown in different e}q)erimental models of rat colitis that altered in vivo colonic fluid absorption can be considered a sensitive measure of injury to the intestinal mucosa, resulting from mucosal leakiness and extensive epithelial necrosis in the acute stages of colitis and from unclear mechanisms in the chronic phase [154, 155,157,158,173,174], Moreover, it has been described that colonic hydroelectrolytic transport is one of the last parameters to recoverfrominflammation [154], being altered even when the inflammatory status has been essentially resolved [175], Different flavonoids, like quercitrin [154], rutin [155], hesperidin [157] and morin [158], have been reported to improve colonic fluid absorption in colitic animals. However, only quercitrin was able to conqjletely restore normal colonic fluid transport in colitic rats, acconq)anied with a reduction in the incidence of diarrhoea compared to non-treated animals, one of the symptoms that characterize intestinal inflammatioa Alternatively amelioration of colonic transport can be viewed as a consequence, rather than a cause, of a quicker recovery of mucosal barrier function and/or mucosal protectionfrominflammation. Nevertheless, a role for flavonoids in the regulation of hydroelectrolytic transport has been envisaged, as previously discussed. Thus a possible inhibitory effect of quercitrin on the secretory response to inflammatory mediators, including PGE2 [161, 176,

628

177], cannot be excluded, given the antisecretagogue properties of this flavonoid as well as those of its aglycone quercetin [119, 141], which is supposed to be released after hydrolysis of the glycoside by bacterial enzymes in the colon [27]. It is important to note however that this response is tipically downregulated in the inflamed intestine, so that the relevance of this mechanism may be less than previously thought. Data obtained from a nmnber of in vitro assays suggest other possible lines of actuation of flavonoids, albeit not substantiated as yet by in vivo experiments. In particular, flavonoids may interfere with the effect of several cytokines, like tumor necrosis factor-a (TNF-a), which is considered of central pathogenic importance in intestinal inflammation [172]. In fact, a monoclonal antibody against TNF-a has been found to be a promising new therapeutic approach in IBD, providing long-term remission at least in Crohn's disease [178, 179]. In this regard, Habtemariam [180] studied the ability of seventeen flavonoids as modulators of the cytotoxicity of TNF-a in a tumor cell line, and reported that all flavonols tested, including quercetin, morin and rutin, protected these cells from TNF-induced cell death. Quercetin was the most potentflavonol,being sbc and and twelve times more potent than morin and its glycoside derivate rutin, respectively. The mechanism whereby flavonoids inhibit the cytotoxicity is yet to be established, but it may be associated with inhibition of the enzyme activities upregulated following receptor activation or with their antioxidant properties. On the other hand, it has been shown that severalflavonoidsare able to inhibit the expression of adhesion molecules stimulated by TNF and other cytokines at the transcription level [181], an early process in the cascade of events leading to derangement of intestinal mucosal homeostasis in IBD [172]. Considering that the TNBS model of rat colitis has shown to have an upregulated proinflammatory cytokine pattern, it is probable that the effects of flavonoids on cytokine production contribute to their intestinal In conclusion, the beneficial effects offlavonoidsin experimental IBD are mostly related with a preservation of intestinal fimction and/or with their ability to interfere simultaneously with different steps in the inflammatory cascade; i,e. eicosanoid generation, oxygen and nitrogen reactive metabolites production and proinflammatory cytokine release. The interest of flavonoids as potential drugs in the treatment of IBD is growing, and recently the intestinal antiinflammatory effects of the derivate flavonoid DA-6034 (7carboxymethyloxy-3',4*,5-trimethoxy flavone) has been reported in three

629

experimental models of rat colitis [182], suggesting that it could be a promising drug in IBD therapy. ANTICANCER ACTIVITY Epidemiological evidence collected in the past decade has consistently established a link between the dietary intake of vegetables and tea and a reduced risk of certain diseases including cancer. Flavonoids have been proposed as one of the phytochemical groups responsible for the cancer preventive effect of foodstuflFs of vegetal origin. In particular, the low incidence of breast and prostate cancer in Asian countries has been attributed to the high consumption of tea and soy beans, which are rich sources of catechins and isoflavonoids, respectively [183, 184]. It is important to note that there is a great deal of controversy on this subject, since while some epidemiological studies have confirmed the protective effect of flavonoids against gastrointestinal and other types of cancer [185-187], others seem to rule out this hypothesis [4,188]. Nevertheless, there is sufficient evidence to warrant further studies offlavonoidsas antineoplastics in the gastrointestinal tract. Although there are preliminary data supporting the antitumoral activity of quercetin, the most commonflavonoid,in humans in the course of a Phase I clinical trial [189], direct evidence of the anticancer effect offlavonoidsis derived almost exclusively from studies performed in animal models as well as studies performed on cultured cell lines. Fig. (2). Most animal studies on gastrointestinal cancer have focused on colon cancer using the azoxymethane (AOM) model in rats or mice [190-197]. There are also available reports on models of cancer of the stomach (induced by benzo[a]pyrene [198] or N-methyl-N*-nitro-N-nitrosoguanidine [199]), oesophagus (N-methyl-N-amykiitrosamine [200]), and the tongue/oral cavity (methyl-(acetoxymethyl)-nitrosamine [198], 7,12-dimethylbenz[a]anthracene -DMBA- [201] and 4-NQO [202,203]). The anticancer effect of soy, the only relevant dietary source of isoflavones and the main candidate foodstuff responsible for the cancer protection conferred by Asian diets, has been tested in a number of animal studies. Messina et al [204] reviewed 26 such studies published up to 1994 (gastrointestinal and non gastrointestinal). Seventeen of them (65%) reported a protective effect and none found a potentiation of carcinogenesis. One study has examined the effect of feeding a soy protein diet in a genetic

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model of intestinal cancer, the Apc(Min) mouse, which is heterozigons for a nonsense mutation in the Ape (Adenomatous Polyposis Coli) gene [205]. No differences in the incidence of spontaneous adenomas were observed. Soybeans are rich in isoflavonoids, namely genistein and daidzein, a fact that may account for their putative cancer preventive effect [197]. In fact genistein, administered as a pure compound, has been reported to reduced AOM-induced colonic foci of aberrant crypts in rats, an early marker of neoplasia [191, 193, 197]. On the other hand. Booth et al [206] found genistein (administered subcutaneously) to have no effect on small intestinal homeostasis. Genistein may not be the only factor involved in cancer prevention by soy diets. Thus Hawrylewicz et al [207] found that dietary methionine supplementation reversed the growth inhibiting effect of soy protein diets in non gastrointestinal rat tumours, suggesting that the low content in methionine in soy is also involved. Genistein has been shown to significantly reduce the incidence of experimentally induced gastric cancer [199] in rats. A number of studies carried out with other flavonoids. Table (1), have shown their effect on oral [198, 201-203], oesophageal [200], gastric [198,208] and colonic cancer [190,192,209,210]. Little information about the mechanism of action of flavonoids is anticipated from in vivo studies. The mechanism of catechin and morin seems to be related to an increase of the activity of detoxifying enzymes like glutathione-S-transferase and NADPH:quinone reductase [198, 211], Similarly, EGCG effect at the colonic level is associated to an increase in tissue superoxide dismutase levels, suggesting that it may act through a potentiation of the antioxidative defense [210]. Another experimental approach is the use of cultured cell lines of gastrointestinal origin to test the antiproliferative or pro-apoptotic activity of flavonoids. The main drawback of this model is that cell lines are generally cancerous in nature, making it critical to distinguish an unspecific toxic effect from true antitumoral activity. This is of special relevance if we consider the vast array of biochemical processes that may be potentially modulated by flavonoids [24]. Few investigators have compared the effects of these compoxmds on both cancer and 'normar cell lines, and even in these cases one has to take into account that the latter display a transformed phenotype rather than the normal, physiological scenario. In this regard, Kawaii et al [212] recently evaluated the antiproliferative effect of 27 Citrus flavonoids against several tumor and normal cell lines; seven of them (in decreasing order of potency: luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3*,4*,5,6,7,8-heptamethoxyflavone) showed

631

specific activity against the tumor cell lines while exerting a weak antiproliferative effect on normal cells. The deducted structure-activity relationship included an 0-catechol moiety in ring B together with a C2-C3 double bond and a 3-hydroxyl group for maximum activity. In another recent study. Booth et al [213] examined the antiproliferative activity of genistein and other isoflavonoids on both normal (IEC6, IEC18) and cancerous (SW620, HT29) intestinal epithelial cell lines and compared it with that of estradiol, tamoxifen (a estrogen receptor antagonist), and tyrphostin (a tyrosine kinase inhibitor). They concluded that all isoflavonoids, but mainly genistein, inhibited proliferation and stimulated apoptosis similarly in all four cell lines and that this effect was associated to tyrosine kinase inhibition. Musk et al [214] compared the antiproliferatice effect of quercetin on naive and detransformed/differentiated HT29 cells and found that the flavonoid had a more pronounced effect on the former. Chen et al [215] reported a modest selectivity of EGCG towards cancer cell lines (12-fold). Conversely, in a study utilising normal, dysplastic or cancerous oral cell lines, Khafif e/ al [216] found that EGCG showed a weak antiproliferative effect which was lower in cancer cells than in the normal or dysplastic counterparts. Along the same path is the report of Kuo [217], who found no specificity of quercetin or genistein against cancerous (HT29, Caco-2) as oppossed to normal (IEC6) cell lines of intestinal origin. Taken together, these studies suggest that flavonoid selectivity for cancer cells is modest at best. PROCARCINOGENIC ri-% 4I ^^''© O '

Citochrome l

P450

» ^

^ © T P-glycoprotdii CARCINOGENIC — - _ _ » Detoxication or Extrusion GST ROM ^ © - ^

©

DNA INTERACTION

1

MUTAGENIC

• Apoptosis

Neoplastic cell

Fig. 2. Schematic diagram delineating some of the multiple stages of mutagenesis and the interference by flavonoids. 1. Flavonoids induce apoptosis and enhance mutagen detoxification and extrusion from the cell. 2. Flavonoids interfere with the metabolic activation of mutagens and protect DNA by means of their antioxidative action. GST: glutathi(me-S-transferase; ROM: reactive oxygen metabolites.

632

On the other hand, there is strong evidence that the effects of flavonoids on cultured cells are not merely toxic but via induction of apoptosis or inhibition of proliferatioa Thus signs of apoptosis, such as induction of caspase-3, fragmentation of DNA and chromatin condensation, are frequently detected upon cell exposure to flavonoids [213, 217-221]. In addition, the effects of flavonoids are generally reversible upon removal or addition of serum [222-224]. Kunz et al [221] attempted to establish a structure-activity relationship for the antineoplastic potency of flavonoids in gastrointestinal cell lines, examining more than 30 flavonoids for antiproliferative activity against Caco-2 and HT29 cells. In contrast with the findings reported by Kawaii et al [212], the authors found no apparent relationship between activity and either 'core' subclasses {le.flavones,flavonols,flavanones,isoflavones) or substituents. However, most studies point to quercetin, genistein and EGCG as the most potent compounds overadl. In this regard, it is important to note that although some flavonoids are active at low concentrations in vitro, like genistein at 6 |iM [225] or quercetin at 10 nM-10 |iM [222], the anticancer effect is morefrequentlyreported at relatively high concentrations, i.e. in the 1-100 ^M range [218,221,224,226]. Different mechanisms have been postulated to contribute to the antitumorigenic activity of flavonoids at the gastrointestinal tract, taking into consideration evidence collectedfromexperiments with both gastrointestinal and nongastrointestinal cell lines. A brief review of these activities with a special emphasis on their relevance in cancer is provided betow. a) Antioxidant/antimutagenic activity Flavonoids have well characterized antioxidant and free radical scavenger properties. This is relevant in view of thefeetthat oxidative stress may cause oxidative modifications in DNA and originate mutations. Many investigators have demonstrated the antimutagenic/anticlastogenic activity of flavonoids on both prokaryotic models of spontaneous and induced mutagenesis [227229] and eukaryotic cells [230-235]. The antimutagenic effect of flavonoids may be also achieved via other mechanisms: first, some flavonoids inhibit the metabolic activating process of dietary carcinogens such as benzo[a]pyrene, aflatoxin Bl, 2-amino-3-methylimidazo[4,5-f|quinoline (IQ), MelQ, Trp-P-1, etc. [227, 233, 236]. In particular, flavonoids have been shown to inhibit the citochrome P450-monooxygenase system, which is

633

involved in the oxidative activation of mutagens such as benzo[a]pyrene [236, 237]. Second, flavonoids may confer protection through activation of transporters that mediate the extrusion of mutagens from the cell, like Pglycoprotein [238] and the multidrug resistant protein, MDRP [239], or detoxifying enzymes like NADPH:quinone reductase [240] or glutathione-Stransferase [241]. Third, reactive nitrogen metabolites have also been involved in mutagenesis, particularly ui the context of chronic inflammation; flavonoids inhibit inducible nitric oxide synthase [166], the main source of NO and subsequent nitrogen reactive substances in inflammation, and are capable of scavenging these reactive molecules [167]. And fourth, myricetin has been shown to stimulate DNA repair via activation of DNA polymerase p [234] in hepatocytes, an action that may contribute to its antimutagenic effect. The fact that flavonoids behave predominantly as antimutagenic/antioxidants in vivo must be reconciled [241] with their well documented properties as pro-oxidants and mutagenic agents in their own right in vitro [242-253]. In this regard, it is possible tiiat environmental conditions, e.g. the low concentration of metal ions in vivo [232] or the interaction with other dietary polyphenols, like curcumin [198, 216] or even carcinogens [227, 233, 236], are determinant. Zhu et al [254] have also suggested that rapid metabolization of flavonoids may prevent their toxic effects. The dose may be another important factor; thus quercetin has been reported to increase the multiplicity of AOM-induced colonic adenocarcinomas in one study [194], in which the doses were quite elevated (16.8 and 33.6 g/kg chow, i.e, about a hundredfold tlK)se exerting antiinflammatory effects in rat colitis in our laboratory [154]. This carcinogenic behaviour of quercetin may be related to the knoAvn mutagenic properties of manyflavonoids.Genistein has also been reported to increase non-invasive and total adenocarcinoma multiplicity, without affecting incidence, in this model [196]. In addition, some flavonoids may actually favor mutagen activation [236]. However, the predominant effect of flavonoids in vivo seems to be antimutagenic/anticarcinogenic. b) Inhibition of enzymes involved in signal transduction Flavonoids exert a modulatory activity on a nimiber of enzymes that participate in signal transduction, like protein kinase C, phosphatidylinositol 3-kinase, and tyrosine protein kinases (see [24] for review). Many of these

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enzymes are upregulated in neoplastic cells, evidencing a state of activation, and therefore inhibition byflavonoidsmay play a role in their anticancer effects [255, 256]. In particular, some tumors are highly dependent on an increased activity of receptor tyrosine kinases like the epidermal growth factor receptor, and may especially benefit from this approach [225, 257, 258]. Quercetin has been slK)wn to downregulate the e5q)ression of K-, Hand N-ras in both colon cancer cell lines and primary colorectal tumors [259]. c) Modulation of DNA-related enzymes and cell cycle factors Someflavonoids,including quercetin, inhibit eukaryotic topoisomerase I at the religation step [260]. Topoisomerases are involved in DNA replication, transcription and recombination and play therefore a key role in cell proliferation. Genistein inhibits topoisomerase n [261, 262], suggesting that it may be useful to treat rapidly proliferating carcinomas expressing high levels of this enzyme. Constantinou et al [261] also reported inhibition of both topoisomerase I and n by myricetin, quercetin, fisetin and morin. On the other hand, EGCG has been shown to inhibit telomerase, an enzyme necessary to preserve the chromosome tips of proliferating cancer cells, in HT29 cells [263]. Flavonoids seem to induce the expression of the antioncogene/755 [264]. In support of this hypothesis, quercetin is ineffective in reducing txmK)rigenesis mp5S -A knockout mice [265] and in arresting cell cycle in p53 knockout cells [264]. However, one study carried out in a lung cancer cell line found no evidence ofp53 induction by flavone [266]. Instead, the authors reported the induction of p21, an inhibitor of cyclin-dependent kinases, as well as dephosphorylation of RB protein, leading to cell cycle arrest in Gl. Genistein has been also shown to induce the expression of p21 as well as to downregulate cyclin Bl specifically [262]. Finally, Heruth et al [267] showed that genistein could downregidate the oncogene c-myc in colonic cancer cell lines as a consequence of tyrosine kinase inhibitioa It is noteworthy that EGCG, quercetin andflavonols/flavonesseem to block the cell cycle at the Gl or Gl/S phase [216, 222-224, 268], whereas genistein, an isoflavonoid, causes blockade at G2/M [223]. Only one study offers conflicting results, showing cell cycle blockade at G2/M for quercetin, apigenin and luteolin [264].

Table 1. Summary of the experimental studies showing the effect of flavonoids on animal models of gastrointestinal cancer. The model of cancer, the animal species and the main effects observed are shown.

rutin liquiritin

oesophageal colonic colonic

MNAN AOM AOM

rat CFI mice F344 rat

hesperidin 12031 & incidence of squamous cell carcinoma. 4multiplicity of carcinoma, papilloma and preneoplasia [200] -1hyperproliferationand focal areas of dysplasia [I901 & hyperproliferative markers [192]

. OI

01 u

636

d) Antiestrogenic activity Flavonoids exhibit weak estrogenic/antiestrogenic properties [269]. In particular, flavonoids have been shown to bind type II but not type I estrogenic binding sites in the cytosol and nucleus [270]. Type 11 estrogen binding sites (EBSII) were initially described as receptors distinct from the 'classical* ones with a lower affinity (--10-20 nM) and which were highly correlated with hypertrophy and hyperplasia of the rat uterus, an effect which is selectively blocked by flavonoids [270]. Subsequently flavonoids, particularly quercetin and genistein, were shown to have antiproliferative effects on the human breast cancer model cell line MCF-7 and this activity was correlated to the blockade of EBSII [270, 271]. Furthermore, quercetin was found to induce EBSII in these cells [272]. The expression of EBSII is not limited to breast or ovarian cancer cells, since Ranelletti et al [222] and Di Domenico et al [273] described their occurrence in several cell lines of colonic origin and in human colorectal cancer biopsies. Moreover, the antiproliferative activity offlavonoidsagainst these cell lines was correlated to the number of EBSII. In Caco-2 cells estradiol but not other estrogens stimulates cell proliferation and activates c-src, c-yes, erk-1 and erk-2 [273]. Inhibition of cell proUferation by genistein was associated to downregulation of erk-2. Thus antagonism of EBSII seems to be involved in the anticancer activity offlavonoids.Nevertheless, this is clearly not absolutely required sinceflavonoidsare active also on cell lines that do not express estrogen receptors [274-276]. Some studies have proposed additional antiestrogenic actions of flavonoids, such as inhibition of aromatase [277] and of 17phidroxysteroid [278]. e) Others The flavonoids fisetin and genistein exhibit antiangiogenic activity as evaluated in in vivo studies [279]. Flavonoids may also exert a limiting effect on tumor metastasis by way of their inhibitory activity on proteolytic enzymes such as trypsin, leucine aminopeptidase and other methalloproteinases [280, 281]. Bergan et al [282] examined the effect of genistein, a putative antimetastatic agent, on cell adhesioa They showed that the isoflavonoid caused cell flattening, associated to the specific accumulation of focal adhesion kinase in contact areas and the formation of a

637

complex with Pi-integrin. Finally, flavonoids have been proposed to act as antineoplastic drugs sensibilizing agents via inhibition of the heat shock activating cascade, since heat shock proteins are thought to confer resistance to chemotherapy [283,284]. It is important to bear in mind that many of these activities have not been studied in gastrointestinal cells and that they do not apply to all flavonoids. The only purpose of the above compilation is to serve as a reference for the possible mechanisms of action of flavonoids as antineoplastics in the gastrointestinal tract. Further investigation is needed to clarify the respective contributions of the many activities proposed. Finally, we would like to mention two synthetic flavonoid derivatives with gastrointestinal anticancer activity for the sake of completeness: flavone acetic acid andflavopiridol.Flavone acetic acid (FAA, LM975) is the active principle of a parent esterified compound (LM985) which emerged from a series offlavonoidssynthesized by Lyonnaise Industrielle Pharmaceutique and screened by the National Cancer Institute [285]. FAA showed dramatic activity against solid tumors in mice including colonic transplantable tumors* Its mechanism of action was found to be complex, including antiangiogenic and TNFa releasing properties, as well as activation of natural killer cells. Unfortunately it showed no effect in humans for unclear reasons. On the other hand,flavopiridol(L86-8275) is a potent inhibitor of cdc-2 kinase (at least 250-fold more potent than quercetin or genistein), an enzyme involved in cell cycle regulation, as well as of protein kinase C [286]. Flavopiridol is capable of promoting mitomycin C-induced apoptosis in gastric cancer cells [287] and induces cell cycle arrest and apoptosis in oesophageal cell [288]. It has undergone Phase I clinical trial. In summary, the evidence pointing to the possible use offlavonoidsin the treatment or prevention of gastrointestinal cancer, as derived from epidemiological, in vivo and in vitro studies, is still promising but inconclusive. Further investigation about the effects of these compounds on gastrointestinal cancer is needed, particularly with regard to their mechanism of action. ABBREVIATIONS 4-NQO = 4-nitroquinoline 1-oxide AOM = azoxymethane cAMP = cyclic adenosine monophosphate

638

CFTR = Cystic Fibrosis Transmembrane conductance Regulator cNOS = constitutive Nitric Oxide Synthase DMBA = 7,12-dimethylbenz[a]anthracene DMH: 1,2-dimethylhydrazine EBSn = Estrogen Binding Site 11 ED50 = Eflfective Dose 50 EGCG = (->Epigallocatechin Gallate GST = Glutatliione S-Transferase IBD = Inflammatory Bowel Disease iNOS = inducible Nitric Oxide Synthase IQ = 2-amino-3-methylimidazo[4,5-fjquinoline LTB4 = leukotriene B4 MAN: methyl-(acetoxymethyl)-nitrosamine MDA = malondialdehyde MDRP = Multidrug Resistance Protein MelQ = 2-amino-3,4-dimethylimidazo[4,5-f|quinoline MNAN: N-methyl-N-amylnitrosamine MNNG: N-methyl-N'-nitro-N-nitrosoguanidine MPO = myeloperoxidase NADPH = Nicotine Adenosine Dinucleotide Phosphate, reduced form NS AIDs = Nonsteroidal Antiinflammatory Drugs PAF = Platelet Activating Factor PGE2 = prostaglandin E2 PMA = Phorbol 12-Myristate 13-Acetate PPIs = Proton Pump Inhibitors ROM = Reactive Oxygen Metabolites TNBS = Trinitrobenzene Sulphonic Acid TNF-a = Tumor Necrosis Factor a Trp-P-1 = 3-amino-l,4-dimethyl-5H-pyrido[3,4-b]indole VIP = Vasoactive Intestinal Peptide ACKNOWLEDGEMENTS The authors acknowledge funding by the Spanish Ministry of Education and Culture (SAF98-0157 and SAF98-0160).

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BIOACTIVITY OF THE PHENOLIC COMPOUNDS IN HIGHER PLANTS JUAN M. RUIZ and LUIS ROMERO Departamento de Biologia VegetalFacultad de Ciencias, Universidad de Granada, 1807 l-Granada,Espana. ABSTRACT: Phenylpropanoid compounds encompass a wide range of stmctural classes and biological functions. Among plant polyphenols, of which several thousand have now been described, flavonoids form the largest group. However, phenolic quinones, lignans, xanthones, coumarins and other groups exist in considerable numbers and there are also many simple monocyclic phenolics. In this review, we examine the principal biosynthetic pathway of phenolic compounds in higher plants, and we define the different enzymes responsible for the regulation of phenolic metabolism. The central focus, however, is on the different factors, both biotic and abiotic, which influence the synthesis or degradation of phenolic compounds (bioactivity), analysing the physiological and ecological implications of these compounds in tlie adaptation by plants to adverse conditions. Finally, we related bioactivity of phenolic compounds with tlie appearance of physiological disorders in different crops.

GENERAL PHENYLPROPANOID PATHWAY Higher plants use amino acids not only as protein building blocks but also, and in even greater quantities, as precursors for a large number of secondary metabolites [1]. Phenylpropanoid compounds are among the most influential and widely distributed secondary products in the plant kingdom. These compounds serve a range of important functions in plants, providing structural components (such as lignin), protection against biotic and abiotic stresses (anti-pathogenic phytoalexins, antioxidants and UV-absorbing compounds), pigments (particularly the anthocyanins), and signalling molecules (e.g. flavonoid nodulation factors) [2]. Limiting this discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds that are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant [3,4].

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Biosynthesis of phenylpropanoid compounds The general phenylpropanoid pathway links the shikimate pathway to the lignin branch pathway. The latter pathway leads to the formation of a series of hydroxycinnamic acids and hydroxycinnamoyl-CoA esters varying in their degrees of hydroxylation and methylation [5]. There are diffent pathways by which all phenolic compounds are synthesized [6,7]. The shikimate/arogenate pathway leads, through phenylalanine, to the majority of plant phenolics, and therefore we shall centre the present revision on the detailed description of this pathway. The acetate/malonate pathway leads to some plant quinones but also to various side-chain-elongated phenylpropanoids (e.g. the group of flavonoids). Finally, the acetate/mevalonate pathway leads by dehydrogenation reactions to some aromatic terpenoids. The shikimate/arogenate pathway leads to the formation of three aromatic amino acids: L-phenylalanine, L-tyrosine, and L-tryptophane. This amino acids are precursors of certain homones (auxins) and of several secondary compounds, including phenolics [6,7]. One shikimate/arogenate is thought to be located in chloroplasts in which the aromatic amino acids are produced mainly for protein biosynthesis, whereas the second is probably membrane associated in the cytosol, in which L-phenylalanine is also produced for the formation of the phenylpropanoids [7]. Once Lphenylalanine has been synthesized, the pathway called phenylalanine/hydroxycinnamate begins, this being defined as "general phenylpropanoid metabolism" [7]. The general phenylpropanoid pathway begins with the deamination of Lphenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL), Fig. (1), the branch-point enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism [5-7]. Due to the position of PAL at the entry point of phenylpropanoid metabolism, this enzyme has the potential to play a regulatory role in phenolic-compound production. The importance of this is illustrated by the high degree of regulation both during development as well as in response to environmental stimuli. The enzyme PAL was first detected by Koukol and Comm [8], and is found in most higher plants as well as many microorganisms. This enzyme has an optimal pH of 8.8 and does not require a cofactor for its activity. Some preparations of PAL show activity towards the tyrosine although PAL

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is especially specific towards the substrate from which it draws its name [6,7].

Phony lalan mo

SaJioylic acid (SA)

CiMnam»c acid

\^^

4-coumaric a o d

4 -coumaroyl-CoA

Ftnuiic acio n3C3{[]> sinapic ac:d Feruloyl-CoA

Sinapyl-alcohol

Guaiacyl.siibunils

Syringytsubunits

Lignin

Coumartns Isoflavonoids (via C H R and CHS)

Naringenm chalcoru)

c:K::n

Naringomn ÛF3H "^ OH 0«hydrokaomp(erol

ILX^^^î

Au

r^;^t{]> Phlobapher c-s 1L_^ FlavonoH

C3c=)t{> Flavonois

^.

Loucopoiargonidin

•'' Anthocyanins Tannins

Fig. (1). Schematic view of some branches of phenylpropanoid metaboHsm. Solid arrows indicate enzymatic reactions with the respective enzyme indicated on the right. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; CHS, chalcone synthase; CFI, chalcone flavavone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol reductase; CHR, chalcone reductase. Broken arrows indicate metabolic branches towards several classes of phenylpropanoids, or several subsequent enzymatic steps. In some cases the enzymes indicated are also involved in other reactions, not shown. •Taken from Weisshaar and Jenkins, 1998, [2].

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All phenylpropanoids are derived from cinnamic acid, which is formed from L-phenylalanine by the action of PAL. Several simple phenylpropanoids (with the basic C6-C3 carbon skeleton of phenylalanine) are produced from cinnamate via a series of hydroxylation, methylation, and dehydration reactions, including /7-coumaric, caffeic, ferulic, and sinapic acid and simple coumarins, Fig. (1). The free acids rarely accumulate to high levels inside plant cells, but instead are usually conjugated to sugars (e.g. salicylate-glucose conjugates), cell-wall carbohydrates (e.g. ferulate esters), or organic acids (e.g. chlorogenic acid) [4]. Salicylic, benzoic, and /7-hydroxybenzoic acids, although not strictly phenylpropanoids themselves because they lack the three-carbon side chain, originate from the phenylpropanoids cinnamate and /7-coumarate, Fig. (1) [9]. Lignin and suberin, complex polymers formed from a mixture of simple phenylpropanoids, Fig. (1), vary in composition from species to species [5,10]. A large number of stress-induced phenylpropanoids are derived from the CI5 flavonoid skeleton, which is synthesized via the chalcone synthase (CHS)-catalyzed condensation of/?-coumaroyl-CoA and three molecules of malonyl-CoA, Fig. (1) [6,7]. In most plant families, the initial product of CHS is a tetrahydroxychalcone, which is fiither converted to other flavonoid classes, such as flavones, flavonones, flavonols, and anthocyanins [11]. In a number of species (e.g. pine, grapevine and peanut) the condensation ofpcoumaroyl-CoA or cinnamoyl-CoA with three malonyl-CoA molecules can also give rise to stilbenes by action of stilbene synthase (SS), Fig. (1) [12]. In legumes, isoflavone synthase (IFS) rearranges the flavonoid carbon skeleton, leading to the accumulation of a wide range of simple isoflavonoids, coumestrans, pterocarpans, and isoflavans. Structural diversity among the phenylpropanoids is brought about by a variety of modifications, including regro-specific hydroxylation, glycosylation, acylation, prenylation, sulfatron, and methylation [4]. Oxidation of phenolic compounds The metabolism of phenolics is regulated by the activity of various enzymes. As indicated above, the main and determining enzyme of phenolic synthesis is PAL, while in oxidation processes, the enzymes involved are peroxidase (POD) and primarily polyphenoloxidase (PPO).

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PPO (known also as catecol oxidase, phenolase or diphenol-oxygen oxidereductase) and POD catalyse the oxidation of o-diphenols to odiquinones, as in the hydroxylation of monophenols [13]. PPO is located exclusively in the plastids of healthy tissues, while most phenolic compounds are localized in the vacuoles, the two compounds thus being physically separated [13]. PPO apparently, however, exists free in the cytoplasm in degenerating or senescent tissues such as ripening fruit [14]. Reports of PPO in other organelles may be artifactual due to a POD activity mistakenly identified as PPO, solubilization of the enzyme during organelle isolation, or fragmentation of plastid parts containing PPO into other organelle [13]. Three lines of experimental evidence are used to support the idea that PPO is solely a plastid enzyme: fractionation studies, histochemistry, and chemical or genetic modifications of the plastid [13]. Futher confirmation of a plastid localization was reported by Henry et al. [15] after coupling fractionation techniques with PPO cytochemistry. The only structures with cytochemically detectable PPO activity were recognized as plastid parts. Because PPO can readily convert monophenols to o-diphenols in vitro [16], some investigators have assumed that the same process is involved in phenolic compounds synthesis in vivo. There are several arguments against this assumption. As discussed above, PPO is located exclusively in the plastids of healthy tissues and is apparently not even activated until it crosses the plastid envolope. The vast majority of phenolic compounds in higher-plant cells are located in the vacuole. One might argue, however, that the plastid location of PPO is necessary to provide a strong reducing environment in order to prevent fiirther oxidation of o-diphenols to odiquinones by PPO [13]. Although some investigators have found relationships between phenolic compound content and extractable PPO activity from cells or tissues, the relations may be due to POD activity, given that several works have demonstrated close relationships between POD activity and the oxidation of phenolics [17]. Moreover, there is considerable evidence that PPO is not active as a phenol oxidase in chloroplasts, but is limited as a phenol oxidase by latency or lack of substrate [18]. The latent form of the enzyme can be activated by a wide variety of treatments, including detergents [19], fatty acids [20] trypsin [21] and Ca^"" [22]. The results of Tolbert [21] indicated that light could activate latent PPO because polyphenols can be oxidized photochemically by chloroplast membranes in the absence of other

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activators. The only clear roles that PPO has in phenolic metabolism are in those cases in which plastid and vacuole contents are mixed. These cases can be divided into two categories: 1) senescence and 2) injury. In the first case, PPO has been invoked to explain the development of pigmentation in black olives, and other dark brown or black, usually dead, plant tissues. The increase in extractable PPO activity often seen during senescence is due to activation of previously synthesized enzyme [23]. Many researchers believe, however, that there is a fiinctional significance to the rapid production of quinones caused by injury [16]. Whether due to mechanical injury or to cellular disruption from disease, the quinones produced by the resulting PPO-phenolic compounds interaction are very reactive, making them good candidates for involvement in protection from other organisms. Thus, disruption of the plastid results in activation of latent PPO, which reacts with phenolics released from the vacuole. Finally, recent research suggests that different types of stress caused either by biotic or abiotic factors activate PPO and POD, thereby stimulating phenolic boactivity [24-27]. PHYSIOLOGICAL IMPLICATIONS: COMPOUNDS VS. PLANT HORMONES At present, it is still not known definitively whether phenolics play a physiological role in the growth and metabolism of plants. Many phenolic compounds are capable of exerting significant effects on growth and developmental processes in plants, when these compounds are applied to tissues at physiological concentrations [6]. With so much variation in structure, it is unlikely that phenolics as a group of substances have one particular universal role in regulating growth and development. Rather, it is possible that individual classes or individual subtances may carry out significant activities in certain physiological processes. Many of the observed physiological interactions may be incidental to the wider fiinction of phenolics as protective agents in plants [6]. Even though most phenolics are not hormones themselves, they may affect plant growth by interaction with one or other of the major kinds of plant hormones, such as the auxins. Physiological studies have suggested

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that several phenolic compounds regulate auxin, indole-3-acetic acid (lAA), and others affect polar transport of auxins. Hydroxyphenolic, chlorogenic acid, was shown to be a protector of auxin against oxidation by lAA oxidase in sunflower leaves, thereby stimulating plant growth [28]. Dihydroxyphenolic compounds have been considered inhibitors of lAA oxidation, whereas monohydroxyphenolic derivatives stimulated lAA oxidation and thus have a potentially inhibiting effect on growth [29]. Certain flavonols, such a quercetin, were found to inhibit polar transport of lAA [30]. Changes in the levels of lAA as well as abscisic acid had been measured during growth and ripening in the pericarp tissue of two cultivars of tomato fruit [31]. Recently, Buta and Spaulding [32] found that the decline of chlorogenic acid and rutin levels during fixiit ripening paralleled the decline in lAA levels measured previously in the pericarp tissues of two varieties of tomato fhiit during maturatio (Lycopersicon esculentum fruit var. Ailsa Craig and Pik-Red). These phenolics are among the ones that have been suggested as regulators of auxin metabolism. Another hormone that can be affected by phenolics is ethylene. Coumaric acid is necessary as a cofactor for the biosynthesis of ethylene [33]. On the other hand, caffeic acid inhibits an enzyme (peroxidase type) for which the cofactor is p-coumaric acid. Therefore, the balance between /7-coumaric and caffeic acid can in theory influence the regulation of ethylene biosynthesis [6]. Phenolics can react with other hormones by synergism or inhibition, and both situations can be observed in the case of gibberellin. There is evidence that dihydroxyconiferyl-alcohol gives rise to synergetic effects over gibberellin, stimulating hypocotyl elongation [34]. On the contrary, the replacement of hydroxyconiferyl-alcohol for one or several hydroxycinnamic acids results in the inhibition of effects caused by gibberellin [34]. Finally, in view of the above examples, phenolics appear to interact specifically with certain plant hormones which effect growth. ECOLOGICAL IMPLICATIONS AND FUNCTIONS OF PHENOLIC COMPOUNDS

PHYSIOLOGICAL

There is increasing evidence that the functional aspects of phenolics must be considered in making any distinction between primary and secondary

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compounds. For instance, it is known that many secondary compounds are valuable storage vehicles and indispensable elements in anatomical and morphological structures. However, there is increasing knowledge that they are also of prime ecological importance for the improvement in plant survival [7]. First of all, phenolics are of great importance as cellular support materials. They form an integral part of cell wall structures, mainly in polymeric materials such as lignins and suberins. Lignin is the second most abundant plant polymer after cellulose and forms a major component of terrestrial biomass [35]. Lignin is the term given to a group of complex phenolic polymers that provide important strengthening and waterproofing properties to plant cell walls. It is not suprising, therefore, to find lignin playing fiindamental roles in mechanical support, solute conductance, and disease resistance in higher plants [5]. The prospect of manipulating the quantity and composition of lignin in plants has several attractions, particularly in the modification of the digestibility of plant material and in the development of resistance to microbial pathogens. Lignification is ultimately dependent on phenylpropanoid metabolism for the supply of the basic building blocks of lignin and this dependency has led to the targeting of phenylpropanoid metabolism as a suitable point at which to manipulate lignin biosynthesis. Phenolics are of great ecological importance along with various toxic nitrogen-containing compounds as well as attractant or repellent terpenoides. The most significant fimction of the phenolic flavonoids, especially the anthocyanins, together with flavones and flavonols as copigments, is their contribution to flower and finit colours [36]. This is important for attracting animals to the plant for pollination and seed dispersal [6]. On the other hand, phenolics may protect plants against predators. Herbivores react sensitively to the phenolic content in plants. The rise in cumestrol and cumarin levels can be toxic for herbivores, causing anti-coagulation and estrogenic effects [37]. Phenolics may influence competition among plants, a phenomenon called allelopathy. A series of experiments in both the field and the laboratory have indicated a role for a number of phenolic derivatives (hydroquinone, hydroxybenzoates and hydroxycinnamates) as allelopathic agents. These are chemicals excreted by the plant, which may be autotoxic or affect the growth of other plants in the environment [6,7]. A role in recognition and signalling has been determined for a variety of

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phenolics. Thus, phenolics induce the germination and development of haustorium in parasitic plants [38], the expression of v/> genes responsible for T-DNA transfer from Agrobacterium to the plant cell [39], and regulate the expression of "nod" genes during the formation of the legumeRhizobium symbiosis [40]. Li the last case,flavonoidsand isoflavonoids from seed and/or root exudates or extracted from roots, were identified as regulatory molecules [40]. The processes of isoflavonoid exudation and accumulation are partly regulated by environmental factors. The isoflavonoid exudation and accumulation is inhibited by phosphate [41] and various N forms with nitrates being a stronger inhibitor than ammonium or urea [40]. However, most of the literature available relates phenolic compounds to resistance against the following types of stress: (i) pathogen attack, (ii) woimds, (iii) ultraviolet radiation, (iv) environmental pollution (specially ozone), and finally (v) thermal stress. Due to the importance and incidence of these types of stress for the survival and adaptation of plants, this topic will be explored in more detail in the forthcoming sections. Bioactivity of phenolics in resistance to pathogen attack Three groups of phenolic compounds are involved in defense responses: (1) the signal molecule salicylic acid [42], (2) phenylpropanoid compounds (phenolics and phytoalexins) [43], and (3) lignin and related polyphenolics [44,45]. Plants react to pathogen attack through a variety of active and passive defense mechanisms. At the site of infection, a hypersensitive response is often initiated in resistant plants, which isfrequentlymanifested as necrotic lesions resultingfromhost cell death. In addition, the distal uninfected parts of the plant can develop systemic acquired resistance, which results in enhanced long-lasting defense against the same or even unrelated pathogens [45]. Both the hypersensitive response and systemic acquired resistance are associated with increased expression of a large number of defence or defence-related genes [46]. Examples of the defence reactions include the lignification and suberization of the plant cell wall, deposition of callose, de novo synthesis of pathogenesis-related proteins, production of active oxygen species, and biosynthesis of secondary metabolites (phytoalexins) [47-50]. Much evidence suggests that the increases in salicylic acid levels are

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essential for the induction of systemic acquired resistance [42]. Pathogen-induced necrosis on the leaves of many plants results in the production of a mobile signal in the phloem that triggers systemic resistance to subsequent pathogen [51]. The development of systemic acquired resistance depends on the rate at which the pathogen causes necrosis in the infected leaf Pathogen-induced necrosis in the inoculated leaf is accompanied by the accumulation of salicylic acid at the site of inoculation, in phloem fluids, and in healthy, uninoculated leaves [52]. Several reports suggest that PAL is a key regulatory enzyme in the synthesis of salicylic acid. Fig. (2), and the establishment of systemic acquired resistance. Mauch and Slusarenko [53] showed that in Arabidopsis, PAL activity was essential for the accumulation of salicylic acid and expression of the hypersensitive response. Recently, it was also reported that tobacco plants epigenetically suppressed in PAL activity were unable to express systemic acquired resistance [54].

Fig. (2). Proposed pathways of SA and 4HBA biosynthesis. Enzymatic steps for which the enzymes have been identified include PAL, CA4H (cinnamic acid 4-hydroxylase), and BA2H (benzoic acid 2hydroxylase). *TakenfromSmith-Becker et al., 1998, [55].

Smith-Becker et al. [55] have shown that the first measurable effect of the mobile signal for systemic acquired resistance in cucumber inoculated

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with P. syringae is the transient stimulation of PAL activity in the petiole of the inoculated leaf and in the stem above the inoculated leaf The transient increase in PAL activity precedes a transient increase in salicylic acid and 4hydroxybenzoic acid in phloem fluids, and suggests that the two compounds are produced de novo in stems and petioles, perhaps in vascular tissues.

Host Defense Activation by H.O, Elevation

Fig. (3). Hypothetical representation of host defense activation by extracelluiarly produced H2O2 in GO-transgenic plant. Salicylic acid biosynthesis is elevated through stimulation of benzoic acid 2hydroxylase activity. The expression of defense-related proteins is induced by increased salicylic acid and perhaps also by H2O2 itself via a separate pathway. •TakenfromWu et al, 1997, [42].

A metabolic engineering approach has now provided direct evidence for the role of salicylic acid in systemic acquired resistance. Transgenic tobacco plants were produced expressing the "nah G" gene from Pseudomonas putida, which encodes a salicylate hydroxylase that converts salicylic acid

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to catechol [56]. These plants had greatly reduced salicylic acid levels and were unable to establish systemic acquired resistance. Moreover, not only did they fail to exhibit resistance to virulent challenges following inoculation with avirulent pathogens, but also they were no longer able to express hypersensitive resistance against the primary avirulent challenge [56], reflecting an important role for salicylic acid in the expression of local resitance. Fig. (3). A range of defence response genes, including those encoding the so-called pathogenesis-related proteins, are activated in systemically protected leaves and in response to exogenously applied salicylic acid [51]. Plants have envolved a wide array of chemical defences against pathogens. These include secondary metabolites with anti-microbial properties [57]. Some secondary metabolites are constitutively present in normally developed healthy plants, whereas others are induced by pathogen invasion [58]. The latter include phytoalexins, which are synthesized de novo after a pathogen attack [43], and phytoanticipins [59]. The first direct demonstration of the potential significance of phytoalexins in plant defence was provided by the introduction of a grapevine SS gene into tobacco plants [60]. The foreign gene product was able to divert a portion of the substrates of chalcone synthase to the synthesis of the stilbene phytoalexin resvaratrol, resulting in plants with increased resistance to thefiingalpathogen Botrytis cinerea. Generally, the increase in phenolic content is the response associated with plant disease resistance. Diibeler et al. [61] found a direct relationship between, on the one hand, the accumulation and qualitative composition of phenolic compounds in Fagus sylvatica and resistance against Cryptococcus fagisuga, Li addition, Valette et al. [62] carrying out histochemical and cytochemical investigations of phenolic compounds in roots of banana infected by the burrowing nematode Radopholus similis showed the importance of phenolic accumulation to differentiate plants which were resistant from tiiose sensitive to infection by this nematode. Using Neu*s reagent, these researchers indicated the presence of flavonoids and caffeic esters in parenchyma and vascular cells and of ferulic acid in walls of parenchyma cells in the resistant cultivar, while these compounds were not detected in susceptible roots. Finally, Valette et al. [62] concluded that, in resistant banana, phenols incluidingflavonoids,caffeic esters, and dopamine may limit root penetration by the nematode, and the high level of vascular lignification and suberization of endodermal cells restricts xylem

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invasion by, and prevents multiplication of, the pathogen in the vascular tissues. As indicated above, PAL, the first enzyme of phenylpropanoid metabolism, plays a significant role in the regulation of phenol biosynthesis in plants as a response to pathogen infection [55,63]. The phenylpropanoid skeletons serving as building blocks for lignin induce lignification, which has been proposed as a mechanism of disease resistance in plants against the invasion of fungal pathogens [64]. Lignin precursors and free radicals are known to inhibit fungal enzymes, and lignin accumulation may act as a barrier and check to the translocation of host nutrients into the pathogen. Nagarathna et al. [63] suggested that an increased amount of PAL increases lignin synthesis, leading to a hypersensitive response at the site of infection in the incompatible interations and thereby restricting fungal growth. Bioactivity of phenolics against wounds Woxmds may be inflicted by severe weather, insects, large herbivores, the activities of man, or even during the normal development processes of the plant, such as abscission or growth cracks [65]. In commerce, even minimal processing of fresh fruit and vegetables involves many mechanical processes (e.g. abrading, cutting and peeling), which injure the tissue [66]. Such wounding induces alterations in many physiological processes, which often make the processed item more perishable than the unprocessed fresh product and diminish the shelf life of thefinalminimally processed product [67]. Wounding also elicits several physiological responses associated with wound healing [68]. Foremost among these reactions are alterations in phenolic metabolism and the concomitant increase in the propensity of the wounded tissue to brown [66]. The activity of PAL and the concentration of phenolic compounds (e.g. chlorogenic acid, dicaffeoyl tartaric acid, and isochlorogenic acid) increase in excised iceberg lettuce midrib segments after wounding [69]. In addition, Thypyapong et al. [25] provide evidence that wound-responsive expression of potato PPO is regulated at the levels of transcriptional activity or mRNA stability. Similarly, wounding increases the steady-state PPO mRNA levels in apple [70]. Phenylpropanoid biosynthesis gives rise to caffeic acid, lignins.

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flavonoids, and salicylic acid. The coordinated regulation of PPO and phenylpropanoid biosynthesis underscores the importance of PPO in wound responses. Wound induction of the phenylpropanoid pathway leads to intracellular accumulation of phytoalexins and the extracellular polymerization of phenolics, e.g. lignification, providing barriers to pathogen ingress [4]. Once pathogens overcome these barriers and disrupt plant cells, PPO activity diverts phenolics to quinone production, causing cell death and providing additional polymerized phenolic barriers to sencondary infection [71]. PPO catalyses the dependent oxidation of phenolics to quinones. The secondary reactions of quinones lead to the formation of polymeric brown or black pigments, which are responsible for significant post-harvest losses offiiiitsand vegetables [72]. Finally, induced PPO activity consists of both systemic and localized components. Systemic induction of PPO in tissues in response to all types of injuries may represent a broad, defensive role for PPO in protection of juvenile tissues from subsequent attack by a broad spectrum of pathogens and pests [71]. Bioactivity of phenolics in resistance to ultraviolet rays In recent years, there has been considerable concem over reductions in stratospheric ozone concentrations resulting from human activities. Since stratospheric ozone is the primary screen of solar ultraviolet radiation, ozone reduction would increase ultraviolet radiation (UV-B, wavelengths between 280 and 320 nm) reaching the earth's surface. UV-B is biologically significant, since it is potentially mutagenic and its influnce is increasing due to ozone depletion [73]. Among the most important physiological and biochemical processes affected by UV-B exposure is photosynthesis. UV-B radiation generally results in reductions in net photosynthesis, coinciding with ultrastructural damage to the chloroplast. UV-B radiation also affects stomatal resistance, chlorophyll concentration, soluble leaf proteins, lipids, and carbohydrate pools [74]. Finally, exposure to UV-B give rise to variations in anatomical and morphological plant characteristics. Commonly observed changes include plant stunting, reductions in leaf area and total biomass, and alterations in the pattern of biomass partitioning into various plant organs. In sensitive plants, evidence of cell and tissue damage often appears on the upper leaf

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epidermis as bronzing, glazing and chlorosis [74]. Interception of UV-B by epidermal flavonoids is often proposed as an adaptive mechanism preventing UV-B from reaching the mesophyll and affecting photosynthesis [75], Flavonoids have high absorption capacity for UV-B and they may be present in mature leaves at levels exceeding 50 |imol g"* f w. [76]. When higher plants are subjected to supplemental UV they tend to synthesize flavonoids and anthocyanins [77], and some fimgi also react by forming such pigments [78]. Liu et al. [79], analysing the effect of UV-B radiation on phenolic metabolism, growth and photosynthesis in barley primary leaves, indicate that UV-B significantly slows rates of leaf elongation with a simultaneous, possibly related, increase in wall-bound ferulic acid esters in the epidermis. UV-B also markedly increases flavonoid accumulation in both the lower epidermis and underlying tissues. On the other hand, since UV-B had no significant effect on photosynthesis rates, photosynthetic pigments, fresh weight or dry weight, this authors conjecture thatflavonoidsin the barley primary leaf provide considerable constitutive, as well as inducible, resistance to damage from this type of radiation. Finally, Liu et al. [80] showed that UV-B sharply boosted PAL activity while reducing POD activity; this would explain the synthesis and accumulation of flavonoids and ferulic acid in barley primary leaves. Bioactivity of phenols against ozone exposure Phenolic compounds take part in protection, regeneration and degradation processes caused by toxic pollutants [81]. An increase in the phenolic level is commonly observed after exposure to hydrogen fluoride [82], sulphur dioxide [83] and especially to ozone [84]. Ozone in recognized as one of the most damaging secondary gaseous air pollutants essentially generated from nitrogen oxides produced by anthropogenic activities [85]. Among its numerous impacts on the environment, it has been shown to affect crop yield significantly [86]. Currently, to indicate phytotoxic environmental concentrations of ozone, the assessment of visible ozone injury (necrotic spots and bronze-coloured symptoms that follow a chlorotic fecking of leaves) of more or less ozone sensitive plant varieties is becoming widely used [85]. Tobacco, Nicotiana tabacum [87], and clover, Trifolium sp., [88] are the most important ozone

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bioindicators. From a physiological standpoint, molecular and biochemical studies have suggested that ozone stimulates phenolic metabolism and the biosynthesis of lignin or other substances partly derived from coniferyl alcohol [89]. Lignification of mesophyll cell walls might confer some protection against oxidation, and thus be a defence response against ozone [90]. Studies have shown that phenylpropanoid metabolism can be stimulated by ozone. The activity of PAL increased in soybean [91], Scots pine {Pinus sylvestris L.) [92], and parsley {Petroselinum crispum L.) [93] soon after treatment with 150-200 nmol O3 mor\ Rapid increases in transcript levels for PAL in response to ozone have been observed in parsley [93], Arabidopsis thaliana L. Heynhold [94] and tobacco {Nicotiana tabacum L.) [95]. Transcript levels for 4-coumarate:CoA ligase (4CL), the last enzyme in the general phenylpropanoid pathway, increased commensurately with PAL transcripts in ozone-treated parsley seedlings [93]. Phenolic compunds reported to accumulate in leaf tissue in response to ozone include hydroxycinnamic acids, salicylic acid, stilbenes, flavonoids, furanocoumarins, acetophenones, and proanthocyanidins [85, 92, 93, 96, 97]. A rise in transcript levels and activity of cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in lignin biosynthesis, were observed in Norway spruce {Picea abies L.) needles [84] and parsley leaves [93] after treatment with ozone. Increased lignin in sugar maple {Acer saccharum Marshall) foliage following exposure to ozone has also been reported. However, higher levels of lignin were not detected in the foliage of several ozone-treated conifers and other hardwood species [96]. Booker and Miller [98], studying the phenylpropanoid metabolism and phenolic composition of soybean leaves following exposure to ozone, found that the activities of general phenylpropanoid pathway enzymes (PAL and 4CL) were stimulated by ozone 6h after their application, whereas the activity of the key enzyme in lignin synthesis, CAD, was stimulated 27 h after ozone exposure. In addition, these authors report that the levels of cellwall-bound total phenolics, acid-insoluble lignin and lignothioglycolic acid extracted from leaf tissue from ozone-treated plants increased on the average by 65%. However, histochemistry, UV and IR spectra, radiolabelling and nitrobenzene oxidation assays all indicated that lignin and suberin did not increase with ozone treatment. Finally, taking into

667

account these results, Booker and Miller [98] concluded that ozone-induced incrases in phenolic metabolism, resemble certain elicited defense response, and thus occur in concert with effects characteristic of the browning reaction and wound responses. Bioactivity of phenolics in resistance against thermal stress Most plants suffer damage, both physiological and biochemical by exposure to temperatures higher or lower than optimal for growth [99]. The results of these injuries, which are reflected in most metabolic processes may be a reduced growth capacity of the crops and therefore lower commercial yield [100]. It has been demontrated that thermal stress induces the production of phenolic compounds [3,4,101], Phenolic compounds may be involved in plant responses to cold stress and in plant acclimation to low temperature. Acclimation of apple trees to cold climates was found to be associated with a seasonal accumulation of chlorogenic acid [102]. Strengthened frost tolerance in a variety of plants were attributed to thicker cell-wall lignification or suberization [102]. Thickening of cell walls and increased production of suberin-type lipids were observed in cold-acclimated winter rye leaves [103]. The presence of suberin in cell walls may favour membrane cell-wall adhesion, a major factor in the resistance of plant cells tofreezing[104]. Stimulated of PAL activity as a response to chilling treatment was observed in chilling-sensitive tissues such as potatoes and sweet potato tubers, as well as in apple fruits [105]. Solecka and Kacperska [106] studying in the leaves of winter oilseed rape plants grown for 3 weeks at 2^C and then exposed to a brief freezing and thawing, found pronounced changes in PAL activity. These changes included: a) a marked boost in the total and specific activities of the PAL, noted during thefirst2 days of plant treatment with chilling temperature, b) a sharp, swift and transient rise in PAL activities noted directly after thefreezingand thawing treatment. Recently, working in our laboratory with tomato and watermelon plants exposed to different temperature, we found that the optimal growth temperatures were 25 and 35 °C respectively, while over 35 ^C and below 15 °C, respectively, caused thermal shock . The results of this experiment indicate that in plants the response against thermal stress was similar regardless of the temperature. With regard to phenolic metabolism, thermal stress in tomato and watermelon plants is characterized by

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increased PAL activity and foliar accumulation of phenolic compounds, and also by a sharp drop in the activity of enzymes governing phenolic oxidation (PPO and POD)^ These results, together with those of the other works described above indicate that phenylpropanoid metabolism may play an important role in the development of plant acclimation to thermal stress. ABIOTIC FACTORS COMPOUNDS

AND

BIOACTIVITY

OF

PHENOLIC

Given the scope of the processes in which phenolic compounds are involved (e.g. interactions with plant hormones, resistance to pathogen attack, wounds, UV rays, ozone and thermal stress, implications for agriculture), knowledge of the factors that regulate the metabolism of these compounds could enable the manipulation of their synthesis or degradation, depending on the conditions chosen or the results desired. Among the abiotic factors most widely used to manipulate and vary the metabolism of phenolics, are biocides, primarily herbicides and fungicides, and different nutrients. Biocides Herbicides are the biocides most likely to affect the metabolism of plants, including secondary metabolism [107]. The synthesis of hydroxyphenolics and anthocyanin in plants can be influenced by a variety of environmental and chemical stimuli. Some herbicides were found to raise the levels of these compounds in plants [108] whilst others had the opposite effect [109]. The products of secondary metabolism are controlled by enzymes, including PAL and chalcone isomerase (CI), and several herbicides appear to intensify the activities of those enzymes involved in the accumulation of hydroxyphenolic compounds and anthocyanin biosynthesis in several plant species [109-111] whereas others depress this activity [112]. For example. Rivero, R.M.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; S^chez, P.C.; Romero, L. Resistance to thermal stress: accumulation of phenolic compounds in tomato and watermelon plants (in consideration).

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PAL activity in soybean seedlings was increased by several herbicides such as DPX-4189, glyphosate and acifluorfen [112,113]. Also, the activities of PAL and tyrosine ammonia lyase (TAL) in both maize and soybean seedlings were also increased by alachlor [110] and metolachlor [111]. Depending upon the type and concentration of the herbicides, a range of activity from little impact on plant growth to severe symptoms of toxicity were found [114] and these varied effects proved to be associated with marked changes in secondary metabolism. Nemat Alia and Younis [115] studing the effects of different herbicides (trifluralin, fluometuron, atrazine, alachlor, and rimsulfuron) on phenolic metabolism in maize {Zea mays L.) and soybean {Glycine max L.) seedlings, detected various modes of action on this metabolic process, depending on the herbicide applied. The results of that work showed that the activities of PAL and CI were greatly enhanced in both species by alachlor and rimsufuron, but diminished by trifuralin. Moreover, dydroxyphenolic compounds were increased in both species by alachlor and rimsulfuron and decreased by trifuralin and atrazine. Similarly, anthocyanin content was augmented in both seedlings by alachlor and rimsulfuron, but reduced by trifluralin and fluometuron, whereas atrazine decreased the anthocyanin content in maize only. As indicated above, although less data is available that for herbicides, fungicides also have been implicated in the appearance of variations in phenoUc metabolism. Molina et al. [116] have demostrated that the systemic acquired resistance signal transduction pathway, a salicylic aciddependent plant-defence mechanism, mediates fungicide action in the plant. Ruiz et al. [27] found that the application of the fungicide carbendazim (bencimidazol 2-il methyl carbamete, C9H9N3O2) in tobacco plants not infected by pathogens caused significant variations in the bioactivity of phenolics with respect to control plants. The foliar application of 2.6 mM carbendazim boosted phenol biosynthesis and accumulation, since PAL activity was stimulated and, in addition, the oxidative enzymes PPO and POD were inhibited. According to these researchers, this may imply an increased resistance of plants to infection by pathogens, given the essential role of phenolic compounds in the lignification and suberization of the plant cell wall.

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Nutrients Different works show that the nutritional status of certain nutrients, such as boron (B), calcium (Ca), nitrogen (N), phosphorus (P) and iron (Fe) can trigger changes in phenolic metabolism. Of these nutrients, B is attributed with a clear and significant effect on the metabolism of these secondary compounds. As we shall discuss below, the relationship between B metabolism and phenolics is complex and depends largely on the sensitivity of the plant to B deficiency or toxicity. Boric acid has the particular ability to form stable complexes with compounds that present cis-hydroxyl groups (cis-diol groups) [117]. Several compounds, such as sugars and their derivatives and some phenolics (o-diphenols) have these cis-diol groups and therefore can form stable complexes with B [117]. The cell wall is a structure rich in compounds with a cis-diol configuration. On the other hand, it has been found that B is a predominant element in this structure, principally under deficiency conditions [118]. It has been estimated that the B present in the cell wall represents roughly 96% of the total of cellular B in carrots (Dancus carOta) [119]. In tobacco cells, the B in the cell wall reportedly makes up 90% of the total B in the cell with deficient levels and 60% with normal (or adequate) levels of this micronutrient [119]. Increases in the phenolic concentration under conditions of B deficiency are observed particularly in plants with high requirements of this element, such as sunflower {Helianthus annuus) [120]. The formation of cis-diol complexes with B, in some sugars and phenolic compounds, play a decisive role in the accumulation of phenolics in the B-deficient tissues. This element forms complexes with 6-phosphogluconic acid, inhibiting the activity of 6-phosphogluconic dehydrogenase activity, which augments the concentration of 6-phosphogluconic acid in the tissues deficient in B [121]. Consequently, under conditions of B deficiency the substrate is moved from glycolysis to the penthose phosphase shunt, which increases the synthesis of the phenolics [121]. The formation of B-phenolic complexes can also affect the quantity of these compounds in the tissues. As indicated by Pilbeam and Kirkby [122], the bonding of B and caffeic acid blocks the formation of quinones, and therefore facilitates the synthesis and accumulation of phenolics. The accumulation of phenolics in B-deficient tissues is a critical step in

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the start of different cell responses, since a characteristic of these is their phytotoxicity, even at low concentrations (l|iM) [123]. In B-deficient plants, caffeic, chlorogenic, ferulic and vanillic acids can accumulate [124], this phenomenon being most pronounced with stronger light intensity [125]. The stimulation of phenolic synthesis by intensifying luminosity has also been demonstrated, and is attributed to inducing PAL by light [126]. However, several authors have corroborated that PAL activity, regardless of light intensity, is increased by B deficiency [120]. Phenolic accumulation in B-deficient tissues can activate a group of enzymes that use these compounds as substrates. In B-deficient sunflower leaves, PPO activity progressively augments as symptoms characteristic of this deficiency appear. Plants with sensitivity to B deficiency differ in PPO activity. The accumulation of phenolics and PPO activity are reportedly greater in more sensitive plants, such as sunflower, than in less deficiency-sensitive plants, such as com {Zea mays) [125]. Phenolic oxidation by PPO and POD in B-deficient leaves leads to the production of quinones. These compounds are known for their high toxicity, and for being responsible for the production of oxygenated radicals such as: H2O2 and O2". The accumulation of quinones in plants that act as indicators of the deficiency has been considered as the prime cause of cell damage and of growth reduction [127]. In most cases, B deficiency is associated with brown coloration in foliar tissues. In sunflower plants, Cakmak et al. [128] demonstrated that the foliar pigmentation intensified in B-deficient plants exposed to intense light. This pigmentation may be caused in B-deficient tissue by a stronger phenolic concentration and their subsequent oxidation by PPO. The quinones produced by PPO activity are consecutively polymerized, giving rise to this pigmentation. Also, in fruits, the brown colour during ripening or after harvest is associated with high phenolic content and strong PPO activity [128]. As commented above, most works that relate B to phenol metabolism centre on the effect of B deficiency. Nevertheless, other works show the effect of growing dosages of the micronutrient on the metabolism of these secondary compounds. For example, Fawzia et al. [120] found that as the B dosage increased to 500 ppm, the phenolic concentration declined, as did the PAL and PPO activity, while the contrary effect was evident in POD activity. The high POD activity, together with the low PAL activity perhaps decreased the phenolic content. These same authors found

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completely opposite responses under conditions of B deficiency. In addition, Ruiz et al. [26] reported a foliar accumulation of phenolics under the lowest B dosages (0.5 jiM) and the highest (20 |iM), while intermediate dosages (5 and 10 |iM) resulted in oxidation of these compounds. The explanation of these results may be based on different forms in which B is found in the leaves, either forming complexes with the phenols or in free form. According to these authors, the lowest and highest dosages reflect, respectively, deficiency and high levels of B. Under these conditions, the proportion of free B would be minimum, while the proportion of B forming complexes with phenolics could be high. Different works indicate that the application of B at deficient or excessive dosages causes the formation of complexes between B and compounds such as pectins and phenols (more than 90% of the total B) [118,130]. Therefore, the lowest proportion of free or metabolic B in these treatments would account for the high PAL activity, the low availability of phenolics for oxidation due to their bonding with B, the low PPO and POD activities, and the highest concentrations of phenolics [26]. On the contrary, the intermediate B dosages (5 and 10 (iM) gave rise to adequate levels of this micronutrient, possibly raising the levels of free or metabolic B. These may inhibit PAL and increase phenol oxidation, due primarily to two causes: i) a greater availability of uncomplexed phenolics with B, and ii) an effect of free B boosting the activities of PPO and POD. Finally, Ruiz et al. [27] observed that the foliar application of fiingicide carbendazim B dosage of 8 mM strengthens synthesis (PAL) and subsequent oxidation of phenols (PPO and POD). The oxidation of phenols under this treatment could generate active oxygen species, which perform multiple important fiinctions in early defence responses of the plant. In short, these authors conclude that joint application of carbendazim with certain dosages of B could imply an increase in the resistance of plants to infection by pathogens, and suggest that the application of carbendazim-B could reduce the recommended applications of fimgicide without decreasing effectiveness. As opposed to the case of B, very few works investigate the direct effect of Ca on phenolic metabolism. Castaneda and Perez [131], working with lemon trees, observed that the application of 10 juM of CaCb increased PAL activity one hour earlier than control only if the trees were treated with cell walls isolated from the fungus Alternaria alternata, or

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when the trees were inflicted with injury. Therefore, the authors suggested that the external application of Ca favours the transduction signals produced by the inductors or by the wound, for two responses: one related to plant defence and the other to injury repair. According to the results of the above work, Ca appears to act as a second messenger. However, as the authors indicate, it is difficult to determine whether the increase in cytosol Ca, which would act as a second messenger, is due to the ingress of extracellular Ca or to the exit of Ca from other cell organelles. To address this latter doubt, the authors administered seedlings with 50 |iM of Varopimil, a compound well known for its effective blockage of Ca channels. The result was a sharp drop in Ca absorption, and also a decline in PAL activity. On the other hand, the increase in cytoplasmic Ca from the uptake of the culture medium would involve the appearance of a series of transduction signals which would cause the rapid and efficient initiation of response against mechanical damage or pathogen attack. Finally, these researchers indicate that different CaCk concentration added to the reaction medium of PAL did not alter the activity of this enzyme, suggesting that the Ca participates in the cell response and not directly in the activity of the enzyme [131]. Another number of works [22,69,132] showed the effect of Ca on the enzymes responsible for the oxidation of phenolics. This subject is surrounded by controversy, since, as we shall discuss below, some researchers claim a positive effect of Ca on PPO and POD activities, while others maintain the contrary: i) Soderhall [22] indicated that the presence of CaCb stimulated PPO activity. Taking into account that this enzyme is normally found in latent form, this researcher explained the increased PPO activity on adding 6 mM of CaCb by the conformational alteration of the active site of the PPO to make itself accessible to the substrate. ii) Lastly, other workers have reported that an increase in the Ca concentration depresses the PPO and POD [69,132]. Another of the nutrients which has been related to the bioactivity of phenolics has been N. In present-day agriculture, the main types of stress commonly generated as a consequence of the heavy use of inorganic fertilizers are related to the nutritional status of certain nutrients, primarily N, given its extensive use. In general, although only scant literature is available on the relationship between N and phenol metabolism, it appears

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that the relationship between the N availability and the accumulation of phenolic compounds is usually positive [133, 134]. Recently, we found that the toxicity of N (27 mM) in green bean plants is characterized by the inhibition of the oxidative enzymes PPO, POD and catalase (CAT), and by the stimulation of PAL, thus resulting in a foliar accumulation of phenolic compounds . On the other hand, and studying the N deficiency in green bean plants in relation to phenolic metabolism, we found that N deficiency (1.35 mM) is characterized by a stimulation of the oxidative enzymes PPO, POD and CAT, which inhibited PAL activity, resulting in the lowest foliar accumulation of phenolic compounds . Finally, several works have also implicated the nutrients P and Fe as possible inductors of changes in phenolic metabolism. However, studies of these relationships have been scarce. With regard to the former nutrient, P deficiency has been observed to raise the level of anthocyanins, but the reason for this rise remains unclear [4]. Meanwhile, low levels of Fe can increase the release of phenolic acids, presumably to help solubilize metals and thereby facilitate their uptake [135]. IMPORTANCE OF BIOACTIVITY PRESENT-DAY AGRICULTURE

OF

PHENOLICS

IN

The accumulation or oxidation of phenolic compounds principally in stored agricultural products normally gives rise to the physiological disorders that result in a major loss of commercial value of the products. Among the most common of these disorders in which phenol metabolism is involved is fruit browning and the russet spotting (RS) in harvested lettuce. Due to the importance of visual appearance as a parameter of produce cosmetic quality, tissue browning has long gained the attention of horticultural researchers [136]. Fruit browning, as a consequence of bruising, is due to phenolic oxidation [136,137]. The destruction of fruit Sdnchez, E.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; Rivero, R.M.; Romero, L. Response to the bioactivity of phenolic compounds and oxidative metabolism in green bean plants undergoing nitrogen toxicity (in consideration). ^ Sanchez, E.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; Rivero, R.M.; Romero, L. Behaviour of phenolic and oxidative metabolism as bioindicators of nitrogen deficiency in green bean plants (Phaseolus vulgaris L. Cv. Strike). Plant Biol., 2000 (in press).

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cellular compartmentation allows the phenolic substrates to be accessible to PPOs which catalyze phenolic oxidation [137]. The concentration and composition of phenolic compounds and/or activity of PPOs are often the major factors governing the development and intensity of tissue browning [137]. Infruitsof apples [138] and avocado [139] positively correlated with the tendency of the fruit to tum brown was total phenol content and PPO activity. Cheng and Crisosto [24] found that browning in the buffer extracts of peach and nectarine skin tissue depends on the presence of PPO activity and chlorogenic acid, which are major contributors to enzymatic browning. RS is a physiological disorder in iceberg lettuce that manifests itself as oval, brownish spots or lesions, mainly on the achlrophyllous midribs, although in advanced stages may spread over the entire leaf blade [140]. Several studies have described a relationship between the activity of PAL in iceberg lettuce leaf tissue and the development of RS symptoms [141-146]. Hyodo et al. [141] observed that an ethylene-induced increase in PAL activity parallelled the appearance of RS symptoms. These researchers also measured an increase in total phenolic compounds. It has been proposed that ethylene induces PAL activity and the resulting accumulation of phenolic compounds in cells leads to their discoloration and eventual death [141,143,145]. Taking into account the essential role of PAL activity in the appearance of RS, Peiser et al. [146] studied whether the application of various inhibitors of PAL activity could prevent the development of RS lesions in lettuce midribs. The use of the inhibitor 2-aminoindan-2-phosphonic acid (AIP) sharply diminished the formation of phenolic compounds, although this did not reduce the number of lesions associated with RS [146]. In short, according to the results of Peiser et al. [146], the early development of RS lesions is independent of the increase in PAL activity and phenolic compounds, rather than resulting from these increases as previously suggested. However, the accumulation of phenolic compounds does contribute to the subsequent browning symptoms indicative of RS. FUTURE As indicated throughout this review, phenolic compounds play an essential part in different physiological processes, in the adaptation of plants to adverse conditions, as well as in the commercial quality of many

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agricultural products. Therefore, future research should centre on the different stages that define the metabolism of these phenolics, in addition to studying the regulation of this metabolic process in order to control synthesis or oxidation of phenolics, depending on the conditions chosen or the result desired. With regard to the first point, most of the final enzymes of phenolic metabolism, that is, those that give rise to the formation of lignin, have not been characterized nor are their biochemical properties known, and therefore in the future it would be useful to elucidate these stages of phenylpropanoid metabolism. Also, in studying the regulation of phenolics, there are two options. Firstly, through genetic engineering, the genes that determine the synthesis or oxidation of phenolics can be strengthened or inhibited. However, at present, the use of transgenic plants best adapted or lease sensitive to some type of stress would be quite difficult due to the social controversy surrounding the commercialization of agricultural products derived from these types of plants. An altemative, which is possibly more rapid and useful in the short term, and which is currently generating a great number of studies, is to explore and define a series of abiotic factors which serve primarily activate or inhibit some of the stages of synthesis or oxidation of phenylpropanoid metabolism. ABBREVIATIONS PAL CHS SS IFS POD PPO lAA 4CL CAD CI TAL CAT RS AIP

= = = = = = = = = = = = = =

phenylalanine ammonia lyase chalcone synthase stilbene synthase isoflavone synthase peroxidase polyphenoloxidase indole-3-acetic acid 4-coumarate:CoA ligase cinnamyl alcohol dehydrogenase chalcone isomerase tyrosine ammonia lyase catalase russet spotting 2-aminoindan-2-phosphonic acid

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Eckey-Kaltenbach, H.; Ernst, D.; Heller, W.; Sandermann, Jr.H.; Plant Physiol, 1994,104,61-1A, Sharma, Y.K.; Davis, K.R.; Plant Physiol, 1994,105,1089-1096. Bahl, A.; Loitsch, S.M.; Kahl, G.; Environ. Pollul, 1995,89,221-227. Booker, F.L.; Anttonen, S.; Heagle, A.S.; New Phytol, 1996,132,483-492. Loponen, J.; Ossipov, V.; Lempa, K.; Haukioja, E.; Pihlaja, K.; Chemosphere, 1998, 37, 1445-1456. Booker, F.; Miller, J.E.; J. Exp. Bot., 1998,49,1191-1202. Grace, S.C; Logan, B.A.; Adams III, W.W.; Plant Cell and Environ., 1998, 21, 513-521. Anderson, M.D.; Prasad, T.K.; Martin, B.A.; Steward, C.R.; Plant Physiol, 1994, 705,331-339. Bharti, A.K.; Khurana, J.P.; Photochem. Photobiol, 1997, 65,765-776. Chalker-Scott, L.; Fuchigami, L.H. In Low temperature stress physiology in crops, Paul, H.L., Ed.; CRC Press Inc., Boca Raton, FL., 1989; pp. 27-40. Griffith, M.; Huner, N.P.A.; Esspelie, K.E.; Kolattukudy, P.E.; Protoplasma, 1985,125, 53-59. Bartollo, M.E.; Wallner, S.J.; Plant Physiol, 1986,80,122 (abstract). Graham, D.; Patterson, B.D.; Annu. Rev. Plant Physiol, 1982,33, 347-372. Solecka, D.; Kacperska, A.; Plant Physiol Biochem,, 1995,33, 585-591. Lydon, J.; Duke, S.O.; Pestic. Scl, 1989,25,361-373. Falco, J.M.; Vilanova, L.; Segura, J.; Agrochim., 1989, 33, 166-173. Hoagland, R.E.; Pestic. Biochem. Physiol, 1990, 36,68-75. Scarponi, L.; Mansour, F.A.; Nemat Allah, M.; Agrochim., 1991,35,91-100. Scarponi, L.; Nemat Allah, M.; Martinetti, L.; J. Agric. Food Chem., 1992, 40, 884-889. Hoagland, R.E.; WeedSci., 1989,37,743'141. Hoagland, R.E.; WeedSci., 1989,37,491-497. Hart, S.E.; Saunders, J.W.; Penner, D.; WeedSci., 1992,40, 378-383. Nemat Alia, M.M.; Younis, M.E.; J. Exp. Bot., 1995,46,1731-1736. Molina, A.; Hunt, M.D.; Ryals, J.A.; Plant Cell, 1998,10, 1903-1914. Shelp, B.J. In Boron and its role in crop production, Gupta, U.C, Ed.; CRC Press, Charlottetown, Canada, 1993; pp. 53-85. Hu, H.; Brown, P.H.; Plant Physiol, 1994,105,681-689. Loomis, W.D.; Durst, R.W.; Curr. Topics Plant Biochem. Physiol 1991,10, 149178. Fawzia, S.A.; Al-Whaibi, M.H.; El-Hirweris, S.O.; J. Plant Nutr., 1994,17, 10371052. Dugger, W.M. In Encyclopedia of plant physiology, new series', Lauchli, A., Bieliski, R.L., Eds.; Springer-Verlag, Berlin, 1983; Vol. 15, pp. 626-650. Pilbeam, D.J.; Kirkby, E.A.; J. Plant Nutr., 1983, 6,563-582.

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Marschner, H. Mineral nutrition of higher plants. Academic Press, Inc., San Diego, 1995. Chattopadhyay, S.; Datta, S.K.; Mahato, S.B.; Plant Cell Rep., 1994,13, 519-522. Pillinger, J.M.; Coope, J.A.; Ridge, I.; J. Chem. EcoL, 1994,20,1557-1569. Cakmak, I.; Kurz, H.; Marschner, H.; Physiol. Plant., 1995,95, 11-18. Cakmak, I.; ROmheld, V.; Plant Soil, 1997,193,71-83. Hu, H.; Brown, P.H.; Labavitch, J.M.; J. Exp. Bot., 1996, 47, Hi-Ill. Castafleda, P.; Perez, L.M.; Phytochemistry, 1996,42,595-598. Kawai, T.; Hikawa, M.; Ono, Y.; J. Jpn. Soc. Hort. Sci., 1995, 64, 79-84. Stout, M.J.; Brovont, R.A.; Duffey, S.S.; J. Chemical EcoL, 1998,24,945-963. Spencer, D.F.; Ksander, G.G.; J. Aq. Plant Man., 1994,32,1\-1?>. Marschner, H. In Plant roots, the hidden half, Waisel, Y., Eshel, A., Kafkafi, U., Eds.; Marcel Dekker, Inc., N.Y., 1991; pp. 503-528. Vamos-Vigyazo, L.; CRC Crit. Rev. Food Sci. Nutr., 1981,15,49-127. Mayer, A.M.; Harel, £.; Phytochemistry, 1979,18,193-215. Harel, E.; Mayer, A.M.; Lemer, H.R.; J. Sci. FoodAgric, 1970,21,542-545. Golan, A.; Kahn, V.; Sadovski, A.Y.; J. Agria Food Chem., 1977,25,1253-1260. Lipton, W.J.; Stewart, J.K.; Whitaker, T.W.; US Dept. Agric. Mark. Res. Rep., 1972, 950. Hyodo, H.; Kuroda, H.; Yang, S.F.; Plant Physiol, 1978, 62,31-35. Ke, D.; Salveit, M.E.; HortScl, 1986,21,1169-1171. Ke, D.; Salveit, M.E.; Plant Physiol, 1988,88, 1136-1140. Ke, D.; Salveit, M.E.; J. Amer. Soc Hortic. Sci., 1989,114, A12A11, Ke, D.; Salveit, M.E.; J. Amer. Soc Hortic. Sci., 1989,114, 638-642. Ritenour, M.A.; Saltveit, M.E.; Physiol Plant., 1996, 97, 327-331. Peiser, G.; L6pez-G^lvez, G.; Cantwell, M.; Saltveit, M.E.; J. Amer. Soc. Hort. 5c/., 1998, 725,687-691.

[126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] [145] [146] [147]



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BIOACTIVE NATURAL PRODUCTS FROM MARINE SOURCES M J . ABAD* AND P. BERMEJO Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040Madrid, SpainTel: ^34-l'3941871; Fax: +34-1394J 764; E-mail: mjahaddeticmos, sim. vcm. es ABSTRACT: Natural products from plants and microorganisms have traditionally provided the pharmaceutical industry with one of its most important sources of "lead" compounds in the search for new drugs and medicines. Although twenty thousand plant species are used in traditional medicines, most species have not been thoroughly examined chemically or pharmacologically. Natural product research is increasingly tiu^ning to marine animals, plants and microbes as source organisms. The oceans with their millions of species are a rich source of marine plants and animals. In recent years, a number of potential therapeutic agents have been isolated from marine flora and fauna. Several marine natural products are currently in preclinical and clinical evaluation, others show promising biological activities in vitro and in vivo assays, and others are making significant contributions to our understanding of cellular processes at the biochemical level. Although only initiated in the late 1970s, natural drug discovery^ from thev world's oceans has been accelerated by the chemical uniqueness of marine organisms, and by the need to develop drugs for contemporary, difficult to cure, diseases. The isolation, structure, biological activities, chemical properties and synthesis of compounds from marine soiwces, have attracted the attention of chemists, biologists and phannacists. Current research activities have generated convincing evidence that marine drug discovery has an exceedingly bright future. This article deals principally with bioactive constituents characterized in tha past decade from marine sources in order to obtain a better understanding of the biological significance of marine flora and fauna. The structural diversity of the medicinal constituents is discussed.

INTRODUCTION It is well knovv^n that plants are an exceptional source of biologically active products w^hich may serve as commercially significant entities in themselves, and v^hich may provide lead structures for development of modified derivatives possessing enhanced activity and reduced toxicity. It is likely that many compounds still await discovery. However, in the last decade the source of natural drugs has expanded to include lower plants, microorganisms and animals as well as marine organisms.

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The oceans cover more than 70% of the earth's surface, which represents over 95% of the biosphere. The oceans are therefore an unexplored area of opportunity for the discovery of pharmacologically active compounds. Although it has been one of man's principal sources of food for thousands of years, the sea was not considered as a supply of biologically active substances until forty years ago. In the last two decades, the search for marine-derived natural products has been extended to all oceans of the world. The enormous potential of the sea as a source of energy, food and chemicals has led to its being the subject of intense research. The results of this search had been reported in numerous reviews [1-8], Marine organisms have been shown to be a very rich source of unique and biologically active secondary metabolites that have attracted the interest of both chemists and pharmacologist. Plant and animal marine life forms have been studied with a view to obtaining such products. In particular, in pharmacognosy, the centre of interest is biologically active substances with therapeutical possibilities. Marine natural products represent a vast potential source of new drugs with diverse and often unique stmctures, many associated with interesting biological properties. Among the properties which have been reported for different marine natural products are very diverse: toxicity, antiviral, antibacterial, antimalarial, antifungal activity, antitumor, anti-inflammatory, analgesic, hypocholesterolemic and hypolipidemic activity. Success in these areas is demonstrated by the agents now in pre- or clinical evaluation. Biologically active natural products, or secondary metabolites, have become fine tools for pharmacologists and biochemists. Several marine natural products have entered pharmaceutical development, and others are making significant contributions to our understanding of cellular processes at the biochemical level [9,10]. As ligands for cellular receptors, they are used to explore fundamental processes that elicit behavioral responses in living systems, both in homeostasis and in disease states. Biological investigations of marine secondary metabolites have already yielded promising candidates for fliture drugs, e.g., didemnin B and bryostatin in the area of cancer chemotherapy [11], or have proven, useful as biological probes in studying cellular events, e.g., saxitoxin as a sodium channel blocker [12]. The marine environment, comprising approximately half of the total global biodiversity, offers a rich diversity of species, which is in many ways

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comparable to that of tropical rain forests. This environment also contains a great number of organisms for which there are no terrestrial counterparts, and which offer an enormous source of novel and biologically active compounds. Biological and chemical investigations of marine ecosystems have provided insights into a wonderful and complex underwater world. As a direct result of these investigations, the structural classes which can be obtained from certain taxonomic groups is to some extent becoming predictable, and some emphasis is now being placed on the biological properties of extracts, fractions and isolated pure metabolites. Ecological pressures on marine organisms, which include competition for space, maintenance of an unfouled surface, deterrence of predation, and the ability to reproduce successfully, may have led to the evolution of unique secondary metabolites, which are responsible for the chemical components of these actions and interactions. The oceans support a stable and thriving community of sponges, corals, echinoderms and many other invertebrates, that have adapted to the freezing temperatures, low nutrient levels and periodic low light levels. It has been suggested that marine habitats lack sufficient predation pressure to drive sessile invertebrates to produce defensive metabolites. Sessile marine organisms possess various defense systems against predators, larvae of other sessile organisms and pathogenic microorganisms. Since marine invertebrates do not produce antibodies, their defense mechanisms are based primarily on phagoc>1:osis by leukocytes, aided by producing and exuding secondary metabolites. The presence of endogenous secondary metabolites is believed to endow marine organisms with a chemical means of defense. Both chemists and biologists have been intrigued for many years as to the role of secondary metabolites of terrestrial and marine origins. Dudley Williams (1989) [13] proposed that "secondary metabolites are a measure of the fitnees of the organism to survive by repelling or entrapping other organisms". The accumulation of biologically active substances in marine invertebrates has been observed as a general phenomenon which reflects the defensive strategy of these often sedentary filter-feeding organisms. Although only initiated in the late 1970s, natural drug discovery from the world's oceans has been accelerated by the chemical uniqueness of marine organisms, which have the highest probability of yielding natural products with unprecedented carbon skeletons and interesting biological activity. Marine organisms, specially sponges, have attracted the attention

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of natural product chemists because of their versatility in using different metabolic pathways, that have no counterpart in the terrestrial world. Because life began in the ocean, it is hardly surprising that marine organisms have not only adapted to the high salt concentrations in the ocean, but have incorporated halogens into their chemical constitutions [14]. While ocean water is universally known for its chloride ion content, it is also an abundant source of bromide, and to a lesser extent iodide. An important consequence of halogen ion availability has been the extensive utilization of halogenation reactions by various marine organisms in their evolutionary biosyntheses of defensive and other necessary constituents. Additionally, during the past decade interest in the secondary metabolites of marine microorganisms and fungi has been increasing at a slow but definitive pace. Marine microorganisms have become recognized as an important and untapped resource for novel bioactive compounds [15-17]. The vast area of research into marine microorganisms, comprising marine bacteria, ranging from archaebacteria to glinding bacteria, fungi, and a whole range of microalgae, e.g., dinoflagellates, diatoms and protozoa, is just emerging and already showing immense potential. Microbiological investigations of marine environments have yielded a number of new biologically active microorganisms [18-22]. Moreover, symbiotic marine organisms, e.g., sponges and algae and/or microorganisms are common in all marine environments, and are believed to be of great importance in the biosynthesis of biologically active natural products within these organisms. Cases where secondary metabolites have been intimated as products of the microorganisms-containing symbionts of a sponge or algae have attracted much attention. However, it is difficult to rigorously sort out which compounds are metabolites elaborated by the marine organisms or by the symbiont, and most of the suggestions on this point are based on circumstantial rather than hard experimental evidence [23]. In this review, data have been presented to illustrate the diversity of organisms living in the sea and the plethora of chemical compounds that have been discovered from them. Since the late 1970s, there has been a veritable explosion of activity and many new marine metabolites have been isolated and identified. Therefore, the present review will cover only recent development in this area. The information was obtained from a review of the scientific literature, and the sources are referenced at the end of the chapter. Bioactive compounds found in marine environments include

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terpenoids and steroids, alkaloids, peptides and proteins, phospholipids, poliketides, carbohydrates, macrolides and toxins. TERPENOroS The diverse, widespread and exceedingly numerous class of natural products that are derived from a common biosynthetic pathway based on mevalonate as parent, are synonymously named terpenoids, terpenes or isoprenoids, with the important subgroup of steroids, sometimes singled out as a class in its own right. Monoterpenes, sesquiterpenes, diterpenes and triterpenes are ubiquitous in terrestrial organisms and play an essential role in life, as we know it. Although the study of terrestrial terpenes dates back to the last century, marine terpenes were not discovered until 1955. Sponges remain the primary target in the search for "drugs from the sea". It is known that sponges produce the greatest variety of secondary metabolites of any animal group. Diterpenes are one of the most abundant non-steroidal secondary metabolites isolated from marine sponges, with a wide range of biological properties. Structurally, the diterpenes from sponges possess polycarbocyclic skeletons, which are sometimes very degraded with loss of one or two carbon atoms to give nor- or bis-nor-derivatives.

,0H H3C CH3 .CH3

CN3

m

CH3

Fig. (1). Structure of agelasimines

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From the Thai marine sponge belonging to the genus Mycale, two labdanes diterpenes, mycaperoxides A and B were isolated. Both compounds exhibited antibacterial and antiviral activity, and showed significant cytotoxicity against tumor cell lines [24]. The marine orange sponge Agelas maiiritiana and others of the same genus, yielded two novel derivatives of a biclycic diterpene, agelasimine A and B, Fig. (1), both exliibiting a wide range of biological activity [25]. Cytotoxicity was reported from both compounds, and both caused relaxation in smooth muscle of rabbit gut and bovine coronary artery. Recently, these marine diterpenes have been reproduced by chemical synthesis using sigmatropic rearrangement and Ritter reaction [26]. Novel diterpenoids, including nakamurol A with unique thelepogane skeleton, were isolated from another Agelas sponge species, Agelas nakamnrai [27]. A Philippine marine sponge of the genus Strongylophora yielded new meroditerpenoids with antimicrobial and antiflmgal activity [28], while sponges of the genus Diacarnus yielded epidioxy-substituted nor-diterpenes with antimalarial properties [29]. Additionally, marine organisms have provided a large number of compounds of mixed biogenesis, originating partly from mevalonate and partly from a benzenoid precursor. A number of linear or cyclic prenylhydroquinones have been described with a terpenoid portion from one to eight isoprene units. Many sponges belonging to the familiy Spongiidae are chemically characterized by a series of terpenoids containig 21 carbons and displaying two (J-substituted fliran moities at the end of the molecule [30,31].These unusual compounds are probably biogenetically derived from higher terpenoids. These marine sponges are a well known source of novel furanoterpenes. Hippospongia sp., from the southern Australian sea, produced hippospongins A-F, with antibiotic activity [32]. The sponges Spongia officinalis and Fasciospongia cavernosa yielded fliranoditerpenes, including the novel ambliofuran 2 [33]. Ircinia sp. yielded bioactive fliranoterpene sulfates, which specifically inhibited the neuropeptide Y receptor in vitro, and also showed cytotoxicity against KB cells [34]. The fliranoterpene ircinin has been shown to inhibit phospholipase A2 (PLA2) activity and to affect human neutrophil functions like superoxide generation and degranulation [35]. Recently, this marine natural product has been synthesised [36]. Sponges of the genus Acanthella have previously been shown to be rich sources of terpenes having various nitrogen-containing groupings, with

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antimicrobial and antifungal activities [37,38]. From the Okinawan sponge Acanihella cavernosa, novel kalihinane diterpenoids. Fig. (2) were isolated, with potential antimalarial activity [39,40]. Recently, 15 diterpenes which contain isonitrile, isothyocianate and isocyanate groupings were also reported from the tropical marine sponge Cymbasiela hooperi. The majority of them demonstrate significant and selective in vitro antimalarial activity [41,42].

HO.,

Fig. (2). Structure of kalihinol A

Besides sponges, other marine organisms such as corals and algae are begining to receive attention from natural product chemists. Soft coral are symbiotic associations of coral animals with their algal partners. They are a rich source of terpenoids, notably cembranoid diterpenes with cytotoxic and antifungal activity [43,44]. Their abundant production and accumulation of diterpenoids is intriguing, as it seems unlikely that these compounds act solely as repellents against predators. Recently, new bioactive cembrane-type diterpenoids have been isolated from octocorallia [45,46].

HO

1

OH

Fig. (3). Structure of pseudopterosin E

Pseudopterosins are a series of tricyclic diterpene glycosides from the

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Caribbean coral Pseudopterogorgia elisabethae discovered by Fenical et al [47]. Pseudopterosin E, Fig. (3) is the one with the best pharmacological profile, combining low toxicity and potent antiinflammatory activity. In human neutrophils, pseudopterosin E inhibits degranulation and formation of leukotrienes [2]. In 1991, phase I clinical trials were initiated with pseudopterosin E as a topical anti-inflammatory agent. Recently, novel anti-inflammatory natural products have been isolated from this Caribbean soft coral [48,49]. Japanese researchers reported the isolation of a group of compounds designated helioporins AE, which are related to the pseudopterosins [1]. Several other soft: corals have been investigated in recent years. Eleiitherobia aurea yielded two novel diterpenoid glycosides, elenthosides A and B [50]. Seo et al. [51] isolated three pigments of the guaiazulene class from the gorgonian Calicogorgia granulosa. From the Japanese soft coral Similaria imnolobata^ new amphilectane-type diterpenoids with cytotoxicity activity were isolated [52], while a sample species of Sinularia genus from the Indian Ocean yielded aromadendrane diterpenoids with larvicidal activity [53]. Another Sinularia species also showed interesting biological properties, such as antispasmodic activity from Sinularia flexibilis [54]. New cytotoxic and antitumor diterpenes were isolated from the Caribbean gorgonian Eunicea toiirneforti [55], the Formosan gorgonian coral Briareum excavatum [56,57], the Okinawan soft coral of the genus Xenia [58], and the European Eunicella cavolinii [59]. Brown algae of the family Dictyotaceae yielded diterpenes of the dolabellane, xenicane, crenulide as well as extended germacrane and hydroazulenoid types. Some of these compounds were identified as capable of demonstrating appreciable selectivity as antimalarial agents [60], and are being synthetised in the laboratory [61]. The brown alga Dilophus ligulatus yielded diterpenoids with cytotoxic activity [62]. The novel xenicane diterpenoid dilopholide, was also obtained in this study. New secospatane diterpenes were recently isolated from another Dilophus alga, Dilophus okamurai [63]. From the marine alga Stypopodium flabelliforme^ several diterpenoids with interesting biological properties were isolated. The diterpenoid epitaondiol exhibited a potent anti-inflammatory activity related to inhibition of human PLA2 activity and leukocyte accumulation [64]. Additionally, epitaondiol has been shown as a potent calcium antagonist in

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a study of the cardiovascular system [65]. The diterpenoid 14-ketostypodiol diacetate, isolated from this alga, inhibited the proliferation of human prostate cells [66]. This compound and several derivatives are being synthetised in the laboratory as the racemate in a stereoselective manner [67]. More recently, new terpenoid compounds have been reported from another Stypopodium species, Stypopodium zonale, as tyrosine kinase inhibitors [68]. From the blue-green alga Tolypothrix nodosa, an anti-inflammatory diterpenoid, tolypodiol, was isolated [69]. Tolypodiol showed strong anti-inflammatory activity in the mouse ear edema assay. Another anti-inflammatory diterpene, pheophytin, was isolated from the edible green-alga Enteromorpha prolifera [70]. Bioactive diterpenoids were also isolated from marine microorganisms, such as phomactin derivatives. Fig. (4) reported from the marine fimgus Phoma sp. as platelet activating factor antagonists [71]. OHO

Fig. (4). Structures of phomactin derivatives

Marine organisms have also been intensively examined for their sesquiterpene content. Dysidea herbacea is a sponge species which has yielded new metabolites for more than 20 years, and no doubt further collectionsfromdifferent locations will continue to reveal new chemistry.

Fig. (5). Structure of herbadysidolide

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The terpenoid metabolites reported from Dysidea sp. are predominantly sesquiterpenes [72]. They possess a spiro moiety as in herbadysidolide, Fig. (5), herbasolide. Fig (6) and spirodysin. Fig. (7), or they are fUranosesquiterpenes such as forodysinin. Fig. (8). However, Dysidea herbaceafromtwo collection sites on the Great Barrier Reef less than 120 km apart also yielded enantiomericfiiranosesquiterpenes[73].

.O Q

Fig. (6). Sructure of herbasolide

These results suggest that samples of this sponge differ in their enzymatic capabilities concerning the cyclization of geranyl-geranylpyrophosphate.

Fig. (7), Structure of spirodysin

More recently, two new isonakafuran-type sesquiterpenes were isolated from this sponge species [74]. These types of compounds possess interesting antitumor and antifungal activity, and attempts to synthesize them are being conducted [75]. Other bioactive metabolites, such as antifouling sesquiterpenes, have also been recently isolatedfromDysidea herbacea [76].

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Fig. (8). Structure of furodysinin

From another Dysidea sponge species, Dysidea avara, the sesquiterpenes avarol. Fig. (9) and avarone. Fig. (10), which show a wide variety of biological activities, were first isolated. Both compounds are potent antileukemic agents in vitro and in vivo. They were determined to be neither direct mutagens nor premutagens, and they displayed antimutagenic activity

Fig. (9). Structure of avarol

Both avarol and avarone inhibit replication of the etiological agent of acquired immuno-deficiency syndrome (AIDS) [77]. Additionally, avarol and avarone effectively control acute inflammation in experimental models after either oral or topical administration. Their anti-inflammatory activity may result from inhibition of eicosanoid release and depression of superoxide generation in leukocytes [78]. Several studies reviewed the structures and bioactivity of compounds related to avarone as an antihuman immuno-deficiency vims (HIV), antitopoisomerase II activity and as proteinkinase C (PKC) inhibitors [3, 79].

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Fig. (10). Structure of avarone

From two sponge samples, Luffariella sp. and Accmthella klethra , and from red algae of the genus Laurencia, several sesquiterpenes with an isonitrile or isothiocyanatefixnctionalitywere obtained [80]. Some of them exhibited detectable cytotoxic activity against cultured tumor cells [81,82], as well as antibacterial [83] and antimalarial activity [84]. More recently, these types of compounds with antimalarial and antifouling activities were also isolated from marine sponges of the g^mxs Axinyssa [85,86]. Additionally, sesquiterpene-substituted quinones and related compounds constitute an important class of cytotoxic natural products of marine origin. Natural products of mixed sesquiterpene and quinol biosynthesis are common to marine algae and sponges. For example, several sesquiterpenoid/quinols have been isolated from a deep water collection of the marine sponge Siphonodictyon coralliphagum [87]; cyclorenierins from the sponge Haliclona sp. [88] and a Philippine sponge of the genus Xestospongia [89]; dactyltronic acids from the sponge Dactylospongia elegans [90]; two sesquiterpene hydroquinones from Polyfibrospongia australis [91]; vinylfurans from Euryspongia de lieu lata [92]; and several sesquiterpene quinones and hydroquinones from Thorecta choanoides, a marine sponge from the southern Australian sea [93], and from Perithalia caudata, an Australian marine brown alga [94]. In many cases, these compounds showed other interesting biological properties, such as antibiotic [95], anti-inflammatory [96,97], antiviral activity, e.g., peyssonols A, Fig. (11) and B, two anti-HIV sesquiterpenes hydroquinones isolated from the Red Sea alga Peyssonelia sp. [98], and cardioactive properties, e.g., halenaquinol, recently isolated from the sponge Petrosia seriata [99].

695 JCMO

Fig. (11). Structure of peyssonol A

From the marine sponge Haliclona sp. (also known as Adocia sp.), a family of hexaprenoid hydroquinones called adociasulfates, have been recently reported as inhibitors of kinesin motors [100,101]. These types of compounds were also found in several soft corals, such as Lemnalia africana [102], Okinawan soft coral of Nephthea sp. [103], and the gorgonian Alertogorgia sp., which yielded the cytotoxic tricyclic sesquiterpene, suberosenone [104]. Marine organisms, specially sponges, have also provided a large number of biologically active sesterterpenoids. The sesterterpenes are the smallest class of terpenoid compounds and consist of alcohol, aldehyde and ketone derivatives of terpene hydrocarbons. The occurrence of sesterterpenes in nature is somewhat uncommon, but for the last two decades an increasing number of examples have been reported. Interestingly, many of the recent additions have been isolatedfi*ommarine sponges of the order Dictyoceratida. These metabolites may be listed in two main groups: linear sesterterpene molecules terminated by a fiiran ring at one end and by a tetranoic acid or lactone ring at the other end, and tetra-or pentacyclic-sesterterpenes which are analogues of the scalarane skeleton. The scalaradial group of marine metabolites exhibit potent biological activity, mainly anti-inflammatory properties [2,105]. Scalaradial, Fig. (12) and other scalaranes were found to completely inactivate the enzyme PLA2 from bee venom directly and irreversibly. Marine sponges are a wellknown source of bioactive scalaradial sesterterpenes. Phyllospongia sp., collected in the South China Sea, yielded two new scalarane-type sesterterpenes, phyllactone H and I [106]. Scalarolide and scalarin were reported from Cacospongia and Ircinia sponges, besides other scalarane

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sesterterpenes [107,108].

Aco . cno f

CHO

Fig. (12). Structure of scalaradial

These types of compounds also showed other interesting properties, such as antineoplasic and cytotoxic activity reported from scalarane-type sesterterpenes of the Indian Ocean sponge Hyrtios erecia [109,110], and antituberculosis properties [111]. In recent years, many marine sesterterpenes which are promising candidates for new drugs have been discovered. The sesterterpenoid manoalide. Fig. (13), obtained from the sponge Luffariella variabilis^ was detected in a program searching for new anti-inflammatory compounds. Manoalide proved to be a potent inhibitor of PLA2 and has become a usefiil biochemical tool. Inhibition of phospholipase C and the ability of manoalide to function as a calcium channel blocking agent allows this compound to be used in the study of the role of calcium mobilisation in inflammatory processes, and in a more general sense, in signal transduction pathways [105,112]. A significant number of manoalide derivatives has been isolated and evaluated for their biological activity [113,114]. A total synthesis of manoalide employs an organometallic coupling strategy [115]. Clinical trials are currently underway with some of these and synthesised derivatives, and it is probable that a manoalide-inspired derivative will reach the market [116].

Fig. (13). Structure of manoalide

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New and interesting anti-inflammatory sesterterpenes have been reported in recent years from marine sponges. Petrosaspongiolides, Fig. (14), isolated from the Caledonian marine sponge Petrosaspoiigia nigra [117], were found to potently inhibit PLA2 on acute and chronic inflammation [118]. In a similar manner, cacospongiolide B, Fig. (15), a sesterterpene isolated from Fasciospongia cavernosa [119,120], was shown to be a potent inhibitor of human synovial PLA2 [121].

'

GMaOAc

Fig. (14). Structure of petrosaspongiolides

Fig.(15). Structure of cacospongiolide B

Several other marine sponges have been investigated in the last decade, in the search for novel bioactive sesterterpene molecules. A sample of the sponge Dysidea herbacea from the Red Sea is unique in that it contains cytotoxic sesterterpenes with a scalarin skeleton, e.g., scalardysin. Fig. (16) and the C2i-furanoterpene flirospongolide. Fig. (17).

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Fig. (16). Structure of scalardysin

This compound also showed antispasmodic activity [122]. A variety of cytotoxic compounds was isolated, including a bishomosesterterpene and dysidiolide from another Dysidea sp., a sulfated sesterterpene hydroquinone from a Hippospongia sp., and two new sesterterpenes, lintenolides F and G from the Caribbean sponge Cacospongia linteiformis [123,124].

Fig. (17). Structure of furospongolide

From the Maldives' Black marine sponge Hyrtios erecta^ several cytotoxic sesterterpenes were isolated, such as the penytacyclic sesterterpenes designated sesterstatins [125-127] and puupehenone. Fig. (18) with a quinone-methide system [128]. Three novel cytotoxic norsesterterpenes, rhopaloic acid A, Fig. (19), B and C, were recently isolated from the sponge Rhopaloeides sp. These compounds also inhibited the gastrulation of the starfish (Asterina pectmifera) embryo [129,130]. Both racemic and enantiomeric forms of rophaloic acid A have been synthesised by very diflFerent strategies [131,132].

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Fig. (18). Structure of puupehenone

Additionally, during the search for biologically active sponge metabolites belonging to the sesterterpenoid class, a sulfated sesterterpene hydroquinone, halisulphate. Fig, (20), was isolated from the dark brown sponge Halichondriidae sp.

OOOH

Fig. (19). Structure of rophaloic acid A

It demonstrated in vitro antimicrobial, antifungal and anti-HIV activities [24,133]. Recently, a halisulphate derivative with antithrombin and antitrypsin activity was isolated from the marine sponge Coscinoderma mathewsi [134], The absolute configuration of halisulphate has been determined by application of the chiral amide method coupled with chemical degradation procedures [135]. NaOaSO'

Fig. (20). Structure of halisulphate

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Fig. (21). Structures of malabaricane triterpenes

Triterpenoids are a minor group of sponge metabolites; of these malabaricane/isomalabaricane triterpenoids. Fig. (21) are known from Jaspis and Stelletta sponge species. All of this group shows cytotoxicity and anti-HIV activity [136-139].

Fig. (22). Structure of sodwanones

Other cytotoxic triterpenes, designated as sodwanones A-M, Fig. (22), were recently reported in Axinella weltneri^ a marine sponge from the Indian Ocean [140]. The investigations of small samples of the Mediterranean sponge Raspaciona actileata revealed the presence of raspacionins. Fig. (23), triterpenoids containing two perhydrobenzoxepine systems [141]. Besides sponges, other marine organisms have been reported to produce bioactive triterpenes, including algae from Lanrencia genus [142], and the \\o\o\h\xn2inPsolusfabricci [143].

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OCOCHa

Fig. (23). Structure of raspacionins

As we indicated in the introduction section, halometabolites frequently occur in marine organisms and are known to have basic functions related to the survival of the living creatures producing them. Bromine is by far the halogen most frequently found in these metabolites. Halogenated marine terpenes were first isolated only in 1963. Since most marine organisms have been found to contain halogenated compounds, there are certainly thousands of different, new organohalogenated terpenes in marine organisms awaiting discovery. Among marine organisms, red algae, particularly species oiLaurencia and Plocamium, have provided a rich and diverse collection of halogenated terpenes over the past 25 years [14]. Red algae of the genus Plocamium have been shown to be a rich source of acyclic and cyclic halogenated monoterpenes that vary for a given species depending on collection location and season. These algae can be found in many locations ranging from Antarctica to tropical waters. Numerous chemical studies of these species show the presence of bioactive halogenated monoterpenes, whose structure and yield vary greatly [4]. Ahhough the red algae Plocamium have been investigated for its chemical content for many years, several new bioactive compounds have been identified recently from these species. For example, plocamadiene A is a polyhalogenated monoterpene which causes histamine release from mast cells of the guinea-pig and rat in vitro [144]. The species Plocamium cartilagineum found on the Portuguese coast, produced acycUc polyhalogenated monoterpenes [145]. More recently, new halogenated monoterpenes were isolated from Plocamitmi costatimi. These compounds have been shown to deter settlement of barnacle larvae, suggesting a potential ecological role [146]. An array of new and unusual halogenated terpenes have been isolated and characterized from Laurencia red algae [14]. This genus is well known as a source of halogenated sesquiterpenes. New chamigrane-type derivatives were isolated from Laurencia species, some from Laurencia

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nidifica [147] and some from Laurencia nipponica growing in Japan [148]. Examples of iodinated terpenes, which are quite rare and interesting, were found in Laurencia majuscula collected in the South China Sea [149]. More recently, Rovirosa et al [150], reported the isolation of new halogenated sesquiterpenes from Laurencia claviformis, a species endemic to Easter Island.

Fig. (24). Structure of halomon

Several other red algae have been investigated in recent years. Among them, it is interesting to point out the halogenated monoterpene halomon. Fig. (24) and related compounds, isolated from the red alga Portieria hornemannii^ which exerts potent antitumor activity in vitro and in vivo [151-153]. Species of sea hares, a marine moUusk, have also provided a rich source of halogenated terpenes [14]. In many cases, these compounds are derived from the sea hare's algal diet. New chamigrene-type halogenated sesquiterpenes were isolated from Aplysia dactylomela [154], and from the Spanish sea hare Aplysia punctata^ which exert potent cytotoxic activity [155]. STEROIDS Since the start of the twentieth century, steroids have continued to be the focus of the research activities of natural product chemists, synthetic chemists, biochemists and clinicians. The reasons are several-fold and related to the fascination of the chemical complexity of sterols and their biochemical functions in living organisms. Sterols and steroids are excellent compounds for the organic chemists to practise their skills upon in the development of new reactions and synthetic procedures. The biological functions of sterols, for example as an essential constituent of membranes, have proved thought-provoking to lipid biochemists. Marine organisms have been found to be storehouses of sterols.

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particularly in terms of unique side-chain structures and unusual fiinctionalization. For example, marine sponges are a rich source of steroids with highly functionalized nuclei and modified side chains. Numerous new sterols have been isolated from marine sponges. Although most have side chains that are polyoxygenated or alkylated, other occurring sterols are known to contain a methylether. Extensive studies on sterols from marine sponges during the past decades have resulted in the identification of a plethora of unusual forms with interesting biological activities [156]. For example, topsentinols A-J, Fig. (25), new sterols with unusual polyalkylated side chains, were isolated from the Okinawan marine sponge Topsentia sp. [157], while sponges of the genus Ircinia yielded new epoxy sterols [158]. Sterol composition has also been reported from the sponge Faciospongia cavernosa growing in the Adriatic, Aregean and Tyrrhenian Seas [159]. Recently, a new sterol containing an unprecedented seven-membered cyclic enol-ether has been isolated from the Australian Euryspongia arenaria [160]. Most of these compounds showed interesting biological properties, such as antiplasmodial and cytotoxic activity of the steroids from Agelas oroides, a Maltese marine sponge [161]; novel cytotoxic steroids from sponges of the genus Xestospongia sp. [162,163], Biemna sp. [164] and Scleritoderma sp. [165]; an antifouling epidioxy sterol from Lendenfeldia chondrodes, a Palauan marine sponge [166], and antiviral sterols from the marine sponge Petrosia weinbergi [167]. It is interesting to point out the cytotoxic activity of camptothecin and related compounds, with which clinical developments have recently been initiated [168-170].

Fig. (25). Structure of topsentinols

Additionally, sulfated sterols have been described from a wide variety of marine organisms, particularly sponges and echinoderms, and several of these steroidal sulfates have exhibited a broad range of activities. Halistanol sulfates are a group of sulfated polyhydroxysteroids from

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Sponges, which are very attractive because of their biological activity. Halistanol disulfate B, isolated from the marine sponge Pachastrella sp., was shown as a potent inhibitor of endothelium converting enzyme [171], while halistanol trisulfate, a sulfated steroid derivative isolated from the marine sponges of the genus Topsentia, inhibits protein tyrosine kinase activity [172,173]. New trisulfated trihydroxysteroids were also isolated from two different collections of the sponges Trachyopsis halichondroides and Cymbastela coralliophila [174], while tamosterone sulfates, new polyhydroxylated steroid sulfates, have been reported from a new oceanapiid sponge genus [175]. Acanthosterol sulfates A-J, isolated from the Western Japan sponge Acafithodendrilla sp., exhibited antifungal activity [176]. More recently, some cytotoxic bis-steroid sulfates called crellastatin, were isolated from the Vanatua marine sponge Crella sp. [177,178]. Fom the marine sponge Jaspis sp., several steroidal sulfates have been reported as inducers of larval metamorphosis and inhibitors hatching enzyme activity in the ascidian Halocynthia roretzi [179]. Additional unusual steroid derivatives have been isolated from marine sponges, e.g., polymastiamides, steroid/aminoacid conjugates isolated from Polymastia holetiformis, a Norwegian marine sponge [180], and an aminoimidazolium sah of steroid trisulfate from Topsentia sp. [181]. Some of these marine sterols are being reproduced by chemical synthesis in the laboratory [182]. Besides sponges, other marine organisms have been investigated in recent years for their steroid content, such as octocorallia [183]. Sarcoaldesterols A and B, two new polyhydroxylated sterols together with novel epoxy steroids were isolated from the soft coral Sarcophytum sp. [184,185], Gorgonian of the genus Mtaicella sp. from Jaejn Island of Korea, yielded calicoferols. Fig. (26), new secosteroids with significant cytotoxicity and inhibitory activity against PLA2 [186,187]. Recently, chemical examination of the soft corals Cimndaria viridis, Nephthea chahroli and Simdaria disseda yielded novel polyhydroxy steroids [188190]. Red algae are known to be important sources of cholesterol and desmosterol in the marine environment. Some of these compounds, e,g., oxygenated desmosterols and clerosterols isolated from the red algae Codinm arabicum and Galaxaiira marginata, showed interesting cytotoxic properties [191,192]. More recently, a new sterol amide with antimicrobial activity, boophiline, was isolated from the cattle tick Boophihis micropliis [ 193 ].

705

Fig. (26). Structure of calicoferols

ALKALOIDS AND RELATED COMPOUNDS Alkaloids are extremely difficult to define because they do not represent a homogeneous group of compounds from either the chemical, biochemical or physiological viewpoint. All do occur in plants, but some are found in animals, and practically all have been reproduced in the laboratory by chemical synthesis. Most possess basic properties due to the presence of an amino nitrogen, and many, specially thoses pertinent to pharmacy and medicine, possess marked physiological activity. Marine organisms are known to be a rich source of alkaloids with unique chemical features and pronounced chemical activities, which suggest potential value as lead structures for the development of new pharmaceuticals [194], Extensive studies on alkaloids from marine organisms during the past decades have resuhed in the identification of a plethora of compounds, sometimes with interesting biological activities. For example, indole alkaloids isolated from marine sponges such as Raphisiapallida [195], Ircinia sp., a Okinawan marine sponge [196], and Hamacantha sp., with antifungal activity [197]. Imidazole alkaloids such as leucettamine A and related compounds isolated from the marine sponge Leucetta microrciphis^ have been shown as potent antagonists of leukotriene B4 receptor [198], and antitumor agents [199]. Recently, new imidazole alkaloids were reported from an Australian marine sponge Axinella sp., with interesting bactericidal activity [200,201]. Stellettamide A and B, Fig. (27), indolizidine alkaloids isolated from sponges of the genus Stelletta have been reported as inhibitors of

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calmodulin [202,203]. The absolute configuration of these molecules have been established by synthesis of their enantiomers [204]. From this sponge species, new alkaloids named stellettazoles B and C, which exhibit antibacterial activity have recently been reported [205]. From the Caribbean marine sponge Agelas dispar, novel betaines alkaloids which also exert antibacterial activity have recently been isolated [206], Cytotoxic guanidine-alkaloids have been reported from different samples of marine sponges [207-210].

Fig. (27). Structure of stellettamides hAeO

PR^

MeO Fig. (28). Structure of lamellarins

Another cytotoxic compound, a sulfur-containing alkaloid, was isolated from the ascidian Polycarpa aurata [211]. Recently, another series of ascidian alkaloids, the lamellarins. Fig. (28), have been shown as selective inhibitors of HIV virus replication in cell culture [212], together with new indolocarbazole and ergoline alkaloids isolated from the ascidians Eudistoma toealensis and Botryllns leachi, which showed moderate cytotoxic activity [213-215]. From a Philippine marine sponge, Oceanapia sp., an unusual sesquiterpene alkaloid, oceanapamine, was isolated [216], while marine sponges of the genus Corticium sp. yielded unusual steroidal

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alkaloids [217]. Additional cytotoxic alkaloids were reported from other marine sponges, e.g., peptide alkaloids from Lissoclinum sp. [218], and tryptophan-derived alkaloidsfromthe Okimy^^n Aplysina sp. [219]. From the Southern Australian sponge Spongosorites sp., a new class of marine alkaloids, dragmacidins, have been reported as potent inhibitors of protein phosphatases [220]. Some of these cytotoxic marine alkaloids are promising candidates for new drugs. For example, ecteinascidins. Fig. (29) are a family of tetrahydroisoquinolone alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata, which have been selected for clinical development. These compounds are presently in pre-clinical and clinical trials for human cancers [221-225]. A series of totally synthetic molecules that are structurally related to the ecteinascidins is currently being prepared and evaluated as antitumor agents [226]. 0CH3 H>,CO.

H3CO

Fig. (29). Structure of ecteinascidins

However, pyrroloquinolines and pyridoacridines are the alkaloids of major interest as metabolites in sponges and ascidians [227]. Many of these compounds have generated interest both as challenging problems for structure elucidation and synthesis as well as for their cytotoxicities [228230]. A family of alkaloids characterized by a pyrroloquinone skeleton has been isolated in recent years from several sponges. Included in this family are the batzellines, isobatzellines, damirones, makaluvamines, discorhabdins, prianosins and wayakin. These alkaloids have shown a

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variety of biological activities including cytotoxicity against human tumor cell lines, in vivo tumor inhibition and inhibition of topoisomerase I and II. Among these, the makaluvamines. Fig. (30) are the most potent inhibitors of topoisomerase II, suggesting their efficacy as anticancer agents. The principal structural feature of these alkaloids is the core of a planar iminoquinone moiety which can intercalate into DNA and cleave the DNA double helix, or inhibit the action of topoisomerase II [231]. This family of makaluvamines alkaloids was mainly isolated from the Philippine marine sponge Zyzzya fidiginosa [232-234]. Recently, these alkaloids are being reproduced in the laboratory by chemical synthesis employing a strategy based upon intramolecular nucleophilic substitution reactions [235-237].

Fig. (30). Structure of makaluvamines

Discorhabdin alkaloids. Fig. (31), in contrast, are of high cytotoxicity, but they exhibit no inhibition of topoisomerase II. They were isolated from the Anthartic sponge Latnmculia apicalis [238], and more recently from a deep-water marine sponge of the genus Batzella sp. [239]. The new discorhabdin derivative isolated from this sponge showed in vitro cytotoxicity against tumor cell lines.

Fig. (31). Structure of discorhabdin

Marine sponges of these genus Batzella sp. also yielded novel

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pyrroloquinolines alkaloids, batzelladines. Fig. (32), with interesting biological properties [240,241]. Many of these types of alkaloids were also isolated from other marine sponges, e.g., Agelas sp. [242], and tsitsikammamine A and B reported from a South African marine sponge, which exhibited antimicrobial activity [243]. As representative of the derivatives of pyridoacridine, eilatin, a marine alkaloid inhibits in vitro cell proliferation in chronic myeloid leukemia patients [244]. Other members of the pyridoacridines, such as alkaloids isolated from a Cystodytes sp. ascidian, inhibit topoisomerase II [245]. Additionally, analogues derivatives of these type of alkaloids showed interesting anti-HIV activity [246].

Fig. (32). Structure of batzelladine A

Fused tetracyclic and pentacyclic alkaloids constitute a relatively new class of natural products isolated mostly from ascidians and sponges. Cytotoxic, antimicrobial and antiviral activities have been reported for many of these compounds. The manzamine alkaloids. Fig. (33) are characterized by a complex pentacyclic diamine linked to C-1 of Pcarboline moiety. Manzamine have been isolated mainly from six different genera of marine sponges: Haliclona, Pellina^ Xestospongia, Ircinia, Pachypellin and Amphimedon.

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Fig. (33). Structure of manzamines

Haliclonacyclamines, manzamine alkaloids with pronounced cytotoxic activity, were isolated from Haliclona sp., a tropical marine sponge [247249]. Other manzamine-type alkaloids with cytotoxic and antibacterial activity were isolated from the Philippine marine sponge Xestospongia ashmorica [250]. Some of these compounds are very attractive because of their biological activity, e.g., manzamine alkaloids isolated from another Xestospongia sp., also reported in the marine sponge Agelas novaecaledoniae^ which are potent somatostatin and vasoactive intestinal peptide inhibitors [251]. These compounds could be promising agents in the research on compounds for therapeutical interventions in cystic fibrosis, Alzheimer's disease and some tumors.

Fig. (34). Structure of norzoanthamine

Manzamine-type alkaloids were also reported in another samples of marine sponges, e.g., the Okinawan Amphimedon sp. [252-254], Pachypellina sp. [255], and a novel alkaloid called hyrtiomanzamine from Hyrtios erecta, with interesting immunosuppressive activity [256]. From

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the colonial zoanthid Zoanthus sp., a zoanthamine-type alkaloid. Fig. (34) has been reported as a good candidate for an osteoporotic drug [257], while the marine bacteria Agrobacterium sp. yielded agrochelin, a new cytotoxic thiazole alkaloid [258]. Recently, investigations have been conducted to reproduce these type of compounds by chemical synthesis in the laboratory [259-261].

X = Br{9S/^R = 6:4) X = H (9S^f^ = 1 1)

Fig. (35). Structure of tauroacidins

Although very few terrestrial plant alkaloids contain halogen, brominated alkaloids have been reported from the marine environment. From the Okinawan marine sponge Hymeniacidon sp., several bromopyrrole alkaloids have been described, e.g., tauroacidins A and B, Fig. (35) [262], konbuacidin A, Fig. (36) [263] and spongiacidins A-D [264]. Several species of sponges contain hymenialdisine. Fig. (37), which has been shown as a potent inhibitor of nuclear factor kappa B and interleukin-8 production in vitro [265,266].

H;^.

.N

Bf Fig. (36). Structure of konbuacidin A

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New bromopyrrole alkaloids were also isolated from different species of Agelas sp., such as the Caribbean Agelas dispar [267], Agelas nakamurai^ si Papua New Guinean marine sponge [268,269] and Agelas wiedenmayeri [270]. Two samples of the marine sponge Stylissa carteri collected in Indonesia, yielded two new bromopyrrole alkaloids [271]. Brominated indole alkaloids have been reported from the Caledonian marine sponge Orina sp. [272], while bromotyrosine alkaloids with cytotoxic and antitumor activity have been isolated from several marine sponges, such as Aplysina aerophoha [273], the Okinawan Psammaplysilla purea [274] and Psendoceratina verrucosa [275].

Fig. (37). Structure of hymenialdisine

Additionally, marine organisms have proven to be a rich source for a wide variety of modified nucleosides considered worthy for clinical application. For example, arabinoside-nucleosides, constituents of the Caribbean sponge Cryptotethya crypta^ have led researchers to synthesise analogues with improved antiviral and anticancer activity [4].

Fig. (38). Structure of tubercidin

Other bioactive nucleosides have been reported from the marine

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environment, e.g., pyrrolo-pyrimidine nucleosides such as tubercidin. Fig. (38) and analogues derivatives from the ascidian Didemmim voeltzkowi [276]; caissarone. Fig. (39), a sea anemone iminopurine with adenosine receptor antagonist activity [277]; phenethylguanidine analogues from Petrosia contignata, a Indo-Pacific marine sponge [278]; and bioactive bisguanidines from Stylotella miraritium, with potent cytotoxic, antibiotic and immunosuppressive activity [279]. More recently, nucleosides have also been reported in the Australian marine sponge Carteriospongia sp. [280].

II

H

0:Vo Me

M«

Fig. (39). Structure of caissarone

PEPTIDES AND PROTEINS The "term" peptide includes a wide range of compounds varying from low to very high molecular weights, and showing marked differences in physical, chemical and pharmacological properties. The lowest members are derived from only two molecules of aminoacids, but higher members have many aminoacid units and form either peptides, simple proteins or more complex proteins, conjugated proteins, for example, lipoproteins in which proteins are combined with lipids. Marine organisms are a well-established source of unique and biologically active peptides. Complex cyclic peptides and depsipeptides have emerged as an important new class of metabolites present in extracts of marine organisms. Many of these peptides have been found to be extremely potent cytotoxic and /or enzyme inhibitors.

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Fig. (40). Structure of didemnin B

Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. Didemnin B, Fig. (40), one member of this family obtained from the tunicate Trididemnum solidum^ has antiviral, immunosuppressive and potent cytotoxic properties [281-283]. The compound is too toxic to be useful as an antiviral or immunosuppressive agent, but has been in phase I clinical trials as an anticancer agent, and phase II clinical trials are currently underway [284]. Arenastatin A, Fig. (41) is another potent cytotoxic depsipeptide isolated from the marine sponge Dysidea arenaria, which shows selective toxicity against tumor cells [285]. This compound have been reproduced by chemical synthesis in the laboratory [286]. In a similar manner, hapalosin and aplidine, marine cyclic depsipeptides with inhibitory activity against human tumor cell lines, have been obtained by chemical synthesis by a route involving a macrolactamization as an important ring-forming step [287-289]. Other cytotoxic and antiproliferative depsipeptides were recently isolated from the Vanatua marine sponges Axinella carteri [290], Jaspis splendcms [291], Geodia sp. [292], and the Papua New Guinea sponge Cymhastela sp. [293]. Marine depsipeptides also showed other interesting biological properties, such as antiviral [294], antifimgal [295,296] and hemolytic activity [297]. It is interesting to point out the biological activity of papuamides A-D, new cyclic depsipeptides isolated from the Papua New Guinea sponges Theonella sp., which showed interesting anti-HIV and cytotoxic activity [298].

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A ? ^-^=^M Fig. (41). Structure of arenastatin A

Marine organisms, specially sponges, have provided a large number of other biologically active peptides in the last decades. Cyclotheonamides, Fig. (42), a family of cyclic pentapeptides isolated from marine sponges of the above mentioned genus Theonella sp., have been shown as potent thrombin, trypsin and other serine proteases inhibitors [299-302]. From the marine sponge Theonella swinhoei a highly cytotoxic peptide, polytheonamide B, was recently isolated [303].

O

^N O^

^

NHCOMe

H^ ^N. -. ^ .

Fig. (42). Structure of cyclotheonamide B

Phakellistatin, Fig. (43) is a series of cyclic hepta and octopeptides isolated from the Indian Ocean marine sponge Plakellia sp., with interesting antineoplasic activity [304,305]. These compounds have recently been reproduced in the laboratory by chemical synthesis using a combination of stepwise coupling and segment condesation [306]. New cytostatic heptapeptides, isolated from marine sponges, were also chemically synthetized using a new synthetic method to elaborate peptide bond [307309]. Additional biologically active peptides of marine sponge origin include dipuupehedione, a cytotoxic compound from the New Caledonian

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Hyrtios sp. [310,311], and the antifungal peptides halicylindramides from Halicoridria sp. [312].

Fig. (43). Structure of phakellistatins

Besides sponges, other marine organisms have been reported to produce bioactive peptides, which are promising candidates for new drugs. For dolastatins, e.g. dolastatin 10, Fig, (44), potent antineoplasic peptides isolated from the Indian Ocean molusk Dolahella amictilaria, clinical trials are pending [313]. Recently, a structural derivative of dolastatin called auristatin, has been evaluated in human tumor cell lines and has undergone clinical trials [314].

iHgCO H

O

Fig. (44). Structure of dolastatin 10

Microcolins, Fig. (45) are lipopeptides isolated from a strain of the blue-green alga Lyngbya majtiscula^ which revealed interesting cytotoxic and immunosuppressive activity [315]. Several synthetic derivatives are also being evaluated [316]. These compounds resemble majusculamides, which were isolated from another chemovariant of the same species and from marine sponges [317]. Dendroamides, new cyclic hexapeptides, were isolated from another blue-green alga [318].

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b

d^

o Fig. (45). Structure of microcolin A

Marine microorganisms have also been reported to produce bioactive peptides, such as marinostatin from the marine bacterium Alteromonas sp. [319], pentapeptides from the cydinohdiCttnum Anahaena cylindrica [320], and new anti-inflammatory cyclic peptides from the marine Strepiomyces sp. [321]. From the marine fungus Hypoxylon oceariicum, several lipodepsipeptides with antifungal activity have recently been reported [322,323]. Besides peptides, marine organisms have been reported to produce biologically active proteins, which are probably involved in the protection of organisms against physiological and stress conditions. Recently, these molecules have been cloned from sponges [324] and marine microorganisms [325]. The marine protein variabilin. Fig. (46) has been shown as a potent dual inhibitor of human secretory and cytosolic PLA2 with anti-inflammatory activity [326]. An interleukin-6 cytokine family antagonist protein was reported from the marine sponge Callyspongia sp. [327]. From the marine sponge Pachymatismajohnstonii, a, cytotoxic glycoprotein, pachymatismin was isolated [328,329]. Another active glycoprotein, niphatevirin, isolated from the marine sponge Niphates erecta was reported as an HIVinhibitory agent [330], together with cambrescidins, proteins isolated from marine invertebrates which also exert antiviral activity [331]. The activities of the purple fluid of the sea hare Aplysia dactylomela, such as toxic, antimicrobial and hemagglutinating properties, have been attributed to a substance of protein nature [332]. Proteoglycans and adhesive glycoproteins present in the extracellular matrix of vertebrates, have also been reported in sponges. These molecules are probably involved in the cell adhesion systems of sponges [333]. Recently, novel marine proteins have been reported, such as silicatein from sponge biosilica [334], and a metallothionein protein from the marine alga Fiicus vesiculosus [335]. Metallothioneins have also been isolated from Arctic

718

bivalves as possible indicators of the availability of trace metals in the Arctic [336]. Additionally, proteins were also isolated from other species in the marine environment, e.g., from the Kuruma prawm Penaeus japonicus [337,338], and the shore crab Carcirms maenas [339].

OH Fig. (46). Structure of variabilin

Enzymes are also colloidal in nature and consist of protein or contain proteins as an essential part. Several enzymes have been reported from marine organisms, specially sponges and algae, e.g., exopolyphosphatases from the marine sponge Tethya lyncuhum [340], tauropine dehydrogenases from the Demospongia Halichondria japonica [341], and more recently phenylalanine hydrolases from the sponge Geodia cydonium [342]. From this marine sponge Geodia cydonium, oligoadenylate synthetases were also isolated which may be useful as biomarkers for environmental monitoring [343,344]. Additionally, these sponge species contain high levels of telomerase activity, suggesting that they possess a high proliferation capacity [345]. A protease hydrolyzing casein with proteolytic activity has been reported from the Papua New Guinea sponge Callyspongia schulzi [346], while the glutathione-S-transferase activity of the sponge Suberites domuncula has been used as marker of thermal stress [347]. This enzyme was also reported in a marine fish, Pleuronectes platessa [348]. Additionally, isomerases were reported from the marine alga Ptilota filicina [349], while the cyanobacterium Synechocystis sp. yielded pcarotene hydroxylases [350]. Cyanobacteria also yielded oligomeric forms of dehydrogenases [351]. Besides sponges and algae, enzymes were also isolated from marine organisms and microorganisms. For example, polymerases and proteases from marine Vibrio sp. [352], marine bacterium such as Alcaligenes faecalis [353], and from archaeons, such as the psychrophilic Cenarchaeum symbiosiim [354], and the hyperthermophile archaeons Pyrococciisfuriosus [355], Sulfolobus solfataricus [356], and Aeropyrum pernix [357]; transferases from marine bacterium such as Vibrio vulnificus

719

[358], and Photobacterium damsela [359,360]; dehydrogenases from different strains of Nocardioides sp. [361]; novel alginate lyases from marine bacterium Alteromonas sp. [362], and phenoloxidases from the colonial ascidian Botryllus schlosseri [363], and the marine bacterium Marinomonas mediterranea [364]. More recently, enzymes of the lysozyme family were purified from marine bivalves and conchs [365,366]. Additionally, the marine sponge SpirastreUa sp., in symbiotic associations with marine fungi and bacteria, produces enzymatic activities, e.g., serine-type acetylcholinesterase with the marine bacterium Arthrobacter ilicis [367]; urethanase activity with Micrococcus species [368]; and asparaginase and amylase activity produced by the ftingus Mucor sp. associated with this sponge [369,370]. Aminoacids occur in plants and animals, both in the free state and as the basic units of proteins and other metabolites. Aminoacid derivatives have been reported in marine environment, such as from marine sponges of the genus Jaspis sp. [371,372], fi-om Suberea creba^ a Coral Sea marine sponge [373], and the marine ascidian Leptoclinides dubius [374]. Some of these compounds have been shown to possess interesting biological properties, e.g., cytostatic activity exhibited by axinastatin-4, an aminoacid derivative isolated fi'om a marine sponge [375]. However, tyrosine-derived halometabolites frequently occur in marine organisms. Marine sponges of the order Verongida are of much current biological and chemical interest. An unusual secondary metabolites containing up to four bromotyrosine residues has been isolated from sponges belonging to this order which includes, among others, the genera Aplysinia^ lanthella^ Psammaplysilla^ Pseiidoceratina and Verongida. Bastadins, Fig. (47) are a family of bromotyrosine-derived metabolites isolated from different samples of the marine sponge lanthella basta, which exhibit a wide range of biological activity, such as antineoplasic [376], antimicrobial [377], and inhibitory activity of the endothelin A receptor [378]. These types of compounds have recently been reported from another marine sponge, Psammaplysilla purpurea, together with two new dibromotyrosine-derived metabolites [379]. This sponge also afforded brominated benzenoacetonitriles, unusual dibromo-tyrosine derivatives [380]. From the marine sponge Verongida gigantea, a bromotyrosinederived metabolite, verongamine, has been reported as a potent histamine receptor antagonist. This compound and new acetylenic derivatives are being developed by chemical synthesis. [381].

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Fig. (47). Structure of bastadin 8

Other biologically active bromotyrosine-derived metabolites of marine origin include aeroplysinin. Fig, (48) as cytotoxic and tyrosine kinase inhibitor [382,383], fistularin isolated from Aplysina archeri which exhibited antiviral activity [384], and ceratinamides A and B, antifouling metabolites iromPseiidoceratinapurpurea [385].

Fig. (48). Structure of aeroplysinin

PHOSPHOLIPIDS Lipids are esters of long-chain fatty acids and alcohols or of closely related derivatives. The chief difference between these substances is the type of alcohol; in fixed oils and fats, glycerol combines with the fatty acids; in waxes, the alcohol has a higher molecular weight, e.g., cetyl alcohols. Several monounsaturated phospholipid fatty acids exist in nature, but few cases are known of very long-chain monounsaturated acids longer than 22 carbons. However, marine sponges are unusual in that they have very long-chain fatty acids in their phospholipids. Sponges have provided the most interesting examples of long-chain phospholipid fatty acids since

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acids with chain-lengths between 24 and 30 carbons have been reported. This unusual ability of these marine invertebrates to biosynthesize very long-chain fatty acids has been responsible for the many interesting structures which have been reported without counterpart in the terrestrial world. Sponges have long been recognized as a rich source of structurally novel lipids including unique fatty acids, phospholipids and triglycerides. The fatty acids of sponges have attracted considerable interest because of their unique characteristics, such as increased chain length, branching and unusual unsaturation patterns, and because of the implications the structural variations may have, when present in phospholipids, for membrane fiinction. Common phospholipid fatty acids from marine sponges include 5,9 hexacosadienoic, which occurs in most know^n sponges, 5,9 heptacosadienoic and 5,9 octacosadienoic. For example, marine sponges of the class Demospongiae contain high levels of characteristic C24-C30 fatty acids and are unique in that they seem to be able to biosynthesize these compounds with amazing ease. Studies have shown that many of these "demospongic acids" possess unusual unsaturation and/or methyl branching not found in the fatty acids of other more common organisms. Many of these compounds have been shown to possess interesting biological properties, e.g., antifungal [386], amidolytic [387], inhibitory activity of PKC and anti-inflammatory activity [388,389] and topoisomerase I [390], and inhibitory activity of HIV reverse transcriptase reported from taurospongin A, Fig. (49), a fatty acid derivative isolated from the Okinawan marine sponge Hippospongia sp. [77,391].

o Fig. (49). Structure of taurospongin A

Branched fatty acids of longer than unusual chain-length have also recently countered in several other sponges. Sphingosine derivatives, such as plakoside A and B, Fig. (50), two unique prenylated glycosphingolipids isolated from Plakortis simplex , have been reported as potent immunosuppressive agents [392]. The Okinawan marine sponge Age las

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mauritianus yielded a family of new glycosphingolipids named agelasphins, which have been shown as strong antitumor compounds [393]. These lipids have been recently reproduced by chemical synthesis in an efficient manner [394]. Glycosyl ceramides are a family of agelasphin derivatives also with antitumor and immunomodulating activity, which have been reported fi*om different Age las sp. such as the above Agelas mauritianus [395] and Agelas dispar [396]. These compounds were also isolated from other marine sponges, e.g., Haliclona koremella, as an antifouling substance against macroalgae [397], and Spirastrella abata as inhibitors of cholesterol biosynthesis [398]. Recently, these compounds have also been reproduced by chemical synthesis [399]. From marine sponges of the genus Peirosia sp., several glicerol derivatives have recently been isolated, showing interesting biological properties, such as cytotoxicity against human tumor cell lines [400], inhibitory DNA replication [401], and inhibitory activity of HIV reverse transcriptase [77]. New glycerol derivatives were also isolated from the sponge-associated hdiCiQrmm Micrococcus hiteiis [402].

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BIOLOGICAL ACTIVITIES OF NATURAL HALOGEN COMPOUNDS GERHARD LAUS Immodal Pharmaka GmbH, Bundesstrasse 44, A-6111 Voiders, Austria ABSTRACT: Algae, bacteria, fiingi, lichens, sponges, plants, and mammals produce a wide variety of halogen-containing secondary metabolites. Biological activities also cover a wide range. Many natural halogen compounds exhibit antibiotic activity, for example the altematamides, the pyralomycins, the longamides, clathramides, celenamide, axinellamides, and the macrolide flurithromycin. Cytotoxicity is displayed by the polychlorinated phenolethers russuphelins, the macrolide phorboxazoles, the cyclopropane derivatives grenadadiene and callipeltosides, the aurantosides, the aurisides, the rubrosides, the didemnolines, the spiro isoxazole derivates purealidins, and the cyclic peptides geodiamolides. The chloropyrrol-containing hexapeptide cyclocinamide A, and the nostocyclophanes, natural cyclophanes, also belong to this categoiy. Spongistatins are among the most cancer cell growth inhibitory compounds yet discovered. Some natural halogen compounds display a gallery of activities, like the cytotoxic cyclodepsipeptide jaspamide which is also antifungal and insecticidal. The mycorrhizin-related chloroepoxide lachnumone combines antifungal, nematicidal and antimicrobial activities. The chlorosteroid glycoside blatellastanoside is a pheromone of cockroaches. Polyhalogenated monoterpens are insecticides, some deter the settlement of larvae. The volutamides, barbamide, and parguerol show antifeedant properties. Kalihinol A inhibits the settlement of barnacle larvae. Some natural halogen compounds have emerged as potent lead structures for the development of new synthetic drugs, like the analgesic chloropyridine epibatidine, or pyrrolnitrin and dioxapyrrolomycin which have lead to powerful fungicides and insecticides. Even polychlorinated dibenzodioxins have been found in nature. In some cases, the halogen is essential for activity like the antifungal chloroorcinols, ichthyotoxic malyngamides, and cytotoxic makaluvamines. The biological activities of these and related compounds are reviewed, and 173 references are given.

INTRODUCTION Algae, bacteria, fungi, lichens, sponges, plants, and mammals produce a wide variety of halogen-containing secondary metabolites. Natural halogen compounds have long been considered as chemical freaks. The antibiotics aureomycin and chloramphenicol, and the antimycotic griseofulvin are classic examples. Today there are more than 3000 examples known. Most of them are of marine origin. Ocean water is approximately 0.5 M in chloride, 1 mM in bromide, and 1 |aM in iodide. Given this high halogen content, it is not surprising that marine organisms have developed means to incorporate halogens into their metabolites

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which are sometimes passed on to symbionts mid possibly modified for defensive purposes. The origin of marine metabolites can be obscured by these symbiotic associations. Recent reviews concern origin and occurrence of natural organobromine compounds [1], organochlorine compounds [2,3], the diversity of natural organohalogen compounds in living organisms [4,5], and natural chemistry of chlorine in the environment [6]. Marine sponges continue to be a rich source of secondary metabolites with novel structures and desirable biological activities [7]. Various aspects of this field, e.g. marine haloperoxidases [8], ascidian metabolites [9], marine invertebrate chemical defenses [10], bioactive metabolites of symbiotic marine microorganisms [11], marine bacteria [12], biosynthesis of marine natural products [13], microalgal metabolites [14], bioactive sponge peptides [15], marine pyridoacridine alkaloids [16], and marine toxins have been reviewed earlier [17], The present review is divided into major areas of activity although some overlapping does occur due to the many and diverse bioactivities of some natural halogen compounds. It emphasizes the newer literature and refers to older publications only when they are of special relevance. A discussion of the more striking structural features is given where appropriate. BIOSYNTHESIS OF HALOGENATED ORGANIC COMPOUNDS Haloperoxidases are enzymes that catalyze the oxidation of a halide by hydrogen peroxide, a process which results in tlie concommitant halogenation of organic substrates. The nature of the oxidized halogen intemiediate has been shown to depend on the nature of the organic substrate [18]. Haloperoxidases have been isolated from all classes of marine algae and many other marine organisms. Two general types of marine haloperoxidases have been identified: vanadium haloperoxidase and Fe-heme haloperoxidase. A remarkably thermostable iodoperoxidase has been isolated fi-om the sea weed Saccorhiza polyschides [19]. In addition, other terrestrial haloperoxidases are known. Mechanistic considerations of the vanadium haloperoxidases have been reviewed recently [20]. CYTOTOXICITY Marine tunicates are a rich source of intriguing structures and interesting biological activities. Eudistomidin A (1) fi"om the Okinawan tunicate Eiidistoma glqucus possesses powerfiil calmodulin antagonistic activity (IC50 3 X 10'^^ M). Eudistomidins B-D (2-4) show potent cytotoxicity

759

against murine leukemia L1210 (IC50 3.4, 0.36, and 2.4 |ig/ml) and L5178Y (ICsq 3.1, 0.42, and 1.8 jig/ml). In addition, eudistomidm B activates rabbit heart muscle actomvosin ATPase by 93% at 3 x lO'*' M, while eudistomidin D induces Ca'^^ release from the sarcoplasmic reticulum, about 10 times more potent than caffeine [21].

3 (eudistomidin C)

4 (eudistomidin D)

The remarkably antineoplastic spongistatins have been obtained from a Maldivian Spongia sp. Spongistatin 1 (5) possesses two spiro ketals and is extremely potent against highly chemoresistant tumor types (GI50 typically 3 x 10"^^ M) [22]. Spongistatins 4 and 5 have been obtained from the African sponge Spirastrella spinispirulifera. Mean growth inhibition (GI50) values of 10'^ M are found in the National Cancer Institute's (NCI) 60 tumor cell lines. Human breast cancer cell lines are even more sensitive (GI50 down to 10" M) [23,24]. The names altohyrtin and cinachyrolide A have also been used for spongistatins 1 and 4, respectively [25]. Thus, cinachyrolide A from a sponge of the genus Cinachyra is highly cytotoxic against L1210 murine leukemia cells with an IC50 of »

XY = -O-CH2-O. l,Et743R, = Me,R2 = OH 63,Et729Ri = H,R2 = OH

Ecteinascidin 736-type

64,Et745Ri = Me,R2 = H

XY = .O-CH2-O-

65, Et 759B Ri = Me, R2 = OH, S-oxide

68, Et 722 Ri = H, R2 = OH

66,Et770Ri = Me,R2 = CN Me 67, Et743N*^-oxideR2 =0H, Ri = i^

oV

H NH2

70, Et597Ri = Me,R2 = OH X - O M e , Y = OH 71,Et583Ri = H,R2 = OH X = OMe,Y = OH

o 72, Et 594 Ri = Me, R2 = 0H X Y = -O-CH2-O73,Et596 Ri = Me, R2=0H X = OMe, Y = OH

828

In relation to their biosynthesis, it was proposed that A-B units of Et's are most likely formed by condensation of two Dopa-derived building blocks and that the tetrahydroisoquinoline ring in unit B is closed by condensation (Pictet-Spengler) with a serine(or glycine)-derived aldehyde, as in the case of the related saframycins. SAdenosylmethinonine is the likely source of the methyl groups. A plausible route for the formation of unit C was proposed later [75]. This was partially demonstrated by incorporation of radiolabeled tyrosine and cysteine by Kerr and Miranda [80] and by incorporation of labeled methionine, glycine and tryptophan by Morales and Rinehart [81]. Sulfide Diketopiperazines Several sulfide diketopiperazines were isolated from marine bacteria. Gliovictin (74) composed 27% of media extract of cultured broths of the marine deuteromycete Asteromyces cruciatus [82]. Compound 74 was previously found in terrestrial fungi of the genera Helminthosporium and Penicillium [83]. Maremycins A and B (75 and 76) were additional diketopiperazines obtained from the cultured broth of the marine Streptomyces sp. and the structures of these compounds were elucidated by spectral methods and chemical means [84].

r

Me

T

IN

O

SMe

75, maremycin A R= ••••••OH 76, maremycin B R= —OH

The Structure of cyc/o-(L-Pro-L-Met) (77), obtained from a bacteria strain of Pseudomonas aeriginosa associated with the bioactive Antarctic sponge Isodictya setifera, was determined by spectroscopic methods and confirmed by synthesis [85]. Although the sulfur-containing diketopiperazine cyc/o-(L-Pro-L-thioPro) (78) was isolated from the Bermudian sponge Tedania ignis, a bacterium origin of this metabolite was suggested. The structure of 78, including the 5,5 absolute stereochemistry, was elucidated through spectral analyses and confirmed by synthesis from commercially available L-amino acids [86].

829

NH SMe

o 77, cvc/o-(L-Pro-L-Met)

78, cvc/o-(L-Pro-L-thioPro)

Miscellaneous Sulflde-containing Marine Metabolites Several structurally diverse marine alkaloids bearing a sulfide flinctionality were isolated from sponges and nudibranch. The sulfurcontaining phloeodictines (79-81), isolated from the New Caledonian sponge Phloeodycton sp., belong to a group of alkaloids characterized by the presence of a unique 6-hydroxy-1,2,3,4-tetrahydropyrrolo[ 1,2ajpyrimidinium skeleton. The structures of these systems were determined by extensive spectroscopic analysis and they exhibited in vitro antibacterial activity against Gram-positive and Gram-negative bacteria as well as being moderately cytotoxic against KB tumor cells [87, 88].

79, phloeodictine CI n = 2 80, phloeodictine C2 n = 1 81, phloeodictine B n = 1, A

The Structure of corallistine (82), a new polynitrogen compound from the New Caledonian sponge Corallistes fluvodesmus, was determined by X-ray single crystal analysis of its 6-isobutyloxycarbonyl derivative [89]. Compound 83 {9-[5*-deoxy-5'-(methylthio)-p-D-xylofuranosyl])adenine} was reported as the first naturally occurring purine carrying a xylosederived substituent and is an analog of methylthioadenosine (MTA), the ubiquitous biological natural methyl donor. This compound, along with its epimer at C-3' (84), were isolated from the nudibranch Doris verrucosa and their structures were deduced by spectroscopic methods [90]. Radiolabeled experiments identified adenine and methionine as the

830

biological precursors of nucleoside 83 and demonstrated that is produced by oxidation-reduction of its epimer [91, 92]. p

NH2 N

N N"^N

MeS

NH2

MeS

83, Ri =0H,R2 = H 84,R, =H, R2 = 0H

82, corallistine R2 OH

A methyl sulfide group is present in the cyclic heptapeptides phakellistatin 5 (85), obtained from the Micronesian sponge Phakellia costada [93], and hymenamide F (86), which was isolated along with its S-oxide form [Mso] (87) from the Okinawan sponge Hymeniacidon sp. [94]. Their structures were determined by spectral and chemical methods. The structure elucidation of hymenamide F (86) was carried out mainly on the basis of the spectroscopic data of [Mso] hymenamide F (87), which appeared to have been generated from hymenamide F through autoxidation of the methyl sulfide residue during purification. Compound 85 exhibited an unusual pattern of selective growth inhibition against the US NCI human cancer cell line panel [93]. NH2

MeS

HN^NH O^s^N

85, phakellistatin

86, hymenamide F R = SMe 87, [Mso] hymenamide F R= S—Me O

831

The macrocyclic lactones thiomycalolides A and B (88 and 89), which are characterized by a glutathione adduct, were isolated from a Japanese sponge Mycale sp. [95]. Their structures were determined by interpretation of spectral data and chemical transformations. Compounds 88 and 89 exhibited cytotoxic activity against P388 cells with an IC50 value of 18 ng/ml each. It is not known whether thiomycalolides are produced by an enzymatic reaction in the sponge or are simple adducts of co-occurring mycalolides and glutathione. H O'^ N AcO

R

Meb

6 ^ / 0 OMe

V.

,

^" O

N-\

OH

^

Jl y\ ^^y^

, O

88, thiomycalolide A R = O 89, thiomycalolide B

N/ OMe

H OMe

Umbraculumin C (90), characterized by the presence of a trans'3(methylthio)-acrylic acyl residue, is a diacylglycerol obtained from the skin of the opisthobranch mollusc Umbraculum mediterraneum [96]. Taylor and co-workers determined the absolute configuration of 90 by total synthesis [97] while Sodano et al. reached the same conclusion by derivatisation of the natural compound [98]. Compound 90 displayed ichthyotoxic activity against the mosquito fish at 0.1 |ag/ml and should represent the deterrent of this organism against predators [96].

OH O 90, umbraculumin C

91,R = CH20H 92, R = COjMe

The fouling bryozoan Dakaira subovoidea has been shown to contain two thiophene compounds, 1 -hydroxymethyl-6-oxo-6//-anthra[ 1,96c]thiophene (91) and its 1-methoxycarbonyl derivative 92 [99]. Their structures were determined by spectral and crystallographic analyses. The

832

acetylated derivative of 91 displayed antioxidant activity, showing 99.5% inhibition of lipid peroxide formation in rat liver microsomes at 10 |ig/ml. An unprecedented sulfur-containing yellow pigment, namely benzylthiocrellidone (93), was isolated from the Australian sponge Crella spinulata [100]. This compound represents the first recorded example of a natural product containing a dimedone unit. The structure of this compound, determined by spectroscopic methods in conjunction with an X-ray crystal study, was confirmed by total synthesis [101]. The first example of a naturally occurring 5-thiosugar, 5-thio-D-mannose (94), was reported from the Australian sponge Clathria pyramida [102]. This compound had to be isolated as its peracetylated derivative, after treatment of the crude material with Ac20/pyridine, and then be deacetylated with methanolic ammonia.

HO^ OH OH 00

94, 5-thio-D-mannose

93, benzylthiocrellidone

Additional pharmacological studies on the cytotoxic compound acanthifolicin (95), the 9,10-episulfide derivative of okadaic acid obtained from sponge Pandaros acanthifolium [103], showed it to be an inhibitor of phosphatases 1 and 2a (PPl-1 and PP-2a) with similar potencies to okadaic acid [104].

" 95, acanthifolicin 9,10^^^^^''" okadaic acid 9, 10

C=C

" 6 H Me

833

DISULFIDES AND POLYSULFIDES Tunicates are by far the most important source of marine metabolites containing disulfides and polysulfides, followed in importance by marine microorganisms and, to a lesser extent, sponges and algae. Dopamine-derived Polysulfides A series of sulfur-containing dopamine-derived metabolites has been isolated from tunicates, predominantly belonging to the genus Lissoclinum. Varacin (96), the first reported naturally occurring benzopentathiepin, was isolated from Lissoclinum vareau [105]. The evidence for a benzopentathiepin system was provided by tandem MS in the (-) FAB mode on the A^-trifluoroacetyl derivative. The structure of 96 has been confirmed by several syntheses [106]. A closely related compound, lissoclinotoxin A (97), which was obtained from the tunicate Lissoclinum perforatum, was originally reported to have a cyclic benzotrithiane structure [107] but it was subsequently shown to be a benzopentathiepin [108]. The synthesis of lissoclinotoxin A and its regioisomer 98 (referred to as isolissoclinotoxin A) led to a second revision of its structure [109]. Varacin (96) and lissoclinotoxin A (97) were found to be chiral, displaying unusual stereoisomerism due to restricted inversion about the benzopentathiepin ring. However, their optical activity may be lost during isolation [110, 46]. Two tunicates belonging to Lissoclinum genus have been shown to contain additional members of this series. The benzopentathiepin lissoclinotoxin B (105) was isolated as a minor component of L. perforatum [108] while the dithiomethyl lissoclinotoxin C (106) was obtained, along with the dimeric lissoclinotoxin D (107), from Lissoclinum sp. [46]. The energetically more favorable dimeric "head-totail" situation for 107 was proposed for lissoclinotoxin D but the alternative "head-to-head" cannot be excluded. Faulkner, Carte, and coworkers described the isolation of five additional varacin-lissoclinotoxin derivatives and used extensive spectral analysis in their structural determination [111]. These compounds include A^,A^-dimethyl-5(methylthio) varacin (99) and 3,4-dimethoxy-6-(2'-iV,A^-dimethylaminoethyl)-5-(methylthio)benzotrithiane (102) from L. japonicum, an

834

inseparable 2:3 mixture of 5-(methylthio)varacin (100) and the corresponding trithiane (103) from a different unidentified Lissoclinum species, and 3,4-desmethylvaracin (101) from Eudistoma sp. The tunicate Polycitor sp., another source of this type of compounds, yielded a benzotrithiepin, varacin A (104), and two benzotrithiepin S-oxides, varacins B (108) and C (109) [112]. ^H-NMR and MS studies of varacin A (104) and varacin (96) (also isolated from this tunicate) revealed that they readily equilibrate to give a mixture of both compounds. As a matter of fact, equilibrium reactions between the benzopentathiepin 96 and the benzotrithiepin 104 + Sg were observed to occur in CHCI3, MeOH, and pyridine. For example, 96 generated 104 when it was dissolved in MeOHd4. More recently, lissoclin disulfoxide (110) was obtained from an unidentified species of Lissoclinum [113]. It was proposed that these natural products derived from an overlapping manifold of tyramine biosynthesis coupled with hypovalent sulfur metabolism [46]. Benzopentath iepin 96, varacin Ri = R2 = Me, R3 = R4 = H

'sylp^N^^^ RsO-^Y^Rj

97, lissoclinotoxin A Rj = R3 = R4 = H, R2 = Me 98, isolissoclinotoxin A Ri = Me, R2 = R3 = R4 = H 99, N, N-dimethyl-5-(methylthio) varacin Ri = R2 = Me, R3 = SMe, R4 = Me

OR,

100,5-(methylthio)varacin Ri = R2 = Me, R3 = SMe, R4 = H 101,3,4-desmethylvaracin Ri = R2 = R3 = R4 = H

Benzotrithiepin S'^S

Sv JV^.,X-V^N; ^

II

^

102, Rj = R2 = R4 = Me,

R3 =

SMe

103, benzotrithiepin 5-(methylthio)varacin Rj = R2 = R4 = Me, R,= H 104, varacin A Rj = R2 = Me, R3 = R4 = H

835

MeO SMe

MeO

OH

S-S'^ y MeO NH2

106, lissoclinotoxin C

OH

NH2 107, lissoclinotoxin D MeO O OMe MeO^ . A . ^S-Y'^Y'^^®

105, lissoclinotoxin B

-S^y^SMe

MeS 109, varacin C

NH2

NH2

110, lissoclin disulfoxide

The polysulfides showed a wide range of biological 108, dopamine-derived varacinB activities: antimicrobial (96, 97, 99, 102, 105, acetates of 104, 108, and 109), antifungal (96, 97, 107, acetates of 104, 108, and 109), cytotoxic (96, 97), and antimalarial (97) [105-113]. Furthemiore, a differential toxicity of 1.5 for varacin (96) toward the CHO cell lines EM9 (chlorodeoxyuridine-sensitive) versus BRl (BCNU-resistant) indicated that varacin's mechanism of action involves the formation of single stranded DNA breakage [114]. Lissoclin disulfoxide (110) was found to be a potent inhibitor against both IL-8Ra and IL-8RP receptors with IC50 values of 0.6 and 0.82 \xM, respectively, and also had activity against PKC (IC50 1.54 |ag/ml) [113]. Furthermore, compounds 99, 100, 102, and 103 showed selectivity for inhibition of PKC in comparison to PKA [111]. A number of structure-activity relationships were deduced. The importance of the presence of a benzopentathiepin/benzotrithiepin ring in the activity of these compounds was suggested since lissoclinotoxin C (106) [46] and other tris(methylthio) derivatives [111], which do not contain such a system, were inactive. On the other hand, the activity of these compounds seems not to depend upon the presence of a free amino group in their side chains since varacin (96) and its iV-acetylated derivative displayed similar

836

activities [112]. Finally, the structural relation of this system to dopamine was also found to be related to the potent activity. Polycarpine and Polycarpamines Tunicates belonging to the Polycarpa genus are another source of disulfide metabolites. The isolation of the dimeric disulfide alkaloid polycarpine (111) from P. aurata was reported by Schmitz et al [115] and from P. clavata by Kang and Fenical [116] at the same time. Compound 111, as its dihydrochloride salt, was converted into the free base by silica gel chromatography, a process that also gave several degradation products (e.g. I l i a and 111b) that appear to be artifacts of the isolation process. Spectral studies, chemical transformations, and X-ray crystallography allowed the structural elucidation of these compounds. The synthesis of 111 in three steps from /7-methoxyphenacyl bromide confirmed its structure [117]. Polycarpine inhibited the enzyme inosine monophosphate dehydrogenase, but this inhibition could be reversed by addition of excess dithiothreitol, suggesting that 111 reacts with sulfhydryl groups on the enzyme [115]. Furthermore, 111 showed cytotoxicity against HCT-116 cells (IC50 0.9 |ig/ml) [116] and significant antitumor activity in vivo (white mice) against P388, L1210, carcinoma Ehrlich cells, and high inhibitory activity against reverse transcriptases from Raus sarcoma and avian myeloblastosis viruses in vitro (IC50 = 3.5 x 10"^ M) and Na^, KÂTPase isolated from rat brainof (IC50 = 5.0 x 10"^ M) [117]. The tunicate P. auzata has also been shown to contain the polycarpamines A-E (112116). These are benzenoid compounds with uncommon sulfiir functionalities and were characterized through interpretation of their NMR data and MS fragmentation patterns. Only the first collection of this organism yielded polycarpamines, suggesting that the tunicate may not be the true source of these compounds. Polycarpamine B (113) exhibited significant antifungal activity in vitro [118]. s

ixy^y^ ^-w-

MeO-

111b

837 NMe2

MeS^"

"

112, polycarpamine A Rj = H, R2 = OMe

MeO 116, polycarpamine E

113, polycarpamine B Rj = R2 = O 114, polycarpamine C Rj = R2 = S 115, polycarpamine D Rj = COMe, R2 = OMe

Other Disulfldes/Polysulfldes from Tunicates A number of other disulfides and polysulfides have been obtained from tunicates. Citorellamine (117) was the first indole disulfide dihydrochloride isolated from a marine organism - the tunicate Polycitorella mariae. The previous structure proposed for citorellamine [119] was revised and confirmed by total synthesis [120]. Compound 117 possesses both cytotoxic (against L1210 cells with an IC50 value of 3.7 |Lig/ml) and potent antimicrobial activity [119]. An unidentified New Zealand tunicate species of the Aplidium genus was the source of the trithiane 118 [121]. Spectroscopic methods and chemical degradations established the structure of 118 as c/5'-5-hydroxy-4-(4'-hydroxy-3*methoxyphenyl)-4-(2"-imidazoyl)-1,2,3-trithiane. Thus, in neutral or slightly basic solution this compound interconverted to give the trans isomer 119 and 2-vanilloyl imidazole 118a, which could arise from thione 118b. Furthermore, conformational analysis of the trans isomer showed a chair conformation for the trithiane ring with the hydroxyl functionality equatorial and the imidazole ring axial. Compound 118 displayed antimicrobial, antifungal, and modest cytotoxicity (P388, IC5o= 13 and 12 l^g/ml for 118 and 119, respectively) activities [121]. Namenamicin (120) was the first sulfur-containing enediyne compound structure to be isolated from a tunicate {Polysyncraton lithostrotum) [122]. The structure of 120 was deduced from its spectral data and by comparison to those of the

838

known enediyne antitumor antibiotics calicheamicins and esperamicins [123, 124]. Compound 120 exhibited potent in vitro cytotoxicity with a mean IC50 of 3.5 ng/ml and in vivo antitumor activity in a P388 leukemia model in mice (ILS 40% at 3 |ag/Kg). Namenamicin (120) also showed potent antimicrobial activity and cleaved DNA with a slightly different recognition pattem than calicheamicin yi^ [122]. The fact that all of the enediyne antitumor antibiotics previously isolated were products of actinomycites, as well as namenamicin's extremely low and variable yield from the tunicate, lend support to the hypothesis of a microbial origin for this natural product. HO,

HO, rH>

0.020/0NaOH ^^^.

. MeO J - ^ " " N^ N

orCD30D MeO

119

118

118a X = 0 118b X = S

OMe

3,XX^"

-''^Xx'S-)2

117, citorellamine

Me

MeS

^S

«=s-ziô;^!>l,o/o' "°

HO

9

I MeO 120, namenamicin

Disulfides/Polysulfides from Marine Microorganisms Marine microorganisms have been an important source of disulfide and polysulfide marine metabolites. Among these compounds are the leptosins and the thiomarinols, which form a well-defined group of natural products.

839

Leptosins The polysulfide leptosins (121-135) belong to a series of epipolythiodioxopiperazines produced by a fungal strain Leptosphaeria sp. OUPS-4, isolated from the marine alga Sargassum tortile [125, 126, 127, 128]. HOv.^

\ ^

oOH

^ ^

O^^^Me

..OH

I

O ^ ^ M

121, leptosin A x = 4, y = 2

128, leptosin K n = 2

122, leptosin B x = 3, y = 2

129, leptosin Ki n = 3

123, leptosin C x = 2, y = 2

^Ô, leptosin K2 n = 4

124, leptosin G x = 4, y = 3 125, leptosin Gi x = 3, y = 3 126, leptosin G2 x = 2, y = 3 127, leptosin H x = 2, y = 4

1 "^.-^^^ ^"'^

a

—k—f

133, leptosin D x = 2

131, leptosin I Rj = CH2OH, R2 = H

134^ leptosin E x = 3

132, leptosin J Rj = H, R2 = CH2OH

135^ ^^^^^^^^ p ^ ^

840

The structures of these systems have been elucidated by spectroscopic analysis, some chemical transformations, and by X-ray crystallography in the cases of 128-130 [127]. These compounds can be structurally divided into four main groups: the dimeric systems, including leptosin A type (121-127), leptosin K type (128-130), leptosin I and J (131 and 132), and the monomeric systems 133-135. All of these compounds showed potent cytotoxicity against P388 cells that range from ED50 values (in |ag/ml x 10"^) of 1.13 for leptosin I (131) to 8.60 for leptosin D (133). The dimeric leptosins displayed more potent activity than the monomeric ones and the number of sulfur atoms in the dioxopiperazine rings was not found to influence the activity [125-128]. Furthermore, leptosins A (121) and C (123) showed antitumor activity against the Sarcoma-180 ascites tumor in mice with a ratio of (T/C%) of 260 and 293 and at doses of 0.5 and 0.25 mg/Kg, respectively [125] Thiomarinols Thiomarinols A-G (abbreviated as TMA-G) (136-142) are a group of antibiotics isolated from the cultured broth of marine bacterium Alteromonas rava sp. no. SANK 73390 [129, 130, 131, 132]. Their structures were elucidated by NMR spectral analysis, chemical degradation, and by X-ray analysis in the case of 141 [131].

136, thiomarinol A Rj = R2 = OH, R3 = H, n = 5 137, thiomarinol C Rj = H, R2 = OH, R3 = H, n =5 138, thiomarinol D Rj = OH, R2 == OH, R3 = Me, n = 5 139, thiomarinol E Rj = OH, R2 = OH, R3 = H, n = 7 140, thiomarinol F Ri = OH, R2 = O, R3 = H, n = 5

841

All of these compounds showed excellent in vitro antimicrobial activity, with 136, 139, and 141 being the most active. Furthermore, compounds 136, 138, and 141 were found to inhibit the isoleucyl-transfer RNA synthetase in bacteria [132].

HO

OH

141,thiomarinolB

Me

Me^ ^

^^Y^Yy^^'

.^r>^ ^

Ô

Me

O

OH 142, thiomarinol G

Additional disulfide metabolites have been isolated from marine bacteria. The unique pyridone-oxazole B-90063 (143) was isolated from the culture supernatant of Blastobacter sp. SANK 71894 [133]. The structure of 143 was determined to be bis[6-formyl-4-hydroxy-2-(2'-Ai-pentyloxazol-4'-yl)4-pyridon-3bis[6-formyl-4-hydroxy-2-(2*-«-pentyl-oxazol-4*-yl)-4pyridon-3-yl]disulfide on the basis of spectral data and chemical reactions. Compound 143 inhibited human and rat endothelin-converting enzyme (ECE) with an IC50 of 1.0 and 3.2 |aM, respectively. B-90063 also inhibited the binding of endothelin-1 to rat endotheliuA and bovine endothelin receptors, although its antagonist activities were weak. These activities led to the proposal that 143 might abolish the physiological actions of endothelin through the ECE inhibitory and receptor antagonistic mechanism [133]. The thiodepsipeptide thiocoraline (144), isolated from the mycelium Micromonospora sp. L-13-ACM2-092, was found to be a potent antibiotic and cytotoxic compound. Its structure was deduced on the basis of spectroscopic methods [134].

842

O

Hi,

C5H,,

?\

C5H1

Y

O ^

Me-N

SMe

r

s-s oo 143, B-90063

0=
iCS

HO/

HO/ A = O .UNC CN ^ ^ ^ - ^ ^ ^

NCS

HO' = H SCN "

227

SCN

h^-^""

229

228

230, Ri = NCS, R2 = R3 = CH2 jjQ, ,

J. J "4^^ R2 H=^^\oo(--R3

231, Ri = NC, R2 = NCS, R3 = Me 232, Ri = NCS,R2 = NC,R3 = Me

CN

233, Ri = R2 = NCS, R3 = Me Cis decalin-tetrahydrofuranyl

kalihinol

OHCHH

H iNC

NCS

CN

HO 235

NC

"A!)

236

|N( NCS

856

Table 3.

Diterpene Isothiocyanates Belonging to the Kalihinol Family Isolated from Sponges Name

INO!

Origin

Ref.

Biological activity

222

Kalihinol I

Acanthella cavernosa

[168]

223

Kalihinol J


[168]

Anthelmintic

224

(-)-kalihinol X

Acanthella spp.

[189]

Antimicrobial

225


[175]

Antifouling

1

226

(+)-lOP-formamido-5 Pisothiocyanatokalihinol-A (-)-10-ep/-kalihinolI

Acanthella sp.

[188]

Antimalarial

1

227

(-)-lO-epi-kalihinolH

Phakellia pulcherrima

[187]

228

(-)-5,10-bisisothiocyanokalihinol G

Acanthella sp.

[188]

Antimalarial

1

229

(-)-kalihinol L


[187]

230

(-)-lO-isothiocyanatokalihinol C


[187]

231

(-)-kalihinol G

Acanthella spp.

[189]

Antimicrobial

1

232

(+)-kalihinol H

Acanthella spp.

[189]

Antimicrobial

1

233

(-)-lO-isothiocyanatokalihinol G


[187]

234


[186]

235

6-hydroxy-10-formamido15-isothiocyano-kalihinene (-)-lO-epi-isokalihinol H


[190]

236

(-)-15-isothiocyanato-1 -ep/-kalihinene


[190]

1

Compounds 237 and 238 are the only isothiocyanate diterpenes with a biflorane carbon skeleton that do not belong to the kalihinol family. They are diastereosiomers of 10-isothiocyanatobiflora-4,15-diene. A planar structure was reported for compound 237, which was obtained from an unidentified sponge of the Family Adocidae [191]. A (15*,6/?*,7i?*,105*,l li?*) configuration was proposed for 238, which was isolated from the sponge Cymbastela hooperi [192]. From the latter sponge, three diterpene isothiocyanates that have an amphilectane framework (239-241) have been identified. Extensive spectroscopic experiments led to their structures being proposed as (15,35,4i?,75,85,115,125,135,15/?,20/?)-20-isocyano-7-isothiocyanatoisocycloamphilectane for 239, (15*,3S*,4/?*,75*, 85*,125*,135*)-7isocyano-15-isothiocyanatoamphilect-11 (20)-ene for 240, and

857

(IR *,35*,4i? *,75*,85*, 127? *, 13R *)-12-hydroxy-7-isothiocyanatoamphilecta-ll(20),14-diene for 241. Compounds 239-241 show significant and selective in vitro antimalarial activity [192].

NCS 237 (planar structure)

239

NCS

238

Non Terpene Isothiocyanates The only non-terpene isothiocyanate compounds reported from marine organisms are two quinoline alkaloids, namely cylindrines I (269) and J (270) [193] (they will be discussed in the thiocyanate section) and the long chain aliphatic isothiocyanate metabolites. Eighteen long chain a,cobisisothiocyanates [eight diolefinic (242-249) and ten monoolefinic (250259) and three a-isothiocyanate-co-formyl (260-262) compounds have been reported from the Fijian sponge Pseudaxinyssa sp. [194]. The absence of the corresponding isocyanate analogs suggests a different biogenesis for these compounds.

SCN 242,n=14 243, n = 8 244, n = 9 245, n = 1 0

NCS 246, 247, 248, 249,

n=ll n = 12 n=l3 n=14

SCH

'l^r^^NCS n

250,n=16 251, n = 9 252,n=10 253, n =11 254,n=12

255,n=l3 256,n=14 257,n=15 258,n=17 259,n=18

SCN^^

CHO ^ '

260,n=15 261,n = 9 262,n=16

858

THIOCYANATES Although marine organisms produce a large number of isocyanates, isothiocyanates, and formamides, the corresponding thiocyanates have rarely been encountered. Indeed, the thiocyanate functionality has only been found in six sesquiterpenes (263-268), in four tricyclic quinoline alkaloids (271-274), and in psamaplin B (172) (included in the bromotyrosine derivatives discussed in the disulfide/polysulfide section). They have been found in marine sponges, as well as in nudibranches and tunicates. The presence of a thiocyanate group can be very easily deduced from the IR spectrum, which contains a sharp, but weaker in intensity, IR band around 2150 cm~^ and an NMR carbon resonance between 112 and 115 ppm. In the case of psamaplin B (172), ^"^C-NMR additivity effects applied to the methylene carbon linked to the thiocyanate were used to distinguish between a thio- and isothiocyanate group [142]. Chemical proof can be provided by LAH reduction of the thiocyanate, which gives a thiol rather than a methylamine group as the reaction product of the reduction of the isothiocyanate [195]. Sesquiterpene Thiocyanates In 1989, the research groups of Faulkner and Clardy reported compound 263, the first sesquiterpene thiocyanate to be isolated from a marine organism - the sponge Axinyssa (= Trachyopsis) aplysinoides [165]. The structure of this compound was determined by X-ray analysis to be (15'*,45*,6iS'*,7i?*)-4-thiocyanato-9-cadinene. In 1991 the research groups of Scheur and Higa reported the isolation of two isomeric sesquiterpene thiocyanates, 2- and 4-thiocyanatoneopupukeanane (264 and 265) from two sponges, the Okinawan Phycopsis terpnis and an unidentified species collected in Pohnpei, respectively [195]. The structures of 264 and 265 were determined by spectroscopic methods and chemical transformations. A recent enantiospecific synthesis of (-)-4-thiocyanatoweopupukeanane from (i?)-carvone allowed the determination of the absolute configuration of 265 [196]. Subsequently in 1992, Fusetani et al reported the isolation of cavemothiocyanate (267) from the sponge Acanthella cf. cavernosa and its nudibranch predator, Phyllidia ocellata [197]. In the same year, Faulkner et al published the isolation of (15*,25*,3i?*,6i2*,75*,9/?*)-2thiocyanatoweopupukeanane (266) and (l/?*,2/?*,3/?*,5/?*,65*,75*)-2-

859

thiocyanatopupukeanane (268) from a Pohnpei specimen of the sponge Axinyssa aplysinoides, elucidating their structures through extensive spectroscopic studies [160]. Compound 264 displayed very modest cytoxicity against KB at 0.01 mg/ml [195]. In relation to the biogenesis of this type of compound, Garson et al. demonstrated the biosynthetic origin of the thiocyanate carbon in 2-thiocyanatOAzeopupukeanane (264) by incorporation of sodium [^^C] cyanide and [^"^C] thiocyanate into Axinyssa n.sp.[198].

A R,

r^^4^ ''

H =

^\

/^

cb r^"S--v

KySCN

1H

-SCN 263

264,Ri = SCN,R2 = R3 = H 267

268

265, R3 = SCN, Ri = R2 = H 266, R2 = SCN, Ri = R3 = H

Perhydroquinolines These non-terpene thiocyanates have been isolated from tunicates. The cylindricines constitute a series of reduced pyrrolo[2,l-y]quinolines and pyrido[2,l-y]quinolines isolated from the Tasmanian tunicate Clavelina cylindrica [193, 199]. Cylindricines I and J (269 and 270) are isothiocyanates while cylindricines F, G, and H (271-273) are thiocyanates. They represent the first examples of the isolation of iso- and thiocyanates from tunicates. Compounds 269-273 were identified by a combination of spectroscopic methods and their relative stereochemistries were assigned by using molecular modeling. A closely related compound, fasicularin (274), was isolated from the Micronesian tunicate Nephteis fasicularis [200]. Compound 274, whose structure was elucidated primarily by interpretation of its spectral data, was found to be active in a DNA-damaging assay and cytotoxic against to Vero cells with an IC50 of 14 |ig/mL

860

oOAc SCN

"SCN

SCN-^ 269, cylindrine I

270, cylindrine J

274, fasicularin

271, cylindrine F Rj = R2 = O, n = 3 272, cylindrine G Rj = R2 = O, n = 1

SCN

» ; \

273 cylindrine H Rj = OAc, R2 = H, n = 1

SULFONES, SULFOXIDES, SULFONIC ACIDS AND THEIR DERIVATIVES In this section a very structurally diverse group of compounds bearing oxygenated sulfur functionalities is covered. Sulfones Most of the reported sulfone-containing marine metabolites have been obtained from sponges and bryozoa. The hypotaurocyamine-containing agelasidines, some adociaquinone derivatives, and the euthyroideones constitute small groups of compounds bearing a sulfone group. Agelasidines Agelasidines A~D (275-279) are a series of terpene derivatives of hypotaurocyamine and were isolated from sponges of the genus Agelas. Agelasidine B (276) and (+)-€ (277) were obtained from the Okinawan A. nakamurai [201] while agelasidines (-)-C (278) and D (279) were isolated from the Caribbean A, clathrodes [202]. The structures of these compounds were elucidated by interpretation of spectral data and by

861

chemical degradation experiments. The structure of agelasidine A (275), the first reported marine natural product containing a sulfone unit [203], was confirmed by an efficient three-step synthesis based on a biomimetic approach from famesol [204]. Compounds 275-277 showed antimicrobial and antipasmodic activity, inhibitory effects on contractile responses of smooth muscle, and a potent inhibition of brain Na"^- and K'^-ATPase [201, 205]. NH

NH ''. -S—^

NH2

\S-^

276, (-) agelasidine B

275, (+) agelasidine A NH d\

NH2

.OH - ^ - ^ N

d'^0 ^

277, (-I-) agelasidine C

NH2

T

NH2

NH

279, (-) agelasidine D

278, (-) agelasidine C

Adociaquinones A series of sulfur-containing quinone sesquiterpenes, structurally related to xestoquinones and halenaquinones [206], have been isolated from sponges belonging to the Adocia and Xestospongia genus. Adociaquinones A and B (280 and 281), along with 3-ketoadociaquinone A (282), were obtained from a Pacific Adocia sp. and are characterized by the presence of a l,l-dioxo-l,4-thiazine ring [207]. Their structures were determined by comparison of their spectral data to those of xestoquinone and halenaquinone, and by chemical correlations. A subsequent total synthesis of 280 and 281 confirmed the proposed structures and determined the XAhS absolute stereochemistry for these compounds [208]. Xestoquinolide B (283), a metabolite that is structurally closely related, was isolated from the Indo-Pacific sponge Xestospongia cf carbonaria. Unfortunately, the taurine annulation regiochemistry could not be established in this case [209]. Secoadociaquinones A and B (284 and 285), isolated from the Philippine sponge Xestospongia sp., bear a taurine moiety instead of the l,l-dioxo-l,4-thiazine ring present in the closely structurally related compounds 280 and 281 [210]. Compounds 280, 281, 284, and 285

862

showed inhibition of topoisomerase II in catalytic DNA unwinding and/or decatanation assays. Furthermore, compound 281 showed activity in a KSDS assay, suggesting that 281 inhibits the enzyme by freezing the enzyme-DNA cleavable complex [210]. Compound 281 also displayed mildly cytotoxic activity [207].

O 280, adociaquinone A

O

281, adociaquinone B

282,3-ketoadociaquinone A

HO3S HO3S' O 283, xestoquinolide B

O

284, secoadociaquinone A

O

O

285, secoadociaquinone B

X=NH, Y = S 0 2 or X = S 0 2 , Y=NH

Euthyroideones and other sulfones A sulfone group incorporated within a l,l-dioxo-l,4-thiazine ring is also present in 6-(p-hydroxypheny l)-2i/-3,4-dihydro-1,1 -dioxo-1,4-thiazine (286), a compound obtained from the sponge Anchinoe tenacior [211], and in euthyroideones A-C (287-289), which are brominated quinone methides isolated from the New Zealand bryozoan Euthyroides episcopalis (order Cheilostomatida, suborder Ascophotina, family Euthyroididae) [212]. The spectral data of euthyroideone A (287), secured by X-ray analysis, was used for the structural determination of euthyroideones B (288) and C (289). Compound 288 showed a weak cytotoxic activity towards the BSC-1 cell line. Very simple sulfones have also been isolated from marine organisms. The isolation of sulfolane (290) from the sponge/tunicate composite Batzella spJLissoclinum sp. was the first report, either terrestrial or marine, of this compound as a natural product [213]. The pyrogallol Phenol B (291), obtained from the red alga Grateloupia filicina, was

863

reported as the first example of a sulfone derivative from marine algae. Its structure was deduced by spectroscopic means and confirmed by synthesis. Phenol B exhibited antibacterial activity against Bacillus subtilis at a concentration of 1 mg/disk [214]. Compound 34 is another sulfone-containing metabolite isolated from a bryozoan and has already been cited along with the sulfide P-carbolines. MeO

0

^ . N ^ J L Br II

"AN

0

OH

Q

'1

0 I

0

jlft

HO^yÔH OH

0

1

Me

Q

290, sulfolane

291, phenol B

287, euthyroideone A 288, euthyroideone B , A 2 289, euthyroideone C,A^

Sulfoxides The sulfoxide group is present in very few marine natural products. We have already described eudistomin K sulfoxide (26), eudistomidin F (29), didemnolines C (32) and D (33), [Mso] hymenamide F (87), and lissoclin disulfoxide (110). Additional examples have also been reported. The red alga Laurencia brongniartii was found in 1978 to be a rich source of polybrominated methylthioindoles [7]. Two additional methylthioindoles and two methylsulfinylindoles (292-295) were isolated in a subsequent study of an Okinawan specimen of this algae [215, 216]. The structures of itomanindole A (294) and the methylthio bisindole 293 were determined by X-ray analysis. The X-ray analysis not only showed the location of the sulfoxide group at C-2 in itomanindole A (294) but also revealed that a racemic pair of molecules are joined centrosymmetrically by two strong hydrogen bonds [216]. Br SMe Br 292

SMe N

293

Y

Br % - M e

SMe

Br^Ns^N

Me

294, itomanindole A

rVVsMe 295, itomanindole B

864

Two sulfur-containing brevetoxin analogs were isolated from two New Zealand bivalves. Brevetoxin Bi (BTXBl, 296), characterized by the presence of a taurine moiety, was obtained from the toxicated shellfish Austrovenus stutchburyi [217], while brevetoxin B2 (BTXB2, 297), characterized by the presence of a cysteine sulfoxide unit, was isolated from the hepatopancreas of greenshell mussels, Perna canaliculus [218]. The structures of both compounds were elucidated by comparison of their spectral data with those of brevetoxin B (BTXB), the major toxin produced by the Florida red tide organism, the dinoflagellate Gymnodinium breve, whose structure was secured by X-ray crystallography in 1980 [219]. Compound 297 seemed to be a diastereoisomeric mixture having two chiral centers at C-41 and the sulfoxide. Attempts to separate the diastereoisomers by HPLC, after acetylating the amino and hydroxyl groups or by methylating the carboxyl groups, were unsuccessful. A plausible biosynthetic route to Brevetoxin B2 from BTXBl was proposed [218]. Compound 296 (BTXBl) has a minimum lethal dose in mice of 0.05 mg/Kg (i.p.), producing very similar symptoms to those caused by other brevetoxins [217]. On the other hand, compound 297 (BTXB2) displayed a similar potency to BTXB in activating Na"^ channels [218],

-v:

ff

NH2

N"X^S03Na

296, brevetoxin B1 (BTXB 1)

297, brevetoxin B2 (BTXB2)

Sulfonic Acids and their Derivatives Marine natural products bearing a sulfonic acid/sulfonate group are very abundant, with a huge diverse array of structures known. In most cases, this functionality is present in the discodermin-halicylindramide depsipeptides, in sulfonolipids, as well as in assorted metabolites.

865

Depsipeptides bearing a cysteic acid residue Polydiscamide A (309), discodermins A-H (298-305) and halicylindramides A~E (306-308, 310 and 311) form a series of depsipeptides composed of 13 or 14 amino acids and bear a sulfonic acid group in a cysteic acid residue, with Cys(03H), with the //-terminus blocked by a formyl group. Their total structures, including absolute stereochemistries, were determined in most cases by a combination of spectral and chemical methods.

298, discodermin A Ri = R2 = H, R3 = R4 = Me, R5 = X, R6 = Tr,R7 = Me, 299, discodermin B Ri= R2 = R3 = H, R4 = Me, R5 = X, R^ = *Pr, R7 = Me 300, discodermin C Ri = R2 = R4 = H, R3 = Me, R5 = X, R^ = *Pr, R7 = Me 301, discodermin D Ri= R2 = R3 = R4= H, R5 = X, R^ == 'Pr, R7 = Me 302, discodermin E Ri = R2 = H, R3 = R4 = Me, R5 = Y, R^ = 'Pr, R7 = Me 303, discodermin F Rj = R2 =H, R3 = Me, R4 - '^., R5 = X, R^ = 'Pr, R7 = Mc 304, discodermin G Ri = R3 = R4 = Me, R2 = H, R5 - X, R^ = *Pr, R7 = Me 305, discodermin H Rj = H, R2 = OH, R3 = R4 = Me, R5 = X, R^ = *Pr, R7 = Me 306, halicylindramide A R| = R3 = R4 = H, R2 = Br, R5 = X, R6 = Ph, R7 = Me 307 halicylindramide B R,= R3= H, R2 = Br, R4 = Me, R5 = X, R^ = Ph, R7 = H 308, halicylindramide C Ri = R3 = H, R2 = Br, R4 = Me, R5 = X, R^ = Ph, R7 = Me

O

866 O^^NH2

O

=

Br

0

0 N^

^

^

/SO3-

O ^=

O \

"

\

N'H X+

Me,^^^

N^ ^^

O ^ ^ Y ^ O^ O

309, polydiscamide A

HN

03*)- r.2*-diacylglycerol, namely KM043 (317), from the red alga Gigartina tenella, which was found to be a potent inhibitor of eukaryotic DNA polymerases and HIVre verse transcriptase type 1 [234]. Illustrative examples of sulfonoceramides are the flavocristamides A and B (318 and 319), obtained from the marine bacterium Flavobacterium sp. that was isolated from the marine bivalve Cristaria plicata. The structures 318 and 319, including absolute configurations, were determined by spectroscopic data and chemical means and both compounds were found to be DNA polymerase a inhibitors [235].

869

318,flavocristamideA A^ 319, flavocristamide B

Marine Sulfur metabolites bearing a Taurine Residue Taurine is present in a wide range of marine organisms [7]. The taurine residue (HO3S-CH2-CH2-NH-), whether joined directly to a compound or through an amide link, has been found as a portion of a very wide range of marine metabolites and is not specific to any particular phylum. The taurine residue can be easily identified by the NMR diagnostic signals at 3.0-3.9 ppm (t, y-- 6 Hz)/35-39 ppm (t) for the methylene attached to the sulfur and 2.5-3.0 ppm (t, 7 --^ 6 Hz)/48-52 ppm for the methylene attached to the nitrogen. In many cases it is possible to observe the m/z peak corresponding to [M - CH2CH2SO3H] fragment in the MS. Melemeleones A and B (320 and 321) possess a 4,9-friedodrimane sesquiterpene array linked to a quinone bearing a taurine and they are structurally related to avarol. Compounds 320 and 321 were isolated from the sponge Dysidea sp. and identified by analysis of their spectroscopic data. Compound 321 showed a moderate inhibitory activity against the pp60^"^'^^ protein Tyrosine Kinase with an IC50 of--28 |aM [236].

xvs^SOjH x^/SOjH

320, melemeleone A

321, melemeleone B

870

Several bromopyrrole alkaloids of the oroidin family, containing a taurine residue, have been isolated from the same sponges that biosynthesize this type of alkaloid. Mauritamide A (322), the first member of the oroidin alkaloid class having a taurine moiety, was isolated from Agelas mauritiana and characterized by spectroscopic methods [237]. Tauroacidins A and B (323 and 324), the taurine derivatives of the antihistaminic dispacamides C and D [238], were isolated as racemic compounds at C-9 {9SI9R = 6:4 and 9SI9R = 1 : 1 , respectively) from Hymeniacidon sp. Their structures, including the absolute configuration at C-9, were elucidated on the basis of spectral data and by chemical means. Compounds 323 and 324 exhibited inhibitory activity against EGF receptor kinase and c-erbB-l kinase (IC50 of 20 |ig/ml, each) [239]. Compound 325 is the taurine derivative of clathridine (a very stable zinc complex obtained from Clathrina clathrus) [240] and was isolated from the sponge Leucetta microrhaphis. Compound 325 is (9£)clathridine 9-A^-(2-sulfoethyl)imine and its structure was deduced by spectroscopic and chemical analysis, and confirmed by single crystal Xray analysis. The taurine residue appears to sterically inhibit the formation of the zinc complex [241].

r\

Br

N. Br" N Me O

V^

NN"^NH

/ MeOaS

HO HN

NH

O 323, tauroacidin A R = H

322, mauritamide A

324, tauroacidin B R= Br Me

{tXi>V.

"t.N

325

HO3S

•Me

S(

871

Several fatty acid derivatives bearing a taurine residue have been isolated from diverse sources. Tauropinnaic acid (326) is the taurine derivative of the mono-fatty acid containing a 6-azaspiro[4.5]decane unit and, along with pinnaic acid itself, was isolated from the Okinawan bivalve Pinna muricata and characterized by extensive 2D-NMR spectroscopic analysis. Pinnaic acid and tauropinnaic acid (326) inhibited CPLA2 activity in vitro with IC50 values of 0.2 and 0.09 mM, respectively [242]. Taurospongin A (327) is an acetylene-containing marine metabolite consisting of a taurine and two fatty acid residues and was isolated from the Okinawan sponge Hippospongia sp. [243]. Its structure was deduced by spectral data and chemical means while its absolute configuration was established by applying the Mosher methodology to a degradation product. Enantioselective total synthesis of taurospongin A confirmed the proposed structure [244]. Compound 327 showed weak inhibitory activity against cerbB-2 kinase but exhibited potent inhibitory activity against DNA polymerase p and HIV reverse transcriptase, with IC50 values of 7.0 and 6.5 |iM (Ki values of 1.7 and 1.3 jiM), respectively [243]. This inhibitory activity has been frequently associated with the sulfonic acid function [235].

CI OH 326, tauropinnaic acid HO3S C15H31

327, taurospongin A

872

The taurine residue can also be found as an amide derivative of the 26carboxylic acid function in the 3p,5a,6p,15a-polyhydroxylated steroids 328 and 329, which were obtained from the starfish Myxoderma platyacanthum [245]. The structures of both compounds were determined from spectral data and chemical correlations. The bile of the sunfish Mola mola has been shown to contain a new bile acid conjugated with taurine (330) together with sodium taurocholate. Compound 330 was identified as sodium 2-[3a,7a, 11 a-trihydroxy-24-oxo-5p-cholan-24-yl]amino]ethanesulfonate on the basis of its physicochemical data and chemical transformations [246]. SOsNa

.^\^S03Na

328, R = H 329,R = Me, A'22

Closely related to these compounds are the carolisterols A~C (331333), which were isolated from deep-water starfish Styracaster caroli [247]. Carolisterols A-C are characterized by a polyhydroxycholanic acid moiety in which the 24-carboxylic acid function is found as an amide derivative of i)-cysteinolic acid. D-cysteinolic acid has been found in fish, alga, and other marine organisms [248].

SOjNa

331, carolisterol A Ri = R3 = OH, R2 = H 332, carolisterol B Rj = R2 = O, R3= OH 333, carolisterol C Ri = R3 = H, R2 = OH

873

Several types of microbial lipids containing a taurine residue have been described in the literarure. One example to illustrate this type of compound is the glycolipid l,2-diacyl-3a-D-glucuronopyranosyl-5«glycerol taurineamide (334), obtained from the seawater bacterium Hyphomonas jannaschiana [249]. The structure of this glycolipid was determined using a combination of chromatographic, spectroscopic, enzymatic- and chemical-degradation methods. Mycosporine-like amino acids (MAAs) are a family of compounds characterized by a cyclohexenone or cyclohexenimine chromophore conjugated with the nitrogen substituent of the amino acid. These systems have been found in taxonomically varied symbiotic and non-symbiotic marine invertebrates. Several studies have demonstrated the protective function of these compounds against the damaging effects of UV radiation, a mechanism that involves filtering harmful wavelengths. An MAA bearing a taurine group, designated mycosporine-taurine (335), has been isolated as the major MAA component of the sea anemone Anthopleura elegantissima. Taurine constitutes more than 90% of the amino acid pool in this organism and, by incorporating taurine into a UV-absorbing compound, the anemone may exploit the ready availability of the most concentrated component of its free amino acid pool [250]. The presence of a taurinelike moiety has also been found in the portion linked to the pyridinium nitrogen in pyridinebetaine B (336), obtained from the Caribbean sponge Agelas dispar [251 ]. o

^^-v^soaH O

H03S^ ^^ HO3S' ^

o={

.Ac

•O"^*'*^'^^ OH -^ o==(^ O ^ ^ A ^ O H HO3S

334

JLÔMe HO'H^^^'^^-^N ^^

^SOjH

o

335, mycosporine-taurine

'—^^^3 336, pyridinebetaine B

874

Finally, the sulfonic acid group can also be found into an isoethionic acid portion in didemnaketal C (337), isolated from the Palauan tunicate Didemnum sp. [252]. The authors suggested that the previously reported metabolites isolated from this source are actually artifacts of 337 resulting from prolonged storage in methanol at 4 ^C. o \ô

\ o'^^

MeOOC

NaOaS^^^^

337, didemnaketal

THIAZOLIDINONE-CONTAINING METABOLITES The latrunculins (abbreviated as Lat) form a well-defined group of marine natural products characterized by the presence of a 2-thiazolidinone unit. After the isolation of Lats A (338) and B (339) in 1980 from the Red Sea sponge Latrunculia magnifica [253, 254], additional members of this group were discovered later on. Lats C (340) and D (341) [255], 6, 7epoxy-Lat A (342), and Lat M (343) [256] were obtained from subsequent studies of the same sponge, while Lat S (344) was isolated from the Okinawan sponge Fasciospongia rimosa [257]. The total synthesis of (4-)Lat A [258] and (+)-Lat B [259] confirmed the proposed structures for these compounds. Furthermore, a single crystal of Lat A (338) suitable for X-ray analysis confirmed its absolute configuration [260]. Compounds 338 and 339 have been shown to disrupt microfilament organization and exert profound effects on the morphology of nonmuscle cells without affecting the organization of the microtubular system [256]. Compound 338 was found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 complex with G-actin. This phenomenon affected different components of the actin-based cytoskeleton [256]. Comparison to cytochalasin showed 338 to be an order

875

of magnitude more potent and represented the only alternative to cytochalasins in pharmacological studies of both actin polymerization in vitro and actin organization and function in living cells [261]. Compound 338 was also found to inhibit macrophage phagocjôsis without interfering with cell viability, strengthening the case for participation of microfilaments in the mechanism of phagocytosis [262]. All these results illustrate the important contribution of this compound to the study of microfilament- microfilament-mediated process. Furthermore, 338 showed excellent in vitro anthelminthic activity at 50 jag/ml against A^. brasiliensis [263] and cytotoxic activity towards HEP-2 and MA-104 cells at 0.072 and 0.23 |ag/ml [264].

338, Lat A

O 339, Lat B

340, Lat C

'-

342,6,7-epoxy Lat A

-

OMe

343, Lat M

876

THIAZOLINE- AND THIAZOLE-CONTAINING METABOLITES Marine bacteria are known to produce thiazoline-containing metabolites: the antimitotic curacins and the metal chelators anguibactin and agrochelin. Thiazoline-containing Metabolites Curacins Curacins (345-348) are a small family of bioactive compounds with a unique thiazoline-containing lipid bearing a cyclopropane unit. From the cyanobacterium Lingbya majuscula, Gerwick reported the isolation of curacin A (345) in 1994 [265], curacins B (346) and C (347) one year later [266] and, finally, curacin D (348) in 1998 [267]. The structures of these compounds were determined by detailed spectroscopic analysis. The previous absolute configuration proposed for 345 by chemical degradation [268] was confirmed by several total syntheses [269, 270]. The absolute configuration of 346 was deduced by its thermally induced interconversion with 345 [266]. Compound 345 was found to be cytotoxic against several tumor cells and inhibited tubulin assembly by binding with high affinity to the colchicine site, which is one of the two distinct drug-binding locations on tubulin [271]. This result is intriguing because curacin A did not show any structural homology to known natural and synthetic colchicine-site Hgands. A later report by Hamel et al indicated that 345 affected the morphology of the microtubules that managed to form in its presence [272]. Studies on structure-activity relationships showed that the cyclopropane-thiazoline moiety appears to be necessary, but not sufficient on its own, for repressing tubulin polymerization [273]. A 95:5 mixture of 346:347 displayed a similar cytotoxic activity to 345, indicating that the geometrical isomerization of the C-7 and C-9 olefins in curacin A has little effect on their biological activities [266]. Compound 348 was comparable to 345 as a potent inhibitor of colchicine binding but it was 7 times less active in its ability to inhibit tubulin polymerization [267].

877

R

345, curacin A

OMe OMe

346, curacin B

R= X

OMe

.

.H

347. curacin C

348, curacin D OMe

Metal chelators Anguibactin (349) is a siderophore (microbial iron transport compound) that has been isolated along with 350 from the iron-deficient cultures of a fish pathogenic bacterium, Vibrio anguillarum 775 (pJMl) [274]. The structure determination of 349 was based on extensive spectroscopic data, chemical degradations, and single crystal X-ray diffraction studies of its anhydro thiazole derivative. For this reason it was not possible to determine the stereochemistry of the side chain on the thiazoline ring. Compound 349 was shown to be a very important factor for virulence, as demonstrated by its ability to cross-feed a siderophore-deficient receptorproficient mutant of V, anguillarum, which allows the establishment of this organism in the host vertebrate. Moreover, the anguibactin receptor system has been shown to be highly specific for 349 and inert toward a range of bacterial, fimgal, and synthetic iron chelators [274].

HO

OH

O OMe

349, anguibactin

350

878

Another thiazoline compound, agrochelin (351) has been isolated from the fermentation broth of a marine unicellular bacterium belonging to genus Agrobacterium. Compound 351 showed cytotoxic activity against mouse (P-388) and human (A-549, HT-29, MEL-28) tumor cell lines from 0.05 to 0.2 |ag/ml, and chelating properties to Zn^^ JQ^S [275].

OH •

HH^'^M^^'^*^ N-

351, agrochelin

Thiazole/Thiazoline-containing Cyclic Peptides The thiazoline and thiazole rings are present in many cyclic peptides isolated from marine organisms. Most of these types of compound have been isolated from tunicates belonging to the Lissoclinum and Didemnum genus, from sponges of the genus Theonella, and from the sea hare mollusc Dolabella auricularia. The isolation of closely related compounds from cyanobacteria pointed out the symbiont origin of these metabolites. Lissoclinum Cyclopeptides Tunicate Lissoclinum species, mainly L. patella^ produces a prodigious range of novel and unusual cyclic peptides that show considerable promise as potential antineoplastic agents. They are distinguished from other natural cyclic peptides by the presence of thiazole, thiazoline, and oxazoline rings in the macrocyclic skeleton. Thirty members of this series have been isolated so far and they belong to four general families: the octapeptide patellamide (352-366), the heptapeptide lissoclinamide (367377), and the hexapeptide bistratamide (378-381). These compounds are listed in Tables 4-6.

879

Table 4. Thiazole-containing Octapeptides Isolated from the Tunicate Lissoclinium patella.

^ s X ^ N ^ J ^ ^ Thr2 R3

1 No.

O

^

Ref. 1

Name

X

Y

'352" Pateliamide A

H

Me

D-Val

L-Ile

D-Val

L-Ile

[277]

Ri

R2

R3

R4

353

Patellamide B

Me

Me

D-Ala

L-Leu

D-Phe

L-Ile

[278]

354

Pateliamide C

Me

Me

D-Ala

L-Val

D-Phe

L-Ile

[278]

356

Patellamide E

Me

Me

D-Val

L-Val

D-Phe

L-Ile

[280]

357

Patellamide F

Me

H

D-Val

L-Val

D-Phe

L-Val

[281]

358

Patellamide G ( l )

Me

Me

D-Ala

L-Ile

D-Phe

L-Leu

[282]

359

Ascidiacyclamide (3)

Me

Me

D-Val

L-Ile

D-Val

L-Ile

[283, 284]

360

Me

Me

D-Ala

L-Leu

D-Phe

L-Ile

[285] 1

361

Prepatellamide B formate (2) Ulithiacyclamide

Me

Me

D-Leu

L-1/2 Cys

D-Leu

L.l/2Cys

[286]

362

Ulithiacyclamide B

Me

Me

D-Phe

L-l/2Cys

D-Leu

L-1/2 Cys

[287]

363

Ulithiacyclamide E (1,2) Ulithiacyclamide F (1) Ulithiacyclamide G (1) Preulithiacyclamide (1,2)

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-l/2 Cys

[282]

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-1/2 Cys

[282]

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-1/2 Cys

[282]

Me

Me

L-Leu

L-1/2 Cys

L-Leu

L-1/2 Cys

[288]

364 365 366

(1) Thfi has not cyclized to an oxazoline; (2) Thr2 has not cyclized to an oxazoline; (3) Ascidiacyclamide has been isolated from an unidentified tunicate species

880

Table 5. Thiazole/Thiazoline-containing Heptapeptides Isolated from the Tunicate Lissoclinium patella Q

Thri

Ri

'^^^\^

No!

Name

X

Y

R2

Ri

ReE

367

Lissoclinamide 1

thiazole

thiazole

L-Val

D-Ile

[289]

368

Lissoclinamide 2

thiazoline

thiazole

D-Ile

D-Ala

[289]

369

Lissoclinamide 3

thiazoline

thiazole

D-Ile

L-Ala

[289]

370

Lissoclinamide 4

thiazoline

thiazole

L-Val

D-Phe

[290]

371

Lissoclinamide 5

thiazole

thiazole

L-Val

D-Phe

[291]

372

Lissoclinamide 6

thiazoline

thiazole

D-Val

D-Phe

[279]

373

Lissoclinamide 7

thiazoline

thiazoline

Val

D-Phe

[292,293]

374

Lissoclinamide 8

thiazole

thiazoline

Val

Phe

[292]

375

Ulicyclamide

thiazole

thiazole

L-Ile

D-Ala

[294]

376

Prelissoclinamide 2(1)

thiazoline

thiazole

D-Ile

D-Ala

[285]

377

Preulicyclamide(l)

thiazole

Thiazole

L-Ile

D-Ala

[285]

(l)Thri iias not cyclized to anI oxazoline

881

Table 6. Thiazole/Thiazoline Bistratamide Hexapeptides Isolated from the Tunicate Lissoclinium bistratum O

[TJo!

Name

Ri

Ri

383

Bistratamide A

Ala

Phe

384

Bistratamide B

Ala

Phe

385

Bistratamide C

L-Ala

L-Val

R,

A ring oxazoline R3 = Me oxazoline R3 = Me oxazol

Cring

thiazoline X = S

thiazoline

'[295]i

thiazoline X = S

thiazole

[295]

thiazole X = S

thiazole

oxazol X = 0

thiazole

[296, [297] [296]

R3 = H

386

Bistratamide D

L-Val

L-Val

oxazoline R3 = Me

Ref. 1

B ring

The identification and absolute configurations of the amino acids, except for the thiazole residue, were established in most cases by GC retention time correlation of the methyl ester trifluoroacetyl (ME-TFA) derivatives on a chiral column. The problem of racemization of thiazole amino acids during acid hydrolysis can be solved by treatment of the peptide with singlet oxygen prior hydrolysis and, by doing so, the stereochemistry of the side chain can be preserved. Total synthesis has played a dominant role in establishing the structures of these compounds, thus allowing the revision of some previously proposed ones. Most Lissoclinum cyclopeptides displayed moderate to high levels of cytotoxicity. Ulithiacyclamide (361) is the most potent cytotoxic molecule among the related peptides from this tunicate and is followed by lissoclinamide 7 (373). Structure-activity relationship studies have revealed that the oxazoline ring is vital for the cytotoxic activity of these compounds and that the bridging disulfide unit (or the corresponding reduced dithiol moiety) greatly enhances cytotoxicity. One possible determinant of the cytotoxic activity could be the combined effects of the molecular conformation (enforced by the disulfide link in ulithiacyclamide) and the chemical reactivity of the side chains

882

(nonbonding interactions of the disulfide group). For this reason, molecular conformation studies of some Lissoclinum cyclic peptides were performed in order to obtain additional information for the structureactivity relationship. The capacity of these cyclic peptides for metal complexation has also been studied. In relation to the true origin of these metabolites, Prochloron algal symbionts associated with L. patella have been implicated in the transfer of amino acids to their tunicate [276]. The patellins 1-6 (382-387) and trunkamide A (388) [298, 299] along with tawicyclamides A (389) and B (390) [300] represent another variation of the cyclic peptides produced by Lissoclinum species. All of these compounds lack the characteristic oxazoline ring present in most Lissoclinum cyclic peptides. Compounds 382-386 and 388 were inactive in a series of in vitro cytotoxicity assays while 387, 389, and 390 showed modest cytotoxicity, supporting the importance of the presence of the oxazoline ring for cytotoxic activity [298, 300].

382, patellin 1

383, patellin 2

384, patellin 3 Rj = L-Leu, R2 = L-Leu 385, patellin 4 Rj = Val, R2 = Leu 386, patellin 5 Rj = L-Phe, R2 = L-Val 387, patellin 6 Rj = L-Val, R2 = L-Phe

883

388, trunkamide A

389, tawicyclamide A R = L-Phe 390, tawicyclamide B R = L-Leu

Other Thiazole/Thiazoline-containing Cyclic Peptides Another tunicate, Didemnum molle, has been shown to contain a novel series of cyclic peptides. They include the hexapeptides comoramides A (391) and B (392) [301] and the heptapeptides, moUamide (393) [302], cyclodidemnamide (394) [303], and mayotamides A (395) and B (396) [301]. The structures of these compounds were elucidated from spectral data, degradation experiments and, in the case of moUamide (393), by Xray analysis. The total synthesis of cyclodidemnamide led to the revision of its structure [304]. All of them exhibited cytotoxic activity and 393 also inhibited RNA synthesis [302].

)^N

J-'-'

rVvô

391, comoramide A

o\

N-^""" ^ ^ J !

i-v-vô

392, comoramide B

N-HT^

^ w '~~r\ 393, moUamide

884

O^yy

9

1

394, cyclodidemamide

395^ mayotamide R = H 396, mayotamide R = Me

Sponges of the genus Theonella are another source of thiazolecontaining cyclic peptides, which to date include: keramamides F, G, H, J, and K (397-401) [305, 306, 307] from a specimen collected on Okinawa, and oriamide 402 [308] from another specimen collected in Sodwana Bay. Ise O

i,ej O S ^

Dpr Ala O I

I

Trp 397, keramamide F Rj = R2 = R3 = H (135. A "'^ ) 398, keramamide G Rj = R2 = R3 = H (13/?, A "'^) 399, keramamide H Ri = OH, R2 = Br, R3 = H (135) 400, keramamide J Ri = R2 = R3 = H (135) 401, keramamide K Rj = R2 = H, R3 = Me (135)

885

The keramamides have unique structural features containing some unusual amino acids such as isoserine, A-Triptophan, and (Omethylseryl)thiazole. Their structures, including in most cases the absolute stereochemistry, were elucidated by spectroscopic methods and degradation experiments similar to those used for the patellamides or lissoclinamides. A recent total synthesis of the simplest member of this series, keramamide J (400), indicated that its structure should be revised [309]. Compounds 397 and 401 exhibited cytotoxicity against L1210 and KB [305, 307]. Ircinia dendroides is another sponge that has been shown to contain a thiazole-containing cyclic peptide, in this case waiakeamide (403) [310].

OH

OH O

O ,J

O Q-J*kÔ

402, oriamide

403, waiakeamide

Thiazole-containing Dolastatins The sea hare Dolabella auricularia has been the source of the powerful cytotastic and antineoplastic constituents designated as dolastatins [311]. Some of these compounds are thiazole-containing cyclic peptides. They were found in very small quantities in the animal (ca. 1 mg each from 100 Kg), making the isolation and structural elucidation of these peptides exceptionally challenging. A review on the dolastatins, written by G. R. Pettit in 1997, covers the reported literature regarding their isolation, characterization, biological activity, and synthesis [312]. Pettit and coworkers reported the thiazole-containing dolastatins: dolastatin 3 (404) [313], 10 (2) [314], and 18 (407) [315]. The most important dolastatin and the most potent antineoplastic and tubulin-inhibitory substance known to date is the unique linear pentapeptide dolastatin 10 (2).

886 y

.

/—CONH2 / ^^^^"2

R

•-^ V Oy-^^^J^/^

\

I

2, dolastatin 10 R = H 405, symplostatin 1 R = Me

o

404, dolastatin 3

O^N

O

406, symplostatin 2

o X) 407, dolastatin 18

Compound 2 inhibited murine and human bone marrow cell colony fomiation with and ID50 of 0.1-1 pg/ml, with complete inhibition occurring at 10-100 pg/ml. It was found to be more potent than vinblastine or taxol, with an ICsoof 0.23 nM against human ovarian cancer and colon cancer cell lines. Furthermore, dolastatin 10 was shown to be powerfully effective at binding to tubulin, inhibiting polymerization and it also non-competitively inhibits the binding of vinca alkaloids to tubulin,

887

and most resembles phomopsin-A in its activity [1]. Dolastatin 10 has been reported to be in advanced preclinical development as an anticancer agent [316]. Japanese specimens of Dolabella auricularia afforded additional thiazole-containing metabolites: the cyclic hexapeptides dolastatin E (408) [317] and dolastatin I (409) [318], and the linear bisthiazole dolabellin (410) [319]. These compounds all exhibited moderate cytotoxicity activity. More recently, the isolation of symplostatin 1 (405), a dolastatin 10 analogue [320], and symplostatin 2 (406) [321], a dolastatin 13 analogue, from the marine cyanobacterium Symploca hydnoides supported the proposal of the cyanobacterial origin of the dolastatins. Symplostatin 1 (405) exhibited a cytotoxic effect against KB cells with an IC50 value of 0.3 ng/ml as opposed to < 0.1 ng/ml for dolastatin 10 (2). Since 405 induced 80% microtubule loss at 1 ng/ml when tested on A-10 cells, its mechanism of action must be similar, if not identical, to that of 2, The isolation of 405 from a cultivable source is significant, as this potentially allows the study of its biosynthesis and the isolation of further quantities for more rigorous biological evaluation [320]. A thiazoline-containing cyclic hexapeptide, keenamide A (411), was also isolated from a mollusc, the notaspidean Pleurobranchus forskalii [322]. The structure of 411 was deduced by spectral methods and chiral amino acid analysis and it showed weak cytotoxic activity against several tumor cells.

N Ô

408, dolastatin E

409, dolastatin I

Y MeO'

y^ LV

410, dolabellin

411,keenamide A

Thiazole-containing Linear Peptides Virenamides Thiazole-containing linear peptides have also been isolated from tunicates. Virenamides A-E (412-416) were obtained from the didemnid tunicate Diplosoma virens, which contains symbiotic prokaryotic algae in its cloacal cavity. The structures of virenamides A~C were determined by HPLC analysis using Marfey's procedure [323] while the absolute stereochemistry of virenamide E (416) was proven by its synthesis from virenamide A (412) [324]. Compounds 412-416 showed modest cytotoxicity toward a panel of cultured cells and 412 also exhibited topoisomerase II inhibitory activity.

412, virenamide A R = CH2CH=CMe2 414, virenamide B R = *Pr 413, virenamide D R = H

415, virenamide C R = CH2Ph

889

Thiazole-containing Polychlorinated Peptides The sponge Dysidea herbacea and cyanobacteria Oscillatoria spongeliae (a symbiont of that sponge) and Lyngbya majuscula have been shown to contain a series of linear polychlorinated peptides bearing a thiazole residue. They can be divided into three groups: the dysidenin, the isodysidenin, and the dysideathiazole series. Most dysidenin (e.g. 419) and isodysidenin (e.g. 417) metabolites were reported from D, herbacea before 1985 [7], and only one new member of the isodysidenin family has been published since that date and this is 9,ll-didechloro-13demethylisodysidenin (418), which was isolated from the same source [325]. Metabolites of the dysidenin and isodysidenin series can be distinguished by the chemical shift values of the H-6 and H-7 protons: the isodysidenin 5R series is characterized by chemical shift values close to 5 2.9 and 1.5 for H-6, and 2.7 for the H-7 signal [325]. Furthermore, it was proposed that the stereochemistry at H-5 strongly influences the optical rotation of the metabolite; thus compounds with positive [OJD values are ascribed to the isodysidenin series (e.g. 5R) whereas the negative {a\xy values are ascribed to the dysidenin series (e.g. 5S). All known natural dysidenins and isodysidenins have 25 and 75 configurations [326].

417, 13-demethylisodysidenin X = CI

419, dysidenin

418,9,11-didechloro-13-demethylisodysidenin X = H

The dysideathiazoles (420-424) were also reported from the same sponge and their structures were determined by spectroscopic methods and X-ray analyses [326]. More recently, new variations of this type of unique marine metabolite were obtained. Herbamide A (425) [327] was isolated from a collection of A herbacea, presumed to be rich in cyanobacteria because of the rich presence of chrorophyll, and barbamide (426) [328] was obtained from the cyanobacterium Lyngbya majuscula. Faulkner and

890

Unson [329] have demonstrated by flow cytometry that the polychlorinated metabolite 13-demethylisodysidenin (417) is localized in the cells of the filamentous cyanobacterium Oscillatoria spongeliae, which is the major prokaryotic symbiont of the sponge Dysidea herbacea. This result provided additional evidence regarding the symbiont origin of these compounds. Several reports on the biosynthesis of barbamide have recently been published. Feeding experiments with differently ^^C-labeled L-leucines have found the origin of the trichloromethyl group [330] while the use of [2-^'^C/^N] glycine demonstrated that cysteine is the origin of the thiazole ring in barbamide [331]. R K^ O N^S

"

420, dysideathiazole Rj = H, X = Y = CI 421, A^methyldysideathiazole Ri = Me, X = Y = CI 422,10-dechloro-//-methyldysideathiazole Rj = Me, X = CI. Y = H 423, 10-dechlorodysideathiazole Rj = H, X = CI. Y = H 424,9,10-didechloro-iV-metyldysideathiazole Ri = Me, X = Y = H

cai XJ

Me

425, herbamide A

OMe CCI3

426, barbamide

Dysidenin (419) has been shown to have inhibition activity of the iodide transfer in thyroid cells through a different mechanism than ouabain [332]. Dysideathiazoles (42(M24) were strongly deterrent in fish-feeding experiments [326] and barbamide (426) has shown moUuscicidal activity [328].

891

More recently, another cyanobacterium belonging to Lyngbya genus, L bouillonii, has been shown to contain a new tetrapeptide, lyngbyapeptin A (427), which contains the rare 3-methoxy-2-butenoyl moiety and a thiazole ring [333]. OMe

OMe

427, lyngbyapeptin A

Thiazole-containing Macrolides A series of macrolides bearing a thiazole moiety have been isolated from tunicates and sponges. The tunicate Lissoclinum patella has afforded the patellazoles A-C (428-430), which were found to be potent cytotoxins in the NCI human cell line protocol with mean ICso values of lO'^-lO"^ |ig/ml as well as exhibiting antifungal activity [334, 335]. Furthermore, patellazole B (429) exhibited very potent antiviral activity against Herpes symplex viruses [334].

428, patellazole A Rj = R2 = H 429, patellazole B R^ = H, R2 = OH 430, patellazole C Rj = R2 = OH

892

The theonezolides A-C (431-433) were isolated from the sponge Theonella sp. They are 37-membered macrocyclic compounds consisting of two principal fatty acid chains with various functionalities such as a sulfate ester, an oxazole, and a thiazole [336, 337]. Compounds 431-433 displayed cytotoxic activity against L1210 and KB cells and were found to have a unique bioactivity in terms of induction of rabbit platelet shape change and aggregation [338].

431,theonezolide A n = 3 432, theonezolide B n = 1 433, theoiiezolide C n = 5

A thiazole-containing macrolide with a rare dilactone functionality, pateamine (434), was isolated from a New Zealand sponge Mycale sp. [339]. Its relative and absolute stereochemistry was confirmed by total synthesis [340]. Pateamine was shown to be very potent and selective against P388 cell lines (although inactive in vivo) and displayed in vitro antifungal activity [339]. Furthermore, compound 434 displayed potent immunosuppressive properties (mixed lymphocyte reaction) with low cytotoxicity through inhibition of T cell receptor-mediated ILtranscription, indicating that its mode of action is similar to those of FK506 and CSA but distinct from that of rapamycin [340].

893

HoN'

434, pateamine

Thiazole-containingPyridoacridines Thiazole-containing pyridoacridines are a group of fused ring alkaloids having a pyrido[4,3,2-m,n]thiazolo[3,2-Z>]acridinium skeleton related to shermilamines, varamines and diplamine, which were discussed in the sulfide section [37]. The pyridoacridines have a characteristic UV absorption pattern [A.max(MeOH) 245, 307, and 361 nm], which is highly sensitive to the pH of the medium. Dercitin (435) was the first compound of this series to be discovered and it was obtained from an unidentifed deep-water sponge belonging to the genus Dercitus [341]. Its structure was tentatively deduced by a combination of long-range ^H-^^C and ^'^C-^'^C NMR correlations on the parent compound and its tetrahydro derivative but the regiochemistry of the thiazole moiety was assigned incorrectly. Subsequently, the same regiochemistry was proposed for cyclodercitin (436), a minor metabolite of the sponge Dercitus sp., and for nordercitin, dercitamide, and dercitamine (437-439), all of which were obtained from another deepwater sponge, Stelleta sp. The same regiochemistry was put forward because the proposed structures were based on long-range ^H-^^C correlation information and spectral comparison to dercitin [342]. The regiochemistry of the thiazole moiety was correctly assigned by interpretation of the HMBC experiment and the VH-C-N-C values in the kuanoniamines A-D (444, 440, 438, and 441), which represent the following compounds of this series and were isolated from an unidentified tunicate and its mollusc predator Chelynotus semperi [343]. Single crystal X-ray diffraction together with long range ^H-^ C coupling constants of stellettamine (445), obtained from a tunicate tentatively identified as a

894

species of Cystodytes, allowed th*^ reported regiochemistry of the thiazole moiety of dercitin and the other four related alkaloids to be corrected [344]. Compound 446 was also isolated in this study. The total synthesis of dercitin and other related compounds confirmed their structures definitively [345]. Additional members of this series were isolated from sponges belonging to the genus Oceanapia: sagitol (447), the first pyridoacridine alkaloid in which the aromatic system has been disrupted, was obtained from O. sagittaria [346] while the iV-deacyl derivative of the kuanoniamines, compound 442, was isolated from Oceanapia sp. [347]. Furthermore, compound 443 was obtained from the Fijian tunicate Cystodytes sp. [41]. The presence of similar polyaromatic alkaloids possessing a common tetracyclic ring system (a pyrido[4,3,2-/w,«]acridine skeleton) in unrelated phyla led to a symbiont origin being proposed for these compounds [343].

NMe2

435, dercitin

436, cyclodercitin

437, nordercitin R = NMe2 438, dercitamide (kuanoniamine C) R = NHCOEt 439, dercitamine R = NHMe 440, kuanoniamine B R = NHCOCH2CHMe2 441, kuanoniamine D R = NHCOMe 442, iV-deacylkuanoniamine R = NH2 443,R = NHCOCH RO«+OH + Me'^ ROOH + Me'^ -> ROO«+ir + Me'^ ROOH + ROOH ^ ROO'+ROO'+HÔ Termination; R*+ R» -^ nonradical products R»+ R00» -^ nonradical products R00»+ ROO* -^ nonradical products According to the theory of free radical oxidation, antioxidizing activities of chlorogenic acids are stipulated by their participation in reaction with free radicals. Chlorogenic and caffeic acids have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of tocopherol [48]. Considering

934

that trolox traps 2 peroxyl radicals per molecule, the stoichiometric number of peroxyl radicals trapped by each compound is as follows: chlorogenic acid, 3.1, caffeic acid, 2.7 [48]. The stoichiometric number of chlorogenic and caffeic acids show a good correlation with their scavenging activity on the l,l-diphenyl-2-picrylhydrazyl (DPPH) radical of our examination [38]. A free radical scavenger will come to possess an unpaired electron once it has contributed an electron to neutralize a free radical. Paradoxically, the free radical scavenger becomes a free radical. When chlorogenic acids neutralize a free radical they become phenoxyl radicals. However, products of chlorogenic and caffeic acids formed by reaction with free radicals are rapidly broken down further to products that are not able to generate any free radicals. This is the beneficial nature of antioxidants, because other antioxidants are not necessary for the reduction of one-electron oxidation products of these compounds [39]. It is currently believed that the oxidative modification of low density lipoprotein (LDL) is an important initial event in pathogenesis of atherosclerosis [50]. LDL peroxidation initiated by metmyoglobin/hydrogen peroxide is inhibited by chlorogenic and caffeic acids [51]. This is accomplished by a mechanism involving the oneelectron transfer reaction between chlorogenic acids and ferrylmyoglobin, with formation of metmyoglobin and corresponding phenoxyl radicals from chlorogenic acids [52]. Regarding the suggested role of ferrylmyoglobin as a contributing factor in the pathogenesis of ischemia reperfusion and atherosclerosis, and the suggested antioxidant activity of the chlorogenic acids in vivo, these findings provide new insights into the biochemical mechanisms underlying their antioxidant activity. In another demonstration of the ability of chlorogenic acid to reduce (in a dose-dependent manner) the level of lipid peroxidation products, carbon tetrachloride (CCI4, a widely used solvent and cleaning chemical) or CCI4 plus chlorogenic acid are administrated. When ingested, CCI4 severely damages liver tissue, and this damage is considered to be freeradical based. When chlorogenic acid is administered, however, beneficial results similar to those previously described above are obtained against CCI4 [53, 54] With respect to in vivo studies investigating the antioxidant potential of chlorogenic acid and caffeic acids, oral administration of these

935

compounds have been found to inhibit the elevation of serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase induced by feeding peroxidized oil [55]. Another study has used paraquat, a highly toxic xenobiotic. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which are increased by feeding paraquat, decline to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet [41]. It is well known that paraquat and CCI4 are metabolized by cytochrome P-450, resulting in the generation of the superoxide anion and other active oxygen species [5658]. Accordingly, it is suggested that part of the protection against CCI4 and paraquat hepatotoxicity afforded by chlorogenic acid in vivo may be due to its ability to act as a radical scavenge, analogous to various demonstrated effects in vitro. From these observations it is showed that the antioxidant activities of chlorogenic acids are preserved by inhibiting the formation of reactive oxygen species or by scavenging them. As a result, chlorogenic acids may play a beneficial role in the prevention of some oxidative diseases. Other Evidence ofAntioxidative Properties of Chlorogenic acids

Free radicals can also originate in an exogenous fashion, being formed by X-ray and y-ray ionizing radiation. In an effort to test the antioxidant properties of chlorogenic acids against potent •OH, ionizing radiation has been administered to bone marrow cells with or without chlorogenic acids. Ionizing radiation has been chosen, as it is known to cause the formation of oxygen-based radicals that can damage genetic material. Oral administration of chlorogenic acid (50, 100 and 200 mg/kg) to mice may significantly reduce the chromosomal damage induced by yradiation. The protective effect of chlorogenic acid has been observed in bone marrow cells sampled 24, 30 and 48 h after exposure to yradiation [59]. Intraperitoneal administration of caffeic and ferulic acids to mice has showed the survival effects. Skin injury induced by yradiation has been protected by intraperitoneal injection of ferulic acid [60]. Due to their powerful antioxidative potential, it has been postulated that chlorogenic acids help to preserve the overall integrity of DNA and in so doing reduce the likelihood of cancer.

936

Possible Mechanisms of the Chlorogenic acids Antioxidizing Action

Low-molecular-weight substances are well known to have the ability to strengthen the action of other antioxidant. The mechanism of this phenomenon is an antioxidant radical reduction up to the initial condition. In organisms and in their biological membranes, the natural antioxidants are always present. The antioxidizing effect of chlorogenic acids in vivo and in vitro can consist in strengthening already existing antioxidant actions. In LDL enriched with a-tocopherol, caffeic acid inhibits the initiation of oxidation longer than expected from the sum of discrete periods characteristic of caffeic acid and a-tocopherol. An effect similar to caffeic acid has been observed on a Triton-100 micellar system. These results suggest that caffeic acid may act synergistically with a-tocopherol, extending the antioxidant capacity of LDL by recycling a-tocopherol from the a-tocopherol radical [61]. In the same system, a synergistic antioxidant effect of caffeic and p-coumaric acids with ascorbic acid has been indicated [62]. The antioxidizing effect of chlorogenic acids synergistically increases in the presence of a-tocopherol or ascorbic acid [61, 52]. The other important property affecting lipid oxidation is the chelating effect of chlorogenic acids. It is important to keep in mind that the influence of biometals (Fe, Cu etc.) on lipid free radical oxidation is essential. It is well known that iron can react with hydrogen peroxide by the Fenton reaction (Equation 3). The hydroxyl radical formed in the Fenton reaction is capable of reacting with lipid and PUFA as the initiation stage. Iron can also participate in alkyl peroxide or lipid peroxide decomposition. Therefore, the nature of iron chelation in a biological system is an important aspect in disease prevention. Chlorogenic and caffeic acids have suppressed the formation of hydroxyl radical via the Fenton reaction, probably due to chelation of these acids with iron [44]. Indeed, recent studies report that chlorogenic acid shows chelating activity or reducing activity on iron required for the production of superoxide and hydroxyl radicals, resulting in the inhibition of lipid peroxidation induced Fenton reaction [45, 63].

937

The Antimutagenetic and Anticarcinogenic Activities

Chlorogenic and caffeic acids have been reported to react with reactive species of nitrogen in vitro [39, 49, 64]. In these reports, chlorogenic and caffeic acids have a good relationship between the ability to scavenge free radicals and to inhibit N-nitrosation. Since reactive intermediates of nitrogen have been reported to be important mediators of mutagenesis and carcinogenesis, these studies suggest that chlorogenic and caffeic acids may be effective not only in protecting against oxidative damage in vitro but also in preventing potentially mutagenic and carcinogenic reactions in vivo. It has been reported that chlorogenic acid is effective in reducing the mutagenic activity of the nitrosation products in Salmonella typhimurium TA 1535 [65]. Dietary administration of chlorogenic, caffeic and ferulic acids during the initiation phase of 4-nitroquinoline-l-oxideinduced rat tongue carcinogenesis clearly suppressed tumor development [66]. Chlorogenic, caffeic and ferulic acids inhibit the mutagenicity of the ultimate metabolite of benzopyrene in Salmonella typhimurium TA 100 [67]. Caffeic acid has been observed to diminish the incidence of forestomach tumors in mice treated with benzopyrene [68] and chlorogenic acid has been shown to block the formation of mutagenic compounds resulting from pyrolysis of protein [69]. Huang et al [70] have evaluated the effects of chlorogenic acids on tumor promotion in an animal study using CD-I mice. Chlorogenic, caffeic and ferulic acids inhibit the induction of ornithine decarboxylase by 12-0-tetradecanoylphorbol-13-acetate (TPA). TPA-mediated DNA synthesis has been weakly inhibited, but TPA-induced skin tumor promotion has been markedly inhibited by these compounds. Caffeic and ferulic acids inhibit superoxide anion production, when phorbol-12-mysristate-13-acetate (PMA) or mezerein interact in vitro with murine peritoneal macrophages. Thus, it seems that there is a close correlation between the ability of tumor promoters to induce the production of superoxide anion by peritoneal macrophages and their tumor-promoting activity [71]. The role of these phenols has been investigated in the promotional phase of carcinogenesis. Topical application of these compounds simultaneously with PMA or mezerein has resulted in significant protection against 7,12-dimethylbenz[a]anthracene-induced skin tumors in mice. Caffeic acid is a strong

938

inhibitor of tumor promotion with respect to tumor incidence at both stages of skin carcinogenesis. Further, caffeic and ferulic acids are stronger inhibitors of PMA- and mezerein-induced superoxide anion radical than ellagic acid in vivo and in vitro conditions [72]. Thus, it is possible that chlorogenic, caffeic and ferulic acids, may even inhibit the production of arachidonic acid metabolites in addition to superoxide anion generation by PMA, thereby reducing the tumor incidence. Immuno-regulatory Activity

Inflammation is always connected with the production of free radicals by inflammatory cells, so that, substances acting as radical scavengers have some anti-inflammatory capabilities. In this section, the activities of chlorogenic acids on cells of the immune system, namely mast cell, macrophage and neutrophil, are discussed. It is well known that a primary reaction of allergy and inflammation is the release of histamine from the tissues. Histamine release from isolated mast cells is a useful in vitro model for studying allergic and inflammatory diseases. Most of the drugs used in the treatment of allergy, asthma and inflammation are effective inhibitors of in vitro histamine release. It is well known that compound 48/80 or concanavalin A plus phosphatidylserine causes histamine release from mast cells. So, the inhibitory effects of chlorogenic acids on the histamine release from rat mast cells induced by compound 48/80, and on the histamine induced by concanavalin A plus phosphatidylserine have been determined by Kimura et al [73]. Chlorogenic and caffeic acids exhibit over 50% inhibition of the histamine secretion induced at compound 48/80 from mast cells at a concentration of 25 |uiM. 3,4-, 3,5and 4,5-di-O-caffeoylquinic acids (DCQAs) exhibit over 50% inhibition of the histamine secretion induced by compound 48/80 from mast cells at a concentration of 50 |jiM. Chlorogenic acid, caffeic acid and 3,5-di-Ocaffeoylquinic acid (3,5-DCQA) exhibit over 50% inhibition of the histamine secretion induced by concanavalin A plus phosphatidylserine from mast cells at a concentration of 10 (jiM. Antioxidants are useful for treating allergic disease [74, 75], since active oxygen species such as superoxide and hydroxyl radicals induce histamine release from mast cells [76, 77]. Therefore, allergic reactions may be prevented by scavenging active oxygen species. Ito et al [78] examined the

939

relationship between anti-allergic capabilities and the radical scavenging activities of chlorogenic acids. Chlorogenic and caffeic acids inhibit the activation of hyaluronidase, which is known as one of the enzymes involved in allergic effects [79], migration of cancer cells [80], inflammation [81, 82], and increased permeability of the vascular system. The superoxide anion radical scavenging activity of chlorogenic acids correlates linearly to the hyaluronidase-inhibitoiy activity. ^-Hexosaminidase release from rat basophilic leukemia cells induced by antigen has been inhibited by chlorogenic and caffeic acids. These compounds do not inhibit phexosaminidase activity itself Oxygenation of arachidonic acid catalyzed by either cyclooxygenase or lipoxygenases initiates the biosynthesis of eicosanoids. Eicosanoids can be synthesized by all cells of the immune system, especially by monocytes and macrophages which are the first line of defense against infection. Macrophages contain both cyclooxygenase and lipoxygenase activities and are capable of generating large amounts of prostanoids, leukotrienes and different hydroxyl fatty acids. In a recent study, the effects of caffeic acid on eicosanoid production by mouse peritoneal macrophages in vitro and in vivo have been investigated [83]. Caffeic acid inhibits the production of leukotriene B4 by isolated mouse macrophage. Caffeic acid also suppresses the synthesis of leukotriene C4 in vivo, Chlorogenic acid has enhancing effects on the spreading and mobility of murine macrophages [84]. Chlorogenic acid, caffeic acid and DCQAs above 10 jxM enhance macrophage spreading in a dosedependent manner. There are no significant differences between monocaffeoylquinic acids and DCQAs. Macrophage spreading and mobility are preliminary steps, which precede macrophage infiltration into tissues affected by injury or infection. Macrophages are the first cells recruited to fight injury and infection and present a first line of defense in most tissues. The spreading and mobility of macrophage are thought to be two important markers of macrophage activation. It is therefore important to identify those compounds, that are active in the early stimulation of macrophages. Moreover, the effects of chlorogenic acids on arachidonate metabolism in human peripheral neutrophils have been investigated [85]. The results show that the formation of leukotriene B4 induced by calcium

940

ionophore A 23187 in human peripheral neutrophil leukocytes has been inhibited by 3,4-, 3,5-, 4,5-DCQAs and caffeic acid. Chlorogenic acid had no effect. Koshihara et al [86] have found that caffeic acid is a selective inhibitor for 5-lipoxygenase and leukotrienes. Consequently, the anti-allergic capabilities of chlorogenic acids are correlated with the radical scavenging activity against the superoxide radical. Also, chlorogenic acids inhibit 5-lipoxygenase in arachidonate metabolism, so that the formation of the pro-inflammatory leukotrienes has been blocked. The recent accumulation of data clearly indicates that chlorogenic acids can exhibit potent antioxidant properties, significantly benefiting the defensive of organisms. Conclusion

Chlorogenic and caffeic acids may play a role in the body's defense against carcinogenesis and mutagenesis by their antioxidant properties. The antioxidant and antiradical influences of chlorogenic acids on lipid peroxidation are not yet clear. Research has indicated that chlorogenic acids are an antioxidant, however, their complex antioxidant properties require systematic investigation in diverse directions. To determine the influence of chlorogenic acids on various stage of lipid free radical oxidation in vivo and in vitro, it is necessary to use a highly sensitivity method, such as the chemiluminescence method. Antiviral activity It is assumed that chlorogenic acids in plants possess antiviral properties and the concentration increases after infection. Indeed, chlorogenic acid is an inhibitor of Botjytis spore germination [87]. Previously, chlorogenic acid was reported to have antiviral activity [88, 89]. In a recent investigation of phenolic compounds tested, using herpes simplex virus type 1 (HSV-1) infected Vero cells, caffeic acid has been reported to inhibit virus replication [91]. In a related study [92], chlorogenic acid significantly inhibited acyclovir-resistant HSV-1 replication without any cytotoxicity. However, flavonoids have exhibited cytotoxicity at the same concentration [92]. The human immunodeficiency virus (HIV) has been identified as the

941

cause of acquired immunodeficiency syndrome (AIDS), which is considered a lethal infectious disease. This disease is distributed around the world and is transmitted by sexual contact, by blood or blood products. Drug therapy for AIDS is currently limited to several classes of drugs. Drugs that can effectively treat or prevent AIDS are in demand. It has been reported that extracts of several medicinal plants and foods show inhibitory effects on HIV [93-95]. Also, HIV inhibitory substances have been investigated. Caffeic acid has been exhibited to inhibit HIV-induced cytopathogenicity in MT-4 cells and giant cell formation [96]. Schols et al have reported that this action seems to be due inhibition of interaction of the virus with the cellular target CD4 receptor. In another experiment, chlorogenic acid shows inhibitory activity against HIV-protease [94]. Dose-response study yields IC50 of 100 Jig/ml. Since HIV-protease plays an important role in the process of maturation and infections of the virus, it is considered to be a good target for the development of anti-HIV drugs. Today, most HIV drugs target two essential viral enzymes, reverse transcriptase and protease. Inhibiting these enzymes protects virus replication. In addition to these enzymes, another therapeutic target is HIV integrase. Integration of the cDNA copy of the HIV type-1 (HIV-1) genome is mediated by an HIV-1-encoded enzyme, integrase, and is required for productive infection of CD4+lymphocytes. This enzyme represents a novel target to which antiviral agents might be directed. Robinson et al [97] isolated 3,5-DCQA from an aqueous extract of Baccharis genistelloides and searched for effects against HIV-1 integrase or HIV-1 replication in tissue culture. Anti-HIV-1 activity has been measured as 50% protection against HIV-1-induced cytopathic effect 72 hr after addition of MT-2 cells. The anti-HIV-1 activity of 3,5-DCQA is 1 jxg/ml. Furthermore, it inhibits HIV-1 integrase in vitro and blocked HIV-1 replication in tissue culture. The toxic concentration of this compound is fully 100-fold greater than its antiviral concentration. Thus, 3,5-DCQA represents a potentially important new class of antiviral agents. In a related study [98], 1,5-, 3,4- and 4,5-DCQAs have been tested for inhibition of HIV-1 integrase in vitro and inhibition of HIV-1 replication in tissue culture. The activities of DCQAs against HIV-1 integrase are less than 1 |xM. Indeed, all of DCQAs have been found to inhibit HIV-

942

1 replication at concentrations ranging from 1 to 6 fxM in T cell lines, whereas their toxic concentrations in the same cell lines are all greater than 1.2 |jiM. Mahmood et al [93] reported that inhibition of virus infection by 3,4-DCQA was due to interaction of its compound with gpl20, preventing virus binding to the CD4 receptor. Furthermore, McDougall et al [99] measured the specificity of DCQAs against HIV-1 integrase. Clearly, DCQAs are potent and selective inhibitors of HIV-1 integrase. This result indicates that DCQAs are a potentially important class of HIV inhibitors. In an effort to develop more potent and selective inhibitors of HIV-1 integrase, analogues of DCQAs have been synthesized. The length of the side chains, the spatial arrangement of the phenolic hydroxyl groups, the size and structure of the central molecular core structure, and the requirement of one or more free carboxyl groups have been all studied. The effects of these changes have been assayed against HIV-1 integrase in the disintegration reaction as well as against HIV-1 replication and cell growth in tissue culture [100-104]. Conclusion

Chlorogenic acids are produced by plants as defense mechanisms in response to viral infection. Moreover, dietary intake of these substances may have an antiviral effect in humans. Bioability of Medicinal Plants As described above, chlorogenic acid and DCQAs are widely present in various plants. It is likely that many of the alleged effects of medicinal plants are linked to the functions of their constituents. In this context, several plants have been investigated for their biological activities and their active substances. This section briefly outlines the occurrence and role of chlorogenic acids in medicinal plants. Plants

The herb Cusciita reflexa has traditionally been used to treat blood infections. The active compounds have been isolated from the crude

943

extract and identified as flavanone and caffeoylquinic acid derivatives. Crude water extracts of Ctisciita reflexa exhibit HIV activity. The flavanone and caffeoylquinic acid derivatives isolated from Cusaita reflexa inhibit virus infection by different systems. The anti-HIV activity in crude extract of Ctiscuta reflexa may be the result of combined effects from these different modes of action [93, 105, 106]. For the purpose of finding anti-HIV agents from natural sources, various plant extracts have been screened for their inhibitory activity against HIV-protease, an enzyme essential for viral proliferation by Matsuse et al. [94]. The bark or roots of Swietenia mahagoni are used for treatment of gonorrhea, to halt diarrhea and as a febrifuge in America. The methanol extract of the bark of Swietenia mahagoni has shov^n inhibitory activity against HIV-protease. The butanol fraction of the methanol extract of the bark of Swietenia mahagoni affords chlorogenic acid methyl ester, which inhibits HIV-protease activity. Catechin and gallocatechin have also been isolated from the same plant extract, however, there were no inhibitory effects shown. Chlorogenic acid and its methyl ester inhibit HIV-protease in a concentration dependent manner, giving IC50 of 100 |ig/ml and 40 jxg/ml, respectively. Moreover, the same extract has inhibited the replication of HIV-1 in infected MT-4 cells [107] and moderately inhibited avian myeloblastosis virus-reverse transcriptase [108]. These investigations suggest that the anti-HIV effect of the methanol extract of Swietenia mahagoni seems to be due to inhibition of HIV-protease by chlorogenic acids. An infusion ofPersea americana leaves (Lauraceae) strongly inhibits HSV-1, Aujeszky's disease virus and senovirus type 3 in cell cultures. Chlorogenic acid (14.16 mg/ml) has been reported to be the main constituent in an infusion oi Persea americana leaves (10% w/v) [92]. Chlorogenic acid significantly inhibits HSV-1 replication. However, chlorogenic acid is less active than the infusion. The flavonoids isolated from the leaves of Persea americana show higher activity against acyclovir-resistant HSV-1. However, they are present at very low concentration in the infusion. This study suggests that the antiviral activity of the infusion may be due to a synergistic effect between chlorogenic acid and flavonoids. Extract from artichoke, Cynara scolymus L., has been used in folk medicine against liver complaints and such extracts or several constituents thereof have been claimed to exert a hepatoprotective effect

944

[109, 110]. Artichoke extracts show marked antioxidant and protective potential [HI]. This result suggests that chlorogenic acid and 1,5DCQA account for only part of the antioxidative principle of these extracts. Recently, Maruta et al [112] have found that methanol extracts of roots of burdock show a significant antioxidant activity in an in vitro lipid peroxidation assay, and have isolated five caffeoylquinic acid derivatives (CQAs) from the roots of burdock {Arctium lappa L.), an edible plant in Japan. Antioxidant activities of DCQAs and related compounds have been investigated by measuring the hydroperoxidation of methyl linolate via radical chain reaction. This study indicates that in this particular system caffeic acid and CQAs are more effective than atocopherol. These results approximately agree with our findings [38]. Additionally, CQAs as the principle antioxidative substance in burdock root have been characterized. Moreover, DCQAs (1,5-, 3,4- and 4,5-DCQAs), with antioxidative activity, have been isolated from the leaves of garland {Chrysanthemum coronaritmi L.) [113]. The garland {Chrysanthemum coronarium L.) has been regarded as a health food in East Asia because the edible portions, such as leaf and stem, contain abundant jj-carotene, iron potassium, calcium, and dietary fiber. In addition to these common nutrients, some compounds responsible for the chemoprevention of cancers and other diseases are thought to be contained in garland. The antioxidative activity of DCQAs has been assayed by the decay curves of p-carotene. The antioxidative ability of 1 (xg/ml these compounds are nearly equal to that of 0.1 |ig/ml 3-/^r/-butyl-4-hydroxyanisole (BHA). Both garland {Chrysanthemum coronarium L.) and burdock {Arctium lappa L.) belong to the same family oÂsteraceae. These facts suggest that various DCQA derivatives contributing to the plant protection systems are contained in the family Asteraceae. Chlorogenic acids have been isolated from other plants (flower and leaf) in the family Asteraceae [114]. Okuda et al. [11] have studied the medicinal property of Artemisia family and found that it relates to the composition of chlorogenic acid derivatives (chlorogenic acid and DCQAs) in the Artemisia species. Research on the distribution of DCQAs (3,4-, 3,5and 4,5-DCQAs) in the species oiArtemisia shows that the species used as haemostatics generally contain CQAs of composition similarly to those oi Artemisia montana P. and Artemisiaprinceps P.

945 Propolis

Propolis is a mixture of compounds obtained from beehives and has been a popular folk medicine. Dimov et al. [115] reported on the immunomodulatory function of propolis. They suggest that the water-soluble constituents of propolis contribute to macrophage activation and thus propolis exerted preventive effects against infection. Tatefuji et al [84] isolated six compounds from the water-soluble fraction of propolis and identified these substances as DCQAs which enhance macrophage spreading and mobility. Since chlorogenic components of propolis (chlorogenic acid, caffeic acid and DCQAs) appear to stimulate macrophage spreading and mobility, these properties might partly explain the immunomodulatory effects of propolis. Konig et al. [88] have reported that propolis shows preventive activity against the Herpes viruses. Also, they have described chlorogenic acids (chlorogenic acid, caffeic acid and DCQAs), which have been found in propolis, as antiviral active compounds. The water-soluble fraction of propolis shows a strong hepatoprotective activity against toxicity induced by CCI4 in rats [116]. Chlorogenic acid, caffeic acid, 3,4- and 3,5-DCQAs protect injury CCl4-induced in cultured rat hepatocytes. The active constituents have been found to be chlorogenic acid derivatives (chlorogenic acid, caffeic acid, 3,4-, and 3,5DCQAs). Probably the hepatoprotective activity of propolis may be due to the protective effect of chlorogenic acid derivatives. Elsewhere, the effects of propolis and its components on eicosanoid production during the inflammatory response have been investigated. This investigation demonstrates that both propolis and its component, chlorogenic acids, inhibit eicosanoid production which can strongly affect the immune and inflammatory response. Consequently, the effect on eicosanoid production by propolis may be due to chlorogenic acids of propolis component [83]. Conclusion

The observed pharmacological activities of medicinal plants relate to the chlorogenic acids constituents of the plant. However, medicinal plants are a complex mixture of chemically different compounds.

946

Consequently, study of pharmacological effects should focus on interaction with other components occurring in medicinal plants. Prooxidant and Mutagenic Activity Despite the abundance of biological data demonstrating antioxidant activities of chlorogenic acids, the controversy whether these compounds are potent antioxidants or pro-oxidants, remains. The pro-oxidant characteristics of chlorogenic and caffeic acids have been suggested in the several papers [44, 52, 117-121]. Chlorogenic and caffeic acids stimulate the formation of hydroxyl radicals in the Fenton reaction [117]. However, they have reported that chlorogenic and caffeic acids result in a decrease in hydroxyl radical formation in the different condition [44]. Yamanaka et al [121] have observed that chlorogenic and caffeic acids exert accelerate effects on the propagation phase of Cu^^-induced LDL oxidation at 0.5 jjiM. In contrast, chlorogenic and caffeic acids inhibit LDL oxidation in the initiation phase at the same concentration. Moreover, an elevated concentration of caffeic acid inhibits oxidation even in the propagation phase. Chlorogenic acid indicates the same tendency. In this experiment, these compounds display antioxidant and pro-oxidant activities depending on the oxidation state of LDL. This pro-oxidant activity is also observed for ascorbic acid [122, 123] and a-tocopherol [124]. Indeed, other studies have shown that chlorogenic acid is a potent cocarcinogenic agent [125-129] and an inducer of DNA damage [130, 131]. The cytotoxicity and mutagenicity of chlorogenic acids may be attributed to the generation of hydrogen peroxide, the reduction of transition metals (Cu^^ and Fe^^) and the catalyst of free radical formation [132], Stadler et al. [133] suggest that dual effects of chlorogenic acids are due to the high oxygen tension and large quantities of iron used for measurement. Conclusion

The above-described observations indicate that chlorogenic and caffeic acids switch from anti- to pro-oxidant activity, depending on their concentration, on the presence of free transition metal ions, or on their

947

redox status. CONCLUSIONS The extent of absorption of chlorogenic acids from dietary sources is not completely understood; for instance, data on the chlorogenic acids of coffee, a major dietary source, are virtually absent. Information on absorption is limited to caffeic acid and quinic acid. The absorption of caffeic acid estimated by measuring its urinary metabolites in humans was 24-30%. Caffeic acid gave rise to more than ten metabolites, then chlorogenic acid, which yielded an intermediate number, while coffee was the least effective [15]. Since caffeic acid occurs as a quinic acid ester in chlorogenic acid, this could tend to prevent a sudden increase in the concentration of caffeic acid per se in the animal body when chlorogenic acid is given. This would also be true of chlorogenic acid in coffee. The rate of absorption and total absorption of caffeic acid given in the form of chlorogenic acid could be considerably lower than that for caffeic acid itself The absorption of quinic acid in rats was estimated to be about 33% of ingested radioactive quinic acid based on the amount of radioactivity excreted into urine. The major sites of chlorogenic acids metabolism have been found to be the liver and the colonic flora. Only the liver has been investigated as a metabolic organ. Other tissues such as the intestinal wall and kidneys may play a role. The findings summarized in this review clearly show that gastrointestinal microorganisms can carry out a large number of metabolic reactions with chlorogenic, caffeic, and quinic acids in hydrolytic, reductive, oxidative, and decarboxylative pathways. Nonetheless, new routes of metabolism can be expected to be encountered in the future, especially among reactions of a pronounced degradation. Chlorogenic acids are shown to have some desirable biological activities. Most of their property relates to their function as antioxidants, evidenced by their activity to scavenge free radicals, to inhibit the formation of free radicals, and to block the oxidation reaction. However, other activities based on mechanisms other than scavenging capacity cannot be ignored. Also, further tests for the in vivo bioactivity of chlorogenic acids are to be needed. However, it is expected that chlorogenic acids may be beneficial to human health.

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ABBREVIATIONS /M-HBA /M-HPPA

m-HHA E. coli ATP ROS Eq. SOD PUFA DPPH LDL PMA TPA DCQAs DCQA HSV-1 HIV AIDS HIV-1 CQAs BHA

/w-Hydroxybenzoic acid m-Hydroxyphenylpropionic acid 7w-Hydroxyhippuric acid Escherichia coh Adenosine triphosphate Reactive oxygen species Equation Superoxide dismutase Polyunsaturated fatty acid 1,1 -Diphenyl-2-picrylhydrazyl Low density lipoprotein Phorbol-12-mysristate-l 3-acetate 12-0-Tetradecanoylphorbol-13 -acetate Di-0-caffeoylquinic acids Di-0-caffeoylquinic acid Herpes simplex virus type 1 Human immunodeficiency virus Acquired immunodeficiency syndrome Human immunodeficiency virus type-1 Caffeoylquinic acid derivatives 3-teA'/-Butyl-4-hydroxyanisole

ACKNOWLEDGEMENT We would like to express our thanks to Professor emeritus of Wakayama Medical College, Ryo Kido for critical reading of the manuscript. REFERENCES [1] [2] [3] [4]

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Matsuse, I.T.; Nakabayashi, T.; Lim, Y.A.; Hussein, G.M.E.; Miyashiro, H.; Kakiuchi, N.; Hattori, M.; Stardjo, S.; Shimotohno, K. Phytother, Res., 1997, 11, 433-436. Wigg, M.D.; Al-Jabri, A.A.; Costa, S.S.; Race, E.; Bodo, B.; Oxford, J.S. Antivir. Chem. Chemother., 1996, 7, 179-183. Schols, D.; Wutzler, R.; Klocking, R.; Helbig, B.; Declerq, E. J. AIDS, 1991, 4 677-685. Robinson, W.E.Jr.; Reinecke, M.G.; Abdel-Malek, S.; Jia, Q.; Chow, S.A. Proc. Natl. Acad. Sci. USA, 1996, 93, 6326-6331. Robinson, W.E.Jr.; Cordeiro, M.; Abdel-Malek, S.; Jia, Q.; Chow, S.A. Reinecke, M.G.; Mitchell, W.M. Mol. Pharm., 1996, 50, 846-855. McDougall, B.; King, P.J.; Wu, B.W.; Hostomsky, Z.; Reinecke, M.G; Robinson, W.E.Jr. Antimicrob. Agents Chemother., 1998, 42, 140-146. Nicklaus, M.C.; Neamati, N.; Hong, H.; Mazumder, A.; Sunder, S.; Chen, J. Milne, G.; Pommier, Y. J. Med. Chem., 1997, 40, 920-929. Artico, M.; Santo, R.D.; Costi, R.; Novellino, E.; Greco, G.; Massa, S. Tramontano, E.; Marongiu, M.E.; Montis, A.D.; CoUa, P.L. J. Med. Chem. 1998,41,3948-3960. Desideri, N.; Sestih, I.; Stein, M.L.; Tramontano, E.; Corrias, S.; Colla, P.L Antivir. Chem. Chemother., 1998, 9, 497-509. Vhetinck, A.J.; DeBruyne, T.; Apers, S.; Pieters, L.A. Planta Med, 1998, 64, 97-109. King, P.J.; Ma, G.; Miao, W.; Jia, Q.; McDougall, B.R.; Reinecke, M.G. Cornell, C ; Kuan, J.; Kim, T.R.; Robinson, W.E.Jr, J. Med Chem., 1999, 42 497-509. Mahmood, N.; Moore, P.S.; DeTommasi, N.; Desimone F.; Colman, S.; Hay A.J.; Pizza, C. Antivir. Chem. Chemother., 1993, 4,235-240. Mahmood, N.; Pizza, C ; Aquino, R.; DeTommasi, N.; Piacente, S.; Colman, S. Burke, A.; Hay, A.J. Antivir Res., 1993, 22, 189-199. Otake, T.; Mori, H.; Morimoto, M.; Ueba, N.; Sutardjo, S.; Kusumoto, I.T. Hattori, M.; Namba, T. Phytother. Res., 1995, 9,6-10. Kusumoto, I.T.; Shimada, L; Kakiuch, N.; Hattori, M.; Namba, T.; Supriyatna, S. Phytother Res., 1992, 6, 241-244. Adzet, T.; Camarasa, J.; Laguna, J.C. J. Nat. Prod, 1987, 50, 612-617. Wojcicki, J. Drug Alcohol Dependence, 1978, 3, 143-145. Gebhardt, R. Toxicol. Appl. Pharmacol, 1997, 144, 279-286. Maruta, Y.; Kawabata, J.; Niki, R. J. Agric. Food Chem., 1995, 43, 2592-2595. Chuda, Y.; Ono, H.; Kameyama, M.; Nagata, T.; Tsushida, T. J. Agric. Food Chem., 1996, 44, 2037-2039. Fontanel, D.; Galtier, C ; Viel, C ; Gueiffier, A. Z Naturforsck, 1998, 53c, 1090-1092. Dimov, v.; Ivanovska, N.; Bankova, V.; Popov, S. Vaccine, 1992, 10, 817-823. Basnet, P; Matsushige, K.; Hase, K.; Kadota, S.; Namba, T. Biol Pharm. Bull, 1996, 19, 1479-1484. Iwahashi, H.; Morishita, H.; Ishii, T.; Sugata, R.; Kido, R. J. Biochem., 1989, 105,429-434.

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[118] Gutteridge, J.M.C.; Xaio-Chang, F. FEES Lett., 1981, 123, 71-74. [119] Laughton, M.J.; Halliwell, B.; Evans, P.J.; Hoult, J.R. Biochem. Pharmacol, 1989, 38, 2859-2865. [120] Wiseman, H.; Halliwell, B. FEES Lett., 1993, 332, 159-163. [121] Yamanaka, N.; Oda, O.; Nagao, S. FEES Lett., 1997, 405, 186-190. [122] Fischer-Nielsen, A.F.; Poulsen, H.E.; Loft, S. Free Radic. Eiol. Med., 1992, 13, 121-126. [123] Kasai, H.; Nishimura, S. Environ. Health. Perspect., 1986, 67, 111-116. [124] Maiorino, M.; Zamburlini, A.; Roveri, A.; Ursini, F. FEES Lett., 1993, 330, 174176. [125] Van Duuren, B.L.; Goldschmidt, B.M. J. Natl. Cancer Inst., 1976, 56, 12371242. [126] Hecht, S.S.; Camiella, S.; Mori, H.; Hoffmann, D. J. Natl Cancer Inst., 1981, 66, 163-169. [127] Stich, H.F.; Rosin, M.P.; Wu, C.H.; Powrie, W.D. Mutat. Res., 1981, 90, 201212. [128] Stich, H.F.; Rosin, M.P.; Wu, C.H.; Powrie, W.D. Cancer Lett., 1981, 14, 251260. [129] Morimoto, K.; Wolff, S.; Koizumi, A. Mutat. Res., 1983, 119, 355-360. [130] Nakayama, T.; Kaneko, M.; Kodama, M.; Nagata, C. Nature, 1985, 314, 462464. [131] Borish, E.T.; Cosgrove, J.P.; Church, D.F.; Deutsch, W.A.; Piyor, W.A. Eiochem. Biophys. Res. Commun., 1985, 133, 780-786. [132] Inoue, S.; Ito, K.; Yamamoto, K.; Kawanishi, S. Carcinogenesis, 1992, 13, 1497-1502. [133] Stadler, R.H.; Turesky, R.T.; Mtlller, O.; Markovic, J.; Leong-Morgenthaler, VM. Mutat. Res., 1994, 308, 177-190.



955

EFFECTS OF ETHANOL EXTRACT OF CROCUS SATIVUS L. AND ITS COMPONENTS ON LEARNING BEHAVIOR AND LONG-TERM POTENTIATION H. Saitol, M. Sugiural, K. Abel, H . Tanaka^, S. Morimoto^, F. Taura^ and Y. Shoyama^* Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan ^Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University 3'1'1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Abstract: The increase of errors and the decrease in number of learned mice were significantly ameliorated by Crocus sativus ethanol extracts (CSE) in SD test. The LTP-blocking effect of ethanol was significantly improved by oral-, intravenous-, and intracerebroventricular-administration of CSE, respectively. When a single oral administration of crocin was given 10 min before the ethanol treatment the number of successful mice increased at a dose of 200 mg/kg. Crocin of 50 mg/kg ameliorated the blocking effect of ethanol on the LTP at 84% compared to the control. Qocetin gentiobiose glucose ester also antagonized the blocking effect of ethanol on the LTP dose-dependently indicating about a half of crocin. Crocetin di-glucose ester did not remove the inhibitory effect of ethanol on the LTP. It is concluded that two gentiobiose moieties are necessary for the appearance of pharmacological activity of crocin in the central nervous system.

1. INTRODUCTION Crocus sativus L. (Iridaceae) is cultivated for its red stigmatic lobes that constitute saffron. This plant blooms only once a year and the manual harvest of stigmas should be performed within a very short time. The manual cultivation methods practiced with saffron contribute greatly to its high price. Furthermore, weather conditions affect the quality of saffron. Author corresponding: Y. Shoyama Tel and Fax+92^642-6580 e-mail [email protected] ac.jp

956

Therefore, an indoor cultivation system was established in Japan nearly 80 years ago [1]. The stigmas can be collected from full blooming C. sativus in the room, Fig. (1) resulted that the concentration of crocin reached approximately 30 % in ethanol extracts of saffron [1]. This is the reason why the indoor cultivation method is advantageous for the achievement of a high quality and homogenous saffron and for saving time. Saffron finds use in medicine as well as a flavoring and a coloring agent. Saffron has three main chemical compounds as indicated in Fig. (2). TTie bright yellow coloring carotenoids; a bitter taste, picrocrocin; and a spicy aroma, safranal. The carotenoid pigments consist of crocetin di-(fi-Dglucose)-ester, crocetin-(B-D-gentiobiosyl)-(6-D-glucosyl)-ester and crocetin-di-(6-D-digentiobiosyl)-ester (crocin).

Fig. (1). Blooming of Crocus sativus L. in indoor-cultivation.

.CHO

HOOC

Picrocrocin

.coo

OOC RrOHgi

^^

R2-OH2I 0H2C OH

Ri«Glc R2»Glc

Crocin

Ri«Gic R2«H

Crocctin-(D-D-gcntiobiosc)-(D-D-glucosyl)-cstcr

Ri«H

Crocetin-di-(O-D-glucosyJ).ester

R2»H

Fig. (2). Structures of major compounds in C. sativus L.

Safranal

957

Concerning the pharmacological B ciooetin di-(p-D-g]uoow)-«tt«r activities of saffron components, D ciooetlo dl-(§-D-geiitlobi(Myl) •(p>D-gluoosyl)-e«tei the inhibitory effect for the increase • aoda of bilirubin in blood [2], and the deterioration activities or cholesterol and triglyceride levels in serum by crocin and crocetin have been reported [3]. Anti-tumor activity of saffron on mice transplanted with sarcoma-180, Ehrlich ascites carcinoma and Fig. (3). Seasonal variation of crocetin Dalton's lymphoma ascites glucose esters in C sativus L. tumours [4], inhibitory effects of saffron on chemical carcinogenesis in mice using two-stage assay system [5] and the effect of crocetin on skin papillomas and rous sarcoma [6] have been reported. Recently Escribano et al. [7] reported crocin inhibits the growth of HeLa cells and suggested apoptosis induction. More recentiy we reported the oral administration of ethanol extracts of saffron and crocin demonstrated an inhibitory effect on two-stage carcinogenesis of mouse skin papillomas [8]. From these results, crocetin and/or crocetin glucose esters in saffron are important constituents. Previously, we revealed that crocetin glucose esters increase from the period before blooming and reach maximum in the full blooming period, Fig. (3). They are sensitive for the presence of oxygen, light irradiation cause of the polyene structure, and for an indigenous 6-glucosidase [1] which hydrolyzes crocin to crocetin di-(B-D-glucose)-ester. These artificial pathway was indicated in Fig.(4). Moreover, it is evident that storage of saffron at -20 'C promotes the constant supply of saffron with a homogeneous pharmacological activity [1]. In order to control the quantity of saffron, we have ahready prepared a monoclonal antibody (MAb) against crocin, and set up a competitive ELISA using anti-crociu MAb [9].

Q-Glucosidtse

Unknown compounds Crocetin*(0*D*Kentiobiose)-(0-D>glucosyt)*ester W 0.Glucosid)ise Crocetin>di>(0-D*glucosyl)-ecter

Fig. (4). Artificial pathway of crocin

958

Development of natural products having the alleviation activity for the symptoms of leaming and memory impaimient has been expected. It is well-known that the hippocampus is very important in leaming and memory processes, and tiie long-temi potentiation (LTP) induced from its tissue is believed to be closely related to leaming and memory [10]. One of the authors, Saito investigated that the ginseng extract, ginsenoside Rbl and Rgl protected the increase of failure of ttie retrival of memory and prolonged the extinction of memory in hanging stressed mice, also antagonized the electro convulsive shock-induced inhibition of the retention of memory, and showed a tendency to facilitate the acquisition of short term memory in the step down test [11]. Recently Chang et al. [12] found that red ginseng water extract at 0.5 g/kg improved cycloheximide-induced amnesia in rats. Furthermore, they reported ginsenosede Rbl purified from ginseng at 0.1 mg/kg significantly improved cycloheximide-induced amnesia and at 1 mg/kg completely augmented. Recently several groups found the induction of LTP by natural products. Smriga et al. reported that Hoelen (Porta cocos Wolf) and ginseng {Panax ginseng C.A.Meyer) promoted the hippocampal LTP in vivo [13]. Abe et al. found the differential effects of ginsenoside Rbl and malonyl ginsenoside Rbl [14]. More recently Dunwiddie et al. [15] and Pockett et al. [16] found the LTP stimulation activity of forskolin isolated from Coleus forskohlii, and now drug in Germany, India and Japan. When compared these compounds, it is quite difficult to mle out some theory dependent upon the relation between structure and activity. As our ongoing study on leaming and memory of folk medicines we herein review the effects of ethanol extract of C. sativus (CSE) and its purified chemicals on the central nervous system in terms of leaming behaviors in mice and the LTP in the dentate gyrus of hippocampus in anesthetized rats and in the CAl region of rat hippocampal slices.

2 . MATERIALS AND METHODS 2.2. Extraction and separation of saffron components

Dried saffron (500 g) was extracted with 50% of EtOH. EtOH extracts of C. sativus (CSE) (311 g) were separated by Silica gel column chromatography using EtOAc-EtOH-H20 gradient solution (9:3:1 to 7:3:2) to separate crocetm glucose esters containing fraction (275 g) and noncrocetin glucose ester fraction (36 g). The crocetin glucose ester containing fraction was repeatedly purified by Silica gel column chromatography using EtOAc-EtOH-H20 gradient solution as same with the above, and finally purified by MCI gel column chromatography eluting with H2O-

959

MeOH gradient solution (1:0 to 0:1) to yield crocetin di-glucose ester (0.41 g), crocetin gentiobiose glucose ester (5.10 g) and crocin (5.69 g). CSE was dissolved in saline. Qocin and its analogues were dissolved in saline containing 30% dimethylsulfoxide. Their solutions were demonstrated for animals and hippocampal slices. 2.2. Learning performance test

Step through (ST) and step down (SD) tests were performed using 5week-old male ddY mice according to the method employed in our laboratory [9]. They were used in the experiments after 1 week acclimation to the environment. 2.3. Long-Term potentiation (LTP) experimental procedure

Anesthetized male Wistar rats 7-9 weeks old were used. Extracellular recording of population spike amplitude in the dentate gyrus in hippocampus were performed according to the method employed in our laboratory [10]. CSE was administered orally, ethanol was administered via three different routes, i.e. orally, intravenously or intracerebroventricularly. Crocin and its analogues were injected intracerebroventricularly. To summarize and compare several data sets of time-course curves of potentiation, the area under curve (AUC) from 5 to 60 min after tetanus was calculated. 2.4. LTP experimental procedure using rat hippocampal slice

Hippocampal slices (400-500 fim) were quickly prepared from male Wistar rats (8- to 9-weeks-old) and maintained in a chamber at 35 °C, where they were continuously perfused with artificial cerebrospinal fluid as described in our previous paper [11]. A bipolar tungsten electrode was placed in the stratum radiatum to stimulate Schaffer collateral and commissural afferents. The evoked potential was extracellularly recorded from the pyramidal cell layer of the CAl subfield with a glass capillary microelectrode. A single test stimulation (0.05 msec duration) was applied at intervals of 30 sec. Drugs were delivered by perfusion. To induce potentiation of the evoked potentials, tetanic stimulation was applied at the same intensity through the same stimulating electrode as used for the test stimulation. The magnitude of LTP was evaluated by the population spike amplitude 30 min after tetanic stimulation.

960

3. RESULTS J. 1, Effect of CSE on learning behavior [12]

There were no differences between control and CSE treated groups in either ST or SD test, suggesting that CSE had no effect on memory restoration in nomial mice. It is well-known that the oral administration of ethanol induced impairment of memory acquisition in ST and SD tests Fig. (5). The increase of errors (C) and the decrease (D) in number of leamed mice were significantly ameliorated by CSE in SD test dose-dependently, but not in ST test. The improving effects were dose-dependent.

Step-through test

Step-through test

CSE (mg/kg) + 30% EtOH 10 ml/kg

CSE (mg/kg) + 30% EtOH 10 ml/kg

Step-down test

Step-down test

300H

CSE (mg/kg) • 30% EtOH 10 ml/kg

CSE (mg/kg) •»• 30% EtOH 10 ml/kg

Fig. (5 ). Effect of CSE on memory acquisition in 30% ethanol-treated mice in ST and SD tests. D=Learning trial; attesting trial. A: The latency which indicated the time mice entered the dark compartment in ST test. B: Mice which did not enter the dark compartment within 300 s were termed successful mice. C: The number of errors was the number of times of stepping down to the floor in SD test. D: The number of mice which made no errors in SD test. *p < 0.05. **p < 0.01 vs. control group '*'p < 0.05 vs. ethanol group in Mann-Whitney's Utest. Mean + SEM. n=12.

961 3.2. Effect of CSE on LTP [13]

The induction of LTP after tetanus was significantly blocked by oral administration of ethanol in a dose-dependent manner Fig. (6). Thirty % of ethanol completely blocked the induction of LTP, Fig. (6-A).

H-H4i

30 Time (min) 3000

B c E

K

o
*e«575 Oviz and Rassulova 185 192,200,197,202,203 penegrimis Woh] 185,195,216,225 pterocephaliis Bunge 185,192,193 pycnanthus 185,193,198 qidsqualis Bunge 185 saponins 186,219,224,227

schachirudensis Bunge 185,192,193, 194 siculns 220 sieberi DC 185,92,225 sieversiamis Pall 185,192,193,194, 195,219 simcits L. 185,203 spinosiis Vahl 185,188,192,193,221, 224 synthetic 200 tagacantha 192,197 taschkendicits BungQ 185,194,198 tomentosiis Lam. 185,199,225 tragacantha 185 tribidoides 203,204 trigomtsDC 185,188,193,194,200, 203,226 unirwdus 185,192,193 verrucosus 185,194,195,220 vUlosissimtis 185 Astragalus sp 179 Astragalus saponin I 226 Astragalus s^.\)OgQmx\s 186 Astragersianin VH 193 Astrailienin A 195 Astramembranin I 193,220 Astraverrucin 1 194 Astraverrucin II 194 Astraverrucin III 194 Astraverrucin IV 195 Astraverrucin V 195 Astraverrucin VI 195 Astraversian nl 193 Astraversian nil 193,224 Astraversian nIV 193 Astraversian nV 193 Astraversian nVI 193,224 Astraversian n X 192,224,227 Astraversian nIX 194 Astraversian nXI 194,226 Astraversian nXII 195 Astraversian nXIlI 195 Astraversian n XIV 193,194,224 Astraversian ns 179 Astrojanoside A 203

977

Atherosclerosis 934 Atisane 241 ATP hydrolysase 612 ATPase 780 ATP-binding cassette (ABC) membrane transport proteins 321 Atrazine 669 Atrial natriuretic factor (ANF) 519,540,547 Aulacocarpines A & B 259 Auiacorarpinolide 259,266 Aurainvilleol 787 AurantosideA 793,763 Aurantoside B 763 Aurantoside C 793 Aurantoside F 763 Aurantosides 757,763 Aureomycin 757 Aurilol 763 Auriside 757 Aurisides A and B 762 Auristain 716 Ausantoside E 780 Austrovenus stutchburgi 864 Aiixarthron unberium 19A Auxins 367,379,652,656,657 Avarol 697 Avarone 697 2-AVD treatment 462 AvenacinA-1 304-306,314 Avenacinase 304-306,308 Avirulence gene product 393 Avnacinase cDNA 308 avr (avirulence) gQiXQ 397 avr Bs2 gene 399 avr gene products 398,399 avr gene-encoded enzyme 397 avr genes 399,400 avr proteins 401,399 avr9 peptide 400,401,402 Avrainvillea longicaulis ISl AvrPto recognition 399 Axane skeleton 848,850 Axinella 847 Axinella cannabiena 848,852,853 Axinella carteri 714

Axinella sp 705 Axinella weltneri 700 Axinellamines 778 Axinellamine B 778,779 Axinellida 847 Axinysaa tenestratus 852 Axinyssa (Trachyopsis) aplysinoides 852, 853,858 Axinyssa 694,847,848,859 (+)-Axisothiocyanate-2 853 1-Axisothiocyanate 852 3-Axisothiocyanate 848 4-Axisotiiiothiocynate 852 6-Azaspiro (4,5) decane 871 Azepine-type bromopyrrole 799 Azukisapogenol B 97 Azukisaponin 95 Azukisaponin II 95,105,104,115,116, 226,118,203 Azukisaponin V 104,115,116,118,120, 203,226 Azukisaponin VI 97,104 B-90063 842 Baccatin III 245 Baccharis gaiidichaudiane 251,269 Baccharis genistelloides 941 Baccharis pedunelata 251 Baccharis scoparia 251 Baccharis petiolata 251 Bacillus calmette-Guerin (BCG) 462,497 Baciliis cereiis 259 Bacillus %^ 155 Bacillus subtilis 220,258-260,765,778, 770,776,777,779,782,789,792,863 Bacterial toxins 388 Baekhousia citriodora 132 Baeyer-Villiger monoxygenase 156 Baicalein 593,595 Baker's Yeast 143,145 Balanus amphirib 783,784,785 Baptisiasaponin 95 Barbamide 757,785,786,889,890 Barbatol 250

978

Barbiturates 528 Bartlett method 435 Basidiomycetes 150,129 Bastadin 719,720 Batzella sp 708,760,774,790,822,852 Batzelladine A 709 Batzelladines 708 BatzellineA 822 BatzellineB 822 Batzellines 707 Batzellines-isobatzellines 822 BCA method 433 BCG/LPS induced hepatotoxicity 471 Beesia 180 Bencimidazol 2-il methyl carbamete 669 Benthiozepines 534 diltiazem 534 Benzazepines 534 Benzoacetonitriles 719 Benzodiazapines 528 Benzoic acid 655,660,928,929 Benzoic acid-2 hydroxylase 660,669 Benzopentathiepin lissoclinotoxin B 833 Benzopyranopyrrole chromphore 776 Benzotrithiane 833 Benzotrithiepin 834,835 Benzotrithiepin S-oxides 834 Benzotrithiepin-5-(methylthio) varacin 834 Benzthiazole 896 Benzylisoquinoline alkaloids papaverine 541 isoquinoline 530 Benzyltetrahydroisoquinoline 824 Benzylthiocrellidone 832 Berbamine 472 Berbamine alkaloids 476 Berberine 528 Berberis vulgaris 476 Bethanecol 612 Betulin 60 Betulinic acid 59-61,66,541 BHT 792 Bidem aiirea 614 Bidesethylchloroquine 344

Biemma sp 703 Biflavones 125,126,127,131,539 amentoflavone 539 bilobetin 539 sequoiaflavone 540 ginkgentin 540 Biflustra pregragilis 825 Bioactive oleanane glucumoides 89 Bioactive peptide 367 Bioactive sponge peptides 757 Bioactivity of phenolic compounds 668 Biocides 668 Biological response modifiers (BRMs) 431,434 Biomineralization 335 of heme 334 ofhemozoin 360 process 327 Biomphalaria glabrata 7S5 Bionucleating tempUates (BNTI and II) 334,336,337 Biopholaria glabrata 792 Biosilica 717 Biosynthesis of brassinosteroids 413 enzyme involved in 413 Biota orientalis 275 Biotransformation of triterpenoids 138,125 Bis(6-formyl-4-hydroxy-2-(2'-n-pentyloxazol-4'-yl)-4-pyridon-3-yl] 841 Bisabolane 848 Bisbenzylisoquinoline alkaloid oxycanthine 530 Bisbenzylisoquinoline alkaloids 530 berbamin 530 decricine 530 isochondroendrine 530 macoline 530 rodiasine 530 Bisbenzyloisoquinoline alkaloids tubacurarine 530 Bisdesmosyl saponin 108,120 (-)-5,10-Bisisothiocyanakalihinol G 857 a,co-Bisisothiocyanates 857 Bismuth citrate 617

979

Bisobolane skeleton 849 ^/i:-quinoline antimalarial compounds 346 5/5-quinolines 345 Bis-t\\\?LZo\Q metabolite 790 Bistratamide D 903 Blastobacter sp 841 Blatella germanica 795 Blatellastanoside 757,795 Blepharispenum zangubariciim 251 Blossom oils 165 Blue cheese flavour 129 Blumenkrantz method 435 Boophiline 706 Boophilus microplus 706 Bordetella pertussis 521,538 Boric acid 674 Borjatriol 249,250 Bomeol 128,154 Botrylus leactis 706 Botrylus schlosseri 719 Botrytis cinerea 138,305,313,314,316 Botryodiplodia malorum 143 theobromae 320 Botryospheria obtusa 318 Botrytic cinerea 136,137,142,293,307, 662 Botzella sp 790 Bovine aortic endothelial cells 598 Bovine endothelin 841 Bovine aortic endothelial cells (BAEC) 593 Bovine famesyl-proteine transferase 790 Bovine serum albumin 334,337 BrachyosideA 197 BrachyosideB 192 BrachyosideC 197 Brassica (SRK) 368,386 Brassica napus 413 i5ra55/ca5-locus receptor kinase 384 Brassin 413 Brassino steroid precursors 415 Brassinolide 415-416 Brassinosteroid 316,413,420,425,367 Brassinosteroid biosynthesis 425,414,420

Brassinosteroid hormone 413 Brassinosteroid intermediates 414,417 Brassinosteroid pathway 421 Brassinosteroid precursor biosynthesis 416 Brassinosterol pathway 426 Brevetoxin 864,730 Brevetoxin B i 864 Brevetoxin B2 864 Brewer's wort 139 Briareitm excavatum 670 Brickellia lemmonic 230 BRMs (biological response modifiers) 452-455 Brominated aminoimidazolinylindole 763 Brominated indole alkaloids 712 Brominated phenylethylamine 765 Bromoagelifterin 779,780 6 Bromo-5-hydroxyindole 792 6-(p-Hydroxypheny l)-2H-3,4-dihydro-1,1,dioxo-l,4-thiazine 862 18-Bromo-octadeca-5,7,17-triyonoic acid 802 18-Bromo-octadeca-15,17-diene-15-ynoic acid 802 Bromobenzene 460 p-Bromobenzoyl derivative 816 Bromopyrrole alkaloids 762 5-Bromopyrrole-2-carbamide 780 Bromopyrrols 711,712,779,784 Bromotyrosin derivative 766 Bromotyrosine 642 derivatives 766 Bromotyrosine alkaloids 712 Bryostatin 684,726 derivatives 727 Bryostatin 1 726 Bryozoan 825 Bt2 cAMP 489,496 Bt2 cAMP-treated carrot cells 490 BufadienoHde 535 Bufadienolide Scillaren A 535 Bufotenin 531 Bugiila neritina 726 Bulgarian rose oil 132

980

Bupleuroside IIIJV, Xlli 474 hepatoprotective activity 474 Bupleiirum falicatum 474 Buplenrum Radix 474 Bupleiirum scorzonerifolium 474,476 triterpene glycosides of 474 Butein 546 N-t-Butylamodiequine 344 C-14 reductase 304 C6 oxidation pathway 421 Ca^-ATPase 516 Ca^"^- and phospholipid dependent protein kinase (PKC) 522,547 Ca^"^- dependent protein kinase 547 Ca^'^-and Cu^"*'-CaM-dependent enzyme Ca^^-ATPAse inihibtor 536 Ca2-^-ATPase 492,493,524,536 Ca^'^-calmodulin (CAM-dependent protein kinase) 261,524 Ca^"*'-Cam-activated adenylyl cyclase 538 Ca^'^-CaM-dependent protein (CaM PK) 522,546 Ca^"^-CaM-depdendent protein kinase 522,547,544 Ca^"^-channel blockers 489 Ca^^^-channels 521 Ca2"^-dependent CAM 491,540 Ca^"^-dependent enzymes 517 Ca^'^-dependent process 487 Ca^"*"-dependent K^ channel 534 Ca^"^-induced LDL oxidation 946 Ca^^^-inhibitors 487 Ca2+-ionophoreA23189 490,487,509 Ca2~^-mediated signalling 526 Ca^^^-mediated signalling targets 542 Ca^"^-permeable ion channel 397 Ca2"^-pumping ATPase 492 Ca2+ -tansporting systems 492

Ca2+-translocating ATPase 492 activity of 492,496 Ca^+ZCalmodulin 261 Ca2+/CAM -dependent protein kinases 489 volatge-dependent 498 Cabreuvaoil 162 Cacospongia linteiformis 698 Cacospongiolide B 697 CAD 666 cADPR 527,535 Caenoshabditis elegans 785 Caesalpiniaceae 92 3-0-Caffeoyl-4-0-feruloylquinic acid 920 3-(9-Caffeoylquinic acid 920,921 4-0-Caffeoylquinic acid 920 5-0-Caffeoylquinic acid 919,920 Caffeic acid 919,920,922-927,932-934, 936-938,655,657,663,671 Caffeine 532,539 Caffeoylquinic acid 943,944 derivatives 465,662 in body tissues 924 Caissarone 713 Calarene 170 Calceolareoside A 543 Calceolsareoside B 543 Caledonin 813,814 Calendonian 697 Calendula officinalis 58 Calicheamicin y 796 Caiicheamicin yl 838 Calicheamicin Q 796 Calicheamicins 796,838 Calicoferols 705 Calicogorgia granulosa 670 Callipeltasp 775 Callipltoside A 775 Callipeltosides 757,775 Callyspongia 724 Callyspongia johnstorii 111 Callyspongia truncota 723 CallystatinA 724

981

Calmodulin (CAM) 373,487,509,517, 522,524,547,489,494,543 Ca^"^-binding protein 487 Calmodulin antagonist activity 758 Calmodulin domain-containing protein kinase (CDPK) 544 Calmodulin-dependent phosphodiesterase (CAM-PDE) 509 Calvularia virdis 704 Calyculin derivative 731 CalyculinJ 732 CaM antagonists 489,492 CaM inhibitors 491 CaM dependent kinases 543,544 CaM dependent MLCK CaM-dependent isoformofPDE (CAM-PDE) 490 CaM-dependent PKs I-IV 524 CaM-dependent protein kinases 493,527 Camellia sinensis 472,478 cAMP-dependent protein phosphorylation 494 cAMP-dependent protein kinase (PKA) 522,547 cAMP-gated Na"^ channels 517 cAMP-dependent protein kinase 488,489, 522,548 cAMP-dependent channels 495 cAMP-gated N^"^ channels 523,524,546 cAMP-inducedCa2+-influx 491 cAMP-induced influx of K"^ 497 cAMP-phosphodiesterase 539 cAMP-response element binding (CREB) 523 cAMP-response element binding protein 548 cAMP-sensitive ion channels 490,496,497 Campesterol 415,416,417 formation of 419 Camphor 125,128,154,156,547 D-(±)-Camphor 156 Campylotorpis hirtella 94 Canavalia gladiata 93 Cancer chemopreventive agents 43

Candida albicans 258,759,764,773,780, 790,822 Candida pellicula 308 Cannabis sativa 532 Cannobinoid receptors 533 Canton iensistriol 96 Caprifoliaceae 249,251,259 Carbachol 613 Carbendazein B 6772 Carbendozin 669 4-Carbohydroxypatchoulol 172 (/?)-Carvane 858 8-Carboxylinalool 140 j8-Carboline 33,782,902 /J-Carboline alkaloid eudistomidins 817 /J-Carboline derivative 10,14 /?-CarboIine marine alkaloids 819 j8-Carboline metabolites 818 j3-Cardene 944 jS-Carotene 236 /J-Carotene hydrolase 718 Carbomanoyl oxide 261 Carbon catabolite 313 Carbon tetrachloride 460 Carboxypeptidase 375 Carcinogen MNU 66 Carcinoma Ehrlich cells 836 Carcinus maenas 718 Cardenolide 535 Cardiac steroid 55,59 Caribbean coral 690 Caribbean sponge 698 Carnitine synthetase 525 Carolisterol A-C 872 Carotenoids 44,237 Cairageenan-induced edema 220 Carveol 145,147,149 cis 2iX\A trans 148,150 Carvone 145-149 Caryophyllene 125,168 (-)-Caryophyllene-4,5-oxide 168 Casbene synthesis 486 Casein kinase 545 Casein spleen 431 Casopongia 695

982

Caspase inhibitor 2-VAD 462 Caspase-3 activation 471 Caspase-3 protease 469 Cassane 241 Casteriospongia sp 713 CAT 674 Catabolite repression 313 Catabol ism of haemoglobin 332,348 Catechin 541,612 Catechin derivatives 612 (+)-Catechin 612 Catechin-based flavones 541 Catechol 613,662,929 Catechol oxidase 654 Catecholamines 516 dopamine 520 epinephrine 520 horepinephrine 520 Catechol-O-methyltransferase 923,924,927 Cathasterone 415,420 Cathansterone 23a-hydroxylation 421 Catharantheus roseus 384 Cativic acid 254 Cavemothiocyanate 858 CB2 receptors 532,533 CCl4-induced cytotoxicity 468 CCl4-induced liver injury 466,469 CCl4-induced liver injury model 471 CD 18 antigens 454 CD20 antigen 272 CD25 positive cells 454 CD4 positive cells 454 CD4-antigens 454 CD69 positive cells 454 CD71 positive cells 454 CDg positive cytotxic T lymphocytes 454 Cd-induced liver injury model 471 cDNA clone encoding tomatinase 308 cDNA coding 61 cDNA copy of the HIV type-1 (HIV-1) genome 941 CDPK 542,546 inhibitors of 544 Cefriaxon 258

Ceftazidine 258 Celenamide 757 CelenamideE 778 Cell division protein kinases (CDKs) 526 Cellobiose degradation 308 Celosia argentea 476 Celosian 476 Cenarchaeum symbiosum 118 Cenebellar granule cells 268 apoptosis 268 Centrifugal partition chromatography (CPC) 207 Cephalomannine 245 Cephalosporium aphidicola 153,155 Cephalotoside A 197 Ceratinamides A and B 620,784 Ceratocystis 129 Ceratocytis fimbriata 485 Ceratodictyor spongiosum 902 Ceratosoma brevicaudatum 815 C-erbB-2-kinase 797,799,870,871 Ceric ammonium nitrate 821 Cervical carcinoma 268 /w-Coumaric acid 923,925 cGMPPDEs 523 cGMP phosphodiesterase 521 cGMP synthase 540 cGMP-dependent protein kinase (PKG) 522,523,548,517 cGMP-gated ion channel 541 cGMP-gated Na"^ channels 517,521 a-Chaconinase 305 a-Chaconine 300,305,311 Chaetomium cochiliodes 168,170 Chalcone isomerase 668 Chalcone reductase 653 Chalcone synthase 655 malonyl-CoA 655 Chalcone-synthase 504 Chamaecyparis obtiisa 246 Chamigrane-type derivative 701 Chamigrene-type, halogenated 702 Chemiiuminescence 940 Chemokines 535

983

Chemo-perception system of plant cell 394 Chemotactic tripeptide 275 Chitin 396 Chitosan 396 Chlamys hastata 813 Chloramphenicol 757 Chlorella 429 Chlorella kessleri 430 Chlorella vulgaris 430,431,450,452,453 antitumor activity 452 Chlorella vulgaris strain CK 22 (CVs) 429-433,436,437,440,450,447 antitumor immuno-activity 432 anti-peptide ulcer effect 430 characteristics 429 glycoprotein from 429 host-mediated activity 431 immunopotentiating effects 429 Chlorinated cylindrol Bi 790 Chlorinated malyngamides K 788 2-Chloroethylphosphonic acid 509 Chlorogenic acid 663,655,657,667,919921,930,932-935,937-940,942-947 antioxidative properties of 919, 930,935 Chloroissoclimide 270 Chloroorcinols 757 Chloroperoxidase 767 Chloropyridine 792 Chloroquine 336,337,343,344,345,346, 347,348,349,352,360 Chloroquine Fe(III) PPIX 346 Chloroquine-pyrollidinyl 344 Cholera toxin 521,538 Cholesterol acyltransferase 797 Cholesterol biosynthesis 722 Cholesterol synthesis inhibition 525 Choline 475,476 Choloroquine 335 Choloroquine-resistant parasites 352 Cholynotus senperi 893 Chondocurine 472,476 Chondodendron autumnal 476 Chondria californica 842

Chondria tenuissima 249 Chondromyces sp 730 Chromaggin granules 536 Chromodris hamitoni 735 Chronic viral hepatitis 463 Chrysanthenum coronarium 944 Chrysazine 543,544 Chrysocephalum ambiguum 251 Chrysophanoic acid 543 CoumaroyI CoA Chymo trypsins 388 Cicer arietium 93 Ciguatoxins 732,733 Cimetidine 612 Cimicifuga 180 Cinachyrolide A 759 Cinchona officinalis 476 1,8-Cineole 140 Cinnamate-4-hydroxylase 653,660 Cinnamic acid 925,652,653 Cinnamomum camphora 140,156 Cinnamonin 395 Cinnamoyl CoA 655 Ciona savignyi 784 Cis and /ra«5-deethyleburnamonines 19 Cis decalin-tetrahydrofuranyl kalihinol 855 C/5,c/^-ceratospongamide 904,905 C/5-5-hydroxy-4(4'hydroxy-3'-methoxyphenyl)-4-2"-imidazoyl)-l,2,3-trithiane 837 Cw-deethylburnamonine 6 C/5-deethyldihydroebumamenine 6 Cistaceae 249,252,253 Cistanche deserticola 468,476 Cistus clusii 250 Cistus creticiis (cistaceae) 246,249,250, 252,255,256,257,266 Cistus creticiis subsp erioocephatus essential oils of 259 Cistus incanus sub sp. creticus 266,258 Cistus ladaniferus 252 Cistus laurifolius 250 Cistus palinhae 250 Cistus symphylifolius 250

984

essential oils 255 subspecies 255,257 Citral 132,133,136,137,143 Citranellae 132,140 Citranellol 132,133,136,137,140 Citrellamine 837 Citrol 136,137,140 Citronellal 128 Citronellic acid 133,136 Citronellyl acetate 136 Citrus limonum 594 Citrus sp 128,266,267 Citrus aurantium 476 Citrus peel oils 145 Cladosoporium sp 145 Cladosporium cucumerinum 116 Cladosporium coccodes 305 Cladosporiumfulvum 302,308,398,399, 400 Clarithromycin 617 Clary sage oil 252 Clathramides 151,111 Clathrina clathrus 870 Clavelina cylindrica 859,793 Clerodane 241 Cliona chilensis 11S Clonidine 530 Clostridium perfringens 925 Clostridium tyrobutyricum 147 Cnemidocarpa bicormita 165 CoA ligas 653,666 Corynantheine-type oxindole alkaloids 26 Cochliobolus lunatus 318 Codium arabium 704 Codium dworkense 725 Colchicum autumnale 473 hepatoprotective effect 472 serum ALT activity 473 Coleosol 252 Colestane 49 Coleusforskohlii 256,252,261,958 Coleiis sativus 956 Collectotrichum coccodes 307 Colon-38 tumor cells 761

Cowbretaceae 180 Combretum 180,183 C/.s-Communic acid 275 Comoramide A 883 Comoramide B 883 Complogenin 101,184,185,186,204 Comploside I 95 Comploside II 95,203,104 Comploside III 97 Comploside IV 97 Comploside V 97 Compositae 921 Compylotropis hirtella 93 Con A 227,271,272,436,463,464,455, 477 Con A agarose 436 Con A hepatotoxicity 464 Con A induced liver injury 464,465 Con A model 463 Condensation of/?-coumaroyl-CoA 655 Conifery 2 alcohol 666 Coniine 528 Coniiim macidatiim 528 Conyza steiidellii 252 Conyza trihecatactis 252 Conns 732 Copaiba oWs 168 Copaifera L. 168 Corallistersfluvodesmus 829 Corallistine 829,830 Corculigo 180 Coriloiis versicolor 452 Coronarins (A-D) 265,266 Coronary artery ligation-reperfusion 594 Coronopifolia 111 Corriander oil 140 Corticum sp 707 Cortisone 516 Corymbiu villosum 251 Corynantheidol 24 Coiynantheine-type indole alkaloids 31 Coryne cassiicola 149 Corynebacteinim sp 145 Coiynebacterium kutschei 452 Corynespora cassiicola 149,162,166

985

Corynoxine 27 Corynoxine B 27 Coscinoderma mathewsi 699 Costasiella ocellifera 1^1 4-Coumarate 653,664 p-Coumaric acid 655,657,920,936,925 w-Coumaric acid glucuronide 923,925 4-Coumaric acid 652,660 4-Coumaroyl-CoA 652 3-0-Coumaroylquinic acid 920 4-(9-Coumaroylquinic acid 920 5-O-Coumaroylquinic acid 920 Coumarine isoepoxypteryxin 476 Coumaryl glycosides vanicosideA 543 vanicoside B 543 Counter-current chromatography (CCC) 205 Coumaric acid 657 Creatine kinase 527 Crella sp 704 Crella spinulata 832 Crellastatin 704 Creulide 690 Cribicellina cribraria 819 Cribochalina sp 900 Cristaria plicata 868 CRO 46 Crocin 964 effect of 964 Crocetin 958 Crocetin di-glucose ester 955 Crocetin gentiobiose glucose ester 959, 966 Crocetin di-(jS-D-digentiobiosy)-ester (crocin) 956 Crocetin di-(j3-D-gentiobiosyl)-(j3-Dglucosyl)-ester 956,957 Crocetin di-(/J-D-glucose)-ester 956,957 Crocetin gentiobiose glucose 955 Crocetin glucose esters 957 Crocetin-di-glucose ester 959 Crocin 956,957,959,964,965,968 Crocetin glucose esters 968 Crostis sativa L. 955,956

Crotalaria albida 93 Croton 242 Croton oil 45 Croton oil-induced ear edema 556 Croton oil induced edema (CRO) 45 Croton oil induced edema inhibition 46 Croton tigUiim 242 Cryptotrienolic acid 259 Cryptochlorogenic acid 921 Cryptococcus fagisiiga 662 Cryptogein 395 Cryptomeria japonica 257,275 Cryptophycin I 789 Cryptophycins 789 Cry^ptotethya crypta 712 CSF (colony stimulating factor) 455,960, 963 Cubebane skeleton 51,848 Cucumis melo 390 Cucumisin 390 Cucurbitacin F 59 Cucurbitane 50,57,59 23,24-dihydrocururbitacin F Cunnighamia lanceolalta 251 Ciinninghamella 172 Cimmnghamella blakesleana 172,173 Cupressaceae 246,249 Cupressus sempervirens 253 CuracinA 732,733,876 Curacin B 876 CuracinC 876 Curacin D 876 Curacins 876 Curculigo orchioides 227 Curculigosaponin G 227 Curcuma longa 476 Curcuma zedoaria 172 Curcumin (turmeric yellow) 44,469,470, 541 Curcimua longa L. 469 hepatoprotective activity 469 serum transaminase activity 469 Curvularia liinata 254,261,260,263 Cuscuta reJJexa 944,945

986

CVS 443,445,447,450 antimetastatic effect 441 CVS induced antitumor immunity 454 antitumor effect 440 restortive effect on progenitor cells 448,449 tumor specificity 442 Cyanobacteria 728 Cyasterone 59 Cycas cricinalis 529 Cycleanine 472,476 Cyclic adenosine monophosphate 260, 509,598 Cyclic adenosine-5'-diphosphateribose 547 Cyclic adenosine-5'-diphosphateribose (CADPR) nicotininc acid adenine dinucleotide phosphate (NAADP) 516 Cyclic ADPR 517 Cyclic AMP phosphodiesterase 539 3*,5'-Cyclic AMP-dependent protein kinase (PKA) 512 Cyclic AMP-dependent protein phsophorylation 483 3'-5'-Cyclic AMP-sensitive cation channels 483 Cyclic GMP-phosphodiesterases 541 Cyclic nucleotide phosphodiesterase 512 Cyclic dependent kinase 4 798,799 Cyclo-(L-Pro-L-thiopro) 829 Cyclo-(L-Pro-Met) 829 Cycloacanthaside F 197 Cycloalpegenin 179,184,187,190,201 Cycloalpigenin A 184,201 Cycloalpigenin B 201 Cycloalpigenin C 201 Cycloalpioside A 201 Cycloalpioside B 201 Cycloalpioside C 201 9,19-Cycloanost-24-en-ol 190 Cycloanthogenin 187 Cycloaraloside A 194 Cycloaraloside B 195 Cycloaraloside C 195 Cycloaraloside D 195

Cycloaraloside E 194 Cycloaraloside F 195 Cycloarbicoside A 202 24/?-Cycloartan-3j3,6a,16j3-24,25-pentaol, 6-dehydroxycycIoastragenol 184 Cycloaitane 61,52 Cycloartane glycoside 221 Cycloartane saponins 183 Cycloartenol ferulate 61 Cycloasgenin C 185 Cycloastragenol 179,184,185,186,187, 190,191,192,215,217,225 Cycloastragenol-6-O-glucoside 224 Cyclocanthaside D 197 Cyclocanthogenin 184,185,197 Cyclocanthoside A 197 Cyclocanthoside G 197 Cyclocanthoside E and G 227 Cyclocarposide 193 Cyclocarposide B 193 Cyclocephalosdie I 200 Cyclocephaloside 11 193 Cyclocinamide 757 Cyclocinamides A 761,762 Cyclodercitin 893,894 Cyclodesipeptides 768,884 Cyclogalegenin 179,184,187,191,196, 218,225,227 Cyclogalegenoside A 196 Cyclogaleginoside B 196 Cyclogaleginosides 191 Cyclohexane-l,2,-diamine 345 Cycloheximide 58 Cycloheximide-induced amnesia 958 9j3,19-Cyclolanost-24-en-3/3-ol 181 Cycloorbicoside G 202 Cycloorbigenin B 185,187,202 Cyclooxgenase 597,616,790,939 Cyclooxygenase pathway 263,593 Cyclooxygenase inhibitor 593 Cyclooxygenase/Lipooxygenase 615 Cyclophosphamide 431 Cyclophosphamide treatment 447 Cyclopycnanthoside I 198 Cyclorenierins 694

987

Cyclosieversioside A 193 Cyclosieversioside B 194 Cyclosieversioside C 193 Cyclosieversioside D 194 Cyclosieversioside E 192 Cyclosieversioside F 193 Cyclosieversioside G 194 Cyclotheonamide B 715 Cyclotheonamides 715 Cycloxygenase pathway 274 Cyctodytes 894 Cyditol 920 Cylindricine A and B 793 Cylindricines I and J 859,860 Cylindricins 859,857 Cylindrines F,G and H 859,860 Cylindrol B] 790 Cylindrospermopsis raciborskii 732 Cymbastela 847 Cymbastela coralliophila 704 Cymbastela hooperi 689,814,856 Cymbastela si^ 714,768,792 /7-Cymene 145 Cynara scolymus 943 CynidinldB 613 a-Cyperone 170,171 Cysteine sulfoxide 854 Cysteine protease falcipain 331 Cysteine proteinase 375 D-Cysteinolic acid 872 Cystodytes genus 820 Cystodytess^ 820 Cystodytes violatinacus 820 Cystodytins 821 Cytochalasin 874,875 Cytochrome P-450 421,424,935 Cytochrome P-450-dependent monooxygenase 413 Cytochrome P-450 monooxygenase 318, 597 Cytochrome oxidase-inhibiting cyanide 513 Cytokine gene 470 Cytokines 463

Cytokinins 367,379 Cytolytic lymphocytes (CTL) 273 Cytolytic T cells 453 Cytolytic T lymphocytes 271 Cytomegalo virus infections 431 Cytoplasmic guanylate cyclase 382 Cytosolic PIA2 717 Cytosolic dehydrogenase activity 423 Cytosolic sphingosine kinase 264 Cytostatic T cells 453 Cytotoxic guanidine-alkaloids 706 Czapek-Dox medium 154 D-3-(3,5-Dihydrophenyl) alanine 824 Dactylospongia elegans 694 Dakaira subovoidea 831 Dalbergia hupeana 93 Dalbergia parviflora 162 Damarane 50 Damirones 707 Damnacanthal 546 Damacits carota 377,669 Dansyl-CaM fluorescence 544 Daphnane 246 Daphnoretin 545 Dasyanthogenin 199 Dalton's lymphoma ascites 957 3,4-and 3,5-DcQAs 945 1,5-DCQA 944 3,5-DCQA 942,941 l,5-3,4-and4,5-DCQAs 941 3,4,-3,5-4,5-DCQAs 940 De novo DNA synthesis 227 D^wovo generation of thymocytes 454 De «ovo-biosynthesis 374 Denovo synthesis 128,129,294,300, 329,401,659,661,662 of bioflavours 125 10-DeacetylbaccatinIll 245 10-Deacetyl cephalomannine 245 10-Deacetyltaxol 245 Debromoeudistomin K 816,817 Debromohymemialdisine 791 10-Dechlorodysideathiazole 890

988

10-Decholoro-N-methyldyrideathiazole 890 Decriamide 893 Decumbesterone A 59 Defence gene activation 396 Defense proteins 370 serine proteinase inhibitor I 370 Dehydro 7-o:-hydroxy-tomatidenole (D^-d) 318 3-Dehydro-24-e/>/-teasterone 422,423 3 -Dehydro-24-^/7/-teasterone-reductase 422 3-Dehydro-6-deoxoteasterone 415 Dehydro-6-hydroxymellein 498,499 Dehydroeburiconic acid 57 Dehydroepiandrosterone 419 5-Dehydroepisterol 415 Dehydrojuvabione 546 Dehydrometenolic acid 57 Dehydropachymic acid 57 Dehydroquinic acid 928 Dehydrosaponin 1 105 3-Dehydroshikimic acid 928 Dehydrosoyasaponin I 97,104,115,116, 118,120 3-Dehydro-teasterone 415,422,423 4'-Dehydroxy-4'-flourotebquine 344 4'-Dehydroxytebuquine 344 3-Dehyrdo-6-deoxoteasterone 415 13-Demethylisodysidenin 889,890 Demissidine 536 Demospongic acid 721 Dendrimeric multiple antigenic peptides 335 Dendrimedric peptide 336 Dendroamides 716 6-Deoxocastasterone 415 C6 oxidation 424 6-Deoxoteasterone 415 3-Deoxotyphasterol 415 3-[5-Deoxy-5(dimethylarsinoyl)-/J-Dribofuranosy loxy]-2-hydroxypropene-1 sulfonic acid 867 3-Deoxy-7-phospho-D-arabinoheptuiosonicacid 928

6-Deoxyandalusol 250 Deoxyglycyrrhetol 58 Deoxygylulose 130 Deoxyparguerol acetate 788 Dephosphoglycogenyl synthase 525 Dephosphory lation of retinoblastoma protein 270 Dercitamide 893,894 Dercitin 893,894 Dercitus 893 Dermasterias imbricate 824 A^-Desacetyllappaconitine 528 Deserpindine 8 4-Desmethyl sterols 56,57 3,4-Desmethylvariacin 834 Desmal 546 Desm odium styracifloiiim 93 2-Desoxy-D-glucose 613 Destruxin-A4 chlorohydrin 800 Det2-cDNA 419 Detoxification of heme 334,335,343 Detoxification of tomatine 313 3- Deuteroisoreserpine 8 Deuteroporphyrin IX (DPIX) 355 Dextran induced edema 46 24-Dexyoxytrogenin 97 Dezoxiben 593 D-Gal N/LPS model 468 D-GalN/LPS 459-461 liver intoxication with 462 D-Gal N/LPS-induced liver injury 460, 467,472-474 D-galN/TNF-a 460,461 liver intoxication with 462 ^1,6-D-galactopyranose 429 D-galactosamine (D-GalN) 219,473,465, 477,461,463,468,471 D-GalN/LPS model 464 D-GalN/LPS-intoxication 468 D-GalN-induced cytotoxicty 468 D-GalN-induced liver injury 463,469 j3-l,2-D-glucosidase 306 DI dopamine receptor 529 Diabrotica iindecimpimctata 792 Diacarms 688

989

Diacyglycerol (DAG) 271,483,487,488, 516,542, l,2,-Diacyl-3a-D-glucuronospyranosyl-snglycerol taurieamide 873 Diacylglycerol acyltransferase 797 Diacy Iglycerol-1 -oleoly 1-2-acetyl-mc glycerol 488 Diarylheptanoids 543 Diatriba 186 Dibromoageliferin 779,780 Dibromophakellstatin 770 Dibromosceptrin 779 Dibromo-tyrosine derivatives 719 Dibromotyrosine-derived metabolites 719 4,5-Dibrompyrrole-2-carbamide 784 DibutylcAMP 271,488,509 Dicaffeoyl tartaric acid 663,667,932 9,10-Dichloro-N-methyldysideathiazole 890 2,4-Dichlorophenol 767 2,6-Dichlorophenol 765 Dicholorolissoclimide 269,769 Dictyopteria genus 841 Dictyostelium discoideum 523 Didemnum sp 765 Didemnoline A 765 Didemnoline C 765 Didemmun voeltakowi 713 Didemnin 684,714 Didemnin B 713,757,816 Didemnoline 818,863 DidemnolinD 818,863 Didemnolines 757,816 Didemnum 878 Didemnum conchylatum 802 Didemnum rodriguesi 813,897 Didemolin A 818 Didenumsp 818,874 Didemum molle 883 Dideoxyforskolin 269 10,12-Dideuterated reserpine 10 Didemnimides B 802 Didemnaketal 874 8,13-Di-^/?/-sclareol252 Dietyosphaeriafarulosa 898

Diginea symplex 846 Digitalis lanata 535 Digitalis purpurea 535 /^-Digitatum 148,149 Digitonin 308 Digliicoside 200 Digioxigenin-3-O-tridigitoxide 535 23,24-Dihydrocucurbitacin F 59 (S)-(-)-9,10-Dihdyroxygeranylacetone 168 Dihydro alpha-agarofuran 171 2,3-Dihydroapigenin 541 Dihydrobetulinic acid 61 Dihydrocaffeic acid 923,926 (+)-Dihydrocarvone 145,146,147 Dihydrocinchonamine 28 Dihydrocitronellol 133 Dihydrocorynantheine 31,32 Dihydroeperutic acid 248 Diiiydroferulic acid 922-925 2,3-Dihydrofisetin 541 Dihydroflavanoids 469 (+)-ampelosin 469 dihydromyricetin 469 Dihydroflavonol reductase 653 Dihydrokoempferol 653 2,3-Dihydroluteolin 541 5,6-Dihydro-PGl2 595 Dihydropyridines 534 2,3,-Dihydroquercetin 541 6a,25-Dihydroxy-3,16-dioxo-9j3,19cyclolanestane 184 2,3-Dihydroxy-24-norfrieadela-l,3,5,7tetraen-29-oic acid 59 diacetoxy derivatives 59 l,3-Dihydroxy-3,7-dimethyloct-6-ene-2one 134 Dihydroxyconiferyl-alcohol 657 (S)'{-)-10,11 -Diiiydroxyfarnesol 166 5,4'-Dihydroxyflavone 541,544 5,7-Dihydroxyflavone 544 5,7-Dihydroxyflavone 541 10,11-Dihydroxynerolidol 163 Dihydroxyphenolic compounds 657 Diketopiperazine 901

990

Dilodia gossypina 131,149 Dilophus 690 Dilophus ligulatus 690 Dilophus okamurai 690 3,4-Dimethoxy-6-(2'N,N-dimethylaniinoethyl)-5-(methylthio)benzotrithiane 833 (2E,5E)-3,7-Dimethyl-2,5-octadiene-1,7diol 138 3-Dimethylsulfoniompropionate(DMSP) 846 7,12-Dimethyl-benz [a]anthracene 937 3,7-Dimethyl-(E)-2,6-octadien-l -al 132 3,7-Dimethyl-l,6-octadien-3-ol 140 3,7-Dimethyl-l,7-octanediol 133,136 2,6-Dimethy 1-1,8-octanediol 138,135 3,7-Dimethyl-l-octene-3,8-diol 138 (E)-2,6-Dimethy 1-2,7-octadiene-1,6,-diol 138,142 (Z)-2,6-Dimethy 1-2,7-octadiene-1,6,-diol 142,138 (+)-(R)-3,3-Dimethylsulfonio-2-methoxypyropanoate 846 (E)-2,6-Dimethyl-6-hydroxy-2,7octadienoic acid 140 (E)-3,7-Dimethyl-2-octene-l,8-diol 138 (E)-3,7-Dimethy 1-5-octene-1,7-diol 13 8 (Z)-2,6-Dimethyl-2-octene-1,8-diol 136 (Z)-3,7-Dimethyl-2-octene-3,8-diol 138 3,7-Dimethyl-6-octene-l-al 132 3,7-Dimethyl-7-octene-1,6-diol 138 8-Y,Y-Dimethylallylwighteaone 541 3'-Y,Y-DimethylalIylwighteone 541 7-12-DiiTiethylben[a] anthracene (DMBA) 44 4,4,-Dimethylcholestane-3a,5a-diol 56 2E-6E-2,6-Dimethylocta-2,6,-diene-1,8diol 138 2E-6Z-2,6-Dimethylocta-2,6,-diene-1,8diol 138 3,7-Dimethylet-6-ene-1,2,3,-triol 134 4,4,-Dimethylsterols 56 5-Dimethylsulfonio-2-methoxypropanoate 846

3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide method (MTT) method 266 Dimethyl-j3-propiothein 846 (E)-2,6-Dimethyl-2-octene-l,8-diol 138 7,12-Dimethylbenz [a] anthracene (DMBA) induced skin tumors 46 4,4-Dimmethylcholestane 56 Dimnoline B 818 Dinophysisfortii 729 1,3-Di-O-Caffeoylquinicacid 920 3,4-Di-O-Caffeoylquinic acid 920 3,5-Di-O-Caffeoylquinic acid 920 3,6-Di-O-Caffeoylquinic acid (3,5-DCQA) 938 4,5-Di-O-Caffeoylquinic acid (AQAs) 920,938,939 Di-0-Caffeoylquinic acids 921,947,948 Di-0-hemiphtholates of oleana-12- ene-3 j3,30-diol (deoxyglycyrrhetol) 58 Diosmetin 618 2,7-Dioxabicyio [4,3.o] honane 803 Dioxapyrrolmycin 757,794 l,l,-Dioxo-l,4-thiazinering 861 0-Dipehnols 654,670 Diphenol-oxygen oxidereductase 654 1,1 -DiphenyI-2-picrylhydrazyl (DPPH) 934,948 Diplamine 820,821,893 Diplodia gossypina 162,166,168,170 Diplosoma sp 821 Diplosoma visens 888 Dipolar learsiae 788 Discodermia 867 Discodermia calyx 731 Discodermia polydiscus 763 Discodermin E 867 Discodermin-halicylindramide 864 Discodermis A-H 665,865 Discorhabdin 824 Discorhabdin A 760,761,822 Discorhabdin alkaloids 708 Discorhabdin B 760,761,822,823 Discorhabdin C 760,776 Discorhabdin D 822,823

991

Discorhabdin P 760,761 Discorhabdin Q 760,761,822,823 Discorhabdins 823 Discorhaines 707 Dismosin 469 Dispuupehedione 715 Distratamide A 881,882 Distratamide B 881,882 Distratamide C 881,882 Distratamide D 881,882 Distyophaeria sericea 722 Distyostelium cAMP receptor protein 526 Distyota ciliolata 722 1,3-Disubsituted tetrahydro /J-carboline 16,17 Diterpene isothiocyanates 853 Diterpenoid alkaloid aconifine 534 aconitine from Aconitum 534 ajacine 534 avadharidine 528 bikhaconitine 534 cassaidine 535 cassaine 535 condephine 528 delcoline 528 delcorine 528 delphinine 534 falaconitine 534 indaconitine 534 jesaconitine 534 lappaconitne 534 mesaconitine 534 N-deacetyllappaconitine 534 pseudoconitine 534 raynodine 535 Dithiofurodysinin disulfate 815 Dithiomethyl lissochinotoxin C 833 Dithiothreitol 836 Dittrichia viscosa 613 DMBA 45,48,61,62,65 743-DNA 826 DNA electophoresis 267 DNA polymerase 895 DNA polymerase B 871

DNA polymerase inhibitors 868 DNA synthesis 526 DNA-damaging antitumor 796 Docetaxel 245 Dodeahydro benz[/]indolo [2,3,-a] quinolizidine 20 Dodonaea viscosa 276 DolabeUa auriculana 716,760,763,878, 887 Dolabellane 690,790,791,888 Dolastatin 10 716,802,886,887 Dolastatin F 887 Dolastatin I 887 Dolabella 724 Dopamine 516,530,536,535,538,662 Dopamine Dl receptors 531 Dopamine D2 receptors 532 Dopamine signalling 531 Dopamine receptors 531 Doris verrucosa 829 Down-regulators of TNF-a 465 PGE2 463 dexamethasone 463 DPY-4189 669 Dragmacidin 765 Dragmacidine D and E 798,799 Droplet counter current chromatography (DCCC) 205,206 Drosophila 389 Drupellafragiim 792 Deserpindine (11-demethoxyreserpine) 8 Dt-B 118 Dt-C 118 Dt-E 118 Ducan's multiple range test 93,960, 964-966 Dumas ia truncata 106 Dyrideathiazole 890 Dysamides 801 Dysamides K 801 Dysidea arenaria 714 Dysideaavara 693,814,815 Dysidea herbacea 691,692,697,776,801, 815

992

Dysidea sp 264,692,693,815,869,896,698 Dysidea sxiongQs 815 Dysideathiazole series 889 Dysidenin 889,890 Dysidia chlorea 801 Dysinin 814 EBA-EA activation inhibition assay 58 Ebumamonine 19 EBV 46 EBV assay 45 EBV-EA activation induced by TPA 59 Ecdysone 515 Ecdysone synthesis 242 Echmoclathria sp 814 Echinoclathrine B 814 Echinoclathrine C 814 Echinocystic acid (cochalic acid) 60 Echinodictyum sp 900 Echinosulfone A 900 Echinosulfonic acid A 900 Ecteinascidia turbinata 707,825 Ecteinascidin 736-type 827 Ecteinascidins 707,825 Ectinoscidin 812 Ectoprotein CD38 535 Edman degredation 437 Effective concentration (EC50) 265 EGF receptor kinase 797,870 EGTA 490 EGTA-Ca^-^ buffer system 490 EGTA-containing buffer 491,492 EGTA-treated plasma membrane 492 Ehrlich ascites carcinoma 957,939 Eicosanoid 274,520,693,939 Eicosanoid production 615 Eicosanoid signalling 374 eIF4E-dependent mRNA translation 526 Eilatin 532 Elatin 709 Eleutherobia aiirea 690 Elicitin receptor 396 Elicitins 395,396 Elicitor activity 397

Elicitor-active structure 394 Elicitors 393,394 Ellagicacid 545,938 inhibitor of PKA and PKC 550 Em eric alla iwiquis 780 Emodin 543,544,547,546 Enantiopinifolic acid 252 Enantio-j8-e7?/-lalbdanolic acid 252 Endogenou defense signal system 397 Endogenous hepatoprotective factor 471 Endogenous ligand anandamide arachidonylethanolamide 532 Endogenous ligands 384 Endogenous peptide signals 401,383,402 for plant defense 402 Endoplasmic reticulum 548 Endoproteinases 375 Endoproteolytic proceesing 387 Endothelin-1 841 EndothelinA 841 Endotoxemia 463 Endo-/J-l,4-galactanase 438 Endo-)3-l,6-galactanase 438 Endo-j3-galactosidase 436 Endorphin ligand 533 Enediyne chromophore 796 £-Nerolidol 164 Enkephalin ligand 533 Enkephalins 533 ENOD40 368,379 ENOD40gene 379 ENOD40 peptide 381 £«/-13-^)t?/-12a-acetoxymanoyl oxide 273 £/7/-13-epi-ketomanoyl oxides 260 £A7r-13-e/7/-manoyl oxide 267,253,261, 266 £A7M5,16-epoxy-9aH-labdane-13(16)-14diene 3j8,3a-diol 276 Ent' 16-hydroxy-13-epi-manoyl oxide 250 £A7r-19-hydroxy-13-^/)/-manoyl oxide 253 £m-3a-hydroxy-13-e/?/-manoyl oxide 250 £A7/-3/3,6j3-di hydroxy-13-ep/-manoyl oxide 261 £'w/-3/J-hydroxy 13-e/?/manoyl oxide 253,254,267,263

993

£^/-8alpha-hydroxy-lamba-13, 14dien 263 £m-8,9-di-6?/7/-sclareol 252 £A7/-8-e/?/-sclareol 252 £w/-8a-hydroxy-l 3,14-dien-18-oic acid methyl ester 261 Ent'Sa-hydroxy-labda-13 (16), 14diene 273,274 Entagenic acid 60 Enterobacter sp 170,171 Enterobacter cloaceae 258,259 Enterococcus faecalis 116J1S Enterococciis faecium 116 Ent'hydxoxy and e«/-acetoxy-3j3-monyl oxide 256 £«r-labd-8,13(£)-dien-15-ol 249 £«/-labdane oxides 249 £/7/-labdanes 251 £m-manoyl oxide-16-hydroxy-l 8-oic acid methyl ester 261 £/7/-manoyl oxides 264 £w/-j3-e/?/-concinndiol 249 Epialloyohimbane 5,20 Epibatidine 757,791,792 24-£/?/-brassinolide 425,426 24-£/?/-catasterone by Bayer-Villager oxidation 425 (-)-Epicatechin 541 (-)-Epicatechin gallate 612 13- Epicorynantheidol 24 Epidermal growth factor (EGF) 766 Epidennoid carcinoma 266 (-)-Epigallacatechin gallate (EGCG) 470, 546 Epidioxy-substituted non-diterpenes 688 Epidithodioxopiperazines 901 (-)-10-£/7/-isokalihinol4 856 (-)-10-£/?/-kalihinolI 856 (-)-10-£/?/-kaliinolH 854 13-£/>/-manool 252 Epimephrine 896 Epimerization of/3-carboline-type indole 5 Epimorphinan alkaloid from Sinomenium acutiim 412

Epinephrine 516,536,538,896 Epipedobates tricolor 791 (+)-Epiplasin-B 852 Epipolases kushimotoensis 898 Epipolasin-A 853 Epipolasis kushimotoensis 853 Epipolythiodioxopiperazines 839 Epiquinine complexes 347 13-£p/-sclareol 252 xylopyranoside 252 Episterol 415 Epitaondiol 690 24-£p/-teasterone 422,433 Epithelial NO synthase 525 24-£/?/-typhasterol 422,423 Epoxide hydrolase 150 (20(/?),24)5-Epoxy-9A 19-cyclolanostan3Al6i3-25,tetrol 217 (20(S),24)/?-Epoxycycloartane 227 8,13-Epoxy-labd-14-en-oic acid 249 6,7-Epoxy-Lat A 874 6,7-Epoxylinalool 141 EPR Spectroscopy 340 Epstein-Barr virus (EBV) 45 Ergine 531 Ergoline alkaloids 706 Ergostane 49,61 Ergosterol biosynthetic enzyme 304 Ergosterol peroxide 61 Ergotamine 531 Ergothioneine 813 Erica andevalensis 612 Ericaceae 246 Ericaceae family 242 ER ryanodine receptor Ca^"^ channels 517 Erwinia amylovra 394 Erwinia carotovora 394 Erwinia chrysanthemi 394 Erwinia coli 220,258,259,394,450, 821,928 Erythrina isoquinoline alkaloids 528 dehydro-j5-erythroidine 528 erysinine 528 eiysotrine 528

994

erythratidine 528 a-erythroidine 528 j8-erythroidine 528 Erythrocyte agglutination 464 Erythrodiol 57,60,64 Escherichia chrysanthemi 394 Escherichia coli 251,319 J65,115,925, 948 Escherichia faecilis 259 Esteinascidin 825 Estrogen-receptor antagonists 797 Et 743 N^2 oxide 825,827 Et722 826 EtN^^.oxide 826 Eteromorpha prolifera 691 [^H]-Ethanolamine 342 S,S'-l,2-Ethendiyl 0,0'-diisobutyl 898 Ethyl catechol 925-927 l-Ethyl-4-methylsulfone-p-carboline 819 Ethyl phenylpropiolate (EPP) induced edema 46 Ethylene biosynthesis 657 Etlatol 787 Et's 595,581,592,826 Eucalptus citriodora 132 Eucaryote signals-regulated protein kinase (PK's) 521,522 Eudesmane 848,849 Eudistoidin E and F 818 Eiidistoma divaceum 902 Eudistoma sp 820,834 Eudistoma glaiwiis 758,817 Eudistoma olivaceum 816 Eudistomidins 816 Eudistomidin A 758 Eudistomidins B 759 Eudistomidins C-D 758 Eudistomln C,E 782 Eudistomins C,E,F and L 816,817 Eudistomidin D 759 Eudistomidin F 863 Eudistomin K 815 Eudistomin K sulfoxide 863 Eudistomin sulphide 816,817

Eudistomins 815,817,782 Eudistomins D,J,N and O 782 Eugenol 126 Eiinicea toiirneforti 690 Eimicella cavolinii 690 Euphane 50 Euphol 61 Euphorbia 242 Euphorbia maddeni 594 Euphorbiaceae 242,243,245 Euryspongia arenaria 703 Euryspongia deliculata 694 Euthyroideones A-C 862,863 Euthyroideones and other sulfones 862 Euthyroides episcopalis 862 Evasoteria troschelii 813 Exifone 349,350,351 2,3,4,3',4',5'-hexahydroxybenzophenone 349 Exogenous peptide signals 393 Exogenous peptide el icitors 401 receptors 401 Exophilin A 723 Exophiola pisciphila 723 Exopolyphosphatase 718 Exoproteases 331 Exo-a-galactosidase 438 Exo-a-mannosidase 436 Exo-/J-galactosidase 438 Extracellular LRR domains 401 Extracellular signal regulated kinases 526 Fabaceae 92,168 Fabaceous crude drugs 98 j3-Fabatriose 94 Fabatrioside 89,109 Faboideae 92 Factor-a-dependent inflammatory liver injury 459 a-Fagarine 539 Faglis sylvatica 662 Falcarindiol 471,476 Falcipain 330,352 Famotidine 612

995

Faradiol 57,58,62,63 Faradiol-3-O-myristate 58 3-O-palmitate derivate 58 Farensyl pyrophosphate 237 Farmosan gorgonian coral 690 a-and j3-Famesene 546 Farnesol 125,162,165-168 Famesyl isothiocyanates 854 Fasciospongia cavernosa 688,697,703 Fasciospongia remosa 874 Fasicularin 860 Fatty acid oxidation 525 Fatty acid synthase type-I 500,501 Fe(IlI) protoporphyrin 332 a-Fenchol 154 Fenton process 931 Fenton reaction 634,644 Ferrylinyoglobin 934 Ferulic acid 56,57,653,662,665,671,920 Ferulic acid ester 655,665 Ferulic and p-coumaric acid 920 FeruloylCoA 653 3-(9-Feruloyl-4-0-caffeoylquinic acid 920 Feruloyllycine 923 3-O-Feruloylquinic acid 920 F-gitonin 308 Filter-sterilized pectinase 485 Fischerella ambigua 792 Fischerella major 802 Fischerella muscicola 802 Fisetin 541,543,544 Flagellin 394 Flagellin protein 394 Flavin-adeninedinulceotide-dependent oxidoreductase 417 Flavobacterium sp 868 Flavocristamides A and B 868,869,903 Fig 22 epitope 394,395 5-Flourouracil (5FU)-induced lethality protective effect of CVS 447,448 5-Fluorouracil-inducedmyelosuppression 429 5-Fluorouracil prophlylactic effect against mylosuppression 447,448

Flourinated erythromycin 780 Flow-cytometric analysis 442 Fluometuron 669 Fluorescent Ca^'^'-indicator (fluo-3) 489 Fluoresein 5'-isothiocynate (FITC) 446 Follicular mantle B Cells 272 (-)-10j3-Formamido-5 /J isothiocyanatokalihinol-A Formaldehyde induced arthritis 46 Formation of j3-hemetin 357 Formosanine 25,26 Formyl methionyl-leucyl phenylalanine (fMLP) 275 Forskolin 256,261,262,264,265,268,270272,275,276,489,490,496,539 Forsyth iaside 543 4-0-Freuloylquinic acid 920 5-0-Freuloylquinic acid 920 4,9-Fridodriane 867 Friedelane 54,59 Fructose-2,6-bipohsphate 523 Fructose-6-phosphate-2-kinase 523 5FU (5-Flourouracil) 450,453 5FU treatment 449 5FU-induced adverse effect 449 5FU-induced suppression of the bone marrow hematopoiesis 454 Fuciis vesiculosiis 711,125 Fungal glycoprotein 396 Fungal tomatinase 316,321 Fungal-tomato pathogens 295,312 Fungicide thiazole 321 Furanocoumarines 666 Furanoid 7/J-acetoxylabdanes 251 Furanoid linalool oxide 141 CIS and trans 143 Furanoterpene sulfates 688 Furanoterpenes 688 Ci2-Furanoterpene fiirospongolide 697 Furin 389 Furodysinin 815 Furodysinin lactone thioacetate 815 Furospongolide 698

996

Fusarhim oxysponim 293,295,296299, 305,311,312,313,317,319,320,322 f. sp gladioli 307 f. sp melonis 307,411 f. sp niveum 307 f. sp tuberosi 307 sp lycopersici 295,299,307, 312-314,316,318 Fusarium oxysporum tomatinase 312 Fiisariiim sambucinum 317 Fusarium solani 303,305,307,314,316, 317 Fusicoccin 374 GABA 30,536,537 GABA A receptors 529,895 GABA agonist 528 GABA gated Cl-channels 528 GABA receptors 531 GABA transporter 537 Gaeumannomyces graminis 304 jS-Galactosidase 210 Galangin 541 Galaxura marginate 704 Galeopsis angustifolia 251 Gallocatechin 943 Gamblerdiscus toxicus 730 Gametocyte 329 Gastric H^,K+-ATPase 612 Gastrin receptor 611 Gastrodia elata 529 Gastrointestinal disorders effect of flavonoids 607 Gastroprotective agents 614 Gastroprotective effect of flavonoids 615 Gaudichaudioside F 251 Gaudichaudol 269 Genanial 133 Genatiana macrophylla 476 Genista nimelia 612 Genistein 544,546,593,613 Gentiana sp 471 Gentianaceae 471 Gentiobiose 968

Gentiopicroside 471 serum ALT activity Gentiopicroside 471 serum transaminase activity 471 Geodia 768 Geodia cydonium 718 Geodiamolide A 769 Geodiamolide C 769 Geodiamolide D 769 Geodiamolide E 769 Geodiamolide F 769 Geodiamolides 757,768 Geodiamolides A-T 768 Geotrichum penicillatum 129 Gentiopioroside 476 Geranicacid 132,133,134 Geraniol 125,132,134,136-138,140,142, 143,163,165 Geranium oil 132 GeranyI acetate 136,164 Geranyl geraniol 239 Geranyl linalool 254 Geranyl linalyl pyrophosphate coaplyl PP 239,240 Gerany lacetol 168,169 (5H+)-Geranylacetol 163,164 GeranyIgeranyl pyrophosphate 237,240, 244 Germacrone 172,173 Gemaiol 132 Gernayl geranyl pyrophosphate 248 Geseneriaceae 251 Gibberella cyanea 162,166 Gibberelafujikuroi 253,255 Gibberella gaminis 313 \?ixavenae 306,308,310,304,305 Gibberella pluicaris 293,305,311,317, 319 Gibberella pulicaris 307 Gibberellane 241 Gibberellic acid 245 Gibberellin 367,657 Gibbonsia elegans ISl Ginseng 182 Ginsenoside Rbl 958

997

Ginsetioside Ryl 958 Gissampelos insular is 478 Galactose rich acidic glycoprotein 453,454 GLC method 435 Gleicheniajaponica 251,276 y-Gliadin 320 Gliovictin 828 Gloemerella cingulata 164,166-16 8 Glucans 396 Glucocorticoids 463,516 Gluconeogenic enzyme 523 6-0-j3-D-Glucopyranoside 224 jS-Glucoronidases 313 /J-Glucoronidase inhibitor 263 Glucosaccharo-l,4-lactone 263 j3-Glucosidase 210,211,308,957,968 Glucosinolates 513,514 j5-Glucosyl hydrolases 308 Glucuronic acid 89,94,102-104,119,465 j3-D-Glucronides 263 Glutamate (N-methyl-D-aspartate receptor (NMDA) 519 (non-NMDA receptors) 519 Glutamate transporters 536 Glutamic pyruvic transaminase 935 Glutamine 320 Glutathione 219,614,831,930 Glutathione-S-transferase 718 Glutin-5-ene 59 Glutinane 53,59 Giyceraldehyde-3-phosphate (GAP) 134 Glyceroglycolipids 431 Glycine as co-agonist 529 Glycine max L, 93,105,669 Glycine receptors 530 Glycine max cv. Kuromame 93 Glycine receptors 530 Glycine soya 93,101 Glycine-gated Cl-channels 530 Glycine-rich protein 394 Glycoaldehyde 163,164,296,314,318 Glycoalkaloid 305 from potato 305 Glycoalkaloids 296,311,317-319 Glycogen synthase kinase 3 525,546,548

Glycosyl hydrorolases 306,308,321 glycosylated 543 Glycycrrhizin 114 Glycyrrhetic acid 57,59,60,65 18a-Glycyrrhetinicacid 64,551 G18-j3-lycyrrhetinicacid 551 Glycyrrhiza glabra 465,476 Glycyrrhizin 90,99,103,465,467 hepatoprotective effect 466 \so\diXQA ixom glycyrrhiza glabra 466 Glyphosat 669 j3-Glycosyl hydrolase 304 Gm ENOD46 380 Gnathotricus retusus 143 Gnathotricus sp 143 Gnathrotriciis sulcatus 143 Gnidimacrin 543 Gnoderma applantum 131 Gogonane skeleton 850 Golgi-resident enzyme 389 Gomeraldehyde (ent-8,13,epoxy-labd-15-al) 249 Gomericacid 249,268 Gomerol 249 13-epigomerol 249 Gomojosidae 259 Gomojosides A-J 251 Gonyaulax polyedra 845 Gorgonane 848 Gossypol 541,543,544 G-protein-linked receptors 530,531,539 o2-adrenergic 530 dopamines (D2) 530 GABA(B) 530 opioid (m and d) 530 serotonin 530 somatostatin 530 Gracilaria 769 Gracilaria coronopifolia 729 Gralactose-rich acidic glycoprotein 451, 452 Gramine 531 Granulocyte macrophage colonystimulating factor (GM-CSF) 449 Grateloupiafilicina 862

998

Grayanotoxin 242 Greany lactone 166 Green fluorescent protein (GFP) 380 Green tea polyphenols 470 Grenadadiene 151 Jl\ Grindalia haverrdii 252 Grindella sp. 251 Griseofulvin 757 GTP-binding protein 385 GTP-bound stimulatory G protein 262 Guaiacol 126 Guaiacyl subunits 653 Guaiane skeleton 850 Guaiazulene 690 pigments of 690 Guaione 848 Guanosine triphosphate 598 Guanylyl nucleotide exchange factor 548 Gutierrezia grandis 251 Gutierrezia spathulata 251 Gutierrzia sphacrocephala 251 Gymnodinium 864 H"^, K"^-ATPase inhibitors 612 H"^,K"^-ATPase 613 Haber and Weiss reactions 931 Haemonchus contortus 794 Haemophilus influenzae 781 Halenaquinol 694 Halenaquinones 861 Halichlorine 799 Halicondria cylindrata 867 Halichondriajaponica 718 Halichondria okadii 799 Halichondria sp 716,847,848,852,854 Halichondria panicea 852 Halichondridae sp 263,699 Halichondrin 728 Halichondrin B 727 Haliclonacyclamines 710 Halicona oxigua 732 Halicona kormella 722 Halicona sp 694,695,710,725,709,728 Halicylindramide A 867

Halicylindramide B 867 Halicylindramides D 865,867 Halicylindramides E 866,867 Halimium genus 250 Haliotis discus hannai 788 (-)-Halipanicine 852 Halistanol disulfate B 704 Halistanol sulfates 703 Halistanol trisulphate 704 Halisulphate 263,799 derivative 699 Halocynthia roretzi 704,784 Halogen-free malyngamide J 788 Halomon 702 Halopappum parvifolus 251 Halopappus parvifolus {AstQvacQdLQ) 255 Halopappus sp 251 Haloperoxidase 758 Hamigera tarangaensis 803 Hamigeran B 803 Hamioea cymbalum ISl Hanacantha sp 705 Hansemda mrakii 129 Hapalosin 714 Hapatocyte necrosis 462 Hapatotoxins 461 Haplosamate A 898 Haplosamate B 898 Happlopappus arbutoides 251 Happlopappus pulchellus 251 Hapten-carrier protein conjugated 434 Haimaline 533 Harmine 533 Heat-inactivated proteases 439 Hederagenin 60 Hedychium coronarium (Zingiberaceae) 265 HeLa 301 cells 268 HeLa assay 45 HeLa cells 45,46 inhibition 45 HeLa S3 Cells 790 Helianthiis annuus 391,670 Helianthus occidentalis (Asteraceae) 251 Heliantriol C 57,62

999

Heliantrol B2 57 Helichrysum rupestre 259 Helicobacter pylori 611,617,618,778 Helicobactor pylori infection 617 Helicostylium piriforme 317 Helioporuns A-E 690 Heliothis verescens 768 Helix pomatia 210 Helminthosporium 828 jS-Hematin 343 Hematoporphyrin IX 355 Hematoporphyrin 354 Heme aggregation 329,343,349,351,352, 353,355,356 Heme aggregation activity 341 Heme aggregation assays 348,352,354 Heme aggregation efficacy 355 Heme aggregation inhibitor 352,329,343, 359 Heme aggregation inhibitors 327 Heme detoxification 329,331 Heme polymerase 30,333 Heme polymerization 334,348 Heme polymerization assay 334,354 Heme polymerization inhibitor 327,343, 347,348 Heme polymerization inhibitory activity 345 Heme proteolysis 331 Heme trophozoite 332 Heme-acetate complex 343 Heme-metalloporphyrin complex 358 Heme-thiolate monooxygenase 414 dehydrogenases 414 j8-Hemetin aggregation 357 jS-Hemetin assay 355 j3-Hemetin 31,38,332,333,340,356,358, 360, 11,15-Hemiacetal 266 Hemibiotrophic tomato pathogen 302,308 Hemin 337 Hemoglobin 330 Hemoglobin catabolism 327-331,341, 342,343,345,347-349,352,353,357,360

Hemoglobin proteolytic enzyme 360 Hemoglobinase 330 Hemolysis 180,464 Hemozoin aggregation 334,337 Hemozoin aggregation inhibition 342 Hemozoin aggregation inhibitors 342 Hemozoins composition 338 Hemozoin inhibition 341,342,356 Hemozoin inhibitor drugs 360 Hemozoin nucleation 334 Hemozoin polymer 347 Hepatic apoptosis 468 Hepatic DNA fragmentation 466,467,468, 469,472 Hepatic information 479 Hepatic intoxication with D-GalNandLPS 466 Hepatic mRNA induction 468 Hepatitis B virus 114 Hepatocellular uridine phosphate UDPandUTP 461 Hepatocyte apoptosis 459,460,462,464, 468,469 inhibtion of 477 Hepatocyte necrosis 466,468,476 Hepatoprotective activity of ornithine glucuronides 90,103 Hepatoprotective activity of palustroside III 106 Hepatoprotective agents 462,465 Hepatoprotective drugs 100,103 Hepatoprotective effects 106 of oleanolic acid-type glucuronides saponins 106 of plants 459 Hepatoprotective natural products 465 Hepatoprotective phytoconstituents 477 Hepatoprotective proteins 461 Hepatoprotective saponin 465 Hepatotoxicity 471 2,3,4,5,2',3',4'-Heptahydroxybenzophenone 351 5,9-Heptacosadienoic 721 Heptapeptide lissodinamide 878 Heptapeptides 715,883

1000

HerbamideA 900 Herbasolide 692 Herbicides 668 Herpex simplex 815,891 Herpex simplex vims 121,779,782 Herpex simples virus type 1 948 Hesperetin 469,618,541 Hesperidinase 210 Hetero-metalloporphyrin inhibition complex 359 Heterotrimeric proteins 520 hormones receptors linked to 520 Heteroyohimbine alkaloids 30 5,9-Hexacosadienoic 721 12-O-Hexadecanoyl-16-hydroxyphorbol13-acetate(HHPA) 45 induced edema inhibition (HPA) 46 Hexadentateethylenediammine-N,N'-bis [2-hydroxy-R-benzylimino] ligand (ENBPl) 351 Hexadeutrarated reserpine 9 Hexapeptide biotratamide 878 Hexapeptides 887 j3-Hexosaminidase 939 3-Hexyl-4,5-dithiacycloheptanone 842 /w-HHA 925 HHPA induced edema (HPA) 45 HHPA-induced inflammation 56 9,11 -Hidechloro-13-demothey lisodysidenin 889 Hippospongia A-F 688 Hippospongia sp 698,721,871 Hippuricacid 928,929 Hirsutanonol 543 Hirsutenone 543 Hirsutine 31,32 chromotropic and oxidole alkaloids 32 Histamine 516,520,532,536,538,612, 613,701,719 Histamine induced edema 46 Histamine production 611 Histamine and peptide leukotriene C4 (LCT4) 275 Histamine receptor 611,34

Histamine secretion 936 Histidine 334,335,526,813 Histidine decarboxylase 612 Histidine kinases 526 Histidine-rich protein (HRP) 334,342,360 Histidine-rich proteins II and 111 334 Histone HI 526 HIV inhibitors 942 HIVintegrase 941,942 HlV-protease 941,942 H202-induced apoptosis 269 Ho oils 140 Holoenzyme 522 Homanindole A 863 /J-Homochelidone 539 Homodolastain 3 902 Homphymia conferta 793 Hop-n-ene 59,65 Hordenine 531 Honnone-activated guanylate cyclase (GC) 519 Hormone-activated receptor tyrosine kinase 518 Hovenia dulcis Thunb. 469 Hoxaxinella sp 762 HPA 46 Y-HPKC 61 w-HPPA 925,927 H2 receptors 612 HRP II inhibition 336 HRP II mediated hemozoin synthesis assays 352 5HT3 class receptors 529 Huangquiyegenin A 195 Huangquiyegenin B 202 Huangquiyegenin I 195 Huangquiyegenin II 202 Huangquiyenin D 192 Huangquiyenin 184 Human colon adenocarcinoma cell cultures 903 Human immunodeficiency virus type-1 948 Humulene 168,170 Hyalurnidase-inhibitory activity 939

1001

Hyaluronidase 939 10-Hydroxypatchoolol 171,172 16a-Hydroxytrametenolic acid-3-Oaceate 61 Hydalhodes 391 Hydrazinolysis 438 Hydrocortisone 56,57 Hydrolysates from carrot cells 486 Hydrolyzable tannins 541 2-Hydroxydiscorhabdin D 823 6-Hydroxy-1,2,3,4-tetrahydropyrrole (1,2-a) pyrimidinum skeleton 829 6-Hydroxymellein biosynthesis 499 6-Hydroxymellein formation 503 6-Hydroxymellein synthase-catalyzing reactions 503 6-Hydroxymellein synthase 499-504 mechanisms of substrate channeling 505 7-Hydroxy-6-methylhept-5-en-2-one 138 7-Hydroxy-6-methylhept-5-ene-2-one-1 -ol 136 Hydroquinone 658 22a-Hydroxycampesterol 418 Hydroxy cinnamoyl-CoA esters 652 8-Hydroxy citronellol 136 4-Hydroxy isopiperitenone 151 Hydroxy methylglutarylcoenzyme A synthetase 525 3/J-Hydroxy sterols 306 3^Hydroxy[6a-acetoxy,23-ethoxy, 16^,23 (R) epoxy-24,25,26,27-tetranor]-9i8,19cyclolanostane 185 3pja 22-Hydroxy-16,23-epoxy-24,25,26, 27-tetranor-9/J, 19-cyclolanostane 184 3-Hydroxy-2,2,6-trimethyltetrahydropyran 144 C/5(3S,5R)-2-Hydroxy-2,2,6-trimethyltetrahydropyran 143 1 - Hydroxy-3,7-dimethyl-6-octen-2-one 133 l-Hydroxy-3-methoxyxanthone 467 3-Hydroxy-3-methylglutaryl CoA 130 5-Hydroxytryptamine 529, 531

(E)-IO- Hydroxy-6,10-dimethyl-5undecen-2-one 168 7-Hydroxy-6-methylheptan-2-one 138 (1R*,3S*,4R*,7S*,8S,12R*,13R*)-12Hydroxy-7-isothiocyanatoamphilectall(20),14-diene 853 4-Hydroxy-7[ 1 -hydroxy-7-[ 1 -hydroxy-2methy lamino)ethyl]-1,3-benzothiazole2(3H)-one 896 2-Hydroxy-8-p-mehthen-7-oic acid 149 Hydroxyxanthone-based drugs 349 Hydroxybenzoate 660 Hydroxybenzoic acid 655,660,661,922, 923,947 3jS-Hydroxybiformene 249 22a-Hydroxycampesterol 415,420 conversion of 418 6a-Hydroxycatasterone 424 Hydroxycinnamates 656 Hydroxycinnamic acid 652,657,666,919, 920 2-Hydroxydiscorhabdin D 823 20-Hydroxyecdysone 413 co-Hydroxy-£-nerolidol 162 (225'-24/?)-22-Hydroxyergos-4-en-3-one 418 12-Hydroxyfamesol 165,166 5-Hydroxyflavone 543 8-Hydroxygeraniol 140 11 -Hydroxygeranylacetone 165,168 3a-Hydroxy-gomeric acid 249 Hydroxyhexadecanoic acid (tricolorin A) 543 Hydroxyisopiperitenone 151 C/5(3S,5R)-2-(2'-Hydroxyisopropyl)-5methyltetrahydrofuran 143 3j3-Hydroxyl, A^'^ steroids 419 Hydroxylation of tomatidine 318 co-Hy droxylation 136,13 8,162,163,166, 168 8-HydroxyIinalool 136,140,142 3a-Hydroxymanool 275 6-Hydroxymellein synthase 483,506 6-Hydroxymellein 498,499,506,507

1002

6-Hydroxymellein-O-methyltransferase 483 Hydroxymenthol 155 5-Hydroxy-N,N-dimehtyl tryptamine 531 7-Hydroxypeganine 532 Hydroxyphenolic acid 657 /w-Hydroxyphenylpropionic acid (m-HH A) 922,923,947 7a-Hydroxysitosterol 56 diacetate 56 3j3-Hydroxy-sterols 96,299,303 7-a-Hydroxy-tomatidine 318 16a-Hydroxytrametenolic acid 3-O-acetate lanostanes 63 [^H]-5-Hydroxytryptamine 31 Hydroxyxanthone 327,349,354,361 6-Hydrxy-10-formamido-15-isothiocyanokalihinene 856 Hymenaceae sp 252 Hymenaea ablongifolia 252 Hymenaea parvifolia 252 Hymenamide F 830,863 Hymeniacidon sp 713,797,798,799,830 Hymenialdisine 711,787,795 Hymeriacidon sp 870 Hypercytokinemia 463 Hyperforin 537,538 Hypericum perforatum 551 Hyperkinetic ventricular arrhythmias 594 Hypersensitive response (HR) 402,394 Hyphomonas jannaschiana 873 Hypocholorous acid 931 Hypolactin-6-glucoside 613 Hypolactin-8-glucoside 612,616 Hypooxidaceae 180 [G-H] Hypoxanthine 348 Hypoxylon oceanicum 1\1 Hypselodoris nana 815 Hyrtiomanzamine 710,819 Hyptis spicigora 276 Hyrtios altum 727 Hyrtios erecta 696,698,710,819 Hyrtios sp 716 Hystiomanzamin 819

125I-AVR9 400 lAA oxidase 657 lanthella 719 lanthella basta 719 Ibhaynid 802 Ibotenate 531 Ichthyotoxic malyngomides 757 Ichtyotoxic activities 768 Ichtyotoxic agent 730 Icosanoid 945 IFN-Y 465 IgE/anti Ig E complexes IL (Interleukin) 455 Imberic acid derivatives 183 Imidazole 335 Imidazole [4,5-e-]-l,2-thiazine 897 9-N-[2-Sulfoethyl) Imine 870 Immune-mediated cytotoxicity 467 Immuno-active acidic glycoprotein from C. vulgaris 455 Immunoaffinity chromatography 382 Immunoblotting 499 Immuno-deficiency virus 734 Immunohistochemical staining 442 Immunoreactive polypeptide 383 Immunosuppressive drugs 464 FK508 464 dexamethasone 464 amphetamine 537 calebassine 528 carcurine 528 coronaridine 533 eseramine 536 eseridine 536 harmaline 537 harmine 537 ibogaine 533,537 ibogamine 533,537 physostigmine 536 physovenine 536 reserpine 538 sapargine 528 strychnine 530 tabernanthine 533

1003

toxiferine 528 yohimbine 530 Indolizidine alkaloids 190 Indole hallucinogens 531 psilocin 531 Indoloquinolizidine 4,28 Indolo [2,3-a] quinolizidine 7,10,11 conformations 22 Indomethacin 56,58,593,595,613,616 Infetroban 593 Ingenane 243,246 Ingenol diterpenoids 17-hydroxy ingenol-20-hexadecanoate 543 ingenol-20-hexadecanoate 543 Inguinal lymph nodes 444 Inhibit LDL oxidation 946 Inhibition of 4-(methylnitrosamino)-1 -(3-pyridyl)1-butanone (NNK) induced lung tumors 46 5-lipoxygenase 47 12-lipoxygenase 47 15-lipoxygenase 47 adenylyl cyclase 532 adjurant induced polyarthritis 46 arachidonic acid 66 arachidonic acid induced edema 46 j5-hematin aggregation 342,354 bradykinin induced edema 46 capsaicin induced edema 46 carrageenan induced edema 46 cotton pellet granuloma 46 cycloxygenase 47 Epstein-Barr virus early antigen (EVB-EA) activitaion induced by TPA 46 eukaryate signal transduction componenets 512 falcipain 331 heme polymerizations 348 heme sequestration 348 hemozoin 354 histamine release 47 human leucocyte elastase 47

hylauronidase 47 inducible cyclooxygenase 47 inducible nitric oxide synthase 47 DMBA/TPA induced skin tumor 48 lyso PAF acetyltransferase activity 615 Inhibitors of 544 /J-hemetin 357 cahnodulin 706 human synovial PLA2 697 lAA oxidation 657 kinesin motors 695 PKA 541 PMA 938 protein phosphatases 707 sterol biosynthesis 303 Inhibitory effects on HHPA 55 TPA 55 croton oil-induced inflammator edema 55 Inones 125 Inositol-1,3,4,5-tetraphosphate acid 516 Inositol triphosphate (IP3) 483,487,488 radioactive 487 Inositol triphosphate (IP3) 509,524 Inositol-1,4,5-triphosphate (IP3) 522, 547,548 Insositol-1,4,5-triphosphate (IP3) receptor 534 Insulin B chain 390 Insulin signalling activated protein kinase 522 Insulin-responsive protein kinase (PKB) 517 Interaction of THS with a-TNF 468 Interferons 519 Interferon Y (IFN-y) 431,477,464 Interleukin-2 536 Interleukin 2-dependent (IL-2) 271 Interleukin-6 cytokine family 717 Interleukin-8 711 Interleukines 271,540 Intraerthrocytic antimalarial activity 343

1004

Intraerythrocytic cycle 329 Intraerythrocytic phase 327,328,329 Intrasinusoidal hematosis 464 ^^^ I-iodotyrosine 373 125 I-Tyr2, Ala 16-systemin 373 lodoperoxidase 758 Ion gradient generating ATPase 512 IonophoneA23189 275,274,940 lonophores 374 lP3-lonositol-1,4,5-triphosphate 516 lonotropic glutamate receptors 528 lonotropic receptors 528 lonotropic serotonin receptors 529 losobutylmethylxanthine 275 IP3 receptors Ca^"^ channel 534 IP3-gated Ca^"^ channels 517 lP3-induced release of Ca^"*" 488 Ircinia dendroides 885 Ircinia sp 688,703,705,709 Ircinia sponges 695 Ircinin 688 Ischaemia reperfiision 610,614 (15*,35*,4/?*,75*,85'M25*,135*)-7lsocyano-15-isothiocyanatoamphilect11(20), 14-diene 856 {\SMARJS,%SMSMSA2>S,\5Ra^R)20-Isocyanao-7-isothiocyanato-isocycloamphilectane 856 Isoamyl acetate 129 Isoastragaloside I 193 Fsobatzelline A 822,790 Isobatzelline B 822,790 Isobatzelline D 822 Isobazellines 707 Isobomeol 154 6-Isobutyloxycarbonyl derivative 829 Isochromophilones 797 Isococculidine 528 Isocycloamphilectanes 853 Isodaucane 848,850 Isodictya setifera 828 Isodysidenin 889 Isoepoxyteryxin 471

Isoflavone synthase 655 Isolaurenterol 787 Isoliquirtogenin 537,539 Isolissoclinotoxin A 833,834 Isomenthol 155 Isomitraphylline 25,26 Isonakafuran-type sesquiterpenes 692 Isonovaldal 152 Isopentyl diphosphate (IPP) 130 Isopiperitenol 150 (+)-lsopiperitenone 150 Isoprenoid unit 513 jS-Isopropenyl pimelic acid 145 j3-Isopropyl pimelic acid 154 Isoprostane ¥2a 596 Isoproterenol 594,896 Isopteryxin 41 \,416 Isopulegol 136 Isoquinoline 4,824,825 Isoquinoline alkaloids (-)-salsolinol 531 atherosperminine 540 dihydropyranocoumarins 540 dopamine 531 narceine 533 phenylpropanoid glycosides 540 Isoreserpic acid lactone conformation 24 Isoreserpine 3,13,29 conformation 23 Isorhynchophylline 26 (-)-(15,6/?,7/?, 1 OR)' 10-Isothiocynato-4amorphene 852 10-Isothiocyanato-5-amorphene-4-oI 852 Isothiocyanates 514 (-)-l 5-Isothiocyanato-l-e/?/-kalihinene 856 (+)-10-Isothiocyanato-4,6-amorphadiene 852 7-Isothiocyanato-7,8-dihydro-a-bisabolene 848 (+)-4-1 sothiocyanato-9-amporphene 852 (+)-1 -Isothiocyanatoaromadendrene 853 lO-lsothiocyanatobiflora-4,15 diene 854, 856

1005

(-)-13-Isothiocyanatocubebane 853 (+)-Isothiocyanatocubebane 853 (-)-10-Isothiocyanatoguaia-6-ene 852 (-)-lO-lsothiocyanatokalihinol C 852 (-)-lO-Isothiocyanatokalihiniol G 856 (+)-5-Isothiocyanatopapukeanane 853 (-)-9-Isothiocyanatotrachyopsane 853 (+)-(1 R,6SJS, I OR)' 10-Isothiocynato-4amoq^hene 852 (+)-10/^-Isothiocyanoallo aromadendrane 853 (+)-6a-lsothiocyano-5 aH,7aH, 1 Oaendesm-4-(14) ene 852 (-)-1 Oa-Isothiocyanoallo aromadendrane 853 (-)-4a-Isothioyanatogorgon-l 1-ene 852 (-)-11 -Isothiocyano-7-/JH-endesm-5ene 852 2-Isothiocynoto-6-axene 850 p-Italicum 145 IvyPNP 383 JAK/STAT- linked receptors 520 JAK/STAT pathway 519 Janus kinase 3 (JAK 3) 271 Janus kinase 522,547,548 Japonenyne A and B 802 Jasmonates 367,373 Jasmonic acid 374 Jaspamide 768,757 derivative 768 Jaspis 700,714 Jaspis coriaceae 725 Jaspis splendans 768 Jaspisamides 727 Jaspsamide A 727 Jessie acid 182 Jessie acid arabinoside 182 Jessie acid xyloside 182 Jinbricatime 824 Jospissp 704,719,727,731 Julibrosidel 208 Julibroside II 208 Julibroside III 208

Jimgennanniaceae 252 Jimiperns procera 257,259 Juvabione 546 Juvenile hormone 546 K"^ and Ca"^ channels 531 K"^ channels 530,534 K"^-channeI mediated ion transport 497 Kaempferol 541,544,546,594,615 Kaikasaponin I 111,112,115,116,118 Kaikasaponin III 95,99,104,115,116,118 Kainic acid 529 Kainic acid-binding glutamate receptors 529 Kakkasaponin I 95,104 Kakkasaponin III 111,112 Kalihinane diterpenoids 689 (-)-Kalihinol G 856 (-)-Kalihinol L 856 (+)Kalihinol 856 KalihinolA 757 Kalihinol metabolites 854 Kalihinols 854 Kalinanes 782 Kalinihol I 856 Kalinihol J 856 (-)-Kalinihol 856 Kalipyran B 783 Karacoline 528 p-Ketoamide 785 K A T P channels 534

Karounidiol 61 Kaurane 241,246 Kaurenic acid 242 Kedarcidin 796 Kedarcidin chromophore 797 KeenamideA 887,888 Keramadine IIIJIS Keramamides 885 Keramamides F 884 Keramamides G 884 Keramamides H 884 Keramamides J 884

1006

Keramamides K 884 j3-Ketoacid 165 3-Ketoadociaquinone A 861 6-Keto-PGF2a 595 Ketoreductase 501 19-Keto-soyasapogenol B 184 N-Keto-stypodiol diacetate 591 Kexin 390 Kexin-like subtilases 389,390 3-Ketodociaquinone A 862 Kievetone 546 Kiheisterone C 795 Klebsielapneumoniae 258,259 Kloechera corticis 143 Konbuacidin 798 Konbuacidin A 711,798 Kororamels 902 kP6 preprotoxin 389 KSD assay 862 Kudzusapogenol A 95 Kudzusapogenol B 97 Kuduzusapogenol C 96 Kuanoniamines A-D 893,894,995 Kudzusapogenol A glycoside 103,105 Kudzusapogenol Bi 97 Kudzusaponin Ai 95 Kudzusaponin A2 95,104 Kudzusaponin A3 95^104 Kudzusaponin A4 95,104 Kudzusaponin A5 95,104 Kudzusaponin C] 96,103 Kudzusaponin PA3 118 Kudzusaponin SAj 95 Kudzusaponin SA2 104 Kudzusaponin SA3 95 Kudzusaponin SA4 104 Kudzusaponin SBi 95 Kuepaloxane 787 Kupffer cells 464,475 Kyurenic acid 529 L(+)-Muscarine 530 Laurencia rigida 783

Labd-13(E)-en-8a,15-diol 267 8( 17), 12(E)-Labdadiene-15,16-diol 265,266 Labdadiene-15,16-diol 11,15-hemiacetal (ll-OH-8(17),12(E)-) 266 Labdadienoic acid 248 5RMM. 107?-Labdan-13(6)-ene-8d, 15diol 258 Labdan-8( 17), 13-dien-3j3-15,18-triol 251 Labdane 13-0-glycosides 251 Labdane 241,244,259,276,275,277,256, 266,261 activities 257 Labdane glycosides 251 Labdane S 246,249 Labdane xylosides 252 Labdane type diterpenes (Labiateae) 263, 265 isolated from Hytis spicigora 276 nitrogenous 269 Labdanum oil 253 Labiatae 235,246,252,256 Labiataetannin 921 Labiate 921 Labopghora variegata 729 Labophorins A and B 729 Lachmim papyraceiim 781,785 Lachnumol 785 Lachnumon 781,782 Lachnumore 757 Lactase phlorizin hydrolase 609 Lactobocillus sp 257,924,927,928 16-Oxo-8( 17), 12(E)-ladadiene-15-oic acid 266 Lamellarins 706 Laminae eae 171 Laminaria pedicularioides 868 Lam in aria sp 724 Langmuir-Blodgett films 334 Lanosprazol 612 Lanostane 49,57 16a-Lanostane carboxylic acids hydroxydehydrotrametenolic acid 57 dehydrotumulosic acid 57 dehydropachymic acid 57

1007

16a-hydroxtrametenolic 57 pachymic acid 57 Lanostanes 0-16a-hydroxytrametenolic acid-3acetate 61 pachymic acid 61 poricoic acid B 61 abiesonic acid methyl ester 64 LantadeneA 64 LantadeneB 59,64,66 LantadeneC 59 Z-antibiotic peptidase 388 Lappaconititine 528 jL-arginine-analogues 471 Lappaconitine 528 Laser desorption mass spectrometry 330,331 (+)-LatA 874 (+).LatB 874 L a t e 875 LatD 875 LatM 874,875 LatS 874,875 Laternula elliptica 723 Lathyrus satives 529 Lathyrus palustris war.pilosus 93 Latrunculi B 732 Latrunculia 822,758 Latrunulia monocytogenes 450 (cytomegalovirus) 450 Latrunculia apical is 708 Latrunculia brevis 822 Latrunculia magnifica 874 Latrunculia purpurea 760,822 Latrunculin A 733 Latrunculins 874 L a t s A a n d B 874,875 Laucopolargonidin 653 Lauraceae 943 Laurencia brongriartii 863 Laurencia clavidormis 110 Laurence genus 249 Laurenciajaponensis 803 Laurenica TQd Q\gaQ 701 Laurencia saitoi 788

Laurencia sp 701 Laurenica majusciila 702 Laurenica nidifica 702 Laurenica nipponica 702 LDL peroxidation 934 L-Dopa 824 LeSBTl 392 Leguminosae 92,180,186,188,246,248,465 Leguminous isolflavone reductase (IFRs) 319 Leishmania donovani 254,260 Lemnalia africana 695 Lendenfeldia chondrodes 703 Lens esculenta 93 Lentinan 450 Lentinus edodes Sing 450 Leonunis hereteropyllus 272 Lepidium salivum 781 Leptoclinides dub ius 119 LeptosinA 839,840 Leptosin B 839 Leptosin C 839,840 Leptosin D 839 Leptosin E 839 Leptosin F 839 Leptosin G 839 Leptosin Gi 839 Leptosin G2 839 Leptosin H 839 Leptosin 1 839 Leptosin J 839 Leptosin K 839,840 Leptosin Kl 839 Leptosin K2 839 Leptosins 838,839 Leptosphaeria sp 839,901 Leucetta microraphis 105 Leucettamine A 705 Lucosceptoside 543 Leucine 331 Leucine aminopeotidase 375 Leucine rich repeat (LRR) domain 398, 402

1008

Leukemia P-388 760 leukopenia induced by 431 Leukotriene B4 (LTB4) 275,617,815, 939 Leukotriene B4 production 615 Leukotriene B4 receptor 705 Leukotrienes 274,275,940 Leukotrinene 939 Leyssera guaphaloides 250 Libhim maximowiczii 788 Ligand-gated ion channel neurotransmitter receptor 527 Liginine 126 Ligmncola laenis 899 Lignothioglycolic acid 668 Lilium longiflonim 391 Limocitrin rhamnoside 594 Limonene 44,125,133,140,145-147,149, 150,151 (+)-Limonene-1,2, (-)/m«5-diol 147,150 Limonene-1,2-epoxide 150 Limonene-1,2-epoxide hydrolase 150 Linalool 125,136,140,142,162 Linalool epoxide 143 (R)-(.)-Linalool 143 Linalool-8-carboxylic acid 141 O-Linalool 141,147 Linalyl acetate 136,140,141 Lingbya majusciila 876,902 /3,1 -Linked galactose 295,316 Linone-1,2-diol 146 Linonene epoxide 146,147 Lintenolide F & G 700 Lipoperoxidation assay 940 Lipoperoxidation process 614 Lipooxygenase pathway 263 Lipooxygenase derived fatty acid hydroperoxide 595 Lipooxygenases 597,939,940 Lipopollysaccharide (LPS) 477,460 Lissoclin disulfoxide 834,863 Lissoclinamide 1 880 Lissoclinamide 2 880 Lissoclinamide 3 880

Lissoclinamide 4 880 Lissoclinamide 5 880 Lissoclinamide 6 880 Lissoclinamide 7 880 Lissoclinotoxin A 834 Lissochnotoxin B 835 Lissoclinotoxin C 835 tris(methylthio) derivatives 835 Lissoclinotoxin D 833,835 Lissoclins 821 Lissoclimim 707,821,833,834,862,878 Lissochlinum bistratum 881,882 Z/5'5oc//>7i/Aw cyclopeptides 881

L issoclinum fragile 182 Lissoclimim genes 833 Lissoclinum japonicum 833 Lissoclimim lisschlinotoxin A 833 Lissoclimim patella 878,891 Lissoclimim perforatum 833 Lissoclimim vareaii 871,833 Lissoclimim voeltakowi 769 Lissoclimim voeltzkowi Michaels on (Urochor data) 269 Lissodinum 833,834 Listeria infection 431 Listeria monocytogenes 431 Listeria-specific cell mediated immunity 431 Lithistida (theonelld) 847 L-itronellol 125 Litsea ciibeba oil 132 Liver-derived NO 471 L-norepinephrine 530 Longamide B UIJIS Longamides 757 Lotus corriculatus 93 Low density lipoprotein 948 Lowry method 435 L-Phenylalanine 652,655 LPS hepatotoxicity 461,462 LPS stimulation 475 LPS induced liver and lung injuries 475 LPS toxicity 461 effects on liver injury 461 LPS-beated macrophages 473

1009

LPS-elicited hepatocyte apoptosis 463 LPS-elicited liver injury 463 LPS-induced elevation of [Ca^+j 475 LPS-induced nitric oxide 471 LPS-induced production of TNFa 470 LPS-related liver injuiy models 463 LPS-stimulated TNF-a production 470 LTP-blocking effect ofethanol 966 L-Tryptophane 652 L-Tyrosine 652 Luffariella variabilis 696 Lupane triterpenoids 60,61 Lupanes betulin 61 Lupenyl cation 176 Lupeol acetate 62 Lupinoside PAi 95,104,105,118 Lupinoside PA2 95 Lupinoside PA3 118 Lupinoside PA4 95,104,105,118 Lupinoside PA5 95 Lupinus polyphylus 94 Lupinus polyphylus hydride 93 Luteolin 541,543,544,593,595,615 Luttariella sp 694 Lycopersicon esculentum 294,298,657 Lycopersicon penivianum 372-375 Lycopersicon pimpinellifoliiim 294,298 Lycopersicon sp 293 Lycotetraose 316,317 j3-Lycotetraose 295,296,297,300,306,315 Lymphocyte transformation 224,225,226 Lymphokines 271 Lyngbya bouillonii 891 Lyngbya genus 891 Lyngbya lagerheimii 868 Lyngbya majuscula 722,732,716,761, 785,787,889 Lyngbyapetin A 891 Lypooxygenase inhibition 617 Lypooxygenase pathway 274 Lysergic acid diethylamide (LSD) 531

Lysozyme 334 Maaline skeleton 851 mAb (monoclonal antibody) 465 Madura pomifera 391 Macrolactamization 712 Macrophyllosaponin A 199 Macrophyllosaponin B 198 Macrophyllosaponin C 199 Macrophyllosaponin D 199 Magnoflorine 528 Maititoxins 730 Majusculainides 716 Makaluramines 822,823 Makaluvamin F 824 Makaluvamine A-E 700 Makaluvamine F 700 Makaluvamine N 700 Makaluvamines 707,708,757 Malabarican triterpenoids 700 Malabaricane triterpenes 700 Malampsori lini 397 Malaria pigment 332 Malondialdehyde (MDA) 219 Malonic acid esters 251 Malonyl-CoA 499,501,503,506 Malonylcoenzyme A 525 Malyngamide K 787 Malyngamide L 787 Malyngamide N 787 Mann-Whitney's U Test 962 Manauealide A 771 Manauealide B 771 Manauealides 729,730 a-Mangostin 544 y-Mangostin 544 Manool 253,254,259 biosynthesis ManoyI oxide 245,248-250,253-255 fragmentation 255 derivatives 256 ManoyI oxide isomer 259 13-e/?/-Manoyl oxide 250 ManoyI oxide series 258,259 ManoyI oxides 255 ManoyI oxides epigomeric acid 267

1010

DL-Menthyl chloroacetate 155 l-/?-Manthene 145 [4,6-Meo-ENBPI]Fe(lII) complex 352 Manzamine 709 Mepacrine 344 MAPK 545 2-Mercaptobenzothiazole 816 MAPK Kinase 526 6-Mercaptopurin 266 phosphorylation 526 Meridian B 764 MAPK kinase kinase (Raf) 522,547 Meridian C 764 Marchantia polymorpha 423 Maremycins A and B 828 Meridian D 764 Marine pyridoacridine alkaloids 758 Meridian E 764 Marinomonas mediterranea 719 Merozoite stage 328,329 Marinone 780 Mescaline 531 MauritamideA 870 a-Mating factor pheromone 387 Mayotamide 884 Metabolism of caffeic acid by Mayotamides A 883 microorganism 924 Meciadanol 618 Metabotropic glutamate receptors 531 Medicago polymorpha 93 Metal free porphyrins 354,355,356 Medicago trunuclata 379 Metal homeotasis 327 a-Megaspermin 395 Metalloporphyrins 347 /J-Megaspermin 395 Metalloporphyrins complexes 354 Megnaporthe grisea 322 Metalloprotease 330,331 co-Methyl hydroxylation 158 Metalloprotoporhyrins 357,358 Melemelenose A and B 869 Metallothioneins 717 Melilotus officinalis 93,132 Metal-N402 Schiffbas complexes 327 Melittin 274 Metamorphosis 784 Mellilogenin 97 Meth A 433 Mellilotus-saponin O] 95 Meth 1 tumor cells 432 Mellilotus-saponin O2 97 Meth A tumor 432 Melodinus monogynus (Apocynaceae) 276 Meth A tumor cell antigen 444 Membranipora perfragilis 825 cytotstatic activity 433,434 j8-Menene 131 Meth A tumor growth 453 /7-Menth-l-ene-9-ol 138 /?-Methane-3,8-diol 136 ;?-Menth-8-ene-l,2,-diol 148 2-Methlbutyl acetate 129 Mentha piperita 155 4'-0-Methl-kaempferol 544 Mentha pulegium 156 Methotrexate 266 /7-Mentha,l,8-diene-4-ol 148 1-Methoxymethyl-'6-oxo-6H-anthra /7-Mentha-2,8-diene-l-ol 148 [l,9-bc]thiophene 831 cisdinA trans 148 6-Methoxymellein 486,498,499 l-/?-Menthen-6,9-diol 145 biosynthetic sequence of 498 1-Menthene 149 6-Methyl-5-hepten-one 163 8-/?-Menthene-l,2-c/5'-diol 145 6-Methoxy mellein synthesis 486 8-/?-Menthene-l-ol-2-one 145,147 Methoxy thiofurodysinin acetate lactone Menthol 125,133,155 815 (±)-Menthol esters 155 6-Methoxymellein biosynthesis 505 Menthyl acetate 156

1011

6-MethoxymeIlein production 485,487, 488 biosynthetic enzymes involved in 498 6-Methoxymellein 483,484-486,506,507 biosynthetic activity 483 stimulation in carrot cells 484 6-Methoxymellein-O-methyltransferase 506,507,508 (5)-Methoxyphenylacetic acid 813 Methoxy-substituted dihydrocinchonamine 28 2 Methyl-6-methylisoquinoline-3,5,8(2H)trione 825 3-Methylbutyl acetate 129 10-(/?)-O-MethyleudistomidinC 817 AR,9R, 10/?-[ 1 -(5-Iso-quinolinesulfoxy 0-2Methyl piperazine 488 Methyl 18-carboxy-labda-8,13-(£)-diene15-oate 260 24-Methylcholesterol 417 4'-(9-Mettiyl-kaempferol 541 4-Methyl sterols 56 3-0-Methyl-2,3-dihydroluteolin 541 (+)-2-Methylthio-1,2,-/mAW-cyclopropane dicarboxylic acid 845 3-Methyl-2-butenoic acid 133 3,4(4-Methyl-3-pentenyl)-3butenolide 134 4- (Methylnitrosamino)-1 -(3-pyridyl)-1 butanone (NNK) 65 5-Methylthiovaracin 834 O-Methylseryl thiazole 885 2-Methyl-2-vinyltetrahydrofuran-5-one 141,140 (iEr)-4-Methyl-3-hexenoic acid 140 7-Methyl-3-oxo-6-octenoic acid 134 6-Methyl-5-heptane-2-one 134 6-Methyl-5-heptanoic acid 133 6-Methyl-5-hepten-2-ol 143 6-Methyl-5-hepten-2-one 136,13 8,140, 143,144,145,165,166 (S)-(+)-6-Methyl-5-hepten-2-ol 143 (Z)-2-Methyl-5-isopropylhexa-2,5dien-1-al 152

Methyl-5-methylthio-3-oxopentanoate 845 l-Methyl-5-thiol-L-histidine disulfate 814 (24/?)-24-Methyl-5a-cholestan-3-one 419 2-Methy 1-6-methy len-7-octen-2-ol 131 7-(9-Methylapigenin 541 A^-Methylated systemin derivative 376 (9-Methylbufotenine 531 Methylcholanthrene-induced fibroscoma 432 (24/?)-24-Methylcholest-4-en-3-one 419 24-Methylcholesterol 416,417 1-Methylcyclohexene 150 24-Methyl-demosterol 417 N-Methyldysideathiazole 890 Methylene blue 349 24-Methylenecholestrol 417 Methyl lycaconitine 528 N-Methyl-nitrosourea (MNU) 65 (±)-Methyl-0-acetyl-isoreserpate 18 Methylsulfinylindoles 861 1 -Ethyl-4-methylsulfone-j3-carboline 819 Methyllycaconitine 528 Methylthioadenosine (MTA) 829 Methylthiotidine 813 0-Methyltransferases 505 Methylxanthine theophyline 532 2-Methyl-Y-butyrolactone 138,136 (+)-Methy-0-acetyl-isoreserpine 24 Metmyoglobin/hydrogen peroxide 934 Metolachlor 669 Metronidazol 617 Mevalonic acid 130 [^H] Myo-inositol 487 Mezerein 937 Mezerin-induced superoxide anion radical 938 Microalgal metabolies 758 Micrococcus sp 48,79,94,719 Micrococcus liiteus 258,722,777,778 Microcolins 718 Microcyctis aeruginosa 799 Microcystin 733 Microcystis microcystins 543

1012

Microgenin 299-A 799 Microginin 299-B 799 Micromonospora echiospora 796 Micromonospora sp 841 Microtubule-depolymerization 473 Mimosa hostilis 248 Mimosamycin 825 Mimososideae 92 Minalemines D-F 897,898 Mineralcorticoid aldosterone 516 Misoprostol 616 Mitaria coccinea 251 Mitariosides A-D 251 Mitomycin C 268 Mitogen-activated protein kinase (MAPK) 522,547 Mitogen-induced mouse splenocyte proliferation 271 Mitraphylline 25,26 MLCK 546 MNU inuduced mammary tumors 46 Molamola 872 Mollamide 883 MoUicacid 182 MoUic acid arabinoside 182 Mollie acid derivatives 183 Mollic acid glucoside 182 MoUic acid xyloside 182 Monamine oxidases (MAO-A and MAO-B) 536,537 Moneses iinijlora 111 Mongholicoside I 202 Mongholicoside V 202 Mono and di-oleolglycerol 333 Monoalide 696 Monoalkyl-substituted polysulfides 843 Monoamineoxidase activity 594 Monocaffeoylquinic acid 939 Monoclonal antibody (MAO) 464,955 Monocyte chemoattractant protein 1 598 Monodesethylamadiaquine 344 Monodesethylchoroquine 344 Monodesmosyl saponin 108 Mononuclear cells (MNCS) 462,477 Mono-p-bromobenzamide 847

Monophosphate dehydrogenase 836 Moracenins 594 Moretenane 54 Morin 541,544 Morphinan isoquinoline alkaloids morphine 533 3-methylmorphine ecodeine 533 /3-codeine neopine 533 Moms alba 594 Mosher's method 467 Mossbauer spectroscopy 357,340 mrTNF-a-induced hepatic apoptosis 468 MTT method 266 Mucor circinelloides 166 Mucormiehei 259 Miicor plumbens 253 Mucor sp 723 Mucuna sempervirens 93 Multiflor-8-ene 59 Multiflor-9-ene 59 Multiflorane 53,59 Muricella sp 704 Murine acquired immunodeficienty syndrome (MAIDS) 433 Muscarinic receptors 530,611 Miissaenda 180,183 Mutagen-induced genetic variation 395 c-wj'c and c-7WW pathways 271 Mycalanmide derivative 733 Mycale 698,727,831,892 Mycalolides 831 Mycaperoxide 260 Mycaperoxide A 598 Mycaperoxide B 598 Mycoarbizins 781 Mycobacterium rhodochorus 156 Mycobacterium tuberculosis 779 Mycobacterimi intracelluar 259 MycorrhijinA 782,785 Mycosporine taurine 873 Mycothiazole 896 Myocardial infaction 598 Myocardial ischaemia 594 Myosin light chain kinase (MLCK) 522,523,524,547

1013

Myrcene 125,130,131,541,544,593 Myrtenic acid 154 Myrtenol 153 Myxoderma platyacanthus 872 N(10)-methyleudiotomin E 902 N,N'-Thiocarbonyldiimidazole 250 N,N-Dimethyl-5-(methylthio) varacin 833,834 N,N-Dimethyltryptamine 533 N-(p-hydroxyphenthyl) actividine 538 N-diethybiitrosamine induced hepatic tumor 46 N-formyl methionyl-lucyl-phenylalanine (FMLP) induced superoxide 47 N-methyl-N-nitrosurea (MNU) induced colon tumors 46 N ^ jN^-Bis (7-chloro-quinoline-4-yl) phenylenen-l,2-diamine 346 N4O2 SchifF-base coordination complexes 361 N4O2 Schiff-based metallodrugs 351 Na"^ and K"^-ATPase 517,535,542,836, 861 Na+,K+ ATPase inhibitor 535 Na"^/Ca2"^ antiporters 517 Na"^/Cr-linked monoamine neurotransmitter transporter 537 N-acetyl-beta-D-glucosaminidase 274 NAD (P)-02-oxidoreductase activity 811 NADDP receptor Ca^"^ channels 539 NADDP receptors 534 N ADH-dependent keto-reducing activity 500 NADPH synthase 502 NADPH-depdendent reduction 499 NADPH-dependent 6-hydroxy mellein production 502 NakmurolA 688 Nallanin 541 Namenamicin 837,838 Napyradiomycin Bi 798 Napyrodiomycins A and B 797,798

Naringen 615 Naringenin 614,613,618,653 Naringenin chalcone 653 Naringin 469,541,544,613,615,618 Na-sulfanomido ^carbolin 6 Natriuretic peptides (NPs) 367,381 Natural bolaphile 813 Natural cyclophanes 757 Natural killer cells G^K cells) 273 N-desacetylappaconitinee 528 Neamphine 897,898 Necrotizing agent 615,613 Neohop-12-ene 59 Neohop-13-ene 59 Neohop-13(18)-ene 65 Neoisomenthol 155 Neomenthol 155 Neomycin 925,928 Neoplastic B cells 270 Neosiphonia superstes 731 Neovasularization of solid tumors 593 Nephtea chabroli 708 Nephteis brasiliensis 875 Nephtesis facicularis 859 Neral 125,132,133,136,137,138 Nerol 140,143,165 Nericacid 133 Nerolidol 125,162,163,164,166,168 Nerylacetone 162,165,166,168,169 Netimicin 258 Neiirasapora crassa 304 ergosterol mutants of 304 Neurolathyrrism 529 Neuropeptide receptor 688 Neuropeptides 387,387 Neurotransmitter antagonists 515 Neurotransm itter receptors 512 Neurotransmitter signalling 517 Neurotransmitter-gated ion channels 519 Neurotransmitters 516 Neu's reagent 662 Neurotransmittor coverting protein 512 Neurotransmittor transporting protein 512 Neutraceuticals 597 NF-^B activation 470

1014

NF-A:B Nuclear factor kappa B 470,711 Nicandra physaloides 300 Nicotinic acetylcholine receptors 528 Nicotiana tabaccum L. 665,666 Niphates erecta 1X1 Niphatessp 732 Niphatevirin 717 Nippostronglyolus brasiliensis 815,896 Nippostronglyolus dubias 815 Nitraria tangutoriin 20 Nitric oxide (NO) 470,477,598 Nitrobenzene oxidation assays 666 Nitrogen oxides 665 Nitropropionic acid-glucose derivatives 190 3-Nitropropyl-gluosides 190 selniferous derivatives 190 4-Nitroquinoline-l-oxide 937 Nitrosamine 65 NMDA receptor-mediated responses 966 A^-Menthene 152 N-Methyl chloropyrrole 761 A^-Methylamino-L-alanine (L-BMAA) 529 N-methyl-D-aspartate binding glutamate receptors 529 N-nitro-L-arginine methyl esters 595,598 N-nitrosodiethylamine 66 NNK-induced lung tumorigenesis 65 NO production 471 NO synthase inhibitor 595 NO synthesis 595 Nocardia sp 153 Nocardioides sp 719 Nodule morphogenesis 379 Nodulin (ENOD) 379 Non-adrenergic imidazoline receptors 530 Non-insulin-dependent diabetes mellitus 534 Non-NMDA binding glutamate receptors 529 Nonsteroidal antiinflammatory drugs (NSAIDS) 610 Nootkatone 128,170,171 Noradrenaline 530,595 Norathyriol 543

Norcardia alba 162 Nordercitin 893,894 Norepinephrine 516,536,537,538 Norharmane 531 Nor-pachoulenol 171,172 Norsesquiteipene 163,166 NOS inhibitors 471 Nostoc linckia 794 Nostoc sp 789 Nostocyclophane D 794 Nostocyclophanes 757 Novalal 152,153 /J-7V-oxalylamino L-alanine (L-BOAA) 529 NSAlDs 613 Nuclear factor k B 598 Nuclear sterol regulatory element binding protein nSSREGP 388 Nucleating scaffold proteins 335 Nucleoidin glucoside 781 Nucleotide-gated ion channels 527 Oceanapamine 706 Oceanapia sagittaria 894 Oceanapia sp 706,894 Ocimene 125,130,131 Ocimenone 143,144 5,9-Octacosadienoic 721 Octalactin 728 Octalactin A and B 728 Octandecanoid pathway 374 Octapetide patellamide 878 Oestrogen 516 Ogebikase 654 Okadaicacid 9,10,731,832 from dinoflagellates 543 Okadaic acid-induced TNF-a-gene expression 470 18a-Olean-12-ene-3j3,23,28-triol 64 Olean- l2-ene-3A23,28-triol 60,64 Olean-12-ene-3A23,28-triol tri-Ohemiphathalate sodium 65,66 Olean-12-ene-3j8-23,28-triol 60 18a-Olean-12-ene-3i3-28-diol 60 Oleanane glucuronoide 89-92,94,121

1015

Oleanane saponins 183,203 Oleanane triterpenoids 60 Oleananes arjunolic acid 64 Oleanane-type saponins 207 Oleanene glucuronosides 117 Oleanene-type triteqêne saponins 103 Oleanolic acid 59,61,541,107 Oleanolic acid 28-O-glucuronide 106, 107,108 Oleanolic acid 3-0-glucuronide 106,107, 108 Oleanyl cation 181 Olearia paniciilata 255 Oleic acid 333 Oleoylacetylglycerol 536 D-Oleoylphosphatidylcholine 333 Di-Oleoylphosphatidylethanolamine 333 Oleuropic acid 141 Olfaction 521 Oligoadenylate synthease 718 Oligogalacturonides 485,486,487 Oligonucleotides 390 Oligopeptide elicitors 396 Omeprazol 612 O-Methyltranferase 498,499 Oncogenes 545 Oomycete 299,303 phythhim 299 phytophthora 299 Open readingframes(ORFs) 379 Opiate receptors 533 Orange oils 169 Oregon in 543 Organochlorines 767 Oriamide 884,885 Orientin 594 Orina sp 712 Orinthine 937,66 Oripanine 533 agonist and antagonist effects 533 Ornithine 66 Ornithine decarboxylase (ODC) 45,66 TPA induced inhibition 45 Oroidine 783,785

Oroidin-family 870 Oryza sativa 311 Oscillatoria agardtii 799 Osteoporotic drug 711 Ostodes 242 Ouabagenin 3-0-L-rhamnoside 535 Ovoperoxidase 813 Ovothiol A-C 813 4-Ovothiol ARl 813 5-Ovothiol RBI 813 6-Ovothiol CRl 813 Oxaloacetic transminase 935 Oxazine alkaloid 542 Oxazoline 881 j3-Oxidation 166 Oxidative burst 396 Oxidative DNA damage 66 Oxinastatin-4 721 Oxindole alkaloids 3,4,25,26,27,33 Oxinellamides 757 6-Oxo-cycloartan-3,16 200 6-Oxocampestanol 415,420 6-Oxocativic acid 250 11-Oxo-manoyl oxide 256,252 Oxycorynia fascicularis 251.727 Oxystigma buecholtzii 165 Oxytrogenin 96 Oxytrpis 180 PI3 (phosphatidyl linositol)-kinaseindependent pathway 264 P-388 lymphocytic leukemia cell 764 P-388 model 823 P69 subtilases 391 Pachastrella sp 704 Pachymatismin 717 Pachymeniopsis lanceolata 722 Pachymic acid 63,64 Pachypellins 709 Pachyrhizziis erosus 93 Paclitaxel 245 Paecilomyces sp 725 PAF antagonist 275,615 Pachypellina sp 710

1016

Palauamine 110,111 Palindromic structure 371 Palmitic acid 333 Palmitoleic acid 333 r-(9-Palmitoyl-3'-0-(6-sulfox-D-quinovo pyranosylglycerol 868 Palmrosa oil 132 Palustroside I 97 Palustroside II 104 Palustroside III 104 Palythoa 735 Pan C auxotroph mutant 319 Panacene 786 Panax ginseng C.A. Meyer 956 Pandaros acanthifolium 832 Pantoisofaranoid A 802 Pantoisofaranoid B 802 Pantoisofaranoid C 802 Pantoneura plocamioides 801 Pantopyranoids A and B,C 801 Pantothenate synthetase 319 Papavarine 561 Papuamides A-D 714 Paramecium 494 Para-nitrophenyl-jS^D-glycopyranoside 306 Parasitmia 329 Parguerol 757 Parguerol triacetate 788 Parkinson's disease 531 Passiflora 180,183 Passiflora quadranglaris 205,206 Passifloraceae 180 Patchaulol 125,170 Patchouli alcohol 170 PatellamideA 879 PatellamideB 879 PatellamideC 879 PatellamideB 879 PatellamideG(l) 879 Patellazoles A-C 891 Pathogenesis-related gene expression 394 Pathogen-induced necrosis 660 PBS (phosphate buffered saline) 455 p, coumarate 655

PDE isoenzyme 490 Pectenotoxin 2 729 Pectin esterase 486 Pectinase 483,485 Pellina sp 709,723 Penaeus japonins 718 Penicillium 828,901 Penicillium digitatum 138-140,145,148, 149 Penicillium italicum 150 Penicillium notatum IIOJSO Penicillium roquefortii 129 Penicillium sp 797,155 Pennyroyal oil 156 2,3,4,5,6-Pentahydroxyxanthone 351,349 Pentabromopseudoline 776 Pentacyclic triterpenoids fridelane 55 glutinane 55 hopane 55 lupane 55 moretenane 55 multiflorane 55 taraxastane 55 taraxerane 55 Pentagastrim 613 2,3,4,5,6-Pentahydroxy xanthone 350 3/3, 6a, 16/J,24a, 25-Pentahydroxy-9/J, l9-cyclolanostane 184 Pentapeptide dolastatin 885 1,2,3,5,6-Pentathiepanes 843 (Poly) peptide hormoens 387 (Poly) peptide ligands 385 Peptide enod 40 379 Peptide elicitors 393,395,397 Peptide prohormones 387 Peptide signals 387,388 Peptidonimetic inhibitors 39 Peptostreptococcus sp 925 Perfragilins A and B 825 Perhydroquinolins 859 Pericentral hepatocyte necrosis 466 Periconia byssoides 790 Pericosine A 790,791 Perillaldehyde 146,147

1017

Perillicacid 145,146,147,150 Perillyl alcohol 146,147,148,149,150 Perna canaliculus 864 Peroxidase (POD) 657,663 Persa americana 943 Petrosapongia nigra 697 Petrosapongiolides 697 Petroselimum crispeum L. 666 Petrosia sp 722,723 Petrosia contignata 113 Petrosia steriata 694 Petrosia weinbergi 703 Petusis toxins 489 Peyssonelia sp 694 Peyssonols A 694 PHA 271 Phakellia 847 Phakellia costada 830 Phakellia mauritiana 770 Phakallistatin 5 830 Phakellistain 715,716 pharmacological effects 429 Phase transfer catalyst 357 Phasecoside IV 97 Phaseogenin 97 Phaseolus coccineus 93 Phaseolus vulgaris 489 Phaseoside I 95,104 Phasesidelll 97 Phenobarbitol induced Hepatic tumors 46 Phendoxidase 719 Phenobarbitol 66 Phenolase 654 Phenolic compounds 651,663 degradation of 651 Phenolic ellagic acid 543,546 Phenolic compound metabolism 651 regulations of 651 Phenolic oxidation 671 Phenolic oxidation (PPO and POD) 668 Phenol-sulfuric acid method 435 Phenylisothiocynate 435 Phenylpropanoid 541,543 curcumin 543

2-Phenylacetyl-^carbolines (R,SandT) 782 Phenylalanine 652,653,660,928 Phenylalanine ammonia lyase 489,652 Phenylalanine hydrolases 718 Phenylalkylamines 534 verapamil 534 (/?)-Phenylethanoid glycosides (1,7-Bis(3, 4-dihydroxyphenyl) heptan-3-ol) 543 Phenylethanoids 465 Phenylethylamine 538 Phenyllanine hydroxycinnamate 652 Phenylpropanoid 651 Phenylpropanoid alkaloids 540 D-cathinone 531 D-cathine 531 I-ephedrine 531 pseudoephedrine 531 Phenylpropanoid compounds 651,659 Phenylpropanoid bisoynthesis 663 Phenylpropanoid metabolism 652,658, 663,666,676 Phenylpropanoid pathway 651,652 Phenylpropanoid pathway enzymes 666 Pheophytin 691 Phloeodictine B 829 Phloeodictine C12 829 Phloeodictines 829 Phloeodycton sp 829 Phlomis medicinalis 251 Ph lorn is mendocina 133 Phlomis younghiisbandii 257 Phlomosides 251 Pholagacanthiis thyrsiflorus 251 Phoma sp 691 Phomopsin-A 886 Phorbas sp 759 Phorbol-12,13-dibutyrate(PDB) 542 Phorboal ester 726 Phorbol 242 Phorbol ester promotors 66 Phorbol esters 243,246,524,536,542,543 Phorbol l2-myristate j3-acetate 598 Phorbol 12-mysristate-13-acetate 488, 937,948

1018

Phorbol-12-myristate-13-acetate (PMA) 242 Phorbolbased pKC activators 524 Phorboxazoles 756 Phorboxazoles A & B 759,760 Phormidiiim temte 868 Phosphate buffered saline 434 Phosphatidyl inositol-3-kinase (P13K) 522,525 L-Of-Phosphatidylcholine 333 L-a-Phosphatidylethanolamine 333 Phosphatidylinositol-3,4,5-triphosphate (PI347P3) 516,517 Phosphatidyl inositol-3,4-kinase (PIP2) 522,547 Phosphatidyl inositol phosphate-activated protein kinase 522,547 Phosphatidyl linositol 475 Phosphatidylinositol -4,5-biphosphate (IP3) 521 Phosphatidylinositol 487 Phosphatidylinositol cycle 483,494 Phosphatidylinositol phosphate-activated proteins 517 Phosphatidylinositol-3,4,5-triphosphate (PIP3) 522,547 Phosphatidylinositol-3,4-biphosphate (PI34P2) 516 Phosphatidylserine 938 L-a-Phosphatidy-L-serine 333 Phosphodiesterase (PDE) 507,597 Phosphodiesterase (PDE) inhibitor 488 Phosphodiesterase inhibitor 275 Phosphoenol pyruvate carboxykinase 523 6-Phosphogluconic acid 670 6-Phosphogluconic dehydrogenase 670 Phosphoglycogen synthase 525 Phospholipase A2 263,270,374,734,688, 842,867,903 Phospholipase A2 inhibitor 47,593 Phospholipase B 333 Phospholipase C (PLC) 522,521,530, 548,696 Phospholipase C-y (PLCy) 545

Phosphoprotein (P-Pr) 522,548 Phosphoprotein phosphatases (PPs) 517,521 Phosphorylase kinase 264 Phosphorylated phsophotidylinositol 487,488 Phosphoserine 517 Phosphothreonine 517 Phosphotyrosine binding SH2 domains 525 Photobacterium damsela 719 Phthalide isoquinoline alkaloids (+)bicculine 528 bicuculline 532 N-methyl bicuculline 528 narcotoline 533 norbicuculline 537 a-narcotine 533 tetrahydropapaveroline 533 Phycornyces blakesleeanus 318 Phycopsis terpnis 858 PhyllactonH 695 Phyilacton I 695 PhyUidia ocellata 858 Phyllidia pmtulosa (nudibranch) 852 Phylloquinone 236 Phyllospongia sp 695 Phytoaiexins 484 Phytoalexin production 494 biosynthesis in carrot-cells 483 Phytoalexin r is hit in 319 Phytoalexin synthesis 507 Phytoaiexins 484 Phytoanticipin 294,321,662 Phytocanticipin-degrading enzymes 312 Phytochrome proteins 529 Phytoecdysone 55,59,546 Phytohantiones 367 Phytohemaglutinin (PHA) 267 Phytol 245 Phytolexin in carrot cells 483 biosynthesis 483 Phyto-pathogenic oomycete 395 Phytophthora 303, 395,396 Phytophthora elicitins 395

1019

Phytophthora megasperma 303,396 Phytoprotectants 320 Phytosulfokines 368,380 Phytosulfokines signal 379 Phytosulfokines receptor 378 Phytoxin pisatin 322 a-Pinene 125,145,149,151 ^Pinene 145,154 a-Pinene epoxide 153 Piceaabies L 666 Piceatannol 541,543,546 Picrocrocin 954 Pigment-producing parasites 25 Pilocarpine 530 Pilosine 530 Pimarane 241 Pinaceae 245,246,249 Pinane monoterpenoids 153 Pinifolic acid 258 Pinna muricata 871 Pinocarveol 153 Piniis nigra 249 Pinus caribaca 151 Pinus palustris 151 Pimis strobus 245 Pinus sylvestris 249 Pinusolide 275 Piocarvone 153 Piperidine alkaloids 528 (-)-lobeline 528 âlanine-3-aminopropionic acid 537 anabasine 521 guvacine 537 isoguvacine 528 nicotine 528 nipecotic acid (3-piperidinecarboxylic acid) 537 nomicotine 528 Piperitenone 150 Piperidine alkaloids pseudoconhydrine 528 y-coniine 528 (+)-N-methylconiine 528 Pisumsatium 93,417

Pithomyces sp 172 (2/?,55)-Pitoyl 144 Pityrophtors pitygraphiis 144 PK or PP targetting proteins 521 PKA inhibitors 527,538,542 acridine based 542 isoquinoline based 542 phenanthrene based 542 PKA-catalyzed phosphorylation 529 PKA-gated Na"^ channels 521 PKC isozymes 61,517,524,726 PKC 271,526,536,543,721,726 diacylglycerol activated 66 PKG 523,527 PLA2 669,695,696,704,902 Plakelliasp 715 Plakoside A and B 721 Plakosides 722 Plamepsin I and II 330 Plankortide 724 Plankortis lits 724 Plankortis simplex 721 Plant proteases 389 Plant pyrolysins 392 Plant peptide signals 402 Plantainoside D 543 Plasminogen activator (t-PA) 221 Plasma membrance located receptors 518 Plasma membrance potential 374,396,397 Plasma membrane H"^-ATPase 374 Plasma membrane-located receptors 522 Plasma transaminase 472 Plasma-membrane-located signals receptors 518 Plasmepsin 352 Plasmepsin I 331 Plasmepsin II 331 Plasminogen 725 Plasminogen activator 221 Plasmodia 348 Plasmodia biochemical pathway 337 Plasmodial organelles 334 Plasmodial protQ'm^SQS 330 Plasmodium 327

1020

Plasmodium berghei 333,338,341 Plasmodium CD?K 544 Plasmodium falciparum 328,330-332, 334,336,338,340-342,345,348,350352,359,527,814 Plasmodium malariae 328 Plasmodium vivax 328 Platelet activating factor (PAF) 275,615 Platelet antiaggregant 595 Platelet-derived growth factor (PDGF) 593,598 Plaustroside I 104 Pleckstrin homology 525 Plectranthus {Ld!o\?iX2iQ) 238 Pleiotropic effect of TNF-a 460 Pleurobranchus forskalii 887 Pleuronectes platessa 118 Pleurotus glabellatus 131 Pleurotus sajor-caju 131 Pleurozia acinosa 252 Plocamadiene A 701 Plocamium 701 Plocamium cartilagineum 701 Plocamium castatum 701,781 /?-Methoxyphenacyl bromide 836 PM-located voltage-gated Ca^"^ channels 534 Podocarpaceae 246 Podocarpane 241 Podocarpus ferrugineus 246 Podocarpus macrophyllus 246 Poecillastra sp 731 Pogostemom cablin 111 Pokweed mitogen (PWM) 271 Polauamine 784 Polio vaccine 817 Polio wirus 782 corosolic acid 543 poliumoside 543 Polyalthia macropoda 260 Polyamine metabolism 66 Polyamines 66 Polyasparagine 334 Polybrominated methylthioindoles 863

Polycarpa 836 Polycarpa aureta 706,836 Polycarpamine A 836 Polycarpamine B 836,835 Polycarpamine C 836 Polycarpamine D 836 Polycarpine 836 Polychlorinated dibenzodioxins 757 Polychlorinated phenyl ether 792 Polycitorsp 834 Polycitorella mariae 837 Polyclonal antibodies against ARS2 435 Polydiscamide A 865,866 Polyene antibiotics 299 Polyfibrospongia australis 694 Plygalacturonase 374 Polygalacturonides 486 Polyhistidine 337 Polyhydroxylated phytohormone brassinolide 414 3 j3,5a,6 a, 15 a-Polyhydroxylated steroids 872 Polyketide synthase 502 Polymastiamides 704 Polymerase activity 826 Polymerase chain reaction (PCR) 390 Polymoi*phic S locus 386 Polymorphonuclear leukocytes 432 Polymostia boletiformis 704 Polyol ingenol 242 Polypeptide ligands 368 Polyphenoloxide (PPO) 657 Polyphenols acteoside 476 (+)-ampelopsin 476 curcumin 476 (-)-epigallocatechin gallate 476 gomisin A 476 hovenin 1 476 lithospermate B 476 tetrahydroswertianolin 476 naringin 476 Polypodine B 59 Polysine 334 Polystyrene latex particles 270 Polysyncaraton lithostrotum 837

1021

Polytheonamide B 715 Polyunsaturated fatty acid (PUFA) 932,948 Polyuronides esterification of 486 Ponax notoginseng 476 Poniciretin 618 Pontoneureins A and B 801 Porcirin 618 Porta cocos Wolf 956 Poricoci acid 64 Poricoci acid A 57 Poricoci acid B 57 Porphyrin 338,340 Porphyrin complexes 354,355 Porphyrins 327,359,361 Portieria harnemannii 702,767 CD4-Positive helper T cells 450 Post translational modification 367 Potato-saponin a-chaconine 319 Potomogetonnodosus 260 PP inhibitors 521 PPl inhibitors 543 PP2A inhibitors 543 PPO activity 656 PPO cytochemistry 654 PPO phenolic compounds 656 Purealidins J, K and Q 766,767 Pregnenolone 419 Prehispanolone effect on B cell proliferation 273 Prelissoclinamide 1 880 Prelissoclinamide 2 880 Premna oligotricha (Verbenaceae ) 257 Prenylated xanthones a-mangostin 541 y-mangostin 541 PrepANP 381 Prepatellamide B formate 879 Prepro proteins 400 Prepsammaplin A 844,845 Prepsammaplin B 844,845 Prepsammaplin C 844 Prepsammaplin D 844

Preulithiacyclamide 879 Priano melanos 822 Prianos sp 760,822 Prianosin A 823 Prianosins C and D 823 Prianosinsdischabdins 802 Prianosins-discorhabdins 802 Primaquine 349,350 Primaiy biliary cirrhosis 463 Proanthocyanidins 666 Probit-graphic interpolation method 47 Procyanidin 544 Procyanidin glycoside 594 Procyanidin dimer 541 Prodelphenidin 544 Progesterone 139,421 Proinflammatory cytokines 461 Prolactin (PRL) 265 Proleiis mirabilis 259 Proline 320,371,372 Prolysine 388 prophylactic effects 429 Propionibacterliim acnes/LPS'inducQd hepatocyte apoptosis 463,471 liver injury 463 Propionibacterium acnes 462,465 Propionibacteniim acensll.?^ model 462 Propionibacterium acneS'XndncQd mononuclear cell infiltration 463 Propolis 945 Proprotein coveitase (PC) 402,388 Proprotein processing protease 392 Prosapogenin 1 209 Prosapogenin 2 209 Prosapogenin 3 209 Prosapogenin 4 209 Prosapogenin 5 209 Prosapogenin 6 209 Prosapogenin 7 209 Prostacyclin 598 Prostaglandin E2-dependent cytoprotective effect offlavonoids 607 Prostaglandin D2 (PGD2) 275

1022

Prostaglandin E2 (PGE2) 58,271,273, 598,616 Prostaglandin endoperoxide receptor antagonist 593 Prostaglandin H2 (PGH2) 595 Prostaglandin production 617 Prostaglandin synthesis 616 Prostaglandins 274,367,463,516,616 related eicosanoids 516 Prosystamin cDNA 370 Prosystemin 373,374,375 Prosystemin polypeptide 375 Protaneous lignads 368 Protease 65,483,941 Protease hydrolyzing casein 718 Proteases 483 Protein (SpA) mitogens 271 Protein kinase 261,518 Protein phsophotyrosine phosphatase (PTPase) 264, 522,548 Protein kinase C (PKC) 60,61,243,598, 734 Protein kinase B (PKB) 264 Protein kinase C inhibition 46,488 Protein kinase domain 384 Protein kinase inhibition (PKC) 45,513, 540,545 Protein phosphotyrosine phosphatases 518 Protein tyrosine kinase 519 Proteinaceous receptor 371 Proteinase K 388 Proteolysis assocaited proteins aspartic proteinase 370 carboxypeptidase 370 cysteine proteinase 370 leucine aminopeptidase 370 ubiquitin-like protein 370 Proteolytic inactivation of systemin 375 Proton pump inhibitors (PPIs) 612 Protooncogenes 47,269 Y-myc and bcl-2 267 Protophyrin IX (PPIX) 335,354,355 Protopine alkaloid allocryptopine 539

Prototropic cyclization 141 Prymnerium parvum 794 Prymnesin-2 794,795 Psamaplin B 858 Psammaplysilla purea 712 Psammaplysilla purpurea 719 Psammaplysilla sp 833 Psammaplysin A 784 Psammocinia 761 Pseudaxinyssa sp 857 Pseudaxinyssa pitys 854 Pseudoceratidine 785 Pseudoceratina verrucosa 712 Pseudoceratina purpurea 720,774,784 Pseudoceretina 719 Pseudodistoma aurum 782 P, pseudomallai 154 Pseudomonas 145,146,148,155,929 Pseudomonas aeruginosae 133,257,258, 259, 394,828 Pseudomonas citronellolis 132,133,165 Pseudomonas clavata 836 Pseudomonas convexa 133 Pseudomonas eruciviae 168 Pseudomonas fluorescens 152,394 Pseudomonas incognita 140,133,141,147 Pseudomonas medicinalis (Labiateae) 257 Pseudomonas pseudomallei 140 Pseudomonas putida 131,15 6,661 Pseudomonas putida-arvilla 153 Pseudomonas sp 152,153 Pseudomonas syringe 399,394,398 Pseudo-nitzschia multiseries 729 Pseudoplatanus 376 Pseudopterasin 689,690 Pseudopterogorgia elisabethae 690 Pseudopterosin E 689 Pseudoyohimbine 29,30 Psilocybe mexicana 531 PSK derivatives 378 PSK in mitosis 377 PSK precursor 377 PSK purification 378 PSK-receptor 378 PSK-a activity 378

1023

PSK-a analog 378 Psoitis tabricci 701 Ptilotafilicina 718 Pto-avrPto system 399 PtO'OvrPto complex 399 Pueraria lobata 93 Pueraria thomsonii 93 Puerariae Flos 91,94,99,100 Puerarlae lobata 91,103 PUFA 933 Puleagone 125,156 Pupukeanane skeleton 851 Purealidins 757 Purealidins N,P and Q 766,767 Purine receptors 532 Purpurin 543,544 Purpurogallin 546 Putative receptor proteins 398 Putrescine 66 Puupophenone 698,699 Puwainaphycin C 793, 794 Pyranocoumarins decursin 543 decursin angelate 543 Pyranoid linalool oxide cis and trans 143 Pyridinebetaine B 873 Pyrido [4,3,2,-m,n] thiazolo [322-b] acridinum skeleton 893 Pyrido [4,3,2,-ni,n,]acridine skeleton 894 Pyridoacridine 707,709 Pyridone-oxazole B 90063 841 Pyrogallol 613 Pyroloiminoquinones 824 Pyrolomycins 757 Pyrolysin 390 Pyronaridine 344 Pyrrol [2,1-j] quinolines 859 Pyrrolintrin 757 Pyrrolinyl-j8-carbolines G,H,J,P & Q 782 Pyrrolomycin antibiotics 794 Pyrroloquinolines 705,707 Pyrrolyl-)8-carbolines A and M 782 Pythium 303 Pytressin-induced coronary spasm 594

Quadranguloside 206 Quassine 59,548 Quercetagenin 541 Quercetin 44,56,541,543,544,546,593, 595,597,608,610,613-616 glycosylation 544 Queretaroic acid 60 Quidine 344 Quinacrine 593 Quinalizarin 543,544 Quinic acid 920,922,924,928,929 by microorganism 927 metabolism of 929 Quinic acid esters 919,947 Quinidine 347 Quinine 344,478,347,546 Quinizarin 544 Quinoline antimalarial drugs 329,348,349 Quinoline drugs 348 Quinoline family of drugs 343,360 Quinoline polymerization 348 Quinoline-based antimalarials 344 Quinoline-ring antimalarial drugs 327 Quinolines 327,345,347,348 Quinolin-heme complex 348 Quinolizidine alkaloid (7-hydroxy peganine) vasicinol 536 Quinolizidine alkaloid spartein (lupinidine) 534 R(resistance) gene 397 Race-specific elicitor 397,398 Race-specific peptide elecitors 398,399, 400,401 Radioligands 373 Radopholus similis 662 Raji cells 58,59,60 Ranitidine 612,617 rANP-triggered signal transduction 382 Rapamycin 892 Raphisia pallida 705 Raspaciona aculeata 700 Raspacionins 700,701 Raubasine (ajmalicine) 30,31

1024

Raunculaceae 180 Rauwolfia alkaloids 8 Reactive oxygen species 948 Reactive oxygen metabolites (ROMs) 614 Receptor-like protein kinases 383 Receptor protein phosphotyrosine phosphatase (RPTP) 522,548 Receptor serine/threonine protein kinase (RSTK) 518,522 Receptor tyrosine kinase (RTK) 512,522, 548 Receptor-like kinases 384 ai-Receptors 30 a2-Receptors 29,30 Redox-active metals 933 reduction of adverse effects of 5FU 454 Reinera sp 723 related compounds 538 Renealmia alpinia 266 Reserplc acid lactone 18 conformation 24 Reserpine 3,11-13,16,22,29,33,610 conformation 23 total synthesis 17,23 Resiniferatoxin 242 Response decay mechanism 491 Resveratol 546 Retinoicacid 59,60,515 Retinoids 44,515 Reverse transcription-polymerase chain reaction (RT-PCR) 322,477 Reverse transcriptase 941 Rhamnacease 469 3-O-Rhamnosyl-quercetin 541 Rhizobia 395 Rhizobiaceae 379 Rhizobium meliloti 395 Rhizobium symbiosis 659 Rhizopus nigricans 253,261,318 Rhodococcus erythropolis 150 Rhodoccus rubropertinctus 162 Rhodococcus robropertinctiis 165,166 Rhododendron sp. 242 Rhoifolin 594,595

Rhopaloeides sp 698 Rhopaoloic acid A B and C 698 Rhynchophylline 26 Ribavirin 31 Ribenol 250 Ribosomal 56 protein phosphorylation 525 Rimsulfuron 669 Ritterella sigillinoides 782,816 RNA synthetase 841 Robinia pseiido-acecia 93 Robinioside A 97 Robinioside B 96 Robinioside C 96 Robinioside D 96 Robinioside E 96 Robinioside F 96 Robinioside G 96 Robinioside H 96 Robinioside I 95 Robinioside J 95 Ropholic acid A 698,699 Rosa bourbonia oil 132 Rose oxide trans dindcis 138 Rosmarinic acid 921 Roylea calycina 252 RTK tyrosine kinase 545 Rubiaceae 180 Rubrolides A,B and C 789,790 Rubrosides A-H 763 Rubusfoliolosiis 251 Rudea aurea 93 Rufigallol 349,350,351 1,2,3,5,7-hexahydroxy anthraquinone 349 RuleofRuzika 130 Russula siibnigricans 765 Russuphelin A 765 RussupheUn B 765 Russuphelin C 765 Russuphelin D 765 Russuphelins 757 Rutin 469,608,610,613,615 Ryanodine receptor (RyR) 534,535

1025

Ryanodine receptor Ca^"*" channels 535 S-(4-Hydroxybenzyl) glutathione 820 13-e/7/-Sclareol 246 S. locus cysteine-rich protein (SCR) 387,402 S locus genes 386 S locus glycoprotein 386 S /ocw5 receptor kinase 386 S locus receptor-like kinase (SRK) 384,402,387 Saccharomyces carlsbergensis 759 Saccharomyces cerevisiae 143,258,780, 782 Saccharomyces fibuligera 308 Saccorliza polyschides 758 S-Adenosyl-L-homocysteine (SAH) 509 S-Adenosyl-L-methionine (SAM) 509 Safracins 826 Saframycin 826 Safranal 956 Sagitol 894,895 Saikosaponins 90,474, Salicyclic acid 391,653,655,659-662, 664,666,669 Salicylate hydrosylase 661 Salicylihalamides A and B 728 Silymarin 614 Sanguinarine 541 Salmo salar 723 Salmonella typhimurium 837 Salvia officinalis 12 8 Salvia sclarea 246,253 Sapium 242 Sapogenol 89,94,116 Saponin avenacin A-1 320 Saponin detoxification by fungi 304 Saponin detoxyfying enzyme 313,295, 304 Saponinase 312 Sarcoaldesterols A & B 704 Sarcoma 182 tumors 432 Sarcophytum sp 704 Sarcosine (N-methylglycine) 537

Sargassum lacerifolium 867 Saxitoxin 684,730 Sayasaponin 1 120 Scalaradiol 695,696 Scalardysin 697,900 Sceptrin 779 Schaffer collateral 959 Schizophrenia 531 Schizophyllum commune Fries 452 Scilla maritima 535 Scillarenin 3-O-glusyl rhamnoside 535 Sclareol 246, 252,253,255,259,267 biosynthesis 254 Sclareol glycol 265 Sclareol isomers 266 Sclareolide 253 7a-hydroxy derivative 253 Scleritoderma 703 Scoparia dulics 263 Scoparic acids A,B and C 263 Scots pine (Pinus sylvestris) 666 SCR gene experession 387 SDS buffers 338 SDS denaturation 333 SDS PAGE gels 370 SDS-PAGE analysis 315 SEB staphylococcal enterotoxin B 465 Secoadociaquinones A and B 861 Secoiridoid glycosides 471 Secolactam 33 Second messenger 516 2,3-Secoreserpine 12,33 3,4-Secoreserpine 12,33 (-)-Securinine 528 Seed oil (Croton oil) 242 Self-incompatibility (SI) 402 Self-incompatibility in pollen 386,393 Senieramycin 826 Sepedonium ampullosporum 318 Septoria lycopersici 293,295,301,304, 302,307,308, 314-316 erg-3 gene 304 Serine proteases (subtilases) 388

1026

cysteine proteinase inhibtor 370 poly phenol oxidase 370 serine proteinase inhibitor IT 370 Serine/theorine or tyrosine-specific protein kinases 528 Serine/threonine protein kinases 399,545 Serine/threonine phosphorylation 520 Serine-threonine phosphatase 799 Serine-threonine-specific receptor protein kinases (S/T-RPKs) 520 Serine-type acetylcholinesterase 719 Serotonin (5-hydroxytryptamine) 536 Serotonin 516,519,520,536,537,538,610 Serotonin agonists 531 anticomplementary activity 46 cyclic AMP-dependent protein kinase 46 delayed-type allergy suppressant activity 46 serotonin induced edema 46 Serotonin receptors 531 Serum ALT activity 462 Serum ALT and AST levels 466 Serum TNF-a 468,475 Serum transaminase activity 469 Serum transaminase levels 466,477 Serum transminases 467 Serum tumor necrosis factors (TNF-a) 459 Sesquiterpene isothiocyanates 848 Setaria italia 781 Seubert's medium 153 Sheffe'stest 101 Shermilamine B 820 Shermilamines D & E 820 Shigella boytic 260 Shigella lycopersici 306,313 Shigella Shiga 260 Shikimate pathway 328 Shikimate/arogenate pathway 652 Shikimicacid 928,929 Shizandra chinensis 476 Sho-Saiko-To pretreatment 474 ShugChher 275 Sideritis arborescens 249,250,

Sideritis foetens 250,254 Sideritis gomerae 249 Sideritis javalambrensis 250,263,274 Sideritis lycopersici tomatinase 308 Sideritis mugronensis 250 Sideritis nutans 249 Sideritis shiga 260 Sideritis varoi 250 Sidnutol 249 Sieberosidel 191,196,225 Sieberoside 11 196,225 Signal pathway-associated proteins 370 acyl-CoA binding protein 370 calmodulin 370 lipooxygenase 370 prosystemin 370 nucleotide diphosphate kinase 370 Signal regulated protein kinases 540 Signal transduction mechanisms 518 Signal transduction pathways 514,515 Signal transductors and activators of transcription (STATS) 519 Silicatein 717 Siliquariaspongiajaponica 723,759 Silymarin 612,614 Sinapic acid 434,653,655,920 Sinapis alba 781 Sinapyl-alcohol 653 Singmadocia symbioltica 902 Sino-artrial node 32 Sinomenine 472,476 hepatoprotective effect 472 Sinomenium aciitum 476 Sinularia disseda 704 Similariaflexibilis 690 Sinularia nanolobata 690 Site-1 proteases 390 Skimiate pathway 652,653 Smith degradation 452,438,439 Snake 8 kDa polypeptide toxin abungarotoxin 528 Snechocystis sp 718 Sodium channel blocker 684

1027

Sodium dodecyclosulfate-polyacrylamide 499 Sodium metaperiodate 134 Solanaceae 249,369,370,386,400 Solanaceous sp 304,368 a-Solanine 300,305,311 Solarium sp 293 Solon 618 Somato tropin (STH) 265 Sophorajlavescens 93 Sophora subprostrata 93 Sophoradin 612 Sophoradiol 95,111,112,115,116,118 Sophoradiol OG 94,99 Sophoradiol glucoronides 111,114,119 Sophoradiol glycoside 115 Sophoradiol monoglucuronide (SoMG) 111,112,113,114,115,121 Sophoraflavoside 96 Sophorodiol glucoroides 119 Sophoroflavoside III 96 Sophoroflavoside IV 96 Sopongiacidin B 798,799 Sorangium cellulosum 779 Sorbitol dehydrogenase 472 Sorgassum tortile 839 Souliea 180 Sowdanones 705 Soyabean saponins 222,223 hypocholesterdemic effects 222 Soyapogenin III 95 Soyapogenin IV 95 Soyapogenin V 95 Soyasapogenol A glycoside 103 Soyasapogenol B 94,95,102-104,111, 112,115,118-120,184-186,203 Soyasapogenol B glucuronides 114,119 Soyasapogenol B monoglucuronide (SBMG) 102,121 Soyasapogenol B OG 99 Soyasapogenol E glucuronides 114 Soyasapegenol E 97,115,120 Soyasaponin 90 Soyasaponin A3 95,104

Soyasaponin B 115,116 Soyasaponin 1 95,99,102,103,112,114, 115-118,120,203 Soyasaponin I and II 222,223 Soyasaponin II 103,115,116,119 Soyasaponin III 103,110,112,114,116-118 Soyasaponin IV 103,203 Soyasaponin V 104 Soyasaponins I-IV 101 Soyasaponin Me ester II 203 Soyasaponin Me ester III 203 Sphigolipid receptors 534 Sphigosine-1-phosphate 535 Sphigosyl-phosporylcholine 535 Sphingolipid receptor Ca^"^ channels 535 Sphingosine derivative 721 Sphizomeline 333 Spingosine kinase 269 Spiphonodicyton coralliphagem 694 Spirastrella abata 722 Spirastrella sp 719 Spirastrella spinispirulifera 759 Spiro (4,5) decane skeleton 851 Spiro isoxazole derviatives 757 Spirodysin 692 Spiroiridal triterpenoid 28-deacetylbelamcandal 543 Spirostane 55 Spirulina platensis 725 Spodoptera exigea 792 Spongia mycofijiensis 896 Spongia sp 759 Spongia official is 688 Spongiacidin A 798,799 Spongiacidins A-D 711 Spongiidae 688 Spongistatine 1 and 9 759,760 Spongistatins 4 and 5 759 Spongistatins 757 Spongosorites sp 707,799 Spontaneous metastasis 440 Stachytarpheta cayennenis 611,613 Staphylcooccus aureus 253,257,259,260 271, 765,770,776-778,789,791,821

1028

Staphylococcus boUyosiim 305 Staphylococcus epidermidis 116 Staphylococcus haemolyticus 116 Staphylococcus hominis 258 Stearic acid 333 Stellaria media 781 Stelleta 5/70/7ge species 700 Stelletamine 893,895 Stellettanide A and B 705 Stellettazoles B and C 706 Stemphylium botryosum 307 StemphyUum epidermidis 258,259 Stemphylium solani 302,303,307 Stemphylium sonnei 260,305 Stephania tetrandra 476 Steroid hormones 515 Steroidal glycoalkaloid 293,295,297,302 303 Stevia selriana 251 Stigmastane 49 Stigmasterol 61 Stilbene phytoalexin 662 Stilbene synthase 502,655 Stimulus-response mechanisms 515 Stratospheric ozone concantractions 663 Streptococcus durans 259 Streptococcus faecHis 259,260 Streptococcus fecal is var liquifaciens 923 Streptococcusfecium 925 Streptococcus group 925 Streptococcus pneumoniae 780 Streptococcus pyogenes 452,776 Streptococcus sp 257 Streptokinase/streptodornase 271 Streptomyces aldus 781 Streptomyces erythraeus 780 Streptomyces lavendulae 825 Streptomyces sp 717,781,828 Stroglycentrotus purpuratus 813 Strongylocentrotus nudus 788 Strongylophora 690 Strphanthus grains 537 Stylissa carteri 712,791 Styloguanidines 784 Stylotella agminata 110

Stylotella aiirantium 113,783,784 StylotellaneB 783 Stypopodium 691 Stypopodium zondils 691 St)>popodiimi-flabelliforme 690 Styracaster caroli 872 Sitberea creba 719 Suberin 655,658,666 Suberosenone 695 Subprosidell 95 Subprosidelll 97 SubprosidelV 96 SubprosideV 95,104 SubprosideVl 95 Subproside VII 95 Subtilase (SBT) 388,390,391,392,402 Subtilisin 388 Subtilisin/kexin-isozymel (SKI 1) 390 Subtilisin-like serine protease 388 Succinyl betulinic acid 61 Sulcatol 143,144,145,163 5'-0-Sulfanoyl-2-chlorodensine 781 Sulfated pentapeptide 376,377 Sulfer-containing furanosesquiterpenes 814 Sulfide pyridoacridines 819 Sulfide isoquinolines 824 Sulfide pyrroloquinolines 822 Sulfide pyridoacridines 819 Sulfobus soifataricus 720 Sulfonic acid 900 Sulfonoceramides 868 Sulfonoglycolipids 868 Sulfonolipids 868 Sulfoquinovosyl diacylglycerols (SQDG) 868 Sulforhadamine B (SRB) method 267 6-Sulfo-a-D-quinovopyranosyl-(l-7 3)l',2-diacylclycerol 868 Sulfur-containing diketopiperazine cyclo(L-Pro-thiopro) 828 Sulphonylurea drugs 534 Superoxide dismutase 931 Sweroside 471,476 serum ALT activity

1029

Swertia sp 471 Swertiajaponica 476 Swietenia mahagoni 943 Swinholide 730,731 Symbiodinium sp 722 Symploca hydnoides 887 Symplostatin 1 886,887 Symplostatin 2 886,887 Synaptic dopamine transporters 539 Synaptic glycine transporters 539 Synaptic serotonin transporters 539 Synoicum prunum 723 Syringyl subunits 653 Systematic wound reponse proteins 370 in potato plants 370 Systemic acquired resistance (SAR) 40,394 Systemic wound response protein 402 Systemin 85,371,374,375,377,380 derivative 389 Systemin proteolytic cleavage 376 Systemin (II) 370 Systemin maturation 376 Systemin oligopeptide 371 Systemin polypeptide 371 Systemin signal 374 Systemin synthesis 374 Systemin translocation 375 System in-triggered alkalinization 375 Systemin-triggered changes 374 Tagetone 143,144 Tangutorine 20,21,33 Tanichthys alobonubes 794 Taonia atomaria 249 Taraxastanes 59,64 faradiol 61 heliantriole 61 -Taraxastenes 57 \|/-Taraxasterol 57,58,59,64 Taraxerane 59 Taraxastane 57 Tathy lyncuriiim 718 Tatraclinis articidata 246

Taurine 536,537,872,873 Tauroacidin A 798,713,870 Tauroacidin B 798,713,870 Tauropine dehydrogenases 718 Tauropinnaic acid 871 Taurospongin A 721,871 Tawicyclamide A 883 Tawicyclamide B 883 Taxaceae 244 Taxodiaceae 249 Taxol 244,245 Taxotene 245 Taxiis baccata 245 Taxtts brevifolia IÂÎAS T-cell activation 463 T-cell subsets antitumor-effect 442 T-cell mediated cytotoxicity 466 T-cell mediated liver injury 463 Tea tannins 44 Teasterone 415,421,422 Teasterone oxidative reductive epimerization 422 Tebuquine 344 Tedania ignis 814,828,896 Testosterone 419,516 Teleocidin B 59,65 Terpene isothiocyanates 847 a-Tei-pincol 135,140,142,141,154 Terrestrial haloperoxidase 758 Tert-butyl hydrogeroxide 171 3-Tert-butyl-4-hydroxyanisole (BHA) 944,948 7,8,3',4'-Tetrahydroxyflavone 546 Tetaracyclic polyol esters 242 Tetrachloradibenzofuran 767 Tetrachlorodibenzodioxin 767 12-(9-Tetradecanoyl phorbol-13-acetate 44,222,272,542,948 Tetradecanoylphorbolacetate TPA response elements (TREs) 524 12-(9-Tetradeceanoy Iphorbol-13-acetate(TPA-1) induced edema inhibition 46 Tetrahedroswertianolin (THS) 477 Tetrahydroalstonine 30,31

1030

Tetrahydrobellidifolin 467 A^-Tetrahydrocannabinol 533,532 from marijuana 532 Tetrahydroharmine, (+) and (-) 16 1,3,4,5-Tetrahydropyrdo [4,3,2,de] quinoline 822 Tetrahydroisoquinoline 826,828 (-)- Tetrahydroharmine 18 Tetrahydroswertianolin (THS) 467 hepatoprotective activity 467 Tetrahydroxanthone 465 3A6a,16A25-Tetrahydroxy-20(7?),24(S)epoxy-9,19-cycloalanostane 179 3j3,6a, 16/J,24a-Tetrahydroxy-20,25epoxy 9)3,19-cyclolanostane 184 la, 3 A 16A27-Tetrahydroxy-9i8,19cyclolanost-24£-ene 185 cycloanthgenin 184 la, 7j3, 24a,25-Tetrahydroxy-9j3,19cyclolanostane 184 12-(9-Tetradecanoyl phorbol-13-acetate 271 7,8,3',4'-Tetrahydroxyflavone 541 Tetrahydro-A-carboHnes 782,815 3A 6a, 16/J,24a-Tetrahydroxy-20,25epoxy-9/J,19-cycloIanostane 184 Tetralysine 336 Tetramer russuphelol 765 Tetramic acid glucosides 763 Tetrandrine 472,476 Tetrane isothiocyanates 854 Tetranoic acid 695 Tetrapeptide derivative 376,377 Tetrasaccharide 295 1,2,4,5-Tetrathianes 842,843 Thalassiosira weissflogii 731 Thalassoma bifasciatum 7S7 Thalepogane skeleton 688 Thalictrum 180 Thapsia sp 536 Thapsigargin 536,543 thebaine 533 Theobromine 539

Theonella sp 714,715,771,772,775,878, 884,900 Theonella s^ 892 Theonella swinchoei 115,724,728,731, 848 Theonellamide 771,772 Theonellin isothiocynanate 848 Theonezolides A-C 984 Theonhella 11 \ Theophyline 488,539 Thermitage 388 Thermococcus tadyiricus 842 Theronine deaminase 370 Thiazole alkaloid 711 Thiazoline 881 Thiocoraline 842 {lR*,2R*,5R*,6S*,7S*)-2-Thiocyanatopupukeanane 869 (15*,45'*,65*,7/?*)-4-Thiocyanato-9cadinene 858 Thiocyanotoneopupukeanane 858 2-Thiocyanatoneopupukeanane 859 (-)-4-Thiocyanatonepupukeanane 858 Thiocyantoneopupukeanane 858 Thiodepsipeptide thiocoraline 841 Thiofurodysinin 814 Thiofurodysinin acetate 814,815 Thiomarlnol A 840 Thiomarinol C 840 Thiomarinol D 840 Thiomarinol F 840 Thiomarinol G 840 Thiomarinols 838,840 Thiomycololides A and B 831 5-Thiothistidine derivative 813 2-Thiothistidines 813 Thorecta choanoides 694 Thorectopsamwa xana 844 Threonine dephosphorylation 526 Threshold excitory level 491 Throboxane A2 600 Thromboxane B2 (TXB2) 274,275,600 Thromboxane synthase inhibitor 593 Thromolysin 438,439

1031

THS effect on hepatocyte apoptosis 467 THS inhibited hepatocyte apoptosis 468 Thymelaceae 242,243,246 ^H-Thymidine method 266 Thyroid harmones 515 triidothyronine 515 tetraiodothyronine 515 thyroxine 515 Tigliane diterpenoids 542 Tigliane diterpenoids esters 543 4-deoxyphorbol 543 12-deoxyphorbol 543 4,20-dideoxy-5-hydroxyphorbol 543 Tigliane skeleton 242 Time-off-flight (TOP) mass spectrometer 434 Tirucallane 50 Tistularin 720 TMB (3,4,5,-trimethoxybenzoy) 4 cytotoxic action of 477 TNF-a 461,462-465,469,472,474 treated macrophages 473 dependent liver injury 470 oncolytic effects 460 inhibition by THS 468 TNF-a cytotoxicity 475,477 TNF-a dependent liver injury 472,473, 476,477 TNF-a induced lethality 474 TNF-a production 468,470,471-473,475 suppression of 472 inhibition of 475 TNF-a receptor (TNF-R) 477 TNF-a-depdendent inflammatory liver injury 460,461,465,466 TNF-a-depndendent models 476 TNF-a-gene expression 470 TNF-a-induced hepatocyte apoptosis 471 TNF-a-induced hepatotoxicity 469,471 TNF-a-mediated cytotoxicity 466 TNF-a-production 469,470,477 TNF-a-sensitive L929 cells 467 Tobacco protoplasts 380 a-Tocopherol 236,792,930,936,944,946

Tolypathrix nodosa 691 Tomatidine 293 detoxification 293 Tomatidine 295,296,297,300,301,306, 316-318 /J-glucosyl hydrolases 308 enzymatic detoxification 304 from Botrytis cinerea 314 mechanism of action 306 of Fosaniim oxysporum 314 of Sideritis lycopersici 308,314 Tomatinase degrading enzymes 321 Tomatinase gene 313 Tomatinase-encoding genes 293 Tomatinase-induced genes 322 Tomatinase-producing transformants 308 Tomatinases 306 Tomatinase-encoding genes 295 /}2-Tomatinase 306 Tomatine (lycopericin) 532 deglycosilation of Tomatine 295,296,294,297,298,299, 300,301,302,306,311,312,315-322 insecticidal effects 294 glycoalkaloid 304 deglycosylation 295 toxic action 299 detoxification 296 deglycosylation of 317 Tomatine subproducts 317 Tomatine -detoxyfying enzymes 293 jSl-Tomatine 295,314 j82-Tomatine 317 Tomatine-detoxifying enzymes 295,306 a-Tomatine 293,296,297,306 steroidal glycoalkaloid 294 ^3]-Tomatine 295,317 j32-Tomatine 295,301,306 5-Tomatine 296 Y-Tomatine 296,297 Tomato (pro) systemin 371 Tomatosaponin metabolism 293 by phytopathogen fungi

1032

Tomentoside 1 199 Tomentoside 11 199 Topoisomerase (TOPO) Il-mediated decateration of kinetoplast DNA 820 Topoisomerase I 721 Topoisomerase 11 708,709,862 Topoisomerase Il-sensitive (CHO) cell lineXrs-6 824 Toposiomerase I and 11 708 Topsenia sp 703,704 Topsentinols A-J 703 Torpane alkaloid atropine 530 cocaine 537 ecgonine 537 hyoscamine 530 hyoscine (scopolamine) 530 Tondopsis glabrata 258 Totarane 241 Touroacidins A and B 797 Toxic free heme 327 Toxic xenobiotic 935 TPA (12-O-tetradecanoylphorbol-13acetate) 43 TPA induced edema (TPA) 45 TPA induced ornithine decarboxylase inhibition (ODC) 45,46 TPA induced skin tumors 46 TPA stimulated -^^Pi incorporation in HeLa cells 60 TPA-induced assay 58,61 TPA-induced ear edema 57,58 TPA-induced edema inhibition (CRO) 45 TPA-induced inflammatory edema 56 TPA-induced inflammation 47,48,63 TPA-induced ODC accumulation 60 TPA-mediated DNA 937 TPA-stimulated ^^Pi incorporation in HeLa cell inhibition (HeLa) 46 TPA-stimulated Pi 45 TPA-type tumor promotors 61 Trachycladine 775 Trachycladus laevispirulifer 115 Trachylobhim verrucosum 252

Trachyopsane 848,851 Trachyopsis halichondraides 704 Tradescantia nndtiflora 382 7Va/75decalin-tetrahydropyranylkalihinols 855 rra/75,/m^5-ceratospongamid 902,903 Tram-1,2-dihydroxy limonene 150 Transacylase 501 Transcriptase 722 Transcriptional factor 470 Transcriptional inhibitors 461 Transcylase 502 Transducin 521 7>'aA75-epoxysqualene 181 G1 o-Transgenic plant 661 ZVara-pinocarveol 154 D-(+)- Tm^^-sobrerol 152 7>aA75"-squalene oxide 182 Tras Glogi area 114 Traxacum officinale 391 Trelox 933 2-[3a,7a, 11 a-Trihydroxy-24-oxo-5/Jcholan-24-yl] amino ethane-sulfonate 872 3,3',4'-Trihydroxyflavone 544 Triacetic acid lactone 498 Triacetyl-l,2,-dipalmitoyl-3-0-(6'-sulfo-22',3',4'-D-quinovopyranosyl) glycerol 868 Tribrominated bisindole alkaloid 765 l,8-Tribromo-3,4,7-trichloro-3,7dimethyl-l,5-octadine 783 Tricetin 541,544 methylation 544 Trichlorinated orcinol 786 Trichloroleucine metabolite 801 Trichoderma sp 155 Trichoderma longibrachiatum 725 Trichoderma reesei 308 Trichophyton mentagrophytes 780 Trichosporon sp 150 Tricyclic xanthone 349 Tridecagalacturonides 486 Tridecapeptide polydiscamide A 867 Tridentata marginata 896

1033

Tridentatol A,B and C 895,896 Trididemnum solidiim 718 Trididemmm sp 819,820 Trifloiiim sp 667 Triflouroacetic acid (TFA) 6,434 Trifluoperazine 487 Trifluralin 669 Triglyceride lipase 523 Trigonoside 1 192 ,224 Trigonoside II 194,224 Trigonoside HI 194,224 3A 6a, 16/J -Trihydroxy-9,19-cyclolanost-24-ene 185 3/3, 6a, 16j3-Trihydroxy-9Al9-cyclolanost-24-oxo,25-ene 185 Trihydroxylean-12-ene saponins 179,191 3 j3,16/J,22 a-Trihydroxytaraxastene 62 3,7,11 -Trimethy 1-1,6,10-dodecatrien3-ol 161 (2)-3,7-11 -Trimethy 1-1,6-dodecadiene3,10,1 l,triol 164 3,7,11 -Trimethy 1-2,6,10-dodecatrien-1 -o 1 165 (2£,6£)-3,7,11 -Trimethyl-2,6-dodecadien1,10,11-triol 166 Trioloeolglycerol 333 3,4,5-Tri-O-Caffeoylquinic acid 920 2,3,4,Tri-0-methylgalactose 438 3',4',5'-Tri-(9-methyltricetin 541 D-Tryptophan 885 Tristamine 819,820 Triterpene alcohol 43,44 Triterpenes 478 bupleuroside III, IV and XIII 476 ginsenoside Rs and Rgl 476 glycyrrhizin 476 scorzoneroside A,B,C, 476 Triterpenoids and sterols antitumor and antiinflammatory activities 43 Trithiane 837 1,2,4-Trithiolanes 840,843 Trojanoside A 193 Trojanoside B 194 Trojanoside C 198

Trojanoside D 198 Trojanoside E 198 Trojanoside F 198 Tropanyl compounds 29 Trophozoites 332,333 acetonitrile extract of 333 Trophozoite stage 329 Trophozoite extract 334,342,354 Tropono-1,2-dihydro-3,6-phenathroline 820 Trunkamide A 883 Trypsin 483 Tryptophan 823,885,928 Tsitsikammamine A and B 709 Tubercidin 712,713 Tumor metastasis 440,454 Tumor necrosis factor a (TNF-a) 459, 460,461,477,598 Tumor-promoting phorbol ester 190 Tunicate Lissoclinium patella 879,880 Turpentine 151 TXB2 production 593 Type-lII secretion system 394,398 Typhasterol 415,422,423 oxidative reductive epimerization of 422 Tyrosine ammonia lyase (TAL) 669 Tyrosine dephosphorylation 526 Tyrosine kinase inhibitors 720,691 Tyrosine kinase-mediated signalling 545 Tyrosine kinases 519,518,703 JAKl andJAK2 519 Tyrosine kinases 597 Tyrosine phosphatases (PTPases) 519 Tyrosine phosphorylated STAT dimer (P-STAT) 522,548 Tyrosine phosphorylation 519 U2 66 myeloma cell 273 UDP-galactosamine (UDP-GalN) 461 U-endo-^l,4galactanase 436 U-exo-)8-galactosidase 436 Ulicyclamide 880,882 Ulithiacyclamide 879

1034

Ulithiacyclamide B 879 Ulithiacyclamide E 879 Ulithiacyclamide G 879 Viva sp 725 Umbelliferae 471 Umbraculum mediaterraneum 831 Uncarine C,D,E and F 25 a,JÛnsaturated butenolide 276 A^-Unsaturated sterols schottenol 56 spinasterol 56 Urethanase 719 Uric acid 930 Uronic acids 435,486 Uronide elicitors 488 Uronide complex esterification of 486 Ursane 53,57,59 Ursolic acid 59,60,61,66,543 Vstilago maydis 389 Valencene 125,128,170,171 2-Vanilloyl imidazole 837 Vanadium haloperoxidase 758 Vancomycin 791 Vanilla planifolia 126 Vanillic acid 90,671,924,925 /?-Vanilliccinnamic acid 923 Vanillin 126 Vanilloylglycine 90,923 VaracinA 834 VaracinB 834,835 VaracinC 835 Varacin-lissoclinotoxin 833 Varamines 893 Variabilin 717,718 Varamines 821 Vasorelaxant chalcone 540 Veramines 821 Verapamil Ca^^^-channel blocker 487 Verapamil 673 Ca^"^-channel blocker 487 Veratridine 534

Verbascoside 468 Verbenaceae 249 Verbenol 152 C/.s-D-Verbenol 152 Verbenone 152,153 Verongamine 721 Verongida gigantea 721 Veronica persica 781 Verticillium albo-artrum 299,307,302, 311 Verticillium dahliae 305,307,311 Vescictdar stomatitis 779 Vesicular glutamate transporters 538 Vesicular monoamine transporters 538 Vexibinol 612 Viagra (sildenafil) 541 Vibrio angucillarum 877 Vibrio cholerae 521 Vibrio valnificiis 718 Viburnum suspensum 251,257,259 Viciafaba 93 Vicia sativa 93 Vigna angidaris 93 Vigna unguicidata 93 Vinblastine 269 4.Vinylcatechol 925,926 Vinylfurans 694 Vinylogous urethane 10,11,33,34 Viral glycoproteins 388 Virenamide A-E 888 Virenamide 888 Vitamin C,E,K,A,D 236 Vitexin 594,595 Vinylcatechol 927 Voltage-gated Ca^''" channels 517 Voltage-gated ion channels 520 Voltage-gated K"^ channel 531 Voltage-gated Na"*" channels 533 Volutamides 757 Volutamides A-E 786 Waitzia acuminata 251 Walsh-Krebs inhibitors protein 523 Warangolone 541 Wayakin 707

1035

Welwiltindolinone alkaloids 802 Welwitindolinoric A isonitrile 780 Winn assay 432 Wistaria brachybotrys 222 Wistariasapogenol A 97 Wistariasapon n A2 97 Wistariasapon n A3 97 Wistariasapon n B 2 95 Wistariasapon n B 3 95 Wistariasapon n C 222 Wistariasapon n D 97 Wistariasapon n D 116,118 Wistariasapon! n Y C i 96,104 Wistariasapon n YC2 96 Wistariasapon ns 222 Wisterai brachybotrys 93

X-ray absorbance spectroscopy (XAS) 359 X-ray absorption fine spectroscopy (EXAFS) 338 Xylanases 312 9-[5'deoxy-5'(methylthio)-j3-D-Xylofurano syl)adenine 829 6-0-j^D-Xylopiranoside 224 j3-Xylosidase 210

Xanthium spinosum 781 Xanthocephalum linearifolium 251 Xanthomonas oryzae 395,398 Xanthomonas compestris 399 pv vesicatoria 399 Xanthone 350,351 Xanthone a-mangostin 531,536 inhibitor of Ca^"*" ATPase 536 Xanthone hypothesis 351 Xanthone-a-mangostin 536 inhibitor of Ca2"^-ATPase 536 Xenia 690 Xenicane 690 Xenopus 294 Xestaspongins 732 Xestomycin 826 Xestoquinones 861 Xestoquinolide B 862 Xestospongia 694 Xestospongia genus 861 Xestospongia cf carbonaria 861 Xestospongia sp 703, 709, 710,769,859, 898 Xestospongia ashmorica 710 Xanathomonas adaptation 399

Zea mays 671 Zingiberaceae 249,266 Zinnia elegans 317 Zn(n) protoporpyhyrin (IX) 335 Zoanthellatoxin B 722 Zoanthid 711 Zoanthius sp 711 Zymogen activation 391 Zymosan-activated macrophages 274 Zyzzafuliginosa 708,760,770 Zyzza massalis 760,898 Zyzzya cf.marsailis 790,824

Yarrowinina lipolytica 150 Yeast kex 2 protease (Kexin) 387,388, 389 Yohimbine 20,29 Yohimibine-type alkaloids 3 Yunganogenin C 96


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