Studies in Natural Product Chemistry, Volume 27: Bioactive Natural Products, Part H (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry Volume 27 Bioactive Natural Products (Part H)

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

Vol. 1 Stereoselective Synthesis (Part A) Vol. 2 Structure Elucidation (Part A) Vol. 3 Stereoselective Synthesis (Part B) Vol. 4 Stereoselective Synthesis (Part C) Vol. 5 Structure Elucidation (Part B) Vol. 6 Stereoselective Synthesis (Part D) Vol. 7 Structure and Chemistry (Part A) Vol. 8 Stereoselective Synthesis (Part E) Vol. 9 Structure and Chemistry (Part B) Vol.10 Stereoselective Synthesis (Part F) Vo1.l IStereoselective Synthesis (Part G) Vo1.12 Stereoselective Synthesis (Part H) Vol. 13 Bioactive Natural Products (Part A) Vo1.14 Stereoselective Synthesis (Part I) Vo1.15 Structure and Chemistry (Part C) Vol. 16 Stereoselective Synthesis (Part J) Vo1.17 Structure and Chemistry (Part D) Vo1.18 Stereoselective Synthesis (Part K) Vo1.19 Structure and Chemistry (Part E) Vo1.20 Structure and Chemistry (Part F) Vo1.21 Bioactive Natural Products (Part B) Vo1.22 Bioactive Natural Products (Part C) Vo1.23 Bioactive Natural Products (Part D) Vo1.24 Bioactive Natural Products (Part E) Vo1.25 Bioactive Natural Products (Part F) Vo1.26 Bioactive Natural Products (Part G ) Vo1.27 Bioactive Natural Products (Part H)

Studies in Natural Products Chemistry

Volume 27 Bioactive Natural Products (FWt H)

Edited by

Atta-ur-Rahman

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

2002

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FOREWORD The present volume, the 27*^ of this series, is devoted to the chemistry of several exciting classes of natural products. The article by Kinghom and Kim reviews the current state of knowledge of sweet-tasting and sweetness-modifying constituents of plants. Some of these highly sweet natural products are already marketed as sweetners or flavoring agents in some countries. Spiteller has presented an overview of chemical responses to plant injury and plant aging. Interest in sialic acid chemistry is growing, particulariy due to its role in the regulation of a number of important biological processes. The article by Bianco and Melchioni on the structure, chemistry and biological activity of neuraminic acid covers this area comprehensively. Quaternary benzo[c]phenanthridine alkaloids (QBA) are bright coloured compounds which are interesting both for their chemistry and their biological activities. The article by Dostal and Slavfk covers some aspects of the chemistry of these compounds. Sicker and Schuiz have reviewed the field of acetai glycosides of the 2hydroxy-2H-1, 4-benzoxazine-3(4H)-one skeleton which occur naturally in a number of plant families and which can impart resistance in plants towards insects, microbes as well as pathogenic fungi. Structural identification and bioactivity aspects of simple indolizidine and quinolizidine alkaloids isolated from amphibians, ants, fungi, plants and marine sources is covered comprehensively in an article by Lourenco and co-workers. These substances are responsible for the toxic and teratogenic effects observed in livestock and their action can be associated with their affinity for nicotinic and muscarinic receptors. The metabolism of stevioside has been described by Geuns. Monoterpenoids occur widely in various plant and are also

important constituents

in essential oils.

Enantioselective chromatographic analysis and bioactivity of chiral monoterpenoids has been reviewed by Asztemborska and Ochocka. Abscisic acid is the primary hormone which induces adaptive reactions in plants to various environmental stresses. More than 100 abscisic acid analogs of this compound have been reported so far. The scope of using abscisic acid analogs for probing the mechanism of abscisic acid reception and inactivation is presented by Todoroki and Hirai. Kimura and Okuda have reviewed the biochemical and phamriacological studies of natural products isolated from various medicinal plants and foodstuffs including their effects on lipolysis and iipogenesis in fat cells anti-obesity action, lipid and arachidonate metabolism and reduction of side effects of cancer chemotherapy. Astragalus L. is the largest genus in the family Leguminosae and it is widely distributed throughout the temperate regions of the worid, particulariy in Europe, Asia and North America. The structures and biological activity of secondary metabolites of this genus is presented by Pistelli. Plants of the genus Tanacetum (Compositae) have been used for their medicinal properties for over 2000 years.

VI

Chemical characterization and biologicai activities of compounds found in this genus are reviewed by Goren and co-workers. Contreras and co-workers have described bioactive components of Bupleurum rigidum L. subscp. rigidum, some of which have shown interesting anti-inflammatory activity. Olive oil is widely consumed in Mediterranean countries and is considered to be of benefit in reducing risks of coronary heart disease and cancer. DeH'agli and Bosisio have presented the chemistry and bioactivity of minor polar compounds of olive oil. Class III plant peroxidases are a polymorphic group of heme-containing enzymes located in the vacuole and the plant cell wall. The article by Barcel6 and Pomar covers the chemistry and bioactivity of plant peroxidases. The review of Llakopoulou-Kyriakides covers the field of naturally occurring oligopeptides and presents a variety of biological activities possessed by such compounds. The process of phosphorylation and dephosphorylation of proteins in a variety of physiological events is of growing interest. Manez and Recio have described the modulation of protein phosphorylation reactions by different natural products. There has been considerable interest in the chemistry and biological activity of flavonoids. The cytotoxic effects of flavonoids on cancer cell lines is discussed by Ayuso and co-workers. It is hoped that this volume will be another substantial addition to this series which has benefited from contributions by most of the leading natural product chemists of the worid in the past. I would like to express my thanks to Dr. Shakeel Ahmad and Miss Farzana Siddique for their assistance in the preparation of the index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.

Atta-ur-Rahman Ph.D. (Cantab), Sc.D. (Cantab) Minister for Science & Technology Government of Pakistan

April, 2002

Vll

PREFACE Natural Product Chemistry continues to present exciting opportunities for medicinal chemists to discover new bioactive compounds against various diseases. Even in cases where the structures are too complex to be obtained on a large scale by synthesis, the interest is often focussed on those portions of the structures which are responsible for the biological activity and which can serve as simpler phamnacophores for synthesis and study of structure-activity relationships. The enomrious structural diversity offered by natural products of terrestrial and marine origin therefore offers a vast resource to medicinal chemists in search for new bioactivity profiles or mechanisms of actions. The present volume of "Studies in Natural Product Chemistry", the 2 / ^ of this prestigious series, presents the current frontiers in several important fields of natural product chemistry. All-in-all, the volume presents a very interesting collection of comprehensive reviews by leading experts in a number of important fields. It should be though of as a valuable new edition to this important encyclopedic series on natural product chemistry. Prof. Atta-urRahman, the Editor of this series, is now the Federal Minister for Science and Technology of the Government of Pakistan and it is noteworthy that he manages to take out time to continue his interests in chemistry as Director H.E.J. Research Institute of Chemistry at Karachi University, as Editor of this Series, as well as Editor of a number of intemational chemistry journals including "Current Organic Chemistry", "Current Medicinal Chemistry" and Co-Editor of "Natural Product Letters". He deserves congratulations for maintaining the high standard of this excellent series in the field of Natural Product Chemistry, which should be of interest to a large number of natural product and medicinal chemists who wish to keep breast with developments in biologically active natural products.

Steven V. Ley, FRS, C.B.E. Professor of Organic Chemistry and Novartis Research Felllow Department of Chemistry Cambridge University Lensfield Road Cambridge CB2 1EW U.K. April, 2002

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ix

CONTENTS Foreword

V

Preface

vii

Contributors

xi

Sweet-tasting and sweetness modifying constituents of plants NAM-CHEOL KIM AND A. DOUGLAS KINGHORN

3

Chemical responses to plant injury and plant aging GERHARD SPITELLER Neuraminic acid and its Structures, Chemistry, Biological Activity ARMANDODORIANO BIANCO AND CRISTIANA MELCHIONI

59 103

Some aspects of the chemistry of quaternary benzo[c]phenanthridine alkaloids

JIEDOSTAL AND JIM SLAV~K

155

Benzoxazinones in plants: occurrence, synthetic access, and biological activity DIETER SICKER AND MARGOT SCHULZ

185

Indolizidine and quinolizidine alkaloids structure and bioactivity A.M. LOURENCO, P. MAXIMO, L.M. FERREIRA AND M.M.A. PEREIRA

233

Safety evaluation of Stevia and stevioside JAN M.C. GEUNS

299

Abscisic acid analogs for probing the mechanism of abscisic acid reception and inactivation YASUSHI TODOROKI AND NOBUHIRO HIRAI

32 1

Chiral monoterpenoids in plants-enantioselective chromatographic analysis, and their bioactivity MONIKA ASZTEMBORSKA AND J. RENATA OCHOCKA

361

Biochemical and pharmacological studies of natural products isolated from various medicinal plants and foodstuffs YOSHIYUKI KIMURA AND HIROMICHI OKUDA

393

Secondary metabolites of Genus Astragalus: structure and biological activity L. PISTELLI

443

X

Chemical characterization and biological activities of the genus Tunaceturn (Compositae) NEZHUN GOREN, NAZLI ARDA AND ZERRIN CALISKAN

547

Bioactive components of Bupleurum rigidum L. Sub sp. rigidurn S. S h C H E Z CONTRERAS, ANA M. DiAZ LANZA, M. BERNABE PAJARES, C. BARTOLOME ESTEBAN, L. v. CASTILLO, M.R.A. MART~NEZ,P.B. BENITO AND L.F. MATELLANO

659

Minor polar compounds of olive oil: Composition, factors of variability and bioactivity MARIO DELL’AGLI AND ENRICA BOSISIO

697

Plant peroxidases: versatile catalysts in the synthesis of bioactive natural products A. ROS BARCELO AND F. POMAR

735

Naturally occurring oligopeptides with more than one biological activities M. LIAKOPOULOU-KYRIAKIDES

793

Modulation of protien phosphorylation by natural products S. M-Z AND M. DEL CARMEN RECIO

819

Cytotoxicity of flavonoids of cancer cell lines. Strucutre-activity relationship M. LOPEZ-LAZARO, M. GALVEZ,C. MARTIN-CORDER0 AND M.J. AYUSO

89 1

Subject Index

933

XI

CONTMBUTORS Nazli Arda

University of Istanbul, Faculty of Science, Department of Biology, Vezneciler 34459, Istanbul, Turkey

Monika Asztemborska

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland

M.J. Ayuso

Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

A. Ros Barcelo

Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain

Paulina Bermejo Benito

Departamento de Farmacologia, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain

Araiandodoriano Bianco

Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali-Facolta di Scienze Matematiche, Fisiche e Naturali Universita La Sapienza Roma, Italy

Enrica Bosisio

Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

Zerrin ^aliskan

Yildiz Technical University, Faculty of Science and Arts, Department of Biology, Main Campus, Cukursaray, 80750 Besiktas-Istanbul, Turkey

Lucinda Villaescusa Castillo

Laboratorio de Farmacognosia, Departamento de Farmacologia, Facultad de Farmacia, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain

Sandra Sanchez Contreras

Laboratorio de Farmacognosia, Departamento de Farmacologia, Facultad de Farmacia, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain

Mario Dell'Agli

Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

Jifi Dostal

Department of Biochemistry, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, CZ-662 43 Bmo, Czech Republic

Xll

Carmen Bartolome Esteban

Departamento de Biologia Vegetal, Facultad de Ciencias, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain

L.M. Ferreira

Departamento de Quimica, Centre de Quimica Fina e Biotechnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825-114 Caparica, Portugal

M. Galvez

Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

Jan M.C. Geuns

Laboratory of Plant Physiology, KULeuven, Mercierlaan 92, B 3001, Leuven, Belgium

Nezhun Goren

Yildiz Technical University, Faculty of Science and Arts, Department of Biology, Main Campus, Cukursaray, 80750 Besiktas-Istanbul, Turkey

Nobuhiro Hirai

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Nam-Cheol Kim

Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, USA

Yoshiyuki Kimura

Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

A. Douglas Kinghom

Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA

M. LiakopoulouKyriakides

Department of Chemical Engineering, Section of Chemistry, Aristotle, University of Thessaloniki, Thessaloniki 54006, Greece

Ana M. Diaz Lanza

Laboratorio de Farmacognosia, Departamento de Farmacologia, Facultad de Farmacia, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain

M. Lopez-Lazaro

Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

Kard.

Xlll

A.M. Loviren9o

Departamento de Quimica, Centro de Quimica Fina e Biotechnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825-114 Caparica, Portugal

Salvador Maiiez

Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Av. V.A. Estelles s/n, 46100 Buijassot, Spain

C. Martin-Cordero

Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

Maria J. Abad Martinez

Departamento de Farmacologia, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain

Lidia Fernandez Matellano

Laboratorio de Farmacognosia, Departamento de Farmacologia, Facultad de Farmacia, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain

P. Maximo

Departamento de Quimica, Centro de Quimica Fina e Biotechnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825-114 Caparica, Portugal

Cristiana Melchioni

Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali-Facolta di Scienze Matematiche, Fisiche e Naturali Universita La Sapienza Roma, Italy

J. Renata Ochocka

Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland

Hiromichi Okuda

Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

Manuel Bemabe Pajares

Departamento de Quimica Organica Biologica, Instituto de Quimica Organica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

M.M.A. Pereira

Departamento de Quimica, Centro de Quimica Fina e Biotechnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825-114 Caparica, Portugal

L. Pistelli

Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno, 33-56126 Pisa, Italy

XIV

F. Pomar

Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain

Maria Del Carmen Recio

Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Av. V.A. Estelles s/n, 46100 Burjassot, Spain

Margot Schxilz

Universitat Bonn, Institut fiir Landwirtschaftliche Botanik, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany

Dieter Sicker

Universitat Leipzig, Institut ftir Organische Chemie, Johannisallee 29, 04103 Leipzig, Germany

Jifi Slavik

Department of Biochemistry, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, CZ-662 43 Bmo, Czech Republic

Gerhard Spiteller

Organische Chemie I, Universitat Bayreuth, Universitatsstrape 30, 95440 Bayreuth, Germany

Yasushi Todoroki

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

Bioactive Natural Products

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 27 © 2002 Elsevier Science B.V. All rights reserved.

SWEET-TASTING AND SWEETNESS-MODIFYING CONSTITUENTS OF PLANTS NAM-CHEOL KIM^ and A. DOUGLAS KINGHORN* Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, U.SA, ABSTRACT: The demand for new alternative sweeteners has increased due to certain health problems associated with the use of sucrose. Although the currently developed and conunercially used sucrose substitutes are mostly synthetic compounds, the search for such compounds from natural sources is continuing and about 85 plant-derived sweet compounds of 19 major structural types are known, which have been obtained from 25 different families of green plants. Some of these highly sweet natural products are marketed as sweeteners or flavoring agents in several countries either as pure compounds or refined extracts. Several naturally occurring sweeteners have been chemically and enzymatically modified in order to increase their sweetness potency and/or improve their sweetness quality. In addition to natural sweet-tasting compounds, a number of naturally occurring sweetness-modifying compounds which induce or inhibit the sweet taste have also been isolated from plant sources. Several proteins and triterpenoids have sweetness-inducing properties. Over 60 triterpenoid glycosides have been reported from five plant species of the families Asclepidaceae and Rhamnaceae as sweetness-inhibitory (antisweet) principles.

INTRODUCTION The consumption of sucrose as a sweetener has been associated with several nutritional and medical problems, with dental caries being the most well-documented [1]. Sucrose intake may also be a factor in cardiovascular disease, diabetes mellitus, obesity, and micronutrient deficiency [2]. Therefore, there has been a continual demand for novel * Address correspondence to this author at Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, U.S.A. E-mail: [email protected]. ^ Current address: Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, U.S.A.

highly sweet, non-caloric and non-cariogenic sucrose substitutes for the diabetic and dietetic market. Synthetic or natural sucrose substitutes are required to exhibit a sucrose-like taste quality with properties such as demonstrated non-toxicity, non-cariogenicity, lack of any offensive odor, and should exhibit satisfactory water solubility and hydrolytic and thermal stability. The so-called "high potency" sweeteners are at least 50100 times more highly sweet than sucrose [3]. Such compounds are also referred to as "intense sweeteners" and may be placed in a separate sweetener category than the less sweet caloric or "bulk" sweeteners represented by certain monosaccharides, disaccharides, and polyols, which are approximately equal to sucrose in sweetness potency [4,5]. To date, most of the currently available potently sweet sucrose substitutes in the world market are synthetic compounds: these include acesulfame-K, alitame, aspartame, cyclamate, saccharin, and sucralose [5,6]. These synthetic sweeteners are used as sucrose substitutes in most western countries but the regulations for each sweetener vary from country to country [7-12]. At present, in the United States, four are permitted as food additives, namely, acesulfame-K, aspartame, saccharin, and sucralose [5]. In the United States, food substitutes inclusive of the artificial sweeteners account for an approximately $1.2 billion market [6]. However, several problems with these compounds have long been apparent. For example, the general-purpose sweetener aspartame may not be consumed by persons with phenylketonuria because of the formation of a major metabolite, phenylalanine [9], Saccharin has been used as a sweetener for many years, but is now permitted only on an interim basis, owing to an association with bladder cancer in laboratory animals [11]. Therefore, currently, containers of products that include saccharin must have a cancer warning and state the amount of this sweetener [5]. Cyclamate was used in the United States until the Food and Drug Administration (FDA) banned this sweetener in 1969 because a saccharin and cyclamate mixture was found to cause cancer in laboratory animals [13]. However, cyclamate is still used as a sucrose substitute in about 50 countries [7]. A major metabolite of cyclamate is cyclohexylamine, which is somewhat toxic in causing testicular atrophy and untoward cardiovascular effects at high doses [7]. The search for improved sucrose substitutes is continuing, and one of the most potent sweeteners synthesized so far is the Ncyclononylguanidine derivative, sucrononic acid. This is actually the sweetest compound reported in the literature to date with a sweetness

potency of some 200,000 times that of sucrose [14]. Another synthetic compound of interest is superaspartame, which is produced by the combination of the urea derivative, suosan, with aspartame, and is about 14,000 times sweeter than sucrose [15]. Neotame, a A^-alkylated aspartame derivative, has been developed as a non-caloric sweetening agent, and has a sweetness potency of 10,000 times that of sucrose [15]. Besides the naturally occurring saccharides and polyols, there are a number of plant-derived highly sweet compounds, which are mostly terpenoids, flavonoids, and proteins [16-18]. Several of these sweet substances are used commercially as sucrose substitutes, as will be described in the next section. In addition, a number of plant substituents are known to mediate the sweet-taste response, either by inducing or inhibiting the perception of sweetness [19]. Thus far, all of the known natural product sweet-tasting substances and sweetness modifiers have been obtained from green plants [16-19]. In the remaining sections of this chapter, plant-derived sweet compounds with commercial use will be described, followed by a section on recent theories on the sweet taste phenomenon, and then individual descriptions of potent sweeteners, sweetness inducers, and sweetness inhibitors from plants will be presented in tum. The literature has been surveyed for this chapter until the end of 1999. Commercially Used Highly Sweet Natural Products While many isolated natural compounds have a sweet taste [20], only a few of these have been developed for commercial use. Natural product highly sweet compounds with some commercial use include glycyrrhizin (1), mogroside V (2), phyllodulcin (3), rebaudioside A (4), stevioside (5), and thaumatin, which are used as sucrose substitutes in one or more countries [16,21]. Some of these compounds have been modified chemically or biochemically to produce analogs that are more desirable as sweeteners, in being more highly sweet and/or more pleasant tasting. Although a number of commercially available "bulk" sweeteners with approximately the same sweetness potency as sucrose are naturally occurring, these compounds will not be considered further in this chapter. Examples include the monosaccharide, fructose; the monosaccharide polyols, erythritol, mannitol, sorbitol, and xylitol; and the disaccharide polyols, lactitol, and maltitol [5].

Glycyrrhizin (1), also known as glycyrrhizic acid, is an oleanane-type triterpenoid diglycoside isolated from the roots of Glycyrrhiza glabra L. (Leguminosae) and other species in the genus Glycyrrhiza [21]. Glycyrrhizin (1) is 93-170 times sweeter than sucrose, depending on concentration. In Japan, root extracts of G, glabra (which contain >90% w/w pure glycyrrhizin) are used to sweeten foods and other products, such as cosmetics and medicines. The ammonium salt of glycyrrhizin has Generally Recognized As Safe (GRAS) status in the United States and is used primarily as a flavor enhancer [22]. There have been several attempts using various glycosylation methods to increase the sweetness potency of glycyrrhizin (1). The Tanaka group at Hiroshima University in Japan glycosylated glycyrrhetinic acid to afford various glycyrrhizin monoglycoside analogs using a chemical and enzymatic glycosylation procedure [23]. A coupling reaction using mercury(II) cyanide [Hg(CN)2] for chemical glycosylation was effected, resulting in a significant enhancement of sweetness in the analogs obtained, especially the 3-0-pD-xylopyranoside (6) and 3-O-P-D-glucuronide (MGGR, 7). The sweetness intensities of compounds 6 and 7 were rated as 544 and 941 times sweeter than sucrose, respectively. Such chemically modified products of glycyrrhizin also showed improved taste qualities [24]. MGGR (7), in being more than five times sweeter than glycyrrhizin (1), as well as being readily soluble in water, is now used commercially as a sweetening agent in Japan [25]. COOH

1

R=p-glcA2-P-glcA

6

R=|3-xyl

7

R = P-glcA

Mogroside V (2) is a cucurbitane-type triterpenoid glycoside isolated from the fruits of Siraitia grosvenorii (Swingle) C. Jeffrey

(Cucurbitaceae) [26]. An extract of the dried fruits of S, grosvenorii, containing mogroside V (2) as the major sweet principle, is used in Japan as a sweetener in certain foods and beverages. The sweetness intensity of mogroside V (2) has been rated as 250-425 times sweeter than sucrose, depending on concentration [22]. Recently, a major corporation in the United States has filed a patent concerning the use of extract of S. grosvenorii and other Siraitia species as a sweet juice [27].

P-glc^p-glc

p-glc^-P-glc

p-glc

A dihydroisocoumarin-type sweetener, phyllodulcin (3) occurs in glycosidic form in the leaves of Hydrangea macrophylla Seringe var. thunbergii (Siebold) Makino (Saxifragaceae) ("Amacha") and other species in this genus. After the fermentation of the leaves or by crushing, the native glycosides are enzymatically hydrolyzed, and the sweet phyllodulcin (3, x 400 sweeter than 2% sucrose) is produced. The fermented leaves of K macrophylla are used to prepare a sweet ceremonial tea in Japan, especially at "Hamatsuri", a Buddhist religious festival [22].

Rebaudioside A (4) and stevioside (5) are en^kau^ene-type diterpene glycosides isolated from the leaves of the Paraguayan plant, Stevia rebaudiana (Bertoni) Bertoni (Compositae) [28,29], with stevioside (5) being the more abundant compound in this plant part. The sweetness intensity of stevioside (5) has been rated as 210 times sweeter than sucrose, although this value varies with concentration [30]. However, rebaudioside A (4) (the second most abundant 5. rebaudiana e«/-kaurene glycoside with a sweetness intensity rated as about 240 times sweeter than sucrose) is considerably more pleasant-tasting and more highly water-soluble than stevioside (5), and thus better suited for use in food and beverages [30]. Extracts of S. rebaudiana containing stevioside and/or purified stevioside are permitted as food additives in Japan, South Korea, Brazil, Argentina, and Paraguay, and have been used as herbal dietary supplements elsewhere, such as in several European countries, the People's Republic of China, and the United States [30]. In Japan, the largest market for the S. rebaudiana sweeteners to date, three different forms of stevia sweetener products are commercially available, namely, "stevia extract", "sugar-transferred stevia extract", and "rebaudioside A-enriched stevia extract". "Stevia extract" is a powder or granule made by several industrial steps and standardized so as to contain more than 80% of steviol glycosides, inclusive of dulcoside A (3-5%), rebaudioside A (20-25%), rebaudioside C (5-10%), and stevioside (5055%) [31]. "Sugar-transferred stevia extract" is made by transglycosylation of steviol glycosides present in commercially available "stevia extract" with a cyclomaltodextringlucanotransferase (CGTase)starch system prepared from Bacillus macerans [24]. There have been many attempts to improve the taste qualities of the major S. rebaudiana sweet steviol glycoside, stevioside (5), because of its sensory limitations [24,32-36]. Several systematic studies on the structure-sweetness relationship of steviol glycosides have been conducted [35,37]. For example, the sweetness-pleasantness of stevioside (5) may be increased by treating stevioside-galactosyl ester (Sgal), prepared by removal of the 19-0-glucosyl group of stevioside, and replacing it with a p-galactosyl group. Transglucosylation of the intermediate with soluble starch using CGTase prepared from 5. macerans then affords a mixture of mono-, di-, tri-, and tetra-a-glycosylated compounds. The product with four glucosyl units attached at the C-13 position showed an enhanced sweetness (8, Sgal-2) [35]. A rebaudioside A analog (9) with a (sodiosulfo)propyl

group at C-19 in place of a p-glucosyl moiety showed improved sweetness qualities [33]. Stevioside (5) has been converted synthetically to rebaudioside A (4), by removing a glucose unit from stevioside (5) at the C-13 position using amylase and then reintroducing synthetically two glucose units of different linkage to the remaining glucose unit at the C13 position [38]. "Rebaudioside A-enriched extract" is made from improved varieties of S. rebaudiana, which produce more rebaudioside A (4) than the native Paraguayan species [39].

COOR1 19

4

Ri

R2

p-glc

P-glc^-P-glc p-glc

5

p-glc

P-glc'-P-glc

8

P-gal

P-glc^-P-glc^-p-glc^-a-glc

9

(CH2)3S03Na

p-glc^-p-glc p-glc

Thaumatin is a protein sweetener isolated from the fruits of Thaumatococcus daniellii (Bennett) Benth. (Marantaceae) [40]. Five different thaiunatin analogs are now known (thaumatins I, II, III, a, and b), and thaumatins I and II are the major forms with both having 207 amino acid residues [41]. The molecular weights of thaumatins I and II are 22,209 daltons and 22,293 daltons, respectively [42]. The threedimensional structure of thaumatin I, based on X-ray analysis has been reported [43,44]. The sweetness of thaumatin I is rated between 1,600 and 3,000 times in comparison to sucrose on a weight basis [22]. Talin® protein, the trade name of the commercial form of thaumatin protein as an aluminum ion adduct, is approved as a sweetener in Australia, the United Kingdom, and some other countries, and was first permitted for use as a

10

food additive in Japan in 1979 [22]. Talin® protein has GRAS status as a flavor enhancer for use in chewing gum in the United States [22]. Two natural product derived semisynthetic compounds are utilized as a limited basis, namely, perillartine [28] and neohesperidin dihydrochalcone [45]. Perillartine is an a-syn-oximQ and synthesized from perillaldehyde, a constituent of the volatile oil of Perilla frutescens (L.) Britton (Labiatae), and used in Japan as a sweetener for tobacco [28]. Neohesperidin dihydrochalcone is a dihydrochalcone glycoside prepared from a flavanone constituent of Citrus aurantium L. (Rutaceae), which is permitted for use in chewing gum and certain beverages in Belgium and elsewhere [45]. Discovery of Natural Sweeteners Searching for novel high-potency sweeteners from plants requires an initial dereplication stage for the presence of saccharides and polyols, which, as indicated earlier, exhibit sweetness potencies close to that of sucrose. If the combined amount of those saccharides and polyols exceeds 5% w/w in a given plant part, the resultant sweetness can be considered as being due to the presence of these "bulk" sweeteners. A suitable dereplication procedure using gas chromatography/mass spectrometry (GC/MS) has been developed for this purpose to rule out the sweetness contribution from saccharides and polyols in candidate sweet-tasting plants [46,47]. The general approach to the discovery of new sweetening agents of natural origin used at the University of Illinois at Chicago has been described previously [17,20,22]. RECENT REPORTS ON THE THEORY OF SWEET TASTE Many studies have been conducted to elucidate the functional groups present in sweet-tasting molecules that mediate the sweet taste. Initially, Shallenberger and Acree [48] suggested that sweet molecules contain both a hydrogen donor group (AH) and a hydrogen accepting group (B), and that these groups interact at a receptor by hydrogen bonding to exhibit a sweet taste. The existence of a third binding site (X) was proposed to explain the difference in sweetness between D- and L-amino acids, and was termed the AH-B-X model [49]. The hypothesis of an e-n (electrophile-nucleophile) group rather than a AH-B group was put

11

forward because of the lack of hydrogen bonding exhibited by some sweeteners [50]. Nofre and Tinti proposed a multipoint attachment (MPA) model in which the existence of eight optional cooperative recognition sites in the sweet receptor to interact with sweet molecules was suggested [51,52]. The binding of sweet molecules to the receptor site(s) can be achieved by ionic and hydrogen bonding, as well as by hydrophobic interactions in receptor sites which are designated as AH, B, and XH. There are also other sites that are designated as Gl, G2, 03, and 04 which fit sterically with the sweet molecules involved. Another recognition site, designated as D, was suggested as the hydrogen donor group. Even though all of the known sweet-tasting molecules may not bind to all of these binding sites, the most potent sweet taste intensities may result from the cumulative presence of such binding sites [51,52]. Recent work has been conducted to examine sweetener recognition by identifying the receptor molecules in the sweet taste receptor cells biochemically and physiologically. It is thought that "intense" sweeteners react with membrane receptor proteins connected to a O protein system [53]. In more recent studies, the concept of a multiple binding mechanism has been proposed [54]. In this system, calcium ion, inositol triphosphate (IP3), and cyclic AMP (cAMP) are involved as secondary messengers. The reaction of sugar sweeteners with the receptor can lead to an accumulation of cAMP, while interaction with the receptor of non-sugar sweeteners, such as saccharin, may result in the accumulation of IP3, suggesting that the reactions between sugar sweeteners and receptors, and between non-sugar sweeteners and receptors, have different mechanisms [54]. The receptor involved in these two different mechanisms appears on the same sensory cell. After the contact of a sweet molecule with the receptor, three different kinds of subunits of O protein, a, P, and y, may be activated by the receptor protein. In the sugar sweetener mechanism, stimulation by a sugar triggers a cAMP mediated cascade in which cAMP depolarizes the taste cell by reducing K"^ conductance in protein kinase A (PKA). This depolarization initiates the entry of Ca^"^ influx from the extracellular medium through voltage-dependent Ca^"^ channels, which stimulates the release of neurotransmitters in the synapse with sensory nerve fibers that carry the signal to the brain. The a-subunit of the O protein acting on phosphodiesterase (PDE) keeps the cAMP level low before and after sugar sweetener stimulation [54]. In the non-sugar sweetener system, stimulation mediates the a-subunit of the O protein

12

and triggers the phosphoinositide mechanism by transforming phosphatidyl inositol (PIP2) to IP3 by phospholipase C (PLC). This IP3 then stimulates the release of intracellular Ca^^ [54]. The types of G proteins which are involved in these two different mechanisms may be different, such as the Gs-type for sugar sweeteners and the Gq-type for non-sugar sweeteners. A taste-specific G protein, called gustducin, has been cloned, and possesses a, p, and y subunits, and the involvement of this protein in sweet taste transduction was also suggested [55,56]. Gustducin is related to transducin which is involved in vision, and both can activate PDE [57,58]. a-Gustducin may involve the cAMP pathway [59] while the py-gustducin complex stimulates PLC [60,61]. aGustducin maintains cAMP at a low level after the increase of cAMP by sugar sweetener stimulation. In a-gustducin knockout mice, the level of cAMP is high and the sweet taste stimulation is impared [62]. An interesting hypothesis has been postulated in terms of the sweet taste being activated by a receptor-independent mechanism [63]. It has been proposed that some amphipathic molecules, which have both hydrophobic and hydrophilic groups in the same molecule, can activate G proteins, and thus the enzymes responsible for the transduction pathway or channels, directly. Some non-sugar sweeteners are amphipathic molecules. These molecules can by-pass the taste receptors and permeate the plasma membrane, and activate GTPase directly in a concentrationdependent fashion, according to an in vitro experiment [63]. This stimulation penetration through the plasma membrane can also explain the slow taste onset and the lingering aftertaste which sometimes characterizes the taste of non-sugar sweeteners [64,65]. Certain non-sugar sweeteners even show a sweet taste nerve response when they are injected intravenously or intralingually which is independent of taste cell receptors in the tongue [66]. HIGHLY SWEET NATURAL PRODUCTS In this section, the presently known highly sweet substances of natural origin are described. Sweet-tasting compounds are listed in Table 1, with information published subsequent to an earlier chapter [16] then discussed in more detail. The structures of the compounds mentioned will be interspersed in the text, with the following abbreviations used to designate the sugar units of glycosides: api = D-apiofiiranosyl; ara = L-

13 Table 1. Highly Sweet Compounds from Plants Compound type/name*

Sweetness potency**

Plant name

Reference

MONOTERPENE Perillartine (10)'=

Perillafrutescens (Labiatae)

(L.) Britten

370

28

1,500

28

SESQUITERPENES Bisabolanes (+)-Hemandulcin (11)

Lippa dulcis Trev. (Verbenaceae)

4P-Hyclroxyhemandulcin (12)

L. dulcis

N.S.''

67

Sapindus rarak DC. (Sapindaceae)

ca. 1

47,68

Acyclic glycoside Mukurozioside lib (13)

DITERPENES Diterpene acid 4P, 1 Oa-Dimethyl-1,2,3,4,5,10hexahydrofluorene-4a,6adicarboxylic acid (14)'

Pine tree*^

1,300-1,8008

28

eitr-Kaurene glycosides

30

28

S. rebaudiana

242

28

Rebaudioside B (16)

S. rebaudiana

150

28

Rebaudioside C (17)

S. rebaudiana

30

28

Rebaudioside D (18)

S. rebaudiana

221

28

Rebaudioside E (19)

S. rebaudiana

174

28

Rubusoside (20)

Rubus suavissimus S. Lee (Rosaceae)

115

68

Steviolbioside (21)

S. rebaudiana

90

28

Steviol 13-0-P-D-glucoside (22)

R. suavissimus

N-S.**

69,70

Stevioside (5)

S. rebaudiana

210

28

DulcosideA(15)

Stevia rebaudiana (Bertoni) Bertoni (Compositae)

Rebaudioside A (4)

14 Table 1. Highly Sweet Compounds from Plants (continued) Compound type/name'

Plant name

Sweetness potency''

Reference

eif/-Kaurene glycosides (continued) Suavioside A (23)

R. suavissimus

N.S.^

69

Suavioside B (24)

R. suavissimus

N.S.**

69

Suavioside G (25)

R. suavissimus

N.S.**

69

Suavioside H (26)

R. suavissimus

N.S.**

69

Suavioside I (27)

R. suavissimus

N.S.**

69

Suavioside J (28)

R. suavissimus

N.S.^

69

Baiyunoside (29)

Phlomis betonicoides Diels (Labiatae)

500

28

Phlomisoside I (30)

P. betonicoides

N.S.**

20

Gaudichaudioside A (31)

Baccharis gaudichaudiana DC. (Compositae)

55

71

Labdane glycosides

TRITERPENES Cucurbitane glycosides Bryodulcoside''

Bryonia dioica Jacq. (Cucurbitaceae)

N-S.**

28

Bryoside (32)

B. dioica

N.S."

72

Bryonoside (33)

B. dioica

N.S."

72

Camosifloside V (34)

Hemsleya camosiflora C.Y. Wu et Z.L. Chen (Cucurbitaceae)

51

73

Camosifloside VI (35)

H. camosiflora

77

20

Mogroside IV (36)

Siraitia grosvenorit (Swingle) C. Jeffrey (Cucurbitaceae)

233-392*

74

Mogroside V (2)

S. grosvenorii

250-425«

28

1 l-Oxomogroside V (37)

Siraitia siamensis Craib (Cucurbitaceae)

N.S.^

75

15 Table 1. Highly Sweet Compounds from Plants (continued) Compound type/name'

Plant name

Sweetness potency**

Reference

Cucurbitane glycosides (continued)

54

73,74

Scandenoside R6 (38)

Hemsleya panacis-scandens C . Y . W u e t Z.L.Chen (Cucurbitaceae)

Scandenoside Rl 1 (39)

H. panacis-scandens

N.S.**

76

Siamcnoside I (40)

Siraitia grosvenorii; S. siamensis

563

74,75

30

77-79

100

78,79

Cycloartane glycosides Abrusoside A (41)

Abrus precatorius L.; A.fruticulosus Wall et W. & A. (Leguminosae)

Abrusoside B (42)

A.precatorius\ A.fruticulosus

Abrusoside C (43)

A. precatorius; A.fruticulosus

50

78,79

Abrusoside D (44)

A. precatorius; A.fruticulosus

75

78,79

Abrusoside E (45)

A. precatorius

N.S.''

80

Cyclocarioside A (46)

Cyclocarya paliurus (Batal.) Iljinsk (Juglandaceae)

200

81

Cyclocarioside I (47)

C. paliurus

250

82

GypenosideXXJ(48)

Gynostemma pentaphyllum Makino (Cucurbitaceae)

N.S.^

83

Apioglycyrrhizin (49)

Glycyrrhiza inflata Batal. (Leguminosae)

300

84

Araboglycyrrhizin (50)

G. inflata

150

84

Glycyrrhizin (1)

Glycyrrhiza glabra L.

93-170«

28

Pcriandrin I (51)

Periandra dulcis Mart.; P. mediterranea (Veil.) Taub. (Leguminosae)

90

28

Dammarane glycosides

Oleanane glycosides

16 Table 1. Highly Sweet Compounds from Plants (continued) Compound type/name*

Plant name

Sweetness potency**

Reference

Oleanane glycosides (continued) Periandrin II (52)

P. dulcis; P. mediterranea

95

28

Periandrin III (53)

P. dulcis; P. mediterranea

92

28

Periandrin IV (54)

P. dulciSy P. mediterranea

85

28

Periandrin V (55)

P. dulcis

220

85

50

86

Secodammarane glycosides Pterocaryoside A (56)

Pterocarya paliurus Batal. (Juglandaceae)

Pterocaryoside B (57)

P. paliurus

100

86

Osladin (58)

Polypodium vulgare L. (Polypodiaceae)

500

87

Polypodoside A (59)

Polypodium glycyrrhiza DC. Eaton

600

88-90

Polypodoside B (60)

P. glycyrrhiza

N-S.**

89,91

STEROIDAL SAPONINS

PHENYLPROPANOIDS trans-Anetholc (61 f

Foeniculum vulgare Mill. (Umbelliferae)

13

92

50

20

Illicium verum Hook f. (Illiciaceae) Myrrhis odorata Scop. (Umbelliferae) Osmorhiza longistylis DC. (Umbelliferae) Piper marginatum Jacq. (Piperaceae) Tagetes filicifolia Lag. (Compositae) /raw5-Cinnamaldehyde (62)

Cinnamomum osmopholeum Kanehira (Lauraceae)

17

Table 1. Highly Svi^eet Compounds from Plants (continued) Compound type/name'

Plant name

Sweetness potency**

Reference

DIHYDROISOCOUMARIN Hydrangea macrophylla Seringe var. thunbergii (Siebold) Makino (Saxifragaceae)

400

28

Glycyphyllin (63)

Smilax glycyphylla Sm. (Liliaceae)

N.S.*"

28

Naringin dihydrochalcone"^ (64)

Citrus paradisi Macfad. (Rutaceae)

300

28

Neohesperidin dihydrochalcone'^ (65)

Citrus aurantium L.

1,000

28

Phlorizin (66)

Symplocos lancifolia Sieb. et Zucc. (Symplocaceae)

N.S.**

28

Trilobatin (67)

Symplocos microcalyx Hayata

N.S.**

28

3-Acetoxy-5,7-dihydroxy-4'methoxyflavanone (68)

Aframomum hanburyi K. Schum. (Zingiberaceae)

N.S.^

93

2/?,3/?-(+)-3-Acetoxy-5,7,4'trihydroxyflavanone (69)

A. hanburyi

N.S.'*

93

Dihydroquercetin 3-O-acetate 4'-methyl ether' (70)

Tessaria dodoneifolia (Hook. & Am.) Cabrera (Compositae)

400

20

(2/?,3/?)-Dihydroquercetin 30-acetate (71)

T. dodoneifolia', Hymenoxys turneri K. Parker (Compositae)

80

20

(2/?,3^)-2,3-Dihydro-5,7,3',4'tetrahydroxy-6-methoxy-3-0acetylflavonol (72)

H. turneri

25

94

(2/?,3/?)-2,3-Dihydro.5,7,3',4'tetrahydroxy-6methoxyflavonol (73)

H. turneri

15

94

Phyllodulcin' (3)

FLAVONOIDS Dihydrochalcone glycosides

Dihydroflavonols and Dihydroflavonol glycosides

18 Table 1. Highly Sweet Compounds from Plants (continued) Compound type/name*

Plant name

Sweetness potency**

Reference

Dihydrochalcone glycosides (continued)

20

94

Engelhardtia chrysolepis Hance (Juglandaceae)

N.S.**

95

E. chrysolepis

N.S.**

96

Cinnamtannin B-1 (77)

Cinnamomum sieboldii Meisner (Lauraceae)

RS.**

97

Cinnamtannin D-1 (78)

C. sieboldii

N.S.^

97

Selligueain A (79)

Selliguea feci Bory (Polypodiaceae)

35

98

Unnamed (80)

Arachniodes sporadosora Nakaike; A. exilis Ching (Aspidiaceae)

N.S.**

99

Unnamed (81)

A. sporadosora; A. exilis

N.S.^

99

Haematoxylon campechianum L. (Leguminosae)

120

100

(27?,3/?)-2,3-Dihydro-5,7,4'trihydroxy-6-methoxy-3-0acetylflavonol (74)

H. tumeri

Huangqioside E (75)

Neoastilbin (76) PROANTHOCYANIDINS

BENZOl*]INDENO[i,2-2)-p-Dglucopyranosyl derivative] of abrusoside E exhibited about 150 times the sweetness potency of 2% sucrose, making it the sweetest compound in this series. When the aglycone carboxylic acid group was methylated, as in abrusoside E dimethyl ester, no sweetness was apparent [18,110].

COOH 41

R = p-glc

42

R=p-glcA-6-CH3^-P-glc

43

R=p-glc^-glc

44

R=p-glcA^-glc

45

R=p-glc^-glcA

Cyclocarioside A (46) is a dammarane-type triterpenoid glycoside sweet principle from the leaves of Cyclocarya paliurus (Batal.) Iljinsk (Juglandaceae), a plant used in the People's Republic of China in the treatment for diabetes [81]. Recently, another sweet-tasting principle, cyclocarioside I (47), was isolated from the same plant along with two other compounds with the same dammarane-type triterpenoid aglycone structure [82]. Cyclocarioside I was rated as about 250 times sweeter than sucrose [82]. From the crude extract of the vine of Gynostemma pentaphyllum Makino (Cucurbitaceae), which is used to make a sweet tea ("Amachazuru") in Japan, gypenoside XX (48) was isolated [83]. Although the sweetness of this compound was not reported when it was

29

Ri

R2

46

a-ara-5-Ac

a-rha

47

a-ara

P-qui

first characterized, it was later stated to be sweet [18]. The relative sweetness intensity of gypenoside XX (48) to sucrose has not been determined, but this represents the first sweet dammarane-triterpenoid documented from a plant source. CH2OH

R2

48

P-glc^-P-glc

p-glc'-p-glc

1;

a-rha

As mentioned ealier, glycyrrhizin (1) and its ammonixun salts are available commercially for sweetening and flavoring purposes, and glycyrrhizin 3-0-D-glucuronide (MGGR, 7) is a promising new intense sweetener [21-24]. Apioglycyrrhizin (49) and araboglycyrrhizin (50) have been isolated from the roots of Glycyrrhiza inflata Batal. (Leguminosae) [84]. While glycyrrhizin has a C-3-affixed diglucuronate unit,

This Page Intentionally Left Blank

30

apioglycyrrhizin (49) has an P-D-apiofuranosyl-(l->2)-P-Dglucuronopyranosyl group and araboglycyrrhizin (50) an a-Larabinopyranosyl-(l->2)-p-D-glucuronopyranosyl group at the C-3 position of the aglycone, glyc)nThetinic acid. The sweetness intensities of apioglycyrrhizin (49) and araboglycyrrhizin (50) were rated as 300 and 150 times sweeter than sucrose, respectively. COOH

49

R=P-glcA^-P-api

50

R = p-glcA^-a-ara

Periandrins I-IV (51-54) were characterized in the 1980's as oleananetype triterpenoid glycoside sweeteners from Periandra dulcis Mart. (Leguminosae) (Brazilian licorice) by the Hashimoto group at Kobe Pharmaceutical University in Japan [28], and the sweetness potency was determined as about 90 times sweeter than sucrose for each compound. Periandrins I-IV (51-54) were also found in another species, P. mediterranea (Veil.) Taub. [28]. A fifth compound in this series, periandrin V (55), was isolated from the roots of P. dulcis and found to be based on the same aglycone as periandrin I (51) [85]. The terminal Dglucuronic acid residue of periandrin I (51) was substituted by a D-xylose moiety in periandrin V (55). Periandrin V (55) exhibited 220 times the sweetness of 2% sucrose and was accordingly ranked as the sweetest substance obtained so far in the periandrin series [85]. Two novel sweet secodammarane glycosides, pterocaryosides A (56) and B (57), were isolated and structurally determined from the leaves and stems of Pterocarya paliurus Batal. (Juglandaceae) [86]. Pterocarya paliurus Batal. is a preferred taxonomic name for Cyclocarya paliurus (Batal.) Iljinsk (see above). The leaves of P. paliurus are used by local populations in Hubei Province of the People's Republic of China to

31

Ri

R2

51

P-glcA^-P-glcA

CHO

53

p-glcA^-p-glcA

CH2OH

55

p-glcA^-P-xyl

CHO

HOOC

Ri

R2

p-glcA^-p.glc

CHO

p-glcA^-P-glcA

CH2OH

sweeten cooked foods. While pterocaryoside A (56), which has a Pquinovose unit attached to the C-12 position, is 50 times sweeter than sucrose, pterocaryoside B (57), with an a-arabinose unit at C-12, was rated as 100 times sweeter than sucrose [86]. These are the first highly sweet secodammarane glycosides to have been isolated and structurally characterized, and represent interesting lead compounds for synthetic optimization. The steroidal saponin osladin (58) was isolated as a sweet principle from the fern Polypodium vulgare L. (Polypodiaceae) nearly 40 years ago [20,28]. However, the original structure proposed was later revised because the synthetic compound produced was not sweet at all [87]. The correct structure of osladin (58) was characterized by single crystal X-ray

32

HOOCx,^^

56

R = P-qui

57

R = a-ara

crystallography and the stereochemistry of osladin was reassigned as 22/?, 25iS', and 26R [87]. The actual sweetness potency of osladin was revised as being as 500, rather than 3,000 times sweeter than sucrose [87]. Polypodosides A (59) and B (60) were isolated from the rhizomes of North American fern Polypodium glycyrrhiza DC. Eaton (Polypodiaceae) as additional highly sweet steroidal glycosides [88,89,91]. Their aglycone, polypodogenin, is the A'^'^-derivative of the aglycone of osladin. The structure of polypodoside (59) was also revised as 22/?, 255, 26/?, by a chemical interconversion procedure [17,89,90]. Polypodoside A (59) shows a high sweetness potency and was rated as 600 times sweeter than sucrose [88,89].

'

jCX^ 0 Ri

R:

Other

58

p-glc^-a-rha

a-rha

7,8-dihydro

59

p-glc^-a-rha

a-rha

60

P-glc

a-rha

-

33

Phenylpropanoids The phenylpropanoids /ra«5-anethole (61) and /ra«5-cinnamaldehyde (62) are used as flavoring agents in foods in the United States and some other countries [20]. ^ra«5-Cinnamaldehyde (62) was isolated from Cinnamomum osmophloeum Kanehira (Lauraceae) as a sweet principle, while ^ra«5-anethole (61) was isolated as the volatile oil constituent responsible for the sweet taste of several plant species, as listed in Table 1 [92]. These two compounds occur widely in the plant kingdom. Therefore, it is necessary to rule out their presence in any candidate sweet plant by a dereplication procedure in a natural product sweetener discovery program using gas chromatography-mass spectrometry (GC/MS) [46,47].

Ri

R2

61

CH3

OCH3

62

CHO

H

Dihydroisocoumarin The dihydroisocoumarin, 3i?-phyllodulcin (3, obtained from the leaves of Hydrangea macrophylla var. thunbergii via enzymatic hydrolysis), was mentioned earlier in the chapter as having commercial use. Recently, it has been demonstrated that this sweet substance occurs naturally in unprocessed leaves of its plant of origin as a 5:1 enantiomer with the previously undescribed compound, SS-phyllodulcin [111]. Also reported in this study were the novel 3i?- and SS'-phyllodulcin 3'-0-glycosides, although the presence or absence of a sweet taste in these three new phyllodulcin analogs was not disclosed [111]. Flavonoids Glycyphyllin (63), phlorizin (66), and trilobatin (67) are sweet dihydrochalcone glycosides and were isolated from Smilax glycyphylla

34

Sm. (Liliaceae), Symplocos lancifolia Sieb. et Zucc, and Symplocos microcalyx Hayata (Symplocaceae), respectively [28]. Naringin dihydrochalcone (64) and neohesperidin dihydrochalcone (65) are semisynthetic dihydrochalcone glycosides and can be obtained as byproducts of the citrus industry. Neohesperidin dihydrochalcone (65) is regarded as the more promising sweetener of these two compoimds, because it is sweeter and has acceptable hedonic properties, and its longlasting sweetness is useful in chewing gum, candies, and oral hygiene products [16]. There have been a large number of attempts to synthesize improved dihydrochalcones, with such compounds requiring 3-hydroxy4-alkoxy substitution in ring B [16]. No additional sweet-tasting dihydrochalcones appear to have been isolated and characterized from plant sources in recent years.

Ri

R2

R3

R4

R5

63

H

H

H

a-rha

H

64

P-glc^-a-rha

CH3

H

H

H

65

p-glc^-a-rha

CH3

OH

H

H

66

H

H

H

H

p-glc

67

p-glc

H

H

H

H

The seeds of Aframomum hanburyi K. Schum. (Zingiberaceae) are used as an antidote and ingredient in certain medicinal preparations in Cameroon [93]. From an acetone extract of the seeds of this plant, two sweet dihydroflavonols, 3-acetoxy-5,7-dihydroxy-4'-methoxyflavanone (68) and 2i?,3i?-(+)-3-acetoxy-5,7,4'-trihydroxyflavanone (69), were isolated [93]. 3-Acetoxy-5,7-dihydroxy-4'-methoxyflavanone (68) was previously isolated from a different species, Aframomum pruinosum Gagnepain [112]. However, the sweetness intensities of these compounds were not indicated [93,112]. The previously known (2i?,3i?)dihydroquercetin 3-0-acetate (71) which was rated as 80 times sweeter than sucrose, was isolated from Tessaria dodoneifolia (Hook. & Am.)

35

Cabrera and Hymenoxys turneri K. Parker (Compositae) [20]. The sweetness of this compound was increased to 400 times that of sucrose by methylation at the 4'-0H position (70) [20]. Two dihydroflavonols, huangqioside E (75) and neoastilbin (76), were isolated from Engelhardtia chrysolepis Hance (Juglandaceae) [95]. However, their sweetness was not evaluated. A series of three sweet additional dihydroflavonols (72-74) was isolated from K turneri [94]. R3

l ir ^

^0>

HO^ 1 7

11

4'

si

R2^

i

OH

0

Ri

R2

R3

R4

Other

68

Ac

H

H

CH3

1R,ZR

69

Ac

H

H

H

IR.'hR

70

Ac

H

OH

CH3

71

Ac

H

OH

H

2R,3R

72

Ac

CH3O

OH

H

2R,3R

73

H

CH3O

OH

H

2R,3R

74

Ac

CH3O

H

H

2R,3R

75

a-rha^-•p-glc

H

OH

H

2/2,3/?

76

a-rha

H

OH

H

25,35

-

Proanthocyanidins Several doubly linked ring-A proanthocyanidins are known to be sweettasting [97,99]. For example, two proanthocyanidins, cinnamtannin B-1 (77) and cinnamtannin D-1 (78), isolated from the roots of Cinnamomum sieboldii Meisner (Lauraceae) showed sweet properties [97]. Other sweettasting proanthocyanidins with carboxylic acid (80) and lactone (81) ftmctionalities, were isolated from the ferns Arachniodes sporadosora Nakaike and A, exilis Ching (Aspidiaceae) [99]. However, none of these proanthocyanidins was ever quantitatively rated for its sweetness intensity relative to sucrose. A sweet-tasting proanthocyanidin, selligueain A (79) was isolated from the rhizomes of the fern Selliguea feci Bory

36 upper unit

middle unit ll

terminal unit

OH Ri

R2

77

OH

p-OH

78

OH

a-OH

79

H

P-OH

80

37

81

(Polypodiaceae), collected in Indonesia [98]. Selligueain A may be distinguished from the previously known sweet-tasting proanthocyanidins since it has an afzelechin residue rather than an epicatechin moiety as the lower terminal unit of the molecule. When evaluated by a small human taste panel, selligueain A (79) showed 35 times the sweetness of a 2% sucrose solution and was not perceived as astringent when in solution [98]. A further doubly linked ring-A proanthocyanidin, selligueain B, was also isolated from the rhizomes of S.feei, but was not perceived as sweettasting [113]. As a result of the investigation of selligueain A (79) and related compounds, stringent structural requirements seem to be necessary for proanthocyanidins of this type to exhibit a sweet taste. In this connection, it is notable that an epimer of selliguaein A [epiafzelechin-(4p->8,2p^O->7)-epiafzelechin-(4p->8)-epiafzelechin] was astringent without any hint of sweetness [98]. Benzo [b] indeno [l,2'd\ py ran From the extract of the heartwood of Haematoxylon campechianum L. (Leguminosae), a sweet principle was isolated, namely, (+)-hematoxylin (82) [100]. This compound has been used for a long time as a microscopic staining reagent, but the sweetness of this compound was not recognized previously. Also, in the same study, brazilin, the 4-deoxy derivative of (+)-hematoxylin, and a constituent of Caesalpinia echinata Lam. (Leguminosae), was found not to be sweet [100]. In a follow-up study.

38

(+)-hematoxylin (82) was rated as 120 times sweeter than 3% sucrose, while its synthetic (-)-enantiomer was only 50 times sweeter [114].

Amino acid A highly sweet amino acid, monatin (83), was isolated from an African plant, Schlerochiton ilicifolius A. Meeuse (Acanthaceae) [101]. Monatin (83) was rated as being comparable to the synthetic amino acid, 6-chloroD-tryptophan, which showed a sweetness intensity of 1,300 times that of sucrose. Monatin (83) appears to be the only native plant amino acid with a highly sweet taste to have been discovered.

83

Proteins Several plant-derived proteins have been reported previously as sweeteners, inclusive of curculin [103], mabinlin [104,105], monellin [28,106], pentadin [107], and thaumatin, with the latter compound already mentioned as having commercial use as a sweetener and flavor enhancer [22]. Recently, a sixth sweet protein of plant origin, brazzein, was isolated from the fruits of an African climbing vine, Pentadiplandra

39

brazzeana Baillon (Pentadiplandraceae), which grows in Gabon, Zaire, and Cameroon [102]. Pentadin was also isolated from this same plant [107]. Brazzein has 54 amino acid residues and a molecular weight of 6,473 daltons making it a relatively small protein compared to other sweet proteins such as curculin (12,491 daltons), mabinlin (12,441 daltons), monellin (11,086 daltons), and thaumatin (22,206 daltons) [102]. Brazzein has four disulfide bridges and promising thermostability, since its sweetness was not destroyed at 80 °C for 4 hours exposure [115]. Most of the other protein sweeteners are unstable to heat and inappropriate for use at high temperature. The sweetness of brazzein was rated as 2,000 times sweeter than 2% sucrose [102]. Brazzein has considerable potential as a new naturally occurring sweetening agent, because of its favorable taste profile and thermostability.

NATURALLY OCCURRING SWEETNESS INDUCERS A number of compoimds have been known for some time as sweetness inducers, including the caffeic acid conjugates cynarin and chlorogenic acid [116]. Arabinogalactin is also known to enhance the sweetness potencies of saccharin, cyclamate, and protein sweeteners such as thaumatin and monellin [22]. Miraculin, a protein isolated from the fruits of Richardella dulcifica (Schum. et Thonn.) Baehni (Sapotaceae) (miracle fruit) [117,118], and curculin, a protein isolated from the fruits of Curculigo latifolia Dryand. (Hypoxidaceae) [103] (see previous section), have sweetness-inducing activity [41]. While miraculin has no sweet taste per se, curculin has a sweet taste but this dissipates before the sweetnessinducing effect on water becomes evident [41]. Miraculin is a glycoprotein with a molecular weight of about 24,000 daltons and has the property of making sour or acidic materials taste sweet. Miracle fruit concentrate was formerly on the market in the United States, but was removed because prior FDA approval for the scientific claims made had not been realized [22]. Recently, five oleanane-type triterpenoid glycosides, strogins 1-5, were isolated from the leaves of Staurogyne merguensis Wall. (Acanthaceae). Strogins 1, 2, and 4 (84-86) show sweetness-inducing activity [119]. In Malaysia, S, merguensis grows wild and the local people use the leaves of this plant to sweeten rice during cooking [119]. The sweetness-inducing activities of strogins 1-5 were measured by a

40

psychometric method [119-121]. Briefly, using four subjects, 2 mL of a solution of each compound (1 mM) were held in the mouth for three minutes and then expectorated. Next, the subject tasted 5 mL of water. The induced sweetness activity was measured by comparing with a 0.050.4 M standard sucrose solution. A concentration level of strogins 1, 2, and 4 (84-86) of 1 mM exhibited the same perceived sweetness as 0.3 M sucrose solution [119]. Strogins 1, 2, and 4 (84-86) also showed a sweet taste. The sweetness of strogin 1 (84) at a 1 mM concentration was comparable to a 0.15 M sucrose solution. Strogins 2 (85) and 4 (86) tasted sweet, but the sweetness intensities were less than that of strogin 1 (84) [119]. CH2OH ORha-2',3',4'-Ac

CH2OH 84

R=P-glcA2-P-xyl

85

R=P-glcA

86

R=P-glcA'-p-glc

NATURALLY OCCURRING SWEETNESS INHIBITORS It has long been known that a number of synthetic compounds and certain enzymes suppress the sweet taste in humans and animals [22,122-128]. Three plant species, Gymnema sylvestre (Retz.) R. Br. ex Schult. (Asclepiadaceae), Hovenia dulcis Thunb. (Rhamnaceae), and Ziziphus jujuba P. Miller (Rhamnaceae), have been studied extensively for their sweetness inhibitory (antisweet) constituents [19]. In recent years, additional sweetness-inhibiting agents have been isolated from G. sylvestre and K dulcis, as well as two other plant species, Gymnema altemifolium and Stephanotis lutchuensis Koidz. var. japonica (Asclepiadaceae). The presently known triterpenoid sweetness inhibitory agents from these species are reported in Table 2. A 35-amino acid

41 Table 2. Sweetness Inhibitors from Plants Sweetnessinhibitory potency**

Reference

Gymnemic acid V (94) Gymnemic acid VI (95) Gymnemic acid VIII (96) Gymnemic acid IX (97) Gymnemic acid X (98) Gymnemic acid XI (99) Gymnemic acid XII (100) Gymnemic acid XIII (101) Gymnemic acid XIV (102) Gymnemic acid XV (103) Gymnemic acid XVI (104) Gymnemic acid XVII (105) Gymnemic acid XVIII (106)

0.125 0.125 0.125 1 1 0.5 0.25 0.5 0.5 0.5 N.S.'^ N.S.'^ 0.5 1 1 0.5 0.5 1 1 1 1

131 131 131 132 132 132 133 132 134 134 135 135 136 136 136 136 136 137 137 137 137

Gymnema alternifolium^ (Asclepiadaceae)

Altemoside I (107) Altemoside II (108) Altemoside III (109) Altemoside IV (110) Altemoside V (111)

0.25 0.25 0.25 0.25 0.25

138 138 138 138 138

Hovenia dulcis Thunb. var. tomentella Makino (Rhamnaceae)

JujubosideB(112) HodulosideI(113) HodulosideII(114) HodulosideIII(115) HodulosideIV(116) HodulosideV(117) HodulosideVII(118) Hoduloside VIII (119) Hoduloside IX (120) Hoduloside X (121) Hovenoside I (122) Saponin C2 (123) Saponin E (124) Saponin H (125)

0.25 0.25 0.125 0.125 0.125 0.125 0.25 0.25 0.25 N.S." 0.125 0.125 0.125 0.0625

139 139 139 139 139 139 140 140 140 140 139 139 139 139

Stephanotis lutchuensis Koidz. wax.Japonica (Asclepiadaceae)

Sitakisoside I (126) Sitakisoside II (127) Sitakisoside III (128) Sitakisoside IV (129) Sitakisoside V (130) Sitakisoside VI (131) Sitakisoside VII (132) Sitakisoside VIII (133) Sitakisoside IX (134)

0.25 0.25 0.25 0.25 0.5 0.25 0.25 0.25 0.25

141 141 141 141 141 142 142 142 142

Plant name

Compound name*

Gymnema sylvestre R. Br. ex Gymnemasaponin III (87) Gymnemasaponin IV (88) Schult. (Asclepiadaceae) Gymnemasaponin V (89) Gymnemic acid I (90) Gymnemic acid II (91) Gymnemic acid III (92) Gymnemic acid IV (93)

42 Table 2. Sweetness Inhibitors from Plants (continued) Plant name

Compound name*

Sweetnessinhibitory potency**

Reference

Stephanotis lutchuensis Koidz. ydn.japonica (Asclepiadaceae) (continued)

Sitakisoside XI (135) SitakisosideXII(136) Sitakisoside XIII (137) Sitakisoside XVI (138) Sitakisoside XVIII (139)

0.25 0.25 0.25 0.25 0.25

143 143 143 143 143

Ziziphus jujuba P. Miller (Rhamnaceae)

Jujubasaponin II (140) Jujubasaponin III (141) Jujubasaponin IV (142) Jujubasaponin V (143) Jujubasaponin VI (144) JujubosideB(112) Ziziphin (145) Zizyphus saponin I (146) Zizyphus saponin II (147) Zizyphus saponin III (148)

0.5 0.5 0.25 0.25 0.25 0.25 0.5 0.125 0.125 0.25

144 144 144 144 144 144 144,145 144 144 144

" Structures of compounds 87-148 are shown in the text. ^ Potency compared with gymnemic acid I (90) (x 1). *^ N.S. = Sweetness-inhibitory potency not given. ** Plant taxonomic authority not given in the original article.

peptide called gxmnarin has been isolated from the leaves of G. sylvestre^ and has also been found to exhibit a sweetness-inhibitory effect [129,130]. The sweetness inhibitory activity of plant terpenoids is evaluated by placing 5 mL of 0.5 or 1 mM solution of the compound in the mouth for 2-3 minutes. On expectorating, the mouth is then washed with distilled water. Then, different concentrations of sucrose (0.1-1 mM) are tasted. The maximum concentration of sucrose at which complete supression of sweetness is perceived may then be recorded for each tastant [133]. In practice, antisweet compounds of plant origin have tended to be ranked in terms of sweetness inhibitory potency by comparison with gymnemic acid I (90) [19]. Since the initial reports of sweetness-inhibitory oleanane-type gymnemic acids from the leaves of Gymnema sylvestre, plant species of the family Asclepiadaceae have served as bountiful sources of sweetnessinhibitory compounds. The initial isolation and structural characterization of these compounds was very challenging, and these early investigations have been reviewed [19]. In 1989, gymnemic acids I-VI (90-95) were isolated with a common gymnemagenin (149) aglycone structure and a

43

glucuronic acid moiety [132-134]. A different series of antisweet compounds were then isolated, namely, gymnemasaponins III-V (87-89) [131]. These non-acylated compounds show slightly less potent sweetness-inhibitory activities compared with the previously isolated gymnemic acids. Subsequently, the additional sweetness-inhibitory gymnemic acids VIII-XVIII (96-106), have been isolated from G. sylvestre [135-137]. Gymnemic acids XIII (101) and XIV (102) were previously named gymnemic acids VIII and IX when they were isolated by Yoshikawa et al [136]. However, Liu et al independently isolated different compounds designated as gymnemic acids VIII (96) and IX (97) from the same plant species [135]. Therefore, for clarification purposes, gymnemic acids VIII and IX were renamed as gymnemic acids XIII (101) and XIV (102), respectively [137]. The antisweet potencies of gymnemic acids XIII (101) and XIV (102) were rated as about half the potency of gymnemic acid I. For gymnemic acids XV-XVIII (103-106), their sweetness-inhibitory potencies were judged to be as about the same as that of gymnemic acid I (90) [137].

CH20R'

Ri

R2

87

P-glc

P-glc'^-p-glc

88

P-glc*-P-glc

p-glc

89

P-glc'-P-glc

P-glc^-P-glc

Gymnema altemifolium is an evergreen tree growing in the forests of Taiwan and the southem part of mainland China. The roots of this plant have been used for detoxification purposes, and for the treatment of edema and fever [138]. No phytochemical studies had been performed on G. altemifolium until a Chinese group isolated several common compounds including P-amyrin and cycloartenol from the fruits of this

44

^0R2 ^

OR3

r^^^V'^i^^C 1 I 1 H r*CH20R4 i

tg^:

'OR5 nt)a:

0 II —C-

H

0 II

—c- - C H CH2CH3

CH2C)H

Ri

? " ^ /CH3

-c=c.

CH I3 R2

R3

R4

R5

Ac

H

Ac

H

90

p-glcA

tga

H

91

P-glcA

mba

H

92

p-glcA

mba

H

H

H

93

P-glcA

tga

H

H

H

94

p-glcA

tga

tga

H

H

95

P-glcA^-P-glc

tga

H

H

H

96

P-glcA^-p-OG

mba

H

H

H

97

P-glcA^-P-OG

tga

H

H

H

98

p-glcA

H

H

Ac

H

99

P-glcA

tga

H

tga

H

100

P-glcA^-P-glc

tga

H

Ac

H

101

P-glcA

H

H

mba

H

102

P-glcA

H

H

tga

H

103

P-glcA

mba

tga

H

H

104

P-glcA

H

tga

H

tga

105

P-glcA

Bz

H

H

H

106

P-glcA

H

H

Bz

H

149

H

H

H

H

H

plant [146]. More recently, several oleanane-type triterpenoid glycosides, altemosides I-V (107-111), have been isolated as sweetness inhibitors from the roots of G. alternifolium [138]. Complete hydrolysis of altemosides I-V (107-111) yielded a known oleanane-type triterpenoid, chichipegenin (150) [147]. There is no functional group at the C-21 and C-23 positions of the altemosides, as commonly present in the gymnemic acids. The antisweet effects of altemosides I-V have been evaluated using a 1 mM solution of each compound, and found to completely suppress the sensation of sweetness induced by 0.2 M sucrose solution in all cases.

45

The sweetness-inhibitory potencies of altemosides I-V (107-111) were rated as about half those of gymnemic acids XIII (101) and XIV (102) [136].

y 0R2 ^CHaORa

111" r j^ 1

'OR4 0

RiC

tga:

II —c-

23 Ri

R2

R3

R4

107

p-glcA'-p-glc

Ac

a-rha

H

108

p-glcA^-p-glc

H

a-rha

Ac

109

p-glcA'-p-glc

tga

a-rha

H

110

p-glcA

Ac

a-rha

H

111

p-glcA

H

a-rha

Ac

150

H

H

H

H

?"3 /CH3 H

Subsequent to the isolation of the dammarane-type triterpenoid glycosides jujuboside B (112), hodulosides I-V (113-117), hovenoside I, and saponins C2, E, and H (122-125) as sweetness inhibitors from the leaves of Hovenia dulcis Thunb. var. tomentella Makino [139], hodulosides VII-X (118-121) were isolated as sweetness-inhibitory agents [140]. Hodulosides I (113) and II (114) have hovenolactone (151) as their aglycone which is the same compound as in saponins E (124) and H (125). Hodulosides III-V and VII-X (115-121) are based on two different dammarane-type aglycone structures, however [139,140]. The sweetnessinhibitory potencies of hodulosides are shown in Table 2. The sweetnessinhibitory potency of hoduloside X (121) was not determined [140]. Recently, from the stems of Stephanotis lutchuensis var. japonica, an evergreen woody climber growing in forests near the warm coastal areas of Japan, several oleanane-type sweetness-inhibitory triterpenoid glycosides have been isolated, namely, sitakisosides I-IX, XI-XIII, XVI, and XVIII (126-139) [141-143]. Some sitakisosides have a Nsitakisosides VI (131), VII (132), XI, XII, and XIII (135-137) afforded sitakisogenin (152) [142,143], while hydrolysis of sitakisosides II (127)

46

112

R = a-ara^-P-glc^-P-xyl

115

R = a-ara^-P-qui

a-rha

p-glc 116

R = a-ara^-P-glc

l'

P-glc 117

R = P-glc^-a-rha p-glc

122

R = a-ara^-xyl p-glc

123

R = a-ara^-a-rha P-glc

CH2OR2

Ri

R2

118

a-ara^-a-rha

P-glc

119

a-ara

P-glc'-P-xyl

120

a-ara^-a-rha

P-glc^-P-xyl

121

a-ara^-a-rha

P-glc

.3

P-glc

47

Ri

R2

113

P-glc^-a-rha

p-glc

114

p-glc^-a-rha

H

p-glc P-glc^-a-rha

H

125

p-glc

H

151

H

H

124

CH20H 155

and XVIII (139) yielded marsglobiferin (153) [141,143]. In turn, hydrolysis of sitakisoside VIII (132) afforded 3p,16p,2ip,28tetrahydroxyoleanan-12-en-22-one (154) as the aglycone [142]. Sitakisoside IX (134) has a gymnestrogenin-type aglycone structure (155) [142]. The sweetness-inhibitory potencies of the sitakisosides are about 25% of that of gymnemic acid I, except for the most potent analog, sitakisoside V [130, 0.5 of the activity of gymnemic acid I (90)] (Table 2). In the late 1980s, ziziphin (145) was isolated from the Chinese jujube tree, Ziziphus jujuba P. Miller as the first recognized antsweet principle of this plant [146]. Ziziphin (145) has the same dammarane-type aglycone structure as in hodulosides III-V (115-117). Yoshikawa et aL isolated

48

yd-Neu5Ac-<x2Me Ih 4-Oi)henyl-2-propicHiyl-Neu5Ac-a2Me li 4,7-di-0-acetjd-Neu5Ac-a2Me 11 7-deoxy-Neu5Ac-a2Me Im

KodnM)

1

2.1 2.8 >100

6.5

1

Even if the derivatives Ih and li have shown a Uttle effect on the binding with the HA receptorial pocket, they are partially resistant to the action of influenza virus NA, which normally cleaves the a-glycosidic bond. Resistance to NA is an important property for all designed inhibitor that consist on interfering with viral functions. It seems therefore possible that the addition of bulkier substituents at the 4- position might confer complete resistance to NA hydrolytic action, also because this group is situated in a hydrophobic region of receptorial pocket. To verify this hypothesis it has been prepared the 4-0-capriloyl-Neu5Ac-cxMe In according to Scheme D. It has been chosen, as starting material for the preparation of modified sialic acid derivatives, methyl-2a-methoxysialoside 3a, because this compound has the same configuration at C-2 as natural sialic acid linked to polysaccharidic material. Compound 3a has been prepared according to scheme C. The synthetic strategy is that one described by Hanson [27b, 27a,

124

29a] and consists in the esterification of natural sialic acid 1, with diazomethane, to give the methyl sialoside 2, which is then treated with acetyl chloride. Under these conditions the esterification of the fi-ee alcoholic functions occurs together with the substitution of the hemiacetalic hydroxyl at C-2 with chlorine, which occupies the less hindered axial position owing to its steric requirements. The obtained methyl-tetraacetyl-2p-chloro sialoside 2h reacts with methanol in the presence of silver carbonate to give, via a bimolecular nucleophilic substitution, the methyl tetraacetyl-2a-methoxy sialoside 2L In the presence of catalytic quantities of potassium /-butoxide a rapid transesterification of 2i with methanol, allows to obtain methyl 2amethoxy sialoside 3a in very high yields, without hydrolysis of the amidic linkage at C-4,

COOCH,

Dowex 50 H* MeOH

AcCI

OMe

AGO

HO

AcO

COOCK

AcNH

COOCH,

AcHN AcO 2h

3p

AgjCOg

MeOH COOCH, OMe

3a

AcO

COOCH,

t-BuOK AcO "* MeOH

OMe

AcNH AcO 2i

Scheme C

In scheme C it is also reported the obtaining of Neu5Ac-pMe 3p, which

125

can be easily prepared by treatment of NeuSAc 1 with methanol in the presence of strongly acidic resin. These conditions lead to the formation of the thermodynamically more stable ketal Sp, in which methoxy group occupies the less hindered axial position. On the basis of data obtained from molecular modelling studies, Karplus and Miranker (see ref 29b) reported on the possibility to use sialic acid derivatives having epimeric configuration at C-2, as compound 3p, for the mteraction with the HA receptorial pocket. The synthetic strategy for the preparation of In has been described by Hanson [29a] and us [30]; it is described in the scheme D. OH HI

CO2CH3

OH

OCH3 AcNI

PhCH20H t-BuOK

OH HI

OH

OCH3 AcNI

HO

COsCHsPh

3a

HO 4a 2,2-dimethoxy propane acetone • Dowex-SOK

OH AcNH^

COsCHsPh

^-r^o-y^ocH3 0

4c

0 ^ ?

^O,0H,py^

OH

py/DMAP

AcUH".^/.^—^ HO

Y-(CH2)6CH3 0

4b AcOH80%

OH OH

HO

COsCHjPh H2

X . AcNH^ ^Y-(CH2)6CH3 4d

f OH

f ^^^

Pd/C

0

^Y-(CH2)6CH3 0 1 n 4-O-capri toyl-Neu5Ac-aMe

Scheme D

The methyl ester 3a was converted to the benzyl ester 4a by treatment with catalytic potassium /-butoxide in benzyl alcohol. Treatment of the benzyl ester 4a with 2,2-dimethoxypropane and acetone in presence of

126

Dowex-50 (H"^) aflfords the 8,9-0-isopropylidene derivative 4b, which gives subsequently esterified at the 4-position by addition of capriloyl chloride to a solution of this derivative in pyridine in the presence of 4(dimethylamino)pyridine (compound 4c). Removal of the 8,9-acetonide by heating with 80% acetic acid, affords derivative 4d which produces 4-0capriloyl-Neu5Ac-aMe In by hydrogenolysis with 10% Pd/C catalyst to remove the ben2yl ester. As previously reported, the synthesis of derivatives Ih and l i (see fig. 10) has been performed as described in the scheme D, starting fi-om intermediate 4b, using the required acyl chloride. Results of hemagglutination inhibition assays (see table 3 and 4), performed on synthetic intermediates 4a-4d also, indicate a stronger binding with the receptorial pocket of HA. Therefore, to complete this synthetic approach, the research has been continued with another modification of hydroxyl at C-4 which has been esterified with a medium chain carboxylic acid having a polar end. We have synthesised 4-(8-morpholin)capriloyl-Neu5Ac-a2Me l o [28], wishing us a long carboxylic chain with a polar end would form specific interactions with the protein, or, in alternative, to have a compound which shows resistance to the NA. 9HoH

lo

The chemical procedure adopted for the synthesis of compound 2, is the same described in detail in the Scheme D, starting from Neu5Ac-a2Me methyl ester 3a, using 8-morpholin-capriloyl chloride as esterifying agent of the intermediate 4b, Table (4) describes the antigenic specificity, measured by hemagglutination inhibition (HI) assays, referring to long chain ester derivatives. Particularly it is remarkable that at a concentration of 1.22 mg/ml (4.0 mM) (2a-methyl-4-capriloyl sialic acid. In) and at concentration of 1.08 mg/ml (3.5 mM) (compound lo), inhibition of HA is observed, but also at a

127

lower concentration than the natural compound 2 (12.50 mg/ml, see table 1). Table 4. Hemagglutination inhibition (HI) assays (data by Lepetit Research Centre of Gerenzano, Varese, Italy).

1

Sialic acid derivatives 2P-diloro-peracetyl-Neu methyl ester 2h Neu5Ac-|32Me methyl ester 3P peracetyl-Neu-<x2Me acid methyl ester 2i Neu5Ac-a2Me mefliyl ester 3a Neu5Ac-a2Me benzyl ester 4a 4-capriloyl-Neu5Ac-a2Me In 4-(8-morpholin)capriloyl-Neu5Ac-a2Me lo

KD (ms/ml) 0.62 14.50 8.90 0.78 5.60 1.22 1.08

(mM)

|

43.0 17.6 2.3 13.5 4.0

\

35

1

1.2 1

Our inhibition data, also taking into consideration the previous results [27a], show that the modifications on the structure of sialic acid increase the binding with HA, but not in an enough significant amount to use these type of analogues as anti-influenza drugs. Owing to these results, the research work has continued on verifying the role of the hydroxyl at C-7, in the bmdmg with HA. In order to control the hydrogen bond formation between the ligand and the protein, the 7-deoxyNeu5Ac-2aMe (compound Im) has been synthesised. The synthetic strategy is described in scheme E and startsfi*omderivative 4b, whose preparation is described in scheme D. The 4-hydroxyl of 8,9-0-isopropyUdene derivative 4b has been protected as its 4-t-butyl-dimethyl"Silyl ether 4e. Then, the 7-hydroxyl has been converted to its xantate ester 4f, by deprotonation with butyl litium, treatment with carbon disulfide and alkylation with methyl iodide. Deoxygenatiobn of the 7-position has been accomplished by heating with tributyl-tin hydride in xylenes. Then, the cleavage of acetonide and of silyl ether, by heating in 80% acetic acid, followed by hydrogenolysis to remove the benzyl ester, aflford 7-deoxy"Neu%Ac-aMe Im. 7-hydroxyl (see table 3) does not seem to give significant interactions with the protein, as showed by the inhibition data measured for the 7-deoxyNeu5Ac-2cxMe Im, even if the esterification also of this hydroxyl significantly reduces the activity also (compound II in table 3).

128

C02CH2Ph

02CH2Ph f-Bu(CH3)2SiCI OMe

OMe

AcNH tBu(CH3)2Sid

4b

C02CH2Ph OMe

AcNH

Bu3SnH/A

tBu(CH3)2SiO 4g

AcOH 80% A

4f

02CH2Ph OMe

OMe

1m

4h

Scheme £

Therefore, we have decided to apply the same strategy used in the case of 4-hydroxyl to 7-hydroxyl, esterifing this functional group with a fatty acid that has a medium chain as caprylic acid. Compound Ip has been obtained and the chemical procedure adopted for the synthesis of compound Ip is described in the Scheme F. O OACpA^.^^^^ CO2CH3 OCH3 AGO

ip Neu5 Ac-a2Me methyl ester 3 a is the starting product. Compound 3 a has been selectively silylated with r-butyl-chloro-dimethyl-silane in pyridine and

129

in presence of catalytic quantities of 4-(dimethylamino)pyridine to give the pure silyl derivative 5a in 98% yield. Then the silyl derivative 5a has been then acetylated affording the acetyl derivative 5b quantitatively. The usual procedure to remove the protecting silyl group (tetrabutylammonium fluoride in tetrahydrofuran) has caused acetyl rearrangements rather than to give a sialic acid derivative with a free C-9 primary hydroxyl function. Compound 5c, in which the free hydroxyl appears to be the secondary one at C-7, has been obtained as the sole reaction product in a 85% yield, after chromatographic purification. The overall yield of 5c based on 3a has been 83% [30]. HOL

Up V

OH yH

9O2CH3 1

TBDMCS

TBDMSO^

HO ^

OH - "

HO

CO2CH3

HO

3a

5a AC2O

pyr AqP

OH

AcO^ 3 L StL

CO2CH3

JL

"r^^-^T^o^ocHs AcNH-.j>7--^y

AGO OAO

TBDMSO^ X

-Q—^+p:

P

^^^^^^^^ AcN!

9O2CH3

A

AGO

5c

5b

CTH. 7^15

C7H15COCI pyr/DMAP

AcO AcO ^ AcNI

Scheme F

o-^O

9^2CH3 OCH3

AcO 1p

The fi-ee hydroxyl at C-7 has been esterified with caprylic acid by addition of capriloyl chloride to a solution of 5c in pyridine in presence of 4(dimethylamino)pyridine affordmg derivative Ip. Now we have not any result yet of inhibition assays in relation to this derivative.

130

2.2 SYNTHETIC APPROACH: N A INHIBITORS. Enzymatic inhibition by transition-state analogues

An enzyme catalyses a reaction by lowering the energy barrier between substrate and product - the activation energy (Ea); the origin of this catalytic power has generally been attributed to the enzyme capacity to stabilise the transition-state of the reaction, assuming that the catalyst binds to the transition state more strongly than to the ground state of the substrate. Afterwards it is possible to inhibit an enzyme by structurally analogue molecules to the hypotetic transition state of considered reaction. Transitionstate mimics are often potent inhibitors for the catalysing enzyme. The concept of structural similarity to the transition state has found wide application in drug design over the years. The multitude of enzyme-inhibitor interactions are governed by steric as well as electronic factors. In theory, compounds that closely resemble the transition state structure should give high binding aflBnity towards the target enzyme. These potential inhibitors, besides, that have an high affinity for the enzyme, need to form a stable bond with the protein, and they don't need to be transformed in the reaction products. 2.2.1 DANA One of the most potent synthetic inhibitors for NA, commercially available 2-deoxy-2,3-didehydro-N-acetylneuraminic acid 6 (DANA, fig. (11), Ki = 4 and 20 jxM for flu A and B, respectively) [31], has been obtained by the simple dehydration of sialic acid 1 and it has been discovered over 25 years ago [32]. OH

ACHNTN^

6H 2-deo;^2,3-didehydro-Ne5Ac, 6 Fig. 11. DANA

According to the NA mechanism action, DANA is considered a transition state-like analogue binding to the active site of NA.

131

2.2.2 GG167 Several NA inhibitor analogues have been synthesised using structure of DANA 6 as a base molecule. An extremely potent iniBuenza NA inhibitor, with Ki value of 10"^^ M, is 2,3-didehydro-2,4-dideoxy-4-guanyl-Nacetylneuraminic acid 7, Zanamivir, GG167, fig. (12) [33,34,35].

2,3-didehydfX)-2,4-dideoxy-4-guanidyl-Neu5Ac, 7 Fig. 12. GG167

Synthesis of GG167 is described [36] in the Scheme G. The most direct approach to the synthesis of DANA analogues starts from the commercially available N-acetyl-neuraminic acid. Therefore the synthesis of GG167 requires introduction of the 2,3 double bond and particularly a stereospecific introduction of a nitrogen-based C-4 substituent. N-acetylneuraminic acid 1 has been esterified using methanolic HCl to give the methyl ester 2 in high jdeld. Alternatively, Dowex SOW x 8 resin in methanol could be used. The penta-acetoxy compound 7a could be prepared fi'om this by using an excess of acetic anhydride in pyridine with 4-(dimethylamino)pyridine catalysis. Then preparation of the oxazoline 7b has been achieved by treatment of compound 7a with trimethylsilyl trifluoro-methanesulfonate (TMSOTf) in ethyl acetate at 52°C. The reaction of oxazoline 7b with trimethylsilyl azide m rer/-butyl alcohol at 80°C gives the azido compound 7c stereoselectively.

132 AcO-

OAc

DMAP

7a

AcO

trimethyisilyl trifluoromethanesulfonate

PAc

trimethyisilyl azide t-BuOH

AcO CX)2CH3

aOAc

80'C

AcO / ^ \ AcHN-/ ^—ODfiH^

MeONa MeOH

N^7c

7d HO

a)Et3N/H20

.OH AIMSA.NaOH

^.

CO2CH3

b)H2/Undlar c) Dov^x 2x8 resin

CO2H

7,GG167

Scheme G

Liberation of the 4-amino function requires selective reduction of the azido group in the presence of the double bond. This has been achieved by first gaining water solubility by removal of the acetate protecting groups with

133

catalytic sodium methoxide in methanol to afford triol 7d, followed by the hydrolysis of the ester by using triethylamine in water. Then hydrogenolysis with Lindlar catalyst gives the triethylamine salt of free amine which has been desalted using Dowex 2 x 8 ion-exchange resin to give the free amino acid 7e. Amino acid 7e has been eflSciently converted into the 4-guanidino compound 7 (GG167) by reaction with aminoiminomethane-sulfonic acid (AIMSA), the latter synthesised according to the method of Miller and Bischoflf [37]. Treatment of compound 7e with 3 mol equiv. each of AIMS A and potassium carbonate in a portion wise manner over a period of 8 h allowed isolation in reasonable yield (48%) of the crystalline 7 (GG167) following ion-exchange chromatography. GG167 is a transition state analogue, representing the optimal conformational state imposed on the sialic acid unit by the enzyme during the catalytic cleavage of the glycosidic Unkage. It was originally conceived through the application of modelling techniques to the crystal structure of influenza virus NA complexed with sialic acid [34,38]. The use of software such as GRID39 (a program that determines probable interaction sites between probes with various functional group characteristics and the enzyme surface) suggests that the replacement of the 4-hydroxyl group of DANA by the more basic guanidyl group produces an even tighter aflfinity of the substituted DANA for the active site as a result of lateral bindmg through the terminal nitrogens of the guanidino group with both Glu 119 and Glu 227. In several recent papers the researchers have described detailed structureactivity relationship (SAR) studies [35,36,40] based around GG167. The results of these studies have demonstrated that each of the four substituents around the dihydropyran ring is critical in the binding of GG167 and that significant modification or removal of any one of these groups results in a dramatic loss of inhibitory activity. GG167 exhibits potent antiviral activity against a variety of influenza A and B strains in the cell culture assay; it is currently being evaluated in human clinical trials and has shown eflBcacy in phase HI challenge studies in both prophylaxis and treatment of influenza virus infections [41]. However, poor oral bioavailability and rapid excretion preclude GG167 as a potential oral agent against influenza infection and GG167 has to be administered by either intranasal or inhaled routes in clinical trials.

134

In the case of an influenza epidemic, oral administration may be a more convenient and economical method for treatment and prophylaxis. Therefore, it would be desirable to have a new class of orally active NA inhibitors as potential agents against influenza infection. Moreover, the relative chemical sensitivity (e.g., to acid, base, and heat) and the complex stereochemistry (five chiral centres) for this class of compound seem to make the production of next generation agents problematic. The general approach to the structure-based design of NA inhibitors uses the structure of the DANA - NA complex as a starting point and it is based on the development of new classes of lead compounds by using chemically simpler cyclic templates instead of the dihydropyran ring of DANA. 2.2.3 AROMATIC SIALIC ACID ANALOGUES

Whenever DANA 6, a sialic acid transition-state analogue, is bound to the NA active site, the plane of the acetylamino group is perpendicular to the plane of the tetrahydropyran ring and bisects the 0-C=0 bond of the carboxylate. This produces an amide N to carboxylate C distance of 5.5 A and an amide O to carboxylate C distance of 6.2 A. It has been proposed that simple non-carbohydrate analogues containing the carboxylate and the acetylamino groups attached to a cyclic backbone "spacer" would be suflBcient to generate lead compounds for further elaboration as NA inhibitors. The spacer would need to orient correctly these groups as found in bound DANA. It has been also required that such compounds adopt a planar structure near the carboxylate to mimic the transition state and be able to present additional side-chain fixnctionality for interaction with other conserved anfiino acid residues in the sialic acid binding site. Among several chemical classes considered, the benzene ring spacer has been the first choice. Inspection of the X-ray structure of the NA-GG167 complex reveals that the half-chair conformation of the dihydropyran ring m GG167 is almost flat and all equatorial C4, C5 and C6 substituents are on the ring lay on the same flat plane. Therefore, designing a new series of NA inhibitors, has been a try to mimic this planar arrangement of substituents using a benzene ring as a replacement for the dihydropyran ring m GG167.

135

The benzene ring scaffold has advantages of non-chirality, chemical and metabolic stability and increased lipophilicity compared with the dihydropyran ring. These factors may be important to improve the deficient pharmaco-kinetic profiles observed for GG167. Recently, simple aromatic influenza NA inhibitors have been synthesised [42]. Compound 8 is the most important aromatic analogue of DANA. In fact it inhibits influenza A sialidase with a Ki of 2.5 \JM, Compound 8 has been synthesised [43] as described in the Scheme H. The methyl ester 8a, prepared fi-om conrniercially available 3,4diaminobenzoic acid, has been reacted with N,N'-di(ferrbutoxycarbonyl)thiourea in the presence of mercury chloride to provide the C3 guanylated intermediate 8b. The selectivity observed in this reaction arisesfi*omthe decreased nucleophilicity of the C4 amino group due to the electron v^thdrawing para carbomethoxy fimctionality. s

J

^.^s. .CO2CH3 ^ ^ ^ 2

HjN

J I BocHN-^^NHBoc

1 NH2

HgCL. TEA

^ NH

B o c N ^ NHBoc 8b

8a

^,^^CO,CH3 AC2O

H2N

JI

J NH

BOCN'^^^^NHBOC

8c

a)LIOH b) TFA

frv°°""

T

HN'^^^NH2 8

Scheme H

Acylation of compound 8b followed by saponification and acidic removal of ^er/-butoxycarbonyl groups gave product 8 as a trifluoroacetic acid salt. An alternative synthesis of compound 8 is described [44] in the Scheme I. Compound 8 is a micromolar inhibitor of influenza virus NA. Although the NA affinity of 8 is approximately 1000-fold lower than that of GG167, the most interesting feature of this compound consists on it adopts an alternative binding orientation [44].

136

In fact, by using X-ray crystallography, it has been demonstrated that the guanidino group occupies the glycerol-binding subsite of DANA, forming a salt bridge interaction with Glu 275. Unlike the dihydropyran ring of DANA and its analogues, the benzoic add ring is symmetrical. Since there are no structural constraints, the guanidinium group of 8 can bind 4-guanidinoDANA's guanidinium subsite as well as glycerol subsite, requiring only a 180° rotation of benzene ring. COjH NaOAc

KNO, H2SO4

HOI

NH2NH2 Pd/C EtOH

rt CO,H

NHAc

NHjCN HCI. 80' NHAc

8g

Scheme I

In fact, this result reveals that a single guanidinium group, at least when attached to a benzoic acid ring, undergoes more energetically favourable interactions with 4-guanidino-DANA's glycerol-binding subsite on NA as compared to the guanidinium-binding subsite. CO2H

8 Fig. 13 Neuraminic acid aromatic analogues

This situation is confirmed by the evidence that whenever a glyceric chain at C-5 is included in 8, fig. (13), the resulting compound 9 does not show the

137

enzyme inhibitory activity up to 100 |jM. Compound 9 has been obtained as described [43] in the Scheme L. COjH

a)ICI

b)H2SO, NO2

MeOH

H^N^^y

Acp *

AcHN^'^S^

NOp 9b

9a

S"BU3

Pd(PH3P),

OAc

HG^..-—X^^^^^

2

3

3)OsO,.NMO

AcHN

b) ACjO, pyr

OAc CO2CH3

COjH AcO

Scheme L

138

lodination of 4-amino-3-nitrobenzoic acid 9a with iodine monocMoride followed by esterification gives iodide 9b. Acylation of 9b with hot acetic anhydride provides amide 9c. Palladium catalyses coupling of amide 9c with Z-vinylstanne [45], generate cis olefin 9d. Treatment of 9d with osmium tetraoxide followed by acetylation with acetic anhydride in pyridme gives triol 9e. During this two step sequence the acetamide group, highly activated by the orto nitro functionality has been cleaved to the amine. Catalytic hydrogenation of triacetate 9e produced the diamine 9f. Selective guanidylation of 9f to 9g has been accompUshed by the same procedure described for conversion of 8a to 8b. Finally, acylation of the amine 9g with acetyl chloride in the presence of N,N-diisopropylethylamine affords compound 9h. The saponification and acidic removal of the tert-butoxycarbonyl groups of 9h complete the synthesis of 9 as a trifluoroacetic acid salt. On the basis of the lead compound 4-(N-acetylamino)-3-guanidinobenzoic acid 8, which inhibits influenza A sialidase with a Ki of 2.5 jxM, we have synthesised oxygenated analogous (compounds 10 and 11 in fig. 14) of compound 8, utilising as a starting material the suitable hydroxy-aminobenzoic acid [53]. CO2H

AcO y ^ NH-^-NHj NHAc 10 Fig. 14. Neuraminic acid aromatic analogues

AcNI OAc 11

So, two aromatic analogues, compounds 10 and 11, have been prepared as illustrated in the Schemes M and N. Compound 10 has been synthesised utilising as a starting material commercially available 4-amino-3-hydroxybenzoic acid 10a (Scheme M). Compound 10a has been acetylated with acetic anhydride and an aqueous solution of sodium acetate in presence of hydrochloric acid to give the product 10b.

139 COjH

CO2H Ac20/AcONaaq. 2NHCI

10a

10b

CO2H

CO2H

AC2O/HNO3

li^i^iiT

._.^ . _ ....

.„,„

^^, ^

^^^^

NHAc 10c

lOd

CO2H NH2CN •

6NHCI

10 Scheme M

Compound 10b has been treated with a solution of the nitrating mixture made from acetic anhydride and nitric acid in dioxane aflFording nitro derivative 10c, with not very high yields. The catalytic reduction of nitro derivative 10c, carried out with hydrazine in ethanol in the presence of palladium on calcium carbonate, affords compound lOd. The guanidino group at C-5 has been introduced by reaction of compound lOd with cyanamide in solution of hydrochloric acid affording the objective molecule 10. Compound 11 has been prepared by using a similar synthetic strategy (Scheme N).

140 COgH

CO2H Ac20/AcONaaq. 2N HCl

AcHN OAc 11b

CO2H

CO2H

AC2O/HNO3 - •

dioxane

AcHN y ' ^ "NH2 OAc 11c

lid

NHoCN '2^ 6NHC1 11 Scheme N

The main difference between two syntheses regards the nitration step: in fact the compound 10b nitration a£Fords the nitro-derivative 10c in not satisfactory yields, whereas compound l i e has been obtained as the only reaction product from compound l i b in quantitative yield. 2.2.4 CARBOCYCLIC SIALIC ACID ANALOGUES: SIX-MEMBERED RINGS

It has been proposed that the sialic acid cleavage by NA might proceed via the oxonium cation transition state, with the successive release of free sialic acid, fig (8), Zanamivir (GG167) is an analogue of transition state which exhibits the potent NA inhibitory activity.

141

A new class of orally active NA inhibitors as potential agents against influenza infection utilises carbocyclic templates instead of the dihydropyran ring of the DANA system. It is expected that the carbocycUc ring would be chemically more stable than the dihydropyran ring and easier to modify for optimisation of antiviral and pharmacological properties. The validity of this approach has been verified by the discovery of very potent NA inhibitors in this new carbocyclic series. In particular new lipophilic side chains at the C-3 position of the carbocyclic system imparted potent NA inhibitory activity. In fact X-ray crystallographic analysis of the carbocyclic analogue bound to NA confirmed that there is an hydrophobic space in the glycerol-binding subsite to acconmiodate bulky lipophilic groups. The discovery of this hydrophobic pocket in the active site of NA has been exploited to increase the lipophilicity of inhibitors to optimise pharmacological properties for potential oral bioavailability while maintaining potent antiviral activity. 2.2.5 GS4071 In this new class of DANA's analogues, the most potent NA inhibitor is GS4071,12, fig. (15), with IC50 of InM. CO2H

(CH3CH2)2CHO'"

12 Fig. 15. GS4071

This compound has been synthesised as described in the Scheme O using shikimic acid 12a, as a starting material [46]. The synthesis begins with the preparation of epoxide 12b fi-om shikimic acid as described in the literature [47]. Nucleophilic ring opening of MOMprotected epoxide 12c with sodium azide in the presence of ammonium chloride generates azido alcohol 12d. The ring opening of the epoxide is

142

both regio- and stereo-specific, and this could be attributed to the steric and electronegative inductive influence of the MOM group in 12c. Conversion of azide 12d to aziridine 12f has been eflBciently accomplished via a two-step sequence: i) mesylation of the hydroxyl group in 12d and ii) reduction of the azide functionality in 12e with triphenylphosphine in the presence of triethylamine and water. I

T

ref.47

f

T

MeOCHjCI

12c

^^^

12a shikimic acid

NaN3/NH4CI

|

MeOH/HsO

HO^'^Y^'^

|

MsCI, EtaN CHjClj

^j

MSO^'Y^^ N3

12d

12e

NaNj

M0M0,,^^;v,,^^C02CH3

DMF

H.N^ ^ > .^

TrN^ =^^

12. GS4071

Scheme O

T

O

OH

a) TrCI. EtsN CH2a^

f

143

The aziridine ring opening of 12f with sodium azide gives 12g exclusively. This selective ring opening is a consequence of the favoured azide ion attack at the Cs position due to the steric and electronegative inductive effects of the MOM group again. The removal of MOM gives compound 12h. The aziridine 12i has been derived from the trans amino alcohol 12h by the two-step, one-pot process: i) selective protection of the amino functionality with trityl chloride and ii) mesylation of the hydroxyl in the presence of triethylamine. Under these conditions, the mesylate intermediate has been converted to azuidine 12i. Treatment of 12i with /-pentyl alcohol in the presence of 1.5 equiv of BF3Et20 followed by acetylation of the crude product provides the ether 121. Finally, reduction of the azide functionality and saponification of the ester group in 121 give GS4071,12. Subsequently, GS4104 (the ethyl ester prodrug of GS4071) has found to be highly orally bioavailable in several animal species and efficacious in both the mouse and ferret models of influenza infection by oral administration [48,49]. In recent phase II clinical trials, oral efficacy of GS4104 has been demonstrated both m prophylaxis and treatment of human influenza infection. GS4104 is currently being evaluated in phase n/HI human clinical trials. 2.2.6 BM144 Aiming at developing oral agents against influenza infection, we have designed new carbocycUc NA inhibitors able to inhibit NA as transition-state analogue. Considering the oxocarbonium ion described above as a key transition-state mimic, we have chosen the cyclohexene scaffold as replacement for the oxonium ring, and compound 13, BM144, fig. (16) has been synthesised [54]. CO2H NH I H2N NH"

HO CO2H ^

NHAc i3 Fig. 16BM144

13a, quinic acid

144

Quinic acid 13a, has been chosen as a starting material because it is a widely diffused natural compound, easy to find pure and has a biogenetic via similar to sialic acid. The carbocyclic analogue 13 (BM144) has been prepared according to the synthetic strategy depicted in the Schemes P and Q. Quinic acid 13a has been chosen as chiral starting material, because its fimctionalisation is suitable for my synthetic strategy. In addition it is conmiercially available. Quinic acid 13a is esterified by reaction with methanol in presence of a catalytic quantity of concentrated sulphuric acid to give in quantitative yield the methyl ester 13b which is acetylated after with acetic anhydride in pyridine. By controlling the reaction conditions it was possible to afford selectively the acetylation of secondary hydroxyls obtaining the triacetylderivative 13c in which there is the only fi-ee tertiary hydroxyl. Triacetylderivative 13c undergoes to dehydration by treatment with thionyl chloride in pyridine. Such reaction allows the regiospecific achievement of the only isomer 13d which has the double bond in 1,2 position. The selective hydrolysis of acetyl groups, led in methanol in presence of concentrated sulphuric acid, gives the a,p unsaturated methyl ester 13e. Therefore the two hydroxyls at C-3 and at C-4, in sin position each other, are protected by means of formation of isopropylidene function, doing react the methyl ester 13e with 2,2-dimethoxypropane in acetone in presence of catalytic quantity of strongly acidic resin, so obtain the 3,4-0-isopropylidene derivative 13f. Then the hydroxyl at C-5 is protected by means of acetylation carried out with acetic anhydride in pyridine affording compound 13g. The analysis of the ^H-NMR spectrum of acetyl derivative 13g allows to claim surely the regiospecificity of the dehydration reaction: in fact in the acetylation reaction fi-om 13f to 13g, it is showed the usual deshielding of about 1 ppm of proton whose chemical shift (6 5.15) is, of course, attributable to a not allylic position. Since the isopropylidene fiinction blocks the two hydroxyl groups these are in position 3 and 4 in sin of quinic acid, the double bond position must be 1,2 and not 1,6.

145 HO CO2H

HO CO2CH3 CH3OH

HO' " Y "

OH

H2SO4

HQ CO2CH3 AC2O

OH

HO" ^ ^ ^ O H 6H

13a

13b

13c CO2CH3

CO2CH3 CH3OH

SOCI2 pyr

Acd " V ^ ^OAc 6AC

Py

AcO > ^ ^OAc 6AC

2,2-dimethoxypropane CH3COCH3 •

H2SO4

Dowex-50 H"^ 13e

13d

CO 2CH3

1'

&

o-'V ^ O H

H3C^C5 CH3

13f

C02CH3

CO2CH3 AC2O pyr

^^^^

0 S^OAc H3CT-O CH3 13g

ACOH/H2O

^

HO"

OAc OH

13h

Scheme P

To explain this regioselectivity there is not a certain reason; probably the hydroxyls in position 3 and 5 have a important role in the dehydration process. In fact hydroxyl at C-3 has an absolute configuration which corresponds to a equatorial position, while hydroxyl at C-5 has an absolute configuration which corresponds to an axial position. Obviously the transition state, which corresponds to the dehydration that brings to the product 13d, is more energetically favoured than that one that should bring to the other isomer. Then the isopropylidene fiinction in 13g is selectively eliminated by means of an acidic hydrolysis led in a solution of 80% acetic acid at 70^C, to obtain derivative 13h in which the hydroxyl at C-5 is selectively protected. In a first approach we have tried to fimctionalyse selectively the two

146

hydroxyls at C-3 and at C-4, following the synthetic strategy described in the Scheme Q. CO2CH3

CO2CH3 Jones's reagent •

HO" " Y " ^OAc OH

acetone

13h

131 CO2CH3

CH3SO2CI

-^

Et3N/CH2Cl2

J \ O^^V^OAc

CO2CH3 NaN3 NaN3^ DMF/A DMF/A

^ ^

O O ^^ '' NY-' ' '^^O^A G N3

6MS

131

13m

CO2CH3

Scheme Q

This synthetic project makes provision for the regioselective oxidation of allylic hydroxyl of 13h with Jones's reactant in acetone; this reaction provided the keto derivative 13i quantitatively only when we operates with small substrate quantities (few milligrams). Then the hydroxyl at C-4 has been esterified with methanesulfonyl chloride; the mesylation reaction has been led in methylene chloride in presence of triethylamine at 0°C and it has brought to the formation of product 131. Then it has been substituted azide for mesyl group by the treatment of mesyl derivative 131 with sodium azide in dimethylformamide at 80°C, so obtaining product 13m. The azido group reduction and the following acetylation of obtained amine would have allowed us to insert the acetamido function at C-4, so we should have obtained a selective functionalisation of the three hydroxyls at C-3, C-4 and C-5. Nevertheless we have not succeeded in obtaining a

147

considerable quantities of azido derivative 13m. Section A. CO2CH3

CO2CH3

CO2CH3

OAc

CO2CH3 -H2O OAc

Section B. CO2CH3

CO2CH3

CP2CH3

O^^^^^OAc OMs 131

Scheme R

This synthetic strategy already involved a series of difficulties in the oxidation of allylic hydroxyl and in the substitution of mesyl group. Even we have used weaker conditions than Jones's reactant, the oxidation of product 13h is always coupled, working with greater substrate quantities than few milligrams, with the total insaturation of ring obtaining compound 14. Probably this happens because of the elimination type 1,4 of a water molecule, favoured by the acidic environment, according with the mechanism represented in Scheme R, section A [55].

148

In the other hand, although with low yields, we have succeeded in obtaining keto derivative 13i, the following substitution of azido group for mesyl group brings to the predominating formation of totally unsaturated product 15, here after the elimination of an acetic acid molecule (Scheme R, section B) [55]. Probably the aromatisation is due to the stabilisation of the tautomeric enolic form of 131 in the experimental conditions Therefore we have decided to follow an alternative synthetic strategy for the functionalisation of hydroxyls at C-3 and at C-4. Scheme S reports the passed methodology. Derivative 13e, obtained as it is described in Scheme P, has been maken react with thionyl chloride in methylene chloride and in presence of triethylamine, so we have obtained cycUc sulphite 13n as a stable mtermediate in 94% yield after chromatographic purification. Then the hydroxyl at C-5 has been protected by means of acetylation doing react compound 13n with acetic anhydride in pyridine affording product 13o. The sulphite ring opening of 13o has been carried out with sodium azide in dimethylformamide; this reaction allows the introduction of azido group in allylic position and releases hydroxyUc fimction in position 4, providing derivative 13p. This complete regiospecific ring opening is a consequence of favoured azide ion attack at the allylic C-3 position of 13o. Compound 13p is treated with methanesulfonyl chloride in methylene chloride in presence of triethylamine to provide mesyl derivative 13q in which the three hydroxylic fimctions at C-3, C-4 and C-5 have been protected selectively with three difierent groups. Conversion of azido group in 13q to acetamido derivative 13r has been efficiently accomplished in 68% yield via a two-step sequence: reduction of the azide functionality with triphenylphosphine in tetrahydrofiiran and acetylation of the amine with acetyl chloride. Product 13r is treated with sodium hydride in tetrahydrofiiran to provide N-acetylaziridine 13s by means of type SN2 intramolecular reaction. The treatment of compound 13s with sodium azide in dimethylformamide in presence of ammonium chloride allows ring opening of acetylated aziridine and formation of product 13t. The ring opening reaction is completely regiospecific giving azide 13t as the only product.

149 CO2CH3

C02CH3

CO2CH3

scx:i2 ,^^ OH

HO"

AC2O

EtaN / THF 3

OH

o

Pyr

OAc

s-o

OH 13e

13n

CXD-CHo

CO0CH3

1

NaNj N3-^

1 OH

CO2C

i

^^;A>s^

MsQ

x^s^^

DMF

13o

CHjOz

a)Ph3P/-mF

N 3 ^^ ^ ^ O A c

b) CH3COa

6MS

13p

NaN. NH4a

Ng"'

^OAc

LJndlar

NHJ-

NHAc

"V"

^^^^

NHAc 13u

13t

13s

CO2CH3

/V,A/4>is(f-Boc)-

"

CO2CH3 H,

DMF

Hga2 EtsN / DMF

^OAc

13r

CO2CH3

OAc

^ 6MS

13q

CO2CH3

thiourea

AcHir

hydrolysis

NBOC

I

OAc

BocHN NH NHAc 13v

13

Scheme S

Now the azido group in allylic position undergoes to catalytic reduction with hydrogen in presence of Lindlar catalyst obtaining in this way the selective reduction of the azide in amino group without touching the C-C

150

double bond. This reaction affords derivative 13u. Treatment of 13u with JV;iV-bis(/-buthoxycarbonyl)thiourea [50] using mercury (11) chloride and triethylamine in dimethylformamide provides bisBOC protected guanidine 13v. Finally, the saponification of the methyl ester and the acetyl group at C-5 followed by removal of the BOC groups carried out in water in presence of strongly acidic resinfiimishthe attended product 13, 2.2.7 MISCELLANEA

A new NA inhibitor, 16, of influenza sialidase has been recently designed [51], seefig17, whose molecule consists in the combination of an aromatic moiety with a y-lactame. Its structure is that of a l-(4-carboxy-2-(3pentylamino)phenyl)-5,5-bis-(hydroxymethyl)pyrrolidin-2-one.

NHCH(CH2CH3)2 HOHjC^^N

Q f

HOH2C'"\ 16 Fig. 17.

In this compound the N-acetyl group of aromatic inhibitor 8 (scheme H) has been replaced with a 2-pyrrolidinone ring, which has been designed in part to offer opportunities for introduction of spatially directed side chains that could potentially interact with the enzyme. In addition the guanidino group at C-3 in compound 8 (scheme H) has been replaced with a hydrophobic 3-pentylamino group. Compound 16 shows an IC50 of about 50 nM against influenza A sialidase, although the inhibition of influenza B sialidase is 2000-fold less. This compound is unique among known potent inhibitors of influenza neuraminidase for several reasons. It is the first reported benzoic acid with low nanomolar inhibition; it is the first achiral molecule exhibiting low nanomolar inhibition to be reported fi'om any structural class; it is the first low nanomolar inhibitor that does not

151

contain a basic side chain (amino or guanidino). Compound 16 has been synthesised [52] as described in the Scheme T. Methyl-amino benzoate 16a reacts with diethyl bromomalonate to give 16b, which has been acylated with 3-bromo propionic acid and PCI3 to provide 16c CO2CH3

CO2CH3

diethylbromomalonate

/ W

3-bromo propionic a d d PCI3, benzene

NHCH(C02Et)2 16b CO2CH3

CO2CH3 NO2BF4

NaH DMF O o . /NCH(C02Et)2

^

T/B'

EtOjC

16c CO2CH3

^.

CH3OH

KT

Et02C

>cr 16e

16d

a) NaBH4 THF, CH3OH b) H2, Pd/C

CH2CI2

Et02C.^N.^^Q

>Cr°

HOH2' HOK

16f

a) 3-pentanone NaCNBH3, CH3CO2H dichloroethane b) NaOH

NHCH(CH2CH3)2 HOH2Cv>''^>v^O

16

Scheme T

This reaction has undergone cyclization in the presence of NaH to yield lactame 16d in 80% overall yield. The nitration of compound 16d with nitronium tetrafluoroborate or HNO3 / H2SO4 proceeded in greater than 90% yield to give 16e, The aliphatic esters in 16e have been first selectively reduced to the alcohols using NaBHU, then the nitro functionality has been reduced to the amine using catalytic hydrogenation to give compound 16f.

152

The reductive coupling of amine 16f with 3-pentanone proceeds smoothly in the presence of NaCNBHs and acetic acid to give the alkylamine. Saponification of the methyl ester provides the molecule 16. 3. REFERENCES [1] Schauer, R.; Reuter, G.; Roggentin, P., Biochemistry and role of sialic acids. In: Rosenberg A (Ed) Biology of the sialic acids 1995, Plenum, New York, p, 7. 2] Lutz, P.; Lochinger, W.; Taigel, G., Chem Ber. 1968, lOL 1089. 3] Biedl, A., Naturwissenschaften 1971, 58, 95. 4] Gottschalk, A.: Glycoproteins, Their Composition, Structure and Function, 2^^ Edit. Elsevier, Amsterdam 1972. 5] Yu, R.K.; Ledeen, RW., J. Biol. Chem. 1969, 244, 1306. 6] Schauer, K.,Adv. Carbohydr. Chem. Biochem. 1982, 40, 131. 7] Gottschalk, A, The Viruses 1959, 3, 51 (eds Burnet, P.M. & Stanley, W.M.,), Academic, New York. 8] Paulson, J.C, The Receptors 1985, 2, 131 (ed. Conn, P.M.), Academic, Orlando. 9] Paulson, J.C; Sadler, J.E.; Hill, R.L., J. Biol. Chem. 1979, 254, 2120. 10] Bergelson, L.D. et al. Eur. J. Biochem. 1982,128, 467. 11] Suzuky, Y ; Matsunaga, M.; Masumoto, M., J. Biol. Chem 1985,260, 1362. 12] Bunneister, W.P.; Ruigrok, R.W.H.; Cusack, S., The EMBO Journal 1992, H , 49. 13] Lui, K. J.; Kendal, P., Am. J. Public Health 1987, 77, 712. 14] Hay, A.J.; Wolstenholme, A.J.; Skehel, J.J.; Smith, M.H., The EMBO Journal 1985, 4, 3021. 15] Pinto, L.H.; Holsinger, L.J.; Lamb, RA., Cell 1992, 69, 517. 16] Stoof, J.C; Booij, J.; Drukarch, B.; Wolters, E.C, Eur. J. Pharmacol. 1992, 213, 439. 17] Hayden, F.G. et al. New Engl. J Med 1989, 321, 1696. 18] Wiley, T>.C., Skehel, JJ.; Wilson, LA., Nature 1981,289, 373. 19] Rossmann, M.G., Nature 1988, 333, 392. 20] Weis, W.; Brown, J.H.; Cusack, S.; Paulson, J.C; Skehel, J.J.; Wiley, D.C, Nature 1988, 333, 426. 21] MUler, C A , Biochem. Biophys. Res. Comm. 1978, 83, 1479. 22] Chong, AK.J.; Pegg, M.S.; von Itzstein, M., Biochem Int. 1991, 24, 165. 23] Lentz, M.R; Webster, R.G.; Air, G.M., Biochemistry 1989,26, 5351. 24] Chong, AK.J.; Pegg, M.S.; Taylor, N.R.; von Itzstein, M., Eur. J. Biochem. 1992, 207,335.

153 [25] Janakiraman, M.N.; White, C.L.; Laver, W.G.; Air, G.M,; Luo, M., Biochemistry 1994, 33, 8172. [26]Kelm, S.; Paulson, J.C.; Rose, U.; Brossmer, R.; Schmid, W.; Bandgar, B.R; Schreiner, E.; Hartmann, M.; Zbiral, E., Eur. J. Biochem. 1992, 205, 147. [27] a) Sauter, N.K.; Hanson, J.E,; Glick, G.D.; Brown, J.H.; Crowther, R.L.; Park, S.J.; Skehel, JJ.; Wiley, D.C., Biochemistry 1992, 3 1 9609. b) Sauter N.K., Bednarki M.D., Wurzburg B.A., Hanson J.E., Whitesides G.M., Skenel J.S,, Wiley D.C., Biochemistry, 1989,28, 8388 [28] Bianco, A.; Brufani, M.; Ciabatti, R.; Melchioni, C ; Pasquali, V, Molecules Online 1998, 2. [29] a) Hanson, J.E., private cx)mmunication. b) Karplus M., Miranker A:, Proteins, Structure, Function and Genetics, 1991, H , 29 [30] Bianco, A.; Brufani, M.; Melchioni, C , Gazz. Chim. Ital 1996, 126, 805. [31] Holzer, C.T.; von Itzstein, M.; Jin, B.; Pegg, M.S.; Stewart, W.R; Wu, W.-Y., Glycoconjugate 1993, 10,40. [32] Meinal, P.; Bodo, G.; Palese, P.; Schuhnan, J.; Tuppy, H., Virology 1975, 58,457. [33] von Itzstein, M.; Wu, W.-Y.; Kok, G.B.; Pegg, M.S.; Dyason, J.C; Jin, B.; Phan, T.V.; Smythe, M.L.; White, H.F.; Oliver, S.W.; Cohnan, P.M.; Varghese, J.N.; Ryan, D.M.; Wood, J.M.; Bethell, R.C.; Hotman, V.J.; Cameron, J.M.; penn, C.R., Nature 1993, 363, 418. [34] Taylor, N.R; von Itzstein, M., J. Med Chem, 1994, 37,616. [35] von Itzstein, M.; Wu, W.-Y.; Jin, B., Carbohyd. Res. 1994, 259, 301. [36] Chandler, M.; Bamford, M.J.; Conroy, R ; Lamont, B.; Patel, B.; Patel, V.K.; Steeples, LP.; Storer, R ; Weir, N.G.; Wright, M.; Williamson, C, /. Chem. Soc. PerJdn Trans. 11995,1173. [37] Miller, A.E.; Bischoff, J.J., Synthesis 1986, 777. [38] Corfield, T., Glycobiology 1993, 3, 413.

[39] Goodford, P.J., J, Med Chem. 1985, 28, 849. [40] von Itzstein, M.; Dyason, J.C; Oliver, S.W.; White, H.F.; Wu, W.-Y.; Kok, G.B.; Pegg, M.S., J. Med Chem. 1996, 39, 388. [41] Fromtling, RA.; Castaner, J., Drugs Future 1996, 21, 375. [42] Jedrzejas, M.J.; Singh, S.; Brouillette, W.J.; Laver, W.G.; Air, G.M.; Luo, M., Biochemistry 1995, 34, 3144. [43] Williams, M.; Bischofberger, N.; Swaminathan, S.; Kim, C.U., Bioorg. Med. Chem. Lett. 1995, 5, 2251. [44] Jedrzejas, M.J.; Singh, S,; Bouillette, W.ILaver, W.G.; Air, G.M.; Luo, M., /. Med. Chem. 1995, 38, 3217. [45] Corey, E.J.; Eckrich, T.M,, Tetrahedron Lett. 1984,25,2419. [46] Kim, C.U.; Lew, W.; Williams, M.A.; Liu, H.; Zhang, L.; Swanunathan, S.;

154 Bischofberger, N.; Chen, M.S.; Mendel, D.B.; Tai, C.Y.; Laver, W.G.; Stevens, R.C., J. Am. Chem, Soc, 1997,119,681. [47] McGowan, D.A,; Berchtold, G.A., J. Org. Chem. 1981,46, 2381. [48] Mendel, D.B.; Tai, C.Y.; Escarpe, P.A.; Li, W.; Sidwell, RW.; Huffman, J.H.; Sweet, C ; Jakeman, K.J.; Merson, J.; Lacy, S.A,; Lew, W.; Williams, M.A.; Zhang, L.; Chen, M.S.; Bischofberger, N.; Kim, C.U., Antimicrob. Agents Chemother. 1998, 42, 640. [49] Kim, C.U.; Lew, W.; Williams, M.A.; Wu, H.; Zhang, L.; Chen, M.S.; Escarpe, P.A.; Mendel, D.B.; Laver, W.G.; Stevens, R.C., J. Med Chem. 1998, 41,2451. [50] Kim, K.S.; Quian, L., Tetrahedron Lett. 1993, 34, 7677. [51] V.R. Atigadda; W.J. Brouillette; F. Duarte; S.M. Ali; Y.S. Babu; S. Bantia; P. Chand; N. Chu; J.A. Montgomery; D.A. Walsh; E.A. Sudbeck; J. Finley; M. Luo; G.M. Air; G.W. Laver, J. Med Chem. 1999, 42, 2332. [52] B.G. Chatterjee; V.V. Rao; B.N.G. Majmndar, J. Org Chem. 1965, 30,4101. [53] Bianco, A; Brufani, M.; Melchioni, C , // Farmaco, 2001, in press. [54] Bianco, A ; Brufani, M.; Manna F.; Melchioni, C, Carhohyd. Res. 2001, 332,23. [55] Bianco, A; Bonadies, F.; Melchioni, C, Molecules, 2000, .5,1094.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

155

SOME ASPECTS OF THE CHEMISTRY OF QUATERNARY BENZO[c]FHENANTHRn)INE ALKALOroS j m i DOSTAL* A N D JIRI

SLAVIK

Department of Biochemistry, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, CZ-662 43 Brno, Czech Republic ABSTRACT: Quaternary benzo[c]phenanthridine alkaloids (QBA) are a relatively small class of natural products. These isoquinoline-derived alkaloids are conspicuous for their bright colours. They currently number 18, apart from evident artifacts and synthetic derivatives. QBA are widely distributed in a number of plant species of the Papaveraceae, Fumariaceae, and Rutaceae families. The most abundant sources of QBA are Sanguinaria canadensis, Chelidonium majus, Dicranostigma lactucoides, Macleaya cordata, M. microcarpa, and some Bocconia and Zanthoxylum species. Sanguinarine and chelerythrine, discovered in the 19th century, are the two best-known and commercially available QBA. Quaternary benzophenathridine cations display great susceptibility towards nucleophilic reagents. This nucleophilic attack is associated with a number of significant alterations in physical appearance, solubility, constitution, spectral properties, etc. A specific and very interesting chapter in the chemistry of the QBA concerns the formation of free bases. In an alkaline environment, QBA are converted to bimolecular aminoacetal derivatives with an oxygen atom as the linking bridge. This review describes the distribution of QBA in plants. Methods of isolation and separation are included as well as biological activities. The main part of this chapter is devoted to the chemical reactivity of QBA, nucleophilic transformations and the formation offreebases.

INTRODUCTION Quaternary benzo[c]phenanthri(iine alkaloids (QBA) are a relatively small class of natural products conspicuous for their bright colours and interesting particularly for their chemistry and their biological activities. At least 18 QBA, including one quatemary seco-derivative, have been isolated from plants. The general formula of the N-methylbenzo[c]phenanthridinium cation including the numbering is depicted in Fig. (1). The four fiised aromatic rings are denoted A, B, C, D. The nitrogen atom at position 5 bears a methyl group and a positive charge. Thus, in plant tissues, QBA

156

are cationic species associated with any physiological anions present. The bond between the atoms N5 and C6 is usually called the iminium bond and it is the most reactive site in the molecule. Various combinations of oxygen substituents, namely methoxy and methylenedioxy groups, and one hydroxy group in phenoUc alkaloids, have been found at positions 2, 3, 7, 8, 9, 10 and 12.

Fig. (1). Tlie numbering of the N-methylbQizo[c]phenanthridinium cation

The QBA are divided into the following groups according to substitution: A. 2,3,7,8-Tetrasubstituted (sanguinarine type): sanguinarine (1), chelerythrme (2), isofagaridine (3), fagaridine (4), and punctathie (3 X OMe, 1 X OH). B. 2,3,8,9-Tetrasubstituted (nitidine type): nitidine (5), avicine (6), fagaronine (7), and terihanine (8). C. 2,3,7,8,10-Pentasubstituted (sanguinarine type): sanguilutine (9), sanguirubine (10), cheUrubine (11), cheUlutine (12), 10-hydroxysanguinarine (13), and lO-hydroxychelerythrine (14). D. 2,3,7,8,10,12-Hexasubstituted: macarpine (15) and 12-hydroxychelirubine (16). E. Quaternary B-secoalkaloid: usambanohne (17).

+ XH3 X

Sanguinarine (1): R^ + R^ = CH2 Chelerythrine(2): R^ = R2 = CH3 Isofagaridine (3): R^ = H,R^ = CH3 Fagaridine (4): R^ = CH3,R^ =H

Nitidine (5): R^ + R^ = CH2,R^ = R^ = CH3 Avicine(6): R^ + R^ = R^+ R^ = CH2 Fagaronine(7): R^ = H,R2 = R ^ = R^ = CH3 Terihanine (8): R^ + R2 = CH2,R^ = H,R^ = CH3

157

Sanguilutine(9): R' = R' = R^ = r = R' = CH3 Sanguirubine (10): R^ = R^ = R^ = CH3, R^ + R^ = CH2 Chelirabine (11): R^ + R^ = R^ + R^ = CH2, R^ = CH3 Chelilutine (12): R^ + R^ = CH2, R^ = R^ = R' = CH3 10-Hydroxysanguinarine (13): R^ + R^ = R^ + R^ = CHi, R^ = H lO-Hydroxychelerythrine (14): R^ + R^ = CH2, R' = R^ = CH3, R^ = H

Macaipine (15): R=CH3 12-Hydroxychelirubine (16): R = H

Usambanoline (17)

A number of reviews on various aspects of QBA have been published. The principal information can be found in the two books on isoquinoline alkaloids by Shamma [1,2] and the reviews by Santavy [3,4] and Simanek [5] in The Alkaloids series. A highly valuable review of the physical and spectral data and the occxirence of the 88 benzophenanthridine alkaloids has been con5)iled by the Shamma group [6]. Preininger has summarized the distribution of QBA in plant species of the Papaveraceae and Fumariaceae famUies [7]. A review by Dostal and Potacek specifically devoted to the in vitro nucleophiUc conversions of QBA appeared in 1990 [8]. Surveys on the biological activities [9-11] and biosyathetic formation [12,13] of these alkaloids have been pubUshed. Well-designed reviews of the syntheses of the QBA are also available [14-16]. Hanaoka and Mukai presented a treatise on the biomimetic syntheses of the benzophenanthridhies fi'om the protoberberines m volume 14 of the Studies in Natural Products Chemistry series [17]. Quite recently, a book by Bentley treating isoquinoline alkaloids also involved benzophenathridines [18].

158

DISCOVERY OF THE ALKALOIDS Sanguinarine (1) and chelerythrine (2), the two best-known and commercially available quaternary benzophenanthridines, were discovered in the 19th century (Table 1). As early as 1827 James Freeman Dana isolated crude sanguinarine from the rhizomes oi Sanguinaria canadensis L. [19]. However, due to the primitive isolation procedure, the material isolated was later shown to be a mixture of all the alkaloids occurring in sanguinaria, i.e. the QBA plus some protopine and allocryptopine [20]. Pure sanguinarine was obtained for the first time by Gadamer and Stichel from Chelidonium majus L. [21]. The bright red colour of the sanguinaria latex (from Latm sanguis, blood) is caused by the colours of all the QBA present. The crystalline salts of sanguinarine are copper red. The opinion that colourless sanguidimerine (31) is the principal alkaloidal conq)onent of the S. canadensis rhizomes and that sanguinarine is formed from this compoimd during the isolation process [22,23] is therefore hardly acceptable. In 1839, after isolation from Ch. majus., chelerythrine (2) was described by Probst as an alkaloid yielding red salts (from Greek erythros, red) [24,24]. In fact, it was a mixture with sanguinarine [26]. The pxire alkaloid was isolated by Konig, who left it the original name, chelerythrine, in spite of the bright yeUow colour of its salts [26]. The alkaloid toddahne, isolated from Toddalia aculeata Pers. [27], was later proved to be identical with chelerythrine [28,29]. Up to 1954 no other QBA were known. The discovery of the minor alkaloids chelirubine (11), chelilutine (12), sanguihitine (9), sanguirubine (10), and macarpine (15) was achieved following the introduction of column chromatography. Chelirubine and chelilutine were isolated for the first time in 1954 from the roots of Ch. majus as minor components (0.013% and 0.002%, respectively) [30]. Later they were obtained from a number of Papaveraceae species and one species of Fumariaceae, Dicentra spectabilis L. [31]. They are absent from only the genera Argemone, Roemeria, Meconopsis, and Papaver except P. oreophilum Rupr. [32]. The highest content of both alkaloids has been found in S. canadensis rhizomes (0.074% and 0.229%, respectively) [33]. Bocconine [34], described in 1965, is identical to chelirubine [35]. The salts of chelirubine and chelilutine are deep purple and orange-yellow, respectively, as indicated by the second parts of their names (from Latin ruber, red, luteus, yellow).

159

Sanguilutine (9) and sanguirabine (10) were isolated from S. canadensis rhizomes [33] in 1960. Recently, sanguilutine has also been detected in Ch. majus [36]. The salts of sanguirabine and sanguilutine are carmine and golden yellow, respectively. Macarpine (15), a very rare hexasubstituted alkaloid, was first isolated from the roots of Macleaya microcarpa (Maxhn.) Fedde in 1955 with a yield of 0.002% [37] and later found in M cordata (Willd.) P. Br. [38], Ch. majus [36,37], some Eschscholtzia species [7], Stylophorum diphyllum (Michx.) Nutt. [39], and S, lasiocarpum (Oliv.) Fedde [40]. The lastnamed plant is the best known source of this alkaloid (0.023%). In recent years, dihydromacarpine has been isolated from the cell cultures of Eschscholtzia californica ChmxL [41,42]. Tanahashi and Zenk isolated three new phenoUc alkaloids 13, 14, and 16 from the cell cultures ofE. californica after the action of a yeast eUcitor [41]. These alkaloids are iateimediates in the biosynthesis of chelirabine, chelilutine, and macarpine, respectively. Fagaridine (4), a phenoUc QBA of the sanguinarine type, has been obtained from Fagara xanthoxyloides Lam. [43], Zanthoxylum nitidum DC. [44] (designated as isofagaridine), and Z tessmanii (Engl.) Ayafor comb, nov. (synonym: Fagara tessmanii Engl.) [45]. Quite recently, Nakanishi and Suzuki [46] have revealed on the basis of precise UV and NMR studies and synthesis of 4 that fagaridine of the formerly proposed stracture 3 has in fact the stracture 4, originally ascribed to isofagaridine [44]. The isomeric compound 3 is not considered to be a natiural product. However, Chen et al [47] have isolated a 6-methoxy adduct of the compound 3 (7demethyl-6-methoxydihydrochelerythrine) from Z nitidum and supported its stracture by using X-ray analysis (we have not seen the original paper). Punctatine, a phenoUc QBA of the sanguinarine type, has been isolated from Z punctatum Vahl. [48]. The suggested stracture contains three methoxy groups and one hydroxyl in unknown positions. Nitidine (5) was isolated for the first time from Z nitidum [49,50]. AlkaUzation of the quatemary salts leads to a mixture of 5,6-dihydronitidine and 6-oxonitidine [49,50]. The salts are relatively stable as is the pseudocyanide [49]. Avichie (6), first isolated from Z avicennae D C , is also an unstable species [51]. Fagaromne (7), a phenoUc QBA of the nitidine type, has been isolated from Fagara xanthoxyloides Lam. [52] and exhibits a high antileukemic activity.

160

Terihanine (8) was originally isolated as its 6-oxoderivative named oxyterihanine from Zanthoxylum nitidum DC. (synonym: Xanthoxylum nitidum (Roxb.) D C ; Japanese name, teriha-zansho) [53]. Later Ih-Sheng Chen isolated terihanine from Taiwan Z nitidum as a new quaternary alkaloid (private communication from Professor Hisashi Ishii, Chiba University, Japan). The structures of terihanine and oxyterihanine have been estabUshed by synthesis [54,55]. Usambanoline (17) is a recently isolated quatemary B-secoderivative of QBA found in two Kenyan species, Z. chalybaeum and Z usambarense [56]. It represents the first member of this type found in nature. Table 1. The Chronological Outline of QBA Discoveries Alkaloid

Colour'

Sanguinanne (1)

cx3pperred

1827

Sanguinaria canadensis

Chelerythrine (2)

yellow

1839

Chelidonium majus

Ghdimbine (11)

deeppuiple

1954

Chelidonium majus

Chelilutine (12)

orange-yellow

1954

Chelidonium majus

Macaipine (15)

cdmscnred

1955

Macleaya microcarpa

Avidne (6)

yellow

1959

Zanthoxylum avicennae

Nitidine (5)

yellow

1959

Zanthoxylum nitidum

Saaguirubine (10)

canninered

1960

Sanguinaria canadensis

Saaguilutine (9)

golden yellow

1960

Sanguinaria canadensis

Fagaronine (7)

b r i ^ yellow

1972

Fagara xanthoxyloides

Fagaridine (4)

yellow (orange*)

1973

Fagara xanthoxyloides

Punctatine

orange-yeiliow

1977

Zanthoxylum punctatum

Terihanine (8)

yellow''

1984

Zanthoxylum nitidum

Isofagaiidine (3)

orange''

1989

Zanthoxylum nitidum

lO-Hydroxysanguinarine (13)

dark red

1990

Eschscholtzia califomica

lO-Hydroxychelerythrine (14)

li^red

1990

Eschscholtzia califomica

orange

1990

Discovery

Hani spedes

12-Hydroxydielirubme (16) Eschscholtzia califomica orange 1996 1 Usambanoline (17) Zanthoxylum usambarense ' Colour of the crystalline salt; the colours of solutions may deviate sh^itly depending on the coQcentraticn and the solvent. ^ Synthetic pr^aration.

161

DISTRIBUTION The occurrence of QBA in the plant kingdom is restricted to the famiUes Papaveraceae^ Fumariaceae^ and Rutaceae with one exceptional case in the Ranunculaceae family. In most plants, the QBA are frequently accompanied by greater or smaller quantities of their metaboUc satehtes such as the 5,6-dihydro-, N-demethyl- (nor-), and 6-oxoderivatives. Because of their antimicrobial and antiftingal activity, the QBA are beUeved to be compounds plants use for defense in their struggle for life in the ecosystem. The amount of the QBA in plants varies considerably, from traces to several percent depending on a variety of conditions. Their content is usually highest at the end of the vegetation period and in the undergiound parts of the plant, while it is one to three orders of magnitude lower in the aerial parts. The alkaloids sanguinarine and chelerythrine are in most cases accompanied by the minor alkaloids chelimbine, chelilutine, sanguilutine, sanguirubine, and, in several plants, macaipme. These minor alkaloids have never been found in the Rutaceae family. In contrast, alkaloids of the nitidine type occm* exclusively in the Rutaceae and are one of the chemotaxonomic features of some genera. The Papaveraceae family is the most inq)ortant source of the QBA. This family is known as a taxonomic group very rich in isoquinoline alkaloids of great structural diversity [3,4,7] which are biogenetically derived from phenylalanine and tyrosine. The QBA are distributed ahnost throughout the whole family in a number of genera and species [7] in both the subfamilies Hypecoideae (sometimes considered as an independent family Hypecoaceae) and Papaveroideae {Papaveraceae s.str.) with only a few exceptions. In the papaveraceous plants, the alkaloids are locaUzed in lactiferous vessels. The most abundant sources of QBA are some genera and species of the tribe Chelidonieae, particularly the rhizomes of the North American perennial Sanguinaria canadensis (up to 3-7% of the dry material) [33,57]. The major alkaloid of the rhizomes is sanguinarine. Column chromatography [33] and HPLC [57] separations have shown the following composition of the total QBA fraction (in %): sanguinarine 36.5 and 50, chelerythrine 33.4 and 25, sanguilutine 9.1 and 15, chelilutine 7.8 and 5, chelirubine 2.5 and 4, sanguirubine 1.2 and 1, respectively.

162

The second-richest QBA source is Dicranostigma lactucoides Hook. f. et Thorns., a biennial herb native to the subtropical region of the eastern Himalayas. The QBA content m the roots usually varies from 1.4 to 1.8% [58-60] and can reach up to 3.4% accordmg to recent HPLC studies. In contrast to S. canadensis, the major conoponent of the total QBA is chelerythrine (71%), and there are smaller amounts of sanguinarine (21%) and chelirubine (1%) [58]. The other members of the genus Dicranostigma, i.e, D. franchetianum (Prain) Fedde and D. leptopodum (Maxun.) Fedde, exhibit lower contents of QBA [59]. Another good soiu'ce of QBA is the root of Chelidonium majus, a perennial cormnon in Europe [61-64]. The highest contents, up to 0.5% [61] and 0.8% [62,63], have been found in autumn, when the QBA composition was: chelerythrine 63.6%, sanguinarine 32.9%, chelirubine 3.0%, and chelilutine 0.6% [30]. Some cultivated populations with a higher content of QBA have also been described. Minute quantities of macarpine [36,37] and sanguilutine [36] have also been detected. The major alkaloid in Ch. majus is cheUdonine [61] or coptisine [36,64,65]. The other dominant alkaloids are berberine, homocheUdonine, protopine, allocryptopine, and magnoflorine, and there are a great many minor alkaloids including dihydrosanguinarine, dihydrochelerythrine, dihydrocheUrubine, dihydrochelilutine, oxysanguinarine, and N-demethyl-9,10-dihydrooxysanguinarine [36,61,65]. The species of the genus Macleaya, namely M cordata and M microcarpa, perennial herbs from the Far East, are used in Russia as a source of the natural mixture of sanguinarine and chelerythrine (called sanguiritrine), that has been introduced into medicine for the treatment of various diseases. The content of QBA in the leaves of M microcarpa has been found to be imusually high, up to 1.97% [66]. The QBA contents in M microcarpa and M cordata cultivated in central Europe [37,38] are considerably lower. Snnilar low yields have been found by Bulgarian, Japanese, and Russian authors [5,6]. Chelirubine, chelilutine, and macarpine have also been isolated f^omMacleaya species [37,38]. The alkaloid bocconine [35], isolated from M cordata (invaUd synonym: Bocconia cordata WiUd.) in 1965, is identical to chelirubine [34], described for the first time in 1954 [30]. The genus Bocconia consists of about ten species. These are woody plants, shrubs or small trees indigenous to Central and South America. Some of them are significantly rich in chelerythrine, as for exarcqple B. or-

163

borea S. Wats, (aerial parts 0.86%) [67], 5. pearcei Hutch, (bark 0.91.0% together with 0.7% of sanguinarine) [68,69]. Jn B, frutescens L. [70] and B, pearcei [69], chehrubine was found as a nunor alkaloid. Remarkable is the relatively high content of dihydrochelirubine together with a new alkaloid 12-methoxydihydrochelerytlirine found in B. integrifolia Humb. etBonpl. [71]. Among otilier genera of the Papaveraceae, i.e. Pteridophyllum, Hypecoum, Platystemon^ Arctomecon [72], Romneya, Hunnemannia, Eschscholtzia, Stylophorum, Hylomecon, Glaucium, Meconopsis, and Argemone, the concentrations of QBA are much lower and vary between trace amounts and 0.1%. Most of them have been reviewed by Preininger

m.

The genera Roemeria and Papaver are practically devoid of QBA or contain scarcely detectable quantities. There are a few exceptions: sanguinarine has been isolated f^om Papaver radicatum Rottb. [73] and detected in P. oreophilum Rupr. in traces together wdth cheHrubine [32], The technique adopted by Hakim et al. [74] is not specific enough to identify the QBA and quaternary protoberberines. There are many plant products in an extract which may simulate their presence. In several Papaver species, oxysanguinarine has been found in minute quantities [6,7]. It may be of interest to note that in contradistinction to the intact plants, the biosynthesis of sanguinarine, dihydrosangumarine, and oxysanguinarine does take place in callus tissue cultures of P. somniferum [75,76]. In the Fumariaceae, the occurence of QBA is more limited than in the Papaveraceae, the main QBA being sanguinarine, together with chelerythrine in a few species. Chelirubine and chelilutitie have been encountered only in Dicentra spectabilis [31]. In the Fumariacae, the QBA content in imderground parts varies from 0.001 to 0.1%. Only a few Corydalis species exhibit higher contents of QBA, as for example C severtzowii Rgl. [77] and C. caucasica DC. [78] (0.17 and 0.12% of sangmnarine, 0.03 and 0.08% of chelerythrine, respectively). In several species of the genera Dicentra^ Fumaria^ Sarcocapnos, and Dactylicapnos^ sanguinarine has been found as a minor alkaloid. The Rutaceae are known as a large plant family with approximately 150 genera and 1,000 to 2,000 species, chemically characterized by numerous alkaloids of extensive structural diversity. Some isoquiaoHne alkaloids are known ID this family of which especially aporphines, protopiaes, protoberberines, and benzo[c]phenanthridiaes are cormnon in the Papave-

164

raceae and Fumariacae, The occurence of QBA and their derivatives is restricted to the genera Fagara, Fagaropsis, Phellodendron, Toddalia, and Zanthoxylum (synonym: Xanthoxylum), All Fagara species are often considered to be part of Zanthoxylum. Recently, six benzophenanthridine alkaloids (6-acetonyldihydrosanguinarine, amottianamide, bocconoline, decarine, norchelerythrine, and oxychelerythrine) have been found also in Tetradium glabrifolium (Chanq). ex Benth.) T. Hartley [79]. The QBA content in the Rutaceae is relatively low, mostly up to 0.05%; only rarely does it reach over 0.1%, e.g. up to 0.12% ia Toddalia aculeata Pers. [29]. Chelerythrine and nitidine are ahnost always the principal QBA. Alkaloids of the nitidine type are known to occur only in the Rutaceae family. Chelerythrine and nitidine have been isolated from a number of Fagara and Zanthoxylum and one species of Toddalia. In some cases the artifact 6-methoxydihydrochelerythrine has been obtained. In exceptional cases, sanguinarine has been found in Rutaceae^ namely in Zanthoxylum conspersipunctatum Merr. et Perry [80] and probably also in Fagaropsis angolensis (Engl.) Dale [81] (isolated as 6-acetonyldihydrosanguinarine which might be an artifact). Avicine has been foimd in one Fagara and one Toddalia species [82,83], and also some of Zanthoxylum species. In the Rutaceae, QBA are accon5)anied byfiiranoquinohnes,pyranoquinolines, and acridone alkaloids as well as carbazoles, canthiQe-6-ones, P-carbolines and others which are the dominant alkaloids [84-86]. The only occurence of QBA in the Ranunculaceae family known to date is in the seeds of Coptis japonica (Thunb.) var. dissecta (Yatabe) Nakai [87], from which sanguinarine has been isolated together with dihydro-, oxy- and norsanguinarine. ISOLATION AND SEPARATION METHODS It has been well known since the discovery of sanguinarine and chelerythrine [19,20,24,25] that, in contrast to their brightly coloured salts, their free bases are colourless and pass into non-polar solvents even from sUghtly alkaline media along with other tertiary alkaloids. All QBA of the sanguinarine type have the same behaviour. Generally, the procediu:e for the isolation of QBA is the same as for most non-quaternary alkaloids with typical properties, i.e. a classical acid-base process followed by the extraction of the liberated free bases into non-polar solvents.

165

Methanol appears to be the most suitable relatively inert solvent for the extraction of dried plant material either in the cold or at higher temperature. After evaporation of the solvent, the residue is extracted with dUute acid (e.g. 1 to 5% HCl or H2SO4) and fUtered. The insoluble material is the best source of non-basic alkaloids (dihydro-, 0x0-, and norderivatives of QBA) [61,65]. The acidic fltrate is made alkaUne with Na2C03 or NH3 and extracted repeatedly with a non-polar solvent. The most advantageous choice seems to be diethyl ether because its high selectivity yields a crude alkaloid fraction of relatively good purity (up to 95%). Other solvents, especially chloroform, dissolve much more of the tar material so that the crude fraction may contain up to 90% of non-alkaloidal matter. The separation of the pure QBA fraction may be achieved in different ways. In those cases where QBA represent the major con^onents, such as the xmderground parts ofS. canadensis and D, lactucoides, it is preferable to dissolve the total alkaloid fraction in hot dilute hydrochloric acid. After cooling, the QBA chlorides crystallize out almost quantitatively [33,5860]. If in a total alkaloid mixture other alkaloids prevail and their hydrochlorides are sUghtly soluble (cheUdonine, stylopine), it is necessary to separate these first or use another method. The most suitable seems to be the process based on the insolubility of the 6-cyanodihydro derivatives of QBA (pseudocyanides) in dilute acids [88, also 33,40,61]. This procedure is called for when the QBA are present ia the plant in minute quantity only and might escape attention if another method of separation were used. The total alkaloids are dissolved in 1% sulfiiric or hydrochloric acid and an aqueous solution of NaCN is added to make the reaction mixture alkaline. The mixture is then acidified again, and the insoluble whitish precipitate of pseudocyanides is collected. They are converted back iato chlorides by refluxing in a chloroform-ethanol-hydrochloric acid mixture [30,33,40]. The separation of individual QBA is rather difficult, and several methods have been described in the Uterature (procedures based on different basicity, separation in the form of dihydro derivatives, column chromatography of Ae free bases, and others) [5,8], none of which seems to be suitable for the isolation of minor QBA. Column chromatography of QBA acetates on acidic ahiminhim oxide has been very effective [30]. This method has been modified for the separation of QBA chlorides [60]. A recent alternative is the use of column chromatography with a non-polar sorbent and an acidic aqueous mobile phase [41]. Many reports of HPLC detection

166

and quantitative determination of individual QBA have appeared in recent years [8]. The isolation and separation of the nitidine-type alkaloids is substantially different because of their instabiUty in alkaline media in which they easily undergo a disproportionation to dihydro- and oxoderivatives. In order to prevent chemical changes it is necessary to protect nitidine against a strong alkaline reaction. Isolation methods for nitidine and related alkaloids are mostly based on the direct crystallization of the chlorides from crude or purified concentrated extracts [89-92], pseudocyanide formation [50,51], preparative TLC [93,94] or colimm chromatography [95,96]. Nitidine has also been obtamed by extraction with chloroform from a medium sUghtly alkalized with ammonia [53,82] and its mixture with avicine separated in the form of dihydroderivatives after NaBHU reduction [82]. CHEMICAL REACTIVITY The principal feature of the chemical reactivity of QBA is the addition of a nucleophile to the iminium bond C=N^ (Scheme 1). The carbon atom C-6 displays the lowest 7C-electron density [97,98]. This process is associated with a number of significant alterations in the constitution, physical appearance, solubiUty, spectral properties, etc. The quaternary cation is a brightly coloured, polar, water-soluble species. The tertiary-nitrogen adduct has lost the colour and is non-polar and water insoluble. In the case of aminoacetal and aminal derivatives (Scheme 1, Nu = OR, NHR), the reaction is essentially reversible, i.e. the action of acid immediately converts the adduct back to the quaternary salt. Viewed from another perspective: the emergence of colour is a sensitive indicator of the presence of some acid and ipso facto of deconq)osition of the adduct.

Nu"

Scheme 1

167

On the other hand, the adducts with a carbon-carbon Unkage, e,g, 6cyanodihydrobenzophenanthridines (Scheme 1, Nu = CN) are rather resistent to acids. A number of reactions with carbon nucleophiles (cyanide, Grignard reagent, nitromethane, acetone, butanone, acetaldehyde) are known and well documented ia the Uterature [8]. Only a Uttle is known about QBA reactions with oxygen, nitrogen, and sulfur nucleophiles [8]. Some C-adducts of QBA have been isolated from plant species and these often represent the unique structural motifs of natural products. The alkaloid nitrotyrasanguinarine (18), isolated from Hypecoum imberbe Sibth. et SuL, is one of the very few natural nitrocompounds. It is supposed that nitrotyrasanguinarine is formed by the oxygenation of tyramine foflowed by the addition of its a-nitrocarbanion to the iminium bond of sanguinarine [99]. Nxunerous derivatives of dihydrochelerythrine have been obtained from Zanthoxylum species. The alkaloids 6-methyldihydrochelerythrine (19) and simulanoquiiioline (20) with a quuiolinone pendant have been foimd m Z. simulans Hance [100,101]. Tridecanonchelerythrine (21) has been isolated from Z integrifolium Merr. [100]. Ailanthoidine (22) obtained from Z. ailanthoides Sieb. et Zucc. possesses a cyanopyridine moiety [103]. The work-up of the bark of Z spinosum (L.) Sw. (?) has afforded among others 6-carboxydihydrochelerythrine (23), 6-(4-methyl-2oxopentyl)dihydrochelerythrine (24), and chelelactam (25) with attached pyrroHdone [94].

19: R = CH3

23: R = CH2COOH

20: R = CH2-

24: R = CH2COCH2CH(CH3)2 25: R =

T3f

O

NH

21: R = CH2CO(CH2)ioCH3 22: R = CH{CH3;

26: R = CH2COCH3 27: R = CH20H

V\

// CN

168

6-Acetonyldihydrochelerythrine (26) and bocconoline (6-hydroxymethyldihydrochelerythrine) (27) are other exanq)les of such products which have been found in a number of plant species [6]. Some authors consider 6acetonyldihydroderivatives to be artifacts. They may arise from QBA and acetone (present as an impurity m technical solvents) or acetonedicarboxyUc acid (which may be present in some plants) after being alkaUzed. Because of their great susceptibiUty to nucleophiHc attack the QBA easily yield unnatural con5)ounds (artifacts) that may arise during the isolation and purification processes. Many kinds of such adducts have been described. The artifacts most often encountered in thefiteratiu^eare the adducts with alcohols, particularly with methanol and ethanol, i.e, 6methoxy- and 6-ethoxydihydroderivatives, respectively. Although it has long been known that sanguinarine and chelerytiuine free bases "crystallize with one molecule of alcohol" [20,21,26], these adducts (so-called pseudoalcoholates) have been reported sometimes from plants and not rarely considered as genuine natural products or in some cases even declared to be new alkaloids. It should be pointed out that pseudoalcoholates behave as true bases since they are alkahne to Utmus and with acids (even weak ones) they immediately yield coloured quatemary salts. Because of their alkaline character they cannot exist in more or less acidic plant tissues. The 6-alkoxydihydrobenzophenanthridines are formed by single crystallizations of QBA free bases from alcohols or alcohol-containing solvents during the purification process or in the course of column chromatography and elution with solvents containing alcohols. 6-Methoxydi- hydrochelerythrine (angoline) (28) [104] and 6-methoxydihydrosangui- narine (29) have been described several times as natural alkaloids [5,6,84-86]. In recent years, they have been reportedly obtained from Chelidonium majus [105-107], Hypecoum imberbe [99], H. leptocarpum Hook, f et Thoms. [108] and other species. 6-Methoxydihydronitidine (30) has been obtained from Fagara macrophylla (Oliv.) Engl, and declared to be an artifact [109]. 6-Methoxydihydrofagaridine has been reportedly obtained from Zanthoxylum nitidum DC. [110]. The 6-ethoxydihydroderivatives of sanguinarine and chelerythrine have similar origins [5,6].

169

R^

CX^Hs

28:R^ = R^ = OCH3,R^ = H 29:R^ + R^ = CH2,R^ = H 30:R^ = H,R^= R^ = OCH3

A specific aspect of the chemistry of QBA deals with dimeric derivatives possessing two dihydrobenzophenanthridine moieties comiected by a bridge involving carbon-carbon bonds. In 1970, Tin-Wa and co-workers isolated a new alkaloid fi'om Sanguinaria canadensis with a molecular mass of 720 [22]. Later, they estabUshed its structure as optically active l,3-bis(dihydrosangumarinyl)acetone (31), called (+)-sanguidimerine [23].

31 32

CheUdimerine, an optically iaactive alkaloid of the same constitution 31, was isolatedfiromCh, majus. X-ray analysis indicated the existence of the chiral P2i2i2i space group. Therefore, the authors deduce that it might have been a me^o-isomer of 31 [111]. However, no other X-ray data were reported. Recently, optically inactive chelidimerine has been found ia a Kashmir specimen of Corydalis flabellata Edgew. [112] and Turkish C.

170

rutifolia (Sibth. et Sm) DC. [113]. An analogous dimeric derivative of chelerythrine has been isolated from Bocconia arborea [114] and prepared synthetically from chelerythrine and 1,3-acetonedicarboxyUc acid 'm alkaUne medium [114,115]. Another interesting dimeric alkaloid, caymandimerine (32), 2,2-bis(dihydrochelerythrinyl)acetaldehyde has been obtained from Zanthoxylum spinosum (?) collected on Grand Cayman Island [94]. FREE BASE FORMATION A particular and very interesting chapter in the chemistry of the QBA is devoted to their free bases. The term^^^ base has been universally adopted for the product with basic properties which is formed upon alkalization of an alkaloid salt and which reverts to the original salt hi acidic solutions. Thus, the formation of a free base is a reversible acid-base reaction. Generally, a free base can be considered as a kind of artifact because it does not occur in (acidic) plant tissues. The natural form of a QBA is the iminium salt as can be illustrated by the typically bright colours of the organs, fresh latex or alcohohc extracts of plants. There are essentially two methods of preparing the free bases of QBA (and, in principal, of other typical alkaloids). Method A: The salt of the alkaloid is dissolved in water and the solution is made alkaline with a saturated Na2C03 solution. The amorphous white precipitate of the free base is collected, washed with water and dried in an acid-free environment. This method provides a good yield vsdth minimal losses. However, high purity of the starting salt is a necessary condition in this case. Method B: Another approach is a more elegant altemative. The precipitate of the free base is extracted with a non-polar solvent. We prefer to use ethanol-free diethyl ether. The organic layer is allowed to stand at ambient temperature until the base crystallizes. In this case, the free base appears as colourless crystals with a considerably higher and sharper melting poiat than provided by method A. However, the yield is markedly lower and ia some cases we observed the existence of side products of as yet imknown constitutions. It has long been known that the corresponding quaternary hydroxides of QBA do not exist [5,20,21,26]. Alkalization of an aqueous solution of a QBA salt leads to an unstable transient product, the 6hydroxydihydroderivative. This cortq)o\md is called by the historical term pseudobase to enq)hasize the covalently bound hydroxyl group, A pseudo-

171

base possesses the properties of a semiaminoacetal, le, it displays a high reactivity towards nucleophiles. The reactions of QBA with nucleophiles in aqueous alkaline medium may in fact suggest the reaction between a semiaminoacetal and a nucleophile. In the absence of other nucleophiles, two molecules of pseudobase spontaneously eliminate one molecule of water yielding a bimolecular aminoacetal (Scheme 2). The pseudobase structure has been adopted almost universally for the QBA free bases although exact proofs have been conq)letely lacking. However, some properties of QBA free bases favour the bimolecular structures, whose existence was proposed as early as 1924 [21,33].

x^ ROH -H2O

OH*

ll^W OH

Sch^ne 2

The equilibrium between the quaternary heterocyclic cation (Q^) and the tertiary pseudobase form (QOH) is pH dependent and is usuafly formulated as an acid-base reaction: Q'"+

H2O

QOH + H+

The equilibrium is described by the constant PKR+ (or simply pK) which is analogous to the pKa of a Bronsted acid: KR+ = [QOH] [I^] / [Q^]. The constant e:q)resses the value of the pH at which the iminium cation and the

172

hydroxide adduct are present at equal concentrations. The pK constants of six QBA have been determined in aqueous phosphate buffers by both fluorescence [116] and spectroscopy [117] (Table 2). From the data given it follow^s that the most reactive alkaloid is cheUrubine (pK 7.70). The least sensitive to OHT attack is chelerythrine (pK 9.00). Table 2. The pK Constants of Some Quaternary Benzophenathridines Alkaloid

J ^

8.05

Sanguirubine (10)

7.90

9.00

Chelirabiiie (11)

7.70

8.80

Chelilutiae (12)

8.50

Alkaloid

pK

Saaguiaanne (1) Ghderythriae (2) Sanguilutine (9)

We have found that the free base of sanguinarine possesses the structure of bis(6-dihydrosangiiinarinyl) ether (33) although aheady m 1924 Gadamer (Marburg University, Germany) had described the sanguinarine base by using the formula (C2oHi404N)20 [118] which is consistent with the current finding [119] (Scheme 3).

33

Scheme 3

The structure of 33 has been supported by using elemental analysis, mass spectrometry and NMR spectroscopy [119]. However, the NMR spectra provided a more conq)lex depiction. They indicated the presence of five species in a CDCI3 solution of sanguinarine free base: (I) a major bimolecular stereoisomer, the racemate 6R,6'R + 6S,6'S, which is thermodynamically favoured according to AMI calculations [120]; (H) a minor isomer, a

173

meso-form 6R,6^S, which is due to a synunetrical structure identical to 6S,6'R (Scheme 4).

M e N ^ v^AAAAA/«

6S,6'S

6S,6'R

Scheme 4

The Other three derivatives were minor side-products. (HI) 6-Hydroxydihydrosanguinarine (34), a pseudobase, was a product of the spontaneous hydrolysis of the aminoacetal 33 by residual water in the CDCI3. (IV) Oxosanguinarine (35) and (V) dihydrosanguinarine (36) were the products of the disproportionation of the pseudobase (Scheme 5).

33

H20

Scheme 5

Oxosanguinarine (35) has been isolated from the mother Uquor after the crystalization of 33 and identified by m.p. and spectral data [119]. The formation of 0x0- and dihydrosanguinarine was proved in an independent experiment vA&i the composition of the alkaline reaction mixture was monitored using HPLC. As the compounds 35 (a lactam) and 36 (a substituted naphthylanrine) are virtuafly non-basic species they were readily

174

detectable in a strongly acid mobile phase [119]. Thus, the disproportionation of heterocycHc pseudobases seems to be a general phenomenon, known also in other systems [121,122]. It should be pointed out that the disproportionation of the pseudobase of sanguinarine occurs to a very small extent conopared with the 2,3,8,9-substituted QBA nitidine and avicine [50,51] in which this process is extensive. The structure of bis(dihydrosanguinarinyl) ether (33) has also been examined by X-ray analysis. The compound bears two chiral centers, the atoms C6 and C56, Fig. (2).

Fig. (2). Afrontview of bis(dihymcalfomiiila: C40H28N2O9

yff= 105.32(3) «

Molecular ^M^ight: 680.68

Volume = 3141.5(11) A'

0=

Ciystal system: monoclinic

Z = 4

Tenq)erature: 150 K

Space groiq>: P2j/c

A = 1.439 g e m '

Ciystal shape: prism

Z = 0.71073 A 3.36-25.00*'

a = 14.571(3) A

Radiation: Mo Ka

Ciystal size: 0.30 x 0.20 x 0.20 mm

b = 10.260(2) A

Absorption coefficient: 0.103 mni^

Ciystal colour: colourless

c = 21.788(4) A

F(OOO) = 1416

Crystal source: evaporation Irom ether

Table 4. Selected Bond Lengths (A) and Angles Q in 33 C1-C2 Cl-C12a 02-015 C2-C3 C3-C4 C3-016 C4-C4a C4a-C12a C4a-C4b C4b-C10b C4b-N5 N5-C6 N5-C20 C6-019 C6-C6a C6a-C7 C6a-C10a C7-017 C7-C8 C8-C9 C8-018 C9-C10 ClO-ClOa ClOa-ClOb ClOb-Cll C11-C12 C12-C12a 013-016 013-015 014-018

1.367(7) 1.428(6) 1.358(5) 1.386(7) 1.354(6) 1.381(5) 1.427(6) 1.428(6) 1.430(6) 1.376(6) 1.427(6) 1.451 (6) 1.482(5) 1.442(5) 1.502(6) 1.368(6) 1.425(6) 1.373(6) 1.384(7) 1.366(7) 1.366(5) 1.390(6) 1.392(6) 1.474(6) 1.415(6) 1.343(6) 1.413(7) 1.422(6) 1.428(6) 1.421(6)

014-017 019-056

1.436(6) 1.432(5)

015-02-C1 015-02-03 04-03-O16 Ol 6-03-02 O10b-O4b-N5 O10b-O4b-O4a N5-04b-04a 04b-N5-06 O4b-N5-O20 O6-N5-O20 019-06-N5 019-06-06a N5-06-06a O6a-07-O17 017-07-08 09-08-018 09-08-07 018-08-07 O6a-O10a-O10b 016-013-015 018-014-017 02-015-013 03-016-013 07-017-014 08-018-014 056-019-06

129.2(4) 110.0(4) 127.1(4) 108.9(4) 121.9(4) 120.8(4) 117.3(4) 114.5(4) 111.2(3) 111.6(3) 107.8(3) 107.8(3) 111.8(4) 127.6(4) 109.8(4) 128.7(4) 121.3(4) 110.0(4) 117.1(4) 107.5(4) 108.5(4) 105.0(4) 105.0(4) 105.5(4) 106.1(3) 112.5(3)

176

Fig. (3). A side view of the two molecules of bis(dihydrosanguinarinyl) ether (33)

Acids, even weak ones, immediately convert sanguinarine free base into the brightly coloured quaternary cation (Scheme 3). This property brings out another interesting feature: the alkaloid sanguinarine behaves as an acid-base indicator. In acid solutions it is orange (red), in alkahne environment colourless. The analogous structures and properties of the free bases of the related alkaloids chelerythrine, chelirubhie, and chehlutine have been determined [123,124]. The ^H NMR spectrum of chelerythrine free base (37) is very usefiil in understanding the structure.

The singlet of the H-6 atom at 6.60 ppm is a typical marker of the bimolecular aminoacetal constitution together with die resonances of the two OMe singlets. The C-7 methoxy group displays an unusually shielded sin-

177

glet at 2.41 ppm co]iq)ared with the neighbouring C-8 OMe group (3.72 ppm). The shielding is caused by the anisotropic effect of the second part of the bimolecular structure. In contrast, in the monomolecular 6-adducts of chelerythrine, the C-7 and C-8 methoxy groups have chemical shifts very close to the expected value of 3.90 ppm [6]. Recently, 6-hydroxydihydrosanguinarine (34) has been reported as a new alkaloid isolated from Dactylicapnos torulosa Hook f et Thoms. [125,126]. The conq)ound is described as an amorphous soUd of yellow colour, however, the substance exhibits major discrepancies in spectral data with the structure given [127]. 6-Hydroxydihydrochelerythrine has allegedly been isolated from Toddalia aculeata Pers. in the form of its acetic acid ester [128]. However, its ^H NMR data are not in agreement with those previously pubfished for this confound [129]. Universally, 6hydroxydihydroderivatives (pseudobases) are unstable semiaminoacetals with basic character which cannot occur in acidic plant tissues at all and could not be isolated as natural products especially after 5% HCl has been used in the isolation procedure [cf, 125,128]. The study of sanguilutine free base was very interesting. Sanguilutine (9) is a pentamethoxy substituted QBA. The IR spectrum of the free base showed the strong and sharp band at 3478 cm'^ of a hydroxyl group. This finding was confirmed by the ^H NMR spectrum, where we saw a doublet of the hydroxyl group at 2.20 ppm, spUt by 4 Hz with the same coupling for the signal of the hydrogen H-6 three bonds away. Thus, in the case of sanguilutine, the free base is a real heterocyclic pseudobase. A monocrystal was obtained by crystallizing it from benzene. X-ray data revealed that the crystal was bis(dihydrosanguilutinyl) ether (38). Thus, a non-polar environment supports the formation of a less polar derivative. According to the centrosymmetric space group P-1 the crystal was a racemate, not a me^o-form. In each part of the molecule, four of the methoxy groups are oriented in the planes of aromatic rings; the fifth one, 7-OMe, which is closest to the connecting bridge, is nearly perpendicular. As a consequence of this spatial arrangement, the 7-OMe group is heavily shielded in the ^H NMR spectrum (2.28 ppm) compared with the neighbouring 8-OMe (3.85 ppm); the difference is 1.57 ppm [130]. We have also studied the action of concentrated aqueous ammonia on QBA. In sanguinarine, chelerythrine, chehlutme, and sanguilutine, the pro-

178

duct is the nitrogen analog of the bimolecular free base with the NH bridge connecting the dihydrobenzophenanthridine moieties.

39

In sanguinarine, the precipitate formed contained a nuxture of the bimolecxilar ether base 33 and the bimolecular amine 39 in the approximate ratio 1:3 as a result of the con[q)etition of the two nucleophiles present in the aqueous ammonia. Extraction with ether and crystallization from the same solvent afforded crystalline bis(dihydrosanguinarinyl)amine (39) as the sole product. Elemental analysis confirmed the presence of three nitrogen atoms and the IR spectrum revealed the diagnostically significant band of the NH group at 3372 cm"^ of very low intensity [119]. Under the action of acids, bimolecular aminals like bimolecular aminoacetals immediately give the brightly coloured quaternary salts. BIOLOGICAL ACTIVITIES Sanguinarine and chelerythrine display antimicrobial activity agamst grampositive and gram-negative bacteria, antifimgal activity against some dermatophytes, and anti-inflammatory activity [9-11]. Sanguinarine, chelerythrine and their mixture were tested for anti-mflammatory activity in carrageenan rat paw oedema assays. Sanguinarine showed the highest activity, especially after subcutaneous appUcation, and was suggested as a pharmacotherapeutic agent in stomatology [131]. Sanguinarine has Kttle or no activity against Candida species [132-134]. QBA are also effective for the control of some fimgal diseases in plants. They have been sprayed on greenhouse roses infected with powdery mildew {Sphaerotheca pannosa)

179

with good efifect [135]. Sanguinarine is used as an anti-plaque agent in numerous dental-care products [136] although its eflfectivness is usually assessed as being variable or limited [137]. Generally, sanguinarine oral rinses are much more eflfective than toothpastes. This is probably due to the binding of other corq)onents to the chemically reactive site in the sanguinarine cation. Recent studies have revealed a certain cytotoxicity of sanguinarine for rat liver hepatocytes [138] and himian oral-cavity cells [139]. Chelerythrine is a potent inhibitor of protein kinase C [140], and an enormous number of papers from this area are now available. Sanguinarine and chelerythrine are the Jfirst non-peptide species to exhibit affinity for rat liver vasopresinVi receptors [141]. A novel synthetic QBA of the structure 3, coded NK 109, has shown notable anti-tumour effects against several drug-resistent human tumour cells [142,143]. Nitidine and fagaronine are known as potential anti-tumour, antileukemic, and antiviral agents [9,52,98]. Some synthetic 12-alkoxyderivatives of 2,3,8,9-tetramethoxy QBA are potent in vitro inhibitors of the growth of P388 cells [144]. Nitidine, isolated from Kenyan Toddalia aculeata Pers. (synonym: T, asiatica (L.) Lamk.) has shown antimalarial activity [145]. Nitidine and fagaronine have been foimd to inhibit HTV reverse transcriptase [146]. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 27 © 2002 Elsevier Science B .V. All rights reserved.

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BENZOXAZINONES IN PLANTS: OCCURRENCE, SYNTHETIC ACCESS, AND BIOLOGICAL ACTIVITY DIETER SICKER^ and MARGOT SCHULZ* ^ Universitdt Leipzig, Institutfur Organische Chemie, Johannisallee 29, 04103 Leipzig, Germany * Universitdt Bonn, Institutfur Landwirtschaftliche Botanik, KarlrobertKreiten-Str. 13, 53115 Bonn, Germany ABSTRACT: Acetal glycosides of the 2-hydroxy-2//-l,4-benzoxazin-3(4//)-one skeleton are naturally occurring in Acanthaceae, Poaceae, Ranunculaceae, and Scrophulariaceae, i.e. in plants of different taxonomic positions. They act as plant own resistance factors towards pests, like microbial diseases, insects and fungi. Structurally, they are unique because only in their case a nitrogen atom is part of the aglyconic cyclohemiacetal unit. In case of a pest attack, the glycosides undergo a two step degradation, consisting in an enzymatic deglycosylation followed by a chemical ring contraction of the 1,4-benzoxazinone aglycone to form a benzoxazolinon"2(3//)-one derivative. The latter process is taking place, when plants release such aglycones into the environment by root exudation. Both series of degradation products are bioactive towards pests and can also act as allelochemicals The driving force for all investigations is the possibility to make agricultural use of the results in the growing of main cereals, like maize, rye, and wheat (Poaceae). A future goal consists in the gene transfer for the benzoxazinone biosynthesis into other plants of agricultural interest. A detailed overview on natural occurring benzoxazinone acetal glucosides, benzoxazinone aglycones, and benzoxazolinones is presented. Three subjects of the benzoxazinone research are especially emphasised: synthetic access to aglucones and glucosides, medical effects of structures derived from natural product leads and molecular allelopathy, i.e. detoxification strategies of plants coexisting with benzoxazinone forming species in comparison with those belonging to other plant associations. Rapid access to further fields of research, like biosynthesis, plant-pest interaction, and molecular mode of action is given by citation of leading references.

INTRODUCTION The discovery of natural benzoxazinones was based on the finding that rye plants showed an increased resistance towards pathogenic fungi. Hence, the first aglucone and the first glucoside were reported from rye in two successive papers [1,2]. Acetal glycosides of the 2-hydroxy-2//-l,4-

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benzoxazin-3(4//)-one skeleton are naturally occurring in Acanthaceae, Poaceae, Ranunculaceae, and Scrophulariaceae, i.e. in plant species belonging to families of different taxonomic positions. It was the ability to act as a plant own source for the resistance of cereals towards pests, which immediately caused a really interdisciplinary research directed on the understanding of all aspects of this unique class of natural products, which is often named hydroxamic acids or cyclic hydroxamic acids. We will rather shortly call them benzoxazinoids or benzoxazinones for the following reasons. Though most of them contain a cyclic hydroxamic acid and some the related lactam, it is only by the direct combination with the cyclic hemiacetal unit that these compounds receive their unique bioactive properties. As mentioned below, the bioactivity is clearly enhanced by a donor substituent in position 7. Hence, to avoid misinterpretations of the features responsible for biological effects, the neutral terms benzoxazinones or benzoxazinoids, are better suitable to name the class. From the structural viewpoint benzoxazinoid acetal glycosides are unique because only in their case a nitrogen atom is part of the aglyconic cyclohemiacetal unit. They undergo a two step degradation, consisting in an enzymatic deglycosylation followed by a chemical ring contraction of the 1,4-benzoxazinone aglycone to form a benzoxazolinon-2(3//)-one derivative. The latter process is taking place, when plants release such aglycones into the environment by root exudation. Both series of degradation products are known as bioactive plant own resistance factors towards microbial diseases and insects, which can also act as allelochemicals. Today, the driving force for the interdisciplinary investigations in this field is the possibility to make agricultural use of the results in the growing of main cereals, like maize, rye, and wheat (Poaceae). A future goal consists in the transfer of the genetic information for the ability to biosynthesise such benzoxazinoid plant resistance factors from benzoxazinone producing plants into plants of agricultural interest which are not naturally equipped with this possibility. It is the aim of this review to present a detailed overview on natural sources of benzoxazinones and their enzymatic and chemical degradation, on the synthetic access both to aglycones and acetal glucosides, on medical effects and on the molecular allelopathy of these bioactive natural products.

187

In the last decade, several other authors have been dealing with other topics of the benzoxazinoid research in detail, which will be cited here in form of leading references only, which allow an entrance in these fields. Thus, former investigations directed on the biosynthetic pathway from anthranilate to benzoxazinone acetal glucosides have been reviewed [3]. Recently, the biosynthetic pathway from indole as the precursor [4] of the benzoxazinone moiety was elucidated on the level of the responsible gene cluster discovered and the corresponding enzymes [5]. Thus, the stepwise oxidation of indole with molecular oxygen introduced by means of cytochrome P450 monooxygenases in maize was found to be the route [6,7] to a typical benzoxazinoid aglucone as a direct combination of a cyclic hydroxamic acid with a cyclic hemiacetal unit. Both the genetic and biochemical aspects of benzoxazinoid biosynthesis have been reviewed [8]. Recently, a p-glucosidase specific for DIMBOA-glucoside (see Fig. (1)) a main benzoxazinone glucoside in maize [9,10] as well as indole hydroxylases in juvenile wheat [11] have been characterised. The formation, distribution and biological role of benzoxazinoids in the resistance of crop plants, e.g. maize, wheat and rye to pests and diseases has been summarised by several authors [12-14], This is still a field of detailed investigations, as shown by a recent paper on the simultaneous analysis of aglucones and glucosides in Aphelandra species [15], papers on the amount and distribution in several grasses, the content during special stages of plant development and the role of external influences on benzoxazinone formation [16-24], on cereal resistance towards aphids [25-31], the stalk com borer [32], and Asian com borer [33].

OCCURRENCE OF BENZOXAZINONE ACETAL GLUCOSIDES AND AGLUCONES Benzoxazinone Acetal Glucosides and their Position among the Natural Products All natural benzoxazinone glycosides reported are D-glucosides of the (substituted) 2-hydroxy-2//-l,4-benzoxazin-3(4/]r)-one skeleton as shown in Fig. (1). Usually, several acronyms derived from the rational names are used to name the compounds in papers, as it will be done here. E.g. the

188

acronym GDIMBOA, as one of the best studied glucosides, arises from (27?)-2-P-D-glucoside of 2,4-dihydroxy-7-methoxy-2//-1,4-benzoxazin3(4//)-one and is used synonymously for the exact name (27?)-2-P-Dglucopyranosyloxy-4-hydroxy-7-methoxy-2//-1,4-benzoxazin-3 (4H)'0ne of the compound. Analogously, DIMBOA-Glc is used as another acronym for the same compound. GDIMBOA belongs to the group of glucosides with a cyclic hydroxamic acid moiety, with GDIBOA, and GDIM2BOA as other members. A closely related compound derived from GDIMBOA is the corresponding hydroxamic acid methyl ester called GHDMBOA. Further related compounds are glucosides containing a lactam unit, as GHBOA, GDHBOA, GHMBOA, and GHM2BOA. The only natural glucoside which has been also named after the plant of origin is GHBOA, named Blephahn after the plant Blepharis edulis. OH OH

o .. o ^ ^ ^^'^—.OH •^s^.

N

^O

acronym GDIBOA; DIBOA-Glc

R' OH

R^ H

R^ H

GDIMBOA, DIMBOA-Glc

OH

OMe

H

OMe OMe

OMe H

GDIM2BOA, DIM2BOA-GIC OH OMe GHDMBOA, HDMBOA-Glc GHBOA, HBOA-Glc, Blephahn GDHBOA, DHBOA-Glc

H

H

H

H

OH

H

GHMBOA, HMBOA-Glc

H

OMe

H

GHM2BOA, HM2BOA-GIC

H

OMe

OMe

Fig. (1). Natural benzoxazinone acetal glucosides

species (family) [ref.] Secale cereale (Poaceae) [2,34-37] Triticum aestivum (Poaceae) [36,38-41] Consolida ohentalis (Ranunculaceae) [42] Acanthus mollis (Acanthaceae) [43] | Zea mays (Poaceae) [36,44-48] Triticum aestivum [36,38-41,44,45,49,50] Secale cereale [36] Coix lachrymajobi (Poaceae) [36,51 ] Zea mays [52] Zea mays [53,54] Triticum aestivum [50,55,56] Coix lachrymajobi [51 ] Blepharis edulis (Acanthaceae) [57,58] Zea mays [36] Zea mays [59] Coix lachrymajobi [51] Zea mays [36,46,54,60] Triticum aestivum [36,49,50] Secale cereale [36] Coix lachrymajobi [51 ] Zea mays [52]

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The four stereochemical possibiUties to combine both the aglycone and glucose cyclic hemiacetals parts are on principle the (27?)-2-a-, (25)-2-a-, (2i?)-2-P-, and (2iS)-2-P-types of bonds between both anomeric centres. However, in all cases of completely resolved structures of natural origin, only the (2i?)-2-p-linkage between the 2-hydroxy-2//-l,4-benzoxazin3(4//)-one skeleton and the D-glucose unit has been found, hitherto. We want to emphasise this, because this fact requires the appropriate stereochemical drawing of the chemical formula in any papers, especially that of the C-2-O-bond which has to be oriented backwards. The configuration mentioned is the result of a stereospecific glucosylation in the plant cell. As will be shown later, it proved to be a challenge for the organic chemist to find out a suitable synthetic method which gives access to the natural configuration. Recently, the position of benzoxazinoid glucosides within the realm of ca. 110.000 natural products known altogether has been categorised [61]. Hence, benzoxazinoids belong to a subdivision of about 1.000 hemiacetalether compounds. Their common feature is the existence of an ether like linkage arising from the formal combination of two former cyclic hemiacetals by dehydration (Fig. (2)).

Fig. (2). Retrosynthetic analysis of hemiacetalethers

These ca. 1.000 hemiacetalethers can be further subdivided into three groups. A first small group consists of two monosaccharides linked with their anomeric centres, with a,p-galactobiose and a,a-trehalose as well known examples. Another small group is formed by the combination of the anomeric centres of two nonsaccharidic cyclic hemiacetals. By far the majority (ca. 90 %) of natural hemiacetalethers consists in acetal glycosides, arising from the glycosidation of a nonsaccharidic hemiacetalic heterocycle, with (5-D-glucose as the preferred sugar moiety. Normally, other heteroatoms than 0, like N or S, if constituents of the aglucone at all (ca. 10 %), are not part of the cyclic hemiacetal ring, but outside of this ring. Therefore, in contrast to all other classes of acetal glycosides the unique feature of benzoxazinoid natural products is the presence of a nitrogen atom in the cyclic hemiacetal ring of the aglucone. From the viewpoint of organic chemistry this feature is the source of a

190

certain instability which is essential to obtain the chemical reactivity required for the survival of the plant's defence reaction. Besides of Poaceae, benzoxazinoid acetal glucosides have been isolated from various species of the Acanthaceae, Ranunculaceae, and Scrophulariaceae as well. In most cases reported the discovery of benzoxazinoids was made by chance, i.e. a systematic search for them among other species of the families mentioned has not been undertaken nor has such a search been done within other plant families. Therefore, it seems most likely, that benzoxazinoids will be constituents of other wild plants, too, which are not at all as deeply investigated as agriculturally used ones. One reason for this assumption is the circumstance that the benzoxazinoids' protecting effect especially for a young plant should be a favouring selection criterion in the evolution of any plant equipped with this system. Benzoxazinone Aglucones Aglucones are released by P-glucosidase on a pest attack from their preinfectional glucosidic precursors deposited in the plant cell vacuole, as will be shown below. Furthermore, aglucones can be passively set free into the environment by rotting plant material [62] or actively by root exudation [63]. An overview on benzoxazinone aglucones is given in Fig. (3). The list of aglucones is different from the list of glucosides. Hence, TRIBOA has only been detected as an aglucone, not as a corresponding 2-P-D-glucoside. On the contrary, an aglucone HM2BOA corresponding to the glucoside GHM2BOA has not been described from a natural source. Furthermore, the occurrence of the hydroxamic acid ester HDMBOA could only be proven by some spectroscopic methods [64]. Whereas a sample of a pure benzoxazinone glucoside (Fig. (1)) consists of one single diastereomer as mentioned above, a sample of a pure benzoxazinone aglucone (Fig. (3)) is always a racemic mixture of the {2Ry and (25)enantiomers. This fact is expressed by using a normal line for the C-OH bond in Fig. (3). For reasons given below it is impossible to separate a single enantiomer of such an aglucone.

191

acronym DIBOA

R' OH

R^ H

R^ H

TRIBOA

OH

OH

H

DIMBOA

OH

OMe

H

DIM2BOA HDMBOA HBOA Blephahgenin

OH OMe H

OMe OMe H

OMe H H

DHBOA HMBOA

H H

OH OMe

H H

species (family) fref.) Secale cereale (Poaceae) [1,34,35,65-67] Saccharum officinale (Poacea) [38] Thticum aestivum (Poaceae) [16-18,39-41,68] Zea mays (Poaceae) [60,69] Hordeum vulgare (Poaceae) [25] Agropyron repens (Poaceae) [63,70] Acanthus mollis (Acanthaceae) [43] Aphelandra tetragona (Acanthaceae) [71] Consolida orientalis (Ranuncuiaceae) [42] Zea mays [69] Crossandra pungens (Acanthaceae) [72] Thticum aestivum [16-18,39-41,44,45,50,68] Saccharum officinale (Poaceae) [38] Zea mays [26,44,45,60,69,73-79] Agropyron repens [70] Zea mays [69,78] Zea mays [66] Blepharis edulis (Acanthaceae) [58] Zea mays [69,80] Scoparia dulcis (Scrophulariaceae) [81] Aphelandra tetragona (Acanthaceae) [71] Zea mays [69] Coix lachrymajobi (Gramineae) [51,82] Triticum aestivum [48] Zea mays [69,78]

1 |

| 1 1

| 1 |

Fig. (3). Natural benzoxazinone aglucones

DEGRADATION OF ACETAL GLUCOSIDES TO BENZOXAZOLIN-2(3iy)-ONES Natural Compounds by an Enzymatic and Chemical Step of Degradation Benzoxazinoid acetal glucosides are compounds of low toxicity which have been found to be stable under neutral conditions. They can undergo a two step degradation, an enzymatic followed by a chemical one. In the non-injured plant cell, the acetal glucosides and the enzyme p-glucosidase

192

are stored separately in two different cell compartments, the vacuole and the plastid [83,84]. A feature of an external pest attack is an injury of the intact cell structures causing an interaction between acetal glucoside and pglucosidase. As a result of this enzymatic reaction, the hemiacetalic aglucone is released, (Fig. (4)). The enzymatic release of DIMBOA, e.g., from GDIMBOA of maize is complete within half an hour of wounding. The aglucone DIMBOA has been found to be the toxic principle against microbial and insect pests.

-ax OH

OH OH OH

R = H, OMe

P-glucosidase

"ax-'xDG: OH

OH

-HCOOH

"rxV" Fig. (4). Enzymatic and chemical degradation of benzoxazinoid acetal glycosides

Hence, the plant own defensive system is able to liberate a toxic, bioactive weapon from a non-toxic precursor very rapidly and in appreciable amounts, because of the benzoxazinone glucoside content in seedlings can reach concentrations between 1-10 mmoles per kg of fresh weight [85,86]. E.g., 530 mg of DIMBOA have been isolated from 1 kg of frozen maize shoots [79]. Interestingly, only all aglucones containing the 2,4-dihydroxy-2//-l,4benzoxazin-3(4/i/)-one skeleton have been found to be chemically

193

instable, i.e. the direct combination of cyclic hydroxamic acid and cyclic hemiacetal unit is a prerequisite of degradation. As a result of the chemical degradation accompanied by the loss of formic acid (substituted) benzoxazolin-2(3//)-ones are formed in a ring contraction reaction. The half life of DIMBOA in the juice of injured maize cells is about one day [74]. In fact, it was from this end of the cascade that benzoxazinoid acetal glucosides were detected starting with the discovery of benzoxazolin-2(3//)-one (BOA) from rye [87]. Meanwhile, several substituted benzoxazolin-2(3//)-ones (Fig. (5)) have been found in plants.

R' H

R^ H

R^ H

R^ H

MBOA

H

H

OMe

H

DMBOA 4-ABOA 4-Cl-DMBOA

H Ac CI

H H H

OMe H OMe

OMe H OMe

Species jrcf.] Secale cereale [58,87,88] Blephahs edulis [58] Aphelandra tetragona [89] Zea mays [90] Zea mays [44,9\, 92] Coix lachryma jobi[5\] Scoparia dulcis [93,94] Aphelandra tetragona [89] Triticum aestivum [44] Zea mays [90,95] Zea mays [96-98] Zea mays [90] 1

5-Cl-DMBOA

H

CI

OMe

H

Zea mays [99]

1 acronym BOA

|

Fig. (5). Natural benzoxazolin-2(3//)-ones

The glucosidic origin has only been established in the case of BOA, the 6-methoxy derivative MBOA as the most intensively investigated representative, and the 6,7-dimethoxy derivative DMBOA. On the one hand, the pathway reported for their formation strongly suggests the possibility that also 4-ABOA, 4-Cl-DMBOA and 5-Cl-MBOA have corresponding benzoxazinone glucoside precursors. On the other hand, this has not been proven yet experimentally, and other ways of formation like metabolism of BOA by endogenous fungi have to be taken into consideration.

194

Several proposals for the mechanism of the ring contraction reaction have been made [50,100-104]. The rate of degradation is distinctively enhanced in a strongly alkaline medium, which causes a very rapid decomposition of the 2,4-dihydroxy-2//-l,4-benzoxazin-3(4JT)-one skeleton. Hence, it is extremely important to keep this chemical property in mind to design appropriate preparative methods in any attempts to isolate or synthesise such aglucones. Furthermore, the kinetics of ring contraction [74] are dependent on the pattern of substituents at the aromatic ring as has been shown on a series of synthetic 2,4-dihydroxy2//-l,4-benzoxazin-3(4//)-ones with a non-natural pattern of substituents. Donor substitution increasing the electron density of the aromatic ring has been found to accelerate the rate of decomposition [103]. Probing the Oxo-Cyclo Tautomerism of Natural Enantiomeric Cyclohemiacetals with 2,4-Dihydroxy-2^-l,4-benzoxazin-3(4//)-one Skeleton The structural lability of the cyclic hemiacetals is a consequence of their oxo-cyclo tautomerism, which allows a rapid interconversion of the (27?)configurated enantiomer into the (25)-enantiomer and vice versa via the ring-opened oxo form (Fig. (4)). An analogous and well investigated process is the formation of an equilibrium mixture of a-D-glucose and pD-glucose on dissolution of the pure a- or P-isomer in water by mutarotation. D-glucose cyclic hemiacetals: diastereomers CHO OH H OH HO-^T^ ^^-"^^OH Ho-J -O OH L_oH

^

OH

noy^^^J-^^y^on ""^^

^^^^

LoH

OH

^OH Benzoxazinoid cyclic hemiacetals: enantiomers ,0^

.OH

N ^ ^0 OH R= H, OMe

R^ ^ ^

k/

1

OH

Fig. (6). Benzoxazinone aglucones as equilibrium of enantiomeric cyclohemiacetals

^OH

195

Due to the existence of additional four chiral centres to the anomeric centre a- and p-D-glucose are diastereomers and can be analytically distinguished and separated, easily. It shall be pointed out, that the same process has not been investigated yet on the level of enantiomeric cyclohemiacetals, which have no chiral centre besides the anomeric one. Therefore, hemiacetals like DIBOA or DIMBOA have also been of interest from the viewpoint of physical organic chemistry (Fig. (6)). We have studied if the separation of both enantiomeric cyclic hemiacetals by preparative or analytical means is possible. Therefore, some analytical investigations have been undertaken at the free hemiacetals DIBOA and DIMBOA and their racemic methyl acetals. In contrast to the cyclic hemiacetals the latter ones are stable towards inversion of configuration. Thus, in a HPLC procedure on a 5 ^m LiChroCART column using chemically bonded (i-cyclodextrin (ChiraDex) as chiral selector the enantiomeric separation of the methyl acetals of DIBOA and DIMBOA was possible. However, the racemic hemiacetals DIBOA and DIMBOA could not be separated under similar conditions. This is a consequence of the racemisation caused by the oxocyclo tautomerism of the hemiacetal unit, which occurs continuously and rapidly all the time during the separation procedure [105]. Cyclic hydroxamic acids are remarkably acidic (e.g. pKa of DIBOA and DIMBOA ca. 6.9) and form anions in alkaline buffers (pH 8-10). They are therefore suitable subjects of studies with high performance capillary electrophoresis [106]. A HPCE procedure for the enantioseparation of the methyl acetals of DIBOA and DIMBOA could be developed using methanolic borate buffers (pH 9-10 range) and a-, (3-, or y-cyclodextrins as chiral additives to the mobile phase. The size of the optimal chiral additive depends on the shape of the methyl acetal. However, as in the HPLC method free enantiomeric cyclohemiacetals could not be separated by HPCE due to the same configurational interconversion effect leading to racemisation. Because Qnaniioseparation proved to be impossible for DIBOA and DIMBOA attempts to accomplish at least an enantiodifferentiation by means of the chiral solvating agent (CSA) NMR technique have been undertaken because NMR is based upon a rapid measuring and differentiation process in comparison to the chromatographic separation processes. The NMR discrimination of enantiomeric cyclic hemiacetals and methyl acetals was not described yet. On principle, a pair of

196

enantiomers on addition of a chiral soivating agent is able to form a pair of diastereomeric solvation complexes, the NMR spectra of which are different. We assumed that a basic chiral soivating agent should be capable of forming such diastereomeric solvation complexes and cause chemical nonequivalence of hitherto equivalent protons of the (27?) and (25)-hemiacetal [107]. Indeed, CSA both racemic DIBOA and DIMBOA on addition of (-)-quinine formed two populations of diastereomeric complexes which allowed the first enantiodifferentiation of cyclic hemiacetals by means of ^H NMR, despite of their permanent oxo-cyclo tautomerisation. Of course, enantioseparation of DIBOA and DIMBOA methyl acetals has also been possible.

CHEMICAL SYNTHESIS OF AGLUCONES, ACETAL GLUCOSIDES, AND ANALOGUES Synthetic Approaches to Aglucones The challenge in the synthesis of an aglucone like e.g. DIMBOA consists in accomplishing the direct combination of a cyclic hydroxamic acid with a cyclic hemiacetal unit together with a donor substituent in para position to the N atom keeping in mind the principle tendency of the 2,4-dihydroxy-2^-l,4-benzoxazin-3(4/f)-one skeleton to undergo in solution degradation to a benzoxazolin-2(3//)-one as discussed recently [108] and above. On principle, both the hydroxamic acid and the hemiacetal are partially oxidised structures. Thus, the hydroxamic acid should be accessible both from a nitro precursor by reductive cyclisation and from a lactam by Noxidation (Fig. (7)). Similarly, access to the hemiacetal should e.g. be possible by oxidation of a 2-methylene group as well as by reduction of a 2-carbonyl group, and also by hydrolysis of a 2-halogen function. The influence of substituents at the aromatic ring on the synthesis of the 1,4benzoxazinone ring is hardly foreseeable. However, another circumstance has a very rational basis. Due to the fact that the structural instability arises from the cyclohemiacetal (see Fig. (4)) this unit is prepared at the very end of most syntheses. Until 1989 four syntheses leading to DIBOA or DIMBOA have been published (Fig. (7)).

197

a°"

"^V^XJ^^^^"

UL

N02

C

a

NH2

NO2

O^OMe

OH ,COCHCl2

NO2

O^

OMe

NHOH

COCHOz

/^^^\^^"

OH

NO2

O...^CX)2Et NO2

'xxx ax ai 1 ax ax O^ ^OMe

O^ ^OH

N

O^ ^OMe

1

'^NxSS^^^^V^^

OH R = H, OMe

^O

OH

o^ Zh1/2

O^ ^OMe

a

O^ ^Br

SiMej

OH

^ V - < f S f \ ^ O^ .OH

OH

Fig. (7). Early syntheses for the 2,4-dihydroxy-2//-l,4-benzoxazin-3(4//)-one skeleton

The first synthesis (left pathway) affording DIBOA started from a protected 2-nitrophenol [109]. The need to handle a free donor-substituted arylhydroxylamine reduces the yield distinctly and is a serious disadvantage. The second synthesis (middle left pathway), subject of a patent issued in 1975 to Hoffman-La Roche, is also starting from an appropriate orr/20-substituted nitrobenzene [110]. In both syntheses the hydroxamic acid is generated by reductive cyclisation and the hemiacetal by hydrolysis of a chloride precursor. However, only the second procedure is applicable to the synthesis of the 7-methoxy compound DIMBOA. Therefore, this principle has later been developed further. The strategy of the third synthesis (middle right pathway) is different [111]. After alkaline cyclisation of the dichloroacetamide precursor to the hemiacetal unit the hydroxamic acid is obtained by oxidation of the

198

silylated lactam with Mo05(DMF)2. Besides difficulties in the lactam oxidation this procedure has serious problems in the work-up step. Insufficient overall yields of these initial methods prompted a search for more efficient syntheses. We have reported on a synthesis for DIBOA on the multigram scale [112] that avoids some disadvantages of previous methods (right pathway). Unfortunately, this method cannot be transferred to the synthesis of the 7-methoxy analogue DIMBOA, because due to the donor effect of the methoxy group in the corresponding precursor 4-hydroxy-7-methoxy-2//-l ,4-benzoxazin-3(4//)-one bromination takes place regioselectively at C-6 rather than at C-2 required. A first general method tolerating substituents at the benzene moiety was published in 1991 [103], that allowed the preparation of a series of 2,4-dihydroxy-2//-l,4-benzoxazin-3(4//)-ones with a widespread pattern of substituents at the aromatic ring (Fig (8)). Br^

OMe

Y- -

-O"

^

.O^OMe

^^ — • "CX, NO2

""^

OMe

„^, _ „ „ ^

pd/CNaBH,

COzMe NO2

^

^x-s. . 0 ^ ^OH R = one or two substituents

OH

OH

Fig. (8). A synthesis for the 2,4-dihydroxy-2//-l,4-benzoxazin-3(4//)-one skeleton tolerating various R substituents with access to the hemiacetal by acetal cleavage

The improvement results from using a catalytic transfer hydrogenation [113] for the reductive cyclization. The critical step of this method is the demethylation of the methyl acetal. This problem has been a main subject of a first review on the synthesis of benzoxazinoid aglucones [104]. In one of our altemative approaches the lactol unit of the 2,4dihydroxy-27/-l,4-benzoxazin-3(4//)-one skeleton was developed by oxidation of the unsubstituted methylene function (Fig. (9)) [114]. The starting cyclic hydroxamic acids have been prepared by catalytic transfer hydrogenation of appropriate 2-nitrophenoxyacetate precursors with the sodium borohydride/Pt-C method. The oxidative transformation intended caused a need for protection of the hydroxamic acid moiety. From several

199

attempts to protect the 4-position a cyclosilylation procedure proved to be suitable. Thus, two equivalents of LDA allowed the enolisable hydroxamic acids to react with di-tert-butyldichlorosilane in toluene to give stable cyclosilyl enol ethers. They have been transformed into the intermediate epoxides by oxidation with m-chloroperbenzoic acid in methylene chloride. Treatment of both epoxides with tetra-nbutylammonium fluoride in THF caused deprotection of the silyl enol ethers and ring opening of epoxides to yield DIBOA and DIMBOA, resp., after separation from the reaction mixture by column chromatography.

"xx;i„ ^^^^ "xx;\ OH

•xx:> O^.

N o O-Si / tBu/ MBu

mCPBA

^

O-Si „ / "tBu

Bu.NF^

R-^^YV"*"

overall yields: R = H:41% R = OMe: 32%

OH

Fig. (9). Oxidative generation of the lactol unit by a-hydroxylation of a cyclic hydroxamic acid

Run in an one-pot procedure the whole sequence shown in Fig. (9) was the first a-hydroxylation of cyclic hydroxamic acids reported and independent from substituents in the aromatic ring. However, the overall yields of aglucones obtained have not been satisfactory. Prompted by the aim to synthesize for the first time the 7-OH derivatives TRIBOA and DHBOA a general procedure was established in which the crucial lactol unit is obtained by reduction of a lactone precursor (Fig. (10)). Appropriate 5-substituted 2-nitrophenols have been transformed by acylation with chloroformylformate into (substituted) ethyl 2-nitrophenyl oxalates as extremely reactive building blocks for the synthesis of the 2,3dioxo-l,4-benzoxazine skeleton [115]. Due to their extreme sensitivity towards hydrolysis water has to be excluded on reductive cyclisation of these nitrophenyl esters.

200

T c

'XX°J

ClOC-COzEt

NO2

AcOH or MeOH

NO2

R = H, OMe, OBn

2 H2/Pt(S)-C

H2/Pt(S)-C

^^^-^'"^ NHOH

'XX

OH

> ^ o ^ c . .

in MeOH

NH, 84-86%

in AcOH

"x)c;x

-EtOH 88-90%

DT, EtOH 92-93%

"•ax OH

2 DIBAH, toluene, 2h,

COiEt

+ EtOH, reflux

•XIX

R H H OMe R' OMe after H/Pd'CforR = OBn: ^H OH

R' H OH H OH H OH

compound HBOA DIBOA HMBOA DIMBOA DHBOA TRIBOA

Fig. (10). Reductive generation of the lactol unit from a lactone: A general synthesis for the 2hydroxy-2//-1,4-benzoxazin-3(4//)-one skeleton

Hence, an aqueous borohydride transfer hydrogenation was impossible for the synthesis of a hydroxamic acid. However, the hydroxamic acids could be obtained by catalytic hydrogenation of the nitro oxalates in glacial acetic acid over 3% Pt(S)-C in the presence of 2,2dimethoxypropane added for the chemical transformation of the water resulting from the partial hydrogenation of the nitro group. A simple variation gave access to the lactam series. On changing the solvent of hydrogenation to methanol ethyl N-(4-subst.-2-hydroxyphenyl) oxalamide was obtained. The oxalamides underwent lactonization to form

201

the required lactones on heating in a Kugelrohr apparatus to their melting points. Lactones of this type cannot be recrystallised from protic solvents like ethanol, e.g., due to ring-opening and formation of the oxalamide precursors. The lactone unit in both (substituted) 4-hydroxy- and 4//-2,3dioxo-l,4-benzoxazines has been regioselectively reduced by means of diisobutylaluminum hydride in toluene to form (7-substituted)-2-hydroxy2H' 1,4-benzoxazin-3(4//)-ones and (7-substituted)-2,4-dihydroxy-2//l,4-benzoxazin-3(4/f)-ones, respectively [116]. The use of two equivalents of the reducing agent is most suitable, the first one for deprotonation of the acidic functionality, the second one for the lactonelactol reduction. Attempts to make use of a third equivalent of the hydride for simultaneous unmasking of the 7-hydroxy group in case of a 7benzyloxy substitution were unsuccessful. Finally, the 7-hydroxy substituted aglucones DHBOA and TRIBOA have been prepared [117] by standard hydrogenolyses of 7-benzyloxy precursors over Pd-C in acetic acid. All steps of the reductive pathway proceed with good to excellent yields. This method provides a general access to the 2-hydroxy-2//-l,4-benzoxazin-3(4//)-one as well as to the 2,4-dihydroxy-2//-l,4-benzoxazin-3(4/]0-one skeletons as shown by the six natural aglucones synthesized (Fig. (10)). The synthesis of the 7-hydroxy-derivatives required 5-benzyloxy-2nitro-phenol, hitherto unknown. An earlier procedure for the synthesis of the related 5-methoxy-2-nitrophenol [118] showed, that a selective direct nitration of such electronically overheated aromatics is impossible even with dilute aqueous nitric acid. In such a case, using a weaker and more selective N-electrophile like the nitrosyl cation or an equivalent for it should be helpful because a nitroso compound thus prepared can finally be oxidised to a nitro compound. However, special problems result from the insolubility of monobenzyl resorcinol in water which made a classical aqueous nitrosation procedure impossible. This prompted us to develop a novel regioselective nitrosation procedure for resorcinol monoethers, e.g. for monobenzyl resorcinol, as starting material (Fig. (11)). Solid sodium nitrite in anhydrous propionic acid as solvent was an effective system for the completely regioselective nitrosation of the subject compounds. High yields of pure 2-nitroso products were obtained. Attack at the 4-position becomes only competitive in the presence of water in the solvent. Absence of water was ensured by addition of one percent of propionic anhydride. The excellent regioselectivity observed is obviously related to the presence of nitrosyl

202

propionate that is formed in situ in the solutions of sodium nitrite in anhydrous propionic acid and acts as a weakly electrophilic covalent nitrosation agent.

^«^^ R'

53-82

S^NO «-„, R'

V^

NO2

R'

combinations: R = Me, R' = H; R = Me, R' = OMe; R = Bn, R' = H.

Fig. (11). Regioselective nitrosation of resorcinol monoethers and oxidation to nitrophenols

Mesomeric structures of propionyl nitrite show the 0-NO bond with the NO unit having a partially positive charge. Therefore, propionyl nitrite, the mixed anhydride of propionic and nitrous acid formed under the reaction conditions, behaves rather like nitrosyl propionate, i.e. as a polarised covalent source of a nitrosyl cation. Hence, the regioselectivity of this reaction can be understood in terms of the weak electrophilicity of the NO^-generating species. Finally, the substituted 2-nitrosophenols were easily oxidised with nitric acid to yield the substituted 2nitrophenols required as precursors [119]. The aglucone HDMBOA has been reported as present in com whorl surface waxes [64]. A structural assignment was given, however, some doubt remained, at least in the purity of the sample described. We followed two pathways for the preparation of the hydroxamic acid methyl esters HDMBOA and 2-hydroxy-4-methoxy-2//-l,4-benzoxazin-3(4//)one [120] from their precursors DIMBOA and DIBOA, respectively (Fig. (12)). Whereas an independent synthesis for the latter compound has been reported in a patent [110] a synthesis of HDMBOA has not been described yet. Both hemiacetals have been subjected to benzylation with benzyl trichloroacetimidate. Excess boron trifluoride etherate added to the 2-OH and 4-OH groups, acted as a noncovalent protecting group for the 4-OH group and a promoter for the regioselective benzyl transfer to the more nucleophilic hemiacetalic function. 2-Benzyloxy-4-hydroxy-2//-l,4-benzoxazin-3(4^-one and its 7-methoxy analogue have been obtained as novel derivatives of the lead. These hydroxamic acids have been methylated at N-OH by using the standard K2CO3/CH3I method in refluxing acetone to yield the hydroxamic acid methyl esters 2-

203

benzyloxy-4-methoxy-2//-l,4-benzoxazin-3(4//)-one and its 7-methoxy derivative. Finally, the hemiacetalic function was liberated by hydrogenolysis over Pd-C in dry THF to afford 2-hydroxy-4-methoxy2i/-l,4-benzoxazin-3(4//)-one and HDMBOA.

XxX O^

OH

CCI3

BnO

-i ^J'

X Et20, NH CH2CI2, r. t

81-94%

OH

OH

CH3I, K2CO3! 1 91-94»/ acetone

CH2N2 79- 86% in Et20

XxX R=H ^'^ R = OMe: HDMBOA

•ax

O^ ^OBn

Hj/Pd-C, THF 78-82%

OBn

'•ox OMe

Fig. (12). Alternative procedures for the aglucone HDMBOA starting from DIMBOA

Alternatively, DIBOA and DIMBOA in a THF solution have been methylated with a solution of diazomethane in diethyl ether at their more acidic hydroxamic acid unit, regioselectively. As mentioned above, the assumption that e.g. the highly bioactive 4acetylbenzoxazolin-2(3//)-one (4-ABOA), isolated from kernels of a fusarium resistant maize line [96-98] may have resulted from the enzymatic and chemical degradation of the precursor 5-acetyl-4-hydroxy2-p-D-glucopyranosyloxy-2//-l,4-benzoxazin-3(4//)-one is possible, particularly since no means to inactivate P-glucosidase have been undertaken during the isolation process (Fig. (13)). We have therefore been interested in the synthesis of the corresponding aglucone 5-acetyl-2,4-dihydroxy-2i/-1,4-benzoxazin3(4^-one (5-acetyl-DIBOA). However, when the appropriate precursor 5-acetyl-4-hydroxy-2-methoxy-2//-1,4-benzoxazin-3(4//)-one was subjected to a boron trichloride ether cleavage followed by hydrolysis with silver carbonate/water according to a reported method [110], the expected aglucone was not obtained. Instead of, 4-ABOA was isolated [121]. In

204

conclusion, the aglucone 5-acetyl-DIBOA will hardly be isolable from a natural source. OH OH

II >=o C

synthesis:

OH

assumed precursors:

isolated:

^ „

^!^

Fig. (4). Mass fragmentation pathway of 5,8-disubstituted indolizidines [19] CH3

CH3 a-cleavage Diels-Alder m/z \52

m/z no

Fig. (5). Mass fragmentation pathway of 1,4-disubstituted quinolizidines [19]

Further analysis of the GC-FTIR spectra of class A alkaloids is usually helpful for the assignment of the unsaturation pattern of the alkyl side-chains. The most commonly observed pattem consists of a terminal acetylene unit (V=CH 3320 cm'^ and Vc=c 2100 cm"^) or a terminal ethylene unit (v=cH2 3080 cm'^ and 5=CH 990-910 cm"^) and/or an internal c/5-ethylene linkage (V=CH- 3020 cm"^). Traw^-ethylene linkages (5=CH. 965 cm"^) are rarely present. Class B Alkaloids

Class B alkaloids follow the general mass spectra fragmentation pathway illustrated in Fig. (6). While PTX-A alkaloids are characterised by major peaks at m/z 166 (CioHi6NO'^) and m/z 70 (C4H8N^), the mass spectra of a//o-PTX-A alkaloids present significant peaks at m/z 182 (CioHi6N02'^) due to the extra hydroxy 1 group as well as at m/z 70 (C4H8N'^). Accordingly, homo-PTX-A alkaloids usually show fragments at m/z 180

239

(CiiHigNO"') and m/z 84 (CsHioN^), due to the presence of the extra methylene group with respect to PTX-A alkaloids. Cleavage of the side-chain C-15 allylic hydroxyl group in PTX-A and a//o-PTX-A alkaloids is usually responsible for a [M-OH]^ fragment in the mass spectrum of these compounds. OH

"^(CHa^

\-(CH2)n n=l

/w/zl66(R=H) /n/zl82(R=OH) n = 2 m/zl80(R=H)

H-N^ V-(CH2)n ^(CH2)n

n = 1 Wz 70 n = 2 m/z 84

Fig. (6). General fragmentation pathway of pumiliotoxin alkaloids

The IR spectra of PTX-A alkaloids usually show absorption bands at 3650 cm'^ (side-chain hydroxyl group), 3544 cm"^ (tertiary hydroxyl group hydrogen bonded to nitrogen), a characteristic Bohlmaim pattern with a band at 2798 cm'^ and a shoulder at 2750-2700 cm'\ and two double bond absorptions at 990 cm'^ (trisubstituted 13,14 double bond) and 963 cm"^ (trisubstituted 6,10 double bond). The distinctive features of a//o-PTX-A IR spectra are a Bohlmaim pattern with no shoulder absorptions and a band at 2800 cm"\ and an extra band at 1010 cm•^ //omo-PTX-A alkaloids show a Bohlmann absorption region with a shoulder on the higher wavenumber side of the major absorption at 2750 cm"\ rather than on the lower wavenumber side as in PTX-A alkaloids, while the vibration frequency of the C-9 hydroxyl group is shifted to a higher wavenumber (3555 cm"*) when compared to the analogous C-8 hydroxyl group of PTX-A andfl//o-PTX-Aalkaloids (3544 cm"*) [19].

240

Structure Identification Over the past few years, the estabhshment of the absolute configuration of classes A and B alkaloids has been a challenge, due to the trace amounts in which these metabolites are detected. Since mass spectrometry is still a crucial tool in the structure identification of these compounds, the mass spectra of the new alkaloids covered by this review are given at the end of this section (Table 1). Class A Alkaloids 3,5 -Disubstituted Indolizidines

Two new 3,5-disubstituted indolizidines, (-)-myrmicarin 237A and (+)-myrmicarin 237B, Fig. (7), were isolated as the major components of the poison gland secretion of the african ant Myrmicaria eumenoides [22]. NMR data analysis (^H, ^^C, ^H-^^C COSY, COLOC and 2D-INADEQUATE spectra) of the two compounds allowed the identification of a 3,5-disubstituted indolizidine nucleus with an «-butyl substituent at C-3 and a 1-oxopropyl substituent at C-5, whose presence was further confirmed by the ketone IR absorption bands at 1711 cm"^ (237A) and 1714 cm'^ (237B) and the mass fragment at m/z 180 (CiiHigNO^ and C12H22N'') resulting from the two possible a-cleavages of myrmicarines (Table 1).

(-)-myrmicarin 237A (3R,5S,9R)

(+)-myrmicarin 237 B (3R,5RM)

Fig. (7). 3,5-Disubstituted indolizidines (-)-myrmicarin 237A and (+)-myrmicarin 237B [22]

NOESY experiments with racemic synthetic myrmicarin 237A and

241

myrmicarin 237B established their relative configuration while asymmetric synthesis of the four (3i?) isomers and comparison with the natural products by chiral gas-chromatography led to the determination of their absolute stereochemistry. Full spectral characterisation of the natural products as well as of the synthetic compounds is presented [22]. Since the new metabolites, easily epimerise at C-5 at room temperature, some doubts still remain as to the natural occurrence of both diastereomers. Three new diastereomers of 3-hexyl-5-methylindolizidine, (5Z,9Z)-, (5£,9£)- and (5Z,9£), Fig. (8), were identified in different collections of thief ants Solenopsis {Diplorhoptrum) species from California. The identification of these alkaloids resulted from the analysis of their MS spectra and comparison of their GC-FTIR spectra. The authors also emphasised the chemotaxonomic value of the stereochemistry of these alkaloids [23].

(5Z,9Z)

(5£,9Z)

(5£,9£)

Fig. (8). Diastereomers of 3-hexyl-5-methylindolizidine: (5Z,9Z)-, (5£,9£)- and (5Z,9£) [23] 5,8-Disubstituted Indolizidines

The previously described indolizidine 2231 [5] proved to be a mixture of 2231 and 223J, Fig. (9). FTIR spectral analyses revealed that the alkaloid 2231 was not a typical 5,8-disubstituted indolizidine because its Wenkert-Bohlmann band region differs significantly from that of other thoroughly characterised natural 5,8-disubstituted indolizidines, all of which have a 5H-9H cis relationship and show an intense and sharp band. This feature is not present in the FTIR spectrum of 2231, but is present in the spectrum of a synthetic alkaloid identical to 223J. Based on these findings, alkaloid 2231 was tentatively assigned an 8-butyl-5-propylindolizidine structure with a 5H-9H trans relationship and indoUzidine 223J was proposed to be identical to the synthetic alkaloid [24].

242

2231 (tentative structure)

223J (tentative structure)

Fig. (9). Indolizidine alkaloids 2231 and 223J [24] 5,6,8-Trisubstituted Indolizidines

The isolation and characterisation from a Panamian population of Dendrobates pumilio Schimdt of indolizidine 223A [25], formerly identified as 1,4-dipropyl quinolizidine [5], established a new type of class A alkaloids: the 5,6,8-trisubstituted indolizidines. Three homologues of higher molecular weight were detected in extracts of other populations of this species and the analysis of their GC-MS and FTIR data led to the proposed structures of indolizidines 237L, 251M and 267J, Fig. (10).

223A {5S,6R,iS,9R)

251M tentative structures based on MS data

267J tentative structure based on MS and FTIR c

Fig. (10). 5,6,8-Trisubstituted indolizidines 223A, 237L, 251M and 267J [25]

The major GC-MS fragments of indolizidine 223A at m/z 223 (Ci5H29N*^ [Ml), m/z 180 (Cl2H22N^ base peak) and m/z 124 (CgHnN^ suggested the presence of a propyl side-chain at C-5, while a sharp

243

Bohlmaim absorption band at 2784 cm"^ indicated the cis relationship of protons H-5 and H-9 (5Z,9Z), trans antiparallel to the nitrogen lone pair, in accordance with the spectral features of 5,8-disubstituted indolizidines. The existence of three methyl triplets in the ^H NMR spectrum pointed to a trisubstituted indolizidine nucleus and the analysis of the ^H 2D COSY NMR spectrum, as well as the J coupling values observed and molecular modelling of the proposed structure, revealed that H-9, H-6 and H-5 occupied axial positions, according to the relative configuration depicted for indolizidine 223A.) The 5E,9E stereochemistry assignment of indolizidine 267J was based on the dissimilarities found in the Bohlmann absorption band region of the GC-FTIR spectra of indolizidine 223A and indolizidine 267J, while the nature of the alkyl substituents was proposed on the basis of GC-MS and GC-FTIR analysis: the mass fragments at m/z 196 and m/z 124 were in agreement with a propyl side-chain at C-5 and suggested the presence of an oxygen atom at C-8 (lost as butenol in the retro Diels-Alder process) (Table 1); the oxygen presence was further confirmed by HRMS and its hydroxyl nature was confirmed by a non-hydrogen-bonded hydroxyl group stretching absorption frequency at 3651 cm'^ in the FTIR spectrum of indolizidine 267J. The comparison of the mass spectrum of indolizidine 223A with those of indolizidines 237L and 25IM led to their tentative structure assignment: the major peaks at m/z 180 and m/z 124 confirmed the indolizidine nucleus and the identical substituents at C«6 and C-8, while the observed molecular ions suggested, respectively, «-butyl and «-pentyl side-chains at C-5 (Table 1). The alkaloid 249H, Fig. (11), was isolated from the skin extracts of the poisonous frog Dendrobates auratus of the Isla Taboga of the Pacific coast of Panama [26]. A sample containing only traces of impurities was obtained by successive column chromatography of the methanolic skin extract. ^H and ^"^C NMR spectroscopy and correlation spectra were only consistent with a branched substituent, the first to be reported for simple indolizidines isolated from frog skin, and an unusual c/.y-fused trisubstituted indolizidine nucleus. An intense NOESY cross peak between H-1 and H-5 is only compatible with a c/5-fused indolizidine structure having an axial A^-lone pair on the piperidine ring. The GC-FTIR spectra showed weak Bohlmann bands.

244

unlike the spectrum of the 5,6,8-trisubstituted indolizidine 223A which exhibited an intense band consistent with H-5 and H-9 having a Z relationship. Both the absence of a NOESY cross peak between H-5 and H-9 and the IR data support that 249H has an E orientation of H-5 and H-9.

" U 'p4 H,C

Fig. (11). 5,6,8-Trisubstituted indolizidine 249H [26]

1,4-Disubstituted Quinolizidines

Recently, the first isolation and fiiU NMR characterisation of a 1,4-disubstituted quinolizidine, quinolizidine 217A, Fig. (12), was reported [27].

14'^=^^"

Fig. (12). 1,4-Disubstituted quinolizidine 217A [27]

Quinolizidine 217A had been previously identified in Mantella species

245

[18] and the proposed structure was now confirmed by NMR data. The J coupling analysis and the connectivities observed in COSY and RELAYH = 1 (long-range) spectra suggest a Z configuration of the 13,14-double bond, and an axial orientation of the H-1, H-4 and H-10 protons, in agreement with the 4Z,10Z relative configuration which can be inferred firom the FTIR data. A (l£,10jE)-configuration is proposed. The C-1 methyl group has an equatorial orientation in close resemblance with the C-8 methyl group of the analogous 5,8-disubstituted indolizidines. The similarity of the spectral features of quinolizidine 217A and those of the formerly assigned quinolizidines 2071, 231 A, 233A [18] and 235E', Fig. (13), [19] led the authors to propose the structures here depicted. A cis{\A)'Cis{\,\Q) configuration was proposed for quinolizidine 2071 based on the GC-MS and GC-FTIR spectra obtained for the trans{\,Aytrans{\,\Q) synthetic alkaloid [24].

= H

.N C6Hii

2071

(non terminal double bond) 235E'

Fig. (13). 1,4-Disubstituted quinolizidines 2071, 231 A, 233A [18] and 235E' [19]

Corrections to the structure assignments of some 1,4-disubstituted quinolizidine alkaloids were also proposed by Jain et al [27], The previous identification of compounds of this subclass relied mainly on the presence of massfiragmentsat m/z 152, m/z 166 or m/z 182 (according to the alkyl substituent at C-1) and, in most cases, an extra fi-agment at m/z 110 in ion-trap spectra (Table 1); of these, those possessing a sharp Bohlmann absorption frequency, such as 231B and 273A, were reassigned a 5,6,8-trisubstituted indolizidine structure, with a (5Z,9Z)-configuration and a methyl substituent at C-6 that, after the retro Diels-Alder rearrangement, can give rise to thefi-agmentat m/z 110, in agreement with the spectral features found for the newly isolated 5,6,8-trisubstituted indolizidme 223A [25]. The 1,4-disubstituted quinolizidines with no significant Bohlmann

246

bands, such as 249C and 263, can either be (4£',10F)-l,4-disubstituted quinoUzidines or (5£,9£)-5,6,8-trisubstituted indoHzidines, while those with weak Bohimann bands and no mass fragments at m/z 110, such as 277A and 289A, should be the object of further studies. More recently, the 1,4-disubstituted quinolizidine 275A was reassigned a 4-methyl-9-nonynyl-l-azabicyclo[5.3.0]decane structure by comparison with synthetic diastereomers. Fig. (14) [16]. Nevertheless complete characterisation has not yet been reported.

C9H15

Former 2 7 5 A

New 275A

Fig. (14). Proposed structures of the alkaloid 275A [16] 4,6-Disubstituted Quinolizidines

A previously unclassified amphibian alkaloid, 195C, was recently found in a brazilian ant {Solenopsis sp. Picea group). A major loss of propyl and a minor loss of methyl were observed in the MS spectrum (Table 1), consonant with a-cleavage of such substituents. The FTIR spectrum showed Bohimann bands weaker than those of disubstituted indoHzidines and hence a 4-methyl-6-propylquinolizidine seemed to be the only reasonable structure. The four possible diastereomers of this compound were synthesised and one of them was identical to the natural alkaloid with respect to GC retention time, MS and FTIR data. The ^H NMR spectrum of 195C (isolated from the skin extracts of nineteen specimens of Madagascan mantelline frog, Mantella basileo) agreed with the proposed structure, Fig. (15). The stereochemistry of the natural 195C quinolizidine was established as being (6Z,10i5r)-4-methyl-6-propylquinolizidine in accordance with the weak Bohimann band pattern observed in the GC-FTIR spectrum and with the stereochemistry of the synthetic diastereomer [28].

247

Fig. (15). 4,6-Disubstituted quinolizidine 195C [28] Class B Alkaloids

A new class of PTX-A alkaloids was discovered by Jain et al. [29] when they isolated and characterised deoxy PTX 25IH from the Ecuadoran poisonous frog Epipedobates tricolor. Fig. (16). The absence of the C-8 hydroxy 1 group was confirmed by the lack of the corresponding absorption frequency at 3544 cm"^ in the IR spectrum and by the replacement of the usual fragment at m/z 166 by a main fragment at m/z 150 in the mass spectrum (Table 1). Full ^H NMR data are presented and the analysis of the J couplings observed suggests the depicted relative configuration. Despite the inconclusiveness of the NOE experiments as regards the assessment of the double bond configuration, the fact that all natural PTX-A alkaloids have a Z-configuration at the C-6/C-10 double bond led the authors to propose the same configuration for deoxy PTX 251H. The same reasoning was used for the assignment of the relative configuration ofC-11. The same authors also isolated the alkaloid 341A from Epipedobates tricolor [30]. This is the first knovm PTX alkaloid that contains a cyclic ether moiety, Fig. (16). HRMS showed that this compound is a tetra-oxygenated C19 alkaloid and MS clearly revealed that it belongs to the PTX class (Table 1), while the GC-MS deuterium-exchange measurement demonstrated that it has only three exchangeable hydrogen atoms. As the GC-FTIR showed no carbonyl absorption, one oxygen atom had to be present as an ether or epoxide. The ^H NMR analysis located this oxygen atom as being in a 6,13-ether linkage, creating a pyran ring spiro-fiised to a 6,10-dihydro-7-hydroxyindolizidine nucleus. The chemical shifts and multiplicities of signals assigned to the H-lOa, H-10|3, H-11, H-12a, H-12p and H-13 protons all indicated lack of free rotation. This

248

hypothesis was confirmed by the agreement of the dihedral angles calculated by molecular modelling using MM2, and the J coupling constants between H-13 and H-12a. The assignment of all the proton signals and the absolute configuration of 341A PTX are also discussed.

Table 1. Mass Spectra of Newly Identified Indolizidine and Quinolizidine Alkaloids from Amphibians And Ants.

Alkaloid

Mass spectra

Ref.

195C

195(1), 194(1), 180(5), 153(11), 152(100), 124(5), 82(5), 69(8), 67(8), 55(15)

217A

217(2), 152(100), 110(16)

[28] 1 [27] 1

223A

223 (1), 222 (1), 194 (2), 180 (100), 152 (2), 124 (11), 96 (6), 70 (9), 55 (7)

[25]

223 (5), 222 (5), 208(1), 194(2), 180(100), 166(1), 150(2), 138(7), 124(26), 110 (1), 96 (10), 84 (2), 70 (10)* 237A

236 (1), 235 (1), 182 (1), 181 (13), 180 (100), 178 (3), 164 (1), 152 (3), 150 (1), 136 [22] (1), 137 (1), 125 (1), 124 (9), 123 (7), 122 (10), 120 (1), 108 (2), 96 (2), 95 (5), 94 (7), 93 (1), 84 (3), 83 (2), 82 (6), 81 (4), 80 (2), 79 (2), 77 (1), 70 (1), 69 (3), 68 (5), 67 (7), 57 (4), 56 (5), 55 (12), 54 (4), 53 (2), 41 (8)

237B i3RMM)

236 (1), 182 (1), 181 (14), 180 (100), 178 (2), 164 (1), 152 (4), 150 (1), 136 (1), 137

[22]

(1), 125 (1), 124 (10), 123 (5), 122 (8), 120 (1), 108 (3), 96 (4), 95 (5), 94 (5), 93 (2), 84 (5), 83 (2), 82 (6), 81 (5), 80 (3), 79 (3), 77 (1), 70 (1), 69 (3), 68 (5), 67 (7), 57 (3), 56 (5), 55 (13), 54 (4), 53 (2), 41 (8)

237L

236 (1), 208 (2), 180 (100), 164 (4), 124 (21), 96 (4), 70 (14) *

[25] 1

249H

249 (8), 234 (4), 220 (10), 206 (2), 192 (2), 190 (2), 178 (5), (166) (100), 148 (4),

[26]

136 (5), 124 (4), 122 (4), 110 (17), 95 (5), 70 (5), 67 (5). 251M

251 (2), 250 (2), 222 (1), 180 (100), 164 (4), 136 (3), 124 (20). 110 (6), 96 (5), 70

[25]

(7)* 251H

251 (3), 250 (4), 236 (3), 222 (1), 208 (1), 194 (1), 178 (10), 162 (1), 150 (100), 136 [29]

267J

267 (1), 266 (1), 196 (100), 180 (3), 152 (4), 138 (3), 124 (19), 96 (3), 70 (14), 55

(5), 122 (3), 108 (3), 93 (3), 79 (5), 70 (20) [25]

(10) 196 (100), 124 (72), 96 (4), 70 (18) * 341A

341(6), 323(10), 306(2), 298(16), 266(9), 254(19), 236(3), 198(3), 184(7), 182(6), 180(5), 166(3), 164(3), 126(16), 125(15), 114(28), 112(100), 97(14), 96(11), 87(13), 84(61), 70(98), 55(18)

* ion-trap MS

[30]

249

251H

341A

Fig. (16). Deoxy PTX-A alkaloid 251H [29] and PTX alkaloid 341A [30]

No reports on new alio- and /zomo-PTX-A structures were published during the time covered by this review. However, many of the previously identified /zowo-PTX-A alkaloids still require confirmation of their proposed structures [16,19]. Biological Activity The biological activity of class A alkaloids as non-competitive blockers for nicotinic receptor-channels has been known for years [5] and the attention has now turned to the biological activity of class B alkaloids. Pumiliotoxins proved to have cardiotonic and myotonic activity apparently due to stimulatory effects on the sodium-channel operation. PTX B (323A), Fig. (17), apparently binds to a unique site on the voltage-dependent sodium-channel, causing an inhibition of channel inactivation in neuromuscular preparation [31]. A radioligand has not been developed and fiirther research on the potential of such alkaloids as cardiotonic and myotonic agents is necessary [16]. The use of synthetic PTX-A and a//o-PTX-A alkaloids allowed the assignment of structure-activity relationships for cardiotonic and myotonic alkaloids. At least three hydroxyl groups seem to be required for high cardiotonic activity, two of which can be provided either by the side chain as in PTX B (323A) or by the ring as in a//o-PTX 323B. Absence of side chain hydroxyl groups appears to eliminate cardiotonic activity [16].

250

R =

/

323B

Fig. (17). PTX B 323A, allo-FTX 323B [16] and PTX 251D [32]

Since commercial insecticides are frequently targeted to act on sodium-chaimels, the toxicity of pumiliotoxins on insects was studied [32]. PTX B (323A) proved to be more active than PTX 251D on Heliothis virescens (specially by contact), in agreement with the importance of the number of hydroxyl groups present. The low amount of PTX B present precluded the quantification of the effects. INDOLIZIDINE ALKALOIDS FROM OTHER SOURCES Other natural indolizidine alkaloids comprise the fungi indolizidines (slaframine type alkaloids), Fig. (18), polyhydroxylated indolizidines (usually isolated from the plant genera Eleocarpus, Astragalus and Iponuga), and marine type indolizidines. OAc

HoN

Fig. (18). Indolizidine alkaloid slaframine

251

Structural Studies Recent developments on the analysis and structure confirmation of previously known alkaloids include the HPLC determination of slaframine in blood plasma and milk, by Imerman and Stahr [33], and the discussion of the optical activity of the natural lentiginoside [34]. Natural lentiginoside, extracted from Astragalus lentiginosus leaves, was reported to have the absolute configuration (15',25',8aS) which resulted from biogenetic considerations. Fig. (19). The absolute configuration of the natural product was confirmed by biological assays of synthetic enantiopure samples of (lS,2S,SaS) and (li?,2i?,8a/?) [34], despite the disagreement in the optical rotation values reported for synthetic (15,25,8aS) (+2.8 ° [35], +3.2 ° [36] and +3.2 ° [34]) and natural lentiginoside (-3.3 °). OH

a>™ Fig. (19). (15,25,8aS)-Lentiginoside [34]

Reports on new structures include the new alkaloid polycanthisine, isolated from the genus Astragalus [37], and a marine indolizidine alkaloid, stellattamide B, isolated from a Korean sponge. Fig. (20) [38].

O-H polycanthisine

stellattamide B

Fig. (20). Polycanthisine isolated from genus Astragalus [37] and stellattamide B isolated from a Korean sponge [38]

The unusual enolic form of polycanthisine is supported by NMR data

252

and its stability is attributed to an intramolecular hydrogen bond with the nitrogen atom of the indolizidine skeleton. Full spectral characterisation is presented together with the MS spectrum of the trimethylsilyl derivative. 2D NMR studies of stellattamide B allowed the identification of the indolizidine nucleus and of the side chain structure. The relative configuration of the indolizidine nucleus was estabUshed by NOESY connectivities and the absolute stereochemistry of the 13'-methyl group was determined via chemical oxidation of the side chain to (iS)-2-methylglutaric acid. The nature of the counterion was determined by energy-dispersive spectroscopic experiments. Biological Activity Polyhydroxyindolizidines and slafi-amine form a group of alkaloids that displays a large spectrum of biological activity [39]. One of the most important members of this group of compounds is swainsonine, a potent and specific competitive inhibitor of cellular maimosidases [40,41]. Its high binding affinity to lysosomal a(l,3)- and a(l,6)-mannosidases leads to the inhibition of the degradation of surplus or damaged oligosaccharides and glycoproteins, resulting in their accumulation in lysosomal vacuoles. In addition to swainsonine-induced lysosomal storage disease, this alkaloid also alters glycoprotein biosynthesis by mannosidase II inhibition. Nitrogen-linked glycoproteins are synthesised via the sequential transfer of A^-acetyl-glucosamine, marmose and glucose to the lipid carrier dolichyl-phosphate, originating the usual Glc3Man9GlcNAc2-pyrophosphoryl-dolichol structure [42]. The oligosaccharide chain is then transferred to growing peptide chains, as they are synthesised on membrane-bound ribosomes. The newly synthesised glycoprotein is then transported through various cellular compartments, where enzymatic systems including glycosidases and glycosyl transferases alter the compound to form a variety of high-mannose, hybrid, or complex oligosaccharide structures. Fig. (21). The inhibition by swainsonine of mannosidase II, a key glycosidase in glycoprotein biosynthesis, leads to increased numbers of hybrid oligosaccharide-glycosylated proteins. Typified procedures have been developed for the use of glycosidases inhibitors in the study of glycoconjugates [42].

253 Glc3Man9GlcNAc2-PP-Dol Man- Man^ \ ^Man Man- Man^ Man-GlcNAcj-Protein GIc-Glc-Glc-Man-ManMan'^

Inhibitors

HQ HO^

H PH| . N ^

Hcf

castanospermine HO

H ^N^

pH 1 y OH

swamsonme

Man-Man, ^^^•^ 3GIc ^Man Man-Man ^Man-GlcNAc2-Protein Man-Man-Man^ i Man^ i ^ 4Man ^Man Man Man-GlcNAc2-Protein Man"^, Man^ ^Man Man Man-GlcNAc2-Protein GlcNAcMan''

- HIGH-MANNOSE CHAINS

HYBRID CHAINS

^ ^ 2Man Man-GlcNAc2-Protein GlcNAc- Man^i Man,

COMPLEX CHAINS

Fig. (21). Glycoproteins trimming showing the action sites of castanospermine and swainsonine [43]

Swainsonine has also been identified as the poisoning agent present in Astragalus and Oxytropis species, commonly known as loco-weed plants ("loco" is the Spanish word for crazy) [43]. Feeding of loco-weed pellets or subcutaneous administration of swainsonine to rats resulted in reduced weight gain, decreased appetite and display of nervouseness comparatively to control animals. Histologically, rats develop a high degree of vacuolar degeneration in several organs. The subcutaneous administration of higher doses of castanospermine, another polyhydroxyindolizidine alkaloid present in loco-weed plants, gave clinically normal rats, even when they showed mild vacuole changes especially in the kidneys and thyroid gland. Because of the large economic losses attributed to loco-weed poisoning, several studies have been conducted to define conditions of maximal contact of bovines and ovines without poisoning [44-50], and to detect the presence of swainsonine in an Australian plant, which produces

254

a neurological disorder when consumed by livestock [51]. Essays to determine possible alimentary supplements to alleviate loco-weed toxicity in rats failed [52]. In a recent review of teratological poisonous plants, the swainsonine present in locoweed was also referred as being responsible for abortions, wasting, right heart failure, skeletal birth defects and foetal right heart failure [53]. Drug induced animal models of neuronal storage diseases have been used extensively in recent years to explore the pathobiology and treatment of the inherited neuronal storage disorders in which neurons chronically accumulate material within lysosomes, most often due to defective activity of a specific lysosomal hydrolase [54]. Despite its high toxicity, swainsonine, as other glycosylation inhibitors, is considered a promising anticancer agent and displays immunomodulatory properties, as reported by Goss et al and Jacob [41,55]. Tumour progression in rodents and humans is commonly associated with changes in glycoprotein glycosilation, leading namely to the expression of larger oligosaccharid structures on cellular glycoproteins. Glycosidase inhibitors can disrupt the ability of malignant cells to change glycoprotein glycosilation [56]. As a potential therapeutic agent, swainsonine has several apparent advantages, most notably low pre-clinical toxicity, when compared with inhibitors that block the process earlier in the pathway. More recently clinical trials of swainsonine on cancer patients with advanced malignancies showed objective tumour remission in one patient with head and neck cancer along with adverse symptoms mainly related to elevated serum aspartate aminotransferase levels and dyspnoea [57-59]. Increased susceptibility of some human cancer cells to natural killer cells activated by swainsonine was also described [60,61]. The large period of action of swainsonine has been explained by retention of the compound by the lymphoid tissue for at least 72 hours [62]. Biological applications of swainsonine include the identification of newly isolated mannosidases [63-77], the testing of the importance of glycoproteins in association processes between cells or cellular components [78-86], and thefimction/structurerelationship evaluation of many glycoproteins [87-107]. Swainsonine also stimulates the growth of the roots of Lupinus species [108]. The first sources of swainsonine were the fiangus Rhizoctonia leguminicula and the loco-weed plants but the increasing interest in its biological activity lead to the search of alternative sources of this alkaloid.

255

Swainsonine can be produced from root cultures of Swainsona galegifolia (with a maximum concentration of 122,9 |Lig of swainsonine per gram of dry weight roots) [109,110] or by fermentation of the fungus Metarhizium anisoplilae^ which can lead to as much as 500 mg per litter of culture medium [111,112]. (+)-Swainsonine, the enantiomer of the naturally occurring swainsonine, was enantiospecifically synthesised and proved to be a very potent inhibitor of naranginase, a (+)-ramannosidase, while (-)-swainsonine does not inhibit the same enzyme. Some other more highly hydroxylated indolizidines were also synthesised but proved to be less efficient as glycosidase inhibitors [113]. Both slaframine and swainsonine were isolated from the fiingus Rhizoctonia leguminicula. The ftmgal infection of red clover hay or pasture is associated with the occurrence of the slobbers syndrome in cattle and horses which consists of excessive salivation (slobbering), lacrimation, feed refiisal, bloating, stiff joints, abortion, diarrhoea and/or violent behaviour. Some of these clinical symptoms are similar to loco-weed poisoning but the first two are not observed upon administration of swainsonine to animals [114]. Slaframine is, however, a unique muscarinic agonist due to its very high affinity for the gastrointestinal tract, low affinity for the cardiovascular system and long period of action, which deem this compound a likely candidate for use in the manipulation of digestion and absorption. Studies on the advantages of the use of slaframine supplementation on steers fed with dry-rolled com [115] or a mixed diet [116] and sheep fed with tropical pasture [117] proved however that slaframine did not increase the alimentary efficiency of the animals. More recently it was concluded that slaframine induces digestive and endocrine changes. Digestive changes can be positive in cattle consuming high-grain diets, since a high salivary production can increase the ruminal pH which is very low with this kind of feed [118]. Endocrine changes seem to be species dependent and a general conclusion caimot be withdrawn [119]. Castanospermine, isolated from the seeds of Castanospermum australe and from dried pods of Alexa leiopetala [6], is another polyhydroxylated indolizidine with a potent, competitive and reversible inhibitory effect on several glycosidases. For this reason it has been largely used as a glycosidase identifier and as an auxiliary in the purification of glycosidases and marmosidases [120-125]. Castanospermine was also used in two other

256

proposes: - the establishment of the role of A^-linked glycosylation on the function of several glycoproteins [107,126-132], including the P-amyloid precursor protein (APP) (a highly glycosilated membrane glycoprotein whose proteolitic cleavage gives (5-amyloid, characteristically found in the brain of Alzheimer's disease patients) [133] - the establishment of the role of N-linked glycosylation on lipases (lipoprotein lipase and hepatic lipase) [134-138], that have a main role on triglyceride hydrolysis [134-137] and on cholesterol reverse transport [138]. Castanospermine deficiency is related with hypertriglyceridemia and increased risk of arteriosclerosis. Castanospermine was also shown to block the binding of calnexin to glycoproteins. Calnexin, a rough endoplasmatic reticulum protein, is thought to be a chaperon in glycoprotein folding although its role is not completely understood [139]. The glycoprotein recognition site of calnexin is thought to result from the removal of two of the three glucose residues from the core oligosaccharide [140,141]. A large number of studies was carried out on this subject [142-167]. Another outcome of glycosidase inhibition by castanospermine is the down regulation of the membrane expression of some glycoproteins generated by the interruption of the intracellular trimming of oligosaccharides. Glycoproteins are assembled intracellularly and become functional once they are expressed on the cell membrane, where they act as adhesion molecules, facilitating cell-to-cell interactions. Predictably, adhesion molecules play an important role in the rejection of allografts and hence interference with their membrane expression may curtail the rejection response [168]. Tests have been conducted in vitro and on animals, and the survival of rats with heart, pancreas and kidney allografts, was shown to be castanospermine dosage dependent. Castanospermine also shows synergetic effects when used together with other immunosuppressant agents [169-171]. Castanospermine and several other polyhydroxyalkaloids known to act as glycosidase inhibitors have been used for the characterisation and differentiation of glycosidase activity in insects [172,173] and degradation of residual oligomannosides resultingfi'omthe cytosolic deglycosylation of newly synthesised glycoproteins [174-176]. Castanospermine displayed activity both against Human

257

Immunodeficiency Virus (HIV) [177,178] and as an anti-inflammatory agent. The latter is due to the inhibition of the passage of leucocytes through the subendothelial basement membrane, which results in a characteristic perivascular arrest of leucocytes at inflamed sites [179-181]. In vitro castanospermine showed action against tumours by preventing angiogenesis (the formation of new capillaries) [182], by the induction of changes on the glycosylation pattem and by several other processes [183185]. The potential activity of castanospermine as an anti-hyperglycemic agent was also studied using induced diabetic mice and a significant reduction of the blood's glucose level was seen for periods of 4-6 hours after castanospermine administration [186]. The low toxicity effects of castanospermine have been attributed to an endomannosidase activity that can bypass the glycosidase trimming enzymes involved in the A^-glycosylation of proteins. Fig. (21), providing an altemate deglycosylating pathway [187,188]. Only tissues deficient on endomannosidase activity are affected by castanospermine [181], allowing the use of this compound as a therapeutic agent, despite the fact that activity at cellular level is smaller than at enzyme level. This reduced cellular activity is thought to result from the poor accessibility to the site of action, i.e. the luminal space of the endoplasmic reticulum [189], and can be overcome by the use of castanospermine derivatives that show increased activity on inhibiting a-glycosidase in vivo [190]. 6-O-butanoyl castanospermine, Fig. (22), in particular, has been assayed against Herpes Simplex [191,192] and HIV [193] and in 1995 was a candidate for clinical tests [194].

Fig. (22). Structure of 6-0-butanoyl castanospermine

258

QUINOLIZIDINE ALKALOIDS FROM OTHER SOURCES LUPINE ALKALOIDS The most common group of alkaloids possessing a quinolizidine nucleus is that of the lupine alkaloids which can simply be classified as bicyclic (lupinine/epilupinine type), tricyclic (cytisine type) or tetracyclic, (sparteine/lupanine or matrine type), Fig. (23). This grouping is made according to structure complexity and without considering biosynthesis, as the detailed biosynthetic pathways are still not completely understood.

A

H

CHoOH I

I

B

CH2OH

(-)-lupinine

(-)-sparteine

(+)-lupanine

(+)-matrine

Fig. (23). Structures of lupine alkaloids

The conformational study of sparteine derivatives has been of great interest due to their use as synthetic chiral auxiliaries and a brief account of the progress made in this area will be presented. A description of the newly isolated and characterised lupine alkaloids, as well as an updated review of the biological activity of these metabolites will be given. Conformational Studies The basic bisquinolizidine system, sparteine, consists of two quinolizidine molecules condensed at the 7,9-positions, and three natural structures are knovm: (-)-sparteine (6i?,75',95',115), (-)-a-isosparteine (6RJS,9S,llR)

259

and (+)-P-isosparteine (65,75,95,115), Fig. (24) [195].

(-)-a-isosparteine

(+)-P-isosparteine

Fig. (24). Bisquinolizidine alkaloids (-)-a-isosparteine and (+)-p-isosparteine [195]

In sparteine, although the A/B rings form a rigid system, the possibility of inversion about the N-16 atom confers flexibility to the C/D rings system, allowing the existence of two conformers, the transoidal and the cisoidal. Fig. (25). The former is characterised by a chair:chair:boat:chair conformation of the A/B and C/D rings, a trans disposition of the two nitrogen atoms lone pairs and trans junctions of both the A/B and C/D rings, while the latter is characterised by an all chair conformation of the four rings, a cis disposition of the nitrogens lone pairs, a trans junction of the A/B rings and a c/.y junction of the C/D rings, Fig. (25).

transoidal

cisoidal

Fig. (25). Transoidal and cisoidal conformers of sparteine [195]

While the transoidal conformer is present in aprotic solutions, the cisoidal conformer is found in sparteine monosalts, due to the formation of an intramolecular hydrogen bond between the N-1 and N-16 atoms, involving inversion and protonation of the latter, Fig. (26). a-Isosparteine is characterised by trans junctions of the A/B and C/D rings in an all chair conformation both in solid state and in solution, while P-isosparteine has cis junctions of the A/B and C/D rings in an all chair

260

conformation in monosalts, a cis junction of the A/B rings and a trans junction of the C/D rings in a chair:chair:boat:chair conformation in disaks and trans junctions of the A/B and C/D rings in a symmetrical chair:boat:boat:chair conformation in solution [195,196].

Fig. (26). Structure of the cisoidal conformer of a sparteine monosalt [195]

Several studies of the conformational equiUbrium of bisquinolizidines and sparteine derivatives in the solid state and in solution have been undertaken by several authors and can be summarized as follows:

Conformational Equilibrium in Solids 2-Dehydro-2-substituted and 14-Dehydro-15-substituted Sparteine Derivatives [197]

2-Methyl-, 2-phenyl- and 2-(p-tolyl)-2-dehydrosparteine derivatives have been shown to crystallise as disalts in a transoidal arrangement, while the analogous 14-dehydro-15-phenylsparteine was curiously found to exist in a cisoidal form and to crystallise as a cisoidal monoperchlorate salt and as a transoidal dichloride di-salt. In the case of the 2-dehydro-2-substituted sparteines, the formation of the disalt is attributed to the protonation of C-3 and N-16 with the formation of a mesomeric hybrid I as illustrated for 2-dehydro-2-phenylsparteine. Fig. (27). Futhermore, the fact that the 2-phenyl-2-dehydrosparteine dibromide, dichloride and diperchlorate salts have different IR spectra led the authors to propose that structure I could exist in 3 mesomeric forms, namely, iminium, carbenium and quinonium, depending on the nature of the

261

counterion. The bulky perchlorate anion will probably not influence significantly the more delocalised structure I, Fig. (28), while the small chloride anion, with its larger acceptor character, will stabilise to a greater extent the iminium and carbenium forms.

Fig. (27). Formation of the mesomeric hybrid I of 2-dehydro-2-phenylsparteine [197]

iminium form

quinonium form

carbenium form

Fig. (28). Three mesomeric forms (iminium, carbenium and quinonium) of 2-phenyl-2dehydrosparteine salts [197]

262

In the case of the 14-dehydro-15-phenylsparteine, the inversion about the N-16 atom is responsible for the cisoidal form, Fig. (29), a situation not possible in the case of the 2-dehydro-2-phenylsparteine analogue, whose inversion about the N-1 atom is virtually impossible. In the former, the existing olefin bond flattens ring D, slightly increasing the distance between the two nitrogen atoms and decreasing their mutual repellence. In the formation of the perchlorate salt, protonation takes place initially at N-1 and the cisoidal arrangement of the monosalt is thus stabilised by means of an intramolecular hydrogen bond. This cisoidal arrangement is further stabiUsed by the co-planarity of the enamine double bond, the phenyl and the N-16 lone pair whose delocalisation further reduces repulsion between N-1 and N-16, Fig. (29).

cisodal form of 14-dehydro-15-pheny Isparteine

hydrochloride salt

Fig. (29). Structures of perchlorate and hydrochloride salts of 14-dehydro-15-pheny Isparteine [197]

In the case of the dichloride salt, the smaller size of the chloride anion (comparatively to that of the perchlorate) and its stronger proton acceptor properties allow it to establish intermolecular bonds with all the proton-donor groups occurring in the vicinity of organic counterions.

263

Consequently protonation takes place both at N-1 and C-14, originating the transoidal disalt. This stereochemical analysis was further confirmed by the close resemblance between the IR Bohlmann bands of sparteine and 2-dehydro-2-phenylsparteine; furthermore the IR spectrum of 14-dehydro-15-phenylsparteine showed distinguishing features in the same region, in accordance with the different orientation of the N-16 lone pair. The proposed stereochemistry is also in agreement with experimental observations that demonstrated that the 2-dehydro-2-substituted sparteine derivatives are reduced by sodium borohydride via the P-plane, while 14-dehydro-15-phenylsparteine is reduced via the a-plane, which means that, in both instances, reduction takes place on the least hindered side of the corresponding iminium cations. 2- And 15-Substituted Sparteine Derivatives

All the reduced 2- and 15-substituted sparteine derivatives have been shovm to exist in a transoidal form in solution and to crystallise as monoperchlorate salts in a cisoidal arrangement, in close resemblance to sparteine. Protonation of N-16 or N-1, in 2- and 15-substituted derivatives, respectively, leads to the formation of an intramolecular hydrogen bond which stabilises the cisoidal salt. These results were later supported by the detailed IR and ^^C NMR analysis of 2-(p-tolyl)-sparteine and its monoperchlorate salt [198]. The same authors also showed that the 2-cyano-2-methyl- and 2-cyano-2-phenylsparteine derivatives crystallise in a cisoidal form, as monosalts, probably due to the reduction of the basic nature of the N-1 atom by the negative inductive effect of the cyano substituent. The protonation of the N-16 atom leads to its inversion and establishment of an intramolecular hydrogen bond, as in the case of the 2-methyl- and 2-phenylsparteine derivatives [197]. 17p-Substituted Sparteine Derivatives [197]

The introduction of a methyl or an isopropyl substituent at the 17(3 position of sparteine has been shown to stabilise the transoidal form of these compounds, both in the solid state and in solution, by what is known

264

as the 'anchor effect': obstruction of the inversion of the N-16 atom thus precluding conformational changes. Unlike sparteine, these compounds are unable to form a monocation and tend to crystallise as disalts. This effect is also observed with the derivatives 2-dehydro-2-phenyH7P-methylsparteine and 2-dehydro-17|3-isopropyl-2-phenylsparteine which crystallise only as disalts in a mesomeric structure similar to that of their parent compounds. Conformational Equilibrium in Solution

A new method for the quantitative determination of the conformational equilibrium of bisquinolizidine alkaloids in solution, by ^H and ^^C NMR, was developed by Wysocka and Brukwicki [199]. Using sparteine and 5,6dehydromultiflorine, Fig. (30), as model compounds for a C ring in boat and chair conformation, respectively, the authors showed that the percentage of conformers with the C ring in boat conformation can be determined on the basis of the experimental chemical shifts of C-12 and C-14 and of the J coupling value of H-7/H-17|3, by the formula: fs = (5-6c) /(6B-6C), where 5 is the experimental value, and 6B and 6c represent the 6 or J coupling values of the model compounds in boat and chair conformation, respectively.

Fig. (30). Bisquinolizidine alkaloid 5,6-dehydromultiflurine with C ring in chair conformation [199]

The main factors identified as relevant in the delocalization of this equilibrium towards the boat or chair conformation of the C ring include: - repulsion of the trialkylamine dipoles, favouring the chair conformer; - interactions arising from skeleton strain between rings B and C, favouring the boat conformation, that may be overcome by the effect of substituents that induce conformational changes in ring B. Such is the case

265

of quinolizidines either with an aromatic A ring (e.g. anagyrine), which leads to flattening of ring B, or with a lactam group in ring B (e.g. aphylline), which forces it to adopt a sofa conformation; in both these instances, the chair conformation of ring C predominates; - Van der Waals repulsion between hydrogen atoms located close to each other in the chair conformation of ring C, namely between H-5a/H-17a, H-8oc/H-12p, H-12p/H-17p and H-14p/H-17P, which favours the C ring boat conformation; - effect of substituents, as exemplified by the intermolecular hydrogen bond between the 13a-0H group and the N-16 atom of two molecules of 13a-hydroxymultiflorine, which increases slightly the percentage of the conformer with the C chair conformation, comparatively to multiflorine, by diminishing the repulsion between the amine and the Y-oxo-a,P-enamine system; - effect of geometry changes in the D ring, as is the case of 13-oxolupanine in which the percentage of conformers in chair conformation is increased with respect to lupanine. The flattening of ring D induced by the presence of an oxo group, leads to increased distances between the H-8oc/H-12p, H-12p/H-17P and H-14p/H-17p protons, thus reducing the destabilisation of the chair conformer; - intermolecular interactions such as hydrogen bonds and crystaUine forces. While investigating the behaviour of tricyclic quinolizidines in solution, using namely 5eco-(ll,12)-12,13-dehydromultiflorine, 5eco-(ll,12)-5,6dehydromultiflorine, angustifoline, cytisine and termisine. Fig. (31), the same authors found that the all chair conformation of the three rings is preferred, along with a rigid quasi-Zrara junction of the A and B rings that prevents ring B from assuming a boat conformation [199]. The involvement of the N-1 nitrogen atom in mesomerism of Y-oxo-a,P-enamine, y-pyridone, lactam and a-pyridone systems seems to exclude the interaction of the trialkylamine dipoles and, consequently, in tricyclic quinolizidines the trans ]\mction of the quinolizidine moiety seems to be responsible for the preferred all chair conformation. In termisine, where the quinolizidine nucleus has a cis junction, the middle ring adopts a boat-like conformation illustrating the importance of the interaction of the H-8oc/H-12p, H12-p/H-17p and H-14p/H-17p hydrogen pairs in sparteine and its derivatives.

266 N

O,

seco-( 11,12)-12,13-dehydromultiflorine

secO'( 11,12)-5,6-dehydromultiflorine

H

H

angustifoline

Fig. (31). Structures of the tricyclic quinolizidines seco-{\ 1,12)-12,13-dehydromultiflorine, seco(1 l,12)-5,6-dehydromultiflorine, angustifoline and termisine [199]

For the above mentioned tricyclic quinolizidines, a comparison of the HCCH dihedral angles determined by ^H NMR J analysis and those predicted by molecular modelling or established by X-ray structures, when available, vs^as also performed, which corroborates the previous stereochemical analysis [200]. The reason indicated for the similarity of conformations of these alkaloids in the solid state and in solution is the partial flattening of ring B, caused by the presence of a flat system in ring A, that diminishes the steric hindrance of ring C in an all chair conformation. A similar structural study was made with angustifoline. The ^H and ^^C NMR spectra of the alkaloid in four different solvents were fully assigned by resorting to 2D ^H-^H and ^H-^^C COSY and 2D J resolved spectra. Conformational analysis in the solid state and in solution is presented [201]. More recently other studies of the conformational equilibrium of diazatricyclic systems based on one- and two- dimensional ^H, ^^C and ^^N NMR experiments were published [202,203]. Newly Isolated and Characterised Structures A compilation of ^^C NMR data of tri- and tetracyclic quinolizidine alkaloids was made in several reviews [196,204,205]. New reports on the characterisation of previously known structures

267

include a vibrational circular dichroism study of (-)-sparteine [206], a mass spectrometry study of 2- and 15-substituted derivatives of sparteine and 2and 14-dehydrosparteine [207], the determination of the relative and absolute configuration of alloperine by X-ray crystallography [208] and several reports on the crystal structures of tetracyclic lupine alkaloid derivatives, such as: episparteine N(16)-oxide dihydrochloride trihydrate [209], 2 - dehydro - 13(3- isopropyl - 2 - phenylsparteine [210], 2-cyano-2-methylsparteine [211], 2-cyano-2-methylsparteine perchlorate [212], 2-cyano-2-phenylsparteine [211], 17P-isopropylsparteine and 17p-isopropyllupanine perclorates [213], 17-oxolupanine [214], 17-oxosparteine perchlorate [215] and ll,12-^eco-12,13-dehydro-multiflorine perchlorate hydrate [216]. The previously isolated 13p-hydroxymamanine had its (7i?,9iS',l IR,13R) absolute stereochemistry resolved by X-ray analysis of the corresponding hydrobromide hydrate and it was postulated that its biosynthetic origin was the oxidative cleavage of the N-l/C-10 bond of (-)-baptifoline. Fig. (32) [217].

HO"

13 p-hydroxymamanine

(-)-baptifoline

Fig. (32). Structures of isp-hydroxymamanine and (-)-baptifoline [217]

The reports of newly isolated structures and a brief description of their most relevant structural features can be summarized as follows: Bicyclic lupine alkaloids

The bicyclic lupine alkaloid, (-)-(3'-methoxy-4'-a-L-rhamnosyloxyciimamoyl)-epilupinine. Fig. (33), was isolated from the aerial parts of Lupinus hirsutus [218].

268

OH

OH

Fig. (33). Structure of (-)-(3'-methoxy-4'-a-L-rhamnosyloxycinnamoyl)-epilupinine [218]

Evidence of the structure proposed for this new glycosidic lupine alkaloid came from the analysis of its positive ion FABMS spectrum that showed peaks at m/z 492 ([M+H]"^) and m/z 346 ([(M+H)-146(deoxyhexose) ]^) and a fragmentation pattem similar to the one found for (+)-(4'-hydroxy-3'-methoxycinnamoyl)-epilupinine. The typical UV absorptions for the cinnamoyl moiety at X^max 226 nm and ?imax 316 nm, as well as the characteristic reaction with j9-anisaldehyde-H2S04 to afford a green spot on silica gel TLC, further suggested the presence of the cinnamoyl and sugar substituents, respectively. Confirmation of the assigned structure came from the acid hydrolysis of the isolated metabolite: reaction with 3% hydrochloric acid gave (+)-(4'-hydroxy-3 '-methoxycinnamoy l)-epilupinine and rhamnose, identified by GC-MS after trimethylsilylation. Further hydrolysis with 7% hydrochloric acid yielded (+)-epilupinine, identified by co-HPLC by comparison with an authentic sample. The NMR spectra of (-)-(3'-methoxy-4'-a-L-rhamnosyloxycinnamoyl)-epilupinine showed the presence of both the trans and cis isomers of the cinnamoyl moiety in a 1:5 ratio, and the a-rhamnoside linkage configuration was determined on the basis of the anomeric C-H J coupling observed (176 Hz). A fiill ^H and ^^C NMR characterisation was also presented. Tetracyclic lupine alkaloids

Several new lupanine derivatives with hydroxy, methoxy, oxo, and ester

269

functions were isolated and/or characterised since 1994. New reports on hydroxy and methoxy derivatives include the isolation of (-)-6a-hydroxylupanine from Lygos raetam var. sarcocarpa [219] and the isolation of two methoxylated derivatives, 4a-hydroxy-13P-methoxylupanine and 3P,4a-dihydroxy-13p-methoxylupanine, from Acosmium panamense, as well as the full spectroscopic characterisation of the previously known ISP-methoxylupanine, Fig. (34) [220]. HO.,, 0CH3

(-)-6a-hydroxylupanine

4a-hy droxy-13 (i-methoxy lupanine

HO/, HO

0CH3

3 p,4a-dihy droxy-13 P-methoxy lupanine

'0CH3

13 p-methoxy lupanine

Fig. (34). Structures of the tetracyclic lupine alkaloids (-)-6a-hydroxylupanine [219], 4a-hydroxy-13 P-methoxy lupanine, 3 p,4a-dihydroxy-13 P-methoxy lupanine and 13 P-methoxy lupanine [220]

The structural identification of (-)-6a-hydroxylupanine was based on the analysis of its spectral data and is in fiiU agreement with the C15H24N2O4 molecular formula determined by HRMS [219]. The mass fragments at m/z 247 [M-OH]"^ and m/z 246 [M-H20]^ suggested the presence of a hydroxyl substituent, fiirther confirmed by an IR absorption band at 3400 cm"\ Further analysis of the IR spectrum also provided evidence for the quinolizidine nucleus (2860, 2810 and 2750 cm'\ trans Bohlmann absorption bands) and the oxo substituent (1640 cm"\ lactam group). The proposed structure was confirmed by ^H-^H, ^H-^^C COSY and

270

NOESY NMR experiments: the oxo substituent was located at C-2 due to the resemblance of its ^^C NMR resonance with the one reported for lupanine, and the D2O exchangeable signal at 5 3.95 ppm further confirmed the presence of a free hydroxyl group; the position of the latter (C-6) was derived from the replacement of the corresponding methyne signal in the ^^C NMR spectrum of lupanine with a new quatemary resonance at 6 85.5 ppm in the ^^C NMR spectrum of 6a-hydroxylupanine. The NOESY cross peaks observed between H-lOa and H-8a indicate a 1,3-diaxial relationship of both hydrogens, and the strong cross peak between H-lOa and C-6-0H indicates the a-axial position of the latter. The absolute configuration of (-)-6a-hydroxylupanine (7R,9R,llR) was determined through chemical transformation to (-i-)-5,6-dehydrolupanine, which was in tum identified by comparison with an authentic sample. The structure of the previously known 13 P-methoxylupanine was assigned on the basis of the similarities it has with 13 |3-hydroxylupanine, in terms of ^H and ^^C NMR data. The presence of an extra methyl group is supported by the methyl resonances at 5 3.34 ppm and 5 55.4 ppm (^H and ^^C NMR, respectively), by the C16H26N2O2 molecular formula and by the mass fragment at m/z 247, corresponding to the loss of a methoxyl group. Further evidence for its location at C-13 came from the observed downfield shift of the C-13 signal and upfield shift of the C-12 and C-14 resonances. The overall resemblance of the NMR spectra of 13 P-methoxy lupanine and 13 P-hydroxy lupanine and the multiplicity of the H-13 resonance were the basis for the assignment of the P configuration [220]. Comparison of the NMR data of 4a-hydroxy-13 P-methoxy lupanine and 3P,4a-dihydroxy-13P-methoxylupanine with that of 13p-hydroxylupanine led the authors to propose the depicted structures: the hydroxyl group of 4a-hydroxy-13 p-methoxy lupanine was evident in the massfi-agmentat m/z 294 (C16H26N2O3) and resonances at 5 3.92 ppm and 5 63.2 ppm, the latter replacing the C-4 resonance (6 19.4 ppm) in the ^^C NMR spectrum of 13 P-methoxy lupanine, and its a orientation was determined on the basis of the H-4 multiplicity and magnitude of the observed upfield shift of the C-4 and downfield shift of the C-3 and the C-5 resonances, when compared to a known 4P-hydroxylupanine derivative (4P-hydroxy-13a-0-(2'-pyrroylcarbonyl)-lupanine). Comparison of the spectral data of 3 P,4a-dihydroxy-13 P-methoxy-

271

lupanine with the previously identified ISP-methoxylupanine and 4ahydroxy-lSP-methoxylupanine, as well as the full ^H an ^^C NMR spectra assignment (achieved by COSY and HMQC), and the presence of mass fragments of the trimethylsilyl derivative at m/z 454 [M]"^, 364 [M-88]^ and 274 [M-176]^, confirmed the proposed structure. The H-4 J coupling observed in the ^H RMN spectrum indicates a /ra«5-diaxial relationship between H-3 and H-4 and supports the assigned 3p,4a configuration. New accounts of oxo- derivatives of lupanine include the (+)-15P-hydroxy-17-oxolupanine, isolated from seeds of Lupinus albus [221], and the two structures proposed for 10-oxo- and 17-oxolupanine, isolated from an East African legume, Dicraeopetalum stipulare Harms, Fig. (35) [222].

Fig. (35). Structure of (+)-15p-hydroxy-17-oxolupanine, an oxo- derivative of lupanine [221]

A sparteine skeleton with one hydroxyl and two oxo substituents was proposed for (+)-15P-hydroxy-17-oxolupanine after analysis of MS and IR data: a molecular formula of C15H22N2O3 was determined by HRMS, while the EIMS spectrum showed peaks at m/z 278 [M]"", 261 [M-OH]^ and 260 [M-H20]^ indicating the presence of the hydroxyl substituent, further confirmed by the presence of a broad band at 3420-3250 cm'\ in the IR spectrum. This spectrum also suggested the presence of two lactam carbonyl groups (1640 and 1620 cm"^). Comparison of the ^^C NMR spectrum with the one reported for (+)-17-oxolupanine, as well as the analysis of ^H-^H and ^H-^^C COSY spectra allowed the full assignment of the ^H and ^^C resonances and confirmed the proposed structure: the large J coupling observed for H-15 supports the equatorial position of the C-15 hydroxyl group, and the downfield shift of the C-11 and upfield shift of the C-12 and C-14 resonances, in comparison with those reported for (+)-17-oxolupanine, suggest a chair-chair conformation of rings C and D,

272

further supported by the absence of cross peaks between H-11 and H-9. The oxolupanine derivatives, 10-oxo- and 17-oxolupanine, were tentatively identified on the basis of their EIMS spectra [222]: for both compounds, the molecular ion at m/z 262 suggested a dioxosparteine structure, which was further restricted to an isomer of oxolupanine due to the presence of a prominent ion at m/z 136 that excludes the possibility of a 10,17-dioxosparteine skeleton. The compound with RI (Kovat retention index) 2204, 17-oxolupanine, was tentatively identified on the basis of the presence of mass fragments at m/z 234 [M-CO]+, 150, 136, 97 and 84, a fragmentation pattern typical of 17-oxosparteines. The compound with RI 2218, 10-oxolupanine, was tentatively identified on the basis of its mass fragmentation pattern which shows fragments at m/z 234 [M-CO]^, 233 [M-C2H5]^ 205 [M-CHsCHsCO]^ 150, 136 and 97, typical of 10-oxosparteines. New reports of lupanine esters concem the isolation and characterisation of the new structures (-)-3P-hydroxy-13a-tigloyloxylupanine from the seedlings of Cytisus scoparius [223] and cineroctine from twigs of Genista cinerea subsp. Cinerea [224], Fig. (36). The full ^^C NMR characterisation of the previously known cineverine was also reported[224]. o

H

R = OH, R" =

Me Me

(-)-3 p-hy droxy-13 a-tigloyloxy lupanine O

OH

R' = H, R" = cineroctine R' = H, R" = cineverine

Fig. (36). Structures of the lupanine esters (-)-3P-hydroxy-13a-tigloyloxylupanine [223], cineroctine [224] and cineverine [224].

273

The structure depicted for (-)-3P-hydroxy-13a-tigloyloxylupamne [223] was proposed on the basis of its mass spectrum: the fragmentation pattern observed in the EIMS spectrum showed close resemblance with that observed for (+)-13a-tigloyloxylupanine and the mass peaks at m/z 362 ([M]"") and m/z 1(^1 (base peak) supported the presence of an extra hydroxyl group, further confirmed by the IR absorption at 3350 cm•^ ^H-^H and ^H-^^C COSY spectra allowed the confirmation and full NMR characterisation of the proposed structure while the proposed 13a-axial position of the tigloyl ester was based on the smallJ coupling observed for H-13|3, which suggests an equatorial orientation. The observed downfield shift of the C-13 signal and upfield shift of the C-12 and C-14 resonances in the spectrum(spectra) of (-)-3|3-hydroxy-13a-tigloyloxylupanine, comparatively to the chemical shifts reported for (-)-3p,13a-dihydroxylupanine, further supported the 13a-axial substitution. Full confirmation of the proposed structure came from the enzymatic synthesis of (-)-3P-hydroxy-13a-tigloyloxylupanine from (-)-3p,13a-dihydroxylupanine, tigloyl-CoA and DHLTase [(-)-3 P, 13 a-dihydroxylupanine 13-0-tigloyloxy Itransferase]. The identification and NMR characterisation of cineroctine [224] was achieved by analysis of ^H-^H COSY and one and multiple bond ^^C-^H ^H-detected correlation spectra, and by comparison of the assigned ^H and ^^C resonances with the ones previously determined for 13a- and 13 p-hydroxylupanine. Three new tetracyclic lupine alkaloids were isolated from Lupinus species and characterised: (-)-13p-hydroxymultiflorine was isolated from seeds of L varius, and (+)-2P-hydroxyaphylline and (+)-13a-hydroxyaphyllidine from seeds of I. hartwegii. Fig. (37) [225]. The structure proposed for (-)-13P-hydroxymultiflorine is in full agreement with the C15H22N2O2 molecular formula determined by HRMS, the UV spectrum typical of y-pyridone rings (X,max 327 nm) which is further confirmed by the IR absorptions of the conjugated carbonyl group at 1630 and 1580 cm"\ and the EIMS fragments at m/z 245 [M-OH]"" and m/z 244 [M-H20]'^ that, together with an IR absorption at 3300 cm'\ confirm the presence of the hydroxyl group. The P-equatorial configuration of the C-13 hydroxyl group was proposed due to the differences observed in the ^^C NMR data of the D ring of this alkaloid and 13a-hydroxymultiflorine.

274

The axial orientation of H-13 was confirmed by the muitipUcity of its ^H NMR resonance and by the NOESY cross peaks observed for H-11 and H-15, which indicate a 1,3-diaxial relationship of these two protons with H-8. A boat-chair conformation of rings C and D is also proposed, based on the chemical shift of the C-8 resonance and the presence of a cross peak between H-9 and H-11 in the *H-^H COSY spectrum.

'OH OH

(-)-13 p-hy droxymultiflorine

O

(+)-2p-hy droxyaphy lline

0

(+)-13a-hydroxyaphyllidine

Fig. (37). Structures of the tetracyclic lupine alkaloids (-)-13P-hydroxymultiflorine, (+)-2P-hydroxyaphylline and (+)-13a-hydroxyaphyllidine [225]

The structure proposed for (+)-2P-hydroxyaphylline is in agreement with the C15H24N2O2 molecular formula determined by HRMS, with the EIMS fragments at m/z 247 [M-OH]^ and m/z 246 [M-HaO]^ and with the IR absorptions at 3375 cm"^ (hydroxyl group) and 1640 cm"^ (lactam carbonyl group). The position of the hydroxyl substituent resultedfromthe comparison of the spectral data of (+)-2|3-hydroxyaphylline and aphylline, which revealed coincident ^^C NMR resonances for rings B, C and D, and from homonuclear spin decoupling experiments (H-2 collapses to a singlet upon saturation) and ^H-^H COSY experiments (H-2 is coupled with the multiplets assigned to both H-3). The absence of diaxial coupling for the H-2 resonance which is seen in the ^H NMR spectrum of the depicted compound confirmed the a-axial position of the hydroxyl substituent. The (+)-13a-hydroxyaphyllidine structure was proposed on the basis of the C15H22N2O2 molecular formula determined by HRMS, IR absorptions at 3270, 1635 and 1560 cm"^ (suggesting the presence of hydroxyl, lactam and ethylene fiinctions), and also of the UV spectrum which indicates a vinyl amide group (Xmax 239 nm). The position of the hydroxyl group was assessed by comparison of the ^^C NMR data of (+)-13a-hydroxyaphyllidine and aphyllidine, which showed similar resonances for the carbon atoms of rings A, B, and C, and also by

275

comparison with similar structures. Its a-axial configuration is in agreement both with the chemical shift and small J coupling observed for H-13 in the ^H NMR spectrum, and with NOE experiments that showed negative effects on H-11 and H-15 upon irradiation of H-13. For all three compounds the complete ^H and ^^C assignment, achieved through the analysis of ^H-^H and ^H-^^C COSY spectra, is presented [225]. The matrine type alkaloid, (-)-14P-hydroxymatrine, was isolated from dry roots of Sophora tonkinensis and its absolute configuration, (5iS',6iS',7i?,117?,14S), was determined by comparison with a synthetic sample. Fig. (38) [226].

Fig. (38). Structure of (-)-14P-hydroxymatrine [226]

The proposed structure of (-)-14P-hydroxymatrine is in agreement with the C15H24N2O2 molecular formula determined by HRMS, EIMS fragments at m/z 247 [M-OH]^ and m/z 246 [M-H20]^ and IR absorptions at 3203 cm'^ (hydroxyl group) and 1626 cm"^ (lactam carbonyl group). The proposed matrine skeleton resulted from the comparison of the mass spectra data of (-)-14P-hydroxymatrine and matrine: the existence of mass fragments at m/z 192, 177, 150, 137, 136 and 96, in close similarity with the known mass fragments of the A/B/C rings of matrine, and the two mass fragments at m/z 235 and m/z 221, instead of at m/z 219 and m/z 205 as reported for the B/C/D rings of matrine, led the authors to place the hydroxyl group in ring D. Comparison of the ^"^C NMR data of (-)-14p-hydroxymatrine and (+)-matrine, showed coincident resonances for the carbon atoms of rings A/B/C, and suggested a 14|3-equatorial configuration of the hydroxyl

276

group. Confirmation of the proposed structure and determination of its absolute configuration resulted from the comparison of the natural product with a synthetic sample, prepared by hydroxylation of (+)-matrine. Biological Activity Lupine alkaloids are characteristic of the Papilionoideae subfamily of Leguminosae and are widely known as toxic metabolites, despite the fact that some exhibit pharmacological action. Although present throughout the legume plant they are found in higher quantities in the seeds. They impart a bitter taste, a characteristic which is thought to be a genetic dominant trait, and constitute a physiological defence against predation in species that produce them. Pyridone quinolizidine alkaloids are more toxic than saturated structures, as seen by the comparison of cytisine and anagyrine. Fig. (39), with sparteine and lupanine [227].

Fig. (39). Structure of (-)-anagyrine

It was proved that quinoUzidine alkaloids present in lupine play an important role in plant defence against invading pathogenic organisms. Changes in the alkaloid composition in different organs of Lupinus albus were observed, after elicitation of seedlings with CuCli or fimgi [228]. Legume seeds have received increasing acceptance as protein sources in African and Asian countries due to their high protein content. However, the human consumption of these seeds cannot be considered without taking into account the bittemess and toxicity of the quinolizidine alkaloids they also contain [229,230]. The quinolizidine alkaloids are the main antinutritional components of the legume seeds, as described for Lupinus species such as Lupinus albus and Lupinus angustifolius. Lupinus albus is traditionally consumed in Mediterranean areas and its bitter extract contains mainly lupanine. The debittering is performed by boiling and

277

leaching the lupine seeds with water/brine, though this process removes part of the soluble protein [231]. The seeds of Lupinus angustifolius grown in Western Australia have a high protein content and also benefit the soil due to nitrogen fixation during growth. The seeds alkaloid content (mainly (+)-lupanine, (+)-13-hydroxyangustifoline, angustifoline and a-isolupanine) appears to be the only antinutritional component of significance and its level is controlled to as little as 0.001% before being used as livestock feed[230]. The rhizomes of blue cohosh, Caulophyllum thalictroides (Barbaridaceae), used in certain dietary supplement products, also contain quinolizidine alkaloids and several cases of adverse birth outcomes have been associated with maternal ingestion of those products. Profound neonatal congestive heart failure was attributed to the maternal ingestion of blue cohosh extract and ingestion of the bitter-tasting seeds has resulted in episodes of acute intoxication, including vomiting and diarrhoea. The ingestion of blue and black cohosh (Cimicifuga racemosa) by the mother leads to breathing difficulties of the neonate, and both basal ganglia and parasagittal hypoxic injury were verified by computerized tomography. Investigation of the teratogenic effects of the quinolizidine alkaloids identified in the rhizomes of blue cohosh was undertaken because of public health concerns; of all the identified alkaloids, //-methylcytisine displayed the most acute teratogenic effects in rat embryo culture tests. A^-methylcytisine was found at various levels in dietary supplement products containing blue cohosh, from a low of 3 ppm to a high of 850 ppm. In addition, this compound is found in other plants used as ingredients in dietary supplements, such as fenugreek (Trigonella foenumgraecum) [232]. Quinolizidine alkaloids of Lupinus species have been known to be toxic and teratogenic to livestock since the 19^^ century and are a problem of major concern to stockmen in different regions of the United States and Canada. The consequences of the ingestion of species containing quinolizidine alkaloids, specially anagyrine (found to be the main agent responsible for the teratogenic effects), by pregnant calves in the period of 40-100 days of gestation, include maternal muscular weakness, ataxia, foetal contracture-type skeletal defects and cleft palate, a disease known as 'crooked calf disease'. Toxic and teratogenic effects have been linked to structural aspects of these alkaloids, and the mechanism of action is believed to be associated with an alkaloid-induced inhibition of foetal

278

movement during specific gestational periods. The fact that anagyrine containing species only caused birth defects in cattle and did not affect sheep or goats, led to speculations about possible metabolism or absorption differences between cattle and small ruminants. It was proposed that cows, by ruminal metabolism, could transform anagyrine into complex piperidines that would be the true teratogenic agents. This assumption was not further confirmed and it is not even in agreement with the absorption and elimination pattems of quinolizidine alkaloids, including anagyrine, by cattle, sheep and goats [233]. The clinical signs of poisoning by quinolizidine alkaloids begin with nervousness, depression, grinding of the teeth, frothing around the mouth, relaxation of the nictitating membrane of the eye, frequent urination and defecation, and lethargy. These progress to muscular weakness and fasciculations, ataxia, collapse, stemal recumbency leading to lateral recumbency, respiratory failure, and death. Signs may appear one hour after ingestion and progressively get worse over the course of 24 to 48 hours, even if further ingestion does not take place. Generally if death does not occur the animal recovers completely [233]. It was proposed that the action of the quinolizidine alkaloids present in Lupinus albus, Lupinus mutabilis and Anagyris foetida could be associated with their affinity for nicotinic and/or muscarinic acetylcholine receptors, //-methylcytisine and cytisine have the greatest affinity for nicotinic receptors, followed by lupanine, and are 100 times more active than hydroxylated lupanines or alkaloids of the multiflorine series. Anagyrine has a 16 times greater binding affinity for muscarinic receptors than for nicotinic receptors [234.] These results did not agree with earlier studies of the binding affinities of lupanine and sparteine but still little is known about the toxicity of the individual alkaloids and their mechanism of action [233]. Studies in vivo on the disposition of lupanine and 13a-hydroxylupanine in humans demonstrated that after oral administration these quinolizidine alkaloids were eliminated in the urine and 95% to 100% of the total alkaloid administered was recovered unchanged within 72 hours [235]. After extensive work on the teratogenicity and toxicity of quinolizidine alkaloids, it was proved that the effects of these alkaloids are related to the dosage, rate of ingestion, and alkaloid levels in the plant [233]. Management strategies have been developed to prevent or minimize the economic impact on stockmen of the crooked calf condition. It is of interest to note that livestock research on lupine toxins is a significant

279

research tool for specific human health problems [53]. Among the known quinolizidine alkaloids of natural origin, only a few are of pharmacological or therapeutic relevance. In the period of time covered by this review, sparteine and cytisine were the only two natural quinolizidines that were reported for their biological importance. Sparteine is a drug with antiarrhythmic properties. It has been deduced from pharmacological and electrophysiological studies that sparteine acts via a reduction of the Na^ inward current, e.g. during the upstroke of cardiac action potentials. This process was elucidated by the determination of sodium currents, in isolated muscle fibbers, by ioose' patch clamp measurements [236]. The IC50 value for half maximal blocking of the sodium current was 168.8 |LiM, which is in accordance with the antiarrhythmic activity of sparteine. The importance of sparteine on Na^ channels inhibition was further analysed because of its potencial strong interference in neuronal transmission, particularly in herbivores. This emphasizes the role of sparteine as a chemical defence compound for the plants that produce it. The oxidative metabolism of sparteine is catalysed by human CYP2D6 (cytochrome P450 2D6) and exhibits genetic polymorphism (for a review of debrisoquine/sparteine polymorphism see Meyer [237]). 5 to 10% of the individuals of Caucasian populations, have a severe incapacity to metabolise sparteine and eliminate in the urine most of the parent drug administered, compared with only 1 to 2% in Asian populations. Although sparteine is no longer in therapeutic use, it is still a useful model and probe for polymorphic drug metabolism by CYP2D6 [238-241]. Two different reaction mechanisms for the fomiation by CYP2D6 of the two human enamine-structure sparteine metabolites, 2,3-dehydrosparteine and 5,6-dehydrosparteine, have been discussed. Mechanism A proceeds via initial enzymatic one-electron oxidation of N-1 followed by deprotonation of tlie aminium radical cation and oxygen rebound of the respective carbon radical. Fig, (40). According to mechanism B, the carbon radical is formed by direct hydrogen atom abstraction Fig. (40). The stereoselectivity and kinetic isotope effect of human sparteine metabolism were investigated by in vitro and in vivo experiments using a range of deuterium-labelled isotopomers of sparteine and the results were further compared with observations pertaining to the chemical oxidation of sparteine to 17-oxosparteine. These experiments

280

revealed that the major human sparteine metaboHte 2,3-dehydrosparteine is formed via highly stereoselective abstraction of the 2p-hydrogen atom. An unequivocal discrimination between the two possible reaction mechanisms was not possible by simple interpretation of the magnitude of the kinetic deuterium isotope effect. However, results of competitive experiments revealed the nondissociative enzymatic mechanism of the formation of the two sparteine metabolites, i.e., the sparteine molecule that is bound to the substrate binding site of cytochrome CYP2D6 performs orientational changes without dissociating from the activated enzyme/substrate complex before the first irreversible product-determining reaction step takes place. These results agree with the hypothesis that sparteine metabolism proceeds via direct carbon oxidation [242].

^H

OH

Fig. (40). Possible mechanisms of sparteine metabolite formation by CYP2D6. Mechanism A (postabstraction mechanism) and mechanism B (preabstraction mechanism) [242]

In 1991, Pabreza, L. A. et al. described cytisine [243] as an agonist of autonomic ganglia. It competes with high affinity for brain neuronal acetylcholine receptors (nAChRs) labelled with several ^H-nicotinic Ugands such as nicotine, acetylcholine and carbachol. It was proved that the binding sites of [^H]-cytisine (the binding is higher in the thalamus, striatum and cortex than in the cerebellum or hypothalamus) were nearly identical to those of other nicotinic ligand agonists, and that its pharmacology is also similar. ["^H]-cytisine differs from other nicotinic

281

ligand agonists in several aspects: it displays higher affinity, very low nonspecific binding, slow rate of dissociationfi-omthe receptor, and higher stability. The binding characteristics of [^H]-cytisine make it a particularly usefiil ligand for the study of neuronal nicotinic receptors and it is widely used for the characterisation of nAChRs and for labelling of specific subtypes of these receptors. nAChRs are found in a variety of tissues, including the neuromuscular junction, central nervous system, autonomic ganglia and adrenal medulla [244]. They are members of a gene superfamily of homologous ligand-gated ion channels that mediate several physiological fimctions, such as synaptic transmission and modulation of neurotransmitter release, and may be involved in the regulation of neuronal development [244]. nAChRs are composed of multiple subunits whose diversity allows for the possibility of a daunting number of subtypes based upon subunit composition [244]. The interest in neuronal nAChRs has increased in the past few years and a great number of papers were published on the establishment of the physiology of nAChRs including the determination of type, composition, and number of nAChRs present in different tissues, as well as on their pharmacological properties. The need to understand the functional implications of nAChRs in the human brain arises from several known facts, namelly, the reduced number of nAChRs seen in both Alzheimer's and Parkinson's diseases, the importance of cholinergic defects in particular forms of human epilepsy and the decreased number of hippocampal nicotinic receptors seen in postmorten brain tissue of schizophrenia patients. The presence of nAChRs in lung carcinoma cells and the potential role of nicotine in learning and memory was also reported. The relative potency of (-)-nicotine and (-)-cytisine at different nAChRs was also established and it was observed that it is variable. This diversity of agonist sensitivity raises the possibility of distinguishing receptor subtypes by labelling them with ligands of different selectivity and then determining pharmacological profiles for a number of reference compounds. Cytisine's competition with different nAChRs agonists and antagonist molecules for nAChRs binding sites was tested using modem imaging techniques, autoradiographic, laminar distributions and concentration-response profile experiments. The unique selectivity profiles displayed by human nAChRs constitute a valuable tool for the

282

development of selective and potentially therapeutic nicotinic analogues. Here are referred only the most recent papers published in this area [245248]. The mode of action of cytisine as an agonist of neuronal nAChRs containing P2 subunits was also discussed [249]. This analysis suggested that cytisine is a true partial agonist of p2-containing nAChRs and that, as such, it could inhibit the response of these receptors to ACh through a competitive mechanism. In the case of a4P2 receptors, cytisine binded with high apparent affinity and low efficacy to a site shared with acetylcholine, and for a3P2 receptors both the apparent affinity and the efficacy of cytisine were relatively low. A basic nitrogen and a hydrogen bond acceptor group separated by 5.9 A are essential elements for a nicotinic agonist and the receptor molecule should have coimterparts to these elements in the agonist binding sites. It is still unclear what residues provide the negative subsite and how the agonist binding couples with the conformational change that gates the channel. It was proposed that the agonist binds in a pocket form to two subunits and that it is the binding of the same molecule of agonist to subsites on both the a and P subunits that is the essential factor for the translation of binding energy to the conformational change associated with channel gating. Cytisine shares with ACh the same two-dimensional orientation of the essential elements of a nicotinic agonist, but cytisine is more rigid than ACh, and there is a difference of 1.35 A in the distance between the binding elements of the two, as shown by the comparison of their crystal structures The perception that cytisine was a partial agonist of a4p2 was based on the importance of this structural difference and on how the P subunits configure a site of agonist interaction in the different conformational states of the receptor [249]. The administration of cytisine to rat spinal cord elicited an elevation in blood pressure, tachycardia and a behavioural response that manifested itself in agitation and nociception [250,251]. In an attempt to explain these evidences, different studies were carried out on the ligand specificity of nAChRs, stimulatory pathways, and sites of action of cytisine. It was thus proposed that cardiovascular and behavioural responses were mediated by independent receptor-mediated pathways, located at distinct regions within the spinal cord. Comparatively to nicotine, cytisine proved to be more potent and to have a longer duration of action in producing the irritation

283

response; on the other hand, appart from these differences, the two agonists have similar pressor potencies. Competitive receptor blockers like a-lobeline and D(3E were almost ineffective in blocking the prolonged responses to cytisine. The cardiovascular responses to intrathecal cytisine administration had two components: the first was mediated through direct sympathetic output and rapidly desensitised to cytisine and the second was coupled indirectly to the nociceptive response and showed a diminished capacity for rapid desensitisation [252]. The cytisine stimulation was described as involving both the spinobulbar pathway and the direct stimulation of preganglionic sympathetic neurons [253]. Cytisine also proved to be important in the control of the dopamine metabolism in Parkinson's disease. This illness is characterised by the progressive degeneration of nigrostriatal dopaminergic neurons, which is a consequence of several factors such as disfimction of mitochondrial respiration, generation of hydrogen peroxide by enzymatic and non-enzymatic reactions, and reduced radical defence mechanisms which lead to oxidative stress. These factors are linked and, as a result, an increased concentration of hydrogen peroxide is observed along with an excess of fi:ee iron. Taken together, these might initiate the Fenton reaction, resulting in increased formation of hydroxyl free radicals. It was at this point that cytisine proved to be important. Cytisine's structure indicated that it could potentially be a new iron-chelator. Fig. (41), and its use in in vitro experiments resulted in a decrease of hydroxyl free radical production; in in vivo experiments it displayed protective abilities against toxicity induced by MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine). A patient with Parkinson's disease has relatively high levels of iron in the substantia nigra and thus cytisine could be a promising drug for neuroprotective treatments of this and other related neurological disorders. Moreover, cytisine was able to cross the blood brain barrier, in contrast with other iron chelating agents, and it was found that a subcutaneous cytisine application rapidly led to substantial concentrations in the brain. In the dosages used, cytisine neither altered the locomotive activity nor showed other behavioural signs of acute toxicity. This seems to be somewhat surprising, since it is well known that cytisine can induce intoxication in children as seen by the ingestion of the cytisine-rich fiiiits of L anagyroides [254]. Cytisine itself induced an increase in locomotive activity, as observed

284

for nicotine, and was used as a nicotinic agonist for the study of the neuronal basis that explains the effect of nicotine on locomotive activity. Experiments involved the intracranial administration of cytisine to the ventral tegmentum, to explore the relationship between the sites of injection and the associated degree of locomotive activation. No direct evidence was obtained for the neurochemical identity of the one or more substrates that mediated the increased locomotion associated with ventral tegmental injections of cytisine but the data were not inconsistent with mesolimbic dopamine involvement in the mediation of the locomotive responses [255].

+ Fe^"'/Fe^''(H20)6= NH

NH

Fig. (41). Proposed reaction scheme between Fe^'^/Fe^'^ and cytisine [254]

Several compounds with a quinolizidine nucleus were synthesised and tested for their biological activity [256], [257], [258]. Quinolizidine derivatives with a more or less extended and planar aromatic moiety displayed antimicrobial activity [256]. Similar compounds were prepared for testing against Mycobacterium tuberculosis, due to the growing concern for the upsurge of new cases of tuberculosis and of nontuberculous mycobacterioses in the western world since 1986. Several quinolizidines (Q) bearing different aryl nuclei substituents (which changed both the electronic distribution and the lipophilic-hydrophilic balance of the molecule) were synthesised. Six of these synthesised compounds (Q1-Q6), Fig. (42), were highly active and it is reasonable to state that

285

activity is related to the lupinylidene- and lupinyldiarylmethanes not bearing hydrophilic groups [256]. CfiH,

Q2 Q3 Q4 Q5

R'=F; R'=H; R'=H; R'=H;

R"=F; R"=H; R"=F; R"=C6H5;

R'"=H R'"=C1 R'"=CF3 R"=H

Fig. (42). Structures of synthetic quinolizidine derivatives that possess an aromatic moiety QlQ6 [256]

A^-aryl glycine amides have been described for long as local anaesthetic agents and a set of A^-[(quinolizidin-la-yl)-methyl]-benzotriazol-2-ylacetamides bearing substituents at positions 5 or 5 and 6 were prepared and tested for local anaesthetic activity. The introduction of substituents on the benzene ring and the presence of the (quinolizidin-la-yl)-methyl moiety (lupinyl moiety), instead of the structures bearing (/^rf-amino)-alkyl chains (also tested), proved to be advantageous in terms of both the intensity and the duration of activity. Q7-Q10 were the most active compounds, Fig. (43). Compound Q8 also exhibited a good antiarrhythmic activity in mice subjected to deep chloroform anaesthesia and aconitine infusion, as well as in electrically driven guinea pig left atria. Substituted benzamides with a bulky quinolizidine moiety, such as Ql 1, were subjected to binding assays to 5-HT3 and D2 receptors on membranes obtained from bovine area postrema ([^H]-GR65630) and rat striatum

286 ([^H]-spiperone), respectively. The new quinolizidine derivatives have the structural requirements necessary for having activity as 5-HT3 receptor antagonists as v^ell as a methylene group inserted between the bicyclic basic moiety and the amide nitrogen. The tested compounds proved to be unsuitable for the recognition of D2 receptors, while some, devoided of 5-HT4 receptor activity, had not only consistent affinity for central receptors but also the ability to potently inhibit the ethanol-induced dopamine efflux from the mesolimbic dopamine terminal region. However, they failed in attenuating voluntary alcohol consumption in rats, as observed with several other chemically unrelated 5-HT3 antagonists, leading to the conclusion that S-HTs-mediated inhibition of alcohol-induced stritial release of dopamine by substituted benzamides is not a requisite for affecting ethanol intake. In more general terms, the purported clinical usefulness of 5-HT3 antagonists in the treatment of alcoholism seems to be, at least, doubtful [258]. T

I

^N—CH2--C-NH—CH2.M../

\

Q^

R-R2=H

Q9 QIO

R'=H; R"=0CH3 R'=CH3; R"=CH3

OCH3 H2N

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

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SAFETY EVALUATION OF STEVIA AND STEVIOSIDE. JANM.C. GEUNS Laboratory of Plant Physiology, KULeuven, Kard. Mercierlaan 92, B 3001 Leuven Tel: +32-16-321510; Fax: +32-16-321509 e-mail: Jan. Geuns@bio. kuleuven. ac. be ABSTRACT : The literature about Stevia and stevioside used as a sweetener is discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this safety evaluation. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. A survey is given of calculated ADI's. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.

INTRODUCTION: STEVIA and STEVIOSIDE Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil). It is Imown to the Guarany people, native to these regions since time immemorial, by several names all of which refer to the sweet taste of the leaf, and especially to its use in "mate" tea {Ilex paraguariensis). It is often referred to as "the sweet herb of Paraguay". The Spanish Conquistadors of the Sixteenth Century sent back news to Spain that the indigenous population used Stevia to sweeten their herbal teas since ancient times, i.e. predating 1500 AD. Stevia has been cultivated or is still cultivated in many countries: Paraguay, the USA, Mexico, Central America, Japan, China, Malaysia, South Korea, Spain, Italy, Belgium and the UK. The main sweet component in the leaves of Stevia rebaudiana Bertoni is stevioside 3 (see Fig.(l)). Its content varies between 4 and 20 % of the

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dry weight of the leaves, and around 11% in many economical crop productions. Other compounds present but in much lower concentration are: dulcoside A 9 (± 0.5 %), steviolbioside 2 (trace), rebaudioside A 4 (±3 %), B 5 (trace), C 6 (± 1.5 %), D 7 and E 8 (traces) (See Fig.(l)). p.R2

CH3 COO-Rl

1 2 3 4

Compound name steviol steviolbioside stevioside rebaudioside A

Rl H H PGlc PGlc

5

rebaudioside B

H

6

rebaudioside C (dulcoside B)

PGlc

7

rebaudioside D

pGlc2-ipGlc

8 9

rebaudioside E dulcoside A

PGlc2-ipGlc pGlc

Fig. (1).

R2 H PGlc2-ipGlc PGlc2-ipGlc PGlc2-lpGlc 3-1 PGlc PGlc2-ipGlc 3-1 PGlc PGlc2-laRha 3-1 PGlc PGlc2-ipGlc 3-lpGlc pGlc2-ipGlc pGlc2-laRha

Structures of stevioside and related compounds. In rebaudioside A, B, C, D and E an additional sugar moiety is added on carbon 3 of the first pGlc.

A typical composition on a dry weight basis of the most important components of the leaves is as follows: - proteins: ± 6.2 % - lipids: ± 5.6 % - total carbohydrates (anthrone): ± 53 % - stevioside: ±11% - rebaudioside A: ±2 % - rebaudioside C: ±2% Stevioside is a diterpene glycoside occurring in Stevia rebaudiana Bertoni leaves. It is a high intensity sweetener that is about 300 times

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sweeter than sucrose. In many countries it is used as a low calorie sweetener in a wide range of food products and beverages. Both the plant, its extracts, and stevioside have been used for several years as a sweetener in South America, Asia, Japan, China and even in the USA it is used as a dietary supplement since 1995. Remarks to toxicological studies: Neither those scientific studies where Stevia extract or solution of pure stevioside were injected in animals, nor those studies employing perfusion experiments of organs, are considered relevant for the use oi Stevia or stevioside as food and are not discussed in this review. METABOLISM OF STEVIOSIDE Compounds used as or added to food must be absolutely safe. This means that not only the added parent compound, but also its possible metabolites must be safe, for the possibility exists that compounds that themselves are not harmful can be taken up by the human body and be metabolised into products that may have some harmful effects. Therefore, the parent compounds as well as their metabolites should be thoroughly tested in toxicological studies. In one such study, steviol, the aglycone of stevioside, showed a weak mutagenic activity [1] and, although in later experiments these results could not be reproduced [2], this has led to a whole controversy in scientific literature. It has been shown that stevioside is not taken up by the human body and none of the digestive enzymes from the gastro-intestinal tract of different animals and man are able to degrade stevioside into steviol 1, the aglycone of stevioside [3]. The lack of metabohsm is due to the fact that the bonds in stevioside are p-glycosidic and we do not have the enzymes to split these P-glycosidic bonds. Stevioside was incubated with salivary a-amylase, pancreatic a-amylase, saliva, pepsin, gastric secretion, pancreatin and intestinal brush border membrane enzymes of mice, rats and hamsters. None of these enzymes digested stevioside. Nevertheless, in feeding experiments with rats and hamsters stevioside was metabolised to steviol by the bacterial flora of the caecum. After several hours steviol was found in the blood of the animals, the maximum concentration occurring after 8 hours [4]. In rodents coprophagy occurs (this means that rodents eat their own faeces and in this way they reabsorb nutrients set free by the bacteria of the caecum). In the cited studies, it was not indicated that

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coprophagy was prevented, so it is not clear if the steviol occurring in the blood was taken up directly from the colon or indirectly from the ingested faeces (after passing through the intestines again). Although bacteria isolated from the human colon are able to transform stevioside into steviol in vitro [3], it has never been proven that this is also the case in vivo nor that the steviol eventually formed is taken up directly from the colon. Moreover, studies with roosters [5] indicate that stevioside is rapidly eliminated from the body, largely untransformed. Roosters resemble humans as they too have a lowfimctioningcaecum. Only the bacteria from the caecum or colon were able to degrade stevioside into steviol (caecum of mice, rats and hamsters; colon of man). The bacteria from the human colon also formed steviol epoxid in vitro, that was again metabolised to steviol. However, in vivo this epoxid formation probably will not occur due to the anaerobic conditions of the human colon. It was correctly concluded that steviol is the only possible metabolite [3]. Anyway, steviol epoxid has been tested in mutagenicity studies and showed to be inactive [1]. MUTAGENICITY STUDIES AND CARCINOGENICITY Each new compound in the food chain has to be extensively tested to be sure that it is not carcinogenic. As studies with animals take several years and become expensive when many compounds need to be tested, so called mutagenicity tests were developed that are much faster and less expensive. In these tests compounds are evaluated to see if they provoke mutations or alterations of hereditary material. It should be emphasised that compounds giving a positive response in mutagenicity tests are not carcinogenic in se. Compounds having a positive score should be tested for carcinogenic activity, but not all mutations lead to cancer. This can be exemplified by the following examples. A point mutation in the gene that codes for the growth hormone receptor in chickens causes dwarf mutants (bantams). A dominant gene produces hairs on the mid-phalanx of the ring finger and its recessive mutation causes lack of hair. In a population about one fourth of the people have the recessive mutation (no hair) but this does not mean that they will have cancer because of this mutation.

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Mutagenicity tests In the Ames test or bacterial reverse mutation test, use is made of mutated bacteria that eg. camiot form an amino acid histidine (his"). Therefore, these bacteria are unable to grow on a medium lacking histidine. When these bacteria are grown on such a medium, most bacteria will die, but in some bacteria a natural reverse mutation occurs, i.e. the original mutation is repaired by a reverse mutation and these bacteria can again form histidine and can grow and form colonies on the medium without histidine. Mutagenic compounds will enhance the number of reverse mutations occurring. As many compounds are mutagenic only after metabolism in the body, the test is also done after adding a so-called metabolic activation system. This is a liver supernatant fraction (S9000g) of animals pretreated with known harmful substances (eg. polychlorinated biphenyls). This S9000g fraction will ensure the further metabolism of the compounds added in the Ames test. In XhQ forward mutation test, bacteria are added to a medium containing a harmfiil substance (eg. 8-azaguanine). Under these conditions most of the bacteria will die, but some will survive because they have adapted their genotype by mutation. In the forward mutation test the development of resistence (by mutation) above the control mutation frequency is studied in the presence of the test compound. This is also done in the presence and absence of the metabohc activation system. In the micronucleus test, mice are given the test substance by intraperitoneal injection. Mice ai*e killed 24 or 48 h after treatment and the femoral marrow cells are smeared on glass slides, fixed, stained and the numbers of micronucleated polychromatic erythrocytes and of micronucleated normochromatic erythrocytes are recorded. The numbers of micronucleated erythrocytes and the proportion of polychromatic erythrocytes relative to the total erythrocytes are evaluated by observing 1000 erj^hrocj^es on the same slide. In the chromosome mutation test Chinese hamster lung fibroblast cell line (CHL) can be used. The cells are treated for 24 or 48 hours with a test compoimd at different dose levels. Both with and without the metabolic activation system, the cells are treated with colcemid (0.2 mg/ml) for 2 h to arrest cell divisions and chromosome preparations are made. The chromosome aberrations are recorded in at least 100 metaphases of each treatment. Five groups of structural chromosome aberrations can be found:

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chromatid and chromosome gaps, chromatid breaks, chromatid exchanges, chromosome breaks and chromosome exchanges. In the mammalian cell mutation test CHL cells are exposed to the test compound for 3 h with and without the metabolic activation system. After 7 days of culture, diphtheria toxine is added and the cells are left again to culture for another 7 days until the number of diphteria toxin resistent colonies are scored. In 1985 it was pubHshed [1] that stevioside was completely safe but that metabolically activated steviol was mutagenic in a "Forward Mutation Test". Steviol had to be appUed together with the microsomal fraction of liver of animals treated with carcinogenic compounds (polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone). This publication has led to a confusing discussion between advocates and opponents of the use of Stevia or stevioside. To unravel the problem we must consider the opinion of authoritative international organisations such as OECD (Organisation for Economic Co-Operation and Development) and ICH (International Council of Harmonisation). To accept new substances as food three different mutagenesis tests are accepted and required by the OECD as well as by the ICH. These can be seen in Table 1. Table 1: Mutagenesis tests required by the OECD and ICH. Test Type 1 Bacteria in vitro test (so-called Ames-Test) 2 Non bacterial in vitro tests

3 In vivo Tests

Recommended Test ^ "Bacterial reverse mutation" Test with Salmonella typhimurium TA 1535, TA1537 or TA97 or TA97a, TA98, TAIOO, £co// WP2uvrA or E coli WP2uvrA/pKM101 or TA102 "Mammalian cell mutation" Test with the following Test-System: "L5178Y mouse lymphoma gene and chromosomal mutation test" or "Chromosomal aberration" Test with e g. "human lymphocytes" "Micronucleus" with bone marrow of mice

During the last years several mutagenicity tests were done with steviol. Steviol did not show any mutagenic activity in the standard tests executed following the prescribed protocol: 1) It was inactive in the Ames test with or without metabolic activation [2, 6,7], 2) It did not show any mutagenic action on chromosome aberrations of human lymphocytes [6], 3) It did not induce micronuclei in bone marrow of mice [8].

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The other tests systems used were not standard prescribed tests. The positive mutagenic results for steviol shown by [1] were contradicted by [2] who were unable to reproduce Pezzuto's results. The positive results by [1] were shown to be due to a wrong experimental setup (a too low number of bacteria used : 10^ instead of the prescribed 10^) and to a misinterpretation of the results. It was shown that so far unknown steviol metabolites in Salmonella typhimurium TM677 after metabolic activation caused mutations, i.e. transitions, transversions, duplications and deletions at the guanine phosphoribosyltransferase (gpt) gene [9]. However, steviol was completely negative in the reverse mutation assays using Escherichia coli WP2uvrAypKM101 or using different S. typhimurium TA strains even when activation S9 mix was present [9]. After metabolic activation of steviol some gene mutation and chromosomal aberration was found in Chinese hamster lung (CHL) fibroblasts [8]. It has to be said that of all animals tested hamsters had the most sensitive response. Moreover, in hamster several metabolites of stevioside were found that are not formed in rats or humans. Therefore, the relevance of experiments with hamsters should be questioned. It has been shown that steviol does not bind to DNA, as claimed by the authors an absolute prerequisite to have any mutagenic activity [10]. Anyway, it would be very peculiar that steviol would have a mutagenic activity, while it only differs from its direct precursor ewr-kaurenate by a hydroxyl at Carbon-13 and that itself is not mutagenic. All plants contain the precursor e«r-kaurenate and other very similar compounds, including the gibberellins, a group of natural plant hormones, that are not mutagenic and of which many contain also a 13-hydroxyl function. These compounds are daily consumed by the whole world population as they are present in all vegetables. Male and female F344 rats were daily fed with a ration containing 0.1, 0.3 or 1 % of stevioside and rebaudioside for a period of 22 (males) or 24 (female rats) months [11]. The animals were then killed, and the researchers conducted biochemical, anatomic, patho-logical and carcinogenic tests on 41 organs following autopsy. In addition they performed ongoing hematologic and urine tests on the same animals. Each of the animals was matched to a control animal that experienced exactly the same treatment, except for the stevioside. No significant dose-related changes were found in the growth, general appearance, hematological and blood biochemical findings, organ weights, and macroscopic or microscopic observations (41 organs were analysed). It was also

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concluded that any neoplasms that occurred were not attributable to the administration of stevioside. Even at the highest dose of 1% no significant effects were found. This high dose is equivalent to 125 times the average daily dose of sweeteners that a normal human would require. The effects of a chronic oral feeding of stevioside were studied in rats during 24 months [12]. The concentrations used were: 0, 0.2, 0.6 and 1.2%. Growth, food utilisation and consumption, general appearance and mortality were similar in treated and control groups. The mean lifespan of rats given stevioside was not significantly different fi-om that of the controls. No treatment-related changes were observed in haematological, urinary or clinical biochemical values at any stage of the study. The incidence and severity of non-neoplastic and neoplastic changes were unrelated to the level of stevioside in the diet. The maximum no-observed-effect level of stevioside was 1.2%, and this was the highest concentration tested by the authors. The authors suggested an acceptable daily intake (ADI) of stevioside for humans of 7.938 mg/kg body wt/day (safety factor 100). For a person of 70 kg this is about 555 mg pure stevioside. However, no higher concentrations were tested and therefore the suggested acceptable daily intake does not mean that a higher intake would be harmfiil! Therefore, this ADI has to be considered as a minimum ADI. In a chronic toxicity study with F344 rats [13] it was concluded that there were no significant increases in the incidence of neoplastic lesions in any organ of tissue in the stevioside treated groups (2.5 and 5 %, i.e. daily dose of 385 and 775 mg per rat, i.e. 1 and 2 g/kg body weight/day, which is a very high dose not farfiromthe LD50!). In male animals the number of testicular tumours had the tendency to decrease. Moreover, the incidence of adenomas of the mammary gland in the stevioside-treated females was significantly lower than that in the controls. The severity of chronic nephropathy in males was also clearly reduced by both stevioside concentrations. The JECFA clearly stated [14]:" Stevioside has a very low acute oral toxicity. Oral administration of stevioside at a dietary concentration of 2,5% to rats for two years, equal to 970 and 1100 mg/kg body weight (BW) per day in males and females, respectively, had no significant effect. Reduced body-weight gain and survival rate were observed at a dietary concentration of 5% stevioside. There was no indication of carcinogenic potential in a long-term study...". Also in favour of the use of stevioside is a study by [14] who found that stevioside did not promote urinary bladder carcinogenesis in rats.

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There have never appeared reports proving that the use of Stevia or stevioside enhances the number of cancers in populations, even after a very long time of use (eg. Paraguay: more than 500 years, Japan: more than 25 years, South-Korea: 15 years, Brasil: 12 years, China: 11 years or the USA: since 1995 admitted as a dietary supplement). Thus the general conclusion is that Stevia and its sweeteners stevioside and rebaudioside A are completely safe. ACUTE TOXICITY Dead is an unambiguous criterion. In acute toxicity tests the dose is determined at which half of the animals die (so-called LD50 or lethal dose 50%). Stevioside and steviol have a very low acute oral toxicity in the mouse, rat and hamster, meaning that the LD50 value is large [16, 17]. Stevioside at a dose as high as 15g/kg BW was not lethal to either mice, rats or hamsters. Hamsters were found to be more susceptible to steviol, the aglycone of stevioside, than rats or mice. LD50 values of steviol in hamsters were 5.2 and 6.1 g/kg BW for males and females, respectively. In rats and mice, LD50 values of steviol were higher than 15g/kg BW in both sexes [17]. An oral LD50 of 17 g/kg body weight was shown for the Stevia extract (20 % of stevioside) and 15 g/kg for purified stevioside (purity of 93.5%). With rats and mice a LD50 of 8.2 g/kg was found for stevioside by oral intake, and given intraperitoneally, a LD50 of 2.99 g/kg of body weight [18]. As stevioside is 300 times sweeter than sugar, a LD50 of 8.2 g/kg corresponds to about 2.5 kg sugar/kg body weight! SUBACUTE TOXICITY, CHRONIC TOXICITY In subacute toxicity studies lower concentrations are given to determine the so-called NOEL (No Effect Level), i.e. the maximum concentration at which no effect of the appUed substance can be seen. This NOEL is important to determine the ADI (acceptable daily intake, see Table 2). This is done by dividing the NOEL by 100 (a factor of 10 to pass from animals to man and a safety factor of lOx). In the subacute toxicity studies

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the test compounds are given during long time periods, hence "chronic toxicity studies". Subacute toxicity studies were performed with purified stevioside supplied to rats' rations in concentrations up to 7% daily during 3 months [19]. In other studies rats were fed with 2.5g/kg/day of purified stevioside, also by adding it to their daily ration during 3 months [18]. Both studies did not produce any effect related with the stevioside doses on the animals tested. This means that the NOEL is higher than 2.5g/kg/day and the ADI can be calculated to be at least 25 mg/kg body weight. Stevioside was fed to F344 rats in doses of 0.625, 1.25 and 2.5 g/kg body weight during 13 weeks [20]. The authors concluded that there was a lack of any clear response and concluded that 2.5 g stevioside/kg body weight was a suitable maximum tolerable dose. From these results an ADI of 25 mg/kg can be calculated. Male and female hamsters were daily force-fed with stevioside (0, 0.5, 1 and 2.5 g/kg body wt./day respectively) [21]. This was done over several generations. The authors concluded that stevioside at a dose as high as 2.5 g/kg body weigth/day did not affect growth nor reproduction in hamsters. Also from these results an ADI of 25 mg/kg can be calculated. Table 2: Survey of ADI's for stevioside calculated from published NOELs. ADI (mg/kg body weight/day)

Animal

7.938* 25 25 25 25 21 (males) 24 (females) 6.25**

Wistar rat rat rat rat F344 hamster Wistar rat Wistar rat hamster

NOEL g/kg BW/d 0.794* 2.5 2.5 2.5 2.5 2.1 2.4

**

Reference [12] [19] [18] [20] [21] [22] [22] [23]

* The ADI calculated by [12] is a minimum value as the authors did not test higher concentrations of stevioside. ** This ADI is calculated from the NOEL of steviol (250 mg/kg BW/day) under very unfavourable conditions as steviol is easily taken up by the intestines and metabolised to various unknown compounds, whereas stevioside is not. Moreover, hamsters are known to be very sensitive animals towards steviol and stevioside.

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FERTILITY and TERATOGENICITY Although it was reported [24] that a water decoction of Stevia leaves was used as an oral contraceptive by Paraguayan Matto Grosso Indian tribes and that these decoctions reduced the fertility in adult female rats of proven fertility, subsequent studies could not reproduce their results. Moreover, [25] reported that the use of Stevia as a contraceptive has never been confirmed [19, 26] and in a field study made throughout Paraguay not any local use of Stevia as a contraceptive could be found. Inquiries were made in Asuncion, Concepcion, Pedro Juan Caballero, and in Cerro Kuatia. In the last mentioned location, interviews were conducted with members of the native Indian group, Pay Tavy Tera, as well as other populations that have recently settled in the area. The fact that Stevia and stevioside do not have any effect on fertility or reproduction is illustrated by the work of the following authors. High doses of 525 mg/kg/day of stevioside equivalent aqueous Stevia extract were administered to male and female mice during copulation and pregnancy periods [19]. The authors could not observe any difference in the copulation and conception averages and any change in the fetus and broods when compared with control groups. Male and female Wistar rats received feed containing 0.15 %, 0.75% and 3% stevioside before and at the start of pregnancy [22]. Consumption of stevioside in the 3 treated groups was 100, 480 and 2100 mg/kg/day in males and 120, 530 and 2400 mg/kg/day in females (i.e. about 20x, lOOx and 400x times the ADI). Males received stevioside from 60 days before mating and females from 14 days before mating to day 7 of pregnancy. No differences could be found between test en control groups regarding sexual cycle, mating or pregnancy, nor was there any abnormality in the test groups regarding implantation and survival of the fetus. There were no abnormalities found in the growth, general appearance, viscera or skeleton of the fetus. The final body weight showed no differences between the groups, although a small reduction in body weight was observed at first in the 3% group in both sexes. This might have been due simply to an adaptation of the rats to the extreme sweetness of the feed, avoiding it in the beginning. The authors conclude that the addition of stevioside to feed up to a concentration of 3%, which is 400 times the ADI, has no effect on mating, fertility or the development and the state of fetuses in rats given the diet prior to and during the early stage of pregnancy.

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No effects of concentrated leaf extracts (0.667 g dried leaves/ml, 2 ml/rat twice a day) were found on male fertility nor on the growth of prepubertal male rats [27]. The effects were studied of 0, 5, 25 and 100 % of Stevia extracts (=2.6 % stevioside) (10 ml extract/kg BW per day for 31 days) on male Wistar rats that weighed 270 g at the start of the experiments [28]. The amounts of stevioside given were 0, 13, 65 and 260 mg/kg BW per day. Clinical examinations revealed that there was a total absence of any signs of intolerance. Not any influence could be found on the weight of the animals. No differences could be found in the weights of the male organs (seminal vesicles, prostate, hypo-physis, testicles). The anatomicpathological examination of the testicles disclosed no evidence of any effect due to the treatment. No atrophy of the seminal tubes was found and the generation of semen was present in all the samples studied. The Leydig interstitial cells were not hyperplastic and did not show degenerative changes. No evidence of inflammatory infiltration was found. One month old male and female hamsters were daily force-fed with stevioside (0, 0.5, 1 and 2.5 g/kg body wt./day respectively) [21]. No abnormalities were found in growth and fertility in both sexes. All males mated with females efficiently and successfully. Females showed normal 4-day oestrus cycles and became pregnant after mating. Each female was mated and allowed to bear three litters during the period of the experiment. The duration of pregnancy, number of fetuses, as well as number of young dehvered each time from females in the experimental groups were not significantly different from those in the control group. The young Fl and F2 hamsters continuously receiving stevioside via drinking water until one month old and daily force-fed afterwards at the same doses as their parents showed normal growth and fertility. Histological examination or reproductive tissues from all three generations revealed no evidence of abnormality which could be linked to the effects of consuming stevioside. It was concluded that stevioside at a dose as high as 2.5 g/kg body weight/day affects neither growth nor reproduction in hamsters.

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Toxicity to reproductive organs Not any significant effect was found on spermatogenesis, nor on the interstitial cell proliferation and tumor formation in the testes [11]. Male rats were force fed (25-30-day-old) for 60 days with aqueous S. rebaudiana extracts corresponding to 0.667 g dried leaves/ml, 2 ml/rat twice a day [27]. This way each rat received the equivalent of about 2.668 g of dry Stevia leaves per day, i.e. 5.34 % of their body weight! (stevioside content not known). This is a very high amount: 53.4 g Stevia leaves/kg BW at the start of the experiments (rats weighing about 50 g) and about 13.75 g/kg BW at the end (rats weighing about 194 g). If we assume a stevioside content of 12% this means that the young animals received 6.4 g stevioside/kg BW and the older ones about 1.65 g/kg BW. The authors observed a decreased seminal vesicle weight by about 60 % but they concluded that if Stevia extract does have some potential to decrease rat fertility at all, this effect is almost certainly not exerted on the male. Unfortunately, only one high concentration was tested and no dose-effect studies were undertaken. In contrast to these results, it was reported [29] that concentrated Stevia extracts similar to those of [27] fed for 60 days to prepubertal male rats produced a decrease in final weight of testes, seminal vesicle and cauda epididymidis. In addition the fructose content of the accessory sex glands and the epididymal sperm concentration were decreased. Stevia extract treatment tended to decrease the testosterone level, probably by a putative affinity of glycosides of the extract for a certain androgen receptor. No alteration occurred in luteinizing hormone level. Whereas [29] suggested a possible decrease of the fertility of male rats, his results are in contradiction with those of [27] who applied extracts with similar stevioside content and these authors stated that there is certainly not an effect on male fertility. The difference between the extracts of [27] and [29] is that the last author completely dried the Stevia water extracts, and it is known that various chemical alterations may occur in the extract during drying, that probably were responsible for the observed effects. So it is not sure that the observed effects were indeed due to the stevioside present in the extract. It should also be mentioned that the used extract concentrations were extremely high, at the start of the experiments even 5.34 % of the body weight (or around 6.4 g stevioside/kg BW, which is near to the LD50)! For an adult person of 65 kg this means 3.47 kg of dry Stevia leaves or about 34.7 kg fresh leaves/day, i.e. more than 50% of his body weight! The significance of

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such experiments where only one extremely high concentration was tested, should be questioned. Even the question may be posed if it is still ethical to force feed animals with such high concentrations. Melis' results are also in contradiction with the above and below cited studies that could not reveal any effect on fertility of male or female animals. Also in populations using stevioside as sweetener (Japan, Paraguay, China, SouthKorea, USA) no adverse effects on fertility were reported. Applied stevioside has no effect on fertility, mating performance, pregnancy, number of fetuses, nor on the growth and fertility of the offspring [21, 22, 27, 28, 30]. However, when steviol (the aglucone of stevioside) was given to hamsters on day 6-10 of pregnancy at doses of 500-1000 mg/kg body weight/day it induced toxicity [23]. The number of live foetuses per litter and mean foetal weight decreased. The maternal kidneys showed a dose-dependent increase in severity of convoluted tubules in the kidneys. The no-effect level (NOEL) for maternal and foetal toxicity was 250 mg/kg BW/day. This study with steviol has nothing to do with the use of stevioside as a sweetener. When stevioside is fed to hamsters, no toxic effects were found even not in 3 successive generations [21]. The problem is that when steviol is given in the feed, it can be resorbed directly by the intestines, whereas stevioside is not. Stevioside is transformed only by the bacteria of the caecum or the colon from which steviol eventually may be resorbed, or taken up by coprophagy (see above). Moreover, hamsters are known to be very sensitive to steviol and stevioside [17], this is the reason that hamsters were chosen in this study. The NOEL of steviol was 250 mg/kg BW [23], which corresponds to 625 mg stevioside/kg BW. Even under these very unfavourable conditions an ADI of 2.5 mg steviol/kg BW, which corresponds to 6.25 mg stevioside/kg BW can be calculated, which is close to 7.9 mg/kg BW obtained by [12] (see also Table 2). Besides the lack of effects of Stevia extracts or stevioside in experiments on fertility, no negative reports on human fertility have appeared since the use of Stevia and stevioside in Japan (since more than 25 years), the USA (since 1995) or in other countries. BIO-AVAILABILITY OF NUTRIENTS FROM THE DIET Some compounds added to the food are able to influence the uptake of other essential elements. This way growth curves may be disturbed or

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Other adverse physiological effects may show up, e.g. natural oils of plant origin contain a lot of lipid soluble vitamins. Replacement of natural oils by synthetic fats exempt of vitamins may influence the uptake of other lipids and lead to a plentitude of physiological disturbances due to lack of essential lipids and vitamins. Stevia used in small amounts has no negative consequenses for the bioavailability of nutrients from the diet and it does not have adverse physiological effects. This can be deduced from the dataused to determine the ADI (see above) and from the use of Stevia in countries as Paraguay (used since more than 500 years), Brasil, China, Japan (more than 25 years) and even in the USA (Stevia allowed as dietary supplement). Studies in which Stevia extracts or solutions of pure stevioside have been injected in the animals or were used in perfusion experiments of organs, are considered as not relevant for the use of Stevia or stevioside as food. As indicated above the sweet components of Stevia used in low concentrations, are not taken up in the human body and are not metabolised by the digestive enzymes from the gastro-intestinal tract. From the work of the authors cited in the chapter on subacute toxicity and the ADI, it can be deduced that Stevia had no effects on the bioavailability of nutrients, as no effects could be found either on the growth or the weight of the animals, nor on other physiological processes. Stevia extracts were extensively tested (low and high concentrations) in rats during 22 months (male rats) and 24 months (females) [11]. No differences were found with the control animals, implicating that all nutrients were taken up and metaboUsed in the same way. The same can be concluded from the experiments of [21] who force-fed one month old male and female hamsters with high and low concentrations of stevioside in their food. This was done over 3 generations. This means also that pregnant hamsters as well as their young litters were forced to eat stevioside. No differences in growth or in other parameters were found. This again proves that no effects on food uptake or metabolism are involved even at relatively high stevioside concentrations. The same conclusions are true for the work of [31] done with Wistar rats fed during 30 days with high amounts of Stevia extract. In chronic experiments with rats during 24 months [12] and in experiments with 1000 broiler chickens [32] no effects were found on food consumption or on the weight gain or loss of these animals. Aqueous extracts of Stevia were chronically admistered to prepubertal male rats [27]. The concentrated extract of leaves (66.7 g dried leaves/100

314

ml final solution) was given twice a day by means of gastric tubing (2ml/rat) during 60 days. In order to improve conditions for gastrointestinal absorption the animals were deprived of food and water 1 h before and 30 min after drug administration. No effects on body weight gain curves were found during the duration of the experiment. No differences were found in the results of glycemia and thyroid hormone determinations. Neither the serum levels of triiodothyronine (T3) and of thyroxine (T4) nor the available binding sites in thyroid-hormone carrier proteins were altered by the treatment. This is relevant in view of the very important relationships of thyroid fiinction with body growth and with the endocrine function of the pancreas. In experiments 60 human volunteers received 27.7 mg or 110.8 mg of pure stevioside per day [33]. The author concluded that concentrated Stevia extracts in normal doses to sweeten could be used without restriction by normal persons as well as by diabetics. Pure stevioside and rebaudioside were tested in Sprague-Dawley rats in a concentration of 0.5%, which is still far above the concentrations needed as a sweetener [34]. No differences were found in food and water intake and weight gain. In all of the above cited experiments, no indications of any influence on the bio-availability of nutrients, nor on physiological effects were found. The animal feed used in the experiments did not contain sugar supplements. Therefore, no reduction of weight gain was observed in the experiments as Stevia or stevioside did not substitute for added sugar. Moreover, Stevia and stevioside are used extensively in countries like Paraguay, Brasil, Japan, China, USA etc. and no indications were found of effects on the bioavailability of other nutrients or on other physiological functions of the human body. Stevia and stevioside have been consumed by hundreds of miUions of people and this during a very long period of time, both by adults and by children without giving the smallest indication of any harmful effect. STEVIA, STEVIOSIDE AND SPECIAL GROUPS OF THE POPULATION: NUTRITIONAL SIGNIFICANCE Stevia and stevioside are absolutely safe for diabetics since it is used in minute amounts. Neither stevioside is taken up by our body, nor, due to the p-glycosidic bonds, is stevioside metabohsed by the digestive enzymes

315

from the gastro-intestinal tract. The omission of excessively added sugar in the food is beneficial to diabetics by lowering the blood sugar content [33]. Stevia and stevioside are also safe for phenylketonuria (PKU) patients as no aromatic amino acids are involved. Obese persons might lose weight by the fact that excessive sugar in the food is replaced by Stevia or stevioside. Omitting added sucrose in foods increases the relative proportion of polymeric carbohydrates. This has a beneficial effect for a balanced food intake and for human health [35]. NUTRITIONAL, MICROBIOLOGICAL, TOXICOLOGICAL, ALLERGENICITY PROBLEMS? Stevia and stevioside are unlikely to give rise to nutritional, microbiological, toxicological and/or allergenicity problems. The use of Stevia by eg. Paraguayan, Japanese, Brasilian, American and other people has never led to the demonstration of problems of this kind. In the literature no reports on detrimental effects of either the living plants, or the dried leaves or stevioside can be found ([6]; see also all the references cited above). A literature search could not reveal any publications about a possible allergenic activity of Stevia plants, dried leaves or stevioside itself Kinghom came to the same conclusions: no published reports have appeared that would suggest that extracts of Stevia leaves are immunologically active when taken intemally [36]. Similarly there is no evidence that any of the constituents of Stevia caused allergic contact dermatitis. More information on the use of Stevia in different countries can be found in the List of Food Additives excluding chemical synthetics (Japanese Ministery of Health and Welfare). The consumption of Stevia leaves in 1989 is given for different countries: Japan (2000 tons/year), Brazil (600 tons/year), China (400 tons/year), South Korea (300-400 tons/year), Thailand (100 tons/year), Taiwan (Formosa) (small quantities), Paraguay (150 tons/year) and Argentina (60 tons/year). Even in Belgium, England and The Netherlands several tons of dried leaves have been consumed. In 1989 the (known) world production was estimated at 4100 tons/year. No problems have been published in scientific literature

316

concerning nutritional, micro-biological, toxicological and/or allergenicity problems. STEVIOSIDE AND CARIES Stevioside and rebaudioside A were tested for cariogenicity in albino Sprague-Dawley rats [34]. Sixty rat pups colonised with Streptococcus sobrinus were divided into 4 groups and fed stevioside, rebaudioside A or sucrose added to the cariogenic diet 2000 as follows: group 1: 30% sucrose; group 2: 0.5% stevioside; group 3: 0.5% rebaudioside A and group 4 no addition. All pups received fresh cultures of S. sobrinus in the drinking water on days 18, 19 and 20. All 4 groups were sacrificed after 5 weeks. S. sobrinus counts were made on plaque samples collected from all the molars and cavities were evaluated. There were no differences in food and water intake and weight gains between the 4 groups. Group 1 (30% sucrose) had a very significant higher caries score and S. sobrinus counts than the other 3 groups. There were no significant differences between the stevioside, rebaudioside A and no-addition groups. It was concluded that neither stevioside nor rebaudioside A is cariogenic. Although rather high concentrations of stevioside and Stevia extracts were shown to reduce the growth of some bacteria, the concentrations used for sweetening purposes are rather low. Therefore, the benificial effect of the use of stevioside would rather be due to the substitution of sucrose in the food by a non-cariogenic substance. PURITY OF STEVIOSIDE The purity of the stevioside is not always the same in the various experiments or unknown in some experiments. However, in most experiments the purity of the stevioside used is over 90 % and often over 95%. The impurities occurring in the different stevioside preparations are due to compounds extracted from the Stevia leaves that themselves have been shown to be completely safe. Therefore, the problem of the purity of the stevioside is more a point for academic debate than a matter of concern of human health. Stevioside is safe as well as Stevia leaves and their crude extracts. Therefore, the occurrence of a slightly varying percentage (510%) of residual leaf extract in the stevioside preparation will not be

317

detrimental to human health, and will influence NOEL and ADI only by a few percent. ABBREVIATIONS ADI BW CHL Glc ICH JECFA

= = = = =

LD50 OECD

= =

NOEL PKU Rha

= = =

^

Allowable daily intake Body weight Chinese hamster lung fibroblast cell line Glucose International Council of Harmonisation Joint FAOAVHO Expert Committee on Food Additives Lethal dose at which 50% of the animal die Organisation for economic co-operation and development No effect level Phenylketonuria Rhamnose

ACKNOWLEDGEMENTS. The author acknowledges the "Onder-zoeksraad KULeuven" for grant OT/00/15, the FWO for grant G.Ol 11.01, Drs. Vincent Wargo for proofreading the manuscript and Christine Vergauwen for the « finishing touch ».

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

Pezzuto, J.M.; Compadre, CM.; Swanson, S.M.; Nanayakkara, N.P.D.; Kinghom, A.D.; (1985): Proc. Natl Acac. Set USA, 1985, 82, 2478 - 2482. Procinska, E.; Bridges, B.A.; Hanson, J.R.; Mutagenesis, 1991, 6 (2), 165-167. Hutapea, A.M.; Toskulkao, Ch.; Buddhasukh, D.; Wilairat, P.; Glinsukon, Th.; J. Clin. Biochem. Nutr., 1997, 23, 177-186. Nakayama, K.; Kasahara, D.; Yamamoto, F.; / Food Hygienic Society of Japan, 1986,27,1-8. Pomaret, M.; Lavieille, R.; Bulletin cie la Societe de Chimie biologique, 1931, 13, 1248-1252.

318 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

Suttajit, M.; Vinitketkaumnuen, U.; Meevatee, U.; Buddhasukh, D.; Environmental Health Perspectives Supplements, 1993,101, 53-56. Klongpanichpak, S.; Toskulkao, C.; Temcharoen, P.; Apibal, S.; Glinsukon, T . ; / Med. Assoc. Thailand, 1997, 80, 121-128. Matsui, M.; Matsui, K.; Kawasaki, Y.; Oda, Y.; Noguchi, T.; Kitagawa, Y.; Sawada, M.; Hayashi, M.; Nohmi, T.; Yoshihira, K.; Ishidate, M.; Sofiini, T.; Mutagenesis, 1996,11(6), 573-579. Matsui, M.; Sofuni, T.; Nohmi, T.; Mutagenesis, 1996,11(6), 565-572. Pezzutto, J.; Che, C.-T.; McPherson, D.D.; Zhu, H.-P.; Topgu, G.; Erdelmeier, C.A.J.; Gordell, GA,', Journal of Natural Products, 1991, 54, 1522-1530. Yamada, A.; Ohgaki, S.; Noda, T.; Shimizu, M.; / of the Food Hygienic Society ofJapan, \9%S,26(2), 169-183. Xili, L.; Chengjiany, B.; Eryi, X.; Reiming, S.; Yuengming, W.; Haodong, S.; Zhiyian, H.; Fd Chem. Toxic, 1992, 30(11), 957-965. Toyoda, K.; Matsui, H.; Shoda, T.; Uneyama, C; Takada, K.; Takahashi, M.; Food and Chemical Toxicology, 1997, 35, 597-603. WHO Food Additives (1999) Series 42: Safety evaluation of certain food additives "Stevioside", Genf, 1999,119-143. Hagiwara, A.; Fukushima, S.; Kitaori, M.; Gann, 1984, 75, 763-768. Medon, P.J. ; Pezzuto, J.M.; Hovanec-Brown, J.M. ; Nanayakkara, N.P. ; Soejarto, D.D.; Kamath, S.K.; Kinghom, A.D. (1982); Fed Proc, 1982 , 41, 1568-1982. Toskulkao, C; Chaturat, L.; Temcharoen, P.; Glinsukon, T.; (1997J; Drug and Chemical Toxicology, 1997, 20, 31-44. Mitsuhashi, H.; (1976) Safety of stevioside. In Tama Biochemical Co. Ltd. Report on Safety of Stevia-, 1981, pp. 1-20. Akashi, H.; Yokoyama, Y.; Food Industry, 1975,18, 34-43. Aze, Y.; Toyoda, K.; Imaida, K.; Hayashi, S.; Imazawa, T.; Hayashi, Y.; Takahashi, M.; Bulletin of National Institute of Hygenie Science, 1991, 109, 4854. Yodyingyuad, V.; Bunyawong, S.; Human Reproduction, 1991, 6 (1), 158-165. Mori, N.; Sakanoue, M.; Takcuchi, M.; Shimpo, K.; Tanabe, T.; Shokuhin Eiseiga Ku Zasshi (J FoodHyg. Soc. Jpn), 1981, 22, 409-414. Wasuntarawat, C; Emcharoen, P.; Toskulkao, C; Mungkomkam, P.; Suttajit, M.; Glinsukon, T.; Drug and Chemical Toxicology, 1998, 21, 207-222. Planas, G. M.; Kuc, J.; Science, 1968,162, 1007 Soejarto, D.D.; Compadre, CM.; Medon, P.J.; Kamath, S.K.; Kinghom, A.D.; Economic Botany, 1983, 37, 71-79. Famsworth, N.R.; Cosmet Perfum., 1973, 88, 27-35. Oliveira-Filho, R.M.; Uehara, O.A.; Minett, C.A.S.A., Valle, L.B.S.; Endocrine Effects. Gen. Pharmac, 1989, 20(2), 187-191. Sinchomi, D.; Marcorities, P.; Plantes medicinales etphtytherapie, 1989, 23 (4), 282-287. Melis, M.S.; J Ethnopharmacology, 1999,167, 157-161. Usami, M.; Sakemi, K.; Kawashima, K.; Tsuda, M.; Ohno, U.; Eisei Shikenjo Hokolu - Bulletin of National Institute of Hygienic Sciences, 1995, 113, 31-35.

319 [31] [32] [33] [34] [35] [36]

Melis, M.S.; Brazilian J. Medical and Biological Research, 1996, 29 (5), 669675. Wood, D.J.; Lirette, A.; Crober, D.C.; Ju-Hy; Can. J. Animal Sc, 1996, 76(2), 267-269. Boeckh-Haebisch, E.M.A.; Arq. Biol TecnoL; 1992, 35(2), 299314. Das, S.; Das, A.K.; Murphy, R.A.; Punwani, I.C.; Nasution, M.P.; Kinghom, A.D.; Caries Res., 1992,26, 363-366. Anonymous in Voedingsaanbevelingen voor Belgie, (D/1996/7795/12), De Backer, G., Ed.; Zevecotestraat 43, B-9830 Sint-Martens-Latem, pp 77. Kinghom, A.D.; Food Ingredient Safety Review: Stevia rebaudiana leaves. Herb Research Foundation, USA, 1992.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

321

ABSCISIC ACID ANALOGS FOR PROBING THE MECHANISM OF ABSCISIC ACID RECEPTION AND INACTIVATION YASUSHI TODOROKr AND NOBUHIRO HIRAI' ^Department of Applied Biological Chemistry, Faculty ofAgriculture, Shizuoka University, Shizuoka 422-8529, Japan ^Division of Applied Life Sciences, Graduate School ofAgriculture, Kyoto University, Kyoto 606-8502, Japan ABSTRACT: More than 100 abscisic acid (ABA) analogs have been reported so far. Some were synthesized to clarify structure-activity relationships, and others were developed as tools for investigating the molecular mechanism of ABA action. These analogs, especially those that can be useful tools for studying ABA reception and catabolic inactivation, are summarized together with their design concepts, structural properties and bioactivities.

INTRODUCTION Abscisic acid [(r*S)-(+)-ABA (1), Fig. (1)] is the primary hormone that induces adaptive reactions to protect plants from environmental stresses such as desiccation and freezing [1], The mechanism of action of ABA has been investigated at the molecular, cellular and whole plant levels since ABA was identified as an abscission and dormancy factor in the 1960's [1-4]. An understanding of the whole picture is important not only as a point of academic interest, but also to promote the agricultural application of ABA. Biosynthesis and signal transduction have been extensively investigated at the molecular level over the past decade. Several enzymes have been identified at the gene level [5-7] to support an indirect carotenoid pathway from zeaxanthin via xanthoxine and abscisic aldehyde, and several mediators have been characterized as ABA signal

CO2H Fig. (1). Structure of ABA (1)

322

transduction elements [8,9]. On the other hand, the initial perception and metabolic inactivation of ABA by target cells remain obscure at the molecular level. ABA receptors are believed to be membrane proteins [10,11], and a cataboHc enzyme that catalyzes the first step of ABA catabolism is likely to be a microsomal cytochrome P-450 monooxygenase [12]. However, the corresponding proteins and genes have not yet been isolated. The application of ABA to agricultural production has not been studied in detail. The role of ABA as a stress hormone could greatly contribute to agriculture if its application to plants in the field could evoke the same responses as endogenous ABA. How^ever, the commercial application of ABA has been limited because of its shortlived activity and the difficulty of identifying an optimal timing and appropriate concentration for its application to plants [13]. The development of an analog with strong bioactivity or the clarification of ABA action at the molecular level may lead to a breakthrough in the agricultural application of ABA. More than 100 ABA analogs have been synthesized in an eflFort to find compounds with strong ABA activity, to clarify structure-activity relationships, or for use as tools for investigating the molecular mechanism of ABA action [1,4,14]. ABA and its analogs can be useful tools for probing the mechanism of ABA action, especially ABA reception and catabolic inactivation, since ABA itself binds directly to a receptor and catabolic enzyme. The structures and activities of the analogs that are referred in this review are summarized in Table 1. PROBING THE MECHANISM OF ABA RECEPTION No ABA-binding protein has yet been isolated. Homberg and Weiler published a report on ABA-binding proteins in 1984 [15]. They demonstrated that ABA photoinductively cross-links in guard cell protoplasts with proteins located at the outward-facing plasma membrane. However, this report was not confirmed. In 1994, microinjection of ABA into cells was performed by several groups to determine the primary sites of ABA reception [16-19]. However, conflicting results were obtained; Allan et al and Schwartz et al suggested an intracellular reception site, while Anderson et al and Gilroy e/ al suggested an extracellular reception site. In 1998, using ABA-protein conjugates as aflfinity probes, Pedron et al showed that ABA-binding proteins of Arabidopsis thaliana were in the microsomal protein fraction [20].

323 Table 1.

No.

Structures and Activities of ABA Analogs Structure

Activity*

Ref.

+++

1-7

++

23

+ - ++

24

++

25

+++

26

CO2H

Nr ^o

CO2H

CO2H

°V^°^\^^^*^ ^^Tf^'N^^*^^

V^yv"-

. - ^ ^ ^ - ^ ^

^^'"

CO2H

27

+ - ++

28

324 Table 1.

Structures and Activities of ABA Analogs (Continued) Structure Activity*

No.

Ref.

OH L OH

fluoroethane; and ethene —> fluoroethene. This substitution reaction by fluorine was more exoergic for ethene than for ethane by ca. 2 kcal/mol. This agreed with the observation that 8'-H0ABA was better stabilized than PA by the C-3' fluorine. This stabilization may have been due to delocalization of electrons or less nonbonded repulsion in sp^ hybridization compared to sp^ The isomerization of S'-HOABA and 37 was observed as a first-order reaction in which the rate was proportional only to the concentration of the 8'-hydroxyl compound. As the pH and temperature increased, the reaction proceeded more rapidly. At 25°C, the half-life of 8'-H0iABA was 30 hr at pH 3, 4 hr at pH 7, and shorter than 1 min at pH 10; that is, 8'-H0ABA was isomerized to PA more rapidly at pH 10 than at pH 3 by a factor of 2,000. The temperature dependence of the rate was greater under alkaline conditions than under acidic conditions. The Arrhenius plots of the rate constants gave the activation energies of Arrhenius and frequency factors, which were converted to the kinetic parameters, i.e. the activation enthalpy {AH^), activation entropy (AS^) and activation free energy {AG^) (Table 4). The AH^ was higher under alkaline conditions than under acidic conditions, whereas the AS^ was negative under all conditions and its absolute value was larger under the acidic as compared to the alkaline conditions. This indicated that the slow isomerization under acidic conditions was dependent on the negative, large AS^. The transition structure under acidic conditions may be so solvated as to cause a decrease in AH^ and a rise in the absolute value oi A^. Alternatively, the reaction under acidic conditions may be associated with a concerted mechanism.

352 Table 4.

Rate Constants (k) and Activation Energies of the Isomerization at 25°C 24

25

37 -> 38 (cal/mol deg)

k (/s)

AG^

AH^

(kcal/mol)

(kcal/mol)

(cal/mol deg)

10.6

-46.9

4.8 X 10-«

27.6

16.2

-39.8

11.2

-44.3

9.0 X 10-«

27.1

17.8

-33.3

24.2

12.2

-40.3

1.6x10"'

26.8

19.9

-25.3

1.8x10-^

23.9

12.2

-39.3

5.0 X 10-''

26.1

20.5

-20.9

5.5 X 10-^

23.3

12.4

-36.4

3.5 X lO"'^

24.9

20.0

-18.6

1.7x10-^

22.6

17.0

-18.9

1.6x10"^

24.1

21.6

-10.2

9

2.5 X 10-^

21.0

18.9

-7.1

3.7x10-^

23.6

23.5

-2.4

10

1.1x10-2

19.9

16.7

-10.9

2.0 X 10"^

21.2

20.2

-5.3

k

AG^

(/s)

(kcal/mol)

(kcal/mol)

6.8 X lO*^

24.5

4

8.7 X 10-*'

24.4

5

1.1x10-^

6 7 8

pH 3

AH^

AS^

AS^

The isomerization of 37 was slower than that of 8'-H0ABA by a factor of 10-100 under acidic conditions and by a factor of 3-10 under alkaline conditions. This indicated that the introduction of a fluorine at C-3' stabilized 8'-H0ABA kinetically in addition to thermodynamically. The pH-dependence of the isomerization rate of 37 was similar to that of 8'-H0ABA; at 25°C, the reaction rate constant at pH 10 was 50,000-fold larger than that at pH 3. At the same pH, the zl//* of the isomerization of 37 was higher than that of the isomerization of S'-HO AB A, while the absolute value of the negative AS^ of the isomerization of 37 was smaller than that of S'-HOABA. This meant that the slow reaction of 37 was caused by an increase of the AH^, unlike that in low pH solution. This suggested a decrease in solvation at the transition state of 37/38 isomerization. However, a better explanation would be less interaction between the 2'-carbon and S'-oxygen of the transition state of 37/38 isomerization compared to S'-HOABA/PA isomerization. This was examined by computer-assisted calculations for the model compounds. Introducing a fluorine into the conjugated system should have a large effect on the charge distribution, as mentioned above. The increase in the negative charge at C-2' will result in the larger filled-orbital-filledorbital repulsion for a nucleophile. The repulsion between C-2' and O8' can be represented by the small overlap population which is calculated in the transition state, so we simulated the nucleophilic addition of a methoxide to models la and 36a [Fig. (12)]. The calculated overlap population, 0.105, between the 2'-carbon of la and the oxygen of methoxide in the transition-state structure was smaller than that for 36a (0.080), as expected. These results suggested that the slow

353

isomerization of 37 is dependent on the electron-donating effect on C-2' by the 3'-fluorine via C-3'. In spite of the stability of 37 compared to 8'-H0ABA, the biological activity of 36 was almost equal to that of ABA in any bioassays. This means that 37 is not potent, or otherwise isomerization to 38 is accelerated by a specific enzyme, cyclase. Indeed, biological activity of 37 was lower by a factor of 5 than ABA. These findings suggest that ABA is inactivated step by step with the 8'-hydroxylation, isomerization and 4'-reduction. Other y-Halogenated ABA Analogs. We synthesized the 3'halogenated ABA analogs with a chlorine, bromine and iodine at C-3' for comparison to the effect of fluorine [91]. The effect of 3'-halogenation on the biological activity was similar among all of the halogens. The rate of decrease rate in the medium of rice cell cultures was similar among ABA, 36 and 3'-chloro-ABA (40), while 3'-bromo-ABA (41) and 3'-iodo-ABA (42) decreased a little more rapidly than ABA. Halogens other than fluorine are not effective for increasing the electron density at C-2'. Despite this, however, the apparent effects on bioactivity and metabolism were not different from those of fluorine, suggesting that 3'fluorine has a weak kinetic effect on isomerization. Hexadeuterated ABA. Experiments that involved the feeding of [3',5,7^HgJ-ABA to tomato shoots revealed that the C~3' protonation in the isomerization to PA took place from the a-face (^/-face) where the chemical protonation-deprotonation is difficult [92]. The isomerization to PA may be catalyzed enzymatically, although the enzyme involved has not yet been identified. 2\3''DihydrO'ABA. Saturation of the 2'-double bond of ABA has little effect on ABA activity if C-7' is cis to the side-chain [93]. However, the metabolism of this analog is significant because the 2'-double bond is indispensable for isomerization into PA. Lamb et al investigated the metabolism of 2',3'-dihydro-ABA (43) in a cell suspension culture of bromegrass [94]. Analog 43 was oxidized to 8'-hydroxylated compound 44 and its hemi-ketal 45. A PA-type structure was not formed due to the absence of the 2'-double bond. The open (44) and cyclized (45) forms existed in equilibrium in a 1:3 ratio. Metabolite 44/45 was inactive at inducing freezing tolerance in bromegrass cells, suggesting that 8'-hydroxylation decreases its the bioactivity even if it is not converted to a PA-type bicyclic metabolite.

354

Catabolic Inactivation Initiated by Conjugation ABA and its metabolites are conjugated at the 1-carboxyl, I'-hydroxyl, 8'hydroxyl, or 4'-hydroxyi groups with glucose in plants [Figure (10)] [1,7]. The conjugated forms of ABA are the 1-0-glucosyl ester (ABA-GE, 46) and the I'-O-glucoside (ABA-GS, 47). Are these conjugates the inactivated metabolites or transport forms? A glucosyltransferase which catalyzes the transfer of glucose from UDP-glucose to ABA with the formation of ABA-GE has been purified [95]. Its optimum pH was 5.0 and it was located in vacuoles [96]. This means that the glucose conjugate represents irreversible compartmentation. Indeed, the amount of conjugated ABA gradually increased with age in the leaves and represented almost 95% of the total ABA pool in the oldest senescent leaf [97]. However, it has also been reported that ABA conjugates are found in the xylem sap of stressed plants [98-102]. Dietz et al. reported that the extracellular p-glucosidase activity in barley leaves is involved in the hydrolysis of ABA-GE [103]. This supports the hypothesis that the glucose conjugate is a form of ABA that is suitable for long-distance transport. The functions of other metabolite conjugates are unknown. No probe for studying the mechanism of such conjugation has yet been reported. Photoisomerization Photoisomerization of ABA to the (2£)-isomer (48), which was extensively investigated by Plancher [104], and Brabham and Biggs [105], is caused by natural sunlight and by UV irradiation to give a 1:1 mixture of (22)- and (2£)-isomers. The (2£)-isomer is inactive in all of the bioassays studied thus far. This means that the orientation of the 1carboxyl group is significant for ABA bioactivity. This inactivation is not a biotransformation, and no probes have been reported to study the mechanism. Instead, ABA analogs that are resistant to isomerization have been reported as follows. ABA Analogs Resistant to Photoisomerization Fixed by Covalent Bonds. Fixing the (2Z)-configuration by covalent bonds is a reliable method for preventing photoisomerization. Analog

355

49, which has a benzene ring in its side-chain was synthesized based on this strategy [106]. However, analog 49 showed ABA-like activity only at a high concentration, probably because of the steric effect of the benzene ring. Analog 50, which has a y-lactone in its side-chain was also inactive, also probably due to the absence of the free acid [107]. Raising the Energy Level of n-n^, Cis-trans photoisomerization of the C-C double bond by light is caused by the TI-TT* transition. Therefore, raising the energy level of the transition state at the C-2 double bond of ABA may protect against isomerization to the (2£)-configuration. Kim et al synthesized 2-fluoro-ABA (51) as a potential photo-stable analog [108]. This analog was theoretically estimated to be stable against photoisomerization based on the torsional energy of the groimd-state structure characterized by the dihedral angle C1-C2-C3-C4 (0 to 180 degrees). However, since cis-trans photoisomerization occurs at the excited state, the calculations must be performed at the excited state. No subsequent report has been made. ABBREVIATIONS ABA BSA NOE NOESY 8'-H0ABA PA DPA Alf AS^ AG^ ABA-GE ABA-GS

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

Abscisic acid Bovine serum albumin Nuclear Overhauser effect Nuclear Overhauser enhancement spectroscopy 8'-Hydroxyabscisic acid Phaseic acid Dihydrophaseic acid activation enthalpy activation entropy activation free energy 1 -O-glucosyl ester of ABA 1 '-0-glucoside of ABA

ACKNOWLEDGEMENTS The authors gratefully acknowledge a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; and from the Ministry of Agriculture, Forestry and Fisheries of Japan. We also thank Professor Junichi Ueda of the University of Osaka Prefecture for supplying lettuce and barley seeds and the spiderwort.

356

Professor Hiroshi Okumoto of Kyoto University for supplying rice seeds, Professor Toshiaki Mitsui of Niigata University for supplying rice cells, and Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan for the gift of ABA.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

361

CHIRAL MONOTERPENOIDS IN PLANTS ENANTIOSELECTIVE CHROMATOGRAPHIC ANALYSIS, AND THEIR BIOACTIVITY MONIKA A S Z T E M B O R S K A ' AND J.RENATA OCHOCKA^ Institute of PhysicalChemistry, Polish Academy ofSciences, Kasprzaka 44/52, 01-224 Warsaw. Poland, Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland ABSTRACT: Monoterpenoids are the components of essential oils, which are produced and accumulated, in large amounts by plants from certain families, including Labiatae, Pinaceae, Cupressaceae, Umbelliferae and Rutaceae. They are of special interest because of their industrial applications and on account of their chemical properties and biological activity. Monoterpenic composition can be useful in genetic and chemotaxonomic studies of coniferous species. The first reports concerning the enantiomeric separations of monoterpenoids in essential oils were published in the early nineties. These studies were concerned with the elaboration of simple analytical methods adequate for chiral separations in these complicated natural product mixtures. The enantiomeric composition of certain monoterpenoids in essential oils is now the subject of several studies. Differences in the relative amounts of enantiomers of some monoterpenoids existing between various species, as well as within one, have thus been reported. Owing to the great numbers of possible isomers within each group of monoterpenoids, their analysis presents great difficulties, which are growing significantly when a chiral discrimination is required. In this article the gas-chromatographic methods adequate for an analysis of chiral monoterpenoids are presented. Recent results on the enantiomeric composition of monoterpenoids from different species of plants are discussed. The bioactivity of selected chiral monoterpenoids is also described.

ISOMERISM OF MONOTERPENOIDS Monoterpenoids belonging to the group of terpenoids are compounds very often found in plants. Their role in Nature is not fiilly explained. It is supposed that they play a defensive role for plants. They are widely used in the perfumery and cosmetics industry, as flavour and fragrance materials in the food industry as pharmacological substances and many others.

362

A general review on the structures and biosynthesis of these groups of compounds has been pubUshed [1]. Among the group of monoterpenes C10H16 and their oxygenated derivatives a lot of isomers can be found, namely: 1. Constitutional isomers (the same molecular formula but a different constitution). Constitutional isomers can be subdivided according to the differences in: a) the chain e.g. myrcene and ocymene

myrcene

ocymene

b) the ring e.g. a-phellandrene, a-terpinene and y-terpinene

r ^ ^

a-phellandrene

Y-terpinene

a-terpmene

c) the functional groups e.g. phellandral and perillyl alcohol CHO

phellandral

CH,OH

perillyl alcohol

363

d) the position of the functional group e.g. menthone and carvomenthone

menthone

carvomenthone

2. Stereoisomers (the same constitution but a different space orientation) Stereoisomers can be divided into: a) diastereoisomers b) enantiomers Enantiomers are in the relation one to another as mirror images, which are not superimposable. They are called chiral compounds from the Greek word xsipOG - hand, because the relation between them is like that between the right and the left hand. In an achiral environment all the physicochemical properties of the enantiomers of a given chiral compound are the same. They can differ only in a chiral environment; they have an opposite coefficient of polarised light rotation. The rest of stereoisomers which are not mirror images are diastereoisomers. The physicochemical properties of diastereoisomers are different. They differ in potential energy of molecules, in dipole moments, melting and boiling points and the coefficient of light refraction. The living cells are characterised by a high configurational homogeneity of primary metabolites - proteins and nucleic acids. The proteins are built from L-aminoacids and nucleic acids containing Dsugars. In the group of secondary metabolites such as monoterpenoids homogeneity of so high a degree is not observed. Different species of plants can produce a given chiral terpenic compound in the (+) and in the (-)-form. The same species of plant can synthesize variable proportions of a chiral monoterpenoid. The reasons for this phenomenon are not clear. The consequences of chirality are very important for the human and all the living organisms. As our receptors are built from chiral proteins, two

364

enantiomers of a chiral substance so similar from the chemical structure point of view can differ substantially in their biological activity. Two enantiomers of a monoterpenoid can give a different olfactometric impression: • (+)-carvone - reminiscent of spearmint, (-)-carvone - reminiscent of dill seeds • (+)-a-terpineol - flowery, sweet, lilac, (-)-a-terpineol reminiscent of cold pipe [2]. Interesting examples of diastereomers and enantiomers of monoterpenoids are menthones and menthols.

(+)-menthone

(+)-isomenthone

OH

(+)-menthol

(-)-neomenthol

(+)-isomenthol

(+)-neoisomenthol

Menthone can exist in two diastereomeric forms: cis- and trans- known as isomenthone and menthone respectively. Both of these forms are chiral and can be found in two enantiomeric forms (+) and (-). When menthone is reduced to secondary alcohol - menthol, the new asymmetry centre appears and menthol with three asymmetric carbon atoms can be found in eight optically active forms. Thus in the group of eight stereoisomers of

365

menthol, four diastereomeric pairs of enantiomers exist known as: (+/-)menthols, (4-/-)-neomenthols, (+/-)-isomenthols and (+/-)-neoisomenthols. ANALYSIS OF CHIRAL CHROMATOGRAPHY

MONOTERPENOIDS

BY

GAS

The main method of analysis of monoterpenoids in essential oils is a capillary gas chromatography. Due to the high volatility of monoterpenoids and the high speed and efficiency of the capillary gaschromatography it is applied nowadays as the most universal method for a qualitative and quantitative analysis of monoterpenoids mixtures. For the resolution of enantiomers, very often existing in the group of monoterpenoids a modification of the method by adding a homochiral selector was necessary to produce a chiral environment. Up till now the most universal and efficient chiral selectors used for GC enantioseparation have appeared to be cyclodextrins. Cyclodextrins Cyclodextrins were discovered at the end of XIX century by Villiers (1891) but they were described as cyclic oligosaccharides by Schardinger in 1904. They are prepared by enzymatic degradation of starch. The enzyme cyclodextrin glucanosyltransferase (CGT) from Bacillus macerans. Bacillus megaterium or other bacterial strains cut the starch helix and join both ends of such destruct forming a cyclic compound. Because enzymes are not very specific, the obtained mixture contains cyclodextrins from 6 to 12 glucose units. The main fractions are a-, p- and y-cyclodextrins which correspond to 6, 7 and 8 glucose units. Cyclodextrins are cyclic compoimds built from (+)-a-D-glucose units connected with an oxygen bridge a-1,4. The overall shape of cyclodextrins is that of a truncated cone with the wider side rounded by a secondary hydroxyl group and the narrow side with a primary hydroxyl group. The number of glucose units decide the size and shape of the cavity of a cyclodextrin. The hydrogen atoms and free electron pairs of glycosidic bond are directed into the cavity of cyclodextrin while hydroxyl groups are directed outside the cavity. As a

366

result of such geometry the interior part of a cyclodextrin is relatively hydrophobic in comparison to water while the outer surface is hydrophilic. OH

OH O-pr^O „ ^ > ^ OH o

OH HO

(OH

OH O

HO

OH

O HO

a-cyclodextrin

(3-cyclodextrin

HO

O

HO

y-cyclodextrin

The main physicochemical properties of a-, p- and y-cyclodextrins are summarized in Table I. The amazingly very low solubility of p-CD in water (about 10 times lower than the solubility of a- and y-CD) is explained by its relatively rigid structure and resulting from this high ability to crystallise [3] or by the unfavourable 7-fold symmetry of p-CD leading to a perturbation of the water structure [4].

367 Physicochemical properties of cyclodextrins

Table I

a

P

Y

973

1135

1297

6

7

8

Optical rotation [a]D25

+150.5

+162.5

+177.4

Cavity volume [nm^]

0.176

0.346

0.510

551

572

540

6

11

17

14.50

1.85

23.20

Characteristics Molecular weight Number of glucose units

Melting and decomposition point [K] Number of 1 (in solution)

included

water

molecules

Solubility in water, temp. 25°C [g/100 cm^]

The physicochemical properties of CDs can be modified by their dervatization. Because they possess many hydroxyl groups they can be relatively easily chemically modified. Derivatization can modify their solubility, hydrophobicity, melting point and inclusion properties. For example the solubility of heptakis-(2,3,6-tri-0-methyl)-P-CD in water is 7 g/100 ml what is almost 10 times higher than that of p-CD. The most important property of cyclodextrins is their great ability to form stereoselective inclusion complexes with a variety of compounds. The inclusion complex is formed when the guest molecule, or at least its part, enters the cavity of cyclodextrin-host and covalent bondings are not formed. The main factors responsible for the stability of such a complex are the geometrical fitting of the guest molecule to the cyclodextrin cavity, the polarity of the guest, the temperature and the solvent. Thanks to their properties cyclodextrins can be applied for the resolution of many types of isomers: constitutional, diastereomers and since they are homochiral themselves they may also separate enantiomers. As the processes of complexation by cyclodextrins in solutions are: • not only selective but also stereoselective, • reversible and hysteresis is not observed, • equilibration processes are very fast,

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they were applied for the modification of many chromatographic sytems to obtain separation of isomeric compounds which are not separable in the classical way. Cyclodextrins were used for the modification of such chromatographic techniques as: gas chromatography, classical liquid chromatography, high performance liquid chromatography, thin layer chromatography and many hyphenated techniques. Since in the study of enantiomeric composition of monoterpenes gas chromatography modified with cyclodextrins is the main analytical tool, this method will be more widely discussed later on. Application of cyclodextrins in gas chromatographic analysis of monoterpenoids The first separations of enantiomers in GC on cyclodextrin modified column were carried out by Sybilska et al. in 1983 [5]. They applied a formamide solution of a-cyclodextrin as a stationary phase in the classical packed column. The column allowed an efficient separation of chiral monoterpenes - a- and p-pinenes into enantiomers. This system of using CDs in GC is characterised by obtaining high enantioselectivity factors, so enantioseparation is still possible for receiving not very efficient packed columns. Unfortunately, the columns appeared to be not very stable at higher temperatures. At the end of the eighties some derivatives of cyclodextrins were applied as a chiral stationary phase for capillary gas chromatography. Konig at al applied perpenthylated-p-cyclodextrin, which was liquid in room temperature, as a chiral stationary phase for the sepaiation of some aldose derivatives [6] and aminoacids, aminoalkohols and hydroxyacids [7]. At the same time Schurig et al. applied dissolved in polisiloxane OV1701 permethylated-p-cyclodextrin in capillary GC for the separation of racemic mixtures of many substances [8, 9]. Armstrong et al obtained derivatives of CDs of medium polarity: 2,6-di-0-penthyl-3-0trifluoroacetyl- and 0-(S)-2-hydroxypropyl-a and p-cyclodextrin. Those CDs applied as chiral stationary phases in capillary GC allowed the separation of more than 100 compounds such as: amines, aminoalkohols, epoxides, sugars, esters, ketones etc. [10, 11]. The next step in the aim to improve stability of the columns was immobilisation of cyclodextrins chemically anchoring them to a polysiloxane backbone [12, 13].

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Although capillary columns covered with cyclodextrins or their solutions were a good tool for the separation of enantiomers of single monoterpenes, the analysis of more complicated multicomponent mixtures as essential oils was still a problem. It was difficult to avoid overlapping of the peaks. When the separation of chiral molecules into enantiomers was possible, many other isomers can not be separated. The solution for this problem was the elaboration of enantioselective multidimensional gas chromatography [14]. This method requires a double-oven system with two independent temperature controls two flame ionization detectors and a "live switching" coupling piece. At an optical pneumatic adjustment of this system definite fractions or single components eluted from the preseparation non chiral column are selectively transferred onto the main chiral column. The review articles dealing with the application of cyclodextrin derivatives for a direct gas chromatographic separation of optically active components in the essential oils have been published by Bicchi et al. [15, 16]. ENANTIOMERIC COMPOSITION OF MONOTERPENES IN SELECTED PLANT SPECIES The knowledge of enantiomeric composition of monoterpenes in essential oils is important fi^om a scientific as well as from a practical point of view. It can give information related to chemotaxonomy, biogenesis and biosynthetic relations between compounds. It can also be useful in quality control genuines of essential oils. In some cases it can also answer the questions of applied extraction techniques and geographic origin of the extracted plant. The first reports concerning the enantiomeric composition of essential oils were published at the beginning of the nineties. The contents of chiral constituents in different commercial essential oils were investigated on a capillary GC column modified wdth derivatives of cyclodextrins [17] and on packed columns modified with a native a-cyclodextrin [18]. It was connected with the elaboration of gas chromatography columns modified with cyclodextrins. The papers dealing with the study of a chiral composition can be divided into a few groups. Depending on the complexity of the given

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essential oil or the contents of a particular fraction (monoterpenic hydrocarbons and oxygenated monoterpenoids) the authors study the enantiomeric ratio of all chiral components or limit their investigations to one or a few chosen chiral monoterpenoids. Monoterpenic Hydrocarbons Monoterpenic hydrocarbons sometimes occur in essential oils in considerable amounts. They are often used as a starting material for a synthesis of flavor or fragrance compounds although they themselves contribute relatively little to the fragrance and aroma. Enantiomeric composition of monoterpenic hydrocarbons in conifers Monoterpenic hydrocarbons are the main fraction of essential oils from conifer trees. The composition of monoterpenes in essential oils in the coniferous can be usefiil in the genetic and taxonomic studies. Monoterpenes are used as genetic markers of clones and varietes. They can be applied for the identification of clones, hybrids and subspecies. Monoterpenic hydrocarbons play an important role in the chemical communication between coniferous trees and forest insects. Complicated relationships between the activity of a pheromone and its enantiomeric composition have been revealed. Some forest insects respond only to one enantiomer or to specific proportions of two enantiomers. Sometimes an inactive enantiomer blocks its active antipode. For example monoterpenic alcohol (-)-terpinen-4-ol of enantiomeric purity >99% is aggregating pheromone of the beetle Polygraphus poligraphus while a racemic composition of this monoterpenoid failed to attract the beetles [19]. The enantiomeric composition of monoterpenic hydrocarbons has been studied in laboratory prepared essential oils from: Pinus sylvestris, [2022], Pinus pence [23], Picea abies [24, 25] and Juniperus communis [21,26, 27]. Seven major chiral monoterpenes have been investigated in different plant tissues. The structural formulas of these compounds are presented below.

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(+)-a-pinene

(-)-a-pinene

(+)-P-pinene

(-)-P-pinene

(+)-camphene

(-)-camphene

(+)-limonene

(-)-limonene

(+)-sabinene

(-)-sabinene

(+)-P-phellandrene

(-)-P-phellandrene

^ ' (+)-3-carene

(-)-3-carene

Large differences in the enantiomeric ratio of monoterpenes have been found between species, between individuals of one species as well as between different tissues of one specimen in studied samples.

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From the investigated chiral monoterpenes 3-carene has been detected in only (4-)-form. P-pinene and p-phellandrene has been identified as (-)p-pinene and (+)-P-phellandrene of a high enantiomeric purity. A small amount of their optical antipodes has also been detected in some samples. The prevalence of (-)-enantiomer or close to racemic composition of limonene has been found in Pinus and Picea species while in Juniperus (+)-limonene is dominant. Camphene has been found in most of the examinated samples as (-) enantiomer of not a very high optical purity over its optical antipode. In some samples from xylem of Pinus (+)-camphene dominate. The most variable compounds in their enantiomeric ratio were apinene and sabinene. An excess of (+) or (-) enantiomer as well as an almost racemic composition of these monoterpenes were observed. In the investigated Pinus samples (-)-sabinene was found with a high enantiopurity, in contrast in Juniper samples only (+)-sabinene was detected. In Picea samples depending on kind of tissue sabinene was found in needles in (+) form, while in flowers as (-) enantiomer. In other tissues the enantiomeric ratio of sabinene was variable. In the case of a-pinene its enantiomeric ratio was different between individuals and between tissues of one individual and it was difficult to find any factor responsible for such a high variability. Enantiomeric composition ofmonoterpenic hydrocarbons in Rutaceae The citrus oils are important because of their practical applications as flavour and fragrance materials in the food and cosmetic industries. In comparison with essential oils from conifers the enantiomeric ratio of monoterpenic hydrocarbons is more stable in citrus oils and thus it can give information about their origin, extraction techniques and genuineness of the oils. The main component of monoterpenes in those types of oils is (+)-limonene of a high enantiomeric purity for most of the citrus oils. The enantiomeric ratio of limonene was used to discriminate genuine mandarin and lemon oils from the reconstituted ones [28, 29]. The enantiomeric excess of (+)-limonene in the lemon peel has been found between 97.1 and 97.4% [30]. Mondello et al. found small differences in the enantiomeric composition of monoterpenes between two varieties of lime oils Citrus aurantifolia Swingle (Key lime) and Citrus latifolia Tanaka (Persian lime) [31]. The enantiomeric ratio of limonene was the

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same in cold-pressed essential oils from both the varieties and was about 97% of (+)-limonene. The content of (-)-limonene increased to about 6% when the oils were distilled. Sabinene and P-pinene were found in the prevalence of (-) enantiomers but in the case of Key lime oils the content of (4-)-p-pinene was 3.5% and (+)-sabinene was 15%, while for Persian lime oils the contents of those enantiomers increased to 10% for (+)-Ppinene and to about 20% for (+)-sabinene. Mosandl et al. [32] found in grapefruit oil (+)-limonene and (+)-apinene of high enantiopurity, and only traces of (-) enantiomers of these compounds were detected. In contrast to conifer oils p-pinene has been found in the prevalence of (+) enantiomer (63-66%). Similar prevalence of (+)-P-pinene has been found for Uruguayan Clementine oils (Citrus Clementine) [33]. In the same oils the authors identified also (+)-sabinene of enantiopurity above 97% and (+)-limonene of enantiopurity 99.4%. Evers et al. revealed that (-)-3-carene can be used for the identification of sweet orange oil because this enantiomer has only been found in traces in bitter orange oil and is not present in lemon and mandarin oil [34]. Juchelka et al. found interesting differences in the distribution of enantiomers of a-pinene and limonene between neroli and petitgrain oils {Citrus auranhtium ssp. amara) [35]. These two oils are produced from different morphological parts of bitter orange tree - neroli oil from fresh blossoms and petitgrain oil from leaves and twigs. In neroli oil a-pinene has been detected in a high prevalence of (-)-enantiomer while in petitgrain oil (+)-enantiomer dominates. (+)-Limonene is a dominant enantiomer in both oils but while in neroli oil the content of (-)enantiomer is small (less than 7%) in petitgrain oil it increases above 30%. p-Pinene has been found in both oils as (-)-enantiomer of a high enantiomeric purity. Oxygenated monoterpenoids Oxygenated monoterpenoids (alcohols, aldehydes, ketones and other oxygen-containing compounds) are flavour determining components of essential oils and more important from practical stspects than monoterpenic hydrocarbons. The number of oxygenated monoterpenoids is much higher than corresponding hydrocarbons but contrary to a hydrocarbon fraction in the oxygenated fraction of given essential oils usually only one or a few

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components are dominant and important. As a result the papers devoted to the enantiomeric composition of this kinds of monoterpenoids often deal with the chiral separation of a single component because it is the most characteristic for the given essential oil or because it is important from another reason e.g. for the study of adulteration. For such reasons this part of paper is classified according to the structural similarities of the given oxygenated monoterpenoids. Linalool and linalyl acetate H3COC

(-+-)-linalool

(+)-linalyl acetate

H3COC

(-)-linalyl acetate

Linalool and linalyl acetate are the intensively studied chiral monoterpenoids. Their enantiomeric ratio had been investigated in the precious bergamot oil [36-38], lavender oils [39], and many other plants used as fragrance and flavour materials mainly from Labiatae and Rutaceae families [40-42]. In all the investigated samples excluding one linalyl acetate has been found in (R)-(-)-form of a high enantiomeric purity. The only exception was cardamon {Elettaria cardamonum) containing pure {S)-(+)-linalyl acetate. In the case of linalool the enantiomeric ratio was more variable between plants. In majority of investigated plants (R)-(-)-linalool dominates. The plants of the highest optical purity of (-)-linalool have been found lavender {Lavandula angustifolia), bergamot {Citrus auranthium Bergamia), thyme {Thymus vulgaris) and basil {Ocimum basilicum). In a group of plants in which (S)-(+)-linalool dominates a practically pure enantiomer has been revealed in Robinia flowers {Robinia pseudoacacia).

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An interesting variability is observed in plants from Rutaceae family. In bergamot {Citrus auranthium Bergamia), neroli and petitgrain {Citrus auranhtium ssp. amara), and lime oils {Citrus aurantifolia) the prevalence of (-)-linalool has been found. In orange {Citrus sinensis) and tangerine {Citrus reticulata) (+)-linalool is the dominant enantiomer, while in lemon {Citrus limon) and grapefruit {Citrus paradisi) the enantiomeric ratio of linalool is close to racemate [31, 40]. Menthone, isomenthone andpulegone CH,

CH,

o CH3

(+)-menthone

(-)-menthone

H3C

CH3

H3C

CH3

(+)-isomenthone (-)-isomenthone

CH,

CH,

CH3

(+)-pulegone

H3C

CH3

(-)-pulegone

Those monocyclic monoterpenoids are characteristic mainly for various mentha oils. (+)-Pulegone of a high enantiomeric purity has been found in different varieties of Mentha piperita, Mentha longifolia, Mentha pulegium and Mentha sylvestris, while (-)-pulegone is present in buchu leaf oils {Barosma crenulata and Barosma betulina) [43, 44]. For the same subspecies of Mentha they also found almost enantiomerically pure (-)-menthone and (+)-isomenthone. Reversely in Geranium Bourbon {Pelargonium graveolens) oils practically pure enantiomer of (-)-isomenthone has been accompanied with (+)-menthone [37,45].

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Camphor and borneol CH3

(+)-camphor

H3C

H,C

CH.

(-)-camphor

HX

(+)-bomeol

(-)-borneol

Camphor and borneol are widespread bicyclic monoterpenoids in plants. The enantiomeric ratio of camphor has been investigated in some herbs and spices [39, 46-48]. Ravid et al. found a high variability of enantiomeric ratio of camphor and borneol in Salvia officinalis. An excess of (+) as well as (-) enantiomers has been observed. No characteristic distribution of those two chiral monoterpenoids has been found in the investigated Salvia officinalis samples. In Salvia sclarea, Salvia glutinosa and Salvia fi'uticosa an excess of (+)-camphor has been observed. (+)-Camphor of a high enantiomeric purity has been detected in essential oils from Ocimum basilicum, while in Coriandrum sativum (-)-camphor is the dominant enantiomer.(+)-Bomeol has been found in lavandin and lavender oils, while (-)-bomeol has been detected in Artemisia genus {Artemisia judaica, Artemisia arborescens, Artemisia herba alba) and Origanum genus {Origanum vulgare, Origanum syriacum). In Chrysanthemum parthenium (-)-bomeol and (-)-camphor of a high enantiomeric purity has been found. Terpinen'4'Ol and a-terpineol

OH "3C

(+)-a-terpineol (-)-a-terpineol

"OH

"3C

(+)-terpmen-4-ol

(-)-terpinen-4-ol

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These two monocyclic terpene alcohols also occur in many essential oils but not in large quantities. Their enantiomeric composition has been investigated in essential oils from Rutaceae family, essential oils from Madagascar and many others [31, 33, 35,49-51]. In a majority of investigated samples (-)-terpinen-4-ol has been found as the dominant enantiomer but its enantiomeric purity was not very high. In this group there were some citrus oils {Citrus aurantifolia. Citrus latifolia. Citrus limon), cinnamon oils (Cinnamonum camphora, Cinnamonum zeylanicum), Helichrysum gymnocephalum. Pelargonium roseum, Ravensara aromatica and Vetiveria zizanioides. In the essential oils from Hedychiumflavumand Lantana camara (+)terpinen-4-ol was the dominant enantiomer of not a high enantiomeric purity, either. Terpineol has been found in enantiomerically pure form as (-)enantiomer in Vetiveria zizanioides and as (+)-enantiomer in Micromeria fruticosa. In the Citrus genus, a-terpineol occurs in the prevalence of (-)enantiomer in the species Citrus aurantifolia. Citrus latifolia and Citrus limon, while it has been found as (+)-enantiomer of a high enantiomeric purity in Citrus Clementine, In neroli and petitgrain oils {Citrus aurantium) (+)-fl-terpineol has been detected as the dominant enantiomer. In Salvia genus {Salvia officinalis. Salvia fruticosa. Salvia sclarea and Salvia dominica) (+)-a-terpineol occurs of not a very high enantiomeric purity. BIOLOGICAL ENANTIOMERS

ACTIVITY

OF

MONOTERPENOIDS

In recent years numerous studies have demonstrated biological activity of the chiral compounds. Enantiomers may vastly differ in their activity. They may differ in [52]: • carrier mediated membrane transport - absorption, cell uptake, excretion • protein and tissue binding • receptor binding and interaction • biotransformation- rates, pathways

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In the pertinent literature there are only very few articles concerning the biological activities of monoterpene enantiomers. All the available results can be divided into several groups: 1. structure-odour relationships 2. influence on intracutaneus permeability 3. antimicrobial and antifungal properties 4. cholelithiasis and ureterolithiasis - drug-therapy 5. anti-neoplasmatic activity 6. insect-plant relationships 7. various types of other activities In the earlier articles concerning enantiomers, the descriptors d and 1 which are not in agreement with the currently applied nomenclature are very often met with. Quoting the data from those articles, they were left in the unchanged version. Odour differences between enantiomeric isomers Enantiomers display a sensory and biological activity differentiation. Their sensory properties are not univocal. Differences in the character of odours of enantiomers confirm the supposition that the enzymatic theory perception is based on the activity of enzymes and odour substances. Systemic enzymes being a chiral catalyst of the highest efficiency act in an enantioselective way, and hence the considerable differentiation in biological effects (various odours). At the beginning of the 1970s there appeared works in Science and Nature concerning the relationships between odours and chirality. The authors have stated that the experimental evidence is in favour of the claim that the optical isomers of carvone have characteristically different odours, (-)-carvone resembling spearmint oil and (+)-carvone, caraway oil [53]. The difference between the odours of two optical isomers of carvone has been characterised by various authors. Russell and Hills have described that 4S-carvone has the odour of caraway and 4R-carvone has the odour of spearmint. They have stated that these observations lend a definite support to stereochemical considerations of odour perception [54]. The results obtained by Fridman and Miller confirmed the above statements [55]. Those authors have described not only the differences in

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the odour of carvone enantiomers but also those between naturally occurring R-(+) and S-(-)-limonene (i.e. orange and lemon respectively). It was interesting to find out that synthetic R-(+) and S-(-)-limonene also emitted these characteristic odours. Our investigations have confirmed that too [56]. (+)-Limonene, (-)-limonene and (-)-a-phellandrene have also been subjected to investigation. The results of sensory properties for the mentioned compound are presented below. Compound R(+)-limonene S (-)-limonene R (-)-a~ phellandrene

Odour description orange - like lemon - like terpene, citrus - like

The estimation of the sensory properties of (+)-limonene, (-) and (+)a-phellandrene in the context of their effect on the flavour of dill herb {Anethum graveolens L.) was also carried out [57-59]. The fitting of space structures of a signal carrier and of a bioreceptor is, as it seems, a decisive factor of a sensory signal occurrence. On the other hand, the formation of unstable effector-receptor complexes is stabilised by non-valency effects. In this way, it is possible to explain a similarity of the character of odour between (-)-a-phellandrene and (-)limonene [56]. Very interesting are also the observations of menthol isomers. The flavour profiles of the four (lR)~-menthols which are the natural constituents of peppermint oil and (1S)~ menthols were described by Hopp [60]. Their unique organoleptic properties, refreshing taste and strong cooling effect, are demonstrated by comparing the flavour profiles of the eight menthol enantiomers. The outstanding flavour properties of (-)-menthol concerning the attributes: cool, fresh, minty and sweet are evident. (H-)-Menthol ranks second with respect to its freshness and cooling properties but it also has some negative features such as phenolic, musty and bitter [60]. Experimental investigations concerning the differences between sensory properties of linalool enantiomers were performed [61]. Its enantiomers display different spectra of sensory properties. The same authors [61] carried out estimations of the above-mentioned compounds on human beings. The therapeutical effects of aroma of each optically active linalool were investigated before and after hearing environmental

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sounds by means of the conventional forehead surface electroencephalography (IBVA-EEG) measurements. The human subjects were examined before and after the inhalation of those compounds in order to test their favourable and unfavourable impressions. This experiment was performed with a view to proving the effect of linalool enantiomers on the level of beta wave. The authors arrived at a conclusion that (-)-linalool influences the decrease of beta wave in contrast with (+)-linalool. Influence on intracutaneous permeability Monoterpenes are most frequently applied as promotors of sorption. The growth of intracellular penetration of a drug in the presence of terpenes is probably connected with an increase of solubility of this drug in the corny stratum of the skin. Monoterpenes can also disturb the regular order of lipids in the intracellular spaces. Monoterpenes as promotors increase the division coefficient of corny stratum/matrix for lipophilic drugs. The penetration speed of lipophilic compounds increases together with their solubility in the promoter (monoterpenes). On the other hand, however, the increase of the penetration speed for hydrophilic compounds increases together with the diffusion coefficient [62-68]. The Japanese group of scientists published a series of articles pertaining to the application of monoterpenes as intracutaneous penetration enhancers. They stated that the increase of intracutaneous permeability of the definite medicinal substance depends on the physicochemical properties of the applied monoterpenes. Monoterpenes with polar groups course increase the permeability of hydrophilic drugs, whereas hydrocarbons are more active in relation to lipophilic drugs [6971]. The Japanese authors investigated the effect of cyclic monoterpenes (among others 1-menthol) on the intracutaneous absorption in the case of a water soluble drug [72]. Their results suggest a significant influence of 1menthol on the above mentioned phenomenon. The same authors were interested in estimating the value of the application of the following monoterpenes: 1-menthol, 1-menthone and 1,8-cineole, trans-p-menthane and d-limonene, as absorption promotors in rats and rabbits [73]. They used ketoprofen on the rat skin together with the above mentioned monoterpenes. The most efficacious of them appeared to be d-limonene. The two enantiomers d-limonene and 1-menthol were applied in

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connection with another drug i.e. diclofenac [74], nonsteroidal antiinflammatory drugs [75], indomethacin [76]. Investigations concerning the effect of 1- menthol on the permeability of indomethacin showed that this process depends on pH. 1-Menthol strongly increases the permeability of indomethacin at pH 5.0 and 7.4 but does that only weakly at pH 3.0 because at this pH this substance appears in a non-ionised character. This phenomenon probably bears evidence that 1-menthol affects the polar way of the drug permeability [69]. The determined quantity of intracutaneous permeability of 1- menthol in vitro was performed by Sugibayshi et al. [77]. Those authors applying the dose of 125 |Lig/cm of the promotor (1- menthol) pointed out that the whole amount of it became completely permeated within 8h. The results obtained by many authors do not make it possible to estimate the differences in monoterpene enantiomers permeability. In the case of monoterpenes the differences in the intracutaneous permeability of their enantiomers can not be significant because of their identical molar weight, lipophility as well as their identical mechanism of activity. Antimicrobial and antifungal properties Many monoterpenes are used as a medicinal means in cases of antifungal and antibacterial infections. The expected pharmacological activity of monoterpenes depends on their structure (optical activity) and on the organisms which they affect. Our investigations concern the differences in bioactivities between the enantiomers of two monoterpenes: a- pinene and limonene [78, 79]. Limonene enantiomers activities were investigated on 25 different bacterial species. The results indicate that the (+) limonene was more active than the (-)-limonene. The (+)-limonene was active against the total of 22 species, whereas the (-)-enantiomer displayed activity only against 16 species [78]. Investigations into the activity of limonene enantiomers in relation to 20 laboratory strains of Listeria monocytogenes isolated from different foodstuffs were also carried out. The (+)-limonene was more active against 14 strains of Listeria monocytogenes and the (-)-enantiomer was only active against 7 varieties of the same organisms. Apart from the antimicrobial investigations, our researches also concerned their antifungal applications. Limonene isomers activity

382

against the following eight fungi: Aspergilllus niger, A. ochraceus, A. flavus. A, parasiticus, Alternaria alternata, Chaetomium sp,, Fusarium culmorum and Penicilium citratum was differentiated in six cases, but there was no consistently high activity for one enantiomer [78]. This is in contrast with the results of Onawunmi [80] who found no difference in the activity between the limonene enantiomers against three Candida species: A. fumigatus, Microsporum gypseum and Trichophuton metagrophtes. The researches pertained to the assessment of different pharmacological tissue preparations in vitro including the rat vas deferens, rat phrenic nerve -diaphragm, rat caecum, rat uterus and the guinea-pig ileum. The pharmacological activity of the (+)-limonene against different tissue preparations was consistently higher except for the uterine effect [78]. The results indicate that the proportion of enantiomers could drastically affect the potency of the drugs in vivo. Similarly as in the case of limonene enantiomers, investigations were carried out into the enantiomers of the a- pinene [79]. Activity investigations were performed in relation to 25 different bacteria species. When comparing the a-pinene enantiomers, we saw that the (-)enantiomer had a greater inhibitory effect on 18 out of the 25 bacteria. The examinations also included 20 strains of Listeria monocytogenes. The (+)-enantiomer displayed a higher inhibitory power in relation to 19 out of the 20 strains in comparison with the (-)-enantiomer [79]. Antifungal investigation results indicate that the (+)-enantiomer was more active against the Aspergillus niger in opposition to the (-)-enantiomer. There was, however, very little difference between the two enantiomers as regards Fusarium culmorum. Pharmacological investigations results showed that the (-)-a-pinene enantiomer was stronger in its spasmodic activity on the smooth muscle than that of the (+)-enantiomer [79]. In conclusion, the above presented results indicating the differences in biological effects in vitro, may suggest that their in vivo effects may also be different. Medical and paramedical means applied in various cases should contain a most suitable kind of monoterpenes enantiomer. Cholelithiasis and ureterolithiasis - drug-therapy Terpenes included in medicinal means find application in the diseases of cholelithiasis and the gallbladder tract as well as in liver diseases [81-86].

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Terpenic compounds are resorbed from the digestive tract and are situated in the hepatic tissues. Thanks to their abiUty to dissolve fats, they prevent the formation of cholesterol gathering inside the liver and they also recover proper colloidal state to the bile. Terpenes also enhance the bile content in the hepatic cells and in the liver tracts. Terpenic hydrocarbons dilating the smooth muscles [78, 79] make the hepatic tracts more distended both inside and outside. It has been pointed out that the terpenes contained in Rowachol dissolve bile stones [87-89]. The mechanism of terpenes activity has not as yet been completely explained. It was explained that menthol and other monoterpenes inhibit the activity of the lecithin-cholesterol acyltransferase in the human serum [90]. They also lower the activity of the hepatic S-3-hydroxy-3-methylglutaryl-CoA reductase, which is responsible for the physiological inhibition of cholesterol synthesis in the liver [82, 91, 92]. Monoterpenes reveal relationships with the renal and urinary tracts. This phenomenon has been taken advantage of in such medicinal means as Rowatinex, Uroterp and Terpinex. Those drugs display distending activity on the blood vessels in the renal glomerulae and because of that increase diuretic effects. Monoterpenes evacuated through the urinary and renal passages have antiseptic and anti-inflammatory activity [93]. Similarly as in the case of Rowachol a treatmentby means of Rowatinex led to the dissolving of renal stones [94, 95]. The enzymatic mechanism of terpenes activity indicates that their enantiomeric content in medicinal drugs may have a basic significance in therapy. The data of enantiomeric content in medicinal drugs including terpenes are very few. In the work [96] concerning enantiomeric content of terpenic hydrocarbons the following drugs have been analysed: Rowachol, Rowatinex (Rowa-Wagner, Germany), Terpichol, Terpinex ( Herbapol, Poland) and Uroterp (Krka, Slovenia). The results indicate large differences of the enantiomeric compositions of all the drugs considered. Since biological systems are largely composed of chiral compounds, it should not be surprising that in such a high chiral environment some drugs with asymmetric centre exhibit a large degree of stereoselectivity in their interactions with macromolecules [96]. Those results indicate that plant drugs containing optically active compounds should be standardised. There is also a necessity to perform investigations into the biological activity of monoterpens enantiomers.

384

Insect-plant relationships The natural plant compounds play a very important role in the natural environment. A particularly important role is played with reference to insects. It is possible to distinguish the following activities of monoterpens in connection with insects: • a scoring activity-repellents • an attractant activity- attractants • an inhibiting activity - antifeedants • a toxic activity - insecticides An analysis of literature data points out that many monoterpenoids display a repellent and attractant activity. The following monoterpenes have a repellent activity: a-pinene, limonene, carvone, 4-terpineol. There are only very few data concerning the enantiomers of the above mentioned compounds. The repellent activity of monoterpene enantiomers has been observed for (+)-linalool and (-)-carvone in connection with the German cockroach {Blattella germanica L.) [97, 98]. The d-isomer of limonene was a repellent while the 1-limonene was an attractant in the studies on the house fly (Musca domestica) [99, 100]. The same compound was an attractant at low concentrations and a repellent at high levels in relation to the German cockroaches [101]. The plant - insect attractant performed the role of pheromones. (+)-Carvone has an attractant activity in connection with Calliphora [102]. Monoterpenes play an important part in the feeding inhibiting activity. Their activity is reduced to blocking the taste receptors. Similarly as in the case of attractants and repellent enantiomers data referring to antifeedants are not many. Limonene inhibiting pine larva parasites feeding {Diprian pint and Dendrolymus pini) [103]. The (+)-3-thujone and (-)-3-isothujone, the main components of Thuja plicata leaves, inhibit the feeding of Pissodes on needle trees [102]. Enzymatic mechanism explaining the olfactory processes are the reasons for eliciting responses between the optical isomers and the receptors. One of the interesting phenomena pertaining to the interaction between the plant -insect and the tree host containing monoterpens is the behaviour of the spruce bark beetle {Ips typographus). The main

385

component of the common spruce (Picea abies (L.) Karl.) is (-)-a pinene. This compound displays attractant properties for the female bark beetle. A very interesting phenomenon which was described by Norin [104] pertains to the transformation of the host terpene a-pinene into verbenol by means of the described insect. This transformation takes place in a strictly stereospecific situation, (-)-a -Pinene is transformed by the female Ips typographus into cis-verbenol.

-7-(-)-a-pinene

(S)-cis-verbenol

Isomeric cis-verbenol is an aggregation pheromone attractant for both the male and female insects of the species. The same author [105] published information concerning a defense secretion of the soldiers of the termites {Nasutitermes nigriceps, K Ephratae and Velocitermes velox from Peru). The enantiomeric compositions of a -pinene and limonene produced by three termite species are as follows: • limonene [%] (+/ -) - 74/26 for A^. ephratae, 88/12 for A^. nigriceps and 4/96 for V. Velox, • a -pinene [%] (+/ -) - >99.9/99.9/hiine (0.05 Mg/mL) + Beigenixi Norqnnephrine (0.05 ug/mL) + Aslilbin III!

0

1

ti I I I m i l

10

100

1000

Concentration( JUL g/ml) Fig. (7). Effects of encryphin, bergenin and astilbin on norepinephrine-induced lipolysis in rat fat cells 100

40

60 30

40

1

20

20

0": 0

InM Insulin + Euciyphin Euciyphin

1

10

100

Concentration (Mg/ml)

1000

InM Insulin + Bogenin Bogenin

0

1

10

100

Concentration (Mg/ml)

1000

20 \

1

10

100

1000

Concentratian ( Mg/nil)

Fig. (8). Effects of eucryphin (a), bergenin (b) and astilbin (c) on lipogenesis from glucose in the presence or absence of insulin in rat fat cells Results are expressed as means ± S.E. of 3-6 experiments. Significantly different from no addition: #pL6Ci/mouse) 2 h after the last administration of stilbenes. The mice were killed by decapitation, and their liver were quickly removed. Results are expressed as mean±S.E. of 8 mice. CH2OH

OH

233,4'-Tetrahydroxystilbene2-0-D-glucx)side

Fig. (21). Structures of Stilbenes

As shown in "Table (4)", it was found that administration of peroxidized com oil for 2 weeks caused liver injury with elevation of GOT and GPT as compared to the control rats. In peroxidized com oil-fed rats, simultaneous oral administration of 2,3,5,4'-tetrahydroxystilbene-2-0-Dglucoside (50 mg or 100 mg/kg) inhibited the elevation of both GOT and GPT levels. The serum GPT level was also found to be reduced in rats orally given piceid (100 mg/kg) as compared to the peroxidized oil-fed rats. As shown in "Fig. (22)", lipid peroxide (LPO) content in the liver was lowered by the oral administration of 2,3,5,4'-tetrahydroxystilbene-20-D-glucoside (50 mg or 100 mg/kg) or piceid (100 mg/kg) as compared to the peroxidized oil-treated group. Stilbenes such as resveratrol, piceid and 2,3,5,4'-tetrahydroxy-stilbene-2-0-D-glucoside inhibited NADPH plus ADP-induced lipid peroxidation in rat liver microsomes dosedependently "Fig. (23)".

408 Table 4. Effects of stilbene glucoside isolated from the roots of Polygonum species on serum transaminases (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in rats fed peroxidized corn oil for 2 weeks GOT (Karmen Unit)

GPT (Karmen Unit)

Mecat ± S.E.

Mean ± S.E.

Control

84.5 ± 5.03*

40.5 ± 4.48

Peroxidized oil-treated group

266.7 ± 31.2

166.7 ± 24.4

+ piceid(50mg/kg)

246.0 ±

16.9

167.0 ± 14.7

(lOOmg/kg)

221.7 ± 27.1

76.5 ± 28.2*

+ 23,5,4'-tetrahydroxystilbene2-0-D-glucoside (50 mg/kg)

172.5 ± 7.83*

82.5 ± 11.3*

1342 ± 20.9*

47.6 ± 16.9*

(lOOmg/kg)

Peroxidized com oil was prepared by heating com oil at 180'C for 1 h while bubbling oxygen through the preparation. The Upid peroxide level of com oil increased from 1.6 nmolAnl to 116.1 nmolAnl during this treatment. Rats were orally administered peroxidized com oil (10 ml/kg body weight) for 2 weeks. Blood was taken by venous puncture 18 hr after administration of the peroxidized com oil. Results are expressed as means ± S.E. of 6-7 rats. * Significantly different from peroxidized oil-treated group, PNAE»>He.3.5,4-Tfltiah>dro3y stilbene-2-O-D-glucoside Peroxidized oil Stilbenes (mg/kg)

50

100

50

100

Fig. (22). Effects of stilbenes isolatedfromthe roots of Polygonum species on liver lipid peroxide in rats fed peroxidized oil for 2 weeks. Rats were orally administered with peroxidized com oil (10 ml/kg) for 2 weeks. Rats were killed 13 hr after administration of the peroxidized oil. Results are expressed as mean± S.E. of 6-7 rats.

Concentration (MM)

Fig. (23). Effects of stilbene derivatives on ADP plus NADPH-induced lipid peroxidation in rat liver microsomes. Results are expressed as mean± S.E. of 4 experiments.

h) Effects of Non-Sugar Fraction in Black Sugar on Lipid and Carbohydrate Metabolism in Rats Fed a High-Sugar Diet [11,12] From ancient times, cmde black sugar (brown sugar) prepared from sugar-cane has been widely used as a sweetener. It is well known that the consumption of a large amount of refined sugar causes hyperlipidameia, obesity, diabetes, and arteriosclerosis. We designed experiments to search for some biologically active substances which protect against such pathological changes induced by refined sugar. In the course of these experiments, we discovered that the non-sugar fraction of crude black sugar caused reduction of serum triglyceride, lipid peroxides and insulin levels in rats fed a high-sugar diet for 61 days "Table (5)". In addition, the non-sugar fraction was found, by oral glucose tolerance test (OGTT), to reduce the plasma insulin in rats "Fig. (24)". The active substances (BS-1) isolated from the non-sugar fraction of crude black sugar were a

410

mixture of 3,4-dimethoxy phenyl-O-D-glucoside and 3,4,6-trimethoxy phenyl-0-glucoside (new compound) "Fig. (25)". As shown in "Fig. (26)", BS-1 reduced theOGTT without affecting the plasma glucose level. Table 5. Effects of non-sugar fraction of black sugar on serum total cholesterol (TC), triglyceride (TG), lipid peroxide (LPO), glucose and insulin in rats fed a high- sugar diet for 61 days.

Control

TC (mg/dl)

TG (mg/dl)

LPO (nmoles/ml)

MeankS£.

Mean±S.E.

Mean±SJE.

84.5±

107.9± 3.10± 34.8± 11.7* 0.03*

3.10* High sugar diet-treated

134.3±

group + non-sugar

257.3± 8.49

fraction

(500 mg/kg)

Mean±S.E.

820 115.8± 4.76

19.5 125.1±

\102± 3.37± 55.6± 27.5* 0.07*

7.52*

9.45

195.5± 3.33± 62.2± 21.4* 0.07*

5.43*

130.4±

Mean±S.E. 113.2±

111.8± 0.04

Glucose (mg/dl)

7.50*

8.88

132.8±

(1000 mg/kg)

3.60± 18.9

Insulin (fiU/ml)

3.82 125.3± 323

The control mice were fed laboratory pellet chow ad libitum. The basic composition of the high-sugar diet was as follows (wt %); sugar 75%, casein 15%, com oil 5%, vitamin mixture 1% and mineral mixture 4%. Results are expressed as means ± S.E. of 5-6 rats. * Significantly different from high- sugar diet-treated group, P-OH OH-^ V-CH=CH-CO-0 z^^ \ ^ OH

3,5-Di-O-caffeoylquinic add

a

HQ. ^COOH

OH-^

V-CH=CH-CO-0

>=/

=

y=/

b-co-CH=cH-^

f-on

4,5-Di-O-caffeoylquinic add

a.

HQ. ^COOH

PH

Ha* ^ f ^0-CO-CH=CH--OH

3,4-Di-O-caffeoylquinic add

Fig. (31). Caffeetaimins of the leaves of Artemisia species

Table 12. (A) Content of caifeoylquinic acid in the extracts of A . Montana

Acetone-water (7:3, v/v) extract

Chlorogenic add(%)

3,4-Di-(9caffeoylquinic add

3,5-Di-Ocaffeoylquinic add

4,5-Di-Ocaffeoylquinic add

3.4

3.2

13.4

1.4

(%)

(%)

(%)

420

Table 12. (B) Effects of the extracts oi Artemisia montana on serum TC, LPO, FFA and TG in rats fed peroxidized com oil for 1 week FFA(mEq/l)

Control Peroxidized oiltreated group + Extract (500 mg/kg)

TC(mg/dl)

LPO(nmoUml)

TG(mg/dl)

Mean± S£.

Mean± S.E.

Mean± S.E.

Mean± S.E.

0.13 ± 0.03*

67.3 ± 6.9*

3.03 ± 0.07*

61.6 ± 6.46*

1.19 ± 0.18

172.7 ± 27.2

10.10 ± 3.34

378.8 ± 86.2

0.79 ± 0.12

139.4 ± 21.7

427 ± 0.94*

171.1 ± 104.8

Peroxidized com oil was prepared by heating com oil at 150'C for 1 h while bubbling oxygen through the preparatioa The hpid peroxide level of com oil increased from 0.56 nmol/ml to 54.7 nmol/ml during this treatment. Rats were orally administered peroxidized com oil (10 ml/kg body weight) for 1 week. Blood was taken by venous puncture 4 hr after administration of the peroxidized com oil. Results are expressed as means ± S.E. of 5-6 rats. * Significantly different from peroxidized oil-treated group, Pr^o

OH CH2OH

complanatin = [3-(9-j3-D-glucopyranosyl-4'-0-(3'"-0-dihydrophaseoyl-)8-D-glucopyranosylrhamnocitrin OH

HO

OH

R=H 3-0-(5'"-0-/?-coumaroyl-^-D-apiofuranosyll( 1"' ~^2"')-j3-D-glucopyranosyl) rhamnocitrin R = OMe 3-0-(5"'-0-feruloyl-)3-D-apiofuranosyll( 1 '"->2'")-j3-D-glucopyranosyl) rhamnocitrin Fig. (7). Acylated flavonoid glycosides from A. complanatus

502

All Astragalus spp., investigated untill now for their flavonoid content, are listed in Table (6). TABLE 6. Astragalus Species A. adsurgens Pall. A. aitosensis M.B. A. alexadrinus Boiss. A. ammodendron Bunge A. arguricus Bunge A. austrosibiricus Schischk A. babatagi Popov A. bachycarpus M. Bieb. A. bommuellerianus B. Fedtsch. A. bungeanus Boiss. A. captiosus Boriss. A. caucasicus Pall. A. centralpinus Braun-Blanquet A. cicer L. A. circassicus Grossh. A. clusii Boiss. A. coluteocarpus Boiss. A. complanatus R.Br. A. dasyanthus Pall. A. dipelta Bunge A. eupeplus Bameby A. falcatus Lam. A.floccosifolius Sumn. A. galegiformis L. A. goktschaicus A. himalayanus Klotz A. kabadianus Lipsky A. kadshorensis Bunge A. karakuschensis Gontsch. A. lasioglottis M. Bieb. A. macropterum DC. A. maximus Willd. A. membranaceus Bunge A. membranaceus Bunge A. miser Hook. var. oblongifolius (Rydb.) Cronq. A. mongholicus Bunge A. mongholicus Bunge A. novasanicus Klokov A. onobrychis L. A. orbiculatus Ledeb. A. polygala Pall. A. propinquus Schischkin A. pubiflorus DC. A. quisqualis Bunge A. sevangensis Grossh. A.shikokianus A. sinicus L. A. subrobustus A. testiculars Pall. A. torrentumi Bunge A. tracicus A. trigonus DC.

Species Containing Flavonoids Organ examined Aerial parts Aerial parts

-

Aerial parts

-

Aerial parts

-

Aerial parts Aerial parts Leaves

-

Aerial parts Seeds

-

Aerial parts Aerial parts

-

Epigeal parts Aerial parts

-

Epigeal parts Aerial parts

-

Aerial parts Roots

Aerial parts Roots

-

Roots

-

Aerial parts Seeds Aerial parts Aerial parts

-

Reference [181,259] [130] [83] [174] [107] [231,232] [310] [22,26,163] [308] [107] [110] [27,29] [210] [23,129,187,188,211] [107] [209] [304] [60,69,70,72,74,301] [170,171] [196] [302] [27] [309] [27,28,30,278] [107] [100] [305] [27] [109] [164] [303] [27, 28] [64,197,275,314] [36,243,246,247,248,259,270, 276,312,313,324] [222] [80,194] [80,254] [78] [45,164] [269] [163] [81] [76,77] [210,306] [107] [300] [96] [108] [244] [105] [135] [83]

503 Astragalus spp.

[163,183,264]

* (-): not specified parts

Others Coumarins have been isolated and characterized in many Astragalus species: the main identified compounds are umbelUferone and scopoletin, as simple coumarins, and skimmin and scopolin, as their glycosides, respectively. Table (7). Table 7. Occurrence of Coumarins in Astragalus

spp.

Coumariiis

Plant source

Ref.

scopoletin, umbelUferone skimmin, scopolin

A. cicerL. A. brachycarpus M.Bieb. A. caucasicus Pall. A. falcatus Lam. A. galegiformis L. A. kadshorensis Bunge A. maximu Willd.

[23] [26] [27] [31] [27] [27] [27]

umbelUferone coumarins

A. sinicus L. A. dasyanthus Pall. A. eximus A. lasiopetalus A. lasiosemius A. schrenkianus A. severtzovii A. sieversianus A. tschimganicus A. ugamicus Astragalus spp.

[96] [172] [260] [260] [260] [260] [260] [260] [260] [260] [50]

Two lignans (+)-lariciresinol and (-)-siringaresinol. Fig. (8) have also been isolated from the roots of A.mongholicus [255], while

504 OMe

MeO.

OMe MeO

(+)-lariciresinol

y OH

OMe

(-)-syringaresinoI

Fig. (8). Lignans isolated from A. mongholicus

phenolic carboxylic acids as /7-hydroxybenzoic acid [106], chlorogenic [45,106,108] and neochlorogenic acid [108], ferulic and isoferulic acid [106,307], caffeic acid [45,106,306], 3-feruloylquinic [106,108] and 3-J9coumaroylquinic [108] have been determined in the species: A. subrobustus [108], A. floccosifolius [307], A. quisqualis [306] and A. onobrychis [45]. Activity Flavonoids are a class of compounds displaying many biological activities. A variety of in vitro and in vivo experiments have shown that selected flavonoids possess antiallergic, anti-inflammatory, antitumor, antiviral and antioxidant activities. Moreover, acting by several different mechanisms, particular flavonoids have been shown to exert significant anticancer activity including anticarcinogenic and prodifferentiative activities. Many flavonoids, including those which are phytoalexins, provide plants with a defense against viral infections. The estrogenic action of many isoflavones is well known and mixtures of flavonoids are conmionly used commercially to reduce capillaryfi-agility[200,213]. Reactive oxygen species (ROS), such as superoxide anion and hydroxy radical, induce lipid peroxidation (LPO) which injuries cell membranes.

505

The diseases induced by LPO involve arteriosclerosis, diabetes mellitus, halotane hepatotoxicity and liver desease. Some antioxidants and scavengers inhibit peroxidation induced by ROS (i.e., a-tocopherol, glutathione and carotenoids). Recently, natural oxidants have been found from many plants, such as spices, vegatables and herbs [276]. Kaempferol and quercetin as flavonols scavenge ROS and inhibit LPO. Flavonoids from Astragalus spp. show a significant antiexudative effect [192], while flavonoid complex from A.centralpinus possess a marked spasmolitic action and account for a moderate, but long-standing reduction of the arterial pressure. The total flavonoid content from A, lasioglottis show a high biological activity decreasing the cholesterol and triglyceride levels in animals with experimental hyperlipidemia [195]. Isoflavonoids are best known for being estrogenic, antimicrobial or insecticidal [113]. They are involved with diverse biological activities, including disease resistance, and, potentially, photosensibilization [188]. Calycosin, 245, and formononetin, 251, as isoflavones isolated from the roots of A. membranaceus, inhibit lecithin peroxidation which was induced both by hydroxy radical generation by interation of haemoglobin and hydrogen peroxide and by superoxide anion generation by xanthinexanthine oxidase [312,276]. Afrormosin, 243, calycosin, 245, and odoratin, 252, isolated from the same source have antioxidative activity and prevent lipid peroxidation (all the tested isoflavones have a methoxy group at 4' position) [243]. Other isoflavonoids do not show inhibitory effects on lecithin peroxidations. These results demonstrated that hydroxyl group at the 7 and 3* positions on isoflavones, which have a methoxy group on the C-4', are necessary for the antioxidant properties, because isoflavones which have a methoxyl group at the 6 and 4' positions, have no inibitory effects on LPOs [276]. An anti-hepatotoxic action has been showed on CCLj-induced cytotoxicity in primary cultured rat hepatocytes by 7-O-^Dglucopyranosyl 3',7-dihydroxy-4'-methoxyisoflavone from the roots of ^. membranaceus [36]. Some isoflavans, expecially isoflavanquinones, show antimicrobial activity. Astragaluquinone and 8-methylvestitol, isolated from A, alexandrinus and A. trigonus demontrate weak antimicrobial activity against Gram positive organisms andfiingi[83].

506

POLYSACCHARIDES Isolation and structure elucidation Polysaccharides belong to the most important immunostimulants isolated from higher plants. Plants often synthetize active polysaccharides in their roots and the composition of these polysaccharides in the plant root may vary to climatic conditions; therefore standardization of the plant extracts can be difficult. The polysaccharides from A. membranaceus Bunge are the best known and several research groups have isolated and characterized them. From the hot water extract of the roots of this plant, Tomoda et al, [277] have been isolated a glycan, designated AMem-P, by treatment of the crude acidic polysaccharide fraction with cethyltrimethylammonium bromide in diluted sodixmi sulphate solution, followed by column chromatography on Toyopearl HW60F and Con A-Sepharose column. A quantitative analyses has shown that the glycan is composed of L-arabinose (8.5%), Dgalactose (15.6%), L-rhamnose, D-galacturonic acid (57.8%) residues, and acetyl (1.8%) and methoxyl (1.0%) groups in carboxylic acid methyl esters and a peptide moiety (2.9%). The molar ratio of these component sugars is 6:9:8:30. Thus, ca. 10% of the hexuronic acid residues in the glycan exist as methyl esters. On the basis of chemical and spectroscopic studies AMem-P show to possess mainly a-l,2-linked L-rhanino-a-1,4linked D-galacturonan structure. Terminal and a-l,5-linked Larabinofiiranose, terminal p-1,3-, p-1,4- and P-l,6-linked and 3,6branched D-galactose, and 2,4-branched L-rhamnose residues have also been identified as the component sugar units. On the basis of the results obtained from the analysis of the products of the original glycan and its O-deacetylated product, subjected separately to periodate oxidation followed by reduction with sodium borohydride, the minimal unit of the polysaccharide results composed of 10 sugar units. By the comparison of different extraction methods of polysaccharide contents in A. membranaceus, the ultrafiltation method gives 20% more yield than the boiling-ethanol precipitation method [190]. During a research in which the effect of the oral administration of "Astragali radix" (AR) on IgM antibody production in mice of various ages was examined, the polysaccharides responsible for the antibody

507

production enhancement have been isolated and purified [160]. The separation method is described in Fig (9). Astragali Radix - extracted with hot water - precipitated with MeOH I

dialyzed against water 1

I—precipitated with 8% cetavlon

precipitate

supernatant

— dissolved in 10 % NaCl

added with 1 % boric acid

— precipitated with MeOH containing 1 % KOAc

adjusted pH 8.8 (2 N NaOH )

— dialyzed against water

F-2 DEAE Sephacel

precipitate -dissolved in 10 % NaCl

F-5

F-6

I

F-9

F-3

adjusted pH 4.4 (acetic acid) precipitated " with MeOH

precipitated with MeOH dialyzed against water

F-8

I

-added with acetic acid

F-7

Sephacryl S-300

supernatant

I

dialyzed against water

F-4

Fig. (9). Separation scheme of the polysaccharide fraction from "Astragali radix

Examination of the chemical properties of the active polysaccharide F8 and F-9 shows that F-8 contained 89.3% carbohydrate as glucose and 7.2% uronic acid as galacturonic acid, while F-9 contained 95.5% carbohydrate and 7.5% uronic acid. These polysaccharide do not contain

508

protein. Both fractions give a single peak on HPLC gel chromatography (retention time: 25.3 min. and 27.3 min. respectively) and their molecular masses have been estimated to be 2.2 10"* and 1.2 10^. The major chemical component of these compounds is glucose. The sugar moiety of F-8 was composed of rhamnose, ribose, fucose, arabinose, xylose, mannose, galactose and glucose in molar ratio of 2:2:1:2:6:2:3:100. That of F-9 is constitued of fucose, xylose and glucose in molar ratio of 1:2:100. A. mongholicus Bunge is one of the species more investigated about its polysaccharide content. Fang et al [91] reports that from the aqueous extract of the roots of A. membranaceus var. mongholicus three polysaccharides, astragalan I, II, and III, have been isolated. Astragalan I (mol. weight 36 300) is composed of D-glucose, D~galactose and Larabinose in the molar ratio of 1.75:1.63:1. It also contains trace of pentose. D-glucose is the only constituent of Astragalan II and III. Their average molecular weights are 12 300 and 34 600, respectively. Astragalan II and III, when treated by periodate oxidation and Smith degradation, produces glycerol and a large amount of erythritol. These results suggest that both astragalan II and III consisted mainly of a(l-^4) linked glucopyranosyl residues and also a small amount of a(l->6) linked glucopyranosyl residues. Two other glucans (AG-1 and AG-2) and two heterosaccharides (AH1, AH-2) have been further isolated and purified from a water extract of the roots of ^. mongholicus Bunge [273]. These four polysaccharides resulted homogeneous by electrophoresis and gel chromatography. AG-1 has been identified as an a-glucan, with ratio of a(l'"^4) and a(l-^6) linkages of about 5:2. AG-2 is identical as a a(l->4) glucan. AH-1 is an acidic polysaccharide and the component sugars have been identified as hexauronic acid (galacturonic acid and glucuronic acid), glucose, rhamnose and arabinose in a ratio of approximately 1:0.04:0.02:0.01. AH-2 contains glucose and arabinose in a ratio of 1:0.15. A novel acidic polysaccharide, designated as AMon-S, has been isolated and identified recently from the water extract of the roots of A. mongholicus. It is homogeneous on electrophoresis and gelchromatography and its molecular mass is estimated to be 7.6 10"*. It showed significant reticuloendothelial system-potentiating activity in a carbon clearance test. The isolation method of the polysaccharide is summarized in Fig.(lO) and consists in a treatment of the hot water

509 root of Astragalus mongholicus I extracted with hot water extract treated with cetyltrimethylammonium bromide

I

1

supernatant

precipitate

poured into ethanol precipitate (fr. CTAB-S)

supernatant

applied to DEAE-Sephadex A-25 (acetate) column eluted with 0.2 M ammonium acetate

I eluted with water eluate A

eluate B dialyzed and lyophilized fr.A applied to Con A-Sepharose column eluted with 1/15 M phosphate buffer

I adsorbate

eluate

applied to Toyopearl HW 60 F column eluted with 0.1 M tris-HCl buffer

fr. 1

I

fr. 2

fr. 3

rechromatographed dialyzed and applied to Sephadex G-25 column eluate lyophilized AMon-S Fig. (10). Isolation scheme of polysaccharide AMon-S from A.mongholicus

extract of the roots with cetyhrimethylammonium bromide in presence of small amounts of sodium sulphate. TTie supematant obtained is poured into ethanol, then the precipitate is applied to a column chromatography

510

of DEAE-Sephadex A-25. The eluate with 0.2M ammonium acetate was subjected to affinity chromatography on Con-A-Sepharose. The passedthrough fraction is applied to gel chromatography column, followed by dialysis and gel chromatography with Sephadex G-25. AMon-S is composed by L-arabinose, D-galactose, D-galacturonic acid and Dglucuronic acid and a peptide mojety. Quantitative analyses shows that it contained 40.6% arabinose, 48.8% galactose, 2.9% galacturonic acid, 3.0% glucuronic acid and 4.7% peptide moiety. The molar ratio of these component sugars is 18:18:1:1. AMon-S has a-l,5-linked L-arabino-P3,6-branched D-galactan type structural units as its major part, based on i^C NMR, methylation analysis and periodate oxidation studies [241]. The component galactose and hexuronic acid residues in the structure of AMon-S are closely similar to those in glycyrrhizan GA. However AMon-S possesses only two kinds of arabinose units and no rhanmose, so its structural features is simpler than glycyrrhizan GA. [242]. The presence of terminal p-D-glucuronic acid residues in these two polysaccharides is characteristic and AMon-S is the second example having terminal glucuronic acid units among the known RES-activating acidic polysaccharides. Other polysaccharides, named polysaccharides A-D, have been extracted and purifiedfi-omthe same Astragalus species (aqueous extract) by Huang et aL [125]. Polysaccharides A and B are both soluble in water. Polysaccharide A is a glucane containing a(l->4) and a(l"~>6) (5:2) glucoside; while polysaccharides B and C are both a(l-->4) glucan; B is soluble in hot water and diluted alkaline solution, whereas C is not soluble in hot water. Polysaccharide D is an heteropolysaccharide consisting of glucose, arabinose and rhanmose at a 9:3:2 ratio. The Astragalus plant species produce exudates when they are tapped or otherwise stressed, perhaps as a protective mechanism. The hydrophylic exudates, termed gums, are composed principally of watersoluble polysaccharides, frequently with a small (1-3%) covalently bound protein residue. Tragacanth gum, the dried gum exudation fi:om A, gummifer Lab., has been approved for use in food and pharmaceutical formulation as stabilized and thickening agents, particularly for stabilizing oil in water emulsion, in acetic salad dressing preparations, and are generally recognized as safe. They are notably nontoxic when taken orally, because these polysaccharides are not absorbed fi-om the

511

gastrointestinal system and do not enter the circulation when ingested. Commercial gum tragacanth is a variable commodity, because commercial samples may legitimately be mixtures, in any relative proportions, of the exudates from Asiatic Astragalus spp.; thus for legislative purpose more detailed chemical data are desiderable. Anderson and Bridgeman [32] have studied the composition of the proteinaceous polysaccharides of three Turkish specimens of the gums from A. microcephalus, A. gummifer and A. kurdicus to identify the major Astragalus spp. involved and to secure reference gum specimens from them. The analytical data obtained shows that the exudates collected from the three major contributing Turkish spp. differed extensively, particularly in terms of their fucose, xylose, galactose, arabinose, galacturonic acid and methoxyl contents and in the relative proportions of their soluble (tragacanthin) and insoluble (bassorin) components. In addition the amino acid composition differs particularly in respect of their proportions of hydroxyproline, histidine, aspartic acid and arginine. Gum tragacanth must therefore be regarded as a proteinaceous polysaccharide, with a protein content of ca. 3-4%. About the polysaccharide composition, other studies are performed on other Astragalus spp., as A. falcatus, A. sinicus and A. aitosensis [35,136,185]. From the seeds of this latter species a galactomannane (mol. wt. 464 kD) with 55% yield (this heteropolysaccharide contains Dgalactose and D-mannose in the 1:1.27 ratio) is obtained. The main chain consists of the 1,4-p-D-mannopyranose residues, and single residues of aD-galactopyranose are linked by C-6 position to 78% of this chain. From the hot water extract of the endosperm of ripe seed of A. sinicus a galactomannane has been isolated. Its molar ratio of D-galactose and Dmannose is 1:2.3. From the results of the gas chromatography of alditol acetate derivatives of methylated sugars of hydrolizated, it is suggested that the chemical structure of galactomannan may be composed of a main chain consisting of (1—>4)- and (l~»3)-linked (J-D-galactose residues linked to the main chain through (1-^6) bonds. In order to study the possibility of a biotechnological production of polysaccharides, some researchers have started to cultivate plant cell and organ culture. Hairy roots of A, mongholicus, A. membranaceus and A. gummifer produce significant amounts of polysaccharides, which are secreted into liquid growth media [133,323,271]. It has been found that

512

the hairy roots secrete polysaccharides in the liquid medium, and, in comparison with dry roots, containe higher polysaccharide amount. Although IH NMR, l^C NMR and 2D NMR have been widely used in structural elucidation of polysaccharides, methylating analysis is still an indispensable method to determine the types of linkage between different glycosidic residues. Characterization of partially methylated alditol acetate (PMAA) derivatives produced from hydrolysis, reduction and acetylation, reveals the position of the unmethylated hydroxyl groups. Carbon atoms earring these free hydroxyl groups are involved in linking the sugar units in polysaccharides. Moreover, the number of residues in the average repeating unit, the nature of terminal units and the units at which branching occurs may also be deduced from the analysis of methyled products. Hakomori's procedure is now almost universally used among various methylation methods [111]. In this procedure, CH3SOCH2-, generated from the reaction of dimethylsulphoxide (DMSO) and sodium hydride (NaH), is considered to be the effective basic agent which plays a critical role in the sugar alkoxide formation. However, it has to be prepared before use and generally the methylation has to be repeated several time for the completion of the reaction. Another simple and fast method for sugar alkoxide formation has been reported using a solid base (NaOH or KOH) in DMSO solution, but it is necessary first to prepare methylsulfinyl methanide [65]. In recent year the lithium salt of methylsulfynil carbanion, obtained by treatment of DMSO in butyllithium, has been showed to have more performance in comparison with sodium or potassium salts. This method is characterized by short reaction period and clean gas chromatograms, but the Li-methylsulfynil carbanion still has to be prepared separately before use. A simplified procedure in which butyllithium is directly added to a solution of a polysaccharide in DMSO has been reported by Chen Li et ah [189]. The CH3SOCH2-, generated in the solution, precipitates in the reaction of sugar alkoxide formation immediately. All operations are performed in one flask with one treatment. This procedure is rapid and convenient.

513

Activity "Astragali radix" (AR) [83] is an important traditional Chinese herbal medicine widely used in China and Japan. It is used to improve naturally weak constitutions, as an invigorating drug to improve the immune response, often for the elderly, and to counter unbalanced nutrition in China. The immunostimulant effect of polysaccharides, isolated from A. membranaceus, has been proved; so many authors recommend that this plant may be used in immunotherapy. It is chiefly used as a decoction. There are many reports in the literature on the protective effect of AR, including its enhancement of antibody production, acceleration of macrophage phagocytosis, increase of tumor necrosis factor production, activity on the reticuloendothelial system and its enhancement of natural killer cell activity. A modification of the immune response could be responsible for the antiviral and anticancer activity of A. membranaceus and studies have shown that astragalan I and II, tha main constituents of the water extract from this species, potentiated the immunological response in mice. After i.p. (intra peritoneal) administration, they increase the weight and cell number of mouse spleen, elevate the response of mouse spleen against sheep red blood cells and stimulate phagocytic activity of peritoneal macrophages. When the polysaccharide fraction is given i.v. (intravenous) or p.o. (intragastrically) the phagocytic fraction of peritoneal macrophages do not change significantly [273]. The natural killer cytotoxicity of lymphocyte effector cells is markedly enhanced when treated with partially purified human interferon-a with extract of Astragalus, They stimulate each other: the natural killer cytotoxicity increase five- to sixfold after treatment of effector cells with both agents [273]. Other studies are reported in the review of Rios and Waterman [234]. Tomoda et al [277] isolated a glycan from the hot water extract of A. membranaceus roots and studied its reticuloendothelial system potentiating activity in the carbon clearence system, when a net positive effect was demostrated. The polysaccharide composed of glucose and arabinose extracted from A. mongholicus administrated i.p. (intraperitoneal) to mice increases the immune response, and the amount of RNA in the spleen, but has no effect on thymus, heart or brain RNA, or on DNA metabolism. [273]

514

More recently Kajimura et aL reports a defensive effect of AR in young mice (5-12 weeks), including its protective effect against Japanese encephalitis virus infection, the enhancement of antibody production by mouse spleen cells, and a beneficial effect on the function of peritoneal exudate cells [160,161]. In this studies two polysaccharides (F-8 and F9), isolatedfi-omthe crude polysaccharides AR fraction, enhance the IgM antibody production in aged mice by oral administration. Tragacanthin polysaccharidesfiromA. brachycentrus (AV208) and A. echidnaeformis (AV212) plants, which are devoid of in vitro antiviral activity, are active inhibitors of Punta Toro virus PTV infections in mice [245]. AV212 is more potent than AV208, causing increases in survival over a broader range of doses and time. Due to the large molecular sizes of these substances, they are not absorbed trough the intestinal tract and are not expected to exhibit oral antiviral activity. AV212 and AV208 haven't direct antiviral effect, and the reason for the anti-PTV activity appears to be the macrophage stimulation. The Astragalus root and its polysaccharide (APS) have a lot of biological activity such as antineoplasm, antihepatitis and antisenility [314]. In this article are reported the therapeutic effect on epatitis of Astragalus polysaccharide: the experiments in vivo showes that the interferon induced in the plasma of patients with hepatitis increased 3 titres after the treatment by oral administration of APS, identical with the reported documents in vitro, ALKALOIDS AND NITRO-COMPOUNDS Alkaloids Polyhydroxyindolizidines are a class of alkaloids, occurring primarily in the family Leguminosae [86]. Within the group the most important members are swainsonine (1,7,8-trihydroxyindolizidine), the toxin of locoweeds {Astragalus and Oxytropis species) of North America and the poison peas (Swainsona species) of AustraUa, and castanospermine (1,6,7,8-tetrahydroxyindolizidine), the major alkaloid of the Moreton Bay chestnut or Black Bean (Castanospermum australe). In contrast to the herbaceous locoweeds and poison peas, the latter is a large tree native of Northeastern Australia, which has been introduced as an ornamental in

515

many subtropical regions of the world. The alkaloid swainsonine is also produced by the fungi Rhizoctonia leguminicola and Metarhizium anisopliae. This compound is biologically very interesting and has been foimd to be the toxic constituent of Astragalus and Oxytropis species that cause locoism in cattle, a chronic neurological disease which is similar to mannosidosis, a disease of both humans and liverstock [162]. Biosynthesis of swainsonine has been investigated only in Rhizoctonia leguminicola, where the alkaloid is formed from lysine via pipecolic acid [120] according the scheme reported in Fig. (11). Detection in the diablo locoweeds {A. oxyphysus) of [li?,8aS'-trans]-l-hydroxyindolizidine and [l,8a-trans-l,2-cis]-l,2-dihydroxyindolizidine, known fungal precursors of swainsonine, provides strong evidence that the plant pathway is very similar, if not identical, to that in the fungus. Additional evidence for the similarity of the pathways was obtained by feeding [G-^H]-DL-pipecolic acid to shoots of A. oxyphysus. As in the fungus, tha acid was incorporated into all three alkaloids.

C

= COOH

Lysine

NH

-

pipecolic acid

OH

1-oxoindolizidine

H ,N
=0

Is

0" 14

1 p-Hydroxy-1-desoxo- i 101 tamirin

1 ^\ / T i oy jv

^ ) = 0

569 (Table 1). contd.. Eudesmanolides Name

Structure

Plant species

Ref. 1

Ha^ II 1 a-Hydroxy-1-desoxo-1 tamirin-4a,5p-epoxide

102

/

^»*** A. = 0

6^

!"

103

II II

.rfw

/ " ^ ^ ^ s v ^ ^ ^ s . »t»»Q = 0

^r^

s^

*

OH

1

50 21

T. vulgare T. psendoachillea T. densum ssp. sivasicum T. argyrophyllum var. argyrophyllum T. densum ssp. amani T. chiliophyllum var. heimerlei T. argenteum ssp. canum wax.canum T. praeteritum ssp. praeteritum T. densum ssp. eginense T. argenteum ssp. flabellifolium

15, 51, 66

OH

OH Tanachin (l-e/?;-Tatridin B) (Deacetyldihydrochrys a-nolide)

T. polycephalum T. densum ssp. sivasicum

**

vv

^

67, 68 36 35 30 37 25 17 26 22

r^x^""\.= o 1

Tanacin

104 '

Tanadin

105 '

69,70, 71

T. pseudoachillea

66

OAng

j^X'^^^U^^ 1

T. pseudoachillea

OAng

=0

570 (Table 1). contd.. Eudesmanolides Name

Structure

Plant species

Ref.

Tanalbin A

106

T. albipannosum

11

Tanalbin B

107

T. albipannosum

72

Tanargrolide

108

wo-TanargyroIide

109

T. densum ssp. sivasicum

73

Tansanin

110

T. santolina

18

T. argyrophyllum var. 35 argyrophyllum

571 (Table 1). contd.. Eudesmanolides Name

Structure

Plant species

OH Tatridin A (Tavul un=Tabul in)

111

=0 1

(11R)-11,13Dihydrotatri- din-A

OH

"^' 3 ^

112 \

T. vulgare T. cinerahaefolium T. argyrophyllum var. argyrophyllum T. densum ssp. sivasicum T. chiliophyllum var. heimerlei T. argenteum ssp. argenteum T. densum ssp. eginense T. argenteum ssp. jlabellifolium T. polycephalum T. ferulaceum T. ptarmicaejlorum

T. cinerahaefolium T. ferulaceum

/ *"0

Ref. 1 sV, 15 32 35 36 37 33 26 22 50 12 13

32 12

=?0 HO

/

OH Tatridin B

113 ^X*^^sUX^^^w ,»»*^ 1^

-

'

OH

=0

^

T, vulgare T. cinerahaefolium T. ferulaceum T. argenteum ssp. argenteum T. ptarmicaeflorum

15 1 32 12 33 13

OH (11R)-11,13Dihydrotatri- din-B

114

T. cinerahaefolium

=0 1

OH

32

572 (Table 1). contd.. Eudesmanolides Name

Structure (llR)-6-0-P-DGlucosyl-11,13dihydrotatridin B

Plant species

Ref.

T. cinerahaefolium 32

115

OR R=|3-I>Glucosyl

Desacetyltulipinolidelp,10a-epoxide

116

T. densum ssp. sivasicum T. densum ssp. amani T. argenteum ssp. argenteum T. densum ssp. eginense T. argenteum ssp. flabellifolium

36 30 33 26 22

T. densum ssp. sivasicum

21

T. parthenium T. vulgare

39 15

OH Tulirinol

117

OAc

Anhydroverlotorin4a,5p- epoxide

118

8aHydroxyanhydroverlotorin

119

T. argyrophyllum var. 35 argyrophyllum 36 T. densum ssp. 30 sivasicum T. densum ssp. amani

573 (Table 1). contd.. Eudesmanolides Name

Structure

3pHydroxyanhydroverlotorin

Plant species

Ref.^

T. parthenium

120

39

0

Seco Germacranolides l,3-Dioxo-7a,lipH2,3- secogermacra4Z,9Z- dien-12,6aolide

| 54 1

121

T. sinaicum

2

10 CHO

p b-i^ 0

1 l,3-Dioxo-7a,lipH2,3- secogermacra4E,10(14)-dien-12,6aolide

122

54

T. sinaicum

^ 0

1 1

Germacrane Type Sesquiterpenes Germacrene D

1 Bicyclogermacrene

1 123

i 124

c ^

!?%

| 74 1

T. vulgare T. parthenium T. sibiricum T. tanacetioides

j

39 75 44

T. tanacetioides 1 T. parthenium

44

1 ^^

574 (Table 1). contd.. Eudesmanolides Structure

Plant species

Name

Isohumulen

125

Tanacetol A

126

OAc

Ref.

T. tanacetioides

44

T. vulgare

76

T. vulgare

76,15

OH

Tanacetol B

127

OAc

OH

Guaianolides

8a-Hydroxyachillin

128

T. macrophillum

56

8p-Hydroxyachillin

129

T. microphillum

11

575 (Table 1). contd Eudesmanolides Name

Structure

Plant species

Ref.

OAng Angeloylajadin

130

T. inducum

78

T. annuum

79

T. parthenium

58,39, 41,80

T. macrophillum T. parthenifolium

56 81

T. inducum

78

'.. OH

Artabsin

131

Artecanin (Chrysartemin B)

132

Arteglasin A

133

Canin (Chrysartemin A)

134

OAc

0.««'

T. cilicium 82 16 T. vulgare 58,39, T. parthenium 41 T. macrophillum 83,56

576 (Table 1). contd.. Eudesmanolides Name

Structure

lO-epZ-Canin

135

Plant species

Ref.

T. parthenium

39

o.«*«

....c^^^vA. Canin 8a-isovalerate

136

T. cilicium

82

Canin 8amethylbutyrate

137

T. cilicium

82

T. santolina T. densum ssp. sivasicum

84,18 36

T. santolina T. densum ssp. sivasicum

84, 1J 36

H LOH •""OAc Cumambrin A

138

H l.oOH

Cumambrin B

139

577 (Table 1). contd.. Eudesmanolides Name

Structure

Ref. 1

Plant species

«UOH Angeloylcumambrin B

140

/

1

V""OAig

78

T. indicum

0

LPH 8-Deoxycumambrin B

141

T. densum ssp. amani 30

0

HQ

3,4-P-Epoxy-8deoxycu- mambrin B

/ ^ ^ ' i i ^

142

38

T. parthenium

0

8a1 Angeloyloxyestafiatin

143

/

V'»"OAng

0

T. parthenium

1

39

578 (Table 1). contd.. Eudesmanolides Name

Structure

8a-Hydroxyestafiatin

144

Plant species

Ref.

T. parthenium

39

T. parthenium

39

T. argenteum ssp. flabellifolium

22

T. argenteum ssp. canum var. canum

85,25

T. argenteum ssp.canum var. canum

85

o.«««

8aI sobutyry loxyestafiatin

OiBut

145 Oo«»

14

H .j/*

' 10 5y

i| Flabellin

146

H 15

Epoxyflabellin

•""OH

147

OvJ2/

t=^13

••«'0H

579 (Table 1). contd.. Eudesmanolides Structure

Name

Plant species

Ref.

lla-Dihydroflabellin

148

•OH

T. argenteum ssp.canum var. canum

85

np-Dihydroflabellin

149

••••OH

T. argenteum ssp.canum var. canum

85

A3(4).15.

150

T. argenteum ssp.canum var. canum

85

A3('^)-15-oxo-Flabellin

151

T. argenteum ssp.canum var. canum

85

lp,2|3-Epoxy3P,4a,10atrihydroxyguaian6a,12-olide

152

T. cilicium

82

Hydroxydihydroflabellin

HO

580 (Table 1). contd.. Eudesmanolides Structure

2-Keto-8a-hydroxy5a-6a-7pH-guaian1(10),3(4),11 (13)-trien-6,12-olide

Plant species

Name

Ref.

••lOH 153

T. vulgare

16

Dehydroleukodin

154

T. cilicium

82

Macrotanacin

155

T. macrophillum

56

Magnograndiolide

156

T. argenteum ssp. canum var. canum

IS

11,13Dehydrodesacetylmatricarin

157

T. cilicium

82

OH

581 (Table 1). contd.. Eudesmanolides Structure

Plant species

Name

Desacetoxy matricin

158

Parishin A

159

Pyrethin (Pyretine)

160

Pyrethrin

161

T. annuum

Ref.

79

T. densum ssp. amani 86

COCHzCP^

T. parthenifolium

57,81

T. parthenifolium

81

582 (Table 1). contd.. Eudesmanolides Structure

Name

Pyrethroidinin

162

Rupicoiin A

163

Rupicoiin B

164

Plant species

Ref.

T. densum ssp. amani 86

HO"

•"OH

T. santolina

18

T. santolina

18

T. parthenium

58,39

T. parthenium

39

PH Tanaparthin-aperoxide

165

PH Tanaparthin-pperoxide

166

583 (Table 1). contd.... Eudesmanolides Name

Structure

Plant species

Ref.

Tanciloide

167

T. cilicium

82

iso-Tanciloide

168

T. cilicium

82

Tanciloide 8amethylbuty- rate

169

T. cilicium

82

Tannunolide A

170

T. artnuum

SI, 19

T. annuum

79

0'***|

•OAc

8a-Acetoxy-6-epitannu- nolide A

171

584 (Table 1). contd.. Eudesmanolides Structure

Name

Plant species

Ref.

Tannunolide B

172

T. annuum

87,79

Tannunolide C

173

T. annuum

79

Tannunolide D

174

T. annuum

79

Tannunolide E

175

T. annuum

79

HO

585 (Table 1). contd Eudesmanolides Name

Structure 8a-Acetoxytannunolide E

Plant species

176

T. annuum

Ref. 79

iiiiiOAc

Tuneful in

177

T. inducum var. tunefol OAng

Seco-Guaianolides 3-Methoxytanapartholide

178

T. cilicium

82

MeO

Seco-tanapartholide A

179

T. parthenium

30,58

Seco-tanapartholide B

180

T. parthenium

58,39

HO

\

586 (Table 1). contd.. Eudesmanolides Structure

Name

i

Tanaphillin

| Plant species

181

Ref. 1

T. macrophillum

^6

T. parthenium T. macrophyllum T. odessanum T. corymbosum T. cilicium

39 89 90 91 92

T. odessanum T. corymbosum T. cilicium T. macrophyllum

90 91 92 89

r. aucheranum

93

T. odessanum

90

186

T. odessanum

90 21

187

T. densum ssp. sivasicum T. densum ssp. eginense

0

1

Farnesols p-Farnesene

^^^^^^'^^^-^\|S^»'^\x^'*5'^Y^^^

182

U

Famesol 3,10-Dihydroxy-5,8-diacetoxy-l(2),ll(12)de- hydrofamesol

1

H

,5

183

r r

f«

184 OAc

12-Acetoxyfarnesol acetate

185 \^SS!!^^X^V,p!^»'^X^'^s^^^'\X^Ac

1

4-Hydroxyfarnesolacetate lO-hydroxy-5,14diacetoxy-11,12dehydrofarnesol acetate

AcO

26

JDCOCH^

ll-Hydroxy-5,14diacet- oxy-9,10dehydrofarnesolacetate

188 1 i

5-Hydroxy-9acetoxynerolidol

T. densum ssp. 1 21 sivasicum T. densum ssp. 26 eginense

on

189

T. polycephalum '

OAc

'

OH

'

50

1

587 (Table 1). contd.. Eudesmanolides Structure

Plant species

Name

Ref.

QH

5,8-Diacetoxynerolidol

190

5,ll-Dihydroxy-8,9dihyd-ro-9J0dehydronerolidol

Sesquiphellandren

T. aucherianum

94

191

T. cilicium

82

192

T. odessanum

90

OAc

Longipinane Derivatives

cis-Longipinane-2,7dione

193

T. vtilgare

95

Trans-Longipinane2,7-di- one

194

T. densum ssp. sivasicum

36

1 -oxo-a-Longipinene

195

T. tanacetioides

44

588 (Table 1). contd.. Eudesmanolides Name

Structure

Plant species

Ref.

Vulgarone A

196

T. vulgare

96,97, 98

Vulgarone B

197

T. vulgare

96,97

Pyrethrins

Cinerin I

99,100, T. cinerahaefolium 101,102,1 103

198 R

H R=R'=CH3

H X

Cinerin II

Jasmolin I

199

R

H o R=COOCH3

o

T. cinerahaefolium 100, 101,

R'=CH3

T. cinerahaefolium 100,101, 102,103

200

R=CH3

Jasmolin II

:x^

H H

R'^CHjCHs

201 R=COOCH,

R'KTH^CHj

T. cinerahaefolium 100,101, 102

589 (Table 1). contd.. Eudesmanolides Name

Structure

Pyrethrin I

H R=CH3

o R'=CH=CH2

203

V

T

R=COOCH3

Ref.

100, 101 T. cinerariaefolium 90,102, T. odessanum 103 I

202 R

Pyrethrin II

Plant species

r

2"

T. cinerariaefolium 101,1001 90,102, T. odessanum 103

R'=CH=CH2

Triterpenes

T. densum ssp. eginense a-Amyrin

204

26 22

T. argenteum ssp. flabellifolium

P-Amyrin

205

a-Amyrin acetate

206 AcO'

T. densum ssp. eginense

33

T. argenteum ssp. flabellifolium

22

r. argyrophyllum var. argyrophyllum

35

590 (Table 1). contd.. Eudesmanolides Name

Structure

Fridelin

207

e/7/-Friedelinol

208

Plant species

r^i

Ref. 1

T. albipannosum 72

T. densum ssp. sivasicum T. albipannosum T. heterotomum

36 72 104

T. heterotomum

104

T. densum ssp. sivasicum

36

HO^^^Y'^I^'^^

J.., Lupeyl acetate

209

J: Magnificol

210

591 (Table 1). contd.... Eudesmanolides Structure

3P-

Plant species

Name

211

AcO

Ref.

T. sinaicum

49

T. sinaicum

49

T. heterotomum T. cinerariaefolium

104 20

Acetoxymalabarican14(26),17E,21-triene 24

3-Oxo-malabarica14(26), 17E,21- triene

212

18^25

24

Taraxasterol

23

213

23

Others

Balsamiton

214

7! balsamita ssp. balsamitoides

105

592 (Table 1). contd.. Eudesmanolides Structure

Name

Plant species

Ref.

.OH

Chrysentunone

215

Dimeric coniferyl alcohol derivative

216

Davanone

Hydroxydavanone

T. indicum var. tunejul

217

218

T. sinaicum (Pyrethrum santolinoides)

49

T. vulgare

106

T. sinaicum (Pyrethrum santolinoides)

49

T. sinaicum (Pyrethrum santolinoides)

49

T. vulgare

106

T. heterotomum

104

OH

O^ ^ O

6',8'- Dimethoxyfeselo!

219

593 (Table 1). contd.. Eudesmanolides Name

Structure

Plant species

Ref.

T. parthenium

107

T. heterotomum

104

CH^O^fi;

Isofraxidin drimenylether

220

CH,0^6;

y^

6-Oxo-drimenol-3aisova- lerateisofraxidin-ether

221

Indicumenone

222

tw

OCH3

iValO* 12

13

T. indicum yar.tuneJUl 108 T. inducum

OH

(+)-Sesamin

223

T. cinarahaefolium

31

T. vulgare

109

lU

Tanavulgarol

224

"YT"

594 (Table 1). contd.. Eudesmanolides Structure

Vulgarolide

(2S*,3S*,6R*)2,6Dimethyl 3,6epoxyocta-7enoic acid

Name

(6S,7S,10R)-2Hydroper- oxy-2,6, 10trimethyl-7,10epoxydodeca-3,11dien-5- one

Ref.

T. vulgare

110

54

226

T. sinaicum (Pyrethrum santolinoides) T. vulgare

106

T. sinaicum (Pyrethrum santolinoides)

54

228

T. vulgare

106

229

T. vulgare

106

T. vulgare

106

HO-.oS'''n' f"v 4

I

2,6,10-Trimethyl-2,5epidi- oxy-7,10epoxydodeca-11-en

(6S*,7SM0R*)-3Hydroxy 2,6,10-trimethyl-7,10epo- xydodeca-1,11dien-5-one

|

225

227

(5S,6S,7S,10R)-2,6,10Trimethyl-2,5epidioxy-7,10epoxydodeca-3,11dien-5-ol

Plant species

5

s

230

231

OOH

595

2.1. Sesquiterpenoids The isolation and the structure elucidation of sesquiterpenoids of the genus Tanacetum have been investigated due to their biological activities and their usage as markers in biochemical systematic studies. The presence of sesquiterpene lactones having an a-methylene-y-lactone moiety is characteristic of the genus, which contain mainly germacranolides, eudesmanolides and guaianolides. In this section the sesquiterpenoids of Tanacetum species growing in Turkey will be discussed, however we wish to point out a few confusion in the literature. Yunusov et al worked on the sesquiterpenoids of Tanacetum species in 70's. They isolated a number of new compounds however, they, sometimes reported only the name of the new compounds with a few data which is impossible to display in the tables. The new sesquiterpenoids tanacin and tachillin were isolated from T pseudoachillea. Although a few spectral data was given for tachillin, the type and the structure of the sesquiterpenoid is not clear [70], therefore the compound could not be placed in the tables. No spectral data was given for pyrethin whose formula presented as 10a-hydroxy-3,4-epoxyguai-ll(13)-en-6,12-olide, and isolated from T parthenifoUum, and it was named first pyretine then pyrethrin. In these works the structure of isoridentin was not given as well [57,81]. So far, thirteen endemic Tanacetum species, containing mainly sesquiterpene lactones, and flavonoids have been investigated by our group. The aerial parts of T heterotomum yielded two sesquiterpene-coumarin ethers (219, 221), one of them being new (221) (Table 1). The structure of the compounds were established by spectral methods (Table 2 and 4) [104]. The aerial parts of T argyrophyllum var. argyrophyllum afforded the known sesquiterpene lactones, 8a-hydroxyanhydroverlotorin (119), tanachin (103), tavulin (111), isospeciformin (98), a germacranolide with an 1,5-ether linkage (63), dentatin A (14) and a new germacranolide, tanargyrolide (108) (Table 1). The ^H NMR spectrum of 108 indicated the presence of an a-methylene-y-lactone. From the results of the spin decoupling, in addition to the lactone moiety three further secondary carbons with oxygene functions were recognised. The acetylation gave 5,6diacetate as the signals for H-5 and H-6 were shifted downfield. Thus, the presence of a 12,8-germacranolide with a 1,4-ether bridge was decided. The stereochemistry followed from the results of the NOE experiments (Table 2 and 4) [35]. The aerial parts of T, densum ssp. sivasicum yielded the known sesquiten)enoids, 8a-hydroxyanhydroverlotorin (119), deacetyltulipinolide-lp,10a-epoxide (116), spiciformin (97), deacetyllaurenobiolide (81), isospiciformin (98), la-hydroperoxy-1-desoxo-chrysanolide (55),

596

tanachin (103), tavulin (111), dentatin A (14), cumambrin A (138), cumambrin B (139) as well as the new eudesmanolide named sivasinolide (40), the new geraiacranolide, isotanargyrolide (109) and two new famesol derivatives (187, 188) (Table 1) [21,36,73]. The structure of the new eudesmanolide, sivasinolide (40) followed from its ^H NMR spectrum (Table 2). A pair of doublets at 6 6.18 and 5.95 were typical signals of an a-methylene-y-lactone (H2-13) protons. The appaerance of H2-I3 signals as doublet of doublets in (i^-acetone and a deshielding effect on one of the protons and the chemical shift of H-6 indicated an a-oriented hydroxyl group at C-6 [111]. All the resonances were assigned on the basis of spin decoupling and COSY experiments. The relative stereochemistry was supported by the results of NOESY experiments [36]. The ^H NMR spectrum of 109 in CDClswas reminiscent of that of the isomeric germacranolide diol 108. Better spectral dispersion for 109 was obtained using CD2CI2 as the solvent wherein all 17 magnetically distinct protons were observed (Table 2). ^^C NMR spectrum showed 15 carbon resonances (Table 3). The germacranolide skeleton of the compound deduced from the homonuclear couplings (COSY and LRCOSY) was confirmed by heteronuclear couplings observed in SINEPT and FLOCK experiments. The trans lactone stereochemistry at C7/C8, was confmned by NOE's observed in NOESY and DNOE spectra. The absolute stereochemistry of trans diol was determined by CD exciton chirality method using the bis-/?-bromobenzoates and the stereochemistry was concluded to be (5R, 6S) [73]. The ^H and ^^C NMR spectra of 188 indicated a farnesol derivative (Table 2-4). The NOESY correlations supported the structure. The structure of 187 was very similar to that of 188 except for the terminal section of the molecule (Table 2 and 4) [21]. T. cilicium afforded five new guaianolides and a new secoguaianolide (178) in addition to known ones; canin (134), dehydroleukodin (154), 11,13-dehydrodesacetylmatricarin (157), lp,2P"epoxy-3p,4aJ0atrihydroxyguaian-6a,12-olide (152), 5.1 l-dihydroxy"8,9-dihydro•-9,10dehydron (191). The *H NMR spectra of the new compounds 136 and 137 were very close to that of canin, except for the presence of the additional 8a-isovaleryloxy and 8a-methylbutryloxy ester groups respectively. In addition to diepoxy-guaianolides, the plant afforded three monoepoxy derivatives as well. The ^H NMR spectrum of the derivative of 167 indicated ip,2a,10a-threehydroxy-3a,4a-epoxyguaian 6a,12olide named tanciolide. Other two derivatives were isotanciolide (168) and 8a-methylbutyrloxytancioUde (169) (Table 2 and 4) [82]. The new eudesmanolide, ip,4a,6a-trihydroxyeudesm-ll-en-8a"12olide (26) and the guaianolides; pyrethroidinin (162) and parishin A (159) were isolated from T. densum ssp. amani together with the known sesquiterpene lactones, deacetyllaurenobiolide (81), parthenolide (87), tanachin (103), 8-deoxycumambrin B (141), peroxyparthenolide (91), dentatin A (14), 8a-hydroxyanhydroverlotorin (119), spiciformin (97),

597

and deacetyltulipinolide-lp,10a-epoxide (116) (Table 1-4) [30,86]. Pyrethroidinin was first isolated from Pyrethrum pyrethroides [112,113] and parishin A was first reported from Artemisia tridentata ssp. tridentata f. parishi [114] and subsequently from an unidentified Eriocephalus species [115]. Neither carbon data were reported for (162) and (159) in the past reports, nor the proton spectra were completely assigned. In the original report of pyrethoidinin the structure was confirmed by X-ray crystallography (relative stereochemistry) and only selected proton chemical shifts were given. In our work full NMR assignments and absolute stereochemistry of pyrethoidinin and parishin are given. Each guaianolide possess two olefmic double bonds, one of which is part of an methylene-y-lactone functionality. The only distinction in the spectra of 162 and 159 was the oxidation of a secondary allylic alcohol group in 162, ^H: 6 4.56 (brd, J=7.6 Hz, H-13), ^^C: 5 80.5 (d, C3), to an a , p unsaturated cyclopentanone group in 159, ^^C: 6 200.6 (s, C3). This was confirmed experimentally by Jones oxidation of 162 to yield 159. The signals in the ^H NMR spectrum of 162 were assigned by COSY experiments. HETCOR spectrum enabled assignment of the corresponding carbons. LR COSY and SINEPT experiments supported the proposed structure. Stereochemical assignments were based on NOEs and coupling constants. A trans lactone was also indicated by the (n—>n*) Cotton effect in the CD spectrum according to Geissmann's general rule. Application of the Mosher-Trost model led to the conclusion that the C3 alcohol is an R center with the full absolute stereochemistry of 162 as (IR, 3R, 6S, 7S, lOR), and that of 159 as (IR, 6S, 7S, lOR), the same absolute stereochemistry as isophoto-a-santonic lactone [116,117] and assumed (but rarely confirmed) for all guaianolides in the Asteraceae [86]. The aerial parts of T densum ssp. eginense yielded a number of known sesquiterpenoids, deacetyllaurenobiolide (81), spiciformin (97), l a hydroperoxy-1-desoxo-chrysanolide (55), deacetyltulipinolide-1 p,10aepoxide (116), ll-hydroxy-5,14-diacetoxy-9,10-dehydrofamesol acetate (188), 10-hydroxy-5,14-diacetoxy-ll,12-dehydrofamesol acetate (187), tatridin A (111), l-ej9/-tatridin B (103), tamirin (99), armexifolin (29), deacetyl-P-cyclopyretrosin (34), in addition to a new eudesmanolide; eginense (16) (Table 1, 2 and 4). The *H NMR spectrum of 16 in CDCI3 showed a methyl singlet (H-14) at 5 1.03 and broadened exocyclic amethylene-y-lactone doublets (H2-I3) at 5 5.08 and 4.92. Since H-5 locates closer to C-15 protons and couples with them ca. 1.5 Hz in case of cisfused decalin ring compared to trans-fusQd decalin ring as we observed on a Dreiding model, the appearance of the H2-I5 (J=1.5 Hz) and the lower chemical shift of H-14 with respect to its trans-isomtT (Bohlmann 1982) indicated c/^-fusion of the decalin ring. All signals were assigned by spin decoupling experiments. Acetylation of the compound gave a diacetyl derivative. Inspection of Dreiding models and a NOE study confirmed the structure [26].

598

The aerial parts of T, chiliophyllum var. heimerlei afforded the known sesquiterpenoids, spiciformin (97), desacetyllaurenobiolide (81), l a hydroperoxy-1-desoxo-chrysanolide (55), tavulin (111), tanachin (103), tamirin (99), and dentatin A (14) in addition to two new germacranoiides; chiliophyUin (53) and heimerlein (79) (Table 1) [37]. Tanachin, tabulin, tamirin and dentatin A are common sesquiterpene lactones in Tanacetum species that we investigated. Tanachin, tabulin and dentatin A were first isolated by Yunusov et.al. [23,66]. There were some confusion about the structures of tabulin, tanachin, tatridin A and tatridin B in the literature [34,66,118-121]. Sanz and Marco reinvestigated the structures of tabulin and tanachin and reported that tabulin was in fact tatridin A and tanachin was l-^/7z-tatridin B [15]. Due to this literature tabulin and tanachin which we have isolated from several Tanacetum species [30,35,36,] must be revised as tatridin A and X-epi- tatridin B, respectively. During our recent works, we have also found some confusion in the structure of dentatin A. Although dentatin A was first isolated and its formula was given by Yunusov, the structural data given in the literature was not sufficient [23]. Therefore we have reinvestigated the structure of dentatin A. The ^H and ^-^C NMR data and NOE experiments were given in the work (Table 2-4) [24]. The structure of the new germacranoiides; heimerlein and chiliophyUin were elucidated mainly by ^H and ^^C NMR data. The stereochemistry of 79 was based on comparing the ^H NMR data with those of spiciformin and by Dreiding models. The C-11 configuration assignment was based on the magnitute of Jy n (5.5 Hz). The configuration of chiliophyUin at C-11 position was deduced from the large value of J7 n (10 Hz) [24]. The aerial parts of T. aucheranum gave only a new famesol derivative as sesquiterpenoids (Table 1). ^H and *^C NMR spectra 184 indicated a famesol derivative having two acetoxy groups, two hydroxyl groups, and a terminal double bond of an aliphatic chain. The two broadened singlets at 5 4.97 and 4.84 indicated an exocyclic methylene group instead of a methyl group (Table 2-4). The proton bearing carbons were assigned by HETCOR experiment. The main problem in this structure was the locations of the hydroxyl and acetyl groups at C-9 and C-10. The hydroxy group could be at C-9 and the acetoxy group at C-10 and vice versa. The fragments in the EI mass spectrum at m/z 71, 95, 97 and 149 indicated that the hydroxy group was at 10. In addition, the cross peaks between H-12, H-10, H-12' and C-10 in the COLOC spectrum supported the proposed structure [93]. Although glaucolides have been isolated almost exclusively from members of the tribe Vemonieae (Compositae), being the largest genus of the tribe [122,123,124,125], they were found in the tribe Anthemidea as well [126]. For the first time glaucolide-like lactones were found in a Tanacetum species. Tanacetum albipannosum gave two new glaucolides; tanalbin A (106) and tanalbin B (107) in addition to some known

599

flavonoids (Table 5). The structure of the compunds were elucidated by means of spectral data as well as NOE experiments (Table 2 and 4) [72]. The aerial parts of T, praeteritum ssp. praeteritum yielded almost only eudesmanolides, douglanin (15), santamarin (37), reynosin (35), arglanin (4), ludovicin A (27), ludovicin B (28), armexin (8), armefolin (6), armexifolin (29), 3 a-hydroxyreynosin (36), epoxysantamarin (38), the germacranolide, l-e-j^^Z-tatridin B (103) and the new ones, la-hydroxy-1desoxo-arglanin (5), 3a-peroxyarmefolin (7), praeteritenolide (31), tanapraetenolide (42), arglanic acid methyl ester (45), l a , 6 a dehydroxyisocostic acid methyl ester (47) (Table 1). The structures of the compounds were elucidated by spectral data (Table 2-4) [17,45]. The aerial parts of T. argenteum ssp. argenteum afforded the known sesquiterpenoids, desacetyllaurenobiolide (81), spiciformin (97), tatridin A (111), l-e/?/-tatridin B (103), deacetyl-P-cyclopyrethrosin (34), desacetyltulipinolide-ip,10a-epoxide (116) as well as two new germacranolides, 8a-angeloyloxy-costunolide (58) and 1 P-hydroperoxy8a-costunolide (59) (Table 1). MH and ^^C NMR spectra of 58 (Table 2 and 3) indicated a costunolide having an angeloyloxy group in a-position at C-8. Treatment of 58 with m-CPBA resulted in the unstable Cl-ClO monoperoxide which underwent frofw^-annular cyclization under mild acidic conditions to give a mixture of 58a and 58b, indicating the transtrans decadien structure of 58 [128]. Compound 58 was isolated previously from Brachylaene nereifolia [129], however on carefull checking of the publication a missprint was observed. The authors indicate that the compound is the 8a-angeloyloxy derivative of salonitenolide [111]. The ^H NMR spectrum of 59 was similar to that of 58, with the exception of the signals at 5 4.12 (H-1, brm), the broadened singlet at 5 8.02 and the exocyclic methylene proton signals at 6 5.48 and 5.28. H-1 appeared as a broadened doublet at 53.75 (J=10 Hz) in C6D6 indicating a p-peroxy group at C-1 and exocyclic methylene group at C-10. Acetylation afforded the keto derivative of 59, indicating the presence of the hydroperoxy group at C-1 [33]. The aerial parts of T. argenteum ssp. flabellifolium afforded a number of known germacranolides, parthenolide (87), desacetyllaurenobiolide (81), spiciformin (97), desacetyltulipinolide-ip,10a-epoxide (116), tatridin A (111), l-ep/-tatridin B (103), tamirin (99), desacetyl-P-cyclopyrethrosin (34), isospiciformin (98), the known eudesmanolides, sivasinolide (40), dentatin A (14) and a new guaianolide; flabellin (146) (Table 1). The ^H NMR spectrum of 146 indicated 8a,10a-dihydroxyguaian-4(15),ll(13)dien-6a,12-olide (Table 2). All signals were assigned by spin decoupling experiments in the ^H NMR spectrum. APT and HETCOR spectra were in accordance with the structure (Table 3) giving one methyl, five methylene, five methine and four quarternary carbon signals. NOE experiments confirmed the location of the exocyclic methylene group. Irradiation of H-5 caused enhancement of the signals of H-15', H-7 and H-

600

1, indicating that H-7, H-5 and H-1 are in the a-position, and H-15' and H-5 are close to each other in space. The relative configuration of the compound was determined by X-ray measurements (Table 4). We named the compound flabellin [22]. The aerial parts of T. argenteum ssp. canum var. canum afforded a number of known sesquiterpenoids, parthenolide (87), peroxyparthenolide (91), dihydroparthenolide (88), l-^p/-tatridin B (103), sivasinolide (40), flabellin (146), lp,4a-dihydroxy-6a-angeloyloxyeudesm-l l(13)-en-8,12olide (21), lp-hydroxy-6a-angeloyloxyeudesm-4(15),l l(13)-dien-8,12olide (19), michelenolide (85), magnograndiolide (156), santamarin (37), douglanin (15), and the new ones, ip-hydroxy-6a-angeloyloxygermacra4(5),10(14),ll(13)-trien-8,12-olide (66), and ip,4a-dihydroxy-6aisobutyloxyeudesm-ll(13)-en-8,12-olide (22) and a series of new guaianolides which are the derivatives of flabellin, 147-151 (Table 1). The % and ^^C NMR spectra of 66 and 22 indicated a 8a,12-lactonised germacranolide having a P-hydroxy group at C-1 and an a-angeloyloxy group at C-6 and 8a,12-lactonised eudesmanolide having a P-hydroxy group at C-1, an a-hydroxy group at C-4, and an isobutyryloxy group at C-6 respectively (Table 2 and 3). The structure of the new germacranolide and the new guaianolides were elucidated mainly by spectral methods (Table 4). The ^H NMR spectrum of 147 was very similar to that of flabellin 146. The main difference between compounds 146 and 147 was the presence of the epoxy group at C-4 in 147, which appeared at 5 3.23 (d, y=4.5 Hz, H-15) and 2.93 (d, J=4.5 Hz, H-15') instead of the exocyclic methylene group at that position in compound 146. The stereoposition of the epoxy group was determined by X-ray analysis. 151 and 150 are derivatives of flabellin having a double bound between C-3 and C-4. Compound 151 has an aldehyde group at C-4, while compoimd 150 has a hydroxymethylene group at that position. This was clearly seen from their mass spectra. The HRMS of 151 exhibited a peak at m/z 248.1950 corresponding to C14H16O4 [M-CHOH]-', whereas the HRMS of 150 afforded a peak at m/z 280.1305 corresponding to C15H20O5 [M]"^. In the ^H NMR of 151 a singlet at 6 9.74 indicated the presence of an aldehyde group, due to this group the olefinic proton signal was shifted downfield to 6 6.94 (brs, H-3). The rest of the spectrum was quite similar to that of flabellin. The ^H NMR of 150 displayed the olefinic proton at 6 5.81 (brs, H-3) and a broadened singlet at 6 4.34 (2H, H2-I5), indicating the presence of a hydroxymethylene group at C-4, showing that the downfield shift of the olefinic proton in compound 151 was caused by the effect of the aldehyde group. The other signals of compound 150 were similar to that of flabellin. Spin decoupling, APT, HETCOR experiments were used to assign the resonances of the two compounds. The Rf values of the compounds 148 and 149 were quite different from each other, Rfus- 0.10 and Rfi49: 0.21 in ether. There were few differences in the 4i NMR spectra of 148 and 149, although their *H NMR spectra were very similar

601

to that of flabellin. In the ^H NMR spectrum of 148, a secondary methyl resonance was observed at 5 1.41 (d, J=7 Hz, H-13) rather than the exocyclic y-lactone methylene protons in flabellin, in addition to a methyl singlet attached to a hydroxyl group at 5 1.30 (s, H-14). The exocyclic methylene protons (H2-15) appeared at 5 5.17 (brs) and 4.99 (brs) in the spectrum of 148. The compound gave a better dispersion in MoiCO-ds for the resonances of hydrogens 1 through 9. In the ^H NMR spectrum of 149 the secondary methyl doublet appeared at 5 1.25 (d, J=7 Hz, H-13) and the tertiary methyl singlet at 6 1.25 (s,H-14). The exocyclic methylene signals were observed at 5 5.18 (brs, H-15) and 5.01 (brs, H-15'). As seen in the Table 2, the chemical shifts of the other signals were also slightly different from each other in each spectrum. Compound 149 also gave a better dispersion in Me2CO-(i5 and the overlapped methyl signals appeared at 5 1.27 (d, J=7 Hz, H-13) and 5 1.14 (s, H-14). Thus, compounds 148 and 149 seemed to be C-11 epimers. NOE experiments were carried out on the acetyl derivative of 148. Irradiation of H-11 at 6 2.45 (m, overlapped with H-1) showed NOE with the p-oriented protons at 5 3.99 (H-6) and 5 5.11 (H-8) indicating a-orientation of the methyl group at the lactone ring. It also showed NOE with H-14 at 5 1.24 due to the overlapping H-1 signal. In addition, a NOE was observed between the neighboring protons, H-11 and H-13. The ^^C NMR spectrum of 148 supported the proposed structure. The proton-bearing carbons were assigned by HETCOR experiments [25,85]. 2.2. Steroids and Triterpenoids P-Sitosterol and stigmasterol are the common steroids in the genus. So far only few common triterpenes such as fiiedelin (207), e/7/-friedelinol (208), a-amyrin (204), P-amyrin (205), a-amyrin acetate (206), taraxasterol (213), lupeyl acetate (209), magnificol (210) were isolated from Tanacetum species. However, T. sinaicum afforded unusual new malabaricane type triterpenoids; 3p-acetoxymalabarican-14(26),17E,21triene (211) and 3-oxo-malabarica-14(26),17E,21-triene (212) (Table 1). Table 2.

The *H NMR data of the new terpenoids isolated first from Tanacetum species C:60 MHz, ':80 MHz, ':90 MHz, MOO MHz,':200 MHz, ':250 MHz, ':270 MHz; *:300 MHz; *:360 MHz; '"400 MHz; "500 MHz, ":not given; -:Me20-d6/: C6D6,': CDCI3, "iCDiCli/: DMSO-ds;':CCl4; «:CDCl3+CD30D, 57"C; Pyridine-ds, 'CDCIj+CDjOD, 25*C; -A: Acetyl derivative of the compound)

Epj^$m^nQlide$ 1 H 1

[2

2.A»''

34,c 3.44 m

6*''

,5.c

3.42 br %

3.81 dd

5.87 dd

2.36 ddd

10>'^ 3.68 m

„l,c 3.62 m

5,c

„5.b

12»''

14*'*

3.61 dd

3.54 dd

3.77 dd

IMm

1.85 ddd

1.82 dddd

1.18 m

16»"'*

18*'*

20-A

23*;*

602 (Table2). contd. 1 H

2-A*'«

3^''

^^

10»''

„l,c

4.31 dd«-'

1.55 ddd

18^-'

20-A

5,c

23^'^ 1

1.65 dddd 2.34 ddd

|3'

„5.b

1.02 m

2.22 ddd

1'

1.87 (1

|6

4.10 1

4.11 dd

3.98 t

4.57 dq

I?

2.88 m

2.59 m

2.55 ddd

2.65 m

2J7d

js'

1.50 dddd

19

2.10 m

4.07 t

4.23 t

5.64 dd

1'' n

1.20 d

1.55 d

1

5.51 dd

4.21 t

1

2.54 dddd 2.57 m

2.33 dddd

2.61 m

2.38 dd

2.78 dd

2.22 dd

1.33 dd

1.37 dd

2.19 brd

2.02 brd

2.00 d

4.04 dd

3.85 dd

4.05 t

2.62 dddd

5.25 t

1.06 d 2.60 m 6.07 d

6.13 d

1

5.23 d

5.96 d

1

0.89 s

0.47 s

0.99 s

1

4.82 d

1.07 s

1.40 s

1

6.17 d

6.11 d

6.05 d

6.18 d

6.14 d

5.97 d

6.18 dd

6.17 brd

6.14 d

5.56 (1

5.45 d

5.42 d

5.50 d

5.60 d

5.50 d

5.99 dd

5.97 brd

5.96 d

1 ^* 1.05 t

0.96 bn

0.88 s

1.08 s

0.92 s

0.95 s

0.82 s

1.03 s

0.84 s

|l5

1.35 t

1.33 s

2.04 brd

1.83 s

1.80 s

5.0 brs

5.08 (

5.02 brs

4.87 b n

4.92 t

4.67 brs

4.33 d

|l3 13'

1.33 «

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1.17 d

8.19 br»

|oOH

2.53 s

;.

.

1-

6.85 m

6.12 m

1 ••"

1.30 s

4.32 m

1.95 dd

1 5"

1.30 s

1.82 s

1.88 brs

0 R : 2 " 2.10 s

1 3"

12.03 s

2.33 m 10.91 d

i

11.03 d

/ f i / z ) : 2 - A : 5 , 6 = 1 1 . 7 ; 6,7=11.3; 7,8=8.9=11; 8,9*=4.3; 7.13=7,13'=3; 3:5,6=11.3; 6,7=10.8; 7,13=3.2; 7.13'=3.1; 6:1.2=5.5; 2,3=10; 5,6=6,7=7,8=11; 7,8'=6; 7,13=3.5, 7,13'=3.0; 7:1.2=4; 1,2'=13; 2,2'=14; 2,3=2; 2',3=4; 3,15=1.5; 5,6=12; 6,15=1.5; 7,13=3.5; 7.13'=3.0; 10 and 11:5,6=6,7=11; 7,13=7,13'=3; 1 2 : 3 " , 4 " = 8 ; 4",5"=--l; 14:1,2=11.5; 1,2'=5; 5,6=6,7=7,8=11; 7,13=3; 7,13'=3.5; 8,9=4.5; 8.9'=12; 9,9'=12; 13,13'=1; 16:1,2=5; 1,2'=2,2'=12.5; 3.2=2; 3.2'=4.5; 3,3'=13; 3',2=5.5; 2*,3'=12.5; 5,6=6,7=10; 7.8=12; 7,13=3.5; 7,13'=3.0; 8,9=4; 8 , 9 ' - 9 . 9 ' = 1 2 . 5 ; 13,13*=1; 5,15=15,15'=1.5; 18:5,6=6,7=11; 7.8=8,9=11.5; 8,9'=3.5; 7,13=7,13'=3; 20-A:5.6=6,7=10.5; 7.8=8.9=12; 8.9'=4; 11,13=7; 15,15*=1; 22:1.2=4.5; 1.2'=5,6=6,7=10; 8,9=4; 8,9'=7,8=13; 7.13=3.5; 7,13'=3; 2 " , 3 " = 2 " , 4 " = 6 . 5 ; 2 3 : 5 , 6 - 6 , 7 = 1 0 ; 7,8=8.9=11.8; 8.9'=3.7; 7.13=3.2; 7.I3'=3;

1 II

a4-A

Svc

«"^'

u"^

»"-

3.»-'

1

3.73 brdd

3.50 m

3.92 dd

3.46 brd

2

1.82 ddd

1.70 m

2.60 dd

2.50 m

2*

2.44 ddd

2.76 dd

2.04 ddd

3

3.15 brdd 1.82 d

1.83 d

1.55 d

2.26 brd

6

5.40 t

3.98 dd

4.28 dd 4.76 d

4Jt3d

7

2.60 m

1.60 m

2.60 dddd

8

4.09 ddd 1.75 m

1.27 m

9

1.08 ddd

3.60 dd

33^

3.60 dd

3.94 iddd

2.80 brm

37'"

^^

3.68 dd 3.73 dd

5.35 m

5.52 t

5.52 1

2.80 ttt

2.80 ddd

2.95 ddd 4.10 ddd 4.0 ddd

1.90 m

42»"

43^ 1

45»-'

47*^' 1 3.35 d

4.16 dd 3.45 brs

3.98 t

5.40 m

5.90 m

5.86 d

5.28 m

2.09 brd

3.25 brd

2.57 d

2.21 brd

4.07 dd

4.25 d

4.00 dd

|2.51 m

3.33 m

2.65 dddddd

4.07 dd

2.20 m

1.65 m

2.50 m 2.47 dd

4.^'

6.60 d

5.38 m

5

8'

si'-*

[1.45 dd

2.20 m

5.9 m

4.15 dd 3.85 dd 2.57 m

4.15 m

2.48 m

603 (Table 2). contd..... 24.A

II

a**'

Z*""

2.68 m

2.26 m

13

1.16 d

1.23 d

2,*"

32'-'

6.16 dd 6.25 d

4.37 s

6.12 d

1.42 %

1.43 k

1.17 d

5.45 d 1.92 b n

2.04 d

15'

1 OH

1 OR

40*"

4/'

2.51 dd

5.98 dd 5.58 d 1.19 %

37''

43^

42»"

45»'

47*--

1.45 m

2.64 dd

13'

I 15

33^

1.50 ddd

1.48 dd

2.26 m

9' 11

3.«-*

4.89 brs

4.89 brs

4.62 b n

4.62 bbrs

6.10 d

6.18 brd

6.08 d

6.14 d

6.04 d

6.36 s

6.30 s

5.44 d

5.95 brd

5.40 d

5.48 d

5.37 d

5.80 s

5.73 d

1.87 s

1.95 brs

5.02 s

2.18 s

1.46 s

1.58 s

1.91 brs 1

5.9 m

3.82 s

3.77 s

5.02 s

2.59 s 2.06 s

2.1SS

2.06 s

1.76 s 1.93 s

J(Hz) : 24-A:5,6=6,7=10.6; 7,8=8,9=12; 8,9'=4; 11,13=7; 25:1,2'=6; 1,2=10; 2,2'=15; 2',3=1.5, 2,3=2; 5,6=6,7=10; 8,9'=4; 8',9'=9,9'=13.5; 11,13=7; 26:1,2=5; 1,2'=11; 5,6=6,7=10; 8,9=4; 7,8=8,9=9,9'=12; 7,13'=3; 7,13=3.5; 13,13'=1.5; 29:1,2=12.5; 1,2'=5.5; 2,2'=16, 6,15=1.5; 6,7=11; 13,13'=3; 31:1,2=4.5; 1,2'=2; 2,2^=14.5; 2,3=4.5; 5,6=11.5; 8,8'=14.5; 8.9=5; 8,9'=1.5; 9,9'=13; 32:1,2=10.2; 1,2'=5.0; 5,6=6,7=10.3; 7,8=8,9=11.6; 8,9'=3.7; 7,13=3.1; 7,13'=2.9; 33: 1,2=10.2; 1.2'=5.0; 5,6=6,7=10.3; 7,8=8,9=11.6; 8,9'=3.7; 7,11=12.0; 11,13=6.8; 37:1,2=6.0; 1,2'=9.6; 5,6=6,7=10; 7,13=3.2; 7,13'=3.0; 40:1,2=8; 1,2'=10; 2,3=3,15=1; 5,6=6,7=10; 7,8=12; 7,13=3; 7,13'=3.5; 8',9'=4; 8',9=9,9'=12; 41:1,2=5.0; 1,2'=5,6=11.3; 7,13=7,13'=3.3; 42:5,6=6,7=10; 7,13=3.5; 7,13'=3.0; 43:13,13'=3; 45:1,2=10; 5,6=6,7=11; 13,13'=3.0; 47:1,2=5; 5,6=6,7=10;

CerffiapranQUg^g »'•'

H

1

u'-

3.95 m

5g''

59''

4.92 brd

4.12 brm

TB—T"

60-A

(120 C)

4.60 brd

«**

44'^'

4.73 bn

«'*

«»-

4.89 brt

4.10 m

«'•'

«•«.' 5.03 dd

70

4.61 dd

7,'«" 1 Zio

brddd

1

2

1.98 m

6 20 ddd

2.25 m

1 ^' 1 ^ 1.8 m 1 ^*

1.5-2.2 m

1.85 ddd

3.12 dd 2.16 ddd 2.18 ddd

2.28 ddd 1

4.60 t

4.48 dd

4.46 brdd 1

3.70 d

4.92 dq

4.78 d

5.10 d

5.09 br d 5.23 brd

5.73 dd

5.34 dd

5.69 dd 5.94 dd

3.03 m

2.14 ddddd

3.08 m

1.60 m

4.80 dd

1.21 m

4.20 m

4.76 brd

5.32 brd

4.60 brd

4.23 d

1 ^

5.13 dd

4.85 dd

4.46 t

5.17 dd

4.43 t

2.25 m

3.00 m

3.04 dddd

3.26 brm

2.75 ddddd

2.62 m

4.05 ddd 5.10 ddd 5.27 brm

1.5-2.2 m

1.53 dddd 1.5-2.2m

2.46 dd

5.76 t

1.82 m

3.08 m

2.0 brd

2.25 dd

6.36 d

6.25 d

6.23 d

6.35 dd

6.26 d

6.25 d

6.36 d

6.34 d

5.88 ltd

1.47 dddd

2.00 m

1.81 m

2.30 m

2.22 m

2.00 m

1.76 m

2.75 m

2.45 ddd 2.57 m

2.08 m

1.90 m

5.23 m

5.36 ddq 2.23 brdd

1.90 m

5.23 m

5.36 ddq 2.62 brdd 1

2.30 m

2.24 m

2.43 m

2.35 m 1

1.22 d

1.22 d

1.22 d

1.24 d

5.88 d

5.58 d

5.63 d

5.64 dd

5.45 d

5.63 d

5.12 d

5.88 d

1 ^*

5.83 brs

1.57 s

5.48 brs

1.32 b n

2.50 d

1.70 s

1.67 b n

5.17 br» 5.79 b n 1.68 dd

14'

5.00 t

5.87 brs

5.28 b n

1.45 d

2.38 d

1.63 d

1.76 d

1.70 d

1.70 b n

1 OR:3" 3.41 s

2.07 8

6.10 qq

6.20 qq

1.18 s 1.69 s

1.82 d

5.02 b n 5.85 b n 1.75 s 2.15 s

1.35 d

1.85 d 6.13 qq

1

•*"

1.85 dq

1.90 dq

1.89 b n

1

5"

1.91 dq

2.01 dq

1.94 dq

5.24 dq

5.90 dd

5.07 brs

1 '^

4.48 dd

2.65 m 4.98 brd

3.51 dd

|

2.34 ddd 1

3.88 dd

4.21 dd

1 13'

1.92 ddd 2.02 m

5.10 m

5.11 brd

1 ^ 3.95 m 1 *' 1 ' 2.70 ddd 1 '* 2.48 dd 1 ** 2.85 ddd 1 *^ 3.87 dd

2.90 dd

1.32 dt

5

1^

2.45 ddd

1

1.73 b n 1.67 d

1

5.32 bn 1 15.13 b n

1.76 d

1

1.78 d

604 (Table 2). contd..

P^^

H

54^'^

5»'>'

OOH 1

59*''

4,b 60-A 0 (120 C)

62*'''

64^^''

65^'"

66*'^

67^''

68>»''

70^''=

"TjTirrj

6.76 bn

8.02 b n

OH 1

5.10 bra

3.60 brd

J(Hz):53:\,2=5; 1,2'=5,6=6,7=7,11=8,9'=10; 5,15=1.5; 8,9=3; 11,13=3; 11,13'=9; 13,13'=9; 9,9'=15; 9,14'=2 54:5,6=9.8; 7,8=11.1; 8,9=5.3; 8,9'=3.1; 5,15=0.8; 7,13=2.9; 7,13'=2.4; 13,13'=0.8; 58:5,6=10; 6,7=8; 7,8=7 8,9=10.5; 8,9'=3; 7,13=3.5; 7,13'=3.0; 9,9'=13; 3",4"=7; 5,15=13,13'=3",5"=4",5"=1.5; 59:1,2=10; 1,2'=3 5,6=6,7=8,9=10; 7,8=8; 9,9'=13; 3",4"=7; 3",5"=4",5"=1.5; 60-A:l,2=5; 1,15=1; 2,3=5,6=7,8=10 2,3'=7,8'=7,13=7,13'=3; 3,3'=13; 6,7=9; 13,13'=1.5; 62:2.3=14; 2',3=5; 3,3'=14; 5,6=6,7=9.0; 7,13=3.3 7,13'=3.1; 8,9=8',9=7.5; 14,14' = 13; 64:5,6=7,8=10; 8,9=4; 7,13=7,13'=3; 65:1,2=1,2'=9; 2,2' = 14 2,3=2'.3=6,7=7,13=7,13'=8',9'=3; 5,6=10; 5,15=1.5; 7,8=2; 7,8'=11; 8,8'=16; 8',9=12; 9,9'=13; 66:5,6=6,7=10: 7,8=8; 8,9=5.5; 8,9'=14; 7,13=3.5; 7,13'=3; 5,15=1.5; OR:3",4"=7; 3",5"=4",5"=1.5; 67:2,2'=4.5; 2,3=5 2',3=3;5.6=10; 6,7=9; 11,13=7; 5,15=1; 68:1,2'=11.5; 1,2=2; 2,2'=14.5; 2',3=2.5; 2,3=5; 5,6=6,7=10; 5,15=1.5 7,8'=8',9=11; 7,8=2; 7,11=12; 8,8'=13.5; 8,9=6; 8\14=9,14=1; 11,13=7; 70:1,2=3.5; 1,2'=4; 2,3=3; 2',3=2.5 5,6=10; 5,15=9,14=1; 6,7=8',9=11; 8,9=6; 11,13=7; 71:1,2=4; 1,2'=10; 2,2'=16; 2,3=5; 2',3=4; 3,OH=6 5,6=6,7=8',9=10; 5,15=1.5; 8,9=8; 9,9'=15; 11,13=7; „io,c

H

73^'*=

,,7,c

75»2''

762''

77^'^

„5,c 5.35 m

84»«''

86^'"

87»«''

89»«''

90»»''

92^'

1 1

3.92 dd

5.40 t

2.79 dd

5.58 brt

5.10 brd 3.85 d

2

2.16 m

2.4-2.0 m

2.55 m

3.08 d

4.23 m

1 ^' 1 ^4.48 b n 1 ^'

2.4-2.0 m

1.75 m

2.4-2.0 m

4.45 dd

1 5

5.10 d

5.45 dd

2.33 m

5.00 t

5.15 d

2.64 bn

2.65 d

5.33 brd

2.78 brd

2.79 d

2.87 d

5.14 dd

4.95 dd

6.05 d

3.57 brd

5.20 dd

5.73 dd 4.12 dd

3.67 t

4.29 m

3.85 t

3.91 t

3.76 t

5.38 dd

3.90 d

1.73 m

2.48 m

2.99 tt

3.09 m

2.73 ddddd

3.26 ddddd

3.07 m

4.35 m

4.86 t

4.29 m

4.15 ddd

1

2.25 m

5.20 brd

5.11 brdd

4 J 7 dd

2.03 m

2.4 m

2.42 m

2.00 m

2.30 m

1.95 m

2.5 m

2.42 m

1.77 m

4.77 dd

2.10 m

3.41 brdd

0.98 ddd

2.5 m

1.11 ddd 2.15 ddd

2.75 brd 5.18 brd

1 ^

5.94 dd

1 7 8

1.75 bnidd 3.42 dd

2.10 dd

2.36 brd

1.45 m

1.73 m

1.75 m

1.99 m

4.00 dd

1 ^'

jl.SO ddd 12.83 dd

4.20 d

1 '' 1 *'2.31 dq

12.83 dd

1 13 |1.26 d

6.15 d

1 *'*' 11.24 s 1 ^*

5.75 d

1 *^'

1 '*|l.80 b n

1.60 s

1.20 m

4.23 s

1.42 m

2.35 m

1.59 m

2.95 dq

2.26 m

1.16 d

1.45 s

2.70 m

15.46 dq

1.67 m

1.77 m

2.5 m

obscured

2.40 m

2.5 m

2.34 brdd

2.40 m

2.4 m

2.13 m

2.25 brdd

1

2.92 ddd

6.26 d

6.30 d

3.85 dd

|6.33 brd 6.33 b n

|6.33 d

6.33 d

6.23 d

6.37 dd

1

5.70 d

6.15 d

3.56 dd

|6.24 brd 6.33 b n

5.62 d

5.62 d

5.52 d

5.92 dd

1

il.65bn

2.10 bra 1.32 s

1.75 s

|l.85d

11.70 s

1.71 b n

5.23 b n

1.30 s

1

1.22 d

11.28 s

|5.44 b n 11.60 s

1.80 b n

1.05 d

1.68 b n 1.60 bn 1.47 s

1 OR

1 OH

2.71 q

2.17 m

2.30 t

2.4-2.0 m

1 4

1 9

2.85 t

4.83 brdd

[1.45 s

11.65 s

3.40 s

2.50 b n

|l.29 s

11.29 s

|l.43 s

|1.85d 2.09 s

1

[6.89 d

yC^z;:72:l,2'=10.5; 1,2=2.5; 5,6=6,7=10; 7,8'=8',9=11; 7,11=12; 8,8'=14.5; 8,9=3; 11,13=7; 73:1,2=1,2'=8 5,6=8.9=10; 6,7=8; 7,8=7,13=7,13'=3; 74:1,2=4.5; 1,2'=5; 2,3=2; 2\3=3; 5,6=7,8=11; 5,15=1; 7,11=8.5 11,13=7.5; 75:1,2=9; 4,15=6.8; 5,6=11; 6,7=3; 7,13=7,13'=1.5; 76:1.2=8; 3,3'=12; 5.6=10.5; 6,7=9; 7,13=7,13'=3 77:1,2=5; 3.2=3,2'=9; 5,6=6,7=11; 11,13=7; 5.15=1; 79:5,6=3.5; 6,7=11; 7,11=5.5; 11.13=11.13'=3; 13.13'=9: 84:1,2=11; 1,2'=5; 5,6=6,7=9.5; 7,8=8,9=9; 9,14=1; 86:1,2=2.5; 1,2'=10; 5,6=10; 87:1.2=7.7; 5,6=4.9: 7,13=7.13'=3; 89:1.2=11.5; 1,2'=5; 2.3=10; 2'.3=7; 5,6=6,7=7,8=9; 7.8'=7,13'=3; 7,13=3.5; 90:1.2=5; 1.2'=11 2,2'=13; 2'.3=12; 2'.3'=1.5; 3.3'=13; 5,6=6.7=9.5; 7,8=10; 7.8'=7.13'=3; 7.13=3.5; 9,14=1.5; 92:5,6=6,7=10 5,15=13,13'=1; 7,13=7.13'=3; 7,8=9; 8.9=6; 8,9'=1;

605

H

95'"-^

1^

„10.g

4.44 m

2

2.30 m

3.27 m

2"

2.15 m

3.27 m

1 ^

2.60-2.30

3'

2.60-2.30 m

4.44 m

loo'"-^ 3.85 dd

lOl'^'^ 3.99 dd

102'«-^ 4.23 brdd

103'«-8 4.02 brd

1.94 m 2.10 m

105*»> 2.76 dd

1.15-2.27 m 2.0-2.15 m 2.30-1.90 m

2.15 m

m

104^'»' 2.73 q

2.25 in

1.15-2.27 m

5.01 bm 5.07 brd

1 ^ 5.68 1 I 7

1 1 1 1 1

1.59 m

* ^' 1.93 m ^2.70 brd '* 2.20 m ' *2.25 m 1.93 m

107»«-^

5.24 brd

2.76 d

5.25 brd

4.15 dd

4.27 t

4.43 1

3.41 I

4.41 dd

5.73 1

5.58 m

2.73 ddddd

2.82 dddd 2.90 dddd 3.U4 dddd

2.88 dddd

3.19 m

3.36 m

3.95 ddd

3.96 ddd

3.94 ddd

4.32 q

4.41 m

3.90 ddd

3.04 dq

4.14 ddd

5.18 brd

10910.C

4.65 m

4.63 dd 1

2.87 dd

2.86 dd

2.23 m

2.04 dddd

3.07 dd

3.10 dd

2.23 m

2.18 dddd 1

2.12 m

2.27 ddd 1

1.73 m

1.78 ddd

3.41 brs

3.53 d

1.30m

5.04 brd

108»««=

5.44 ddd

5.27 q

1 4 5

106'«-^ 5.45 ddd

2.30 ddd

2.20 ddd

1.74 brd

1.86 m

4.90 brd

2.53 ddd

2.75 ddd

2.82 ddd

2.75 ddd

3.82 dd

3.83 dd

4.05 dddd

2.84 m

4.11 ddd

4.93 m

1

3.40 ddddd

2.95 dq

2.95 brdd

3.05 dddd

1.65 q

2.35 ddd

2 50-2,:5 m 2.87 brd

2.39 dd

2.15 dd

2 38 brdd 2.24 ddd

2.44 dd

2.41 dd

2.63 brd

2.30 dd

2.50-2.35 m 2.53 dd

2.93 dd 1

6.37 dd

6.35 dd

6.30 dd

6.43 dd

6.30 dd

6.27 q

6.30 d

4.53 brs

4.52 brs

6.36 dd

6.20 dd

6.17 dd

6.20 dd

6.21 dd

6.28 dd

6.18 dd

5.79 q

5.80 d

4.53 brs

4.52 brs

6.17 bre

6.39 dd

5.04 brs

5.83 d

5.16 brs

5.40 brs

5.34 brs

5.29 brs

1.19 s

1.30 s

1.67 s

1.67 s

5.06 brs

4.95 brs

1 '^ 4.87 brs 1 *^1.59 brs

5.78 d

5.10 brd

5.16 brd

5.26 brs

5.16 d

5.00 brs

4.97 brs 1

1.65 d

1.70 brs

1.63 brs

1.38 s

1.70 d

1 75 d

1.79 s

1.13 d

1.18 d

1.44 s

1.23 s

|OR:3'

5.92 q

5.92 m

1 * 1 ^

1.91 d

1.96m

1.88 s

\IM m

13

1.24 d

13' 14

3.60 1 brs(OH) J

J(Hz): 95:1,2=11; 1,2'=2; 2,2'=14; 2,3=2.5; 2',3=6; 5,6=6.7=9.5; 5,15«1; 7,8=10; 7,8'-1.5; 7,11 = 11.5, 8,8'-14; 8.9=3; 8,9'=12 8'.9=8',9'=4; 9,9'=15; 31,13=7; 99:5,6=6,7=10; 5,15=1; 7.8=5; 8,9=3, 8,9'=11; 9.9'-13; 7,13=7,13*=2.5, 7.9-1 5; 9,14=1; 9,14'-2 100:1,2=10; 1,2*=5; 5,6=6,7=9.5; 7,8=3; 8,9=9.14=9,14'=2; 8,9'=10; 9,9'-14; 101:1.2=9; ].2'=2.5, 5,6-6,7=9.5; 7,8-3, 8,9=9,14--2 8.9'-7; 9,9'=16; 102:1,2=8; 1,2'=2; 5,6-6.7=9 5; 7.8=8.9=3; 8,9'=11; 9,9'=13; 103:1,2=10; 5.15=1.5; 5.6=6.7=10; 7,8=6.5; 8,9=9,14=9,14^=2; 9,9'=14; 8,9'=10; 7,13=7,13'=3; 13,13'=1; 104:1,2=2; 1.2'=10.5; 5.6=6.7=10; 7,8=6.8; 7,13=3.1; 7,13'=2.6; 8,9=8.8; 8,9'=1; 9,9'=13 5 13,13'=1; 5,15=1.3; OR:3".4"=7.5; 105:1,2=2.5; 1,2'=10; 3,2=3; 3,2'=10; 7,13=7.13'=3; 106 and 107:1,2=8; 1,2'=9; 1,14=1; 2,2'=16.5 4,5=3; 4,5'=8; 4,15=7; 5',6=7; 5,5'=15; 8,9=3.5; 8,9'=14; 8,8*=13; 8',9=4.5, 8',9'=3.5; 9.9'=13; 108:5,6=2.5; 6,7=10 5; 7.13=3; 7,8=6; 8.9=2; 8,9*=11; 9,9'=13; 109:1,2'=7.4; 1.2=7.3; 2,2'=13.7; 2,3=8.1; 2,3'=10.; 3.3'=12.7; 2'.3=2.4; 2',3*=9 4; 5,6-7.8; 6,7=8.7; 9,9'=14.5 9,8=8; 9',8=4.9; 7,13=2.7; 13,13'=1.1; 7,13'=3.1;

1 H 110^'*'

,,,io,i

112^''^

„3>0,g

IH^'^

1154,h

U8»0'^

1 5.29 t

4.38 brdd 4.40 brdd

2

2.05-1.85 m

2.97 ddd

2*

2.05-1.85 m

2.78 ddd

3

2.29 brdd

3'

1.78 m

5

4.99 brd

120^0.c

1217,c

122^'^

3.84 brdd 4.00 m 4.20 m

2.30-2.00 m

2.33 ddd

16' ^

3.64 m 4.50 ddd 4.46 brt 4.37 dd

1275'n

5.06 brd

5.06 brd 1

2.29 s

2.34 s

5.40 td

5.37 dq

4.59 m

10.15 s

9.50 s

2.94 dd

2.75 q

2.65 dd

1.64 ddd 4.93 brd 5.04 brd

126^'^

5.30 brd 5.30 brd

2.69 d

5.12 brd

5.59 dd

6.36 brdd

4.40 ddd 4.50 t

3.65 t

4.38 t

5.17 dd

4.96 dd

4.00 t 3.12 dd

2.53 ddd 1

2.34 dd

7

2.80 dddd

8

4.54 dd

2.82 dddd 4.67 ddd 3.96 ddd 4.00 m

4.20 m

2.77 m

2.48 m

2.00 m

2.05 m

1.52 dddd

obscured

2.85 m

1.8 m

1.60 m

1.80 m

1

606 (Table 2). contd..

1 "110^'*'

„iio.i

112^''^

113 JO'S

1154.h

114^'^

1 8'

19- ^

5.32 brd

2.38 dd 2.74 dq

11

1 ^^6.27 1 ^^brs 1 13'

5.19 brd 2.96 dddd

5.52 brs

1.40 s 1 ^^ 115^^' 0.85 d

6.30 dd

1.32 d

6.21 dd 1.83 d

1.34 d

1.43 d

2.17 dddd

obscured

3.03 m

1.8 m

2.50 ddd

obscured

6.63 ddd

2.3 m

2.78 brdd

obscured

6.63 ddd

2.3 m

2.45 dq

2.45 dq

6.28 d

6.24 d

5.56 d

5.49 d

5.01 brs 5.00 brs

6.05 s

5.86 s

5.08 brs

5.91 s

5.72 brd

1.36 s

1.76 d

6.20 dd 1.76 brs 5.16 brs 5.11 d

1.78 brs

120'0-=

2.60 dq 2.75 dq

6.37 dd

1.76 brs 1.70 d

1OH'^'

1.70 s

1.80 brs

,227.c

118'0>c

1217,0

1.26 d

1.79 d

i27n

126^'*^

1.15 s

Ills

1

1.34 d

1.24 s

1.21 s

1

5.80 s

1.72 brs

1.70 brs

1

6.10 s 2.07 d

1.90 d

2.62 brs, 2.85 brs

OR

5.67 brs

5.19 brs

5.58 brs

5.08 brs

1

2.27 br

3.80 br

1

2.02 s

2.00 s

J(Hz): 110:1,2=1,2'=9; 4,15=7; 111:1,2=11; 1,2'=5.5; 2,3=6; 2',3=1; 3,3'=12; 5,6=10.5; 6,7=7,8=9; 6,OH=3; 8,9=10; 7,13=7,13'=3; 13,13'=1 9.14=1.5; 112: 1,2=3; 1,2'=10.5; 5,6=6.7=7,8=8,9=7,11=10.5; 11,13=7; 113:1,2=9.5; 1,2*=6.5; 5,15'=1; 5,6=6,7=10; 7,8=6.5: 8,9=9.14=9,14'=2; 8,9*=10; 9,9'=14; 7,13=7,13'=3; 13,13'=1; 114:11,13=7; 5,6=6,7=7,11=10; 6,OH=4.5; 115:5,6=6,7=7,11=10; 11,13=7: 118:2,2'=14; 2,3=6.5, 2,3'=13; 2',3=7; 2',3''=2.5; 3,3'-13; 5,6=6,7-9.5; 7,8=3; 7,8'-9; 7,13-3.5; 7,13'=3, 8,8'=15, 8.9=3; 8',9=5.5: 8',9'=12; 9,9'=15; 9M4=9,14'=1; I20:2,2'=2,3=12; 2',3=6; 5,6=6,7=10; 7,13=3.5; 7,13'»3; 9,14'=1.5; 121:5,6=6,7=9; 5,15=1; 8,9=8',9=7; 9,14=1; 7,11=11; 11,13=7.5; 122:5,6=6,7=9; 5,15=1.5; 7,11=11; 11,13=7; 9,9'=15; 9M4=9',14*=1; 126:1,2=2,3=9; 2,3'=6; 3,3'=12; 6,6'=14; 6',7=3; 6,7=10; 127:1,2=11; 2,3=10; 2,3'=5; 3.3'=13; 5,6=5,6*=4;

gu^i^nolidgs

1 " 129*'^

rn

1 ^

1 1 1 1

130^'*

rS2m

^ ^ ^' ^

132*'«

3.63 d

135»»''

136»»'*

3.68 bra 3.28 s

,3,10,c

3.28 s

142"'»'

,,3l0,b

144^"''

145"'*

146*'«

147*''^

1.87 dddd 2.78 brddd

3.00 brddd 3.00 brddd 2.23 m

2.58 oi

Obscured

1.54 dd

1.67 dd

2.03 dd

1.80 m

1.61 m

0.80 dd

1.14 ddd Obscured

1.76 ddd

1.53 m

1.48 m

2.44 01

2.58 OI

Z^ 2.37 m 1

1.55 m

1.85 m 1

2.36 m

2.35 m 1

1.34 m 1

6.17 q

3.32 s

3.40 d

3.35 brs 3.68 s

3.68 s

2.68 bn

2.91 bn 3.36 brs

3.37 brs

2.30 m

2J5m

3.40 d

3.08 d

2.91 d

2.62 d

2.85 d

2.09 dd

1.78 dd

obscured

2.40 01

2.91 brdd

2.04 dd 2.81 dd 2.78 m 1

3.64 t

3.93 dd 4.33 dd 4.27 d

1 ^3.86 dd 1 ^2.52 m 1" 3.76 dt

1 *'

1 ' 2.75 m 1 '* 2.43 m

1 *'

3.40 m

3.00 m 4.74 dt

2.87 d

4.24 t

3.00 dd

2.93 dd

4.03 dd

4.10 dd

3.88 dd

4.00 t

3.88 t

3.71 m

3.71 m

3.19 ddddd

2.62 dddd

2.77 m

3.17 dd

3.45 dddd

3.37 dddd

2.45 m

2.78 m

5.25 dd

5.21 ddd

0.84 dddd 4.86 ddd 3.95 m

5.01 ddd

3.88 m

3.90 m

3.78 m

3.90 ddd

2.4 n

2.16 dd

2.17 dd 2.45 m

obs.

2.4*

1.89 dd

1.89 brd 1.87 brd 1.79 m 1

*^ *^' ^*2.30 d *^' *^2.43 s 1.27 d

4.30 t

1 1

1.84 dddd 2.18 m

2.11 m

2.11 m

1.16 ddt

2.05 dd

2.05 dd

1.03 ddd 1.87 dd

2.21 brd obs.

2.23 dd 1

2.45 m

2.78 m 1

6.00 d

6.04 d

6.20 d

6.20 d

6.20 d

6.09 d

6.17 d

6.27 d

6.23 d

6.17 dd

6.20 brd 1.41 d

1.27 d 1

5.40 d

5.58 d

5.47 d

5.53 d

5.55 d

4.90 d

5.37 d

6.15 d

15.60 brd

6.00 dd

6.02 brd

1.66 %

1.42 s

1.26 s

1.14 s

1.11 s

0.73 s

4.77 brd 5.08 brs

5.10 brd

1.33 s

U3s

4.62 brd 4.88 s

4.81 brd

2.90 q

1 1 1 1 1

148*'*

1.60 s

iO.94 s

1.55 s

1.56 s

1.58 s

11.67 s

1.59 s

11.58 s

1.59 s

5.16 bra 3.23 d

1.30 s

1.14 s 1

5.17 bra 5.05 t 1

607 (Table 2 ) . c o n t d

.»••'

H

m^'

U2^'

m'«-'

us'"'

U,'"'

.42"-^

.43»*^

.44'»'*

us'**

1 15'

m*-' 5.02 b n

OH

..*•*

,47»-

2.93 d

4.99 bru

j£J

4.89 t 1

3.62 brd 3.56 brd

1 ^^^'^

6.08 01

0.99 d

0.94 t

5.81 qq

2.63 qq

1.94 dq

2.25 d

1.18 d

2.01 dq

1.22 d

1.80 q

2.10 m

2.40 m

1.85 dq

1.20 d

4"

1 *"

J(Hz): 129:3,15: 5,6=10; 7,8=2,4=10.7; 11,13=8; 130:2,2'=12; 5,6=6,7=7,8=8,9=10; 8,9'=3; 7 , 1 3 = 7 , 1 3 ' = 3 ; O R : 3 " , 4 " = 7 ; 3 " , 5 " = 4 " , 5 " = 1 ; 132:2,3=1; 5,6=6,7=11; 7,13=7,13'=3; 135:5,6=11.5; 6,7=9, 7,13=3.5; 7,13=3; 136: and 137:5,6=9.5; 6,7=10; 8,9=5; 8,9'=3.5; 9,9'=16; 142:1,2=6.9; 1,2'=I1.7; 1,5=7.4; 1,9=1.6; 2,2*=13.2; 5,6=11.2; 6,7=9.2; 7,8=7.1; 7,8'=10.6; 7,13=7,13'=0.5; 8,8'=13.5; 8,9=8.3; 8',9=1.6, 8',9'=-8.0; 9',8=10.9; 1 4 3 , 144 and 145:1,2=7.5; 1,2'=11.5; 1,5=8.5; 1,9=2',3'=9,14=1; 2,2'=14; 5,6=11; 6,7=9; 7,8=10; 7,13=3.5; 7,13'=3; 8',9=2 8',9'=5.5; 9,9'=15; 14,14'=2; OR:3",4"=7; 3 " , 5 " = 4 " , 5 " = 1 . 5 ; 146:5,6=6,7=7,8=11; 8,9=4.5; 8,9'=1.5; 9,9'=16: 13,13'=!.5; 7,13=3.5; 7,13'=3.0; 147:1,5-6.5; 5,6=6,7=8,OH-10.5; 8,9=5.5; 9,9'=16, 7,8=9, 7,13=3.5; 7,13'=3.0: 15,15'=4.5; 148 and 149:1,5=7;5,6=6,7=7,8=7,11=10; 8,9=5 5; 8,9'=4, 9 . 9 ' - 1 6 ; 11.13-7;

H

2

150*'*

2.35 m

ISl***

? 08-2.25 m

2.55 m

2.50-2.75 m

1 ^' 1 ^ 5.81 brs 1 ^ 3.95 t

5

1 7 8

155^'=

2.55 m

2.30 m

2.20 m

ISS^^**

ISl"'"

lee''^^

167»»'*

168*"'*

1 6 9 ^

1.86 ddd

5.85 d

4.11 s

4,18 s

4 10 s

6.41 d

5.70 d

3.70 s

3.70 s

3 70 s

2.34 d

2.40 d

2.48 d

3.06 d

2.60 d

6.67 d

1

2.27 ddd 3.18 brs 4.56 brd

6.94 brs

6.21 t

4.61 t

3.47 m

3.57 brd

2.08-2.25 ro

3.87 t

3.65 t

2.50-2.75 m 4.10 q

4.60 dq

4.27 t

3.12 t

4.31 dd

4 26 dd

4.27 dd 1

3.49 dddd

3.47 m

3.20 m

2.87 m

2.90 ddddd

3.18 ddddd 3.03 ddddd

3..54 m

3 32 m

3.05 m 1

3.90 m

3.90 m

3.97 brt

4.64 t

2.99 dd

1 ^ 2.18 dd 1 ^ 1.94 dd 1 ^^6.21 dd

2.21 dd

1 ^^ 1 ^^'

6.05 dd 1.34 s

IS

4.34 s

15'

4.34 s

OH

3.28 brd

OR

165»»'*

3.29 m

5.07 m 2.17 dddd

2.28 m

1.90 dddd 2.35 dddd 2.05 dddd

1.32 dddd

1.58 m

0.86 dddd 1.45 dddd 1.48 m

8'

13-

162^«'«'

5.48 d

1.99 brd

5,06 m

2.36 m 1

1.71 ddd

i.47 ddd

2,67 ddd

i.99 ddd

1.10 ddd

1.88 ddd

1.76 .n

1.99 hi

\

6.07 d

6.18 d

6.16 d

6.05 d

6.18 d

6,19 d

on d 1

6.28 d

5.49 d

5.51 d

5.43 d

4.85 d

5.47 d

5.44 d

5.65 d

1

1.97 s

4.71 brs 091 s

1.44 s

MOs

1.40 s

1,56 s

136 s

1

1.73 s

1.58 s

1.76 s

1,76 s

1.77 s

1

4.7i s

3.98 s

6.22 brd

6.23 q

5.42 d

6.02 brd

6.57 q

1.38 s

2.48 s

4.90 brs 9.74 s

2.22 brs

4.97 s

1.53 s

1.92 dd

5.36 s 3.20 brd

4.30 s 1.02 t

0.94 t 1.22 d* 12.44 i n ]

J{Hzy. 150:1.5=7; 5,6=6,7=7.8=10; 7,13=3.5; 7,13'=3.0; 8,9=6; 9,9'=16; 8,OH=10.5; 15,15'=14; 151:5,6-6,7=10 8,9=6; 9.9'=16; 7,13=3.5; 7,13'=3.0; 8.0H= 10.5; 7,8=8; 153:3,15=1; 5,6=6,7=7,8=10; 7,13=7,13'=3.0 13,13'=1.5; 155:3,2=3,2'=6.8; 7,8=8,9=10.2; 7,13=3; 7,13'=3.5; 161:5,6=10; 6,7=9; 7,13=7,13'=3; OR: 2",3"=10: 162:1,2=8.0; 1,2'=5.7; 1,15=1.3; 2,2'=14.7; 2,3=2.5; 2',3=7.6; 6,7=10.9; 6,15=1.6; 7,8=1.6; 7,8'=10.8; 7,13=3.3 7,13'=3.1; 8,8'=14.6; 8,9=4.0; 8,9'=3.9; 8',9=13.4; 8',9'=3.6; 9,9'=13.5; 165 and 166:2,3 = 6 5,6=6,7=7,8'=8',9=10; 7,8=8,9=7; 7,13=3.5; 7,13'=3; 8,8'=14; 8,9'=8; 8',9'=2; 9,9'=15; 167 and 168:5,6=11 6,7=9; 8,9=8',9'=7; 8',9=9; 8,9'=11; 9,9'=15; 7,13=3; 7,13'=3.5; 169:8,9=5; 8,9'=3.3;

608

H

2

,70»-

.7,^"

.72^

,73«^

6.55 (1

6.49 d

6.53 d

6.33 d

ns*-'

.74"-'

6.31 d

6.30 d

,7/" 6.29 bbrd

.77'-'

5.93 d

2'

.V"

,V""

2.32 dd

6.07 d

m"- 1

I8O10.C

6.17 d

2 J3 d

2.69 dd

3

6.29 (1

6.24 d

6.28 d

1 -*'

6.56 brdd 5.87 brd

5.86 brd

5.9 brd

5.90 d

3.46 brd 2.89 brd

2.89 brd

2.92 brd

2.85 d

4.20 t

4.30 dd

1

2.78 dd 1

4.30 brd

7.49 d

7.47 d

2.71 d

2.33 d

4.70 brs 1

6

5.55 d

5.13-5.5© 5.71 d m

3.78 t

3.96 dd

4.14 t

4.47 dd

4.58 dd

4.94 d

7

2.70 m

2.25-3.00 2.253.00 m m

2.37 m

2.17-2.33 m

2.13-2.31 m 2.62 dt

3.28 brddd 3.05 dddd

3.47 dddl

3.54 dddC

3.12 m

1 *

1.85 m

5.13-5.50 1.95 m m

1.76 m

1.33 m

1.36 m

5.27 brt

1.8-2.0 m

1.89-1.99 m

1.89 dq

1.90 m

1.32 ddt

1.78 ddt

1.78 m

1.8-2.0 m

1.89-1.99 m 2.55 m

r 9

2.42 m

1 ''

2.25-3.00 2.50 m m

2.29 brdd 2.17-2.33

2.13-2.31 m 2.40 m

2.41 dd

2.5-2.7 m

2.55-2.68 m

2.45-2.62 m

2.25-3.00

2.17 ddd 1.19 m

2.13-2.31 m 2.06 m

2.39 dd

2.5-2.7 m

2.55-2.68 m

2.45-2.62 m

2.68 dq

2.17-2.33 m

2.68 qui

2.81 qui

1.11 d

1.34 d

1.17 d

1.16 d

6.30 d

6.35 d

6.35 d

6.35 d

6.32 d

5.56 d

5.66 d

5.78 d

5.75 d

5.67 d

11

2.96 dq

2.25-3.00 2.253.00 m

1 '^

1.26 d

1.34 d

1

4.79 ddd

1.36 d

1 13'

1 ^*2.19 a

4.97 d

1

1

1

2.23 bn

2.19 s

1.90 brs

1.84 d

1.82 d

1.85 d

1.15 s

2.21 s

2.21 s

2.19 s

2.20 s

1

2.23 bn

2.12 s

5.10 brs

1.40 s

1.41 s

1.44 s

1.47 s

2.15 s

1.58 s

1.60 s

2.15 s

1

W

1 '^ 2.09 s

5.21 brs

15'

1.70s, 3.30s. 3.54s

OH

1 OR: 3"

6.22 dq

2.08 s

3.90 s

3.40 s

2.06 dq

4"

1 '"

1.93 t

J(Hz): 170:2,3=6.7=5.3; 7,11 = 11,13=7.4; 171:11.13=9; 2,3=5.5; 172:2,3=5.3; 6,7=6; 11,13=7; 173:2,3=5.5; 3,15=1.5; 5,6=6,7=10.4; 8*,9=12.0; 8',9'=2.2; 8,8'=13.5; 9,9'=14.6; 8,9'=5.9; 11,13=7.5; 174:2,3=5.8; 5,6=11; 6.7=10; 7,8'=10; 8',9=12.1; 8M4=1.9; 11.13=6.9; 175:2.3=5.8; 5.6=6,7=10.6; 11,13=7.8; 8M4=2.0; 176:2,3=5.8; 5,6=6.7=7.8=11; 8,9=8.7; 8.9'=5.3; 11.13=7.5; 177:2,3=5.8; 5,6=11.5; 6.7=9.6; 7,13=3.5; 7,13'=3; 8.9=15.5; 8.9*=3.6; OR:3".4"=7.3; 3",5"=4",5"=1.4; 178:2,3=2.5; 2',3=6; 2,2'=18; 5,7=5; 7.13=3; 7.13*=3.5; 179:2,3=5.8; 5,6=10.4; 6,7=2.1; 7,8=7; 7,13=2.0; 7,13'=1.7; 180:2,3=5.7; 5,6=9.0; 6.7=2.7; 7.8=7; 7.13=2.2; 7.13'=1.9; 181:2.2'=19.2; 2'.3=7; 6.7=6.5; 7,13=3; 7,13'=3.5;

Farnesols. Pvrethrins and Longipinane Derivatives H

184»»'*

5.34 dd

190^-'

H

4.59 brd 4.63 brd 4.54 brd 4.56 brd 5.18 dd

1

185^'*

186^'*

187'''

188*'*

4.95 dd

3

5.37 bn

5.36 bn

5.81 dd

4

2.30 dd

2.39 dd

1.90 re

2.21 dd

2.21 dd 5.70 ddd

5.08 dd 5.88 dd

2.00 dd

5.45 bn 5.61 bn

2.0 m

4.02 (

1.78 m

6 1 7

5.55 ddd

5.61 ddd

2.0 m

2.28 t

5.69 ddd

5.38 brd

5.11 1

5.10 1

5.35 brd 5.33 brd 5.15 brd

203^''

H

1 95 brs

1.93 brs

1

2.72 d

6.53 dm

6.50 dm

6.48 dm

r

2.54 dd

2.21 dd

2.20 dd

2.21 dd

2

198^-'

200^-'

202^''

199^''

201^-'

1.73 s

1.72 s

1.71 s

1.93 bn

4.93 dm

4.94 dm

4.93 dm

2.10 dd 2.10 dd

2.10 dd

193«''

b

5.84 1 ddq J

5

1.41 d

1.41 d

1.38 d

1.74 d

1.75 d

1.73 d

3

2.49 q

7

1.27 s

1.28 s

1.24 s

1.30 s

1.36 s

1.30 s

4

2.20 m

2.15

8

1.14 s

1.15 s

1.12 s

1.23 s

1.26 s

1.23 s

5

1.44 «

2.08 1

lU

5.68 dm

5.77 dm

5.69 dm

5.71 dm

5.79 dm

5.69 dm

7

>'

2.19 dd 2.20 dd 1 2.19 dd

2.20 dd

2.20 dd

2.19 dd

8

2.66 brm

11

2.89 dd 2.87 dd

2.90 dd

2.93 dd

2.90 dd

9

1.75 d

2.88 dd

I

brd 1 brs I

1.2- 1

1.4 m

1.2- 1

1.4 m

1.2- 1 1.4 m 1

609 (Table 2). contd... H

184>»»^

ISS^''^

186'-'^

ISTS''^

188^**=

190^*^

H

198^*** 200^*^

1994,f

202*'^

201^'^

203**^

H

1938»c

9'

1.79d

195''' b

1 8

5.30dd

2.0m

2.0m

22.15m

2.80dd

4.95ro

15

2.25$

2.05s

2.03brd

2.03s

2.05s

2.03$

1 ^

1.70 m

2.0 m

2.0 m

1.65 m

5.59 d(

2.24 dd

16

2.98 d

3.0 d

3.12 brd

2.98 d

2.99 d

3.12 brd

4. (X) dd

5.35 t

5.07 I

4.95 m

17

5.37 m

5.37 m

5.22 dt

5.37 m

5.37 m

5.22 dt

1 1

2.21 d

2.90 1 brd

18

5.28 m

5.28 m

5.85 brdd

5.28 m

5.28 m

5.85 brdd

12

1.20 d

0.75 s 1

R:r

1.73 s

1.72 s

1.71 s

13

1.10 i

0.78 s

3.74 s

3.75 s

3.75 X

14

1.08 s

0.92 s 1

1.72 d

2.15 m

6.63 ddd

15

0.88 t

1.55 dl

0.%t

5.06 dd

10

4.03 brdd 5.67 brd

1 '' 12

4.97 br»

4.44 s

1.71 brs

1 12* 4.84 brs

1 '^ 1 ^* 1 ^*

1.32 s

1.59 brs

r

4.85 q

1.73 bre

1.57 brs 1.59 brs

1.73 brs

1.32 s

1.75 d

1.68 brs 1.68 brs

4.79 d

4.74 d

4.59 d

4 58 d

1.73 brs

1.74 brd

2.00 s

2.00 s

1.96 s

2.05 s

2.05 s

1.93 s

2.06 s

2.07 s

IS

1.28 s

|0R:2*

2.02 s

|0R:4*

4.93 brs

2.09 s

1.62 brs 1.62 brs 2.03 s

2.05 s

2.05 s

lORi.'S'

1 65 brs R'.V 1.70 d

1.72 d

r\

2.15 m

6.63 ddd

0.%t

5.06 dd

2"l

5.10 dd

5.10 dd

1.20 s

7(7/2): 184:1,2=17; r , 2 = l l ; 5,4=8.5; 5,4'=4.5; 4,4'=15; 5,6=9.5; 8,9=4; 8,9'=9; 10,9=3.5; 10,9'=9; 185 and 186:1,2=5,6=9,10=4,5=7; 187:1,2=7; 4,4'=13.5; 4,5=7.5; 4',5=6; 5,6=9; 9,10=13; 9M0=5.5; 12,13=1; 14,14'=12 12',13=1.5; 188:1,2=7; 4,4'=14; 4,5=7.5; 4',5=6; 5,6=9; 8,9=8',9=6; 8,10=1.5; 9,10=15.5; 14,14'=12 190:1,r=1.5; r,2=17; 1,2=10; 4,5=5; 4',5=7; 5,6=9; 6,14=10,13=10,12=1; 8,9=7; 9,10=6; 198:3,4=8; 4,5=5.5 10,11=2; 10,ir=6; l l , i r = 1 8 ; 16,17=6; 18,1"=6; 200:3,4=8; 4,5=5.5; 10,11=2; 10,ir=6; l l , i r = 1 8 ; 16,17=6 r',2"=7.3; 202:3,4=8; 4,5=5.5; 10,11=2; 10,ir=6; 11,1 r=18; 16,17=7; 16,18=1; 17,18=10.4; 18,1"=10.4 r',2"c=10.4; r',2"t=16.5; 2"c,2"t=1.5; 199:3,4=9; 4,5=5; 10,11=2; 10,ir=6; l l , i r = 1 8 ; 16,17=6; 18,r'=6 201:3,4=9; 4,5=5; 10,11=2; 10,ir=6; l l , i r = 1 8 ; 16,17=6; r',2"=7.3; 203:3,4=9; 4,5=5; 10,11=2; 10,ir=6 l l , i r = 1 8 ; 16,17=7; 16,18=1; 17,18=10.4; 18,r=10.4; r',2"c=10.4; r',2"t=16.5; 2",2"=1.5; 193:3,12=7 1,11=3,4=4,5=5,11=1,11=1.5; l,r=20; 4,11=7; 8,8'=14; 9,9'-15, 195:2,4=1; 2,11=?,15=1.5; 4,11=5.5; H

1963'"

F"

2.53 dd

1 -^

5.43 m

,«ll

r

M«OHEXl.eO% RMMnli(lk«MiOH EilractlMwlki«tOH

_L

AquMusExt.

B1-615 (4.04 g.)

-H

B16* (1005 ma)

IE: B17»

I

SNnfil



4.72 (7u-6.8) 3.76 3.70 3.88 3.47 3.69 3.68

104.5 Sil 74.8 69.6 76.4 62.0

4.61 (yu-7.6) 3.27 3.35 3.36 3.35 3.93 3.74 4.45(Ju-7.8) 3.47 3.70 3.49 3.55

106.1 77.9 77.5 71.4 78.9 62.2 105.0 £L1 78.2 72.8 76.5 176.8

6S6

POLYACETYLENES Poliacetylenic compounds are frequently found in Umbelliferae [29]. Several genera among the Araliaceae and Apiaceae families (which include many common plants and species from Hedera, Schefflera, Panax, Apium, Falcaria, Daucus, Oenanthe, and others) have been shown to contain falcarinol [30], isolated from Falcaria vulgaris by Bohlmann. A polyacetylene compound, panaxynol, was isolated by TakahashifromPanax ginseng roots [31]. The chemical structure of the compound was identical to that of falcarinol and also to that of carotatoxin, isolatedfromDaucus carota [32]. Polyacetylenes have also been described in several species of Bupleurum [1, 29, 33, 34]. From the acetonic extract of the roots ofB. rigidum two polyacetylene alcohols (12 and 13) were obtained. OH

CH,

CH

CH.

CH,

12 Faicailnoi

OH

0H2Z=

CH.

CH.

13 Diynene

w

687

The structure of 12 corresponds to falcarinol, and that of 13 to a diynene isolated from Pituranthus tortuosus and Aegopodium podagraria, and described by the first time by Schulte [35, 36]. PHENOLIC GLYCOSIDES To the best of our knowledge, phenolic glycosides have not been reported previously in the genus Bupleurum. However, glycoside 14 was obtained from the butanolic extract of the roots of B. rigidum.

0CH3

HO

f OH

r

/^^^^^

\

^^

//

V'

\\

'y

OH

OH

14 Osmantoiide

This glycoside had been isolated from Osmanthus asiaticus by Sugiyama [37]. No additional information on this compound (Osmantoiide) has been reported so far. MISCELLANEOUS Some carbohydrate compounds, widely distributed in the plant kingdom, were also found in the roots of B. rigidum^ such as ribitol and sucrose.

688

BIOLOGICAL ACTIVITY Some species from Bupleurum genus are often used in combination with other plants as antihepatotoxic, antipyretic, analgesic, sedative and antidepresive agents. Saikosaponins extracted from B. falcatum are reported to have another variety of therapeutic activities such as alleviating hyperlipidemia, hepatic injury and chronic hepatitis as well as cardiac activities. According to the revised literature, saikosaponins are commonly the active constituents as antiinflamatory agents of Bupleurum genus.. From the aerial parts and roots of this plant, we have recently reported the isolation of bioactive saikosaponins, mainly glycosides of saikogenin F and D [38-40]. These type of compounds have also been identified in other species of the Bupleurum genus, and are considered to be the active principles of these drugs with anti-inflammatory action. Saikosaponin F exhibits a dose-dependent inhibiting effect on carrageenan-induced edema in rats, while saikosaponin D shows anti-inflammatory activity due to inhibition of PGE2 production in a macrophage culture system in vitro [ 41, 42]. Additionally, the related saikosaponin A has been shown to possess an inhibitory effect on platelet activation [43]. Recently, we have reported the in vivo and in vitro anti-inflammatory activity of two saikosaponins isolated from B, rigidum, budlejasaponin IV and sandrosaponin I, in order to establish the possible real value of these kinds of compounds as anti-inflammatory agents [44]. We showed that saikosaponins inhibited the mouse ear edema induced by topical administration of phorbol myristate acetate (PMA). Saikosaponins, at a dose of 1 mg/ear, significantly inhibited swelling and were as potent as the reference drug indomethacin at 3 mg/ear. These findings were supported by vascular permeability analysis [Table 10 and Fig. (4)].

689 Table 10.

Inhibitory Effects of Budlejasaponin IV and Sandrosaponin I on PMA-

Induced Mouse Ear Edema Compounds*

Dosage (mg/ear)

Ve Inhibition Ear Edema

Vascular Permeability

Indomethacin

3

96.75

80.96

Budlejasaponin IV

1

94.02

55.32

Sandrosaponin I

1

96.03

56.21

^^administered one hour prior to PM A

Inhibitory Effects of Sall(osaponins and Indomethacin on PMAInduced Mouse Ear Edema 100

96.76

96,03

94.02

80-1 60

E3 Indomethacin

40

D Buddlejasaponin IV

D Sandrosaponin I

20 0 %lnhibition

Fig. (4). Inhibitory Effects of Buddlejasaponin IV, Sandrosaponin I, and Indomethacin on PMA-Induced Mouse Ear Edema.

The in vitro anti-inflammatory activity has been investigated in cellular systems generating cyclooxygenase (COX) and lipoxygenase (LOX) metabolites, in an attempt to gain insight into the mode of action of those saponins. Saikosaponins inhibited generation of both PGE2 and LTC4 by ionophore-stimulated mouse peritoneal macrophages, v^th a more marked action on LTC4

690

Buddlejasaponin IV was more active than sandrosaponin I against LTC4, with inhibition percentages around 100%, similar to the reference drug NDGA. In the PGEa-release assay, budlejasaponin IV and sandrosaponin I also showed a significant effect, although with less potency than the reference drug indomethacin [Tables 11 and 12, and Fig. (5 - 6)]. Table 11. Inhibition of PGEi-Release from Mouse Peritoneal Macrophages Stimulated with Calcium lonophore A23187 (10~^ M) by Budlejasaponin IV and Sandrosaponin I

PGEiCng/mU'

Compounds Control

% Inhibition

26.0 ± 0 . 2

Indomethacin (100 |IM)

3.40 ±0.4**

Budlejasaponin IV (100 |iM)

6.50 ± 0 . 1 '

Sandrosaponin I (100 |IM)

9.50 ± 0 . 1 '

98.7 75.0 63.5

*all values are mean ± SEM ''significantly differentfix)mthe control group (p < 0.01) 'significantly different from the control group (p < 0.05) Inhibition of PG^-Ftelease from Mouse Peritoneal Macrophages Stimulated with Calcium lonophore A23187 bySalkosaponins

H CoTitro\ • buprofen D Sandrosaponin I O Buddlejasaponin IV

%lnhibition

Fig. (5). Inhibition of PGE2-Release from Mouse Peritoneal Macrophages Stimulated with Calcium lonophore A23187 (10"^ M) by Buddlejasaponin IV, Sandrosaponin I, and Indomethacin.

691 Table 12. Inhibition of LTC4-Release from Mouse Peritoneal Macrophages stimulated with Calcium lonophore A23187 (lO"^ M) by Budlejasaponin IV and Sandrosaponin I

1 CompouiMls Control

LTC4{ng/mL)'

•/• Inhibition

-

36.0 ±0.3

NDGA(25|IM)

1.70 ±0.1"

Budlejasaponin IV (100 |IM)

3.60 ±0.1'*

Sandrosaponin I (100 |INf)

4.30 ±0.8'*

100 90.5 88.0

*all values are mean ± SEM. ''significantly differentfromthe control group (p < 0.01).

inhibition of LTC^-Release from Mouse Peritoneal Macrophages Stimuiated with Caicium ionophore A23187 by Saiicosaponins

B Control • NDGA D Sandrosaponin I D Buddlejasaponin IV

%inhibition

Fig. (6). Inhibition of LTC4-Release from Mouse Peritoneal Macrophages stimulated with Calcium lonophore A23187 (10"^ M) Buddlejasaponin IV, Sandrosaponin I, and hidomethacin.

692

We also investigated the action of saikosaponins on TXB2 release induced by calcium ionophore in human platelets. All compounds assayed presented a dose-related response to TXB2 release, with inhibition percentages slightly lower than the reference drug ibuprofen [Table 13 andFig.(7)]. Table 13. Inhibition of TXBi-Release from Human Platelets Stimulated with Calcium Ionophore A23187 (1.8 x 10"^ M) by Budlejasaponin IV and Sandrosaponin L TXB2(ng/mL)'

Conij^iinds Control

.•/oJ_iiWJW«oii_

28.0 ±1.3

Ibuprofen (100 |IM)

0.1310.2"

Budlejasaponin IV (100 |iM)

3.80 lO.l**

Sandrosaponin I (100 |IM)

1.90 ±0.2'*

99.5 86.4 93.2

*aU values are mean ± SEM. ^significantly di£ferentfromgroup {p < 0.01).

Inhibition of TXBz-Release from Human natelets Stimulated with Calcium Ionophore A23187 by Saikosaponins

110 100 90 80-]

B Control

70-1 eo

C3 buprofen D Sandrosaponin I

50-1

O Buddlejasaponin IV

40 30 20-1 10 %lnhibition

Fig* (7). Inhibition of TXBi-Release from Human Platelets Stimulated with Calcium Ionophore A23187 (1.8 X 10"^ M) by Buddlejasaponin IV, Sandrosaponin I, and Indomethacin.

693

Sandrosaponin I was the most active, with an inhibition percentage of around 90% at the highest dose. Without excluding the participation of other possible mechanisms, including non-eicosanoid mediators, our data support the inhibition of arachidonic acid (AA) metabolism as one of the biochemical mechanisms which may contribute to the anti-inflammatory activity of these saikosaponins. It was evident that both COX and LOX "arms" of arachidonate metabolism were inhibited, suggesting that these saikosaponins might provisionally be classified as "dual inhibitors", wdth greater LOX than COX activity. Other biological activities have been reported in the literature fi'om the saikosaponins found in B, rigidum, Budlejasaponin IV, also isolated fi*om the roots of B. fruticosum, have been shown to possess hemolytical activity, hepatoprotective and phagocytosis and immune system stimulating eflfects [45]. Since most colds and infections are caused by a temporary weakening of the immune defence system, the prophylactic or therapeutic use of drugs which enhance the unspecific immune system, the so-called immunostimulants, can have beneficial effects [46]. The efficacy of many of these preparations, like several species of the Bupleurum genus and compounds isolated from them, has been assessed by immunological in vivo and in vitro experiments. For example, the in vivo effects of saikosaponin D isolated from B, falcatum, have been studied. This compound increases phagocytic activities of mouse peritoneal macrophages such as spreading activity, phagocytosis, lysosomal enzyme activity and intracellular killing activity of living yeast [47, 48]. The effects of saikosaponin D on macrophage Sanctions and lymphocyte proliferation in vitro were also investigated [49-51]. Saikosaponin D and saikosaponin F isolated from the related species B, kaoi , also showed immunomodulatory effect in vivo [52]. Additionally, saikosaponin D isolated fi*om the roots of B, falcatum exhibited a potent anti-cell adhesive activity and a strong hemolytic action [53, 54]. It is suggested that the mechanism for anti-cell adhesive activity of this compound may resemble the mechanisms for its hemolytic action [ 55]. Saikosaponin D has been shown to inhibit the cell growth and DNA synthesis of several human cell lines, including human hepatoma cells [56] . Moreover, saikosaponin F and D have been reported to have high liver protective activity against D-galactosamine- and halothane-induced hepatic damage, reducing significantly the pathological changes in

694

hepatocytes, such as inflammatory infiltration and necrosis in the liver parenchyma [42, 57, 58]. This hepatoprotective activity has also been reported for budlejasaponin IV [45]. In addition, saikosaponins of plant origin, including those of the Bupleurum genus, are a protective group of drugs for the prevention and treatment of diseases of the heart and circulatory system [59]. Saponins exert a positive effect on the function of the heart directly, or they help to treat related diseases. For instance, they inhibit the formation of lipid peroxides in the cardiac muscle or in the liver, influence the function of enzymes contained in them, decrease blood coagulation, cholesterol and sugar levels in blood, and they also stimulate the immune system. Other interesting biological properties reported for saikosaponins of the genus Bupleurum includes bactericidal activity and inactivating effects on some viruses [60, 61]. In a screening for protozoocidal activity of Spanish medicinal plants, B. rigidum has been selected as one of the most promising extracts against parasites [62]. ACKNOWLEDGEMENTS This work was supported by Universidad de Alcala de Henares (Ref E006/2001). We want to thank Luis Nafria for his technique support. REFERENCES [1] Tang, W.; Eisenbrand, G. In Chinese Drugs of plant origin; Springer-Verlag, 1992; pp. 223-232. [2]Pharmacopoeia oft he People^s Republic of China. Peoples's Medical Publishing House: Beijing, China, 1992, [3] Chang, H.; But, P.P. In Pharmacology and Application of Chinese Materia Medica,; Ed.; World Scientific.: Singapore, 1986; Vol. 2, pp. 967-974. [4] Hildebert, W.; Bauer, R.; Peigen, X.; Jianming, C ; Offermann, F. Chinese Drug Monographs and Analysis., 1996, 7, 1-11. [5] Kubata, T.; Tonami, F.; Hinoh, H. Tetrahedron Lett., 1968, 5, 303-306. [6] Ebata, N.; Narajima, K.; Kayashi, K.; Okada, M.; Maruno, M. Phytochemistry, 1996, 41, 895-901. [7] Just, M. I ; Recio., M. C ; Giner, R. M.; Cuellar, M. J.; Mafiez, S.; Bilia, A. R: Morelli, I.; Ghelli, S.; Rios, J. L. Natural Product Letters, 1997, P, 167-175.

695 [8] Luo, S-Q.; Lin, L-Z; Cordel, G. A. Phytochemistry. 1993, 33, 1197-1205. [9] Akai, E.; Takeda, T.; Kobayashi, Y.; Oguihara, Y. Chem. Pharm. Bull, 1985, 55, 3715-3723. [10] Kobayashi,. Y.; Ogihara, Y, Chem. Pharm. Bull., 1981,33, llZO-ll^e. [11] Zhao, Y. Y.; Luo, H. S.; Wang, B.; Ma, L.; Zhang, R. Phytochemistry, 1996, 42, 1673-1675. [12] Pistelli, L.; Bertoli, A.; Rita, A.; Morelli, A. Phytochemistry, 1996, 41, 15791582. [13] Est6vez-Reyes, R.; Estevez-Braun, A.; Gonzalez, A. G. J. Nat. Prod, 1993, 55, 1177-1181. [14] Lopez, H.; Valera, A. J. Nat. Prod, 1996,59,493-494. [15] Heywood, V.H. In Lasplantas conflores; Ed.; Reverie, 1985, pp 509-511. [16] Izco, J.; Barreno, E.; Brugues, M.; Costa, M.; Devesa, J.; Femindez, F.; Gallardo, T.; Llimona, X.; Salvo, E.; Talavera, S.; Valdes, B. In Botdnica; Mcgraw Hill, Ed.; Interamericana, 1997, pp 706-708. [17] Tutin, T.G.; Heywood, V. H.; Burgues, N.A.; Moore, D.M.; Valentine, D. H.; Walters, S. M.; Weed, D. A. In Flora Europaea. (II), Ed.; Cambrige University Press, 1968, pp. 345-350. [18] VaJd^s, B.; Talavera, S.; Femandez-Galiano, E. In Flora Vascular de Andalucia Occidental Ed.; Ketres, 1983; pp. 308-314 [19] Bol6s, O.; Vigo, J. In Flora dels Patsos Catalans (II). Ed.; Barcino, 1990; pp 439-450. [20] Hostettmann, K.; Hostettmann, M.; Marston, A. In Methods in plant Biochemistry; Terpenoids; Charlwood B. V.; Bantliorpe D.V., Eds.; Academic Press: London, 1991; pp 435-466. [21] Hostettmann, K.; Marston, A. In Chemistry and Pharmacology of Natural Saponins; Ed.; Cambridge University Press: London, 1995; pp. 175-232. [22] Kubata, T.; Tonami, F.; Hinoh, H. Tetrahedrom, 1968, 24, 675-686. [23] Yamamoto, A.; Miyase, T.; Ueno, A.; Maeda, T. Chem. Pharm. Bull, 1991, 39, 2164-2166. [24] Pistelli, L.; Rita, A.; Marsili, A. J. Nat Products., 1993, 56, 240-244. [25] Miyase, T.; Matsusima, Y. Chem. Pharm. Bull, 1997, 45, 1493-1497. [26] Shibata, S.; Kitagawa, I.; Fujimoto, H. Chem. Pharm. Bull, 1966, J4, 1023-1033. [27] Kubata, T.; Tonami, F.; Hinoh, H. Tetrahedrom, 1967, 23, 3333-3351. [28] Tan, L.; Zhao, YY.; Zhang, RY.; Hong, SH. Journal of Chinese Pharmaceutical Sciences, 1996, 5,128-131. [29] Bohlmann, F.; Burkhardt, F.; Zdero, C. Naturally Occurring Acetylenes; Ed.; Academic Press: London, 1973; pp. 226-468. [30] Hansen, L.; Boll, P.M. Phytochemistry, 1986, 25, 529-530. [31] Takahashi, M.; Yoshikura, M. J. Pharm. Soc. Jpn., 1964,84,754-755. [32] Crosby, D.G.; Aharonson, N. Tetrahedron, 1967, 23, 465-472. [33] Sohn, H. J.; Jang, G. C ; Ran, H. H.; Len, K. S. J. Korean Agric. Chem. Soc, 1991, 33, 120-124.

696 [34] Morita, M.; Nakajima, K.; Ikeya, Y; Mitsuhaslii, H. Phytochemistry, 1991, 30, 1543-1545. [35] Schulte, K.E.; Wulfhorst, G.Arch. Pharm., 1977, 210, 285-286. [36] Schulte, K. E.; P5tter, B.Arch, Pharm., 1977,3J0, 945-946. [37] Sugiyama, M.; Kikuchi, M. Phytochemistry, 1991, 30, 3147-3149. [38] Sdnchez, C. S.; Diaz-Lanza, A.M.; Fernandez, L.; Bemabe, M. J, Nat, Prod., 1998, 61, 1383-1385. [39] Sdnchez, C. S.; Diaz-Lanza, A.M.; Bemabe, M. Phytochemistry,', In press. [40] S^chez, C. S.; Diaz-Lanza, A.M.; Bemabe, M. J, Nat. Prod,', In press. [41] Ohuchi, K.; Watanabe, M.; Ozeld, T.; Tsunifuji, S. PlantaMed, 1985, 51, 208212. [42] Yen, M.H.; Lin, C.C; Chuang, C.H.; Lin, S.C. Fitoterapia, 1994, 65, 409-417. [43] Chang, W,C.; Hsu, F.L. Prostaglandins Leukot. Essent. Fatty Acids., 1991, 44, 51-56. [44] Bermejo, P.; Abad, M.J.; Silvan, A.M.; Sanz, A.; Fernandez, L.; Sdnchez, S.; Diaz, A.M. LifeSci., 1998, 63, 1147-1156. [45] Guinea, M.C.; Parellada, J.; Lacaille-Dubois, M.A.; Wagner, H. PlantaMed, 1994,60, 163-167. [46] Wagner, H. Z Phytother., 1996,17, 79-95. [47] Ushio, Y.; Oda, Y.; Abe, H. Int J. ImmunopharmacoL, 1991,13, 501-508. [48] Ushio, Y.; Abe, H. Int. J. ImmunopharmacoL, 1991,13, 493-499. [49] Ushio, Y.; Abe, H. PlantaMed, 1991, 57, 511-514. [50] Ushio, Y.; Abe, H. Jpn. J. Pharmacol., 1991, 56, 167-175. [51] Kato, M. et al., Immunopharmacology, 1995,29, 207-213. [52] Yen, M.H.; Lin, C.C; Yen, CM. Phytother. Res., 1995, 9, 351-358. [53] Nose, M.; Amagaya, S.; Ogihara, Y. Chem. Pharm. Bull., 1989,57, 3306-3310. [54] Ahn, B.Z.; Yoon, Y.D.; You-Hui, L.; Kim, B.H.; Sok, D.E. PlantaMed, 1998, 64, 220-224.

[55] Kato, M.; Pu, M.Y.; Isobe, K.; Iwamoto, T.; Nagase, F.; Lwin, T.; Zhang, Y.H.; Yanagita, R; Nakashima, I. Cell Immunol,, 1994,159, 15-25. [56] Motoo, Y.; Sawabu, N. Cancer Lett, 1994, 86, 91-95. [57] Lee, J.; Lee, CK.; Choi, J.W. Korean J. Pharmacogn., 1993,24, 153-158. [58] Nishiura, T.; Marukawa, S.; Ishida, H.; Orita, M.; Abe, H. J. Anesth., 1994,8, 8792. [59] Purmova, J.; Opletal, L. Ceska. Slov. Farm., 1995,44, 246-251. [60] Kumazawa, Y.; Kawakita, T.; Takimoto, H.; Nomoto, K. Int. J. Immunopharm., 1990,72,531-537. [61] Ushio, Y.; Abe, H. PlantaMed, 1992, 58, 171-173. [62] Martin, T.; Villaescusa, L.; Gasquet, M.; Delmas, F.; Bartolom^, C; Diaz-Lanza, A.M.; OUivier, E.; Balansard, G. Pharm. Biol., 1998,36, 56-62.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. Allrightsreserved.

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MINOR POLAR COMPOUNDS OF OLIVE OIL: COMPOSITION, FACTORS OF VARIABILITY AND BIOACTIVITY MARIO DELL'AGLI AND ENRICA BOSISIO* Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, Via Balzaretti 9, 20133 Milan, Italy ABSTRACT: The adoption of the Mediterranean style of diet is recommended for reducing the risk of developing chronic illness, mainly coronary heart disease and cancer. The Mediterranean diet reflects the food pattern of Greece, Southern Italy and Crete in the early sixties and is characterized by high consumption of plant food (fruits, vegetables, breads, cereals, potatoes, beans, nuts and seeds), moderate consumption of fish, poultry and dairy products, low amounts of red meat and low to moderate intake of wine. Olive oil is the dietary fat of choice in Mediterranean countries, but its consumption is increasing elsewhere in USA, Japan, Russia and Canada. Olive oil has a peculiar fatty acid composition rich in oleic acid (56-84% of the total fatty acids) and linoleic acid (3-21%). In addition it contains a variety of minor constituents responsible for the unique organolectic properties and stability to oxidation. Unsaponifiable fraction of olive oil is of a diverse chemical nature. The composition varies depending on cultivars, drupe ripening, climate and enviroment, time of harvesting and storage and processing techniques for the oil production. The highest concentration of these compounds is in the extra-virgin olive oil, obtained by the first cold pressing of the olive paste. The minor components are important not only from a commercial standpoint in order to assess the high quality and good palatability of the olive oil, for but also for their potential beneficial impact on human health. As concerns the nutritional aspects, the antioxidant and free radical scavenger properties of the phenolic portion of the unsaponifiable fraction were seen as highly relevant. The most recent research reports on a variety of biological activities demostrated in in vitro studies (protection of low density lipoproteins from oxidation, inhibition of platelet aggregation and production of proinflammatory mediators etc.). Indirect evidence that olive oil phenols might act also in vivo should stimulate future research for measuring bioavailability and kinetics following dietary intake and confirming in vivo reproducibility of in vitro data.

INTRODUCTION The term "Mediterranean diet" indicates the food patterns of Greece, Southern Italy and Crete in the early sixties and is characterized by high

698

consumption of plant food (fruits, vegetables, breads, cereals, potatoes, beans, nuts and seeds), and moderate consumption of fish, poultry and dairy products (cheese and yogurt). Olive oil as the principal source of fat, low amounts of red meat and low to moderate amounts of wine are consumed [1]. With variations, it is shared by almost all populations living in the areas of olive cultivation in the Mediterranean region. Kushi et al. [2] reviewed the health implications of reducing the intake of red meat, drinking moderate wine with meals and using olive oil as dietary recommendations in order to decrease the risk of developing several chronic diseases. According to epidemiological studies, regular consumption of red meat increases the risk of coronary heart disease (CHD), colon and other cancers and osteoporosis. Possible mechanisms include dietary fat, heme iron, production of carcinogens during cooking and increased urinary calcium loss. Moderate alcohol consumption protects againts CHD, possibly by increasing high density lipoproteins (HDL). One common feature of the mediterranean dietary habit is the use of olive oil as fat source in place of animal fat typical of Northern European and USA diets. As compared to other vegetable oils, olive oil is characterized by the peculiar composition of the trygliceride fraction and by the phenolic and volatile constituents which affect the organolectic properties. Olive oil is rich in monounsaturated fat (56-84% of oleic acid), contains 3-21% of the essential linoleic acid [3], is low in tocopherols [4,5] and therefore the presence of phenols is important to mantain the anti-oxidative stability. Several articles [1,2,6] reviewed the reasons why olive oil should be preferable to other dietary fat, paying particular attention to the fatty acid composition. Oleic acid is antithrombotic compared to saturated fatty acids [7]. Monounsaturated and polyunsaturated fats reduced low density lipoproteins (LDL) levels.

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Substitution of olive oil for carbohydrates increased HDL, leaving unaltered LDL levels [8]. In the Mediterranean countries, where olive oil is the major source of fat, low rates of CHD and breast and colon cancer and total mortality are reported [2]. A non-negligeable contribution to beneficial effects of olive oil is given by the nonsaponifiable fraction and in particular its phenolic constituents. In this article attention is focused on the minor polar components (MPC) of olive oil. While the interest of food chemists in MPC, as important factors for taste and stability of olive oil, dates back to the early seventies, the interest of biologists in the phenolic fraction is rather recent. The intention of the authors is to bring together in this article the research findings on chemistry of MPC, the variables influencing composition and amoimt in the olive oil, the use of MPC evaluation for the quality control of olive oil. The final part will be devoted to covering the latest findings on the biological activities associated to some of the MPC, in relation to human health. 1. Chemical composition of minor constituents of olive oil According to merceologic parameters, olive oils are classified in accordance with quality criteria (European Community, Council regulation 365/92, 1992): extra-virgin, virgin and semi-virgin oils are characterized by low acidity (1.0, 1.5, H3

OH

HO

deacetoxy oleuropein aglycone

CH

HO-

ligustroside aglycone

\J

.O

COOCH3

3S OH

Fig. (1). MPC identified in virgin olive oil

706

deacetoxy ligustroside aglycone

elenolic acid

elenolic acid methylester OH

Fig. (1). Continue

COOCHj

1R = 0H 2R = H Fig. (2). Structure of the dialdehydic forms of elenolic acid linked to (3,4-dihydroxyphenyl)-ethanol (compound 1) and to (;7-hydroxyphenyl)-ethanol (compound 2). Compound 3 is an isomer of oleuropein aglycone.

707

The dialdehydic isomers were also found in olive waste waters [18]. 3,4dihydroxyphenylglycol, a catechol metabolite of noradrenaline, was found in trace amounts in the virgin olive oil derived from the Cipressino and Drifta cultivars, and represented the major component in the vegetation water of the fruits of these cultivars [19]. 2. Factors of variability of minor polar compounds A broad variability in the composition of virgin olive oil is generally acknowledged. Surveys were performed on genotypes, climate, agricultural techniques and post -harvest technology (extraction and oil conservation). 2.1 Influence ofcultivar, geographical areas and ripening

Analyses were performed on olive oils produced in Italy during the years 1991-92 and 1992-93, in different sampling areas and for cultivars representative of those areas [20]. The results of this survey permitted the conclusions that the characteristics of virgin olive oils are influenced by genetic and environmental factors. Total sterol levels are affected by cultivar and region of cultivation. The ripening causes a slow continuous decrease of these compounds. Tocopherols are influenced by genetic and environmental factors, conditions of post-harvest fruit transportation and storage, and oil conservation. The smallest quantities were found in oil samples with high acidity and peroxide values. The levels of MPC (expressed as tyrosol) appear to be cultivar and area dependent and inversely correlated with the harvest season. Acidity and peroxide values are linked (weakly and strongly respectively) to the

708

tocopherol and polyphenol content [20]. Recent studies [21-23] where the polar fraction in different olive varieties was evaluated by HPLC analysis of the single constituents confirmed previous results: varietal qualitative and quantitative changes of oleuropein, demethyloleuropein, verbascoside and anthocyanins were found in several cultivars of different regions in Italy. Demethyloleuropein was suggested as a varietal marker since it was found only in two out of eight varieties [21]. It is important to determine a correlation between cultivar and composition of the polar fraction since phenolic compounds may confer a "marked bitter taste" or a "sweet taste" [24] depending on the cultivars of drupes: fruits from Coratina cultivar are typically bitter and pungent, while olives from Ogliarola salentina have a sweet taste, depending on the amount of oleuropein and related compoimds, which were higher in the Coratina with respect to Ogliarola salentina cultivar [22]. Variations in amount and composition of MPC in olives of several varieties have been monitored during development and ripening of the fruits [25,26]: oleuropein content increased during growth and rapidly dropped during ripening, when demethyloleuropein and elenolic acid glucoside are formed [27-29]. Oleuropein decline is accompanied by an increase in flavonoids [25,30], verbascoside and hydroxytyrosol [31]. Demethyloleuropein was not found in all cultivars analyzed, indicating that this compound might be used as varietal marker [21,29]. The increase in esterase activity during ripening could be responsible for these changes [29]. Therefore time of harvest greatly affects oil content and composition ofMPC. The content of phenols and volatile compounds (€5 aliphatic compounds) is correlated positively with fruit pigmentation up to a value after which a reverse tendency occurs. Maximal concentration was found when drupe pigmentation was not completed (semi-black), a time which

709

corresponds also to maximal accumulation of oil in the fruit [32]. The respiratory rate of olive drupes reaches a minimimi in November at the phase of climacteric crisis of the fruits. In this stage the oil extracted from the drupes gave the best analytical characteristics and was rich in tocopherols, phenols and aromatic volatile substances. The respiration rate was therefore suggested as a ripening index to establish the optimal harvest time [33]. Harvest methods (by hand or brushes) did not influence total polyphenol content in oil when it was processed just after fruit harvest. The use of brushes however accelerated total phenol loss in oil when the fruits were stored for one week before oil extraction. Time interval between harvest and oil processing negatively affects the phenol content [32]. Storage, even at low temperature is also detrimental to the quality of oil since chilling causes injury to olives and deterioration occurs due tofimgalgrowth [34]. 2.2 Influence of oil preparation techniques

The formation of €5 aldehyde and alcohols in plant tissues is related to cell destruction. Disruption of intact cells during crushing and milling results in the release of lipid-degrading enzymes, lipoxygenases and fatty acid hydroperoxide lyase, which cleave the fatty acid moiety to €5 compounds [13,35]. The steps for obtaining oil are preparation of the paste, malaxation and oil separation by pressing, percolation and centrifugation. Techniques employed at each step influence the quality of the obtained oil, in term of taste and anti-oxidant stability [22, 36-38]. The equipment used for paste making (hammer crusher, metal disc crusher or stone mills) might influence bitterness and the color of the oil: crusher generally increases the amount of total phenols, in particular that of bitter secoiridoids, and of

710

chlorophyll [22, 38]. If the composition of total phenols is analyzed milling contributes more to the presence of simple phenols (tyrosol, hydroxytyrosol, phenolic acids), whereas crushing causes higher content of oleuropein and other unidentified compoimds [22]. The duration (15-90 mins) and temperature of water added at the step of malaxation reduce the amount of phenols in the oil [32,36]; depending on water / solvent solubility, partition of compounds in waste waters and oil follows their partition coefficients. The systems of extraction: pressure, percolation and centrifugation, have different effects depending on the quality and ripening of olives. With good quality fruits, the pressure system yielded oil with greater quantity of phenols, while the centrifugation system was preferable when ripe and poor quality olives are processed: in this case, the oil contained less free fatty acids and more acceptable taste than with the pressure system [37]. The procedure of adding water to the " oily must" for oil separation has negative effects on total phenols content in oil. In conclusion it appears that the choice of the processing system for producing oils of greater quality and better taste is determined by the olive cultivar and quality of drupes. Differentiating the systems would be instrumental in achieving better organoleptic qualities of the extra virgin oils, while enhancing their preservation [22]. 3. Antioxidant activity and oil stability Deterioration of food lipids by free radical chain reaction and lipid peroxidation is a major problem for food manufacturers. The main route of deterioration of vegetable oils is rancidity deriving from the oxidation taking place at the insaturation sites of the fatty acids in the triglyceride molecules. In general the higher the number of double bonds, the easier is

711

oxidative deterioration. Oxidation proceeds through a series of steps. A fatty free radical is formed by loss of an hydrogen at the a-methylenic carbon in the fatty acid chain. Oxygen attack to this radical causes the formation of peroxides and hydroperoxides. The decomposition step is the splitting of peroxides into smaller compounds, such as aldehydes, ketons, alcohols and short chain acids, responsible for the disagreable odors typical of a rancid fat. The fatty free radical acts as strong initiator of further oxidation hence oxidative degradation is regarded as an autocatalytic process. Oxidative stability is measured as the number of storage days required to obtain a peroxide values of 70 |Lieq/g oil [39], or by the Rancimat induction time [40-42]. A relationship exists between susceptibility to oxidation and the levels of tocopherols, a, p and y being the forms dominating in vegetable oils [5]. Removal of tocopherols from vegetable oils greatly reduces oxidative stability. Levels of tocopherols in virgin olive oil are rather low when compared to other vegetable oils (table 2) [5], Nevertheless virgin olive oil is characterized by higher oxidative stability with respect to other dietary oils. The resistence to oxidation was related to the high content of polyphenols; removal of polyphenols greatly reduced oxidative stability [39]. The subsequent studies were devoted to identifying the compounds which more contribute to oil stability and the mechanism of protection. Hydroxytyrosol and caffeic acid were better protection factors than BHT; tyrosol and other monohydroxy phenols gave little contribution to oil stability [43]; oil shelf-life was positively correlated to hydroxytyrosol / tyrosol ratio [44]. A good correlation (r = 0.97) was found with the dialdehydic form of hydroxytyrosol-elenolic acid and the isomer of oleuropein aglycone ( c o m p o u n d s 1 and 3). a -Tocopherol acted synergically with hydroxytyrosol and the secoiridoid derivatives [45,46]. Simlight exposure

712

increased tyrosol levels in virgin oil and degradation of secoiridoids, thus reducing the antioxidant activity of these phenolic compounds [45]. The comparison of the antioxidant potency of hydroxytyrosol, tyrosol, oleuropein with respect to vitamin E and BHT and the synergistic effect with vitamin E were studied in thermal initiated oxidation of methyl linoleate in the presence of azo-compounds, in heptanol or propanol:water. The activity of the phenols was expressed as increase of induction period and extent of reaction inhibition [47]. The azo compounds initiated oxidation is a free radical chain process. Confirming the results from a previous study [43], this study showed that hydroxytyrosol and oleuropein were much more effective than BHT and vitamin E in extending the induction period. Tyrosol was devoid of activity and no synergistic effect on the preservation of methyl linoleate was found when vitamin E was used together with tyrosol. Hydroxytyrosol proved to be a good protective factor of lipid peroxidation in ox-brain phospholipid liposomes treated with Fe^"^ascorbate and a scavenger of peroxyl radicals formed by pulse radiolysis of carbon tetrachloride and propan-2-ol (rate constant 8.4 |iM sec") [48]. The antioxidant mechanism of olive oil compounds was examined by evaluating the efficiency of several phenols (caffeic acid, hydroxytyrosol, oleuropein and tyrosol) to scavenge hydroxyl radicals, lipid radicals and superoxide ions [49,50]. Several tests including cell-free systems and activated human neutrophils were employed. The latter is based on the production of superoxide ions in phorbol-12-myristate-13-acetate (PMA)challenged human neutrophils. According to Chimi et al [49], all compounds exhibited various efficiencies towards hydroxyl radicals: the order was oleuropein>tyrosol>caffeic acid>hydroxytyrosol whereas they presented the same order of reactivity towards lipid radicals. Oleuropein, caffeic acid and hydroxytyrosol scavenged superoxide ions both in the

713

xanthine-xanthine oxidase reaction and in PMA-challenged neutrophils, hydroxytyrosol being more potent than oleuropein [50]. The order of potency showed some discrepancies in the two studies, which may depend on the experimental conditions employed. 3.1 Phenols of 'waste waters' as source of natural antioxidants

During malaxation, when water is added to olive paste to wash it, tons of waste water (800.000 in Italy only) are produced from the oil industry and discarded. Water soluble compounds are then transfered from olives into waste waters and could be recovered. HPLC analysis of the waste water extract showed the presence of hydroxytyrosol, tyrosol, p-OH benzoic acid, vanillic acid, caffeic acid, oleuropein, verbascoside and other oleuropein derivatives. The extract had powerful antioxidant activity and could represent a cheap source of natural antioxidants to be used to preserve food and cosmetics from fat oxidation and rancidity [51]. 3.2 Prooxidant effects

It has been proven that antioxidants that protect lipids from free radical deterioration may accelerate damage to other molecules such as DNA, carbohydrates and proteins [52-54]. The prooxidant effect can be evaluated as stimulation of DNA damage in bleomycin-Fe^^ induced DNA degradation or as the ability to accelerate sugar deoxyribose damage by hydroxy 1 radicals generated by a mixture of ascorbate, FeCls EDTA, H2O2. Hydroxytyrosol had a weak stimulatory effect in the bleomycin assay at 0.65 mM. The pro-oxidant effect was concentration dependent in the deoxyribose system and the presence of albumin protected from the damage [48]. In a subsequent study performed in

714

another model system, DNA damage by the complex copperphenanthroline, the same authors found that hydroxytyrosol weakly stimulated copper dependent modification of DNA bases, only at millimolar concentrations [55]. These concentrations may not be achieved in vivo after consumption of extravirgin olive oil. Thus the choice of the model system is critical for the evaluation of anti- and prooxidant capacity of a compound. 4. Biological activity of MPC of olive oil 4.1 Atherosclerosis andLDL oxidation

Oxidation of cellular components by reactive oxygen species (ROS) and free radicals is involved in a variety of serious acute and chronic diseases: inflammation [56], ischemia-reperfusion damage [57,58], lung disease [59], kidney damage [60], atherosclerosis, diabetes, allergies, cancer and aging [61]. Atherosclerosis is a multifactorial pathology, and genetic and enviromental factors contribute to the development of the disease. Endothelial and smooth muscle cells and blood components, including monocytes/macrophages, platelets and LDL play a crucial role in the formation of the atheromatous plaque "Fig. (4)", [62]. High levels of circulating LDL, which are rich in cholesterol and cholesteryl esters, cause cholesterol deposition into the endothelium of the so-called lesion-prone sites of the arterial wall. These areas are characterized by increased permeability to albumin, fibrinogen and LDL cholesterol and by a high monocyte recruitment activity. Monocytes migrate from the endothelium to the intima, and differentiate into macrophages. Recruitment and migration are part of an inflammatory

715

Phase 1 Areas of the arterial wall defined as lesion-prone sites are characterized by high permeability to LDLcholesterol and extensive monocyte recruitment activity.

Phase 2

Monocytes migrate from the endothelium to the intima and differentiate into macrophages. The oxidation of LDL by free radicals generated by smooth muscle cells^ endothelial cells and macrophages, induces modifications to both the lipid and protein moieties of the lq)oprotein particle.

Phase 3 Lipoprotein uptake and cholesterol accumulation in the macrophages proceed unlimitedly, thus leading to foam cell formation. Oxidatively modified LDL increases monocyte recruitment, are cytotoxic towards endothelium causing damage to integrity, are antigenic thus predisposing for the conditions to develop the late stage of the atherosclerotic lesion.

smooth muscle cett

macrophage

monocyte

LDL

endothelial cell

O

Fig. (4). Formation of the atheromatous plaque

ox LDL

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process which is guided by chemotactic factors including monocyte chemotactic protein 1 (MPC-1) and oxidized LDL. The oxidation of LDL by free radicals generated by smooth muscle cells, endothelial cells and macrophages, induces modifications to both the lipid and protein moieties of LDL. Polyunsaturated fatty acids are oxidized to hydroperoxides with concomitant release of aldehydes and malondialdehyde (MDA); aldehydes, in turn, react with lysine residues of the apoprotein, leading to protein fragmentation. Free and esterified cholesterol are transformed into oxysterols, "Fig. (5)", [63]. Once LDL are oxidized, they are no longer recognized by the normal LDL receptor (known as apo B/E receptor), but are recognized by the so-called macrophage scavenger receptor. Unlike the normal receptor, the scavenger receptor is not down-regulated by excess cholesterol [64]; lipoprotein uptake and cholesterol accumulation in the cells proceed unlimitedly, thus leading to foam cell formation. Oxidatively, modification of LDL has several biological consequences: increase of monocyte recruitment by induction of MPC-1 and other chemotactic factor synthesis, cytotoxicity towards endothelium causing damage to its integrity, antigenicity which facilitates the conditions predisposing the development of the late stage (inflammatoryautoimmune) of the atherosclerotic lesion. Most of the atherogenic potential of oxidized LDL is awarded by cholesterol oxidation products. These compounds are immunosuppressant, mutagenic and carcinogenic (for further readings on oxysterol toxicity see ref [63]). Thus, inhibition of oxidative modification of LDL is seen as a new strategy for preventing and retarding atherogenesis, as supported by epidemiological and controlled studies that correlate high intake of antioxidants with low incidence of coronary heart disease [65-67], Accordingly, attention has been given to the minor polar compounds of olive oil as protective agents in oxidative stress and related diseases.

717

IN.

l\

"^"i> 5p,63-epoxide

JN. 5a-0H

7P-0H

Fig. (5). Cholesterol oxidation products

4.2 Effects ofMFC on LDL oxidation in vitro

LDL oxidation can be evaluated in vitro by incubation of LDL with a variety of agents including chemicals such as transition metal ions, cultured cells such as macrophages and endothelial cells and by physical means as UV irradiation. LDL particle contains various antioxidants (tocopherols, p-carotene, ubiquinol 10, criptoxanthine): the addition of oxidative agents causes a loss of vitamin E and the starting of lipoperoxidation. The effect of olive oil polyphenols on LDL oxidation v^as estimated in various models of chemically and cell-mediated oxidation and evaluating

718

different parameters of oxidative damage. Oleuropein 10"^ M delayed the fall of vitamin E and the formation of TEARS (thiobarbituric acid reacting substances, an index of lipid peroxidation), and the accumulation of lipid hydroperoxides in human LDL oxidized with CuS04 [68]. Other compounds were shown to contribute to the protection of LDL from oxidation: hydroxytyrosol and two hydroxytyrosol esters 10"^ M prevented the loss of Vitamin E, the formation of TEARS, the modification of protein moiety and the increase of peroxide levels: in particular the levels of the major PUFA in LDL (linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids) were mantained close to the basal level as in native LDL. The lag time of conjugated-dienes formation was prolonged by the addition of phenols to LDL before starting oxidation either by CuS04 and horseradish peroxidase system (H2O2) [69,70]. Hydroxytyrosol dose dependently (1-10 |xM) prevented peroxyl radical dependent oxidation by the water soluble azo compound AAPH, 2,2'azobis(2-amidinopropane)hydrochloride. At 5 |LiM of the anti-oxidant the lag-time before oxidation took place increased to 2 hrs [55] As summarized in the previous paragraph, LDL cholesterol is oxidized to various oxysterols due with high cytotoxicity and promoting vascular injiuy [63]. Oleuropein and a mixture of phenols extracted from virgin oil inhibited dose-dependently the formation of oxysterols and prevented apoprotein modification in UV irradiated LDL. IC50 are shown in table 4: results indicate that the mixture is more potent than the single compounds and probucol, a hypocholesterolemic drug with well-known antioxidant activity. As expected the contribution of tyrosol to the antioxidant effect was minimal [71].

719 Table 4. IC50 of phenolic components of olive oil for the inhibition of oxysterol formation in human LDL*

Compound

IC5o(jiM)

oleuropein

9.2±0.8

tyrosol

47±9.2

phenol enriched extract

8.2±0.7

probucol

20±9.0

* With the permission from Caruso et al. [71]

4.3 Inhibition of platelet aggregation and arachidonate cascade by MFC

Platelets in hypercholesterolemic patients are more susceptible to aggregation than normal subjects [72]. Aggregation is mediated by several mediators, including cyclooxygenase and lipoxygenase metabolites of arachidonic acid [73]. A deficiency of vitamin E enhanced eicosanoid production in platelets [74,75]. Arachidonic acid lipoxygenases are involved in the synthesis of biomodulators which are strictly related to various diseases: allergy, inflammation, atherosclerosis and cancer [77]. 5-Lipoxygenase catalyzes the first step of reactions leading to 5-hydroxyeicosatetraenoic acid (5HETE) and leukotriens responsible for inflammatory and allergic responses. 12-Lipoxygenase catalyzes the formation of 12-HETE, involved in atherosclerosis and tumor metastasis [76]. Human and animal studies evidenced that polymorphonuclear (PMN) leukocytes are involved in the development of CHD, angina, and in a number of pathophysiological conditions related to chronic inflammation and vascular injury. Leukotriens released by PMN under the challenge of various stimuli, promote chemotaxis, aggregation and degranulation of

720

PMN leukocytes [77]. Most of the described events are free-radical mediated, which prompted the researchers to investigate the effect of MPC on them. Chemical (collagen and ADP) induced platelet aggregation and accumulation of the proaggregant agents thromboxane and 12-HETE were inhibited by hydroxytyrosol (100-400 |LIM), At 100 |iM the effect of hydroxytyrosol (55% inhibition) on platelet aggregation was 5 times higher than that of oleuropein (11% inhibition) and of the same order of magnitude of NDGA (nor-dihydroguaiaretic acid, 52% inhibition), a natural antioxidant. In the same study, the phenolic fraction present in the so-called "waste waters", which was shown to be rich in antioxidants [51], was tested on platelet function and showed potent anti-aggregatory activity (80% inhibition at 20 ppm as gallic acid equivalents, which correspond more or less to 100 |iM) [78]. Contrasting results were obtained by Kohyama et al. [79] and de la Puerta et al. [80]: none of the tested phenols, hydroxytyrosol, tyrosol, caffeic acid and oleuropein inhibited thromboxane generation in rat platelets and peritoneal leukocytes activated with calcium ionophore A23187. The discrepancy might be attributable to the different cell type and to the lower concentration of compounds used in the study by de la Puerta [80]. However it is questionable whether such a high concentration, 100-400 |LiM, as employed by Petroni et al. [78] is attained in vivo . Hydroxytyrosol potently inhibited the synthesis of leukotriene B4 and its metabolites 20-hydroxy and 20-carboxy LTB4 in calcium-ionophore stimulated human PMN leukocytes. IC50 was 1.2 |LiM; zileuton used as reference compound had an IC50 of 1.5 |LLM [81]. Inhibition of 5- and 12lipoxygenases from rat platelets and leukocytes by hydroxytyrosol was shown by Kohyama et al [79]. These authors compaired the inhibition by hydroxytyrosol with other related phenols present in olive oil (tyrosol.

721

coumaric acids,vanillic acid, syringic acid and protocatechuic acid) and found that the catechol structure is necessary to elicit the effect. These results were confirmed by a later study performed with rat peritoneal leukocytes [80]. Four compounds were tested: oleuropein, tyrosol, hydroxytyrosol and caffeic acid. Order of potency for 5-lipoxygenase inhibition was: hydroxytyrosol> oleuropein> caffeic acid> tyrosol. The IC50 for hydroxytyrosol (15 |xM) was one order of magnitude greater than that (1.2 |iM) found by Petroni et al [81]. The discrepancy is likely to depend on the source of leukocytes, man in the former and rat in the latter study. No sign of toxicity was observed in leukocytes incubated with the tested phenols, as estimated by thiazolyl test for mitochondrial integrity. 4.4 Effect of phenols on nitric oxide metabolism

Nitric oxide (NO) is a gaseous free radical with cytotoxic and cytostatic properties, released from macrophages as a defense response to pathogens. It is synthesized from L-arginine by the enzyme nitric oxide synthases (NOSs), both the constitutive (cNOS) and the inducible (iNOS) forms of the enzyme are reported. In addition to antimicrobial activity, nitric oxide is an important cellular messenger involved in vascular and neurological signal trasduction and function. It inhibits platelet aggregation, regulates bood pressure and promotes vasorelaxation in order to facilitate blood flow after ischemia (see refs [82-85] for further readings on the biochemical and pathological aspects). Oleuropein, dose dependently (1-100 |iM) enhanced nitric oxide production in E. Coli lipopolysaccharide (LPS)-challenged mouse macrophages. The effect was mediated by stimulated expression of inducible NOS, determined by western blot analysis. No sign of macrophage cytotoxicity was observed with 100 |LIM oleuropein, which

722

per se did not trigger NO production, suggesting that it does not interfere with the basal process involved in NO generation [86] 4.5 Effect on reactive oxigen-induced cytotoxicity

The ability of phenols to prevent cytotoxicity induced by reactive oxigen species was studied in Caco-2 cells (human epithelial cell line derived from colon carcinoma), rat primary hepatocytes and human erythrocytes. Caco-2 cells were subjected to oxidation by H2O2 and superoxide ions generated by xanthine/xanthine oxidase [87]. Hydroxytyrosol, but not tyrosol, prevented cytotoxicity (evaluated as cell viability and permeability) and liperoxidation, measured as release of MDA. The active concentration depended on the type of oxidative stress: 250 and 100 |LiM respectively in the case of H2O2 and xanthine oxidase. Similarly hydroxytyrosol (50-200 |LIM) inhibited oxidative hemolysis and lipoperoxidation and prevented the decrease of energy-dependent methionine and leucine transport in red blood cells challenged with H2O2 [88]. These cells lack the transcriptional and translational systems, therefore the defense mechanisms are not mediated by induction of protein synthesis. Then the molecular mechanism of the protective effect of hydroxytyrososl is its ability to scavenge peroxyl radical [48], acting as chain-breaking inhibitor of lipid peroxidation. Alternatively it might chelate iron ions, which are known to initiate and propagate lipid peroxidation. Rat hepatocytes were treated with Fe-NTA (nitriloacetic acid) [49]. In this model, all the tested compounds, including tyrosol, were able to inhibit lipoperoxidation in the same concentration range 20-100 |LIM (hydroxytyrosol, caffeic acid and oleuropein). The antioxidant activity of caffeic acid has been already described in erythrocytes, rat isolated

723

hepatocytes and Chinese hamster V79 cells [89-91]. The protective effect of tyrosol, a monohydroxy phenol, was quite surprising, since this compound is almost devoid of anti-oxidant and free radical scavenger activities. The result might suggest that cytoprotection could be mediated by mechanism/s independent of anti-oxidant activity. 4.6 Other effects

Substances capable of interaction with ROS are claimed to play an important role in the prevention of cancer. Some compounds of the unsaponifiable fraction from virgin oil (oleuropein, tyrosol, sterols and triterpenoids) were assayed to determine their cytostatic activity in McCoy cells (originated from synovial fluid from a patient suffering degenerative arthritis) [92]. Oleuropein and the sterol+triterpenoid fraction potently inhibited cell growth at 6 |ig/ml (83 and 89% respectively), a concentration recommended by the protocols of the National Cancer Institute of USA [93]. IC50 for oleuropein and sterol+terpenoid fraction was 4.4 and 0.11 |ig/ml, respectively. Tyrosol exhibited lower inhibition: IC50 was 10 |ig/ml. 4.7 Ex vivo effects ofMFC

Some evidence that olive oil phenols might act in vivo comes from the few animal studies described in the following paragraphs. a) Influence on LDL oxidability

Scaccini et al [94] gave the first indirect demostration that factors other than the fatty acid composition and the vitamin E content of dietary oils

724

determine for the stability of lipoprotein LDL to oxidation. Rats were treated with three diets equalized for vitamin E content containing soybean oil, olive oil, or an oleate-rich mixture of triglycerides for a six week period. TEARS and vitamin E were measured in plasma and LDL+VLDL. Lipoprotein resistence to copper mediated oxidation was evaluated by conjugated diene production, lipid hydroperoxide formation and TEARS release. Despite the fact that plasma and lipoprotein levels of ascorbic acid, vitamin E and sulphydryl groups were similar in the three groups of animals, TEARS were lower in plasma and LDL+VLDL of rats fed virgin olive oil. Lipoprotein fractions from olive oil fed rats were remarkably resistent to oxidation, more than the LDL+VLDL of triolein fed animals. The results suggest that the fatty acid unsaturation is not the only determining factor of lipoprotein stability to oxidation, in this animal model. In this study the content and the nature of anti-oxidants in the olive oil was not determined. A partial confirmation of these observations comes from a subsequent study [95], in rabbits. In this experimental design, rabbits were fed refined olive oil, high oleic sunflower oil and extra virgin olive oil. Polyphenols were detectable only in the extra virgin oil. These authors observed that in rabbits fed extra virgin oil, the lag phase before demostrable oxidation occurred was increased as compared to the groups of animals receiving refined olive oil and high oleic sunflower seed oil. In contrast with the results of Scaccini et al. [94], production of conjugated dienes did not differ in the three groups, indicating that there was no influence on this parameter by the polyphenol content. The observation that plasma MDA was lower in rabbits fed the refined oil compared with the values measured in plasma of rabbits fed the virgin olive oil and sunflower oil was also intriguing. It is not clear

725

from this study what factors in the refined olive oil may be functioning in vivo and not in vitro. Oleuropein antioxidant activity was evaluated "ex vivo", in rats treated with 100 mg/kg of the compound [96]. Plasma and bile were collected and assayed for chemiluminescence reaction. The addition of a solution of antioxidant to a glowing steady-state chemiluminescent reaction temporarily interrupts the light output. The light emission is restored after a time interval that is related to the amount of anti-oxidant added [97], Bile was collected at 30 min interval for 2 hrs; plasma was collected before sacrifice. No difference of light emission was measured with plasma from controls and treated animals. Bile samples of treated animals showed a significant inhibition (90%) of the light emission compared with bile samples from control animals (60%). These results may be explained by taking into account an hypothesis for the fate of oleuropein in vivo. If oleuropein is degraded to hydroxytyrosol and, according to the results reported by Bai et al. [98], this metabolite is rapidly removed from the plasma, then at the time interval of 2 hrs, when plasma was collected in the study by Speroni et al [96], the anti-oxidant molecule would not be present any longer, thus explaining the lack of effect of plasma of oleuropein treated rats on the chemiluminescence reaction. In contrast oleuropein or its metabolites might be eliminated through bile, which responded positively to the test. b) Cardiovascular effects of oleuropein

The decoction of the leaves of Olea europea L. has been used empirically as hypotensive remedy. Early experimental studies showed that extracts of Olea europea L. leaves possess hypotensive, antiarhythmic, vasodilator effects [99-101] and produced some evidence that oleuropein

726

could be credited for the hypotensive activity of olive leaves [102,103]. Recently Zarzuelo et al. [104] showed that the hypotensive effect of the leaves is associated to vasorelaxation, which was independent of the integrity of the vascular endothelium. Oleuropein and possibly other components of the decoction were responsible for the relaxant action. Cardiovascular effects of oleuropein were studied further in anaesthetized dogs [105]. Oleuropein, given at 2 mg/kg, slightly increased the sinusal cycle of the sinoatrial conduction time and the sinus node recovery time, prolonged the atrioventricular conduction and increased the duration of atrial and ventricular monophasic action potential. The electrophysiological effects and the mechanism for the negative chronotropic action of oleuropein have been investigated in isolated guinea pig atria [106]. The amplitude of contraction decreased and sinus node recovery time was prolonged by the drug. Oleuropein decreased atrial contractile force at concentrations two order of magnitude lower than those required to reduce atrial rate. Oleuropein had no effect on the resting membrane potential, amplitude and Vmax of the action potentials. Since Vmax of the action potential is an indirect index of the fast inward Na"^ current in the absence of changes of membrane potentials, it was then excluded that oleuropein inhibits fast inward Na"^ current. The electromechanical uncoupling could not be attributed to an inhibition of Ca2+ entry through L-type channels. Then the authors concluded that the negative inotropic effect might be explained by a mechanism occurring at intracellular levels. Oleuropein was studied in experimental hypercholesterolemia in rats. The drug at doses of 0.5 and 10 mg/kg administered orally reduced the increase of serum cholesterol and triglycerides in diet and Triton WR1339 induced hypercholesterolemias [107].

727 4.8 Bioavailabilty and metabolism ofhydroxytyrosol

The beneficial effects of olive oil phenols have been shown in a number of studies in vitro. Investigations in vivo are rare due to the lack of commercially available compounds. Recently a group in Japan has published the first study on the determination ofhydroxytyrosol in plasma after oral administration to rats by GC-MS quantitative method [98]. Hydroxytyrosol of 98% purity (as estimated from ^H NMR) was synthetized in high yield (90%) and in gram-scale from 3,4dihydroxyphenyl acetic acid. The peak of plasma concentration (0.4-2 |Xg/ml) ofhydroxytyrosol was recorded at 5 min after oral administration of 10 mg of compound suspended in tragacanth gum. The plasma concentration dropped sharply, clearance was rapid and no levels were measurable after two hours. The results are rather preliminary; nevertheless it appears that bioavailability of hydroxytyrosol is rather low, compared to the administered dose, but no conclusions could be drawn as no information is available on whether hydroxytyrosol is metabolized in the gastrointestinal tract. 5. Conclusions A great deal of effort has been dedicated to the analytical characterization of extra virgin oil, focusing the attention on the composition and levels of MPC for assessing quality parameters. Oxidative stress and free radicals are becoming more and more the targets for drugs and minor nutrients, to prevent and retard the development of many pathologies. Thus the presence of phenolic substances in extra virgin oil attracted the interest of biologists to investigate their ability to prevent oxidative free radical reactions and the subsequent tissue damages, under several in vitro

728

conditions. It is now essential to concentrate the efforts of future research on determining bioavailability, kinetics and metabolism after dietary intake. And last but not least, studies should be carried out to show how the results obtained in in vitro studies might be reproduced in and correlated with human in vivo studies. ABBREVIATIONS 5-HETE 12-HETE AAPH ADP BHT CHD CNOS Fe-NTA GC-MS HDL HPLC iNOS LDL LPS MDA MPC MPC-1 NDGA NMR p-OHbenzoic acid PMA

= 5-hydroxyeicosatetraenoic acid = 12-hydroxyeicosatetraenoic acid = 2,2*-azobis(2-amidinopropane)hydrochloride = adenosindiphosphate = butylhydroxytoluene = coronary heart disease = constitutive nitric oxide synthase = Fe-nitriloacetic acid = gas chromatography-mass spectrometry = high density lipoprotein = high pressure liquid chromatography = inducible nitric oxide synthase = low density lipoprotein = lipopolysaccharyde = malondialdehyde = minor polar components = monocyte chemotactic protein 1 = nor di-hydroguaiaretic acid = nuclear magnetic resonance = p-hydroxybenzoic acid = phorbol-12-myristate-13-acetate

729

PMN-leukocytes PUFA TEARS VLDL

= polymorphonuclear leukocytes = polyunsaturated fatty acids = thiobarbituric acid reactive substances = very low density lipoproteins

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

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PLANT PEROXIDASES: VERSATILE CATALYSTS IN THE SYNTHESIS OF BIOACTIVE NATURAL PRODUCTS A. ROS BARCELO AND F. POMAR Department of Plant Biology (Plant Physiology) University ofMurcia, E-SOlOOMurcia, Spain ABSTRACT: Class III plant peroxidases are a polymorphic group of heme-containing enzymes located in the vacuole and the plant cell wall. Class III plant peroxidases are capable of oxidizing a broad range of structurally different substrates. This is determined by the strong oxidizing potentials of the activated forms of peroxidase, compound I and compound II, and makes them suitable oxidizing agents in planta for all chemicals donating one electron (alkaloids) or a hydrogen atom (phenolics). Although the participation of peroxidase in both phenolic and alkaloid metabolism is generally associated with concrete steps related to turnover and catabolism, processes which generally reduce the biological (pharmacological) activity of such compounds, in certain cases the oxidation products of such reactions have remarkable biological properties, having been described as antioxidant, antibacterial and antifungal. Other peroxidasecatalyzed oxidation products show antitumor and antiviral activity, togetlier with a miscellany of beneficial (anti-inflammatory, anti-asthmatic and anti-depressant) effects on humans. This review will focus on the general properties of class III secretory plant peroxidases, and emphasis wiU be put on their broad metabolic plasticity, which makes them versatile catalysts in the synthesis of bioactive plant products. Examples of the role of peroxidase in the synthesis of such compounds are reviewed. INTRODUCTION Peroxidases (EC 1.11.1.7; hydrogen donor: H2O2 oxidoreductase) are heme-containing enzymes, which catalyze the single one-electron oxidation of several substrates at the expense of H2O2: 2 RH + H202-> 2 R* + 2 H2O (1) They are widely distributed in the plant kingdom, having been reported in Chlorophyta [1,2], Euglenophyta [3], Rhodophyta [4], Bryophyta [5], Pteridophyta [6] and all the Spermatophyta studied so far [7].

736

Among the heme-containing peroxidases present in eukaryots and prokaryots, plant peroxidases are grouped in a superfamily along side fungal and bacterial peroxidases, in which three distantly related structural classes have been defined [8]. Class I is composed of mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate peroxidases from higher plants, and bacterial peroxidases. Class II groups all the secretory fungal (manganese) peroxidases, while class III contains all the secretory plant peroxidases which show distinctive features from other plant peroxidases, such as the above-mentioned ascorbate peroxidases. Class III plant peroxidases are glycoproteins, and are located in both the plant cell vacuole and in the plant cell wall [9], where most of the bioactive plant products are located. Class III peroxidases show a broad range of substrates with a moderate but noticeable specificity for phenolic compounds, especially for coniferyl alcohol [10]. All these features distinguish them from class I peroxidases, which are not glycoproteins and which are located in chloroplasts, mitochondria, peroxysomes and cytosol, where they show moderate substrate specificity for ascorbic acid. Furthermore, class I peroxidases are inhibited by thiol reagents such as pchloromercuribenzoate and are, in general, regarded as being strongly labile. Class III peroxidases have been the subject of numerous studies [10] and applications [11], since their extraordinary catalytic properties make them a valuable catalytic tool in the plant cell chemical factory, and in organic synthesis. In fact, class III peroxidases, together with other oxidative enzymes, such as cytochrome P450s and oxygenases [12], appear to be the main driving force in the evolution of plant metabolic pathways because individual enzymes can typically accept multiple substrates and form several products. This metabolic plasticity of class III peroxidases, paradoxically, has frequently led to misunderstanding of its vital function in the plant cell biochemical factory. This review will focus on the general properties of class III secretory plant peroxidases, and emphasis will be put on their broad metabolic plasticity, which is what makes them versatile catalysts in the synthesis of bioactive plant products. Examples of the role of peroxidase in the synthesis of such compounds are reviewed.

737

PLANT PEROXIDASES AS VERSATILE CATALYSTS Class n i peroxidases Class III peroxidases are glycoproteins, whose molecular mass usually ranges from 40 to 45 KDa, and which contain protohemin IX as prosthetic group [10]. In their resting state, an iron ion is present in the oxidation state of+3 (Felll) [10]. The iron is five coordinated to the four pyrrole nitrogens of the heme and to a nitrogen from an axial (proximal) histidine. The sixth coordination position is free, thus determining a high spin state for the iron [13]. The prosthetic group is essential for activity and its removal causes total enzyme inactivation [14]. In fact, the catalytic activity of class III plant peroxidase may be simulated by the heme octaand undeca-peptides (microperoxidases), MP-8 and MP-11. These peptides consist of a heme and a peptide resembling the catalytic active center of peroxidase, and which possess most of the properties of these enzymes, exhibiting a similar catalytic cycle to that shown by horseradish peroxidase [15]. Two structural Ca^^ ions are commonly observed in the folded molecule [16], and are considered necessary for its stability [17]. Besides having this stabilizing effect on protein folding, Ca^^ is also considered as an activator of class III peroxidases [18,19]. In the cationic peroxidase from peanut, the presence of 1.0 mole Mn^^ per mole of enzyme has been reported, in contrast with the anionic isoenzyme which apparently does not contain Mn^^ [20]. An interaction between the different prosthetic groups of peroxidases (heme, Ca^^ and Mn^"^) has been postulated to explain their particular catalytic activities [21]. Whether or not this Mn prosthetic group is a general characteristic of the most basic peroxidase isoenzymes is a question that deserves further attention. In this respect, it is worth noting that the presence of such Mn^"^ ion in basic peroxidase isoenzymes could be responsible for the residual phenol oxidase (H2O2independent) activity shown by most basic isoenzymes [9]. Glycosylation of the enzyme varies between 0 and 25 %, the main carbohydrates being GlcNAc, Man, Fuc and Xyl. Most of the oligosaccharides correspond to the high-mannose type [22,23] and, in the case of soybean peroxidase, the structure of the major oligosaccharide, which accounts for 60-65% of the total [22], is:

738

Mana 1 ->6(ManP 1 ^3)(Xyl|31 ^2)Manp 1 -> 4GlcNacp 1 ^4(Fuca 1 ->3)GlcN Ac Glycosylation is one of the main factors determining the unusual thermal stability of class III peroxidases [24], since deglycosylated forms have frequently been observed to exhibit reduced thermal stability [25]. Partial deglycosylation has also been described as causing a change in the kinetic constants of avocado peroxidase and as favoring activation by Ca^^ [25]. Catalytic cycle of class III peroxidases Peroxidases are known to catalyze the one electron oxidation of a wide range of structurally diverse organic and inorganic compounds, Fig. (1). The catalytic cycle, first described by Chance [26] for horseradish peroxidase, may be described as follows. H2O2 oxidizes the ferric form of the enzyme (Felll), in a two electronic oxidation, to yield the enzyme intermediate compound I (Col): Felll + H2O2 -^ Col + H2O (2) Col, which has been described as an oxyferryl porphyrin n cation radical [27], accepts one electron and one proton from a reducing substrate (RH) to yield the corresponding free radical (R*) and the oxyferryl heme intermediate known as compound II (Coll): k2

CoI + RH ^ C o I I + R*(3) The subsequent one-electron reduction of Coll by a second molecule of reducing substrate yields the ferric form of peroxidase, Felll: Coll + RH -> Felll + R* + H2O (4)

739

RV

HOOC

H2O

COOH

Fig. (1). Catalytic cycle of class III plant peroxidases, illustrating the oxido-reduction changes undergone by the prosthethic group

closing, in this way, the catalytic cycle of the enzyme. This catalytic cycle differs slightly for substrates that donate only electrons instead of hydrogen atoms (i.e., electrons + protons) [28]. In this case, the two protons, which are necessary for the liberation of the ferryl oxygen atom as water, do not come from the substrate but are most likely obtained from the bulk water solution: Col + R + IT -> Coll + K^ (5) and Coll + R + IT -> Felll + R*^ + H2O (6)

740

As may be seen from Eqs. (3) and (4), which represent the main features of the enzyme catalytic cycle, a unique feature of the peroxidasecatalyzed reaction is the oxidation of substrates to diffusible free radical intermediates. These unstable radicals are known to participate in a variety of non-enzymatic reactions, including disproportionation, polymerization and electron transfer (oxidative or reductive). Reactivity of the enzyme intermediates As one may expect from the peroxidase reaction mechanism described in Eqs. (2-4), the reactivity of the enzyme intermediates towards a particular substrate may be estimated a priori on the basis of the thermodynamic driving force of these two electron-transfer reactions, which is directly related with the difference between the oxidation/reduction potentials of both the enzyme active intermediates (i.e.. Col and Coll) and the substrate radicals. Thus, the thermodynamic driving force for the reaction of Col (or Coll) with the reducing substrates is the difference between the mid-point potentials of the CoI/CoII (or CoII/Felll) and the substrate radical/substrate (R*,H^/RH) redox couples: ^E = E (CoI/CoII) - E (R*,ir/RH) (7) The mid-point reduction potential for the enzyme active intermediates, Col and Coll, has been estimated from stopped-flow measurements [29], giving the values of £(CoI/CoII) = + 879 mV and £:(CoII/FeIII) = + 903 mV at pH 7.0, both relative to the Standard Hydrogen Electrode (SHE). These values illustrate that Coll may be regarded as a slightly stronger oxidant than Col. However, the order of reactivity of both compounds is somewhat different. In fact, and for a given reducing substrate. Col reacts several-times faster than Coll [30,31] with, in most cases, k2 » h [32]. Hayashi and Yamazaki [32] initially attributed this apparent contradiction to the suspected higher mobility of a porphyrin 7c-electron than that of an iron valence electron. More recently, Candeias et al. [33] have pointed out that the different reactivities of the two enzyme states can be ascribed to a higher apparent rate of activationless electron-transfer in Col reactions, which may, in turn, be attributed to the shorter electron-tunneling distance involved in the electron-transfer to the porphyrin radical cation in Col, compared with the electron-transfer to the iron ion in Coll. In fact, the

741

most important difference between the reduction of Col and Coll is that, in the former case, the electron is transferred to the porphyrin radical cation, whereas in the latter, it is the iron that changes its oxidation state. Kinetic of peroxidase-catalyzed reactions Several kinetic models have been described in the literature to fit steady state peroxidase oxidation rates as a function of substrate and enzyme concentration and to permit the calculation of kinetic constants [34]. Among the most accepted equation rates is that deduced by Rasmussen et al. [34]: v=

2^5[E][RH][H202]

(8)

{hlh) [RH] + [H2O2] based on the three single steps described in Eqs. (2-4). Eq. (8) assumes that ^2 » h, which can be validated from experimental data [30], and that [H2O2] is in a non-inhibitory range, where the participation of other intermediate forms of the enzyme is negligible. Under these circumstances, peroxidase-mediated oxidation rates fitted well to Eq. (8) [9,35-37], and from these representations it is possible to calculate the microscopic constants, k\ and k^,. To calculate k\, Eq. (8) is written as: v=

A [H2O2]

(9)

B + [H2O2] where A = 2 ^3 [RH] [E] and B = {hiki) [RH]. Double reciprocal plots (1/v vs. 1/[H202]) allow A and B values to be calculated for several RH concentrations. Plotting A vs. B values gives a straight line: A = 2A:i[E]B (10) and from its slope it is possible to calculate the k\ value. Similarly, and starting from Eq. (8), the k^ value is calculatedfi*omthe dependence of v on [RH], which may be written as:

742

A[RH] v=

(11) B + [RH]

where k = 2k\ [H2O2] [E] and B = {kilh) [H2O2]. Double reciprocal plots (1/v vs. 1/[RH]) allow A and B values to be calculated for several [H2O2] concentrations. Plotting A vs. B values gives a straight line: A = 2^3[E]B (12) and from its slope it is possible to calculate the k^ value. These A/B plots allow us to calculate the true reaction constants, h, from the steadystate measurement of the oxidation rate, avoiding their intrinsic dependence on substrate concentration [35]. A striking feature of the peroxidase-catalyzed reaction is the intrinsic dependence of the Ky^^ values for the reducing substrate (RH) on [H2O2]: ^ M ^ = B = (^i/A:3)[H202](13) as may be validated experimentally, Fig. (2), and the intrinsic dependence oiKu "'^' values for H2O2on [RH]: Ku''^''^ = B =

{hlkx)[BH\{U)

Likewise, and from Eq. (8), the catalytic efficacy, k^JKu, for the utilization of [H2O2] would be given by: 2^5[RH] kcJKu =

=2kx (15) ihlh) [RH]

while the catalytic efficacy for the utilization of the donor RH would be given by:

743

2A:i[H202] = 2h (16)

kcJK] cat/^M

ikJh) [H2O2] 1"""

T

1

0

200

s

\

100

-

0

0

0

50

100

[Hydrogen peroxide] (^iM) Fig. (2). Lineal dependence of i^M values for coniferyl alcohol on H2O2 concentration during the oxidation of coniferyl alcohol catalyzed by the Zinnia elegans basic peroxidase at pH 5.0

Peroxidase-mediated reactions of redox mediation As has been discussed above, one of the particularly important reactions involving peroxidase-derived radicals is redox mediation, or mediation by electron transfer [10]. In this mechanism, radicals (R*), which have been generated by a peroxidase-catalyzed reaction, Eq. (1), may act as diffusible oxidants to oxidize secondary substrates (SH): R* + S H ^ R H + S* (17) This type of reaction has been well documented in the case of the peroxidase-mediated redox reactions of phenols [38,39]. This reaction may have important physiological connotations for lignin biosynthesis since peroxidases unable to oxidize the syringyl moiety present in sinapyl

744

alcohol (I) can oxidize this substrate using the coniferyl alcohol radical (n) as redox mediator (Scheme I) [40]. CH2OH

CH2OH

CH2OH

CH2OH

Likewise, when the product of the peroxidase-mediated oxidation is a cation radical, R*^ [Eqs. (5,6)], Eq. (17) may be written: R*"^ + S -> R + S*^ (18) This reaction has been extensively studied in the case of the chlopromazine radical (R*^)-mediated aminopyrine (S) oxidation [41], a typical reaction for xenobiotics, as well as in the case of the vindoline radical (R*^)-mediated catharanthine (S) oxidation [42], a key reaction in the biosynthesis of the anticancer drugs, vinblastine and vincristine, which are obtained from Catharanthus roseus. Furthermore, these types of reactions, Eqs. (17) and (18), may have several connotations when investigating the capability of peroxidases for synthesizing bioactive plant products. Foremost is the fact that compounds which cannot be regarded as good peroxidase substrates [e.g. SH in Eq. (17) or S in Eq. (18)] may be oxidized through this sequence of reactions in the presence of appropriate substrates [RH in Eq. (17) or R in Eq. (18)]. This implies that the rate of secondary substrate radical generation [S* in Eq. (17) or S*^ in Eq. (18)] may be dramatically stimulated in the presence of the redox mediator [R* in Eq. (17) or R*^ in Eq. (18)], in which RH (or R) acts as primary substrate. Under such circumstances, the presence of the primary substrate, RH (or R), may stimulate the oxidation of SH (or S).

745

Substrate specificity As one may expect from the electron transfer reactions catalyzed by peroxidases, the rates of Col and Coll reduction by a substrate series with different substituents correlated well with the oxidizability (i.e., the E value) of the substrates [33]. However, in the case of structurally unrelated substrates, this is not the only factor to be taken in to account in determining the oxidation rate, since a certain degree of substrate specificity, controlled by the enzyme and the reorganization energies of electron transfer within the enzyme-substrate transition species complex, appears to be the determining factor [33]. Nevertheless, the substrate specificity of peroxidases is so wide that secretory plant peroxidases are capable of accepting a plethora of natural compounds as substrates, such as indoles [43], porphyrins [44,45] including chlorophylls [46,47], terpenoids such as lutein [48], unsaturated lipid acids such as linoleic acid [49], alkaloids [50-52] including betacyanins [53], phenolics such as benzoic acids [54,55], DOPA [56], coumarins [38,57], stylbenes [58], catechins [36,59], chalcones [60], flavonols [61,62], isoflavones [63,64], cinnamyl alcohols and cinnamic acids [65,66], anthocyanins [67,68] and ascorbic acid [69,70]. Undoubtedly, this broad range of substrate specificity is mainly determined by the strong oxidizing potentials of the activated intermediates of peroxidases, Col and Coll, which make them suitable oxidizing agents for all chemicals donating one electron or a hydrogen atom. Since the rate-limiting step in peroxidase-catalyzed oxidations is the reduction step of Coll, Eq. (4), k^ constants provide excellent values for comparing the substrate's preference or enzyme's efficacy, Eq. (16), for a range of natural compounds, which a priori may be regarded as potential in vivo substrates of the enzyme. A comparison of the h values for a given number of potential substrates reveals (Table 1) that phenolics (e.g. coniferyl alcohol) are preferential substrates for the enzyme, compared with other potential substrates such as ascorbic acid (Table 1). At this point, it should noted that, although ascorbic acid does not appear to be a good natural substrate for the enzyme (^3 « 10^-10^ M"^ s"\ Table 1), peroxidase may oxidize it to a great extent when a phenolic is used as redox mediator, according to Eq. (17) [71,72]. This observation lends

746

support to the speculation that ascorbate may be the true and, to date, unidentified (secondary) substrate of secretory plant peroxidases [69]. Despite the above reasoning, the property of coniferyl alcohol (a cinnamyl alcohol) to act as the perfect substrate for secretory plant peroxidases is not surprising since other phenolics, such as transresveratrol (a stilbene), myricetin and quercetin (two flavonols), and catechin (Table 1), are also oxidized by the enzyme with high kinetic constants (^3 « 10^-10^ M"^ s"^ Table 1). Table 1. Values of the k^ kinetic constant (in fjM"' s'^) for the reduction of CoU by several peroxidase substrates Structure

Substrate Coniferyl alcohol

Reference 1.0-16.0

[73,35]

2.9

[34]

rr 2 R* + 2 H2O (+)-Catechin radicals, R*, couple to generate dimers, R2: 2R*-^R2 These dimers can be further oxidized by plant peroxidases: 2 R2H + H2O2 ^ 2 R2* + 2 H2O to yield heterogeneous product mixtures of different degrees of polymerization [145,146]: R2*+R*->R3

775

These (+)-catechin polymers arise from repeated condensation reactions between the A ring of one unit and the B ring of another, through a mechanism which is known as 'head to tail' polymerization (Scheme XXI).

HO,

2

OH

W^o„ OH

POD

H,0 2"2

ft' "

:

^

;

OH

T?a.

+

OH

Scheme XXI

N^^V-^^'

776

HO,

OH

HO.

POD I xi2^2

etc. Scheme XXI (Continuation)

777

Class in plant peroxidases, such as that purified from strawberries, are able to oxidize (+)-catechin with k^ values of 0.57 foM"^ s'^ at pH 5.0 [36]. The peroxidase-mediated oxidation of (+)-catechin leads to the formation of dimers such as dehydrodicatechin A and B4, trimers, tetramers and oligomers of different degrees of polymerization [146,147].

HO.

(XLIV) OH

Scheme XXn

778

Dehydrodicatechin B4 (XLIII), a dimer resulting from (+)-catechin "head to tail' polymerization (Scheme XXI, continuation), is the precursor of dehydrodicatechin A (XLIV), which is derived from the former through a single step involving enzymatic oxidation followed by an internal stabilization via two intramolecular nucleophilic additions (Scheme XXII). (+)-Catechin (XLII) is an antimutagenic compound [148], whose oxidation products, especially the vast array of oligomeric compounds, also show biological activity. These oligomeric compounds are capable of inhibiting plant p-glucosidases [149] and bacterial glucosyltransferases [146]. This is especially important in the latter case, since these glucosyltransferases are involved in the pathogenicity of the pathogenic organisms of dental caries in humans, such as Streptococcus mutants and Streptococcus sobrinus [ 146]. PEROXIDASES AS CATALYSTS IN THE SYNTHESIS OF NATURAL BIOACTIVE ALKALOIDS The participation of peroxidase in the metabolism of alkaloids has frequently been described in the literature [42,50,52,150,151]. However, the participation of peroxidase in alkaloid metabolism is generally associated with alkaloid turnover and catabolism, which generally reduces the biological (pharmacological) activity of such compounds [152-154]. This is the case of the oxidation of vincristine (XLV) by peroxidase to lead to N-formyl-catharinine (XLVI) [154] (Scheme XXIII).

779

(XLV)

I QH

\\^ H3CO

^N" CHO

POD

HiO 2^2

CHO

^

"OCOCH3 COOCH3

780

There are, however, a few examples in which the exclusive participation of peroxidase in the synthesis of natural bioactive alkaloids has been reported. These mainly concern the biosynthesis of some monoterpenoid indole alkaloids, especially the synthesis of a-3',4'anhydrovinblastine (XLVII) [76].

(XL VII) I

0C0CH3 C00CH3

Monoterpenoid indole alkaloids Monoterpenoid indole alkaloids are the largest group, about one quarter of known alkaloids, of which over 4100 different structures have been isolated. These compounds are notable examples of the biological activity that alkaloids can show and, therefore, of their usefulness for humans. Thus, strychnine from Strychnos nux-vomica is used as a homeopathic drug and a rat poison, ajmalicine (XLVIII) from Catharanthus roseus is used in the treatment of hypertension and obstructive circulatory diseases, quinine is still the number one drug against malaria, and the antineoplastic chemotherapeutic agents vinblastine (XLIX; R = CH3) and vincristine (XLIX; R = CHO) from Catharanthus roseus are among the most important natural plant drugs used in medicine [155-157].

781

H3COOC OCOCH,

(XLvin)

(XLIX)

The basic structure of monoterpenoid indole alkaloids includes an indole nucleus derived from tryptophan via tryptamine (L) and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (LI). Strictosidine synthase catalyzes the synthesis of strictosidine (LII) from tryptamine and secologanin (Scheme XXIV) [76]. CHO

^

LX^OGlu

H

(L)

H3COOC^^° (LI)

OGlu

(LII) H3COOC

Scheme XXIV

Strictosidine is the common precursor of ajmalicine (XLVni), on the one hand, and of vindoline (LIII) and catharanthine (LIV), on the other, these last two being the precursors of a-3',4'-anhydrovinblastine (XLVn), vinblastine (XLIX; R = CH3) and vincristine (XLIX; R = CHO), in that order [76].

782

(LIII)

H3C0

^^

^ N ^

N

y OCOCH3 C00CH3

,

(LIV)

ct;9^ H3C00C

The biosynthesis of monoterpenoid indole alkaloids has been intensively investigated since the sixties [76]. The great interest in this group of alkaloids is due mainly to the cytotoxic vinblastine and vincristine, which are very useful anticancer drugs, but which are produced by the C. roseus plant in extremely low quantities. It is therefore not surprising that most of the biosynthetic and enzymology studies of monoterpenoid indole bases have been performed to elucidate the biosynthetic pathway of vinblastine and vincristine and its regulation in C. roseus plants. C roseus is, therefore, an amazing chemical factory, which produces more than 100 different monoterpenoid indole alkaloids, many of them possessing notable pharmacological activity [158-159]. The main alkaloids present in C roseus plants are catharanthine, vindoline and a3',4'-anhydrovinblastine. Ajmalicine and serpentine are also present in significant amounts in the plant [160-165]. Class III plant peroxidases have been involved in the synthesis of many of these monoterpenoid indole alkaloids. Serpentine Class III peroxidases isolated from C. roseus are capable of oxidizing ajmalicine (XLVIII) to serpentine (LV) [150]. The k^ value of C roseus peroxidase for this reaction is about 0.0011 \}M^ s"* at pH 6.0 [74]. This constitutes a unique example in peroxidase-catalyzed metabolic reactions since it involves the aromatization of a N-heterocyclic ring (Scheme XXV).

783

.CH,

^2^2

.CH3

(XL VIII) H3COOC

H3C00C Scheme XXV

a-3%4'-Anhydrovinblastine Catharanthine (LIV) and vindoline (LIII) are regarded as the monomeric precursors of the dimeric alkaloids vinblastine and vincristine, via a3',4'-anhydrovinblastine. C roseus peroxidase catalyzes the coupling reaction of catharanthine and vindoline (Scheme XXVI) to lead to a3',4'-anhydrovinblastine (XLVII) or, more properly, to an iminium intermediate (LVI) from which a-3',4'-anhydrovinblastine is directly derivated [52,74,166]. a-3',4'-Anhydrovinblastine is then converted to vinblastine (XLIX, R = CH3) and vincristine (XLK, R = CHO) in C roseus plants [167-169]. a-3',4'-Anhydrovinblastine (XLVII), or the unstable iminium intermediate (LVI) formed during the coupling reaction, is then assumed to be the precursor of all dimeric alkaloids in C. roseus. The C roseus peroxidase capable of coupling catharanthine and vindoline to yield a-3',4'-anhydrovinblastine shows a Mr of 41,040 45,400, and a pi possibly higher than 10.7 [74]. In its native state, at least three conformers, probably differing in their glycosylation pattern, were noted [74]. The VIS spectrum of the protein showed maxima at 404, 501 and 633 nm, indicating that the enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily [74]. The a-3',4'anhydrovinblastine synthase activity of this protein showed an optimal pH at around 6.5, but it also showed significant activity in the 4-5 pH range, which is the range commonly found in plant vacuoles [74]. C. roseus peroxidase is capable of oxidizing both vindoline and catharanthine, but does not discriminate between both substrates. In fact, the k^ value of peroxidase for vindoline is 0.0012 ^iM"^ s^ at pH 6.0 while for catharanthine is 0.0006 ^M"^ s'^ at the same pH [74]. Experimental evidence [42] suggests that (Scheme XXVII) peroxidase oxidizes

784

vindoline (LIII) to its cation radical (LVH), which in turn oxidizes cathatranthine (LIV) to its cation radical (LVIII), with the subsequent recovery of vindoline. Finally, vindoline (LIII) and the cation radical of catharanthine (LK, the mesomer in Ci6 of LVIII) couple to yield the iminium intermediate (LVI), which is the direct precursor of a-3',4'anhydrovinblastine.

/ T T\7\

H3COOC

I

COOCH3

(XLVII)

N I CH3

y OCOCH3 COOCH3

H ^ C O ^ ^ ^ ^ N I CH3

0COCH3 C00CH3

(XLIX) I

Scheme XXVI

0C0CH3 C00CH3

785

(LVII)

(LIV)

I CH3

OCOCH COOCH3

H3COOC

\

POD H2O2

H3C0

(LHI)

POD H2O2

N' ^ OCOCH3 I COOCH3 CH3

H3COOC

I

(Lvin)

H3C00C

(LK) POD

H,0 2^2

H3COOC I

/-

(LVI) OCOCH3 COCX:H3

Scheme X X V n

This last reaction is the best example to illustrate the versatility and complexity of the class III plant peroxidase-catalyzed reaction.

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ACKNOWLEDGMENTS This work was partially supported by a grant from the MEC (project # PB 97/1042). F. Pomar holds a fellowship from the University of La Coruna. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

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NATURALLY OCCURRING OLIGOPEPTIDES WITH MORE THAN ONE BIOLOGICAL ACTIVITIES M. LIAKOPOULOU-KYRIAKIDES Department of Chemical Engineering, Section of Chemistry, Aristotle University ofThessaloniki, Thessaloniki 54006, Greece Abstract: Naturally occurring oligopeptides, and their synthetic analogs that present more than one biological activities are reviewed. Peptides are found in many tissues and biological fluids (serum, blood, urine e.t.c.) and play important biological roles. Peptidehormones and neuropeptides, plant peptides, antibacterial peptides and in general natural oligopeptides and their synthetic analogs that have been found to show anticancer or other biological effect in vitro or/and in vivo are included.

INTRODUCTION Naturally occurring oligopeptides, and their synthetic analogs that present more than one biological activities are reviewed. Peptides are found in many tissues and biological fluids (serum, blood, urine e.t.c.) and are involved in extracellular and intracellular functions, hi this study, are included peptide-hormones and neuropeptides, plant peptides, antibacterial peptides and in general natural oligopeptides and their synthetic analogs that have been found to show anticancer or other biological effect in vitro or/and in vivo are included. Peptide-hormones and neurotransmitters constitute a large class of compounds, that serve as the chemical messengers of intracellular communication. Neuropeptides have been found to be of therapeutic value for small cell lung, colorectal and pancreatic cancers. Cyclic oligopeptides from higher plants were also found to present antitumor activity. The tetrapeptide acetyl-N-Ser-Asp-LysPro known as a negative regulator of hematopoiesis, has been reported as an inhibitor of heamatopoietic plurypotent stem cell proliferation. The effect of Fibrin(ogen)-like peptides and immunoreactive opioid-peptides in human breast cancer are discussed as well. The antiproliferative activity of a series of tetrapeptides analogs of AS-I toxin isolated from fungus Alternaria alternata pathogenic to sunflower has been reported recently. Antiproliferative effect on human breast cancer has been shown by bioactive peptides secreted by frog skin. Antibacterial peptides Cecropin-B, Magainin and their analogs are known for their anticancer activity in vitro

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and in vivo. Further, the antibacterial activity of synthetic GHK, a tripeptide found in human plasma, which prolongs survival of liver cells, has been recently reported. Peptide-hormones and analogs for the treatment of cancer Peptide-hormones like hypothalamus-pituitary, gastrointestinal, parathyroid, neurohormones, Gfs related peptide-hormones cannot penetrate the plasma membrane and their receptors are located on the cell surface and the signal transport to the nucleus is becoming via a second messenger. The main hormone action seems to be DNA synthesis whereas other including mediation of neurotransmission, enzyme synthesis, regulation and synthesis of structural proteins are responsible for the specific characteristics of differentiated cell. Peptide-hormones are involved in the synthesis of: a) structural proteins, such as tubulin, actin, myosin and troponin, that are under hormonal control and their changes are considered responsible for the existence of neoplastic cells, b) proteins of the extracellular matrix that may influence the induction and maintenance of the tumors. The administration of hormone therapy is based on the knowledge from hormone-dependent carcinogenesis and on the hormone sensitivity and dependency of classic tumors (breast, prostate, endometrium cancer) after in vitro and in vivo studies in experimental models [1]. Significant therapeutic experience has been further gained from certain analogs of peptide-hormones that are also reported below: Luteinizing hormone-releasing hormone (LHRH) and analogs This hypothalamic neuropeptide (Table 1) was discovered in 1971. More than 3000 analogs (agonists and antagonists) have been synthesized since then [2-6] with the purpose of producing potent analogs that can be used as therapeutic agents for the treatment of hormone-sensitive tumors. Among the high potent synthetic analogs of LHRH are metallopeptideanalogs of LHRH [7-9]. Another class of synthetic peptides, analogs of LHRH carrying cytotoxic radicals should be mentioned. These analogs include the known antitumor drugs such as merphalan,doxorubicin [7] and are used for the treatment of cancers that contain LHRH receptors. These

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analogs act directly to LHRH receptors, thus having high biological endocrine activity in vivo and in vitro. Five hexapeptides and heptapeptide analogs of LHRH v^ere synthesized and used as carriers for cytotoxic compounds [10]. These analogs were obtained from LHRH [1-9] and LHRH [4-9] containing D-lysine and Domithine and at position six were amidated with ethylamine and acetylated on the N-terminus. Experiments showed that the above analogs present a wide range of receptor-binding affinities to rat pituitaries and cell membranes of human breast cancer and rat Dunning prostate cancer, hi addition some of these conjugates exerted cytotoxic effects on MCF-7 breast cancer cell lines. Decrease in the AgNOR number in Dunning R3327 prostate cancer after treatment with agonists of LHRH as well as histological changes in Dunning prostate tumors and testes in rats after treatment with LHRH antagonist SB-75 were reported by Szepeshazi et al [11,12]. The same researchers [13] have also studied the effect of combination treatment with analogs of LHRH or somatostatin and 5fluorouracil on pancreatic cancers in hamsters. Pancreatic cancers induced with N-nitroso bis(2-oxopropyl)amine in female Syrian golden hamsters were treated with 5-flourouracil(5-FU) and with sustained delivery systems of the LHRH agonist D-Trp-LHRH antagonist (Ac-D-Nal(2)-DPhe(4CL)2-Pal(3)3-D-Cit6,D-AlalO)LHRH (SB-75) and somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) and some combination there of The results showed that both LHRH antagonist SB75 and somatostatin analog RC-160 caused significant inhibition of these tumors and their combination had the strongest inhibitory effect, with the best survival of animals, the lowest tumorous pancreas weight and the highest apoptosis index among groups. Szende et al [14] have demonstrated that pancreatic tumor cells exhibit high affinity binding sites for LHRH, but only in their nuclei, whereas low affinity sites are associated with cell membranes. These binding sites have been found by microscopic immunohistochemical studies to be the LHRH receptors. The therapeutic and endocrine effect of a synthetic LHRH antagonist in premenopausal women with metastatic breast cancer has also been reported [15]. Gonadotropin-releasing hormone (GnRH) GnRH is a decapeptide (Table I) that stimulates production of gonadotropin hormone (GTH) through interaction with specific receptors.

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It Stimulates adenohypophysis to release LHRH and follicle-stimulating hormone (FSHRH) that are collectively known as gonadotropins [16,17]. Matsuo et al [3] synthesized GnRH (LHRH/FSHRH), in 1971. Studies have shown that about eleven GnRH molecular forms have been found in vertebrates and protochordates and more than one forms produced in the brain and peripheral tissues of species from all vertebrate classes. The ability of GnRH to either stimulate or suppress reproductive processes and cell division has been applied to a number of clinical conditions including precocious and delayed puberty, amenorrhea, endometriosis, and hormonedependent neoplasia. The potential for therapeutic utility of GnRH agonists and antagonists has been studied by Vickery [18] whereas some evolutionary aspects on GnRH and its receptors are presented by King and Millar [19]. The antitumor effect of GnRH has been reported. Various studies have demonstrated that pituitary [20-22], breast [23,24], prostate [25], ovary [26,27] tumors have specific GnRH binding sites and respond to GnRH analogs, in terms of growth suppression in vivo and in vitro. Studies, on gene expression for GnRH have been reported [28,29]. It would be of interest to include also the two articles on hypothalamic regulatory peptides and their receptors by Schally et al [30] and Hall [31]. Somatostatin analogs Natural somatostatin is a cyclic decatetrapeptide (SS-14) originally discovered from rat hypothalamus during the purification of growth hormone-releasing factor and subsequently isolated from sheep and pig hypothalamus [32-34]. Synthetic SS-14 (Table 1) has been found to show antisecretory effects on various body tissues including pituitary, pancreatic a and p cells as well as decrease of serotonin and cholecystokinin secretion. Inhibitory effects of tumors by SS-14 has been reported. Though, SS-14 cannot be used as a therapeutic agent, since the peptide exerts simultaneous multiple and non selective effects. It has been found that various other SS14 analogs present more selective and prolonged antitumor activity [31,32]. A cross-linking assay that allows the detection of receptors for somatostatin analog lanreitide in human breast tumors, has been reported by Prevost [36]. High or low affinity SS-receptors have been found and evidence that the binding affinity of SS-analogs is different for the various tumors is presented by Srkalovic [35]. Alterations in cerebrospinal fluid of

797

concentrations of somatostatin in several diseases including Alzheimer's disease and depression are reported by Bissette and Myers [37]. Bombesin and GRP-analogs Bombesin is a tetradecapeptide (Table 1) isolated from amphibian skin, biologically active in both the central nervous system and gastrointestinal tract. Bombesin-like peptides have been identified in rat brain [38,39]. GRP is a 27 amino acid peptide, that appears to be a mammalian counterpart of bombesin since its carboxyl terminal decapeptide is identical, except that number 20 in GRP is histidine instead of glutamine in number 7 in bombesin. Bombesin and GRP-peptides belong to the family of gastro-intestinal peptide-hormones and play a role as autocrine GFs mainly in human SCLC [38-41]. Bombesin, releases gastrin and exerts effects on GI system and colon carcinoma [42,43]. Bombesin and GRP-like peptides have specific high affinity membrane receptors, characterized on pituitary cells, pancreatic acini of rodents, human pancreatic membranes, colon cancer in mouse, human glioma cells, lung carcinoid cells and human colon cancer membranes. Bombesin/GRP is a potent mitogen for Swiss 3T3 cells, SCLC and pancreatic carcinoma [44,45]. Release of bombesin-like peptides and location of receptors in SCLC is reported by Moody and Korman [46] and Moody et al [47]. GRP has been also detected in some other neuroendocrine tumors such as carcinoids and medullary thyroid carcinomas [48] whereas bombesin has been found to be involved in the development of some carcinogen-induced pancreatic and hepatocellular tumors in rats [49]. Bombesin and the C-terminal tetradecapeptide of GRP were found to be growth factors for normal human bronchial epithelial cells [50]. The effect of gastrin growth on human gastric and colonic tumors as well as the effect of bombesin/gastrin releasing peptide antagonist RC-3095 and of somatostatin analog RC-180 on nitrosamine induced pancreatic cancer in hamsters have been also studied [51,52]. The role of gastrin and cholecystokinin in tumors of gastrointestinal tract is reported by Lamers and Jansen [53]. Elevation of cytosolic calcium in SCLC by cholecystokinin has also been reported [54].

798 Table 1. Peptide-hormones and their amino acid sequence Name LHRH Somatostatin

Sequence pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 H-Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-

Bombesin

Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-MetNH2

Gastrin releasing peptide (GRP) Gonadotropin-releasing hormone (GnRH) 14-26

H-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-MetTyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2 H-Asp-Ala-Glu-Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val-OH

Neuropeptides and their analogues Neuropeptides are small regulatory peptides distributed mainly in the nervous system and cardiovascular system and gut. They can act as neurotransmitters and hormones. Neuropeptides have been recently recognized as important mitogens [55,56]. Vasopressin, the known antidiuretic hormone secreted in hypothalamus, w^as the first neuropeptide found to act as a growth factor [57], Its mitogenic effect was studied in Swiss 3T3 cells. It was found that vassopressin acts synergistically with insulin and binds to specific high affinity V receptors in these cells [58]. In addition the demonstration that bombesin which was known to be secreted by SCLC, was also mitogenic, led the investigation on neuropeptides as possible mediators of cancer growth. Neuropeptides growth factors and their relation to cancer are well presented in the review article by Woll [59]. Neuropeptides including substance P (SP), substance K, neuropeptide Y (NPY), GRP, calcitonin (CT), leucine-enkephalin (L-ENK) have been examined for their anticancer activity against various cancer cell lines in vitro and their inhibition of tumor increase. The wide spectrum of action of neuropeptides has attracted much interest from pharmaceutical industry, with the purpose that by altering or blocking the action of these peptides we will succeed control of certain human diseases. Substance P and Substance K are also known as tachykinins. They are a family of closely related peptides responsible for important biological actions including bronchoconstriction, salivation, vasodilatation.

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cardiovascular regulation and transmission of pain. Mammalian tachykinins include also Neurokinin A, and Neurokinin B, which activate the G-protein coupled receptors Neurokinin-1, Neurokinin-2 and Neurokinin-3, respectively [63]. Tachykinins are peptides of 10-11 amino acid residue (substance P and substance K) and all of them have common the C-terminal sequence Phe-X-Gly-Leu-Met-NH2. They are distributed in the brain, spinal cord and gut neurones [60-62]. Substance P has been shown to have direct mitogenic effect on T-lymphocytes, mediated by specific receptor [63]. [D-Argl, D-Trp5, 7,9, Phe]-substance P has been identified out of a panel of novel and known Substance P analogs as the most potent inhibitor of signal transudation and growth in vitro and in vivo in SCLC cells [64]. Pharmacokinetics as well as tumor distribution of the neuropeptide growth factor antagonist-[Arg6]-SP in nude mice bearing H69 SCLC xenographt and the characterization of the enzyme responsible, for the metabolism of the anticancer peptide Arg-D-Trp-NmetPHe-D-TrpMet-NH2 were also reported [65,66] (Table 2a). Neurotensin a 13 amino acid peptide, was found in the central nervous system, pituitary gland and gut [67]. It has been reported that neurotensin is produced by some SCLC [68] and Ca^"*" mobilizing receptors have been found in SCLC cell lines by Stanley, Woll and Nagakawa [69-71]. Galanin (Table 2a a widely distributed 29/30 amino acid long neuropeptide/hypothalamic hormone does not belong to any known family of peptides [72]. Galanin has multiple effects in both the central and peripheral nervous system including inhibition of glucose-induced insulin release and hippocampal acetylcholine release. It lowers spinal excitability and firing of locus coeruleus neurons [72,73]. Additionally, the peptide stimulates fat intake and growth hormone release upon hypothalamic or i.c.v. injection. The galanin receptor is a Gi-protein coupled, membranebound glycoprotein. Galanin receptor antagonists have therapeutic potential in the treatment of Alzheimer's depression and feeding disorders, A number of synthetic high affinity galanin receptor antagonists of the peptide type have been prepared and helped in the elucidation of functional roles of endogenous galanin in several systems [74]. Cholecystokinin (Table 2a) with main effect to stimulate gall bladder contraction and secretion has been implicated in the growth of gut tumors as already reported [53,54] and pituitary tumors [75]. It has been found that Dynorphin A-(l-13) improves galanin induced impairment of memory process in mice [76].

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Table 2a. Name Substance P (SP) Neurokinin A (NKA) Neurokinin B (NKB) Eledoisin (ELE) Physalaemin (PHY) Phyllomedusin (PHYL) Uperolein (UPE) Kassinin (KAS) Vasopressin Galanin Neuropeptide Y (NPY) Gastrin releasing peptide (GRP) Calcitonin (CT)

1 Cholecystokinin(l-21) Bradykinin 1 Neurotensin 1 Vasoactive intestinal peptide (VIP)

Neuropeptides and their amino acid sequence S e q u e n c e | H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2 pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Asn-Pro-Asn-Asn-Phe-Ile-Gly-Leu-Met-NH2 pGlu-Pro-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2 H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-MetNH2

1

| | | | 1 |

r

H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Gly-NH2 H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-GlyPro-His-Ala-Val-Gly-Asn-His-Arg-Ser-Phe-Ser-AspLys-Asn-Gly-Leu-Thr-Ser-OH, H-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-AlaPro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-LeuArg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2 H-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-ThrLys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-GlyHis-Leu-Met.NH2 1 I H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-ThrThr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-GlnThr.Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 H-Lys-Ala-Pro-Ser-Gly-Arg-Val-Ser-Met-Ile-Lys-AsnLeu-Gln-Ser-Leu-Asp-Pro-Ser-His-Arg-OH H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH. Acetate Pyr-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-LeuOH H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-ArgLeu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-AsnSer-Ile-Leu-Asji-NH2

The neuropeptides vasopresin, bradykinin, cholecystokinin, galanin, neurotensin and GRP (Table 2a) stimulate rapid transient increases in cytosolic Ca2+ in SCLC cell lines. Small cell lung cancer constitutes 25% of the total and follows a rapid and aggressive chnical course, despite

801

initial chemosensitivity [56]. SCLC is characterized by the presence of intracryptographic neurosecretory granules and by its ability to secrete a variety of ectopic hormones and neuropeptides [77]. It has been found that, SCLC is an excellent model system for analyzing the role of growth factors in lung cancers. It should be mentioned here, that tumors synthesize and secrete polypeptide-hormones, which could cause endocrine syndromes. According to Bunn et al [78], the three types of ectopic peptide-hormone production are: a) production, secretion and release of ectopic peptidehormones with a clinical endocrine syndrome, b) production, secretion and release of ectopic peptide-hormones without a clinical syndrome, and c) production without secretion and release of the ectopic peptide-hormones. Vasoactive intestinal peptide (VIP) (Table 2a) with a diverse range of biological activities, is a 28 amino acid peptide found in mammalian brain, in gut mucosa and muscla, in salivary glands, pancreas, respiratory and urogenital tracts. VIP has been identified in pancreatic, cervical and neural tumors [79-81] but is not known to be a growth factor for them. Studies on the generation and recognition of VIP by cells of the immune system and its role as a messenger in the neurons have been done [82-84]. Gkonos et al [85,86] have reported the stimulation of prostate-specific antigen secretion by Lncap prostate-cancer cells by the VIP. Neuropeptides are considered to influence cancer cell proliferation and growth as first reported by Gkonos et al [85] and Noordij et al [87]. It has also been reported that human lung cancer cell lines have VIP receptors [88]. The role of VIP as a modulator of lung inflammation and airway constriction has been studied by Said [81]. In addition, VIP stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes [89]. The biological role of these neuroendocrine peptides in lung carcinogenesis is nicely reviewed by Quinnetal[90]. In closing, the number of neuropeptides, that act as growth factors, is increasing. The discovery that some of them are encoded by oncogenes strengthens the speculation that neuropeptides are important regulators of tumor growth. Opioid peptides Opioid peptides including the enkephalins, endorphins and dynorphins (Table 2b) have a variety of actions on inter alia pituitary hormone secretion and the immune system. Since release of endogenous opioids

802

found to stimulate growth of experimental breast cancers and opiate receptor blockers reduce the growth of chemically induced rat breast tumors, it is beheved that opioid peptides may play a role in human breast cancer [91]. SCLC cell lines contain opioid peptides and receptors. Opioids have been found to inhibit and stimulate SCLC growth [92]. In addition, studies on action of the opioid peptide circadian rhythm in cancer patients, has also been reported [93]. SCLCs secrete a wide range of peptidehormones, including some that stimulate tumor cell growth such as gastrinreleasing peptide and insulin-like growth factor [94]. The expression of Pendorphin, Met-enkephalin and Leu-enkephalin in 63 malignant and benign human breast tumors has been studied by Chatikhine et al [95]. It was found that although the neuropeptide expression is not cancer-specific, it could be cancer-related. Casomorphin (Table 2b) and other opioid peptides were found to decrease the proliferation of a series of prostate cancer cell lines (LNCaP, PC3 and DU145) in a dose dependent manner acting in most cases through opioid binding sites [96,97]. Binding of opioids to human MCF-7 breast cancer cells, their effect on growth, as well as the effect of opioid and nicotine receptors on human SCLC have been studied by Maneckzee [98-99]. Kita et al [100] have studied the effect of opioid peptides on the tumoricidal activity of spleen cells with or without tumors. The antimetastatic effect of various enkephalin-like peptides has been studied by Scholar et al [101]. Peptides from plant and animal tissues The antiproliferative effect of bioactive peptides secreted by frog skin towards human breast cancer has been reported by Boyer et al [102] and Vandenberg et al [103]. CycUc oligopeptides from higher plants were found to present antitumor activity [104]. One of them isolated from Rubia akane, known as Ra-III was selected as a candidate for phase-clinical trials. After various derivatisation to obtain more active and less toxic derivatives from cyclic hexapeptide Ras including esterification, etherification and thionation, it was found that one of them TI-356 has strong growthinhibitory activity against in vitro cell lines including KB oral epidermoid carcinoma, P388 leukemia and Lizio Leukemia. In addition, the same peptide (TI-356) had significant in vivo antitumor activity in P388 leukemia and B16 melanoma [104]. Furthermore, a cychc hexapeptide from Rubiae Radix, known as RA-700, was found to exert antitumor activity in experiments in human tumor clonogenic assay [105]. For this.

803

human tumor cell lines of lung cancer (PC-6) were used in vitro phase n study and the results showed that the peptide possesses time-dependent antitumor activity. Kuttan and Kuttan [106] have reported the isolation of a tumor reducing peptide from mistletoe extract (Iscador) with cytotoxic and tumor-reducing activity. Administration of the peptides was found to produce increased natural killer cell activity (NK-activity) in the normal animals and tumor-bearing animals. Further effects of the peptide include increased antibody dependent cellular cytotoxicity (ADCC) and antiboding forming cells in the spleen. Kuttan and Kuttan [107] and Kuttan et al [108] have also found that spleen cells from animals treated with this peptide presented increased response to phytohaemagglutinin and concanavalin-A, indicating that more mature lymphocytes are produced by the peptide administration. Diazepam binding inhibitor (DBI) is a polypeptide with a molecular weight of 9 KD. It has been isolated from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gammaaminobutyric acid (GABAA receptor) and from mitochondrial BZD receptor located on the outer mitochondrial membrane. This peptide is known for multiple biological effects [109]. The antiproHferative effect of various tetrapeptides, analogs of AS-I phytoxin isolated from sunflower plant has also been reported [110]. Table 2b. Opioid peptides and their amino acid sequence Leu-enkephalin (L-ENK) Met- enkephalin (M-ENL) B-endorhin Casomorphin Dynorphin A (1-13)

H-Tyr-Gly-Gly-Phe-Leu-OH H-Tyr-Gly-Gly-Phe-Met-OH H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-GlnThr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-IleLys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-OH H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-LysLeu-Lys-OH

804

Peptides with various biological activities The naturally occurring polypeptides alpha- and beta- Thymosins (Table 3) have been postulated to be involved in important cellular proliferation mechanisms [111] through which they can possibly influence the potential for malignant transformation of cells and provide indications as to their normal/malignant state. Dihydrodidemnin (DDB) is a depsipeptide that has been isolated from Aplidium albicans (Mediterranean tumicates). DDB was studied on Ehrlich carcinoma growing in vivo and in primary cells [112] and found to behave as a very potent inhibitor of protein synthesis. Interesting enough, the laminin al chain (Table 3) carboxyl-terminal globular domain (G Domain) presents multiple biological activities. Recent results showed that biological active regions in the G domain are conserved in the laminin al and a2 chains and play an important role in cell surface receptor interactions [113]. Similar results were obtained with four Drosophila laminin peptides, according to Nomizu et al [114]. Thymic humoral factor-gamma 2 (THF-gamma 2) is a known immunoregulatory octapeptide. It has been reported that THF-gamma 2 immunotherapy reduces the metastatic load and restores immunocompetence in 3LL tumor-bearing mice receiving anticancer chemotherapy [115]. In addition it has been shown that when administered as an adjunct to chemotherapy, THF-gamma 2 immunotherapy is equally effective against immunogenic and non immunogenic tumors. It is worth to mention here, the nice review by Schulof [116] on thymic peptidehormones and their clinical application in cancer. Marine products dolastatins 10 and 15 were found to show growth inhibition of human lymphoma cell lines [117]. Dolastatin 15 (Table 3), is a known antiproliferative peptide isolated from the moUusk Dolabella auricularia. It has been found that Dolastatin 15 inhibits the growth of p388 lymphocytic Leukemia whereas a synthetic derivative of Dolastatin 15, the peptide LUl03793 (NSC D-669356) interacts with microtubules and inhibits mitosis and possibly exerts its cytotoxic activity primarily through disruption of microtubule organization [118]. Further, the effect of dolastatin 10 and 15 on human lymphoma cell lines including nonHodkin's lymphoma has been studied by Beckwith [117] and Maki [119], Yew species (Taxus spp) throughout the word are hosts to the majority of endophytic organisms. Most commonly, these organisms are fimgi, living in a commensal or in a symbiotic relationship with their host plant.

805

Experimental studies have shown that Leucinostatin A may be one of the several potentially toxic peptides produced by Acremonium sp. that contribute to the defense of the host. The host plant is relatively immune to Leucinostatin A, because it has an enzyme which transfers two glycosyl residues to leucinostatin A, thus reducing the peptide's bioactivity. From these results it can also be concluded that glucosylation reactions may play a general role in plant defenses, especially against toxin-mediated disease development [120]. The effect of various peptides and growth factors in small cell lung cancer cells has been reported by Bepler et al [121]. Four peptides isolated from completely different sources with structural similarities represent members of a new family of growth factors [121]. These peptides include 1) a 60 amino acid residue breast cancer- associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7, 2) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and 3) a 49 and 50 amino acid residue peptide predicted from a cyclic DNA isolated from the skin of frog, Xenopus laevis. Matrix metalloproteinases (MMPs) are a family of enzymes that degrade constituents of the extracellular matrix and the basement membrane. They include collagenases, gelatinases, stromelysins and membrane type MMPs. It has been found that the two MMPs most closely correlated with metastatic potential are the gelatinase A (72 KDa MMP-2) and Gelatinase B (92 KDa MMP2). Both degrade denatured coUagens and type IV collagen present in the basement membrane. Metastatic tumor cell lines express higher levels of gelatinases than non-metastatic counterpart [122], Gelatinases are also produced by non malignant cells in tumors [123]. In animal models generic MMP inhibitors prevent tumor dissemination and formation metastasiss [124]. Since gelatinases and other MMPs are potential targets for therapeutic treatment of cancer, there has been much interest in developing synthetic MMP inhibitors, MMPs are generally inhibited by compounds containing reactive zinc-chelating groups such as thiol or hydroxamate [125]. These researchers have isolated cyclic peptides by screening phage libraries. They selected peptides that would inhibit two matrix metalloproteinases that are linked to cancer metastasis. The cyclic peptides contain a common HWGF amino acid sequence and are specific inhibitors of MMP-2 and MMP-3. Among these the cyclic decapeptide CTTHWGFTLC inhibits the activities

806

of these enzymes, suppress migration of both tumor cells and endothelial cells in vitro. These enzymes are to clear the way for cancer to migrate through the extracellular matrix. It was also found that the decapeptide which inhibits both enzyme potently and prevents migration of human tumor cells and associated blood vessels in vitro. In animals the same peptide improves the survival time of mice bearing cancerous human tumors, stem cancer metastasis and targets blood vessels developing around growing tumors [126]. The antiproliferative effect of Acetyl-N-Ser-Asp-Lys-Pro inhibitor of hematoporetic stem cell proliferation has been also reported [127]. Fibrinogen-like peptides have been reported to show anti cancer activity as well [128]. The identification of growth factors-like peptides with structures with common features of the known anticancer peptide (pS2), the pancreatic spasmolytic polypeptide (PSP) and frog skin peptides (spasmolysins) has been reported by Thim [129].

Table 3. Various other peptides and their sequence

Name

Sequence

Cecropin-B

H-Lys-Trp-Lys-Val-Phe-Lys-Lys-Ile-Glu-Lys-Met-Gly-ArgAsn-Ile-Arg-Ala-Leu-NH2

Dolastatin-15

(5S)-l-[(2S)-0-(N,N-Dimethyl-Val-Val-N-Me-Val-Pro-Pro)2-hydroxyisovaleryl]-2-oxo-4-methoxy-5-benzyl-3-pyrroline

Thymosin ai

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-ThrLys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-AlaGln-Asn-OH

Laminin ai G domain

H-Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg-OH

Antibacterial peptides Gly-His-Lys (GHK) is a tripeptide found in normal human plasma that prolongs survival of normal hver cells [130]. The synthetic one has been found to accelerate growth in cultured cells and tissues including hepatoma cells [130], neuron and ghal cells [131], mast cells [132] and lymphocytes

807

[133]. It has been recently reported that the synthetic peptide GHK presents antibacterial properties as well [134]. The antibacterial peptide Cecropin B (Table 3) and two analogs of it have been synthesized and tested in liposomes and cancer cells. Cecropin B-1 was prepared by replacing the C-terminal segment (residues 26 to 35) with the N-terminal sequence of CB (positions 1 to 10 which include five lysine residues). The analog CB-2 which is identical to CB-1 except for the insertion of Gly-Pro residue pair between Pro 24 and Ala 25. It was found that the two analogs having extra cationic residues, present less antiliposome and antibacterial activity and higher anticancer activity compared to the natural CB [135-136]. Magainins are naturally occurring membrane peptides with antimicrobial activity [137]. They have linear helical channel-forming peptides similar to the Xenopus-derived antibacterial peptide. The last decade a series of analogs based on the sequence of naturally occurring peptide, were prepared and some of them showed increased antimicrobial activity. Because of their rapid and direct mechanism of cell killing magainin analogs have been further used in the treatment of ovarian cancer. The anticancer activity of magainin-2 and two magainin analogs has been reported by Baker et al [137]. Synthetic peptides comprising amino acids 85-99 (domain II) and 148161 (domain III) of Bactericidal/ Permeability-increasing protein (BPI) were found to exert strong cytotoxic activity for mycoplasma and L-forms of gram-positive bacteria [138,139]. In closing this chapter, two other interesting papers should be mentioned. Liau et al [140] have reported that the levels of plasma and urinary peptides can be used as an indicator for the evaluation of cancerpatients undergoing antineoplasmatic therapy and Eisenbach et al [141] focused on the importance of gene and peptide therapy of tumor metastasis. Conclusions The purpose of this article was to review peptides with more than one biological activities and to show that peptide-hormones and neuropeptides are implicated in more than one biological function including that of cancer. In addition, many other naturally occurring peptides isolated from animal / human and plant tissues where found to exert at least a second biological effect.

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It is obvious, that the article can not cover all the literature related to the peptides mentioned. It presents though sufficient data to support the idea that peptides with such a diversity of biological functions, may be used for/or be the answer for the treatment of various diseases. Furthermore, these data will encourage Reader to pursue reseash of that direction and screening of more peptides (natural or synthetic ones) for other biological activities.

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ABBREVIATIONS BZD CP DDB FSHRH GRP GnRH GTH

= benzodiazepine = calcitonin = dihydrodidemnin = follicle-stimulating hormone releasing hormone = gastrin-releasing peptide = gonadotropin-releasing hormone = gonadotropin hormone

LHRH = Luteinizing hormone releasing hormone MCF-7 = human breast cancer cell line MMP = matrix metalloproteinase NPY = neuropeptide Y L-ENK= Leucine-enkephaline PS? = pancreatic spasmolytic polypeptide SCLC = small cell lung cancer SP

ss

THF VIP GFs GABAA

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

= substance P = Somatostatin = thymic humoral factor = vasoactive intestinal peptide = growth factors = gamma aminobutyric acid

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. Allrightsreserved.

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MODULATION OF PROTEIN PHOSPHORYLATION BY NATURAL PRODUCTS SALVADOR MANEZ* and MARIA DEL CARMEN RECIO Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Av, V. A, Estelles s/n, 46100 Burjassot (Spain) ABSTRACT: Studies carried out to determine the influence of phosphorylation and dephosphorylation of proteins in a variety of physiological events are of increasing interest. The activity of kinases and phosphatases and their respective inhibition by endogenous mediators and by pharmacological agents regulates a huge number of biochemical pathways involved in cellular proliferation, apoptosis, inflanunation, hormonal activity, and gene transcription, among other processes. This article focuses on the recently described natural products able to interfere negatively with the activity of serine/threonine and tyrosine kinases. These agents are classified, according to their biosynthetic origin and chemical properties in phenolics, terpenoids, alkaloids and miscellaneous substances. The nucleus of the review is preceded by a general overview on kinase activity, followed by a chapter devoted to naturally occurring kinase activators. Finally, a section concerning the advances in phosphatase inhibition research is included. The main sources of novel phenolic kinase inhibitors are tannins, coumarins, polycyclic isopentenyl isoflavonoids, and phloroglucinols. Other phenolics like flavonols or simple isoflavones are also reported, together with some reputed plant active principles such as curcumin, hypericin or resveratrol. Among the terpenoids, the effects of wortmannin, and those of certain triterpenoids like ginsenoside Rbi or Rhi should be mentioned. The alkaloids comprise two main groups of inhibitors: the indole alkaloids, headed by staurosporine and its derivatives, which are potent, selective inhibitors of protein kinase C, and the isoquinoline alkaloids, subdivided into aporphines, benzophenanthridines and naphtylisoquinolines. The scientific panorama regarding the inhibition of phosphatases is dominated by the polyether and cyclic polypeptide environmental toxins, although some new agents such as indole and isoquinoline alkaloids have been described. PREFACE The introduction or removal of a phosphate group in an organic molecule may be the most decisive reaction that occurs in biochemical processes. Many substances of importance for living cells are transformed into their respective phosphates as a previous step to their metabolism, transport, energy transfer, or incorporation into larger macro-structures. Phosphate performs extremely different dynamic and static functions, such as utilisation and storage of glucose, regulation of cell signalling or linkage of nucleotides within nucleic acid chains. Under the broad subject of

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phosphorylation, the phosphorylation of proteins by specific kinases has obviously been an object of extensive research, because of the muhiplicity of ways in which proteins regulate all life events. Although the inhibition of protein kinases by natural products isolated fi-om either higher plants or microorganisms has been known for years, and some kinase natural inhibitors and activators are widely used as laboratory tools in pharmacology and allied sciences, not many reviews have been published to the present, and certainly none have brought together all the recent advances in both kinase and phosphatase interactions by natural products. Our review covers the major research in these fields produced in the 1993-1999 period, although some particularly interesting papers published before 1993 and others in the year 2000 are included. In order to make it clear that the present work is closely related to those published previously we should mention some reviews that in our opinion are of obligatory reading, such as those of Hu [1] on selective protein kinase C inhibitors, and Chang and Geahlen [2], and Levitzki and Gazit [3], on protein tyrosine kinase inhibitory drugs. The works of Cohen [4], and by Hunter [5] are essential if we want to see the entire panorama of the intervention of kinases and phosphatases in cellular signalling. BIOCHEMICAL BASIS OF PROTEIN PHOSPHORYLATION Some essential concepts The term phosphorylation is applied to a reaction by means of which a phosphate group, any of the anions derived form or/o-phosphoric acid (H3PO4), is covalently integrated into the structure of a given molecule to convert it into a phosphate ester, phosphate-anhydride or phosphateamide. In most cases, the donor of this phosphate group is adenosine 5'triphosphate (ATP), although other nucleotides such as cytidine 5'triphosphate and uridine 5'-triphosphate participate in certain biochemical pathways. In the process of releasing one phosphate groupfi-omthe ATP molecule, the rupture of the terminal phosphate needs 418 kJ/mol, but if this phosphate is transferred to one water molecule, or in other words, if hydrolysis is associated with this release, 31 kJ/mol are generated. (AG° = -31 kJ/mol). Consequently, in this kind of processes phosphorylation becomes thermodynamically possible if the theoretical reaction coupled to the ATP hydrolysis has a AG° not higher than 31 kJ/mol. A long list of substances of biological interest are substrates for the transfer of a

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phosphate from a nucleoside triphosphate, and the individual enzymes regulating this reaction receive the name of kinases, or protein kinases when the target is a protein. One of the best-known roles of protein phosphorylation is the regulation of enzymatic activity, given that a lot of proteins acquire their catalytic properties only after phosphorylation. Such enzymes coexist under two forms, a (active) and b (not active or less active), and the passage from one to another is catalysed by other enzymes that are usually also activated by phosphorylation. The conformational changes induced by this process lead to changes not only in the catalytic activity itself but also in the sensitivity to allosteric effectors. The sort of cascade of reactions in which a phosphorylated protein product is the active enzyme (kinase) necessary for subsequent phosphorylation of another inactive enzyme is frequent in primary metabolism, and is found in the glycogen breakdown, yielding glucose-1-phosphate, and also in the signal transduction systems dependent on the activation of G-protein-coupled receptors and cytokine/growth factor receptors linked to tyrosine kinase activity [6]. Structural and mechanistic features of kinases How does an enzyme become active by phosphorylation? What changes occur to facilitate the catalytic activity?. Questions of this kind began to be answered in detail by crystallographic studies on the glycogen phosphorylase (GP) molecule. This is a dimer that acquires its active form by phosphorylation of a serine (mammalian GP) or a threonine (yeast GP) near the NH2 terminal residue. In mammalian GP the presence of a phosphate causes, by ionic phosphate/arginine interactions, the two subunits of the enzyme to approach the phosphorylating serine, which then moves the subunits away to open the catalytic crevice. In the similar yeast GP an analogous "mechanic" effect occurs, though it does not depend on an ionic binding, but on a disturbance of the lipophilic interactions between non-polar residues in the vicinity of both the phosphate acceptor and catalytic sites [7]. The catalytic cores of protein kinases present in eukaryotic beings share the samefimdamentalstructure, best defined for cAMP-dependent protein kinase (PKA) and characterised by an amino terminal P-sheet-rich nucleotide-binding domain and a larger, helical peptide-binding domain. In the nucleotide-binding domain there is a flexible glycine-rich loop, absent in prokaryote kinases, that wraps the nucleotide and whose phosphate-

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interacting tip is only stable when a peptide is bound with high affinity. Another characteristic of the nucleotide-binding domain is that the majority of the known natural and synthetic kinase inhibitors act by recognising this site [8]. In spite of the similarities reported in recent studies on protein sequence and conformation affecting phosphorylation processes, there is a huge variety of structures for protein kinases, and this implies different ways of regulation [9]. For many years ago, scientists have known that protein kinases only catalyse the phosphorylation of some of the aminoacids among the "free" positions existing in a peptide sequence. As an example, only one (Ser^"^) out of 64 serine or threonine hydroxyl residues of GP is replaced by the action of GP-kinase. In this context, the substrate aminoacid sequence in the neighbourhood of the hydroxyl group is of critical importance because of its tendency to undergo phosphoiylation by PKAs. The major and simplest positive influence was determined by the presence of one, or preferably two, arginine residues two positions before the phosphate acceptor serine (Ser*): Arg-X-Ser* or Arg-Arg-X-Ser*, where X is a variable aminoacid residue. Among some of the other best-known kinases, miosin light-chain kinase (MLCK) from smooth muscle recognises in the susbtrate the closely-related motif Lys-Lys-Arg-X-X-Arg-X-X-Ser*-X and multifunctional calmodulin-dependent kinases (CaMKs) act on the sequence Arg-X-X-Ser*. A slight variation represents the favourite substrate sequence for the classical forms of protein kinase C (PKC) : XArg-X-X-Ser*-X-Arg. This sequence is mimicked in a regulatory part of the enzyme, the "pseudosubstrate site", and repeats the same motif with the exception of alanine instead of serine. Enzyme specificities on the substrate primary structure are based on sequences of native proteins and synthetic peptides and should be considered in non-exclusive terms, because other, often quite different, sites may be recognised by kinases [10]. Basis of phosphorylation regulation The inverse pathway, through which a compound, e.g. a protein, loses a phosphate group, is termed dephosphorylation and is catalised by the phosphatases. Thus, protein kinases and protein phosphatases have general opposite effects, and when operating on the same reaction the preponderant activity of each produces a displacement of the equilibrium between phosphorylated and non-phosphorylated forms that can therefore

823

determine the efficacy of a physiological system. This phenomenon, called reversible phosphorylation, is not the only way to regulate cellular events dependent on phosphate transfer. In fact, both kinds of enzymes possess, in certain cases, targeting subunits responsible for recognising the target locus (a subcellular structure or a substance dissolved in the cytosol) and therefore also responsible for carrying the catalytic subunit into contact with the substrate. In the case of PKA, there are two regulatory subunits that function as inhibitory targeting subunits because they maintain the well-known inactive tetrameric form, but other true (positive) targeting subunits of PKA bound to neuronal organelles have been described. This binding is mediated by the presence of the so-called A-kinase anchor proteins (AKAPs). An ulterior level of complexity in such a per se complex scenario is revealed by the fact that the activity of protein phosphatase 1 is modulated by phosphorylation of a targeting subunit [11]. TYPES OF KINASES Introduction Enviromental changes can be perceived by cells through extracellular signals. In addition, cells can communicate with each other because they also produce signals. The extracellular signals can be either physical (light, temperature, etc.) or chemical (food, hormones and neurotransmitters). In this sense, two groups of chemical signals can be distinguished: a) membrane permeable signals such as steroid hormones (estrogens, progesterone and androgens), which can directly regulate gene expression, b) the membrane impermeable signal molecules which include acetylcholine, growth factors, extracellular matrix components, thrombin, lysophosphatidic acid, etc. They are recognised by receptors which are locaUsed in the plasme membrane of the cell. The receptors are specific for one particular signal molecule or a family of closely related signal molecules. If a ligand binds to an ion channel receptor it can directly lead to its altered opening, which results in a charged membrane potential. Binding of a ligand may stimulate an intrinsic enzymatic activity of its receptor or the modulation of a transducing protein. Activity modulation can be achieved by covalent modification at the molecular level. The most common and important modifications are phosphorylation and dephosphorylation of serine, threonine or tyrosine residues [12].

824

Receptors with enzymatic activity Receptor tyrosine kinases Epidermal growth factor (EGF), platelet derived growth factor, and insulin are extracellular signal molecules that bind to receptor tyrosine kinases (RTKs). Upon ligand binding, RTK auto-phosphorylates to give phosphotyrosine residues (PY). These receptors acts as highly selective binding sites for the so-called Src homology domain 2 (SH2)-containing proteins, which transduce the signal by changing the enzymatic activity of other recruited proteins. Examples of SH2-containing proteins are the RasGTPase activating protein (GAP) that activates phospholipase C-y (PLCy), which in turn hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca^^fromintracellular stores and DAG activates PKC. Receptor serine/threonine kinases These receptors have a cysteine-rich extracellular domain and a cytoplasmic serine/threonine kinase activity. Transforming growth factor-p (TGF-P) superfamily induces growth arrest in epithelial cells. In addition to the three TGF-P isoforms, this family comprises activins, bone morphogenetic proteins and other secreted factors. The two component regulatory system: histidine kinases This system is employed by prokaryotic organisms, and homologous pathways have recently been identified in eukaryotes. The prototypical two-component pathway consists of two proteins: A protein histidine kinase (sensor kinase) and a response regulator. Histidine kinases are very distinct from the superfamily of conventional protein serine/threonine and tyrosine kinases. The histidine kinases auto-phosphorylate on histidine residues and are involved in the phosphorylation of aspartate amino acids and their targets.

825

Receptors without enzymatic activity Cytokine receptors The activation of cytokine receptors after ligands binding, provokes tyrosine phosphorylations by non-covalenty associated protein tyrosine kinases (PTKs): the Janus kinases (JAKs), a family that comprises JAKl, JAK 2, JAK 3 and TYR2. Integrins Integrins are the major type of cell surface receptors and are formed by a heterodimer with a and (3 subunits. Most integrins bind extracellular matrix components like fibronectin, collagen or intronectin. It seems that integrins activate PTKs clustering. G-protein coupled receptors The G-protein coupled receptor group (GPCRs) includes adrenergic, muscarinic, serotonin, dopamine, adenosine, angiotensin II, and thrombin receptors. Upon binding of its ligand, a GPCR interacts with a heterotrimeric guanine-nucleotide binding protein (G-protein). GPCRs, like p2-adrenergic receptors, can be desensitised by decoupling from their G-proteins and intemalisation. Endocytosis of GPCR is necessary for the P2-adrenergic receptor-dependent activation of mitogen activated protein kinases (MAPKs). Nine cloned mammalian adenylylcyclases (ACs) can be activated by stimulatory a subunits (Gas) and several are modulated by inhibitory a subunits (Gai) and/or Gp/y complexes. cAMP can activate the PKA, which in turn phosphorylates a wide range of substrates, such as the cAMP responsive element binding protein (CREB). When PKA translocates to the nucleus and phosphorylates CREB, the latter is stimulated to regulate gene transcription. There are three mammalian phospholipase C (PLC) isoforms families: PLC-p, PLC-y and PLC-5. The first is activated by serpentine receptors, while the second is stimulated by RTKs. CytosoUc Ca^^ can modulate the activity of serine/threonine CaMKs via calmodulin. For instance, neuronal CaMK stimulates phosphorylation and activates tyrosine hydroxylase, the limiting enzyme in the synthesis of catecholamine neurotransmitters. Ca^^ also regulates the activation of the conventional PKC isoforms. Increased Ca^^ promotes binding of Ca^^ to

826

inactive PKC in the cytosol. It leads to membrane association of PKC, which then binds DAG. Classification The superfamily of protein kinases can be classified by the nature of their substrate in two groups: kinases that phosphorylate serine or/and threonine residues, and kinases that phosphorylate tyrosine aminoacids. The serineand threonine-specific protein kinases can be further classified by the nature of their activators. For example, cAMP-dependent, cGMPdependent, Ca^Vcalmodulin-dependent and Ca^V phospholipid-dependent protein kinases have been identified. Many tyrosine protein kinases are intrinsic parts of the cytoplasmic domains of growth factor receptors. The activity of the protein kinases are regulated allosterically. The serine- and threonine-specific protein kinases have a regulatory domain which, in the resting state, keeps the catalytic part of the enzyme inactive. When a second messenger or activator (cAMP, cGMP, Ca^^ or DAG) binds to the regulatory domain, the enzyme is activated. However, it seems that the catalytic domains of these enzymes have many common features and a basically identical mode of action, although serine/threonine and tyrosine kinases differ in the residues to which they transfer phosphate. The eukaryotic protein kinases can be divided into five groups according to Hanks and Hunter [13]. The members of each group show similarities in the catalytic domain and frequently display an analogous mode of regulation and substrate specifities. These groups of protein kinases are: 1) The AGC group. The main characteristics are the following: The AGC group are serine/threonine kinases. These kinases are basic amino aciddirected enzymes, and many of them are activated upon the release of second messengers. The group includes nine subgroups, among which are the cyclic nucleotide regulated protein kinase (PKA, PKG) family, the diacylglycerol-activated/phospholipid-dependent protein kinase (PKC) family, the "RAC" (PKB/AK) family; the CPCR kinase family and the ribosomal SG protein kinase family. 2) The CaMK group: This group is also made up at serine/threonine kinases. These kinases are basic amino-acid directed enzymes. Regulation via second messenger pathways is also common and Ca^^ is the main component responsible for the regulation of CaMKs. It

827

includes the CaMK group comprised by kinases regulated by the Ca^Vcalmodulin and SNFl/AMPK families, MLCKS and the plant Ca^^-dependent PK. An important member of the CaMK group is MAPK activated protein kinase 2 (MAPKAP2). 3) The CMGC group: Another type of serine/threonine kinase, but that is not regulated by second messengers. This group includes cyclindependent kinases (CDKs) and the closely related MAPK/ERK family, the glycogen synthase kinase 3 (GSK3) family and the CIK (Ccd-like kinase) family. The members of the CDK and MAPK/ERK families are proline-directed, while kinases belonging to the GSK-3 group are acidophilic. The GSK-3 family contains the GSK-3 and caseinkinase II isoforms (CK2). CK2 phosphorylates more than 160 substrates among which are several transcriptional factors. 4) The PTK group or "Conventional" protein-tyrosine kinases. This group differs from all the other groups in that the kinases only phosphorylate tyrosine residues. PTKs are often involved in the transduction of growth and differentiation signals in metazoa. 5) Other protein kinases (OPK): These kinases fall outside of the major groups. They include the MEK/Ste7p family, p21-activated kinase (PAKySte 20p family, MEKK/Stellp family, the Raf family, the activin/TGpp receptor family, the flowering plant putative receptor kinase family, the casein kinase I family and the LIM kinases (LIMKs). The members of the first four families function in the MAPK family protein kinase cascades. CKl has been implicated in the pathogenesis of Alhzeimer's disease through hyperphosphorylation of the x protein. In lower eukaryotes, individual CKl isoforms are involved in the regulation of repair pathway cell proliferation and morphogenesis. Protein Kinase C PKC has diverse functions in the control of membrane processes, growth, and differentiation. The PKC family includes 11 isoforms. All PKC isoforms contain four conserved (C1-C4) and five variable (V1-V5) regions. CI and C2 are localised in the regulatory domain, while the C3 and C4 regions are situated in the catalytic domain. The regulatory domain contains a pseudosubstrate site, which is involved in blocking the kinase. The association between the pseudo-substrate site and the C4 region resuks in an inactive PKC conformation. The CI region binds compounds such as DAG or 12-tetradecanoylphorbol-13-0-acetate (TPA) which

828

activate the kinase. The C2 region is the site for binding Ca^^, and it is absent in Ca^^-independent PKC isoforms. In this region it is found the socalled "pseudo-anchoring site" which is involved in the regulation of PKC binding to receptors for activated C-kinase (RACKs). The C3 region contains the ATP binding site, while the C4 region is responsible for substrate binding. PKC is rapidly activated by a a transient rise in DAG levels resulting from PLC stimulation. Phospholipase D (PLD) causes phosphatidylcholine hydrolysis, which results in an increase in DAG and PKC activity. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), an other lipid metabolite, can activate PKC. There is a functional difference between PKC activated through different pathways. The different PKC isoenzymes are also believed to have distinct biological functions. On the basis of structural elements and activational characteristics, the PKC family has been divided into three subfamilies: a) Classical or conventional PKCs (cPKCs): Phospholipid-acid Ca^^-dependent protein kinase: PKC-a, -pi, -pll and -y. b) Non-classical or novel PKCs (nPKCs): PKC5, 8, r| and 9 isoforms. c) PKC-|Li: It is an isoform between novel PKCs and atypical PKCs. d) Atypical PKCs (aPKCs): They are independent of DAG and Ca^^ PKC-C and -xlX can be activated by PIP3. Mitogen Activated Protein Kinases MAPKs are a family of well-conserved serine/threonine kinases that have a central role in a wide variety of protein kinase cascades. These cascades consist of three kinases, a mitogen activated protein kinase (MAPK), a MAPK kinase (MAPKK) and a MAPK kinase kinase (MAPKKK), which modulate each other in a chain reaction. In other words, MAPKKK activates MAPKK which in turn activates MAPK. The MAPK family comprises three subfamilies: 1. Extracellular signal regulated kinase (ERK) 2. Stress-activated PK/cJun N-terminal kinase (SAPK/JNK) 3. The p3 8 subfamily.

829

The best studied subfamily is the one that includes ERKl and ERK2, which are involved in cascades consisting of Raf, MEKla/MEK2 and ERK1/ERK2 isoforais. Raf is in turn activated by a dynamic combination of phosphorylation (by PKC and/or other protein kinases) and interactions with Ras-GTP and 14-3-3 proteins. The Raf-MEK-MAPK/ERK pathway has effects on non-proliferating cells, but mitogenic signals especially stimulate the pathway. Proliferation can be blocked by inhibiting it. Activated ERKl and ERK2 can phosphorylate a large number of proteins, among which we find transcription factors and other nuclear proteins. Infibroblasts,ERKl and ERK2 promote entry into the cell cycle. Other substrates for these ERKs are upstream proteins of the MAPK cascade (such as the EGF receptor and Raf-1). Finally, an important group of substrates for this kind of kinase are cytoskeletal elements (such as MAP-1 and MAP-2). Usually, ERKs in activated cells are bound to the cytoskeleton and a basal ERK activity is required for the maintenance of cell-matrix interaction in preference to cell-cell contacts. These findings suggest that ERKs are involved in cytoskeletal reorganisation. Mitogens and many types of stress (cycloheximide, UV radiation, and heat shock can stimulate SAPK/JNK subfamily kinases. This subfamily is activate by SEK-1 (also named MKK4 for MAPK kinase 4) and MAPK kinase 7 (MKK7). SEKl can in turn be phosphorylated and activated by MEKKs and other kinases. The transcription factors cJun, EDC-l and ATF-2 serve as physiological substrates for the SAPKs. ERK and SAPK/JNK ofl:en have opposite roles in the regulation of apoptosis. In some cells, such as PC 12 cells, ERK is protective while SAPK/JNK is facilitative. The effects of ERK, SAPK/JNK on p38 modulation appear to be largely dependent on the context in which these components act. Apoptosis is the resuh of a critical balance between several MAPK pathways. NATURALLY OCCURRING PROTEIN KINASE ACTIVATORS The human genome encodes over 2000 protein kinases that are finely turned off and on by different signals. For instance, the MAPK are activated through complex but controlled kinase cascades in response to a number of factors, including stress and cytokines. Experimental protein kinase activation can be performed by exogenous agents of plant or marine origin such as phorbols or bryostatins, respectively.

830

Despite the large number of protein kinases types, studies on natural activators focus mainly on PKC activation. This kinase, which plays a decisive role in many cellular responses, is generally activated in the cell by lipid second messengers, predominantly DAG, in response to various extracellular agonists (hormones, neurotransmitters, growth factors and cytokines). We now turn our attention to some of the best known PKC activators together with other novel compounds. Phorbol esters and related diterpenes The Euphorbiaceae produce a range of toxic diterpenes belonging to a number of structural types. Esters of tigliane, daphnane and ingenane alcohols, possess diflferent biological effects including tumour promotion and cell proliferation, inflammation, degranulation of neutrophils, etc. Phorbol belongs to the tigliane group, and its esters have been found in the genera Croton, Sapium and Euphorbia (Euphorbiaceae) [14]. One of these, TPA (1), is very potent and is used in many studies as a pharmacological tool [15,16]. Diterpene esters are potent activators of PKC [17]. For instance, TPA is able to activate all PKC isoenzymes except the -C, and -X forms [18,19]. 12-Deoxyphorbol-13-0-phenylacetate -20-acetate (dPPA) and thymeleatoxin are selective activators for PKC-pI (dPPA) and PKC-a, -P and -y but not PKC-5 or -s (thymeleatoxin) in vitro. In intact cells it was observed that these phorbol derivatives translocate and down-regulate PKC isoenzymes, including -5 and -s [18].

H3C(CH2),200

CH2OH

ITPA

831

OCOCgHsj HO

CH2OH

2 gnidimacrin The daphnane-type diterpene gnidimacrin (2), isolated from the root of Stellera chamaejasme (Thymelaeaceae), inhibited the cell growth of human leukaemias, stomach cancers and non-small cell lung cancers in vitro and showed significant antitumoral activity against murine leukaemias and soUd tumours in vivo. It inhibited phorbol-12,13-dibutyrate (PDBu) binding to K562 cells and directly stimulated PKC activity in the cells in a dose-dependent manner (3-100 nM) [20]. It has recently been demonstrated that the antitumoral mechanism of gnidimacrin is related to PKC activation: gnidimacrin binds to K562 cells three times more than to HLE cells. Immunoblot analyses revealed pronounced PKC-PII expression in gnidimacrin-sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKC-a, but not PKC-pII. At the growthinhibitory concentration of 0.0005 (ig/ml, the Gl phase was arrested and inhibition of cdk2 kinase activity was found. Therefore, one of the main determinants of the ability of cells to respond to gnidimacrin is PKC-PII and the antitumoral action might be associated with cell-cycle regulation through suppression of cdk2 activity [21]. The bryostatin family The bryostatins are naturally occurring macrocyclic lactones isolated from marine bryozoa that have antineoplastic activity. These macrolactones exhibit high affinities for PKC isoenzymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. They do not act as tumour promoters. The bryostatins are a class of PKC activators that induce only a subset of the typical phorbol ester responses

832

and antagonise those phorbol ester-mediated responses that they themselves fail to induce. These compounds are isolated from natural sources in a limited manner, and their synthesis is complex. Several synthetic analogues have been determined that bound strongly to a mixture of PKC isoenzymes and exhibited significant levels of in vitro growth inhibitory activity against human cancer cell lines [22].

3 Bryostatin 1 Bryostatin 1, Fig. (3), showed lower affinity for PKC-pI and -y than for PKC-a, -5, -s and -^ [23]. Similar results have been found by Keenan et al in a novel in vivo assay valid for identifying PKC isoform-specific activators and inhibitors: The fission yeast Schizosaccharomyces pombe under a thiamine-repressible promoter expresses either a conventional isoform of PKC (PKC-y) or a novel isoform PKC-5. When the authors compared the effects of bryostatin 1 on the growth of the PKC-y and PKC-5 transformants with those of TPA, they observed that bryostatin 1

833

activated PKC-5 but inhibited PKC-y activity [24]. In contrast with longterm TPA treatment, which induces neuronal differentiation through downregulation of PKC activity, it has been demonstrated that bryostatin 1, under similar conditions, increased the levels of PKC-s. This isoform is implicated in phosphorylation of the microtubule-associated protein tau and in neuritogenesis. Treatment with TPA induces neuritogenesis, while treatment with bryostatin 1 for 72 h increases tau phosphorylation and inhibits neuritogenesis [25]. Other differences between phorbol esters and bryostatin 1 have been observed in intestinal transport and barrier function. Bryostatin 1 reduced CI" secretion, Na^-K^-Cl" cotransporter, and cotransport mRNA expression. Unlike phorbol esters, these effects were largely transient. The barrier function was not affected by bryostatin 1 in contrast with what happened with phorbol esters. These differences imply that bryostatin 1 and phorbol esters affect PKC isoforms involved in junctional regulation and that epithelial transport and barrier function may be regulated by different PKC isoforms [26]. Other activators Daphnoretin (4) is a dicoumarin isolated from Thymelaceous plants, {Wikstroemia indica. Daphne mezereum and D, cannabind) that directly activates PKC, which in turn elicits the respiratory burst in rat neutrophils.

MeO

4 Daphnoretin This effect was greatly reduced by the PKC inhibitor staurosporine, which means that PKC plays a major role in daphnoretin-induced respiratory burst. Daphnoretin, like TPA, increased the membrane associated PKC activity in neutrophil suspension. It is possible that daphnoretin, like TPA, may bypass the membrane receptor and act as a PKC activator. Daphnoretin reduced the [^H]PDBu binding to PKC in a

834

concentration-dependent manner, which implies that daphnoretin may bind to the phorbol ester binding site in the regulatory domain of PKC and lead to its direct activation. The specificity of the dicoumarin for certain PKC isoforms is not yet known [27]. Kazinol B (5), a natural isoprenylatedflavanisolated from Broussonetia papyrifera (Moraceae), induces the stimulation of respiratory burst in rat neutrophils. This effect is probably mediated by the synergism of PKC activation and [Ca^^]i elevation in rat neutrophils. The membraneassociated PKC-a and PKC-0 were increased following the stimulation of neutrophils with kazinol B. The conventional and novel PKC isoforms might contribute to the PKC activation upon exposure of cells to kazinol B. In addition, the novel PKC isoforms may play the major role since these are more sensitive and more rapidly activated by kazinol B than the conventional isoforms [28].

5 Kazinol B

6 Decursin

835

From the roots of Angelica gigas and A. decursiva (Umbelliferae) the pyranocoumarin decursin (6) was isolated. This compound showed cytotoxic activity against several human cancer cell lines and in vitro PKC activation. The PKC from rat brain used in this assay was mainly composed of conventional isoenzymes (PKC-a, -p and -y). 10 |ag/ml of decursin activated in vitro PKC activity in presence of intrinsic activators such as 10 mM Ca^^ and 10 |ig/ml phosphatidylserine [29]. The MeOH extract from the rhizomes of Iris tectorum (Iridaceae) gave a spiroiridal triterpenoid, 28-deacetylbelamcandal (7). This substance stimulated differentiation of human promyelocytic leukaemia (HL-60) cells. It inhibited the specific [^H]PDBu binding to PKC, similarly to TPA in a dose-dependent fashion. In addition, 28-deacetylbelamcandal directly enhanced PKC activity [30].

7

28-Deacetylbelamcandal

The effect of nicotine, the main principle of tobacco, on PKC activity was measured as a function of time. At a concentration of 100 nmol/1, nicotine caused an increase in PKC activity in endothelial cells from human adult CNS. The increase in PKC activity was significant in 30 s and attained maximum levels at 2 min. In order to assess the significance of the nicotine-induced PKC activation in the observed increase in plasminogen activator inhibitor-1 (PAI-1) production, the effect of nicotine was measured in the presence of the PKC inhibitor GF-109203-X. In these conditions, nicotine had no effect on PAI-1 mRNA levels. Similar results were obtained with another PKC inhibitor (calphostin C), demonstrating that in CNS-endothelial cells PAI-1 mRNA expression and protein production are dependent on the activation of PKC [31].

836

NATURAL INHIBITORS OF PROTEIN KINASES Introduction There are different sites in protein kinases that may be considered pharmacological targets for selective inhibitors. a) The binding sites for the activator molecules of the serine and threonine protein kinases. b) The region involved in the inactivation of enzyme in absence of activator molecules. c) The ATP site on the catalytic domain of the protein kinases. This ATP binding site and much of the rest of the catalytic domain of protein kinase C shows striking homology with ATP binding sites of the other serine and threonine-specific kinases and even of the tyrosine-specific kinases. This means that selective inhibitors at this level are unlikely to be found. Although the biochemical functions and the stereochemistry of the enzyme active sites are completely established, and many natural and synthetic compounds have been assayed for their inhibitory activity, there is no an unique or preferred structural type for the highest effectiveness. In this review we have classified the active compounds in phenolics, terpenoids, alkaloids, and other principles. Phenolic compounds Phenylpropanoid and phenylethanoid glycosides One of the most widespread examples of phenylethanoid glycosides is verbascoside, also called acteoside, which has been obtained form sources belonging to different plant families. This compound, isolated from Lantana camara (Verbenaceae), was characterised as an inhibitor of rat brain PKC, with an IC50 value of 25 |LIM. This effect was abolished by adding ATP, which indicated a competitive interaction with the nucleotide. The inhibition was non-competitive with respect to the phosphate acceptor, histones type HIS in this case, but other kinases, such as PTK from a lymphoma cell line or PKA, were not inhibited. In order to translate the biochemical effect to a related cellular event, the ability of verbascoside to reduce the proliferation of the lymphocytic mouse leukaemia L-1210 cells was examined. This compound showed an IC50 value of 13 |LIM [32].

837

A number of closely-related phenylethanoid glycosides isolated from Digitalis purpurea and Penstemon linarioi (Scrophulariaceae) were studied for their inhibitory activity against recombinant human PKCa, using glycogen synthase peptide as a substrate. An IC50 of 9.3 |iM was reported for verbascoside itself, whereas calceolariosides A (8) and B, and forsythiaside, from D. purpurea, were more potent, with IC50 ranging from 0.6 to 4.6 |LiM. Other phenylethanoids oiPenstemon, like leucosceptoside and poliumoside, showed lower potency [33].

8 Calceolarioside A Other PKC-inhibitory phenolic saccharide conjugates, which are not true glycosides and contain cinnamic moieties, are vanicosides A (IC50 = 44 |LiM) and B (IC50 = 32 |LIM), from Polygonum pensylvanicum (Polygonaeceae). These are mono-feruloyl-tri-/7-coumaroyl-esters of sucrose, differing only in that vanicoside B presents an acetyl group on glucose C-2 [34]. Oligomeric catechins and tannins In a study by Polya and Foo, twelve polyphenols of increasing complexity, sharing the catechin or epicatechin structure, were evaluated for their effect on the activity of PKC, PKA-catalytic subunit and MLCK. In general, rat brain PKC was the most sensitive to polyphenol interaction, given that eight of the inhibitors had IC50 values below 10 |jM. The compounds that showed the highest potency were the procyanidins obtained from the bark of Pseudotsuga menziesii (Pinaceae), and among these, the one possessing the highest degree of polymerisation was a tetramer of epicatechin with repeated 4-8 linkages (9) (IC50 = 0.6 |LIM).

838 OH

OH

9 An epicatechin tetramer from Pseudotsuga meinziesii This compound behaved as a non-competitive inhibitor with respect to ATP and to the substrate, EGF-derived synthetic peptide. Other quite potent agents were two single 5-deoxy-6,8-dihydroxyepicatechins differing in the orientation of the 4-hydroxyl group, which were isolated from Acacia melanoxylon (Leguminosae) heartwood, and two catechin esters of 3-hydroxy-5-(3,4-dihydroxyphenyl) pentanoic acid from the cladodes of Phyllocladus trichomanoides (Podocarpaceae). Most of the assayed substances were effective inhibitors of bovine heart PKA-catalytic subunit, and again the highest potency corresponded to compound 9. According to the authors, it seems that the presence of a voluminous substitution embracing C-7 and C-8, or at C-3 or C-4 is detrimental to the inhibitory activity of monomers [35]. In order to identify the mechanism underlying the antiproliferative effect of some naturally occurring polyphenols on

839

vascular smooth muscle cells, epigallocatechin (10) was evaluated for its PKC and PTK inhibitory activities by using cultured rat aorta A7r5 cells. This substance, one of the major constituents of green tea, exerted at 10"^ M a faint inhibition (19.3 %) of membrane-associated PTK activity, but this effect disappeared at 10"^ M. Epigallocatechin was found to be inactive against cytosolic PKC activity both in presence and absence of TPA. However, it managed to reduce substantially the serum-stimulated expression of the protooncogene c-jun, measured by the corresponding mRNA level, which is an important indicator of growth factor stimulation. Furthermore, epigallocatechin acts as an inhibitor of JNK. For that reason, activity of the c-Jun protein itself is limited, and the antiproliferative effect is partially explained [36]. OH OH

HO^ , ^

^^ , ^^ ,

^ ^

. "

^^ , ^" ^

^ ^*OH

10 Epigallocatechin The effect produced by catechin structurally-related tannins (condensed tannins) was determined for eighteen plant tannin preparations. When the results on PKC, PKA and MLCK were considered globally, the most active among the screened extracts were those obtained from the leaves of Grevillea robusta (Proteaceae), Ribes sanguineum and K rubrum (Grossulariaceae), the fruits of Vaccinium corymbosum (Ericaceae) and from Lotus corniculatus (Leguminosae). For each of the tannins studied the potency diminished according to the sequence PKA > PKC > MLCK. It was further observed, with few exceptions, that the potency as PKA inhibitors (IC50 between 9 and 70 nM) correlates positively with the proportion of trans forms in C-2/C-3 and also with the proportion of prodelphinidin units. The most potent product, Ribes rubrum tannin, acts as a competitive inhibitor of the rat liver PKA-catalytic subunit with respect to ATP and non-competitive with respect to the substrate peptide

840

(kemptide). The inhibitory effect on rat brain PKC was expressed by IC50 values below 1 |LIM, with a maximum potency attributed to Phoenix canariensis (Palmae) frond and Ribes nigrum leaf tannins, IC50 = 0.3 |LIM [37]. On the basis of the studies concerning the activity of the hydrolysable tannins from Phyllanthus amarus (Euphorbiaceae), it seems that their effectiveness against common protein kinases is lower than that of condensed tannins. In fact, IC50 values ranged from 0.2 to 1.7 |LIM for the rat liver PKA-catalytic subunit, and was 26 |iM for geraniin (11) and other closely-related compounds against rat brain PKC. Other tannins of the same plant inhibited PKC at the high fixed dose of 167 |LIM [38].

HO

OH

HO

OH

OH

HO

11 Geraniin Some experiments have demonstrated that tannic acid, also called simply "tannin", a natural mixture of gallic esters of glucose classically obtained from Rhus species (Anacardiaceae), interferes with cellular activation of PKC. It inhibited, for example, the phosphorylation catalysed

841

by membrane-bound PKC and DNA synthesis, both of which events are induced by TPA in NIH 3T3 cells. It did not compete with PDBu for binding with PKC and did not affect membrane PKC translocation [39]. Tannin obtained from cotton bracts was able to inhibit histone III phosphorylation in membranes of canine airway cells and to reduce CI" secretion induced by TPA in a tracheal epithelium mucosal preparation, which suggested that inhalation of tannin by cotton manipulators may produce its deleterious effect (byssinosis) trhough PKC inhibition [40]. Flavonoids In this category we include phenolic compounds based on the structure of 2-phenyl-4H-l-benzopyran-4-one (ftavones, flavonols and related dihydro analogues), the isomeric 3-phenyl derivatives (isoflavonoids, including isoflavans) and other non-pyronic C6-C3-C6 plant pigments such as chalcones, aurones and anthocyanidins. One of the most frequent flavonols found in plants is quercetin (3,5,7,3',4'-pentahydroxyflavone), for which interesting inhibitory properties affecting serine/threonine and tyrosine protein kinases were reported in several studies. In addition to the effects on many other kinases, quercetin was characterised as an inhibitor of the oncogene product pp60^'^^, in an ATP-competitive fashion [41]. In another study done prior to the time period covered by the present review, it was found that myricetin (3,5,7,3',4',5'-hexahydroxyflavone) exerted similar effects with even higher potency. Some of the tyrosine protein kinases tested were not affected by the presence of myricetin, namely PTK from platelet particulate fraction. Other ones, such as the oncogene product ppno^"", were competitively inhibited by myricetin with respect to ATP {Ki =1.8 |LiM), and in other cases myricetin inhibited without competing with ATP (rat liver insulin receptor, Ki = 2.6 |LIM). The inhibition of serine/threonine protein kinases was competitive for MLCK and CKI and CKII {Ki =1.7, 9.0 and 0.6 |LIM, respectively). Myricetin also inhibited PKC and PKA, though at Ki values over 10 |iM. Comparison of the potency of seven flavones and flavonols with increasing degrees of hydroxylation, from flavone itself to myricetin, revealed that the number of free hydroxyl groups correlated closely with the potency against the susceptible tyrosine protein kinases, whereas this correlation was not entirely applicable to the inhibition of serine/threonine protein kinases [42]. When the inhibition of

842

pSe^^'^-PTK activity was studied for theflavonoidaglycones and glycosides isolated from Koelreuteria henryi (Sapindaceae), no clear correspondence between hydroxylation and potency was found, although glycosidic forms were the least potent ones [43]. In a study on the effects on rat brain PKC, fisetin, the 5-deoxy analogue of quercetin, was found to be the most active among 15 flavonoids tested at 50 |LiM. Some chemical features such as the presence of orthodihydroxyl groups and a 2-3 double bond, and the absence of methoxyl groups and sugars were recognised to be essential for the activity. The inhibitory effect of fisetin was dose-dependent (IC50 = 10 |j,M) and competitive with respect to ATP {Ki ^4.6 |iM) [44]. The flavone apigenin (5,7,4'-trihydroxyflavone) was studied for its effect against several kinases in mouse NIH 3T3 cells. It competed with ATP to inhibit PKC (IC50 = 10 |LIM). In addition, it inhibited the PTK activity of the fibroblast growth factor with half the potency (IC50 = 20 |LiM), but was not active on the phosphorylation mediated by pp60''"^'^' (IC50 > 200 |LiM). Apigenin also inhibited the expression of c-fos and c-jun induced by TPA in the same cellular system [45]. In a study on human promyelocytic leukaemia HL-60 cells, quercetin reduced cellular growth dose-dependently, and abolished it at a concentration of 80 |LIM. This effect was attributed to the concomitant inhibition of membrane, but not cytosolic, PTK activity, determined on poly Glu-Tyr (4 :1) as a substrate (IC50 = 20.1 fxM). Inversely, quercetin inhibited cytosolic, but not membrane, PKC activity. As a possible result of the inhibition of phosphatidyl-inositol kinases, the production of phosphoinositides in intact HL-60 cells was greatly diminished by quercetin [46]. Activation of PKC is supposed to be a necessary prerequisite for neutrophil respiratory burst, because it mediates the phosphorylation of the proteins involved in the assembly of NADPH oxidase, which at last produces superoxide anion (O2") from external oxygen. The isoprenylflavone cycloheterophyllin (12) isolated from Artocarpus heterophyllus (Moraceae) was known to inhibit that respiratory burst, and its interaction with PKC was therefore studied. Enzyme preparations obtained from rat neutrophil cytosol were inhibited very slightly by cycloheterophyllin, whereas those obtained from rat brain were moderately dose-dependently inhibited by the same compound at doses between 6 and 60 |LIM. Some degree of competition

843

with PDBu for PKC binding (30 % inhibition) was observed with cycloheterophyllin at 60 |LIM, but this did not affect translocation to the membrane [47]. Me>^

^Me OH

12 Cycloheterophyllin Although strictly speaking phosphatidylinositol 3-kinase (PI3K) is not covered by the present review, it deserves particular attention because after stimulation by growth factors, it contributes to the generation of PIP2 and PIP3, which are resistant to the hydrolysis by PLC, and its increased levels are associated with cellular transformation induced by diverse oncogenes. Given that the modulation of other kinases by flavonoids was already known and also presumably involved in their antitumoral properties, the interactions with PI3K were investigated. Quercetin was reported to be a potent inhibitor of bovine brain PI3K (IC50 =1.3 |jM), whereas different chemical modifications on the flavonoid structure led to a decrease in activity [48]. At a fixed concentration of 60 |iM, a short series of structurally dissimilarflavonoidswere tested for their inhibition of PI3K fi-om human platelets, and other kinases. As quercetin and luteolin (5,7,3',4'-tetrahydroxyflavone) produced the highest inhibition (near 90 %) of PI3K, whereas catechin, genistein (13) (an isoflavone) and hesperetin (aflavanone)were inactive, the study was broadened to include other flavones andflavonols.Myricetin practically abolished PI3K activity, while galangin and chrysin, both lacking hydroxyl groups in the phenyl radical, had no effect, and apigenin and diosmetin (4'-methylluteolin) showed significant inhibition of the enzyme activity. On a dose-response

844

analysis, myricetin showed an IC50 value of 1.8 |iM and luteolin of 8 |LIM. An analogous profile was observed for the inhibition of bovine brain PKC, with an inversion in the magnitude of the effects of apigenin and kaempferol (3,5,7,4'-flavone).

However, when the flavonoids were assayed on A431 carcinoma cells for PTK activity associated with EGF overexpression, genistein (13) (as expected) and kaempferol were the most active compounds, ahead of myricetin and diosmetin [49, 50]. One of the most typical chemical characteristics in the Leguminosae is the production of isoflavones and related compounds, which often confer pharmacological or toxicological properties on plants of this family. Species of the genus Derris, for example, have insecticidal activity because of their rotenone content. One of them, D. scandens, is a good source of several prenylisoflavones, which were studied together with other coumarins and simple isoflavones as inhibitors of certain kinases. The most sensitive of the enzymes tested was PKA (catalytic subunit), as it was potently inhibited by warangalone (14) (IC50 = 3.5 |LIM). The isomers 8and 3-dimethylallylwighteone showed IC50 values below 30 JLIM. Nallanin (15) was the most potent of the isoflavones against PKC (IC50 =120 |LIM). Robustic acid (16) was the only coumarin that potently inhibited a kinase activity. Its IC50 for PKA inhibition was 10 |j.M, whereas other coumarins showed IC50 values higher than 100 |LIM for inhibition of any of the kinases tested [51]. Genistein (13) is a compound massively present in some foods, like soybeans, and well known for its estrogenic and PTK inhibitory properties. For these two reasons, genistein is considered to prevent hormonedependent breast cancer and strongly inhibit the mitogenesis induced by growth factors like EGF and TGF-P, with positive implications in the amelioration of osteoporosis and haemorrhagic telangiectasia. However,

845

the range of concentrations at which this isoflavone impairs the growth of tumoral cells is substantially lower than the concentration at which it inhibits PTK activity. This suggests that although the activity of tyrosine protein kinases is involved in the cellular effects of genistein, it does not occur by, or at least not only by, a direct inhibitory mechanism [52]. In a study on the proliferative effects of angiotensin II on rat aorta smooth muscle cells, genistein inhibited the incorporation of [^HJthymidine to cells, which is an indicator of DNA synthesis inhibition. Moreover, genistein was a weak inhibitor of MAPK [53].

Mes

,Me

y

>r°v^k^°\ kAAyA o

®v»

e

14 W ara ngalone

15 Nallanin

846

OMe

OH

OMe

16 Robustic acid Glabridin (17) is an isoflavane derivative present in liquorice root that has been investigated for its ability to modify the chemical degradation of low density lipoprotein (LDL) by macrophages, an event strongly linked to the genesis and progression of atherosclerosis. After incubation with 20 laM glabridin, it is incorporated into the murine peritoneal macrophages and there produces a pronounced inhibition of both cytosoHc and, especially, membrane PKC. This PKC inhibition is thought to be a crucial step in the inhibition of the assembly of NADPH oxidase, an enzyme responsible for the oxidation of LDL by macrophages. Methylation of the two free hydroxyl groups of glabridin leads to a loss of the effect [54].

OH

17 Glabridin Butein is a simple chalcone formed by the union of a phloroglucinol ring with a catechol one through a propenone chain. It has been characterised as a PTK specific inhibitor, because it reduced the extent of the EGF-induced phosphorylation of the EGF receptor localised in human hepatocellular carcinoma cells, and the activity of the soluble EGF receptor and of p60''"'"^^. In contrast v^th other flavonoids, butein was ineffective against serine/threonine protein kinases [55].

847

Coumarins Among the coumarins isolated from species of Artemisia (Asteraceae), esculetin (6,7-dihydroxycoumarin) and its dimethyl-ether scoparone were known to be antiproliferative on vascular smooth muscle cells. This activity was further found in some very simple mono-substituted, coumarins, which were even more potent than esculetin, although less effective. In an attempt to verify its mechanism of action, esculetin was tested for interactions with PTK and PKC. The induction of membrane PTK activity by either foetal calf serum or platelet-derived growth factor (PDGF) was moderately reduced by esculetin, whereas no effect was observed against PKC [56]. Daphnoretin (4) was characterised as an inhibitor of PTK. It managed to inhibit the tyrosine kinase activity of the EGF receptor from A431 human epidermoid carcinoma cells, with an IC50 value of 97.5 |LIM. However, it showed cytotoxicity on four different proliferating cell lines. The authors were cautious in ascribing a particular value to daphnoretin as tumour chemopreventive [57]. The resuks obtained for daphnetin (7,8dihydroxycoumarin) on the same activity on soluble EGF receptor (IC50 = 7.7 |a.M) were of great interest because no inhibition of the EGF-induced phosphorylation of this receptor in hepatocellular carcinoma cells was observed. The inhibition of PTK activity was competitive with respect to ATP and non competitive with respect to the synthetic substrate. Daphnetin was also quite potent against PKA (IC50 = 9.3 \xM) and PKC (IC50 = 25 laM), practically abolishing their activities at 200 |iM. In the same study, the paucity of the effects of esculin and three simple coumarin monohydroxyl derivatives was demonstrated, and this made the importance of the 7,8-dihydroxyl substitution clear [58]. Dicoumarol is an artefactual coumarin formed by putrefaction of ensiled sweet clover and has been employed as an anticoagulant because it inhibits quinone reductase activity and, therefore, the function of vitamin K in the way of synthesising coagulation factors. Given that quinones are involved in redox systems affecting mitogenic kinase cascades, dicoumarol was studied for its interaction with some of these enzymes. In fact dicoumarol prevented the activation of SAPK and of nuclear factor-KB (NF-KB) [59].

848

Xanthones The first natural xanthones described as kinase inhibitors were mangostin and y-mangostin, two diisoprenyl derivatives isolated fi*om the fiaiits of Garcinia mangostana (Guttiferae) and endowed with inhibitory activity against PKAfi-omrat liver in vitro, with IC50 values of 13 |iM and 2 [iM, respectively [60]. Much more extensive is the research on norathyriol (18), which was obtainedfi-omthe aerial parts of Tripterospermum lanceolatum (Gentianaceae) and later characterised as a vasorelaxant, anti-aggregant and anti-inflammatory agent. Norathyriol was demonstrated to inhibit TPA-induced neutrophil respiratory burst and aggregation, because of its PKC inhibitory activity in these cells. The xanthone reduced in a dosedependent manner the activity of rat brain and neutrophil cytosolic PKC, and in the latter had this effect on both complete and trypsin-treated (deprived of its regulatory domain) forms. Although the IC50 values were not calculated, an inhibition of nearly 50 % was observed on the trypsintreated preparation at 100 |LIM. Norathyriol acted non competitively with ATP and was unable to affect PKC-P translocation to the membrane or PDBu binding [61]. Further studies with cultured rat heart endothelial cells revealed microscopically that norathyriol induces notorious changes in the serotonin-induced effects on the localisation of several PKC isoenzymes, particularly a and 0 forms. In addition, it decreased serotonin-induced translocation of PKC-a to the membrane [62].

OH

18 Norathyriol

849 QH = COOMe

MeOOC

OH V"

19 Secalonic acid Secalonic acid (19) is a fungal phenolic metabolite that can be formally considered a bis-tetrahydroxanthone and is found in Claviceps purpurea and Penicillium oxalicum. Due to its demonstrated teratogenic properties, this substance may make foods infested by the mentioned funguses toxic. In a survey of well-known natural food- and environment-related teratogens for their serine/threonine kinase inhibitory activity, Wang and Polya [63] found that secalonic acid was a potent inhibitor of rat brain PKC (ICso = 15 MM) and of the catalytic subunit of rat liver PKA (IC50 = 12 |a,M), and that it was also active against MLCK (IC50 = 60 |LIM). The authors suggested that by analogy with other kinase inhibitors, these effects could be related to the teratogeny, but the link between the two events must be demonstrated. Quinones and related compounds In this chapter, we include certain natural products, such as typical anthraquinones and naphthoquinones, which are present, along with condensed dimeric structural drivatives such as dianthrones, in many medicinal plants. In addition, we deal with the much more infrequent or///o-quinones. Emodin (l,6,8-trihydroxy-3-methylanthraquinone), the active principle of^ Polygonum cuspidatum (Polygonaceae), was reported to be an inhibitor of the p56^''^-PTK activity from bovine thymus, with an IC50 of 18.5 piM. When the hydroxyl functions at C-6 or C-8 were blocked by methylation or glycosylation, respectively, the effect disappeared. The inhibition was competitive with respect to ATP and non competitive with respect to the substrate [64]. In a bioassay-guided separation of the anthraquinones found in rhizomes of another Polygonaceae species, rhubarb {Rheum

850

palmatum), emodin was again the main active principle when tested against the activity of several serine/threonine protein kinases, such as PKA, PKC, cyclin B/cdc2 kinase (cdc2), CKI and CKIL Emodin was highly potent and selective for CKI and CKII, with IC50 values of 7 |LIM and 2 |jM, respectively. Rhein, the carboxyl superior homologue of emodin, showed an IC50 value of 7 \JM for its CKII inhibition. Considering that CKII accumulates in the nuclei of growing cells and seems to be crucial for both normal and oncogenic proliferation, the inhibitory effects of emodin may correlate with its mouse antileukaemic and the traditional anticancer use of rhubarb in Asia [65]. Hypericin (20) is a natural phenolic naphtodianthrone pigment found in Hypericum species and known for its photosensitising, antiviral and inhibitory properties of both serine/threonine and tyrosine kinases. Hypericin inhibited irreversibly the autophosphorylation of EGF receptor in a time-dependent, oxygen-independent manner. Irradiation with fluorescent light caused a large increase in the potency of hypericin (IC50 = 0.044 versus 0.37 |iM). On the other hand, hypericin inhibited PKC, with an IC50 value of 3.4 |LIM, and was not active against other serine/threonine kinases [66]. Further studies by the same authors using fluorescent light irradiation established the selectivity of hypericin against membrane-bound PTKs (EGF and insulin receptors) versus cytosolic PTKs (Lyn, Fgr, c-Src kinase), and also the high potency of this quinone in inhibiting the serine/threonine kinases CKII (IC50 =" 6 nM) and MAPK (IC50 = 4 nM). Unlike what it was described for the perylene quinone calphostin C, a classical specific PKC inhibitor of similar structure, the activity of hypericin was not affected by singlet oxygen quenchers such as 1,4diazabicyclo[2,2,2]-octane or 2,5-dimethylfuran. For this reason it was postulated that light-induced inhibition is not mediated through singlet oxygen generation [67]. Nevertheless, it should be pointed out that the oxygen-quenching interaction with calphostin C was tested using other substances such as lycopene, p-carotene or a-tocopherol [68]. The relationship between photo-irradiation and enzyme inhibitory activity was also examined by other authors, who measured the light-induced increase in the potency of hypericin as a brain PKC activity inhibitor. Light exposure did indeed influence positively the ability of hypericin to interfere with cellular activities dependent on PKC such as the generation of superoxide radical by guinea pig neutrophils [69]. In a study designed to search for the structure-activity relationships of hypericin derivatives, it

851

was demonstrated that di- or tetra-bromination of hypericin decreased its potency as a kinase inhibitor, and that exhaustive methylation augmented its potency and selectivity against PKC, but not against other serine/threonine kinases [70].

20 Hypericin Lapachol, an alcohol that can be converted into p-lapachone (21), a prenyl-naphto-l,2-quinone known for its anti-tumoral, pro-apoptopic and topoisomerase I-inhibitory properties was obtained from the lapacho tree, Tabebuia avellanadae (Bignoniaceae). Although an inhibition of PTKs has not, to our knowledge, been reported, P-lapachone inhibited the activation of JNK and MEK by tumour necrosis factor (TNF). These effects may account for the observed inhibition of the NF-KB-mediated gene expression and the mentioned biological activity [71]. rr-^^

21 p-Lapachone

852

Diarylalkanoids In this section we report on the activity of certain diarylheptanoids, perhaps one of the most recently described type of kinase-inhibitory phenolics. Natural diarylheptanoids, like difemloylmethane (curcumin), are formed by two simple or/Zio-hydroxy substituted aromatic rings, bridged by a seven-carbon linear chain. Curcumin (22) is the main principle of Curcuma aromatica, C. longa, and C. xanthrorrhiza (Zingiberaceae) and possesses anti-tumoral, anti-inflammatory, and free radical-scavenger properties. In a study concerning PKC activity in a purified preparation from NIH 3T3 fibroblasts, curcumin caused little inhibition of enzyme activity, although it induced translocation to the membrane. The inhibition of the activity in the particulate fraction was stronger, 69 % after 30 min incubation with 15 |iM curcumin [72]. When it was tested at 100 ^iM for inhibitory activity against several kinases of different kinds and origin, curcumin blocked selectively and non competitively the in vitro activity of phosphorylase kinase. A dose-dependent inhibition of pp60''"'''^'' PTK, culminating an almost complete inhibition at 0.6 mM, was also observed, but other serine/threonine kinases like PKC, PKA and cytosolic protamine kinase were only mildly inhibited or not inhibited at all [73]. However, it was later reported that curcumin inhibited the PKA-catalytic subunit and PKC, with IC50 values of 4.8 and 15 |iM, respectively, and the inhibition was in both cases competitive with ATP and the peptide substrate [74]. An interesting review of the biological effects of curcumin, apigenin and other phenolics in close relationship with dietary cancer prevention has been published [75].

o MeO^

^^s^

^y-S^

JL

o JL

22 Curcumin

^^V^

^^VW

^OMe

853

HO

HO

23 Hirsutenone Other active diarylheptanoids found in nature were the five obtained from the stems of Pinus flexilis (Pinaceae). They have no unsaturations in the linear chain, except hirsutenone (23), and were potent inhibitors of recombinant human PKCa, with IC50 values ranging from 1.4 to 8.6 [xM [76]. Other phenolic compounds Neolignans dijffer from true lignans in that a bonding between two phenylpropanoid moieties affects positions other than the two p-carbons of the lateral chain. A plain example of neolignans is provided by the structure of magnolol (24), which is known to be present in Magnolia officinalis (Magnoliaceae). This compound possesses a slight activity as an inhibitor of PKC from rat brain and neutrophils, without affecting PKC translocation or PDBu binding [77].

24 Magnolol Rottlerin (25) is a chromene based on a prenyl-phloroglucinol structure and isolated from the ancient vermifuge drug kamala {Mallotus philippinensis, Euphorbiaceae). It demonstrated efficacy in inhibiting PKC activity competitively with ATP, although the potency varied notably depending on the isoforms. The IC50 value was 3 jiiM for the inhibition of

854

porcine spleen PKC-6; 30, 40 and 42 |iM for PKC-a, y and P from baculovirus-infected Sf9 insect cells, respectively, and higher than 80 |LIM for the atypical PKC s, rjand Q. Calmodulin kinase III (CAMK-III), an enzyme that catalyses the phosphorylation of the elongation factor-2 (EF2), was also potently inhibited by rottlerin (IC50 = 5.3 [a,M), and not by the PKC inhibitor staurosporine at equivalent doses [78, 79]. It must be noted that EF-2 is insensitive to phosphorylation by kinases other than CAMKn, participates in the GTP-mediated ribosomal translocation of the peptidyl-tRNA and is therefore a key element for protein synthesis in eukaryotes.

25 Rottlerin More recently, it has been shown that rottlerin inhibits the migration towards the membrane of PKC-a and -5, diminishes the binding to DNA of the transcription factors AP-1 and NF-KB, and subsequently affects cytokine production by monocytes [80]. Some simple stilbenes isolated from the roots of Polygonum cuspidatum (Polygonaceae) showed moderate inhibitory activity of the bovine thymus P56^''^ tyrosine kinase activity, when angiotensin 1 was used as a substrate. The most potent were resveratrol (26) and its cis form obtained by photochemical isomerisation. Both compounds showed similar potency against rat brain PKC, whereas resveratrol monoglucosides were less active [81].

855 OH HO

OH

26 Resveratrol Other active stilbenes of greater chemical complexity were the oligomers (tri- and tetramers) obtained from the roots of Canagana sinica (Leguminosae). They were tested for their in vitro inhibitory activity against different PKC isotypes. (+)-a-Viniferin was the most potent compound on a, y, and 6 isoforms, whereas miyabenol C was the strongest on P and s isoforms; the minimal IC50 values for each enzyme ranged from 7 to 37 |j,M for the stilbenes, and 0.02-0.06 for staurosporine. When cellular functions that are closely linked to PKC activation, like whole blood respiratory burst, neutrophil superoxide generation, and lymphocyte proliferation were studied, (+)-a-viniferin manifested the highest potency of the three active oligomers [82]. Gossypol (2,2'-bi [8 - formyl - 1,6,7 - trihydroxy - 5 - isopropyl - 3 methylnaphtalene]), a compound known for years for its spermatocidal activity, is present in some species of Malvaceae, such as Gossypium herbaceum (cotton) and Thespesia populnea, a plant which is employed as an anti-inflammatory for external use. Gossypol produces apoptosis of cultured rat spermatocytes in the same range of concentrations as that producing inhibition of PKC. As PDBu protected the cells from gossypolinduced DNA degradation, it is thought that the enzyme preserves cell integrity [83]. Terpenoids Sesquiterpenoids The ethyl acetate extract of the sponge Aka coralliphagum was fractionated in order to isolate the components able to inhibit PKC activity. The responsible substances were identified as a mixture of two diastereomeric spirosesquiterpene aldehydes, corallydictyals A (27) and B. The IC50 for inhibition of PKC was 28 |LIM, while the corresponding

856

methyl ethers were inactive. Using four purified recombinant human isoforms of PKC (a, 8, r| and Q it was possible to show that the inhibitor was selective for inhibition of the a isoform (IC50 = 30 |LIM) [84]. HO

0

., PPG, PPX (or PP4), etc., have been reported. PPl consists of a catalytic core bound to at least one subunit that functions as a carrier of the whole protein to reach the appropriate subcellular structures and increase the activity for a given substrate. Diverse forms of PPl participate in liver and muscle glycogen metabolism, calcium movements in sarcoplasmic reticulum and mitosis. PP2A is also formed by a rather dynamic association of two or three different subunits, and the trimeric form is activated by ceramide, which is in turn liberated from sphingomyelin after TNF-a receptor activation. PP2A is very potently inhibited by okadaic acid, and loses most of its activity through phosphorylation mediated by several PTKs. The importance of PP2A arises from its role in the control of the cell cycle, in relationship with the inactivation of the cyclinB-cdkl and cyclinB-cdk2 complexes, and of certain kinases of the ERK/MAPK pathway [122,123]. PP2B, also known as calcineurin, is a calmodulin-binding protein formed by two subunits: calcineurin A (catalytic) and calcineurin B (regulatory). Because of its activation of NF-KB and NF-AT it mediates IL-2 synthesis, and therefore T lymphocyte activation, a process that is blocked by the immunosuppressors cyclosporin and FK506. Calcineurin also participates in cAMP-mediated gene expression, as does PP2A, and in the control of corticotropine secretion [122]. PPl and PP2A-B have a large degree of homology (40-50 %) in their catalytic domains, which contain two a helices flanked by P strands to conform a (3-a-P-a-(3 structure. The active site contains two metallic ions (Ml and M2) that are essential for the enzyme function, each of which is coordinated by five ligands. In the PPl catalytic core. Ml coordinates with Asp 64, His 66, Asp 92 and two water molecules, and M2 with Asp 92, Asn 124, His 173, His 248 and one water molecule. Ml and M2 can also act as a kind of hook for the substrate phosphate group [124]. Tyrosine PPs are classified in four famiUes, depending on their biological function and aminoacid sequence: tyrosine-specific phosphatases (TSPs), vaccinia virus HI (VHl)-like phosphatases, cdc25 phosphatases

874

and low molecular weight phosphatases. Although the four main groups are quite dissimilar in structure, some fundamental features in their catalytic core are maintained: the active site contains a Cys-X-X-X-X-XArg sequence; in most cases cysteine is preceded by histidine, and arginine is followed by serine or threonine. Cysteine is essential for the phosphatase activity because of the formation of an infrequent phosphocysteine intermediate, and when cysteine is supplanted by serine the hydrolytic effect is lost. Arginine retains the phosphate group by hydrogen bonding, although the geometry of the alkyl-guanidino chain seems to be more important than its positive charge, because of the similar slowing in the catalysis observed when arginine is substituted by either lysine or alanine. In addition, it should be stated that tyrosine PPs do not require metallic cations for their activity, are sensitive to sodium vanadate, and are localised in different subcellular structures, notably in the nucleus. Moreover, as it occurs with tyrosine kinases, some TSPs adopt the form of a membrane receptor [125]. Receptor-like protein tyrosine phosphatases (RPTPs) contain a variable extracellular domain that, depending on the RPTP subtype, exhibits immunoglobulin and fibronectin-like regions and other sequence motifs involved in cell-cell adhesion. The exact repercussion of ligand binding to RPTPs is currently being studied. The following subtypes have been defined: a) Subtype I RPTP CD45: Involved in the activation of B- and Tlymphocytes. It stimulates kinases by dephosphorylation. b) Subtype II RPTP|a and %. Modulates cell-cell interaction. c) Subtype IV RPTPa: It binds to Grb2 [12]. For a recent review on the major biological aspects on tyrosine PPs see the work by Li and Dixon [126]. Protein phosphatase inhibitors According to Mackinstosh and Mackintosh [127], the phosphatase inhibitors are classified in two main groups: Those inhibiting PP1/PP2A and those inhibiting PP2B. PP1/PP2A inhibitors are usually high molecular weigth substances produced by aquatic lower organisms as a mean of ecological defence and characterised by either long polyhydroxylated alkylic or cyclic polypeptide chains. The most important inhibitors of PP2B are cyclosporin A and tacrolymus (FK506) which will not be further an object of this study because of their well established therapeutic application as immunosuppressors.

875

Non-peptide polyether toxins As it was mentioned above, okadaic acid (48) is surely the best characterised phosphatase inhibitor. It is a carboxylic acid based on a 38-C chain with multiple internal ether pyrane and furane cycles, whose isolation from sponges of the genus Halichondria was reported in 1981. In fact, it is produced by other organisms, such as certain species of Prorocentrum and Dinophysis, dinoflagellates that are ordinary food for marine invertebrates like sponges and edible molluscs. Okadaic acid makes the food in which it is present, even in the range of |Lig amounts, highly toxic, producing diarrhoea, vomiting and abdominal pain [128].The major mechanism behind its biological activity and toxicity is the interaction with serine/threonine PPs, particularly PPl, PP2A and PP3, which were inhibited with respective IC50 values of 49.0, 0.28 and 3.91 nm. If the carboxyl group of okadaic acid is esterified by a methyl radical or is reduced to the alcohol okadaol, potency is severely reduced and the activity against PPl disappears. Other chemical transformations that lead to a loss of the inhibitory effect on each of the three phosphatases are the reduction to 1-nor-okadaone and tetra-acetylation [129].

48 Okadaic acid Okadaic acid has been demonstrated to enhance the activity of the kinases related to PKA and PKC (RAC-PKs), also termed PKB/Akt alluding to the viral oncogene v-aAt, which is related to the human RAC-a gene. These kinases are activated through a multi-stage phosphorylation process assessed by the parallel decrease in electrophoretic mobility of immunoprecipitated RAC-PK from Swiss 3T3 murine cells. The effect of okadaic acid lies in its inhibition of PP2A, which exerts control over RAC kinase activity by dephosphorylation. The process does not depend on the receptor-PTK-dependent PI3K activation because it is insensitive to 200 nM wortmannin [130]. Using MCF7 cells, it was demonstrated that

876

okadaic acid penetrates the membrane and binds strongly to the catalytic subunit of PP2A (PP2Ac), causing a decrease in the phosphatase activity of the soluble cellular fraction. The fact that okadaic acid provokes demethylation of the carboxymethyl terminal group of Leu^^^ in a protein synthesis-independent manner may relate to PPA2 activity regulation, for this reaction was not affected by the presence of either cycloheximide or puromycin [131]. At present okadaic acid is a widespread laboratory tool used to detect the participation of PPs, or more strictly of PPA2, in many physiological processes. For that reason an increasing number of research studies deal more or less specifically with the activity of this toxin and related ones, such as calyculin A and others. For the purpose of the present review, a selection was made of recent papers focusing on those aspects with major pathological incidence. One field in which phosphorylation and dephosphorylation play an important role is the transcriptional enhancement of inflammatory proteins brought about by NF-KB. This factor is inactivated by the IKB proteins, which retain it in the cytoplasm. This kind of protein is phosphorylated, prior to degradation in the 26S proteasome by a kinase (IKK), whose activity is enhanced by a series of physical, chemical and microbiological stimuli. Consequently, this mechanism is an indirect way of amplifying NFKB signalling. Okadaic acid was reported to increase, at concentrations below 5 nM, the phosphorylation of iKBa [132]. Although it was unable to affect the activity of IKK itself isolated from HeLa cells challenged with TNF, it blocked the inhibitory effect that PP2A has on iKBa phosphorylation. In this way it cooperated in the cytokine-induced inflammatory response [133]. Cell death by apoptosis, a phenomenon that is increasingly considered the ultimate cause of many diseases, is also one of the patent toxic manifestations of okadaic acid. Micromorphological studies have demonstrated a parallel between the hyperphosphorylation of neuronal cytoskeletal proteins in Alzheimer disease and that produced by a treatment of cultured rat neurones with okadaic acid, which is accompanied by rounding of the cellular bodies and neurite degeneration. DNA fragmentation and neuronal death are inhibited by the presence of cycloheximide and by the caspase inhibitor benzoyloxycarbonyl-Val-AlaAsp-0-methoxy-fluoromethyl ketone (ZVAD) [134, 135]. Okadaic acid was reported to be an apoptopic agent in rat thymocytes, because it causes

877

phosphorylation of histones HI and H2A/H3. This was demonstrated by detecting [^^P]-phosphate incorporation into proteins near 15 kDa on gel electrophoresis, and ulterior identification of histones by acetic acid-ureaTriton X-100 electrophoresis. It is suggested that after discrete phosphorylation, these histones may suffer structural changes affecting chromatin fibers, which results in a greater accessibility of DNA to DNAses, in this case the caspase-activated DNAse that mediates DNA breakdown in apoptosis [136]. When the cellular toxicity of okadaic acid against human myeloid leukaemia K562 cells was studied, it was proven that interruption of mitosis and apoptosis are in fact two synchronised PPA2-related events but generated by distinct mechanisms [137]. On the other hand, in HIT hamster pancreatic p-cells it was shown that the resistance to okadaic acid-induced apoptosis, which even led to define a particular cellular subline, was not dependent on changes in phosphatase activity, but possibly on the multidrug resistance phenotype [138]. Concerning involvement of phosphatase inhibition in hormonal activity, important modifications in intracellular trafficking of the glucocorticoid receptor (GR) have been detected. Okadaic acid aflfects the usual recycling of GR out of the nucleus and impairs ulterior activation upon hormone binding. Under habitual conditions, a phosphatase sensitive to okadaic acid regulates the dissociation of the GR binding to heat-shock protein 90 (hsp90) and formation of the hormone-receptor complex (HRC) that then enters the nucleus. As the inhibitory effect of this mechanism by okadaic acid in NIH/3T3 mouse embryo fibroblasts is lost when microtubules are disrupted by colcemide, it has been suggested that the transport into the nucleus normally requires cytoskeletal machinery, in absence of which HRC enters the nucleus by passive diffusion [139]. Calyculin A (49) was discovered to be a toxic compound from the sponge Discodermia calyx. Part of the molecule corresponds, as in okadaic acid, to a long chain (C-26 in this case) polycyclic ether, although instead of a carboxyl it has a nitrile group, four conjugated double bonds, and one phosphate group. Another part of the molecule can be theoretically derived from an oxazole alkylamide. Calyculin A is approximately equipotent as an inhibitor of PP2A and PPl (IC50 = 0.3 and 0.4 nM respectively, referred to phosphorylase-a dephosphorylation) and consequently two degrees of magnitude more potent than okadaic acid as an PPl inhibitor [129]. As was reported before for okadaic acid, calyculin A produces demethylation of the carboxymethyl terminal group of Leu^^^

878

in PP2Ac but, unlike okadaic acid, it decreases immunoreactivity of the carboxyl end of the catalytic subunit of PPl (PPlc), possibly by proteolysis [131].

49 Calyculin A Ri = H, R2 = CN, R3 = H, R4= H 50 Clavosine A Ri = H, R2 = CONH2, R3 = Me, R4= rhamnose When studying the influence of PP activity on the inhibitory effect of adenosine on neutrophil function, it was noted that a pre-incubation with 10 nM calyculin A deprived the adenosine agonist 5'-iVethylcarboxamidoadenosine (NECA) of its inhibitory activity on neutrophil superoxide generation. The fact that okadaic acid was ineffective at 10 |iM led researchers to attribute a principal role in the regulation of adenosine activity to PPl [140]. The direct effect of calyculin A on neutrophils was time-dependent. With a short pre-treatment, a theoretically unexpected induction of tyrosine phosphorylation of the signalling proteins Cbl and Syk was observed, whereas a pre-treatment longer than 5 min produced inhibition of tyrosine phosphorylation and an increase in the serine/threonine phosphorylation of Cbl and superoxide formation after cross-linking of CD32, a neutrophil low-affinity IgG receptor. Okadaic acid showed similar effects with slower kinetics [141]. After Calyculin A, many other calyculins, as well as the related calyculinamides, in which the nitrile group is hydrated to give an amide.

879

have been discovered. Two of the recently identified calyculinamides are clavosines A (50) and B,fi-omthe sponge Myriastra clavosa, which share most of the structure of calyculin A but differ in that both are amides, C31 methylated, and present a permethyl-0-rhamnose moiety at C-21. The C2-C3 double bond has a cis geometry in clavosine A and a trans one in clavosine B [142]. Clavosine A was as potent as calyculin A in inhibiting the y isoforms of PPlc (PPlcy) and PP2Ac (IC50 in the 0.5-0.7 nM), whereas clavosine B showed IC5o= 13 nM and 1.2 nM for the inhibition of PPlcy and PP2Ac, respectively. Studies with mutated PPlcy proteins showed that substitution of the 134-Tyr residue by Phe (hydrophobic residue) enhanced the inhibitory activity by the three calyculins, and that when the 134-Tyr was substituted by a 134-Asp (negatively charged residue) the inhibitory potency was seriously diminished [143]. Tautomycin was obtained fi-om the terrestrial microorganism Streptomyces spiroverticillatus and is chemically characterised as an ester of a carboxylic acid derivedfi^oma dialkyl maleic anhydride, with a long chain (26-C) polyol containing two cyclic ethers. In a simultaneous study of the best-known natural inhibitory compounds of PP2A, in which the non-radioactive malachite green assay was applied, tautomycin was the least potent compound [144]. This is consistent with the findings reported by Honkanen et al. [129] and other authors. Its primary site of phosphatase inhibition is PPlc, as was reported in a comparative study with okadaic acid and calyculin A [131]. Cyclic polypeptide inhibitors The most important of the peptidic phosphatase inhibitors are the microcystins and nodularin. Mycrocystins are heptapeptides characterised by the sequence cyclo(D-Ala-X-D-er);^/iro-P-methylisoAsp-Y-Adda-DisoGlu-A^-methyldehydroAla), where X and Y are different L-aminoacids, and Adda is the abbreviation of the p-aminoacid [25',3iS',8iS',95]-3-amino-9methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid. In the most common microcystin, namely microcystin-LR, X is Leu and Y is Arg. This kind of compounds was considered to be the highly hepatotoxic principle of the cyanobacterial genera Microcystis, Anabaena and Oscillatoria. Apart fi'om the variations represented by X and Y, other differences arising fi-om the demethylation of aminoacids, lead to the existence to more than fifty microcystins. The rare acid Adda is also

880

present in nodularin, from the cyanobacteria Nodularia spumigena, which contains the pentapeptide sequence cyclo(D-p-methylisoAsp-L-Arg-AddaD-isoGlu-A^-methyldehydrobutyrin) [128, 129]. When the polar residue LArg of nodularin is replaced by L-Val, the result is motuporin, a compound that was isolated from the marine sponge Theonella swinhoei, and was reported to be a potent PPl inhibitor and cytoctoxic substance against several human cancer lines [145]. Microcystins show the highest potency among the known inhibitors of PP2A, ranging from 0.04 to 2 nM according to different authors and assay methods [144].The inhibition of PPl and PP2A activity by microcystins occurs through two steps: an immediate blockade of the catalytic site by non-covalent forces and a subsequent adduct covalent formation. With regard to this, it has been demonstrated that in the presence of each of the microcystins LR, RR and YR, the affinity of the substrate phosphorylase a for the complexed PP2A was four times lower than that for the unbound PP2A, indicating a partial reduction of substrate binding, which is compatible with a total abolition of the enzyme activity [146]. A rather close relationship between the apoptosis induced by microcystin-LR and caspase-3 activity was observed. Hepatocytes are extremely sensitive to microcystins and nodularin, as can be deduced from the quick apparition of cellular apoptotic changes such as superficial budding and shrinkage of cytosol and nucleus. Analogous findings were obtained in other kinds of cells, like Swiss 3T3 fibroblasts or promyelocytic IPC.81 cells, provided that the toxins were microinjected. However, in caspase-3 deficient MCF-7 cells, apoptosis developed slowly and was independent of the ZVAD, a compound that ordinarily inhibited apoptosis without affecting the hyperphosphorylation caused by PP inhibition [147]. Alkaloids From the dichloromethane extract of the stems of Rollinia ulei (Annonaceae), three aporphine alkaloids were isolated as the principles responsible for the inhibitory activity on yeast recombinant CD45 tyrosine phosphatase. The most potent alkaloid was nomuciferine (51), with an IC5o=5.3^M[148].

881

MeO'

51 Nomuciferine The bisindole brominated alkaloid, dragmacidin D (52), was isolated from a sponge of the genus Spongosorites found off the South Australian coast at a depth of 90 m. This compound was characterised as a selective inhibitor of PPl, while its isomer dragmacidin E also inhibited PP2A [149]. Another example of brominated alkaloid obtained from a sponge is discorhabdin P (53), which was isolated from a species of Batzella collected in the western Great Bahama Bank, 141 m below sea level. This alkaloid can be considered a condensed monoindole derivative and behaved as an inhibitor of bovine brain PP2B, with an IC50 of 1.1 |iM.

52 Dragmacidin D

882 o

H

.A^"-

53 Discorhabdin P Miscellaneous compounds Cantharidin (54) is the vesicant substance obtained from some species of blister beetles, among them Mylabrisphalerata.M. cichorii and Cantharis vesicatoria. Known since ancient times as a strong rubefacient and used as a wart remover and aphrodisiac drug, it is now arousing new interest for its inhibitory activity on PPs. It was reported to inhibit PPl and PP2A with respective IC50 of 0.16 and 1.7 \xM, in phosphohistone dephosphorylation [150]. In bovine arterial tissue, cantharidin produced a smooth muscle contraction that was independent of cytosolic Ca^^ levels, but was, in contrast, closely linked with the phosphorylation of contractile proteins by inhibition of PPl and PP2A, both of which were detected together with their mRNA in the aortic tissues [151]. The same authors reported that cantharidin inhibited PPl and PP2A activity in bovine aorta endothelial cells and that it was determinant for the strong increase produced in albumin permeability in endothelial monolayers [152].

54 Cantharidin

883

CH2OH OPO3OH2 HO

Me

55 Fostriecin Fostriecin (55) is a phosphoalkyl-pyrone antibiotic obtained from Streptomyces pulveraceus ssp. fostreus that was described as a phosphatase inhibitor, particulariy of PP2A (IC50 = 3.2 nM), and antineoplastic drug. Although it is less potent than okadaic acid, its selectivity towards PP2A is much higher [153] and it showed similar potency in inhibiting PP4 from porcine testes, a phosphatase that has a predominant presence in the nucleus and features in common with PP2 A. ABBREVIATIONS AGC ACs AKAPs aPKCs ANP ATP BNP CaMKs CDKs CDPK CKI CKII CIK CMGC cPKCs CREB DAG DPP A EGF

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

Cyclic nucleotide regulated protein kinase family Adenylyl cyclases A-kinase anchor proteins Atypical PKCs Atrial natriuretic peptide Adenosine 5'-triphosphate Brain natriuretic peptide Ca^Vcalmodulin-dependent kinases Cyclin-dependent kinases Ca^'^-dependent protein kinase Casein kinase I Casein kinase II Dcd-like kinase Cyclin-dependent kinases and close relatives family Classical or conventional PKCs cAMP responsive element binding protein Diacylglycerol 12-Deoxyphorbol-13-O-phenylacetate-20-acetate Epidermal growth factor

884

EF-2 ERK GAP GP GPCRs GR GSK3 GTP HRC JAKs JNK IL-\a IP3 LIMKs LPS MAPK MAPKK MAPKKK MAPKAP2 MARCKS MEK MLCK nPKC OPK PAI-1 PAK PDBu PKA PI3K PIP2 PIP3 PLC PLC-p PLC-5 PLC-y PLD PPs PTKs

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

Elongation factor-2 Extracellular signal regulated kinase Ras-GTPase activating protein Glycogen phosphorylase G-protein coupled receptor group Glucocorticoid receptor Glycogen synthase kinase 3 Guanosine 5'-triphosphate Hormone-receptor complex Janus kinases c-Jun N-terminal kinase Interleukin-la Inositol 1,4,5-trisphosphate LIM kinases Lipopolysaccharide Mitogen activated protein kinase MAPK kinase MAPK kinase kinase MAPK activated protein kinase 2 Myristoylated alanine-rich C kinase substrate MAPK/ERK kinase Miosin light-chain kinase novel PKCs Other protein kinases Plasminogen activator inhibitor-1 p21-activated kinase Phorbol-12,13-dibutyrate cAMP-dependent protein kinase Phosphatidylinositol 3-kinase Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-trisphosphate Phospholipase C Phospholipase C-p Phospholipase C-5 Phospholipase C-y Phospholipase D Protein phosphatases Protein-tyrosine kinases

885

PKC RPTPs RTKs SAPK/JNK SEKl SH2 TGF-p TNF TPA TSPs

= = = = = = = = = =

Protein kinase C Receptor-like protein tyrosine phosphatases Receptor tyrosine kinases Stress-activated PK/cJun N-terminal kinase SAPK/ERK kinase 1 Src homology domain 2 Transforming growth factor-p Tumour necrosis factor 12-0-tetradecanoylphorbol-13-acetate Tyrosine specific phosphatases

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved.

891

CYTOTOXICITY OF FLAVONOIDS ON CANCER CELL LINES. STRUCTURE-ACTIVITY RELATIONSfflP M. LOPEZ-LAZARO, M. GALVEZ, C. MARTIN-CORDERO, MJ.AYUSO* Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain ABSTRACT: The therapeutic potential of flavonoids shown by recent research, makes a greater understanding of these compounds that leads to new drug discovery necessary. An important part of the available literature about anticancer activity of flavonoids shows lots of reports about natural and synthetic flavonoids inhibiting diverse cancer cell lines; but this information is very scattered and there is very little understanding about a possible structure-activity relationship. Throughout our present research we have reviewed this literature and we have compiled the cancer cell lines inhibited by more than 500 natural and synthetic flavonoids and we have discussed structural requirements implicated in the activity to obtain a working hypothesis to help to rationalize the development offlavonoidsas antitumor agents.

INTRODUCTION Flavonoids are one of the most representative classes of plant secondary metabolites, occurring widely throughout the plant kingdom. They are a large group of low molecular weight substances characterised by a C6C3-C6 carbon skeleton in which the C6 components are aromatic rings. Different authors classify the flavonoids into different subclasses, mainly based on variations in a heterocyclic C3 ring. In this way, flavonoids mainly consist of flavones, flavonols, flavanones, isoflavones, catechin, anthocyanidins, proanthocyanidins, flavans and aurones (to see diverse classifications see [1] and [2]. One more widely known classification of flavonoids consists of two main groups, namely flavonoids (in the narrow sense) and isoflavonoids, the first with a C6-C3-C6 skeleton based on 1, 3-diphenylpropane and the latter based on 1, 2-diphenylpropane. So, flavonoids include the above mentioned subclasses (except isoflavones) and other groups such as chalcones, and the isoflavonoid group principally include isoflavones.

892

rotenoids, pterocarpenoids, coumestrans, coumarone-chromones, isoflavanones and isoflavanans [3]. Besides, flavonoids can occur as aglycones (without sugar moieties), glycosides (with sugar moieties) or biflavonoids; so, it is very difficult to classify flavonoids. Fig. (1).

. ^ ^

1,3-diphenyipropane

1, 2-diphenylpFOpane

Fig. (1). Main skeletons of flavonoids studied. Note that flavones, flavonols, flavanones and chalcones are 1, 3-diphenylpropane derivates, whereas isoflavones and rotenoids are 1,2-diphenylpropane derivates.

893

Flavonoids are ubiquitous in all terrestrial plants (except the Anthoceropsidae) but the distribution of isoflavonoids among plants is relatively sparse, probably owing to the sporadic occurrence of the enzyme isoflavone synthasa that stabilises, by an aryl migration, the isoflavonoid skeleton. Accordingly, most of the isoflavonoids have been isolated from leguminous plants [3]. In vitro and animal studies have shown that flavonoids are antioxidants and antimutagenic agents and can inhibit, and sometimes induce, a large number of mammalian enzyme systems, that sometimes are involved in important pathways that regulate cell division and proliferation. Thus, these compounds can be involved in various stages of the cancer process [4]. In fact, some flavonoids such as baicalain and its glycosides have been clinically used in China for many years and, flavopiridol (the first cyclin-dependent kinase inhibitor to be tested in Phase II clinical trials), genistein (whose antibody conjugates, B43-genistein and EGF-genistein, are currently in clinical development for the treatment of acute lymphoblastic leukaemia and breast cancer, respectively), catechin and its galates (major ingredients of green tea extract GTE-TP91, that has been conducted in adult patients with solid tumors as a Phase I study) and quercetin [5] are widely studied flavonoids as potential therapeutic agents. It is important to know that flavonoids are generally safe and without adverse effects and, subsequently, that many flavonoids occur in our diet. We can find flavones in grains and herbs, flavonols and their glycosides in fruits and vegetables, flavanones in citrus juices or isoflavones in legumes [1]. So, a greater understanding of the health benefits of flavonoids is not only leading to new drug discoveries, but will also influence our drinking and dietary habits. Cancer cell lines are a usefiil tool in the anticancer research that facilitates knowledge of new cytotoxic compounds, the potency of a drug or the structure-activity relationship; such knowledge helps to develop new anticancer compounds. In this way, lots of pharmacological studies carried out with natural and synthetic flavonoids show that these compounds can inhibit the growth of diverse human cancer cell lines; but this information has not been compiled and there is very little understanding about a possible structure -activity relationship. Some authors have found some structural requirements (that sometimes

894

disagree with other reports) whereas others authors could not find any relationship, possibly because of the few or not appropriated flavonoids used in their studies. The present work seeks to compile information about the cancer cell lines inhibited by different groups of natural and synthetic flavonoids reported in the literature and try to find and discuss structural requirements implicated in the activity to obtain a working hypothesis to help to rationalize the development of flavonoids as antitumor compounds. METHODOLOGY The data have been obtained after a revision of the literature available by different data basis, such as MedLine or NCI data base, and by the references in this literature. The data are presented in different tables according to the main subclasses of flavonoids. Tables are divided in four columns that include the flavonoid subclass substituent, its trivial name, the type of tumor cell line inhibited and the reference. Cj^otoxic values are not presented because the authors use different protocols and different times of incubation of flavonoids with cancer cells and the results would not be comparable. The order of each flavonoid in a Table is based firstly in the number of substituents, then in their position and finally in the type of substituent. Sometimes, the structure of a flavonoid does not appear in the tables, generally because it has a complicated structure. In such cases, you can find it in the reference or in the NCI database, using the NSCnumber (http://dtp.nci.nih.gov/). The discussion about structure -activity relationship is based on all authors' discussion and on our own global discussion based on their results. RESULTS We have found more than "500" natural and synthetic flavonoids that have shown growth inhibitory activity in different cancer cell lines and are presented in Tables 1-8 according to the main subclasses of flavonoids. Tables 7 and 8 are miscellaneous ones and attempt to include all the flavonoids not presented in Tables 1-6.

895 Table 1. Cancer Cell Lines Inhibited by Flavones. Flavone substituents

Trivial name

Celi type

Reference

None

flavone

5- (OH)

-

5- (OMe)

-

6- (OH)

-

6- (OMe)

-

6-(F) 7-(OH)

-

7- (OMe) 7-(F) 3'-(OH) 3'-(N02) 5, 7- (OH)

-

5- (OH) 7- (OMe) 5,3'-(OH)

-

6,7- (OH) 6,8-(F) 6- (OH) 4'- (NH2)

-

6-(OH)4'-(N02)

-

HeLa A-549 HL-60 HT-29 Caco-2 ZR-75-1 KB HL-60 MOLT-4 WA Caco-2 HT-29 ZR-75-1 MOLT-4 ZR-75-1 ZR-75-1 HL-60 MOLT-4 KB LL HL-60 ZR-75-1 KB HL-60 MOLT-4 Caco-2 HT-29 ZR-75-1 HL-60 KB KB KB NCI-60 HeLa MCF-7 SHEP KB Caco-2 HT-29 KB HT-29 SK-MEL MCF-7 A-549 HeLa KB HT-29 SK-MEL A-549 SK-MEL A-549

[6] [7] [8] [9] [9] [10] [11] [8] [8] [11] [12] [12] [10] [8] [10] [10] [8] [8] [11] [11] [8] [10] [11] [8] [8] [12] [12] [10] [8] [11] [11] [11] NSC-407436 [6] [13] [13] [11] [12] [12] [11] [14] [14] [14] [14] [6] [11] [14] [14] [14] [14]

chrysin

LilJ

896 Flavone substituents

Trivial name

7- (OMe) 3- (Me) 7, 8- (OH) 7- (OMe) 8- (Me) 7, 3'-(OH) 7,4'- (OH)

liquiritigenin

7-(OH) 4'-(NH2) 7- (OH) 4'- (NO2) 7- (OAc) 4'- (NHAc)

7- (OH) 4'- (0Si(Me)2-t-Bu)

T- (OH) 3- (CI) 3', 4'-(OH) 4' - (OH), 7- (OMe) 3- (COOH) 6- (OMe) 4'- (NO2) 3- (COOMe) 6- (OMe) 4'- (OH) 3- (COOMe) 6- (OMe) 4'- (NO2) 3- (COOMe) 6- (OMe) 4'- (OBn) 3- (COOMe) 7- (OMe) 4'- (OH) 3- (COOMe) 7- (OMe) 4'- (NHAc)

3- (COOMe) 7- (OMe) 4'- (NO2) 3- (COOMe) 7- (OMe) 4'- (OBn) 3-(COOMe) 3', 5'-(OMe) 5, 6, 7- (OH)

baicalein

Cell type

Reference

FV HeLa Caco-2 HT-29 KB SK-MEL HL-60 MOLT-4 A-549 HT.29 SK-MEL A-549 HT-29 A-549 MCF-7 HT-29 SK-MEL HT-29 A-549 MCF-7 SK-MEL KB MCF-7 SHEP WAC2 ZR-75-1 MCF-7 SK-MEL SK-MEL MALM-3M A-549 HT-29 SK-MEL HT-29 SK-MEL MALM-3M A-549 MCF-7 HT-29 SK-MEL MALM-3M SK-MEL MALM-3M A-549 HT-29 MCF-7 HT-29 Caco-2 HL-60 MOLT-4 MCF-7 HT-29 MDA-MB-435 ZR-75-1

[H] [6] [12] [12] [11] [14] [8] [8] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [11] [13] [13] [13] [10] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [12] [8] [8] [15] [12] [16]

[101

897 Flavone substituents

Trivial name

Cell type

Reference

5,7,4'- (OH)

apigenin

5, 7- (OH) 8- (OMe) 5,7-(OH) 8-(OMe) 5,7- (OH) 4'- (OMe)

wogonin

5,7- (OH) 4'- (NH2)

-

5, 7- (OH) 4'- (NO2)

-

5, 7- (OAc) 4'- (NHAc)

-

5-(OH) 7,4'-(OMe)

genkwanin

5,4'-(OH) 7-(OMe)

sakuramrtin

5,4'- (OH) 7- (0-Api-Glc)

apiin

5,4'-(OH) 7-(0-Glc) 7, 8,2'. (OMe)

apigenin-7-glucoside

7, 8, 3'-(OH)

-

7, 8- (OH) 4'- (NO2)

-

7-(OH) 3', 4'-(OMe) 2', 3', 4'-(OMe) 3', 4', 5'-(OMe) 5, 6, 7,4'- (OMe)

-

5, 7- (OH) 6,4'- (OMe)

pectolinarigenin

5, 7, 3', 4'-(OH)

luteolin

NCI-60 GLC4 COLO 320 HL-60 MCF-7 SHEP KB Caco-2 HT-29 HL-60 MOLT-4 HL-60 Caco-2 HT-29 HT-29 SK-MEL A-549 HT-29 A-549 MCF-7 SK-MEI MCF-7 A-549 HT-29 SK-MEL KB HeLa KB P-388 Caco-2 HT-29 HTC-15 KB NCI-60 MCF-7 SK-MEL A-549 HT-29 MALM-3M HT-29 MCF-7 MALM-3M SK-MEL A-549 NCI-60 KB NCI-60 KB NCI-60 GCL4 COLO 320 KB HeLa NK/Ly

NSC-83244 [17] [17] [18] [13] [13] [11] [12] [12] [8] [8] [18] [12] [12] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] [19] [6] [19] [19] [12] [12] [20] [11] [21] [14] [14] [14] [14] [14] [14] [14] [14] [14] [14] NSC-123383 [11] [21] [11] [21] [17] [17] [11] [6]

acacetin

-

[22]

898 Flavone substituents

Trivial name

Cell type

Reference

GLC4 [17] COLO 320 [17] HL-60 [18] [23] A549 [23] B16 [23] CCRF-HSB-2 TGBCllTKB [23] MCF-7 [13] SHEP [13] [13] WAC2 [24] P-388 [12] Caco-2 [12] HT-29 diosmetin 5, 7, 3'-(OH) 4'-(OMe) [12] Caco-2 [12] HT-29 5, 7, 3'-(OH) 4'-(0-Rut) NSC-660309 NCI-60 5, 7-(OH) 3',4'-(OMe) [21] NCI-60 5-(OH) 7,3',4'-(OMe) [19] KB hispidulin 5, 7,4'- (OH) 6- (OMe) [21] NCI-60 [17] GLC4 COLO 320 [17] KB [11] [25] P-388 HT-29 [25] [25] A-459 L-strain fibroblasts [26] chrysoeriol 5, 7,4'-(OH) 3'-(OMe) [17] COLO 320 COLO 320 [17] [24] P-388 5-(OH) 2', 3', 4'-(OMe) [21] NCI-60 5,2', 3', 4'-(OMe) [21] NCI-60 5,3'-(OH) 7,4'-(OMe) KB [11] luteolin 7-0 glucoside 5, 3', 4'-(OH) 7-(OGlc) [13] MCF-7 [13] SHEP [17] GLC4 COLO 320 [17] [20] HTC-15 diosmin 5, 3'-(OH) 4'-(OMe) 7-(0-sugar) [12] Caco-2 [12] HT-29 KB 5, 3', 4'-(OH) 8-(C-sugar) (133101) [11] velutin 5,4'-(OH) 7, 3'-(OMe) [19] KB [19] P-388 chrysoeriol-7-O-glucos GCL4 5,4'-(OH) 3'-(OMe) 7-(0Glc) [17] COLO 320 [17] [14] A-549 5,4'-(OH) 3', 5'-(OMe) [14] MCF-7 [21] NCI-60 6- (OH) 5, 7,4'- (OMe) 6-(OH) 2', 3', 4'-(OMe) NCI-60 [21] 6, 2', 3', 4'-(OMe) [21] NCI-60 6-(OH) 3\4',5'-(OMe) A-549 [14] [14] HT-29 SK-MEL [14] MCF-7 [14] 6-(OH) 3', 5'-(OMe) 4'-(OSi(Me)2-tBu) HT-29 _Ui]

899 Fiavone substituents

Trivial name

6,4'- (OH) 5, 7- (OMe) 6,4'-(OH) 3', 5'-(OMe) 7-(OH) 2', 3', 4'-(OMe) 7,2', 3', 4'-(OMe) 7-(OH) 3', 5'-(OMe) 4'-(OSi(Me)2-tBu)

-

7-(0Ac) 3', 5'-(0Me) 4'-(OSi(Me)2-tBu))

-

7,4'-(OH) 3', 5'-(OMe) 3'-(Me)2'-(iPro)3,6-(Cl) 4'- (OH) 5, 6, 7- (OMe) 4'-(Me)2'-(iPro)3,6-(Cl) 5,6, 7, 8, 2'- (OMe) 5,6, 7, 8,4'- (OH) 5,6, 7, 8, - (OH) 4'- (OMe)

-

5- (OH) 6, 7, 8,4'- (OMe) 5,6, 7, 8,4'- (OMe)

tangeretin

5,6, 7, 8, - (OMe) 4'- (OAc)

-

5, 6, 7, 8- (OMe) 4'- (EtO) 5, 6, 7, 8- (OMe) 4'- (iPrO) 5, 6, 7, 8- (OMe) 4'- (AllylO) 5, 6, 3'-(OH) 7,4'-(OMe) 5, 7- (OH) 6, 8,4'- (OMe) 5,7-(OH) 6, 3', 4'-(OMe)

-

5, 7-(OH) 2', 3', 4'-(OMe) 5,7,2', 3', 4'-(OMe) 5, 7, 3'-(OH) 6,4'-(OMe) 5,7, 3', 4'-(OH) 6-(OMe)

-

5,7, 3', 4'-(OH) 8-(C-Glc) 5-(OH) 7, 3', 4', 5'-(OMe) 5, 7,4'-(OH) 6,3'-(OMe)

orientin corymbosin jaceosidin

-

puerarin

eupafolin

Cell type

Reference

SK-MEL A-549 MALM-3M NCI-60 SK-MEL NCI-60 NCI-60 HT-29 A-549 MCF-7 MCF-7 HT-29 SK-MEL A-549 MALM-3M HT-29 KB KB KB NCI-60 KB KB FV WA KB NCI-60 HL-60 A549 B16 CCRF-HSB-2 TGBCllTKB Caco-2 HT-29 KB KB PX KB NCI-60 KB NCI-60 NCI-60 NCI-60 KB Caco-2 HT-29 NCI-60 NCI-60 NCI-60 GCL4 COLO 320 KB UACC-62 KB GCL4

[14] [14] [14] [21] [14] [21] [21] [14] [14] [14] [14] [14] [14] [14] [14] [14] [H] [11] [11] [21] [11] [11] [11] [11] [11] [21] [18] [23] [23] [23] [23] [12] [12] [11] [11] [11] [11] [21] [11] NCS-649411 [21] [21] [11] [12] [12] [21] [21] [21] [17] [17] [11] [27] [19]

-ilZJ

900 Flavone substituents

Trivial name

5, 7,4'-(0H) 3', 5'- (OMe) 5, 3'-(OH) 6, 7,4'-(OMe)

tricin

5, 3', 4'- (OH) 6- (OMe) 7- (OGlc)

eupafolin-7-O-gluc

5,4'- (OH) 6, 7, 8- (OMe) 5,4'-(OH) 7, 3', 5'-(OMe) 5, 5'- (OH) 7, 3', 4'- (OMe) 7, 8,4'-(OH) 3', 5'-(OMe)

lethedocin

7, 8-(OH) 3', 4', 5'-(OMe) 7, 8, 3', 4', 5'-(OMe) 7, 8-(0H) 3',5'-(OMe) 4'-(OSi(Me)2-tBu)

-

7,8-(OAc) 3',5'-(OMe) 4'-(OSi(Me)2-tBu)

-

8, 5'-(OH) 7,3', 4'-(OMe) 2', 4', 6'- (Me) 3, 6- (CI) 3', 4', 5'- (OMe) 6, 7- (O-CHj-O) 3', 4', 5'- (NO2) 6, 7- (O-CH2-O) 3', 5' -(OMe) 4'-(OCH2C6H5) 6, 7-(0CH2-O) 3 ',5 '-(N02)4'-(OCH2C6H5) 6,7-(0- CHj0)

-

4'- (OH) 3', 5'- (OMe) 6, 7- (0- CH2-O)

-

4'- (OH) 3', 5'- (NO2) 6, 7- (0- CH2-O) 5'-(OH) 7, 8, 3', 4'-(OMe) 5, 6, 7, 8,2', 4'- (OMe) 5, 6, 7, 8,2', 5'- (OMe) 5-(OH) 6, 7, 8, 3', 4'-(OMe) 5, 6, 7, 8,2', 5'-(OMe) 5, 6, 7, 8,2', 4'-(OMe) 5, 6, 7, 8,3', 4'- (OMe)

nobiletin

Cell type

Reference

P-388 HT-29 A-459 COLO 320 P-388 KB HT-29 GCL4 COLO 320 FB KB WA KB KB HT-29 SK-MEL MCF-7 HT-29 A-549 P-388 MCF-7 HT-29 SK-MEL A-549 MALM-3M HT-29 SK-MEL MALM-3M P-388 SA L1210 HT-29 L1210 HT-29 L1210

[25] [25] [25] [17] [28] [11] [12] [17] [17] [11] [11] [11] [19] [19] [14] [14] [14] [14] [14] [29] [14] [14] [14] [14] [14] [12] [14] [14] [29] [11] [30] [30] [30] [30] [30]

HT-29 L1210

[30] [30]

HT-29 L1210 HT-29 L1210 HT-29 C0I2 P-388 NCI-60 KB KB NCI-60 NCI-60 NCI-60 NCI-60 NCI-60

[30] [30] [30] [30] [30] [29] [29] [21] [11] [11] [21] NSC-94889 [21] [21]

m

901 Flavone substituents

5,6,7,8,3',5'-(OMe) 5-(OH) 6,7,8,4',5'-(OMe) 5,4'-(OH) 6,7,8,3'-(OMe) 5, 3', 4'-(OH) 6,7,8-(OMe) 5-(OH) 6,7,8,2',4',5'-(OMe) 5- (OH) 6, 7, 8, 3', 4', 5'- (OMe) 5, 6, 7, 8, 2', 4', 5'- (OMe) 5,6, 7, 8, 3', 4', 5'-(OMe) 5, 6, 7, 8, 2', 3', 4', 5', 6'-(OMe)

-

5', 3 " - (OH)-amentoflavone

-

NSC 649890

Trivial name

Cell type

Reference

HL-60 [18] [23] A549 [23] B16 [23] CCRF.HSB-2 [23] TGBCUTKB HTB43 [31] glyosarcoma 9L [31] KB [11] KB [11] FV [11] [21] NCI-60 [21] NCI-60 [21] NCI-60 NCI-60 [21] [21] NCI-60 [21] NCI-60 KB [11] [21] NCI-60 KB [11] KB [11] [21] NCI-60 NSC-683132 NCI-60 bartramia-triluteolin NSC-683135 NCI-60 NSC-293015 flavoneacetic acid ester NCI-60 NSC-347512 flavone acetic acid NCI-60 Pan 03 [32] [32] Colon 26 NSC-683134 philonotisflavone NCI-60 A549 flavopiridol [33] HCT8 [33] T98G [33] MCF-7 [33] HL-60 _[33j

Table 2. Cancer Cell Lines Inhibited by Flavonols. Flavonol substituents

Trivial name

Cell type

galangin

MCF-7 WAC2 Caco-2 HT-29 KB KB KB KB FV KB KB KB HeLa

3-(OH)

3- (NHS03Me) 3- (OH) 6- (Me) 3- (OH) 7- (OMe) 7- (OMe) 3- (Me) 7- (OMe) 8- (Me) 2'-(OH) 3-(CI) 3,5, 7- (OH)

Reference

M.

902 Flavonol substituents

3-(OH) 6-(Me) 4'-(OMe) 3,7,3'-(OMe) 3-(OH) 7,4'-(OMe) 3-(OH) 2', 6'-(OMe) 3-(OH) 3', 4'-(OMe) 3- (OH) 4'- (OMe) 6- (Me)

Trivial name

-

oxyayanin A

-

3, 5, 7, 2'- (OH) 3,5, 7,4'- (OH)

datiscetin kaempferol

3, 5, 7- (OH) 4'- (OMe)

kaempferide

3, 5, 7,4'- (OMe) 3, 7, 3', 4'-(OH)

-

3-(OH) 7, 3', 4'-(OMe) 3- (OH) 2', 4', 6'- (OMe) 3-(OH) 3', 4', 6'-(OMe) 3, 4'- (OMe) 5, 7- (AcO)

-

5- (OH) 3, 7,4'. (OMe) 5- (OH) 3,4'- (OMe) 7- (OAc) 5, 7- (OH) 3,4'- (OMe)

-

5, 7, 2'-(OH) 5, 7, 3'-(OH) 5, 7,4'- (OH) 5, 7,4'- (OH)

datiscetin

3-(OMe) 3-(OMe) 3- (0-Api) 3- (O-p-D-Glu)

5, 7,4'- (OH) 3- (0-Rha) 3, 5, 7- (OH) 6,4'- (OMe) 3, 5, 7,2', 4'-(OH)

flsetin

-

kaempferol-3-O-p-Dglucopiranoside

morin

Cell type

Reference

MCF-7 MDA-MB-435 NCI-60 KB NCI-60 KB NCI-60 NCI-60 KB HeLa MMLV RT HL-60 MOLT-4 GCL4 HeLa KB HTC-15 COLO 320 Caco-2 HT-29 Caco-2 HT-29 KB KB MMLV RT NCI-60 MCF-7 SHEP WAC2 Caco-2 HeLa HT-29 NCI-60 NCI-60 KB NCI-60 CA NCI-60 NCI-60 NCI-60 CA HeLa KB NCI-60 P-388

[15] [16] [21] [34] [21] [11] [21] NSC-19024 [11] [6] [35] [8] [8] [17] [6] [11] [20] [17] [12] [12] [12] [12] [11] [11] [35] [21] [13] [13] [13] [12] [6] [12] [21] [21] [11] [21] [11] [21] [21] [21] [11] [6] [11] NSC-641259 [28]

NCI-60

NSC-641259

NCI-60 MMLVRT HL-60 MOLT-4 NK/Ly Hep-2

[21] [35] [8] [8] [22]

J35]

903 Flavonol substituents

3, 5, 7, 3', 4'-(OH)

Trivial name

quercetin

3-(OH) 5,7,3',4'-(OMe) 3,5,7,3',4'-(OMe) 3,5, 7,4'- (OH) 6- (OMe) 3, 5, 3'-(OH) 7,4'-(OMe) 3, 5,3', 4'-(OH) 7-(OMe)

rhamnetin

3,5,3', 4'-(OH) 7-(OS) (115917) 3,5,4'-(OH) 7, 3'-(OMe)

rhamnazin

Cell type

Reference

Caco-2 HeLa HT-29 NCI-60 MMLV RT HTB-43 NK/Ly OVCAR-5 SK-MEL -28 OVCA-433 MDA-MB-435 COLO-320 DM HT-29 WiDr COLO-201 LS-174T MCF-7 SHEP WAC2 HuCC-Tl SCC-25 C0-K3 SK-Lul SW-900 ChaGo-K-1 H441 H661 A549 GCL4 COLO 320 HL-60 A549 B16 CCRF-HSB-2 TGBCllTKB Caco-2 NCI-60 KB MCF-7 HeLa HCT-15 HT-29 WA NCI-60 KB GCL4 COLO 320 NCI-60 NCI-60 NCI-60 HeLa KB NCI-60

[12] [6] [12] [21] [35] [31] [22] [36] [36] [37] [38] [39] [40] [40] [40] [40] [13] [13] [13] [41] [42] [43] [44] [44] [44] [44] [44] [44] [17] [17] [18] [23] [23] [23] [23] [12] [21] [11] [15] [6] [20] [9] [11] [21] [11] [17] [17] [21] [21] NCS-19802 [6] [11] NSC-678106

904 Flavonol substituents

Trivial name

3, 7, 3', 4', 5'-(OH)

robinetin

3,7,3',4',5'-(OMe) 3,3'-(OH) 5,6,7-(OMe) 5-(OH) 3,7,3',4'-(OMe) 5-(OH) 3,7,3',4'-(OMe) 5-(OH) 3,7,3',4'-(OAc) 5, 7-(OH) 3,4'-(OMe) 3'-(BnO) 5,7,3'-(OH) 3,4'-(OMe) 5, 7, 3', 4'-(OH) 3-(0Me) 5, 7,3', 4'-(OH) 3-(Az) 115918 5, 7, 3', 4'-(OH) 3-(Az) 9220

-

5,7,3',4'-(OH)3-(0-glu) 5,7,3',4'-(OH)3-(0-rutinose)

quercetin 3- glucoside rutin

5, 7,4'-(OH) 3,3'-(OMe) 5,3'-(OH) 3,7,4'-(OMe) 5,3'-(OH) 3,4'.(OMe) 7-(EtO) 5,3', 4'-(OH) 3,7-(OMe) 5,4'-(OH) 7,3'-(OMe) 3-(0-Ara-Glc)

-

3,3 '-Dimethylquercetin dimethylquercetin

-

5,4'-(OH) 7,3'-(OMe) 3-(0-Ara-Glc-Api) retamatrioside 7,3'-(OH) 3,6,4'-(OMe) 8,3'-(OH)7,4',5-(OMe)

4'-(OH) 3,5,7,3'-(OMe)

-

3, 5, 6, 7, 3', 4'-(OH)

quercetagetin

3,5-(OH)6,7,3',4'-(OMe)

-

3,5,7,3', 4'-(OH) 6-(0Me)

patuletin

3, 5, 7, 3', 4', 5'-(OH)

myricetin

3', 4', 5-(OH) 7-(MeO) 3-(Az) 19804 3'-(OH)7,8,4',5'-(OMe)

Cell type

Reference

TK-10 MCF-7 UACC-62 MMLV RT KB HeLa KB NCI-60 NCI-60 KB KB NCI-60 NCI-60 KB KB KB WA HCT-15 MMLV RT NKO^y Caco-2 HT-29 P-388 NCI-60 NCI-60 P-388 MCF-7 UACC-62 MCF-7 UACC.62 NCI-60 Col2 P-388 KB Col2 P-388 P-388 HeLa P-388 MMLV RT KB HeLa A-549 HT-29 KB MCF-7 P-388 GCL4 COLO 320 MMLVRT Caco-2 HCT-15 HT-29

[27] [27] [27] [35] [11] [6] [11] [21] [21] [11] [11] [21] [21] [11] [11] [11] [11] [20] [35] [22] [12] [12] [45] [21] [21] [45] [27] [27] [27] [27] [21] [29] [29] [11] [29] [29] [46] [46] [46] [35] [11] [6] [47] [47] [47] [47] [47] [17] [17] [35] [12] [20] [12]

905 Flavonol substituents

Trivial name

3,3'-(OH) 5, 6, 7, 4'-(OMe) 3,5, 3'-(OH) 6, 7,4'-(OMe) 5,6, 3', 4', 5'-(OH) 3 Az (19803) 5,3', 4', 5' (OH) 3- (O-rhamnose)

-

5,6,4'-(OH) 3, 7, 3'(OMe) 5, 7, 3'-(OH) 3, 6,4'-(OMe)

chrysosplenol C centaureidin

5,7-(OH) 6, 8, 3', 4'-(OMe)

hymenoxin

5,2'- (OH) 6, 7, 8, 6'- (OMe) 5, 2', 3'-(OH) 3, 7,4'-(OMe) 5, 3', 4'-(OH) 3, 6, 7-(OMe) 5,4'-(OH) 3, 6, 7, 3'-(OMe)

skullcapflavone II oxyayanin A chrysosplenol D chrysosplenol B

5,4'-(OH) 3, 7, 8, 3'-(OMe) 5,5'-(OH) 3, 7,2', 4'-(OMe) 6,3'-(OH) 3, 5, 7,4'-(OMe) 7,3'- (OH) 3,4'- (OMe) 5, 6- (Me) 3'- (OH) 3, 7,4'- (OMe) 5,6- (Me) 3, 5-(OH) 6, 7, 8,3', 4'-(OMe) 3-(OH) 5, 6, 7, 8, 3', 4'-(OMe)

-

3,5, 6, 7, 8, 3', 4-(OMe)

-

3,5, 3'-(OH) 6, 7, 8,4'-(OMe) 5-(OH) 3,6,7,^,3', 4'-(OMe) 5-(OH) 3, 6, 8, 3', 4', 5'-(OMe) 5, 7, 3', 4'-(OH) 3, 6, 8-(OMe) 5, 7, 4'-(OH) 3, 6, 8,3'-(OMe) 5, 3'-(OH) 3,6, 7, 8,4'-(OMe)

-

-

5, 3', 4'- (OH) 3, 6, 7, 8,3'- (OMe) 5,4'-(OH) 3, 6, 7, 8,3'-(OMe)

-

myricitrin

chrysosplenol G

-

natsudaidain

erianthin

Celi type

Reference

HeLa KB NCI-60 NCI-60 NCI-60 NCI-60 MMLV RT KB NCI-60 KB HeLa 229 HeLa S3 Hep-2 FL Chang liver Intestine 407 cell L1210 KB KB KB P-388 HT KB-Vl NCI-60 KB NCI-60 NCI-60 NCI-60 KB A-549 HL-60 B16 CCRF-HSB-2 TGBC-11-TKB B16 CCRF-HSB-2 HL-60 TGBC-11-TKB KB KB NCI-60 NCI-60 NCI-60 NCI-60 KB A-549 HCT-8 P-388 PRMI-7591 TE-671 KB NCI-60 KB

[6] [11] [21] [21] [11] NSC-19803 [35] [34] [21] [11] [48] [48] [48] [48] [48] [48] [49] [34] [34] [34] [50] [51] [51] [21] [34] [21] [21] [21] [52] [23] [18] [23] [23] [23] [23] [23] [18] [23] [52] [52] [21] [21] [21] [53] [53] [53] [53] [53] [53] [53] [52] [21]

J^

906 Flavonol substituents

5,7,3'- (OH) 3, 6, 7, 8,4', 5'-(0Me) 5, 7- (OH) 3, 5, ( 8, 3', 4', 5'- (OMe) 5, 3'-(OH) 3, 6, 7, 8,4', 5' - (OMe) 3,5,3'- (OH) 6, 7, 8,4', 5'- (OMe) 408168 408169 408170 408171

-

" -

Trivial name

-

digicitrin 3-0-demethyl-digicitrin

-

kaempherol 3-0-(2,6-diO-P-L-rhamnopyranosyl Kaempherol 3-0-PDapifuranosyl (1-2)- p quercetin 3', 4'- coniferyl sunphenon sylimarin

Cell type

Reference

PRMI-7591 P-388 TE-671 NCI-60 NCI-60 KB KB KB KB KB KB NCI-60

[53] [53] [53] [21] [21] [52] [52] [11] [11] [11] [11] NSC-641258

NCI-60

NSC-641259

NCI-60 HuCC-Tl DU145 A431 MDA-MB-468

NSC-648334 [41] [54] [54] [54]

Table 3. Cancer Cell Lines Inhibited by Flavanones. Flavanone substituents

Trivial name

Cell type

Reference

flavanone

HeLa Caco-2 HT-29 KB KB KB SA KB P-388 HT-29 A-549 CA KB KB KB BCl Lul Mel2 P-388 KB NCI-60 KB NCI-60 MDAMB-435

[6] [12] [12] [11] [11] [11] [11] [11] [25] [25] [25] [11] [11] [11] [29] [29] [29] [29] [29] [11] NSC-661206 [11] NSC-641526 [16]

3- (NOH) 3- (NH2(HC1)) 3- (NHSOaMe) 6- (OMe) 6-(CI) 7- (OAc) 3'-(CI) 6, 2'-(OMe) 8- (OMe) 6- (Me) 8, 5'-(OH)

3, 3- (OH) 2'- (OMe) 3- (OH) 5, 7- (OMe) 3-(OH) 3,2'-(OMe) 4'-(OH) 8,3'-(OMe) 5, 7,4'- (OH)

nanngemn

907 Flavanone substituents

Trivial name

5,4'-(OH) 7-(0Me) 5,4'- (OH) 7- (0-Man-Glc)

-

5,4'-(OH) 7-(OS) (5548) 5- (OH) 4'- (OMe) 7- (O-rut)

-

7,3', 4'-(OH) 4'- (OH) 7- (OMe) 6- (methyl-butenyl)

fustin bavachinin

3,3- (OH) 7, T- (OMe) 3,7,3'. 4'-(OH) 5,7-(OH) 6:7-(1, l-dimethyl-2-methyldihidrofurano)

-

5,7,3', 4'-(OH)

eriodictyol

5, 7, 3'-(OH) 4'-(OMe)

hespertin

5, 7,4'- (OH) 6- (Me) 5, 7,4'- (OH) 6- (Pren)

6-prenyl-naringenin

5, 7,4'-(OH) 3'-(OMe) 5, 5'-(OH) 4'-(OMe) 7-(O-Man-Glc)

homoeriodictyol hesperidin

naringin didymin

fustin

-

-

neohesperidin 7,3\4\5'-(OH)

dihydrorobinetin

Cell type

Reference

MCF-7 Caco-2 HT-29 HeLa KB Caco-2 HT-29 WA Caco-2 HT-29 HeLa Caco-2 HT-29 KB HeLa KB-VI

[15] [9] [9] [6] [11] [12] [12] [11] [12] [12] [6] [12] [12] [11] [6] [55]

[55] LNCaP Lu-1 [55] [55] ZR-75-1 NCI-60 NSC-649412 HeLa [6] [18] HL-60 [23] A549 B16 [23] CCRF[23] HSB-2 TGBCllT [23] KB MCF-7 [13] SHEP [13] [35] MMLV RT MCF-7 [13] Caco-2 [12] HeLa [6] [12] HT-29 MCF-7 [15] [16] MDAMB.435 KB [11] A-431 [55] BCl [55] Col2 [55] KB-Vl [55] Mel2 [55] U373 [55] ZR-75-1 [55] HeLa [6] Caco-2 [12] HT-29 [12] Caco-2 [12] HT-29 [12] HeLa

J2

908 Flavanone substituents

Trivial name

Cell type

Reference

7,4'- (OH) 5- (OMe) 8- (Pren)

isoxanthohumol

5'-(OH) 7, 3', 4'-(OMe)

-

3, 5, 7,3', 4'-(OH)

taxifolin (±)

MCF-7 HT-29 A-2780 KB KB-V Mel2 P-388 NCI-60 MMLV RT HL-60 HeLa TGBCllK B HTB43 glyosarco ma9L HTB43 MMLV RT MMLV RT HeLa NCI-60 NCI-60 KB HeLa KB NCI-60 KB A-431 HT Col2 LNCaP Mel2 U373 ZR-75-1 KB A-431

[56] [56] [56] [29] [29] [29] [29] NSC-36398 [35]

HT Col2 LNCaP Mel2 U373 ZR-75-1 BCl KB-Vl Lul KB KB-V Mel2 P-388 P-388

[55] [55] [55] [55] [55] [55] [55] [55] [55] [29] [29] [29] [29]

3, 5, 7, 3', 4'-(OH)

catechin (+)

3,5, 7, 3', 4'-(OH)

epicatechin (-)

3, 5, 7,3', 4'-(OH) 3, 7, 3', 4', 5'-(OH) 3,3'-(OH) 7, 2', 4'-(OMe) 5, 7,3', 4'-(OH) 6-(Pren)

5,7,3'-(OH) 4'-(0Me)PbCPX 61835 5, 7, 4'- (OH) 3'- (OMe) 6- (Pren)

8-(OH) 7, 3', 4', 5'-(OMe)

gallocatechin epigallocatechin

-

(+) pihydrorobinetin

-

6-prenyl-eriodictyoI

-

3 '-O-methyl-6-prenyleryodictyol

-

^

[18] [6] [23] [31] [31] [31] [35] [35] [6] NSC-674038 NSC-674039 [11] [6] [11] NSC-59266 [11] [55] [55] [55] [55] [55] [55] [55] [11] [55]

JE2]

909 Flavanone substituents

Trivial name

4'-(OH) 5,6,7,8,-(OMe) 5'-(OH) 7,8,3',4'-(OMe)

-

3, 5, 7, 3', 4'-(OH) 6-(C-Glc) 3, 5, 7, 3', 4', 5'-(OH) 3, 5,7,3', 4', 5'-(OH) 5, 7,3', 4'-(OH) 6,8-(Pren)

taxifolin-6C-glu epigallocatechin

5,7, 3', 4'- (OH) 8- (Pren) 6- (2-hydroxyprenyl)

-

5, 7,4'-(OH) 3'-(OMe) 6, 8-(Pren)

hiravanone

8,2'-(OH) 7,3',4',5'-(OMe)

-

2'-(OH) 7,8,3',4',5'-(OMe)

-

5-OH-rhamnosylglucosyloxyflavanone

onychin kurziflavonlactone B kurzifchalcolactone D

-

6,8-diprenyleriodictyol

Cell type

Reference

KB Col2 KB-V Mel2 KB KB KB-V Mel2 P-388 NCI-60 HeLa NCI-60 A-431 HT Col2 LNCaP Mel2 U373 ZR-75-1 BCl KB-Vl Lul KB BCl HT LNCaP Mel2 ZR-75-1 A-431 HT Col2 KB Mel2 U373 ZR-75-1 BCl KB-Vl Lul KB BCl Lul Mel2 P-388 KB BCl Lul Mel2 P-388 HT Col2 KB-V P-388 KB KB

[29] [29] [29] [29] [11] [29] [29] [29] [29] NSC-626436 [6] [21] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [55] [29] [29] [29] [29] [29] [29] [29] [29] [29] [29] [29] [29] [29] [57] [58]

1^8]

910 Flavanone substituents 2'", 3 ' " epoxylupinifolin

Trivial name

Cell type

Reference

dereticulatin lupinifolin

P-388 P-388 P-388 KB

[59] [59] [59]

61835

JUL

Table 4. Cancer Cell Lines Inhibited by Isoflavones. Isoflavone substituents 7-(OH) 7- (OMe) 6, 7- (AcO) 7,4'-(OH)

7,4'- (OMe) 4'- (OH) 7- (OGlc) 5, 7,4'- (OH) 5,7,4'- (OH)

Trivial name

daidzein

daidzin genistein

5,7- (OH) 4'- (OMe)

biochanin A

5,4'- (OH) 7-(0glc)

genistin

Cell type

Reference

ZR-75 ZR-75 NCI.60 ZR-75 HL-60 Caco-2 HT-29 ZR-75 UACC-62 KB NCI-60 HL-60 MOLT-4 HGC-27 WAC2 A-204 NBT-II ML-1 U937 K-562 M07E MCF-7 NIH 3T3 HepG2 Caco-2 HT-29 TK-10 UACC-62 NSCLC MDA-MB-231 PC-3 MAT-LyLu HN4 OVCAR-5 B-16 MDA-MB-435 MCF-7 Caco-2 HT-29 NCI-60 TK-10

[10] [10] NSC-600289 [10] [60] [12] [12] [10] [27] [11] NSC-36586 [61] [61] [62] [63] [63] [64] [65] [65] [66] [67] [68] [69] [70] [12] [12] [27] [27] [71] [71] [72] [73] [74] [75] [76] [16] [77] [12] [12] NSC-5112 [27]

911 Isoflavone substituents

Trivial name

Cell type

Reference

maxima isoflavone A maxima isoflavone C maxima isoflavone D maxima isoflavone G maxima isoflavone G methyl ether

MCF-7 UACC-62 Caco-2 HT-29 NCI-60 NCI-60 NCI-60 KB KB FV KB LL HCT-8 RPMI-7951 KB P388 NCI-60 MCF-7 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60

[27] [27] [12] [12] NSC-600285 NSC-600288 NSC-600290 [11] [11] [11] [11] [11] [78] [78] [78] [78] NSC-600291 [27] NSC-382026 NSC-382027 NSC-382028 NSC-382029 NSC-382030

6,7- (OH) 4'- (OMe) 6, 7- (AcO) 4- (Me) 6,7,4'- (AcO) 7- (OH) 2- (Me) 2'- (Br) 5, 6, 7,4'- (OMe) 5, 7- (OH) 4'- (OMe) 2- (COOEt) 7, 2', 4', 5'-(OMe)

7-(OH) 3', 4' - (OMe) 8- (Me) 6'-(OMe) 3' 4'- (-0CH20-) 7-OGlc

Table 5. Cancer Cell Lines Inhibited by Chalcones. Chalcone substituents

4- (OH) 4'- (OH) 4'.(R3)

2- (CI) 4'- (OH) 3, 5- (t-Bu) 3- (CI) 4'- (OH) 4- (N(CH3)2) 2'- (OMe) 4-(NHCOCH3) 3'-(C2H5) 4-(NHCOCH3) 3'-(CF3) 4- (Br) 4'- (OH)

Trivial name

Cell type

Reference

P-388 HeLa L-1210 P-388 L-1210 P-388 L-1210 P-388 L.1210 P-388 L-1210 NCI-60 P-388 L-1210 HeLa HeLa HeLa P-388 L-1210

[79] [80] [79] [79] [79] [79] [79] [79] [79] [79] [79] NSC-608002 [79] [79] [80] [80] [80] [79]

_I22

912 Chalcone substituents 4- (CI) 4'- (OH) 4-(Cl)4'-(R3) 4- (CF3) 4'- (OH) 4- (F) 4'- (OH) 4-(NHCOCH3) 4'-(CN) 4- (NHCOCH3) 4'- (CONH2) 4- (NHCOCH3) 4'- (OMe) 4- (NHCOCH3) 4'- (t-Bu) 4- (NO2) 4'- (OH) 4- (Me) 4'- (OH) 4- (OMe) 4'- (OH) 4-(OMe) 4'-(R3) 2', 4'- (OAc) 4'-(OH) 4-(R2) 4'- (Br) 4- (OH) 2- (OH) 4, 6- (OMe) 2,4- (CI) 4'- (OH) 2, 5- (F) 4'- (OH) 2,6- (CI) 4'. (OH) 3,4- (CI) 4'- (OH) 4- (OH) 3, 5- (CH2N(CH3)2) 4-(N(CH3)2) 2', 4'-(OMe) 4-(N(CH3)2) 2', 5'-(OMe) 4-(N(CH3)2) 2', 5'-(OMe) a-(Br) 4-(N(CH3)2) 2', 5'-(OMe) a-(CI) 4-(N(CH3)2) 2', 5'-(OMe) a-(CH3) 4-(N(CH3)2) 3', 4'-(OMe) 2', 3', 4'-(OMe) a- (CH3) 3', 4', 5'-(OMe) 3', 4', 5'-(OMe) |3-(CH3) 4'-(OH) 3',5'-(CH2N(CH3)2) 4'. (OH) 3',5'-(R,) 4', 5'-(CI) 4-(OH)

Trivial name

Cell type

Reference

P-388 L-1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 HeLa HeLa HeLa HeLa P-388 L-1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 P-388 A-459 HT-29 P-388 L-1210 P-388 L-1210 NCI-60 P-388 L-1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 L-1210 HeLa HeLa HeLa HeLa HeLa L.1210 B16 HeLa HeLa HeLa HeLa P-388 L-1210 P-388 L-1210 P-388 L-1210

[79] [79] [79] [79] [79] [79] [79] [79] [80] [80] [80] [80] [79] [79] [79] [79] [79] [79] [79] [79] [25] [25] [25] [79] [79] [79] [79] NSC-51351 [79] [79] [79] [79] [79] [79] [79] [79] [79] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [79] [79] [79] [79] [79] [79]

913 Chalcone substituents

Trivial name

2-(CI) 4 ' . (OH) 3',5'-(CH2N(CH3)2) 3 , 4 , 5 - ( O M e ) 4'-(N(CH3)2 3,4, 2 ' - ( O H ) 4 ' - ( 0 M e )

3-(NH2) 3 ' , 4 ' , 5 ' - ( 0 M e ) 3-(CI) 4 ' - ( O H ) 3',5'-(CH2N(CH3)2) 4-(N(CH3)2) 2 ' , 3 ' , 4 ' - ( O M e ) 4-(NHCOCH3) 2 ' , 3 ' , 4 ' - ( O M e ) 4-(N(CH3)2) 2 ' , 3 ' , 4 ' - ( O M e ) a - ( B r )

4-(N(CH3)2) 2 ' , 3 ' , 4 ' - ( O M e ) a-(CI) 4- (N(CH3)2) 2\ 3 ' , 4'- (OMe) a - (C2H5) 4- (N(CH3)2) 2 ' , 3 ' , 4 ' - (OMe) a- (CH3)

(NH2) 3 ' , 4 ' , 5'-(OMe) p-(Me) (N(CH3)2) 3 ' , 4 \ 5 ' - ( O M e ) a - ( B r ) (N(CH3)2) 3 ' , 4 ' , 5'-(OMe) a-(CI) - (N(CH3)2) 3 ' , 4 ' , 5'- (OMe) a- (CH3) 4 ' . (OMe) (N(C2H5)2) 2 (N(C2H5)2) 2 , 3 ' , 4'-(OMe) a-(CH3) (N(C2H5)2) 3 , 4 ' , 5'-(OMe) a-(CH3) (N(CH3)2) 2 ' 4 ' , 6'- (OMe) (Br) 3 ' , 4 ' , 5'-(OMe) (CF3) 3 ' , 4 ' , 5'-(OMe) (CN) 3 ' , 4 ' , 5'-(OMe) (NHCOCH3) 3 ' , 4 ' , 5'-(OMe) (NH2) 3 ' , 4 ' , 5'-(OMe) (NHC4H9) 3 ' , 4 ' , 5'-(OMe) (NHCO2CH2) 3 ' , 4 ' , 5'-(OMe) (N(CH3)2) 3 ' , 4 ' , 5'-(OMe) (N(C2H5)2) 3 ' , 4 ' , 5'-(OMe) (NO2) 3 \ 4 ' , 5 ' - ( O M e ) (OC2H5) 3 ' , 4 ' , 5'-(OMe) (R4) 3 ' , 4 ' , 5'-(OMe) (SCH3) 3 ' , 4 ' , 5'-(OMe) (t-Bu) 3 ' , 4 ' , 5'-(OMe) (OH) 3',5'-(CH2N(CH3)2) 4'-(Br) 4-(Br) 4 ' - ( O H ) 3 ' , 5'-(CH2N(CH3)2) 4-(CI) 4 ' - ( O H ) 3',5'-(CH2N(CH3)2) 4-(CF3) 4 ' - ( O H ) 3',5'-(CH2N(CH3)2) 4-(F) 4 ' - ( O H ) 3',5'-(CH2N(CH3)2) 4-(N(CH3)2) 4'-(OH) 3 ' , 5'-(OMe)

calythropsin

Cell type

Reference

P-388 L-1210 HeLa SK-MEL KG SK-OV-3 BT-549 NCI-60 HeLa P-388 L-1210 HeLa HeLa HeLa L-1210 B16 HeLa HeLa HeLa L-1210 B16 HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa P-388 L-1210 P.388 L.1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 HeLa

[79] [79] [80] [81] [81] [81] [81] [82] [80] [79] [79] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [80] [79] [79] [79] [79] [79] [79] [79] [79] [79] [79]

[80]

914 Chalcone substituents

Trivial name

Cell type

Reference

4- (NO2) 4'- (OH) 3', 5'- (CH2N(CH3)2)

-

4-(Me) 4'-(OH) 3', 5'-(CH2N(CH3)2)

-

4-(0Me) 4'-(OH) 3', 5'-(CH2N(CH3)2)

-

4,4'-(OH) 3',5'-(CH2N(CH3)2) 6-(CF3) 3',4',5'-(OMe)

-

P-388 L-1210 P-388 L-1210 P-388 L-1210 L-1210 HeLa P-388 L.1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 P-388 L-1210 MCF-7 HT-29 A-2780 MCF-7 HT-29 A-2780 P-388 L-1210 MCF-7 HT-29 A-2780 HeLa HeLa HT-29 A-549 NCI-60 MCF-7 HT-29 A-2780 MCF-7

[79] [79] [79] [79] [79] [79] [79] [80] [79] [79] [79] [79] [79] [79] [79] [79] [79] [79] [56] [56] [56] [56] [56] [56] [79] [79] [56] [56] [56] [80] [80] [25] [25] NSC-641522 [56] [56] [56] [56]

HT-29 A-2780

[56] [56]

2,4-(CI) 4'-(OH) 3',5'-(CH2N(CH3)2) 2,5-(F) 4'-(OH) 3',5'-(CH2N(CH3)2) 2,6-(CI) 4'-(OH) 3',5'-(CH2N(CH3)2) 2-(CI) 6-(F)4'-(OH) 3',5'-(CH2N(CH3)2) 3,4- (CI) 4'- (OH) 3', 5'- (CH2N(CH3)2) 4,2', 4', 6'-(OH) 3'-(Geran)

-

4,2', 4', 6'-(OH) 3'-(Pren)

-

4-(OH) 3, 5-(CH2N(CH3)2) 4', 5'-(CI)

-

4,2', 4'-(OH) 6'-(0Me) 3'-(Pren)

xanthohumol

6-(Me) 4-(N(CH3)2) 3',4', 5'-(0Me) 6-(Me) 4-(NH2) 3',4',5'-(OMe)

-

2'-(OH) 3,4,3'-(OMe) 5'-(Allyl)

-

dehydrocycloxanthohumol dehydrocycloxanthohumol hydrate

Table 6. Cancer Cell Lines Inhibited by Biflavonoids. Biflavonoid

Trivial name

Cell type

Reference

calycopterone

BCl HT Lul

[51] [51]

J5]I

915 Biflavonoid

Trivial name

-

4-demethylcalycopterone

6,8-Di-p-Hydroxybenzyltaxifolin

gericudrain A

8-p-Hydroxybenzyltaxifolin

gericudrain B

6-p-Hydroxybenzyltaxifolin

gericudrain C

-

isocalycopterone

robustaflavanone 4'-methyl ether

robustaflavanone 7,4'-dimethyl ether

2", 3 " - dihydrorobustaflavone 7,4'-dimethylcter

hinokiflavone

Cell type

Reference

Col2 SKOV-3 HT-29 KB KB-Vl P-388 A431 U373 BCl HT Lul Col2 KB KB-Vl P-388 A431 U373 CRL 1579 LOX-IMVI M0LT-4F KM12 UO-31 CRL 1579 LOX-IMVI M0LT-4F KM12 UO-31 CRL 1579 LOX-IMVI M0LT-4F KM12 UO-31 BCl HT Lul Col2 KB P-388 A431 U373 KB Raji Calu-1 K562 Vero Wish HeLa Raji Calu-1 K562 Vero Wish HeLa Raji

[51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [51] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [83] [51] [51] [51] [51] [51] [51] [51] [51] [28] [84] [84] [84] [84] [84] [84] [84] [84] [84] [84] [84] [84] [84]

916 Biflavonoid

Trivial name

2",3"-dihydrorobustaflavone 7,4', 7' -trimethyl ether

dimere chalcone-chalcone dimere chalcone-chalcone dimere chalcone-flavan 4',7"-Di-0methylmentoflavone

isocryptomerin

7' '-0-Methylrobustaflavone

Cell type

Reference

Calu-1 K562 Vero Wish HeLa Raji

[84] [84] [84] [84] [84] [84]

Calu-1 K562 Vero Wish HeLa L-1210 L-1210 L-1210 BCl

[84] [84] [84] [84] [84] [85] [85] [85] [86]

HT-1080 Lul Col2 KB KB-V+ KB-VLNCaP ZR-75-1 U373 L-1210 BCl HT-1080 Lul Col2 KB KB-V+ KB-VLNCaP ZR-75-1 U373 L-1210 BCl HT-1080 Lul Col2 KB KB-V+ KB-VLNCaP ZR-75-1 U373 L-1210

[86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86] [86]

917 Table 7. Cancer Cell Lines Inhibited by Other 1,3-Diphenylpropane Derivates. other 1,3-diphenyIpropane derivates

Trivial name

7,4'-dihidrowyflavan

Kazinol H

2', 6'- (OH) 4' (OMe) dihidrochalcone 4,2', 6'- (OH) 4' (OMe) dihidrochalcone cathechin 2', 6'- (OAc) 4,4' (OMe) dihidrochalcone 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1, 3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate 1,3-diphenylpropane-derivate Bartramia-triluteolin

-

kazinol Q

kazinol R kazinol D Bis(2,2-dimethyl-1,3-dioxane-4,6-dionate)flavona Bis(flavone-3, 5, 7-trihydroxy-2', 4' diolate) titani flavonlignan flavonlignan

kazinol K hydrocarpin hydnocarpin

Cell type HT3 SiHa CaSki P-388 HT-29 A-549 P-388 HT-29 A-549 HT-29 Caco-2 P-388 HT-29 A-549 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 NCI-60 PLC/PRF/5 T24 HT3 SiHa HT3 SiHa CaSki PLC/PRF/5 HT3 HT3 NCI-60 NCI-60 P-388 L-1210 Tmolt3 KB Adenocarcinoma Colon

Reference [87] [87] [87] [25] [25] [25] [25] [25] [25] [9] [9] [25] [25] [25] NSC-632573 NSC-632574 NSC-632576 NSC-634045 NSC-634049 NSC-635152 NSC-635894 NSC-635895 NSC-635898 NSC-635899 NSC-637208 NSC-635599 NSC-639600 NSC-647042 NSC-647043 NSC-647044 NSC-647045 NSC-647046 NSC-647048 NSC-683132 [87] [87] [87] [87] [87] [87] [87] [87] [87] [87] NSC-638846 NSC-638847 [24] [88] [88] [88] [88]

918 Other 1,3-diphenylpropane derivates

flavonlignan

Trivial name

hydnowightin

flavonlignan

Neohydnocarpin

flavonlignan 2-cyclohexil-5-hidroxychromone

-

2-cyclohexil-6-hidroxychromone

-

2-cyclohexil-7-hidroxychromone

-

2-cyclohexil-7-methoxychromone

-

sinaiticin

Cell type

Reference

HeLa-S3 Lung Osteosarcoma Glioma L-1210 Tmolt3 KB Adenocarcinoma Colon HeLa-S3 Lung Osteosarcoma Glioma L-1210 TmoltS KB Adenocarcinma Colon HeLa-S3 Lung Osteosarcoma Glioma P-388 HL-60 MOLT-4 HL-60 MOLT-4 HL-60 MOLT-4 HL-60

[88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [88] [24] [8] [8] [8] [8] [8] [8]

J2J

Table 8. Cancer Cell Lines Inhibited by Other 1,2-Diphenylpropane Derivates. Other 1,2-diphenylpropane derivates

Trivial name

Cell type

Reference

Trifolirhizin tetraacetate

P-388 HT-29 A-549 HL-60 RPMI-7951 KB RPMI-7951 A-549 HCT-8 TE671 KB P-388 RPMI-7951 A549 HCT-8 TE671

[25] [25] [25] [8] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78]

2- (OH) 8-(0Me) dihidroisoflavone Rotenoid

amorphispironone

Rotenoid

amorphigenin

rotenoid

12aP-0H-Amorphigenin

919 Other 1,2-diphenylpropane derivates

Trivial name

Rotenoid

dalphanol

Rotenoid

6'-0-D-P- glucopyranosyldalpanol

Rotenoid

l2a3-OH-dalpanol

Rotenoid

rotenone

Rotenoid

tephrosin

Cell type

Reference

KB P-388 RPMI-7951 A549 HCT-8 TE671 KB RPMI-7951 HCT-8 TE671 KB P-388 RPMI-7951 A549 HCT-8 TE671 KB P-388 BC-1 COL-2 HT-1080 LU-1 MEL-1 KB P-388 RPMI-7951 A549 HCT-8 TE671 KB P.388

[78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [78] [89] [89] [89] [89] [89] [89] [89] [78] [78] [78] [78] [78]

.jza

ABBREVIATIONS TO THE TABLES A-2780 A-431 A-549 BCl B16 melanoma A45 Caco-2 Calu-1 CaSki CCRF.HSB-2 ChaGo-K-1 C0-K3 COLO 201 COLO 320

Ovarian Cancer Human Epidermoid Carcinoma Human Non Small Cell Lung Cancer Breast carcinoma Melanin Pigment Producing Mouse Colon Cancer Human Lung Carcinoma Cell Line Human Cervical Carcinoma Human T-Cell Leukaemia Human Non Small Cell Lung Cancer Laryngeal Cancer Human Colon Cancer Human Colon Cancer

920

Colon 26 CRL 1579 DV145 H441 H661 HCT-8 HeLa Hep-2 HL-60 HT-3 HT-29 HTB43 Hucc-Tl KB KM12 K562 L.1210 LOX-IMVI LS-174T Lul MCF-7 MDA-MB-231 MDA-MB-435 MDA-MB-468 Mel2 MOLT-4 MOLT- 4F NCI 60 NK/Ly NSCLC OVCA433 OVCAR-5 P-388 P-815 Pan 03 PLC/PRF/5 Raji RPMI-7951 SCC-25

Murine Colon Adenocarcinoma Skin Human Tumor Cell Human Prostate Carcinoma Human Non Small Cell Lung Cancer Human Non Small Cell Lung Cancer Ileocecal Carcinoma Uterine Carcinoma Laryngeal Cancer Human Promyelocytic Leukaemia Human Cervical Carcinoma Colon Cancer Human Squamous Cell Carcinoma Human Cholangio-Cellular Carcinoma Human Nasopharyngeal Carcinoma Colon Human Tumor Cell. Erythroleukemia. Murine Lymph Leukaemia Skin Human Tumor Cell. Human Colon Cancer Lung Carcinoma Breast Cancer Breast Cancer Cells Breast Carcinoma Breast Carcinoma Melanoma Leukemia Leukemia Human Tumor Cell Panel of 60 Human Tumor Cell Lines Ascites Tumor H-460 Non Small Cell Lung Cancer Ovarian Cancer Human Ovarian Carcinoma Murine Lymph Leukaemia Leukemia Murine Pancreatic Adenocarcinoma Human Hepatoma -Ebv-Transformed B Cell Lines. Melanoma Oral Squamous Carcinoma

921

Siha SK-Lul SK-MEL SW900 TGBCllTKB T-24 TK-10 UACC-62 UCLANPA-87-l(NPA) UCLAR0-81A-1(AR0) UCLARO-82W-l(WRO) UO-31 Vero Widr Wish ZR-75 ZR.75-1

Human Cervical Carcinoma Human Non Small Cell Lung Cancer Melanoma Human Non Small Cell Lung Cancer Gastric Cancer Lymph Node Metastaded Human Hepatoma Renal Adenocarcinoma Melanoma Papillary Carcinoma Anaplastic Carcinoma Follicular Carcinoma Renal Human Tumor Cell. Green Monkey Kidney Tumor Cell Line. Human Colon Cancer Transformed Epithelial Cell Line. Breast Cancer Breast Cancer

STRUCTURE-ACTIVITY RELATIONSHIP Edwards et al. [11] studied several hundred flavonoids derivatives, natural and synthetic which had been tested in the screening program of the NCI, on KB cell line, and they found no structure-activity correlation general enough to serve as a working hypothesis for a rationale of the activity of these compounds. Kuntz et al. [12] studied 30 flavonoid on two human cancer cell lines and they found no obvious structure-activity relationship either on the basis of the subclasses or with respect to kind or position of substituens within a class. On the other hand, others authors have found some structural requirements for flavonoids that enhanced their cytotoxic activity on tumor cell lines, but sometimes, these conclusions seem to disagree with other reports. This limited understanding about a possible structure -activity relationship could possibly be, because of the few or not appropriated flavonoids used in their studies. So, throughout our present study, we have attempted to compile all authors' results and discussion found in the studied literature to obtain a global relationship either on basis of the subclasses of flavonoids or/and with respect to kind or position of substituents within a subclass.

922

Glycosides Although we have found some exception in which the glycoside is more active than the corresponding aglycone [17, 13, 12] we could say, in general, that the sugar moiety reduce cytotoxic activity of the aglycone on tumor cell lines [11, 17, 18, 23, 12, 27]. This suggests that the hydrophilic nature of sugars or the increased volume of glycosides could interfere with the drug entering through the cellular membrane [27]. 2-3 Double Bond This double bond differentiates between flavones (or flavonols) and flavanones, and different studies show that it enhances flavonoid activity [6, 18, 23, 13]. We can say that flavones are more active than flavanones but we could not generalize because we have also found two flavanones to be more active than the corresponding flavones [12]. But this fact could be explained because these two pairs of flavonoids are methoxylated in position 4' and Mori et al [6] said, when they studied their flavonoids, that when a B-ring was methoxilated the 2-3 double bond do not increase the activity. C4 Oxo Group The absence of a C4 oxo group is typical of catechins. We have found few studies that compare cytotoxicity of catechins with the corresponding flavonoids. Fotsis et al [13] show that catechins are less active and say that a C4 oxo group is required for maximal biological activity. Mori et al, [6] show one catechin less active than the corresponding flavonoid but, on the other hand, another catechin with similar activity. But, because of the very few number of cytotoxic catechins found in our review and comparing the large difference of different cancer cell lines inhibited by quercetin and the corresponding flavonoid, catechin, we could say, in general, that the C4 oxo group is very important to increase the flavonoid activity. Nevertheless, although catechins seem to produce low cytotoxic activity on cancer cell lines it does not means that they are not interesting as antitumor agents. In fact, the green tea extract GTETP91, whose major ingredients are catechin and its galates has been administered to adult patients with solid tumors as a Phase I study [5].

923

B-Ring at C3 Position B-ring at heterocyclic C3 position instead of C2 is characteristic of isoflavones. We have found very few studies that compare cytotoxicity of isoflavones with the corresponding flavonoids and we have found in one study that isoflavones are more active [12]. The low number of cytotoxic isoflavones compiled in our work, probably due to the relatively sparse distribution of isoflavonoids among plants, that are mainly in leguminous, does not signify that isoflavones are not as cytotoxic as flavones or other groups of flavonoids. In this way Li et al, [78] found a very cytotoxic isoflavone, and genistein is one of the most studied flavonoids with anticancer properties. In fact, two different antibody-genistein conjugates are currently in clinical development for the treatment of acute lymphoblastic leukaemia and breast cancer. Besides isoflavones, rotenoids are also isoflavonoids (flavonoids with a 1, 2-diphenylpropane skeleton) that have shown strong cytotoxicity against tumor cell lines [78]. C3 Substitution

This is a well studied position and differentiates between flavones (-R) and flavonols (-OR). Beutler et al [21] screened a series of seventy-nine flavonoids in a panel of sixty human cancer cell lines and the three most cytotoxic flavonoids had a -OMe at C3 position. Other results can confirm that flavonoids with -OMe at C3 are more active than with a OH substituent [11, 52] although we have also found the contrary mainly in some polimethoxilated flavones [52, 18, 23]. Other works show that flavonoids with -H at C3 are more cytotoxic than the corresponding one with -OH [17, 12, 13] although there are some exceptions [12]. Cushman and Nagarathnam [14] studied fifty-five flavones in five cancer cell cultures and said that is worth noting that substitution at the 3-position of the flavone with COOMe or COOH groups generally resulted in noncytotoxic compounds. The most cytotoxic flavone had a -H at C3 position (IC50 0.11|ag/mL on SK-MEL); but they did not test any flavonoid with -OH or -OMe substituent at this position. So, the results above could indicate, in a general way, the following order of cytotoxicity of C3 substituents:

924

OMe > H > OH > COOR Other studies show a series of 3-aminoflavones evaluated for cytotoxicity by Dauzonne et al [90] but it only shows moderate activities. Gonzalez de Peredo et al [30] also assayed nitro and chloronitro derivates at C3 position with similar results. C4' Position This position does not characterize any subclass of flavonoid but it is an important one. Results of cytotoxicity of the seventy-nine flavonoids screened by Beutler et al [21], that have mainly -H, -OH and -OMe at C4', show that 11 of the 12 most active flavonoids have a OMe group at C4'. Dauzonne et al [90] indicate that, in the 3-aminoflavone series, the 4' methoxy group is important for cytotoxic activity. However, we can find some flavonoids with -H or -OH more active than -OMe in C4' location [12]. Cushman and Nagarathnam [14] suggest the importance of the modifications at the C4' position to obtain new cytotoxic flavones and show one with 4'[(t-Butyldimethylsilyl)oxi] as the most cytotoxic in their series of 55 flavones. C5 Position We have compared the cytotoxic results of different pairs of flavonoids with the only difference being a -H or -OH group at C5 position and sometimes -H is better for higher cytotoxicity [10, 14, 13, 12] and sometimes it is better a -OH group at C5 [13, 21, 12, 27]. So, C5 position seems to be a confused one. But if we observe the results of Beutler et al [21] we can see that the six most cytotoxic flavonoids are hydroxilated in C5 position and in Cushman and Nagarathnam [14] we can also observe that all the four flavonoids with -OH group at C-5 are significantly cytotoxic in at least one tumor cell line. Besides, the four most widely investigated flavonoids as potential therapeutic agents, flavopiridol, genistein, green tea catechins and quercetin, [5] all have a -OH group at C5. Consequently, the hydroxylation of this position can be considered important for cytotoxic activity. When we continued studying the influence of different substituents in other positions we could not find any clear structure-activity relationship, probably because when you are studying one position one should, at the

925

same time, bear in mind the substitution of other positions. Therefore, we begun to study the interrelation of substituents in different positions. Interrelation of Substituents In A-Ring Different results show that the following association of substituents in Aring are good to increase cytotoxic activity of flavonoids: - 5- OH 6, 7, 8- OMe - 5- OH 6, 7- OMe - 5, 7- OH 6- OMe -Association dihydroxy: 5, 7- OH 7, 8- OH 6, 7- OH Accordingly, the most active flavonoid found in Beutler et aL [21], Ryu et aL [49] and Lichius et aL [52] has the association 5- OH 6, 7, 8OMe and its cytotoxicity values are, approximately, in the range of 0.1 |iM. Besides, four of the six most active compounds studied by Beutler et aL [21] have this substitution pattern and Lichius et aL [52] observed six rather active flavonoids with these substituents. Results by Zheng et aL [47] and Wall et aL [51] show that the association 5- OH 6, 7- OMe produces high cytotoxicity (< 0.5 \xM on several cancer cell lines). The fourth most active flavonoid in Beutler et aL [21] also has these substitutions. The highest cytotoxic flavonoid assayed by Woerdenbag et aL [17] and the second most active found in Beutler et a/. [21] (0.24 \xM) have the substitution pattern 5, 7- OH 6- OMe. The presence of two hydroxyl groups in A-ring in positions 5, 7; 6, 7 and 7, 8 also increase the cytotoxicity of a flavonoid. Thus, the most cytotoxic flavonoid studied by Dong et aL [45] and Hirano et aL [8] has 5, 7- OH, substitution pattern that is noted by Lee et aL [83]. In addition, the four most widely investigated flavonoids, flavopiridol, catechins, genistein and quercetin, present these two hydroxyl groups in their A-ring [5]. Finally, Mori et aL [6] obtained a flavonoid with 6, 7- OH in it's a-ring as the most cytotoxic and the most active in Cushman and Nagarathnam [14] show a 7, 8- OH substitution pattern in the same ring.

926

Interrelation of Substituents in B-Ring. Different results show that the following association of substituents in Bring are good to increase cytotoxic activity of flavonoids: -3'-OH,4'-OMe -3', 5'- OMe, 4'[(t-Butyldimethylsilyl)oxi] One of the most active associations in B-ring is -3'- OH, 4'- OMe. The four most activeflavonoidsin Beutler et ai [21] have this association of substituents and they say that the requirements for the B-ring may be quite stringent and when these substituents are reversed (-3'- OMe, 4'OH) cytotoxicity decreases notably. We can observe the same results in two flavonoids assayed on six tumour cell lines by Shi et ai [53]. The first one has -3'- OH, 4'- OMe in its B-ring and shows, for example, IC50 values of 0.045 and 0.055 \xM on KB and P-388 respectively and the second one, with the only difference of these substituents reversed, show IC50 values of 8.38 and 6.52 |iM on the same cell lines. Cushman and Nagarathnam [14], who studied 55 flavones in five cancer cell cultures, said that all five flavones with -3% 5'- OMe, 4'[(tbutyldimethylsilyI)oxi] were significantly active in at least one of the five cell lines and one of them was the most cytotoxic one. However they do not study any flavonoid with -3'- OH, 4'- OMe in its B-ring , so, we cannot compare these two kind of substitution pattern. An5^way, -3', 5'OMe, 4'[(t-Butyldimethylsilyl)oxi] is a substitution pattern that should be bom in mind. Chalcones They are flavonoids without the C-ring and are not frequently isolated from plants, possibly due to their instability. So, most of the chalcones presented in this review are synthetics [80, 79]. This kind of flavonoids, generally, are rather cytotoxic against human cancer cells. Thus, xantohumol, a prenylated chalcone from hops, inhibited the A-2780 cell line at a IC50 of 0.5 |iM [56] and two synthetic chalcones inhibited the HeLa cell line at IC50 of 0.0038 |Lig/mL [80].

927

Prenyl substituents We have found very few studies of flavonoids with prenyl (dimethylalyl) substituents and, although we have not found any report that compare the cytotoxic activity of a prenylated flavonoid with the corresponding one without this substituent, results show that prenyl groups can increase the cytotoxic activity. In this fashion, results of Seo et al. [55] show cytotoxicity of seven prenylated flavanones inhibiting eleven tumor cell lines in a range of 1-20 |ag/mL. Miranda et al, [56] assayed prenylated chalcones and flavanones on three cancer cell lines with IC50 in the range of 0.5-20 |iM. Li et al. [78] isolated two prenylated rotenoids that produce strong cytotoxicity against 6 human cancer cell lines with cytotoxicities in the range of 0.001-10 |ig/mL. GLOBAL STRUCTURE-ACTIVITY DISCUSSION The present structure-activity relationship discussion shows that we cannot generalise and say that a flavonoid with a determinate substituent will be more cytotoxic than the same flavonoid without it or with another one, because we have always found some exceptions, probably due to the different cell lines studied and the different mechanisms of action implicated in the cytotoxic activity of each cancer cell line. Besides, as the antitumor effect of different flavonoids is not always due to a common mechanism, when we are studying a substituent in a determined position, the presence of another one in positions that we are not taking into account, can totally change the mechanisms of action and consequently the cytotoxicity can also change. Anyway, the present work shows that there are some structural requisites that can increase the cytotoxic activity of a flavonoid on cancer cell lines and that some subclasses of flavonoid can be more active than others. Therefore, we dare to suggest possible cytotoxic flavonoids, that we have not found in the literature, and could be studied. It would be interesting to study aglycones with a 2-3 double bound and a 4 0x0 group, combining the following association of substituents: A-ring i- OH 6, 7, 8- OMe 5-OH 6,7-OMe 5, 7- OH 6- OMe 5, 7- OH

C-ring 3-OMe

B-ring 3'-OH, 4'-OMe 3', 5'- OMe, 4'[(t-Butyldimethylsilyl)oxi]

928 6, 7- OH 7, 8- OH

We have found only three of the twelve possible flavonoids resulting in the combination of substituents mentioned above and they were tested by Beutler et aL [21] in a panel of 60 cancer cell lines with high cytotoxic values: 0.13 |LIM for 5, 3'- OH 3, 6, 7, 8, 4'- OMe; 0.24 |iM for 5, 7, 3'OH 3, 6,4'- OMe and 1.7 ^M for 5, 7, 3'- OH 3,4'- OMe. Isoflavonoids are also compounds that should be bom in mind for future cytotoxic studies with flavonoids. Isoflavones (aglycones with a 23 double bound, a 4 oxo group and the B-ring at C3 position) with the association of substituents mentioned above are not studied and could be. Besides, rotenoids are very cytotoxic isoflavonoids that are not well studied although one of them, 12ap-hydroxyamorphigenin, is the most cytotoxic flavonoid found in our review, with values of less than 0.001 |ag/mL on the six human cancer cell lines tested by Li et al.[7S], CONCLUSION The present work compiles the available literature about flavonoids inhibiting cancer cell lines and studies which structural requisites are important to increase the cytotoxic activity of these kind of compounds, to obtain a working hypothesis to help to rationalize the development of flavonoids as antitumor agents. The limited understanding found in the literature about a possible structure-activity relationship is due, in our opinion, to that when the influence of a substituent in a determined position has been studied, the presence of other substituents in other position has not been taken into account. So, we have studied associations of substituents and, based on the studied literature, we have dared to propose possible cytotoxic flavonoids that we have not found in our review. Finally we suggest that isoflavonoids are little studied compounds that should be bom in mind for future antitumor studies with flavonoids. REFERENCES [1] [2] [3]

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Alhasan, S.A.; Pietrasczkiwicz, H.; Alonso, M.D.; Ensley, J.; Sarkar, F.H.; Nutr. Cancer, 1999, 34, 12-19. Shen, F.; Weber, G.; Oncol Res., 1997, 9, 597-602. Record, I.R.; Broadbent, J.L.; King, R.A.; Dreosti, I.E.; Head, R.J.; Tonkin, A.L.; Int. J. Cancer, 1997, 72, 860-864. Peterson, T.; Barnes, S.; Biochem. Biophys. Res. Commun., 1991, 179, 661667. Li, L.; Wang, H.K.; Chang, J.J.; McPhail, A.T.; McPhail, D.R.; Terada, H.; Konoshima, T.; Kokumai, M.; Kozuka, M.; Estes, J.R.; Lee, K.H.; J. Nat. Prod., 1993, 56, 690-698. Dimmock, J.R.; Kandepu, N.M.; Hetherington, M.; Quail, J.W.; Pugazhenthi, U.; Sudom, A.M.; Chamankhah, M.; Rose, P.; Pass, E.; Allen, T.M.; Halleran, S.; Szydlowski, J.; Mutus, B.; Tannous, M.; Manavathu, E.K.; Myers, T.G.; Clereq, E.D.; Balarini, J.; J. Med. Chem., 1998, 41, 1014-1026. Edwards, M.L.; Stemerick, D.M.; Sunkara, P.S.; J. Med. Chem., 1990, 33, 1948-1954. Ross, S.; Zagloul, A.; Nirod, A.; Mehmedic, Z.; El-Sohly, H.; Planta Med., 1999, 65,194. Beutler, J.A.; II, J.H.C.; Gray, G.N.; Prather, T.R.; Shoemarker, R.H.; Boyd, M.R.; Lin, CM.; Hamel, E.; Cragg, G.M.;^. Nat. Prod., 1993, 56, 1718-1722. Lee, I.K.; Kim, C.J.; Song, K.S.; Kim, H.M.; Yoo, I.D.; Phytochemistry, 1996, 41,213-216. Lin, L.; Kuo, Y.; Chou, C; J. Nat. Prod., 2000, 63, 627-630. Lewin, G.; Bert, M.; Dauguet, J.C; Dolley, J.; J. Nat. Prod., 1992, 55, 16791681. Silva, G.; Shai, H.; Grupta, M.; Famsworth, N.; Cordell, G.; Pezzuto, J.; Beecher, C; Kinghom, A.; Phytochemistry, 1995, 40, 129-134. Ko, H.H.; Yen, M.H.; Wu, R.R.; Won, S.J.; Lin, C.N.; J. Nat. Prod., 1999, 62, 164-166. Sharma, D.K.; Hall, I.H., J. Nat. Prod., 1991, 54, 1298-1302. Blasko, G.; Shieh, H.L.; Pezzuto, J.M.; Cordell, G.A.; J. Nat. Prod., 1989, 52, 1363-1366. Dauzonne, D.; FoUeas, B.; Martinez, L.; Chabot, G.G.; Eur. J. Med. Chem., 1997,32,71-82.

933 Abscisic acid 330 biological activity of 332 Astragalus miser \2ff. oblongifolius 519 in Lemna gibba growth 330 Astragalus miser var. serotinus 521 in stomatal closure 330 Abscisic acid 331,352 *H-NMR spectra of 339 activity of 331 structure of 321 utility of 331 Abscisic acid analogs 321 [3',5,7-^H6]ABA 353 Abscisic acid-binding protein 322 ABA Analogs of Arabidopsis thaliana 322 resistant to photoisomerization 354 Abscisic receptors 322 4-AboA 193,203,204,225 Absorption promotors 380 from Zea mays 193 A centhus mollis 191 formation of 204 Artemisia s^QCiQS 418 effect on Aspergillusflavus225 6a-Acetoxy-16P,23(R)-epoxy-24,25,26, effects on Fusarium culmorum 27-tetranorcycloartan-3-ol 451 225 from Astragalus tomentosus Abrisapogenol B 453 Lam. 451 from Astragalus shikokianus 453 2/?, 3i?-(+).3-Acetoxy-5,7,4'-trihydroxyAbrus fruticulosus 15 flavanone 34,35 abrusoside A from 15 structure of 35 abrusoside B from 15 2/?, 3i?-(+)-3-Acetoxy-5,7,4'-trihydroxyabrusoside C from 15 flavonone 17,34 abrusoside D from 15 from Aframomumhanburyi 17,34 Abrus precatorius 15,27 Acetoxy-5,7-dihydroxy-4'-methoxy abrusoside A from 15,27 flavanone 34,35 abrusoside B from 15,27 from Aframomum pruinosum 34 abrusoside C from 15,27 structure of 35 abrusoside D from 15,27 3-Acetoxy-5,7-dihydroxy-4 '-methoxy abrusoside E from 15,27 flavanone 17,34 Abrusoside A 15,27,28 from Aframomum hanburyi K. from Abrus fruticulosus 15 Schum. 17 from Abrus precatorius L. 15 8a-Acetoxy-6-'epi-tannunolide A 583 structure of 28 1 -Acetoxy-6a-hydroxy-germacran-1(10), Abrusoside B 15,27,28 3(4)-dien-8,12-olide 561 from Abrus fruticulosus 15 3 P-Acetoxy-9,19-cyclolanost-24£-eneftom Abrus precatorius 15 la,12p,16p,27-tetraol 448 structure of 28 from Astragalus mongholicus Abrusoside C 15,27,28 Bunge 448 ftom Abrus fruticulosus 15 12-Acetoxy-famesol acetate 586 ftom Abrus precatorius 15 6-Acetoxyldihydrochelerythrine 168 structure of 28 3 p-Acetoxymalabarican-14(26), 17E,21 Abrusoside D 15,27,28 triene 591 fxom Abrus fruticulosus 15 8a-Acetoxy-tannunolide E 585 from Abrus precatorius 15 2-Acetylamino-3//-phenoxazin-3-one 219 structure of 28 as product of Pseudomonas Abrusoside E 15,27,28 iodina 219 frora Abrus precatorius 15 as product of Waksmania structure of 28 SUBJECT INDEX

934 aerata 219 structure of 219 5-Acetyl-DIBOA 263,204 synthesis of 204 Acetylenic derivatives 659 S-N-Acetylneuraminic acid 104 Acetyltrimethylammonium bromide 509 Acicerone 498 Activation energies ofisomerization 351 Activators 833 from Daphne cannabina 833 from Daphne mezereum 833 fromThymelaceouspldints 833 from Wikstroemia indica 833 Activities 323,333,504,513 for Astragalus spp. 483 from Phyllanthus amarus 840 of Astragali radix 513 of abscisic acid analogs 323-329 ofdiarylheptanoids 852 of enzymes 378 of flavonoids 504 of fluorinated probes 333 of hydrolysable tannins 840 of monoterpenes 384 of odour substances 378 of phosphorylase kinase 852 of protein kinases 826 of serine 850 of threonine 850 of tyrosine Acute toxicity 307 ofstevioside 307 ofsteviol 307 Acylatedflavonolglycosides 478 from Astragalus complanatus 478 Acylneuraminic acids 104 Adaptation 529 of plants to selenium 529 Adenosine 394 from glucose 394 structure of 396 from red ginseng roots 394 Adenosine-5'-diphosphate (ADP) 405 Adenosine-5'-triphosphate (ATP) 820 ADI (acceptable daily intake) 308 for stevioside 308

P2-Adrenergic receptors 825 Aerial parts 665 of Bupleurum rigidum 665 aqueous extract root of Astragalus membranaceus 665 Aframomum hanburyi K. Schum. 17,34 3-acetoxy-5,7-dihydroxy-4'methoxyflavnone from 17,34 Afrormosin isoflavones 498,499 Aglycones 929 Agricultural application 321 of abscisic acid 321 Agroastragaloside I 454 from Astragalus membranaceus Bunge 454 Agropyron repens 191,216 AH-b-X model 10 Ailanthoidine 167 from Zanthoxylum ailanthoides 167 Ajmalicine 780 from Catharanthus roseus 780 Alcoholic extract 476 of Astragalus alexandrinus A16 Alexandroside I 454 f^om. Astragalus alexandrinus Boiss 454 Alkaline hydrolysis 671 Alkaloids 880 from Rollinia ulei 880 Alkyl esters 334 of Abscisic acid 334 Allelopathic interactions 214-218 of Agropyron repens L. 216 of Avena fatua L. 215 of Avena sativa 216 of Avena strigosa L. 215 of Lactuca sativa L. 215 of Secale cereale L. 125,216 of Taraxacum officinale Wigg. 215 of Triticum aestivum L. 215 of Zea mays 215 AUergenicity problems 315 of Stevia 315 ofstevioside 315 AUocryptopine 162 from Chelidonium majus 162

935 Altemosides I-V 41,44,45 as sweetness inhibitors 41 from Gymnema sylvestre 41 structures of 41,45 Amantadine 108 for treatment of influenza A 108 infection 108 Ames test 303,304 3-Aminoflavones 926 for cytotoxicity 926 a-Amylase 330,331 in aleurone protoplasts 331 P-Amyrin 477 from Astragalus complanatus All from Astragalus glycyphyllos 477 a-Amyrin 589 P-Amyrin 589 a-Amyrin acetate 589 Anagyrine 276 structure of 276 Analgesic 660 Analysis 365 by gas chromatography 365 ofchiralmonoterpenoids 365,368 of phaseic acid 351 Anderson & Bridgeman study 511 of proteinaceous polysaccharides 511 from Astragalus microcephalus 511 from Astragalus gummifer 511 from Astragalus kurdicus 511 trans Andholt 16,33 from Foeniculum vulgare Mill 16 from Illicium verum Hook f 16 from Myrrhis odorata Scop. 16 from Osmorhiza longistylis DC 16 from Piper marginatum Jacq. 16 hom TagetesfilicifoliaLdig. 16 structure of 33 Anethum graveolens L. 379 Angelica decursiva 835 Angelica gigas 835 8a-Angeloyl oxyestafiatin 577 Angeloylajadin 575 Angeloylcumambrin 577 8a-Angeloyloxy-ip-peroxy costunolide

560 8a-Angeloyloxycostunolide 559 Anhydroverlotorin-4a,5P-epoxide 572 a-3',4'-Anhydrovmblastine 780-783 a-Anhydrovinblastine 782 Anomeric configurations 469,667 inaglycone 667 Antagonist-[Arg6]-Sp 799 Anthocyanidins 893 Anthracenone abscisic acid analog 331 Anthraquinones 849 Anti-neoplasmatic activity 378 Antibacterial peptides 806 Antibody dependent cellular cytotoxicity (ADCC) 803 Anticancer activity 807 ofmagainin-2 807 Anticancer peptide 799 Arg-D-Trp-NmetPhe-D-TrpMet-NH2 799 Antifungal properties 381 Antihepatotoxic activities 660 Antiinflammatory activities 660,858 Antimicrobial activity 807 Antimicrobial properties 378 Anti-neoplasmic activity 386 Anti-obesity action 398,400,402,403 of natural products 398 of soyasaponins 398 ofodongtea 400 of chitosan 402,403 AntiproUferative effect 802,862 Antitumor activity 803 Antitumor effect 439 of carp extract 439 Antitumor mechanism 831 of guidimacrin 831 Antiviral activity 482 for Astragalus membranaceus 482 Aphelandra tetragona 191,193 Apiaceae family 661,686 Apium sp. 686 Falcaria sp, 686 fromHederasp. 686 from Schefflera sp. 686 Panax sp. 686

936 Apioglycyrrhizin 15,29,30 from Glycyrrhiza inflata Batal 15,29 Apioideae family 661 Apoptosis 86-88 relation with lipid peroxidation 86-88 Application of abscisic acid 322 of cholelithiasis 382 ofcyclodextrinsinGC 368 Aqueous extract 313,508 root of Astragalus memhranaceus 508 of Stevia 313 Araborglycyrrhizin 15,29,30 from Glycyrrhiza inflata 15 Arachidonic acid 422 from phospholipids 422 Arachidonic acid metabolism 693 Arachniodes exilis 18,35 Arachniodes sporadosora 18,35 Araliaceae family 686 ArbusculinA 549 Arglanilic acid methyl ester 557 Arglanine 549 Armefolin 550 Armexine 550 Aromatization 782 of A^-heterocyclic ring 782 Artabin 558 Artabsin 575 Artecalin 550 Artecanin 575 ArteglasinA 575 Artemisia arborescens 376 Artemisia genus 376 Artemisia herba alba 376 Artemisia judaica 376 Artemisia montana 418 Artemisia princeps 418 Artemorin 558 Artemorin-4a,5P-epoxide 558 Ascorbic acid 747 Altemaria altemata fungus 793 PiSidXic Astragalus spY^. 511

Askendoside A-F 454 from Astragalus tashkendicus Bimge 454 Aspergillusflavus 382 Aspergillus niger 382 Aspergillus ochraceus 382 Aspergillus parasiticus 382 Astilbe japonica sp. 395 Astilbe thunbergi sp. 395 Astilbe revularis var. rivularis 395 Astilbe chinensis species 395 Astilbin 395 from Astilbe thunbergii 395 structure of 396,397 Astragalus glycyphyllos 444,453 Astracicerans 256 498 Astragali semen 478 Astragali radix 443,445 Astragaluquinone 505 Astragalus L. genw5 443 Astragalus species 502 biological activity of 443 metabolites from 443-525 phenolic compounds from 483 structure elucidation of metabolites from 467 Astragalus gummifer 444 Astragalus canadensis L. 444,522 Astragalus caryocarpus 444 Astragalus complanatus 445,453 Astragalus edulis 444 Astragalus membranaceous 443,445, 467,468,511,446 Astragalus mollissimus Torr. 444 Astragalus sieberi 470 Astragalus spinosus 468 Astragalus aitosensis 449,511 Astragalus alexandrinus 454 Astragalus alopecuros 449,452 Astragalus bisulcatus 516,527,528 Astragalus brachypterus 454 Astragalus cephalotes 454 Astragalus cibarius 522 Astragalus collinus 522 Astragalus convallarius 525 Astragalus crotolariae 528 Astragalus cycloartane 472 Astragalus dissectus 440

937 Astragalus ephemerotum 449 Astragalus falcatus 511 Astragalusflexuosus 522 Astragalusfloccosifolius 504 Astragalus galegiformis 450,470 Astragalus kuhitangi 455 Astragalus kulabensis 449 Astragalus L. 443,445 Astragalus lasioglottis 505 Astragalus lentiginosus 515,524 Astragalus lusitanicus 524 Astragalus melanophrurius 455,482 Astragalus miser 525 Astragalus mollissimus 516,444,524 Astragalus mongholicus 448,467,508,511 Astragalus oleifolius 448 Astragalus onohrychis 453,466,504,480 Astragalus orbiculatus 452 'l-hydroxyindolizidinefrom 515 1,2-dihydroxyindolizidine from 515 Astragalus pamirensis 449 Astragalus pattersonii 528 Astragalus pectinatus 528 Astragalus polycanthus 517 Astragalus praelongus 516 Astragalus pterocarpus 525 Astragalus pterocephalus 449 Astragalus pycnanthus 450 Astragalus quisqualis 450,504 Astragalus racemosus 528 Astragalus root 445 medicinal use of 445 Astragalus sapogenins 447 Astragalus saponins 472 i^j-rragfl/w^ seeds 445 medicinal use of 445 Astragalus severtzovii 478 Astragalus shikokianus 453 Astragalus siculus 482 Astragalus sieversianus 449,478 Astragalus sinicus 453,511 Astragalus spinosus 483 Astragalus spp. 519,446 of Leguminosae 519 Astragalus subrobustus 504 Astragalus tibetanus 517 Astragalus toanus 516

Astragalus tomentosus 451 Astragalus tragacantha 454 Astragalus trigonus 448 Astragalus trojanus 454 Astragalus wootoni 516 Astragenol 449 ftova Astragalus aitosensis M.B 449 Astraglozid 483 from Astragalus sieversienus 483 Astramembrannin I 480,481 from Astragalus membranaceus 481 Astrasieversianin XI 480,481 fromy45^^aga/w5 sieversienus Atrial natriuretic peptide 870 AurisideAandB 789 Aurones 893 Avenafatua 215-217,218 Avenasativa 216-218 Avicine 156 fagaronine 156 from Zanthoxylum avicennae DC. 159 sanguilutine 156-158,161,162 terihanine 156 1-Azido-abscisic acid 330,331 Bupleurum rigidum 664 Bupleurum scorzonerifolium 675 Baccharis gaudichaudiana 14,24,25 gaudichandioside A from 14, 24,25 Bacillus macerans 8 Bacterial peroxidases 736 Bacterial reverse mutation test 303 Bactericidal/permeability-increasing protein 807 Badlejasaponin IV 693 from Bupleurum fruticosum 693 Baeyer-Villiger rearrangement 74 Baiyunoside 14,25 from Phlomis betonicoides 14 structure of 15 Balanol(44) 868,869 Balsamiton 591 Barosma betuling 375 Barosma crenulata 375

938 Botrytis cinerea 768 Benzodiazepine 803 Benzo[c]phenanthridine alkaloids (QBA) 819 biological activities of 178 chemistry of 155 Benzophenone 331 Benzoxazinoid acetal glucosides 191 -194 chemical synthesis of 196,207-210 enzymatic & chemical degradation of 191-196 Benzoxazinone acetal glucosides 187-190 Blepharin 188 DHBOA-Glc 188 DIBOA-Glc 188 DIM2BOA-GIC 188 DIMBOA-Glc 188 GDHBOA 188 GDIMBOA 188 GHBOA 188 GHDMBOA 188 GHM2BOA 188 GHMBOA 188 GIM2BOA 188 HBOA-Glc 188 HDMBOA-Glc 188 HM2BOA-GIC 188 HMBOA-Glc 188 occurrence of 187-190 Benzoxazinone acetal glucosides 224,225 biological role of 224,225 Benzoxazinoids 211 biological activity of 211 Benzoxazinones 185-225 biological activity of 185-225 biological role of 224 defoxification of 218-222 effects on animal organisms 212-214 effects on plant organisms 214-224 in plants 185-225 synthetic access to 185-225 Benzoxazione aglucones 190-191,195, 196-207 as equilibrium of enantiomeric cyclohemiacetals 194 Blepharigenin 191

chemical synthesis of 196-205 DHBOA 191 DffiOA 191,195,196 DIM2BOA 191 DIMBOA 191,195,196 HBoA 191 HDMBoA 191 HMBoA 191 synthesis of analogues 205-207 TRBoA 191 Benzoxazolin-2(3//)-ones 193 4-ABOA 193 BOA 193 4-Cl-DMBOA 193 5-Cl-DMBOA 193 DMBoA 193 MBOA 193 Benzyloxyisoflavone 499 4-(9-Benzylresacetophenone 499 Beogradolide A 550 Beogradolide B 550 Berberine alkaloid 162 in Chelidonium majus 162 Bergenin 395 Betacyanin alkaloid 745 Bezoxazolinone-detoxification 218-222 by fungi 218-222 by plant 218-222 by microbes 218-222 Bicyclic lupine alkaloids 267 Bicyclogermacrene 573 Bidesmosidic 9 675 Bidesmosidic saponin 671 sandrosaponin 671 Bidesmosidic saponins 664 Binding pocket 33 Bioactive compounds 659 of Bupleurum rigidum L. subsp. rigidum 659 Bioactivity 348 of 8'-hydroxyabscisicacid 349 Bioavailability 312,314 of nutrients 312,314 Biochemical basis 820 of protein phosphorylation 820 Biochemical studies 393 of natural products 393

939 Biogenesis 515 of swainsonine 515 Biological activities 377,431,421,693, 778,801,924 acetyl-N-Ser-Asp-Lys-Pro 806 Artemisiae herha 413 baicalein 433 balanol 869 bombesin 797 flavonoids 431 from Angelica shikokiana 429 from foodstuffs 393 from licorice 425 from medicinal plants 393,405 from Scutellariae radix 431 from the roots of Polygonum sp. 405 in black-sugar 409,410 in Bupleurum rigidum 693 in fat cell 393 in red wine 430 of(+)-caffeicacid 421 of (+)-chlorogenicacid 421 of Artemisia montana extracts 420 of baicalein 435 of bombsin 797 of chitosan 404 of dynorphins 801 of endorphins 801 of endotoxin-induced 435 of enkephalins 801 of flavonoids 425 of free fatty acid 404 of Geranii herba 415 of ginsenosideRgi 424 of green tea 416 of green tea 417,420 of hyperlipdaemia 405 of lipogenesis 393 of lipolysis 393 ofmonoterpenoidenantiomers 377 of natural products 393,405 of non-sugar fraction 409,410 of oligomeric compound 778 of Oolong tea 401 of plasma glucose 412 of pyranocoimiarin 429 of resveratrol 430

of saikosaponins 693 of serum TC 420 of serum transaminase 415,417, 420 of serum transaminases 421 of serum triglyceride 404 of soyasaponins 399 of stilbene derivatives 405,409 of tannin 413,416 of P-glucosidases 778 opioid peptides 801 saponin 859 stilbene glucoside 408 structure of 424,425 structure of 426 of total cholesterol 404 of Geranii herba 413 Biophysical studies 334 of abscisicacid 334 Biosynthesis and signal transduction 321 from abscisic aldehyde 321 from zeaxanthin 321 Biotransformation rates 377 Bioavailability of nutrients 312-314 Bipolaris zeicola 498 Bisabolanes 13 highly sweet compound from 13 from Lippa dulcis 13 Bisquinolizidine alkaloid 264 ^^C-NMRof 264 ^H-NMRof 264 Blattela germanica L. 384 Blepharigenin 191,206 synthesis of aza analogues of 207 Blepharin 188,208,209 diastereoselective synthesis of 208 from Blepharis edulis 188 from Zea mays 188 Blepharis edulis 191,193 Blossom oil 373 BoA 193,218-222 BoA-6-OH 220,221 BoA-6-O-P-D-glucoside 220,221 BoA-N-p-D-glucoside 220,221 degradation by Acinetobacter calcoacetius 218 Q^tcX on Avenasativa 218

940 effect on Triticum aestivum 218 Buddlejasaponin 672,677,688 effect on ViciafabaL, 218 Bupleurum bourgeaei 662 from Aphelandra tetragona 193 Bupleurum chinense 660 from Blepharis edulis 193 Bupleurum falcatum L. 660 from Secale cereale 193 Bupleurum gibraltaricum 660,661 from Zea mays 193 Bupleurum kaoi 660 Boat-boat inversion potential 343 Bupleurum marginatum 660 of 1,4-cyclohexadiene 343 Bupleurum rigidum L. 659 Boccon ia arborea 163,170 bioactive components of 659 Bocconia frutescens 163 biological activity of 688 Bocconia germs 162 Bupleurum rotundifolium 672 Bocconia integrifolia 163 Bupleurum scorzonerifolium 660 Bocconia pearcei Hutch. 163 Bupleurum genus 687 Bocconine 162 Bupleurum bourgeaei spp. 662 Bupleurum chinense 660 from Macleaya cordata 162 Bupleurum rigidum 662,663,665 Bocconoline 168 Bombesin analog 797 roots of 665 from emphibianskin 797 structure of 662 (+)-Bomeol 376 6-0-Butanoyl castanospermine 257 structure of 376 Herpes simplex 257 Botanical / ecological description 661 Bovine thymus 854 C-3 substitution 925 Bowlesia 661 offlavones 925 BrachysoideA 454 offlavonols 925 from Astragalus brachypterus 454 C4 0xo group 924 Bradykinin 800 ofcatechins 924 Brain natriuretic peptide 870 Caesalpinia echinata Lam. 37 Brazzein 18,39 Caffeoylquinic acid 419 from Pentadiplandra brazzeana Caged ABA 331,332 18 Cajanin 498 BrefeldinA(47) 871 Catharanthus roseus 780,782 Bromelain 872 Calcitonin 800 from Ananas comosus 872 Calliphora 384 3'-Bromo-ABA 353 Calmodulin kinase III 854 Bryodulcoside 14 Calycocystis Bunge 444 from Bryonia dioica 14 Calycophysa Bunge 444 Bryonia dioica 14,25 Calycosin 498,499 bryodulcoside from 14 CalyculinA(49) 877,878 bryonoside from 14 as okadaic acid 877 bryoside from 14 from Discodermia calyx Bryonoside 14,25,26 sponge 877 from Bryonia dioica 14,25 CAMP-dependent protein kinase 821, 822,823 structure of 26 Bryophyta 735 Camphor 376 Bryoside 14,25,26 Cancer cell lines inhibition from Bryonia dioica 14,25 bybiflavonoids 916-918 bychalcones 913-916 structure of 26 byflavanones 908-912 Bryostatin 832

941 by flavones 897,898,899-903 byflavonols 903-908 by isoflavones 912-913 by 1,2-diphhenylpropane derivatives 920-921 by 1,3-diphenylpropane derivatives 919-920 10-ep/-Canin 576 Canin-8a-isovalerate 576 Canin-8a-methyl butyrate 576 Cantharidin 882 from Cantharis vesicatoria 882 from. Mylabris cichorii 882 from Mylabris phalerata 882 Capparis masaikai Levi. 18 mabinlin from 18 CaprinusB\m%Q 444 Capsaicin 747,749 Capsicum fruits 749 6-Carboxydihydrochelerythrine 167 4'-Carboxyl group 330 for photoaffinity 330 1-Carboxyl group 354 for abscisic acid bioactivity 354 Carboxylic acid 35,36 from Arachniodes exilis 35 from Arachiniodes sporadosora 35 Carcinogenic study 306 of stevioside 306 Carcinogenicity 302 Carcinoid cells 797 Carcinoma cells 844 Coriandrum sativum Caries 316 Camosifloside V 14,25,26 from Hemsleya camosiflora 14 structure of 26 Camosifloside VI 14,25 frora Hemsleya camosiflora 14 structure of 26 Carotatoxin 686 from Daucus carota 686 (-)-Carvone 364 (+)-Carvone 364 Casomorphin 803 Castanosperimum australe 514

Castanospermine inhibitor 525 ofa,P,-glucosidases 525 Castanospermine 255 from Alexa leiopetala 255 from Castanospermum australe 255 (5-e/?/-Castanospermine 517 Castanospermine 253 activity against human immunodeficiency virus 257 as anti-hyperglycemic agent 257 as an anti-inflammatory agent 257 6-0-butanoyl castanospermine 257 biological activities of 255,256 enantiomers of 255 fiom Alexa leiopetala 255 from Castanospermum australe 255 glycosidase inhibition by 256 Castenea gtnQrdi 755 Catabolic enzyme 322 of abscisic acid 322 Catabolic inactivation 354 by conjugation 354 Catalytic cycle 738 of class III peroxidase 738 structure of 739 (+)-Catechin 416,746,893 (+)-Catechin oligomers 774 Catecholamine-like substances 395 of Astilbe thunbergii 395 structure of 396 Catharanthine 1A1J%1 Catharanthus roseus 782 catharanthine from 783 vindoline from 783 Caymandimerine 170 from Zanthoxylum spinosum 170 Cecropin-B 806,807 Cellular toxicity of okadaic acid 876 Cephalotoside A 454 from Astragalus cephalotes 454 from Astragalus mongholicus 454 Cercidothrix BungQ 444 Cemilton 212 in treatment of benign prostatic hyperplasia 212

942 CH3SOCH2- 512 from dimethylsulphoxide 512 Chaetomium sp. 382 Chalcones 745,928 Chelelactam 167 Chelerythrine 158,163,164,172,863 biological activities of 178,179 from Bocconia arborea 170 from Chelidonium majus 158 from Fagara species 164 from Toddalia 164 from Zanthoxylum simulans 863 from Za«r/ioxy/MW species 164 in Corydalis caucasica DC. 163 in Corydalis severtzowii Rg. 163 pK constant of 171,172 Chelibubine 163 in Dicentra spectabilis 163 from Papaver oreophilum 163 from Papaver radicatum 163 Chelidimerine 169,170 from Chelidonium majus 168,169 in Corydalis flabellata 169 in Corydalis rutifolia 170 Chelidonine 162,164 in Chelidonium majus 162 Chelidonium majus 155,158,159,162, 168,169 Chelilutine 156-158,161,162,158,163,172 lO-hydroxysanguinarine 156,157 lO-hydroxychelerythrine 156,157 12-hydroxychelirubine 156,157 biological activities of 178,179 chemical reactivity of 166 from Bocconia frutescens L. 163 from Bocconia pearcei 163 from Chelidonium majus 158 from Dicentra spectabilis 163 from Dicranostigma franchetianum 162 from Dicranostigma leptopodum 162 from Fumariaceae species 158 from Mflc/eflya species 162 from Papaveraceae species 158 in Chelidonieae 161 vnHypecoideae 161 in Papaveroideae 161

in Ranunculaceae family 161,164 isolation of 164-166 Chemical degradation 846 oflow density lipoprotein 846 Chemical properties 336 of cyclopropane 336 Chemical properties 507 of polysaccahride F-8 507 Chemical versatility of peroxidase-catalyzed reaction 749 Chemopreventive efficacy 386 of limonene carcinogenesis 386 Chemotaxonomy 369 Chemotherapy drug 436 antitumor activity of 436 Chichipegenin 44,45 structure of 45 Chiliophyllin 559 Chiral monoterpenes 372 3-carene 372 Chiral monoterpenoids antibacterial activity of 381 antiftmgal activity of 381 anti-neoplasmatic activity of 386 bioactivity of 361,377 enantioselective analysis of 361 in plants 361 isomerism of 361 Chitin 402 from shell of crabs 402 Chitosan 402 structure of 403 5-Chloro-6-methoxy-2-benzoxazolinone 217 effect on Amaranthus caudatus L. 217 effect on Avena sativa 217 effect on Digitaria sanguinalis (L.) Scop. 217 effect on Lactuca sativa 111 effect on Lepidium sativum 111 effect on Lolium multiflorum Lam. 217 effect on Phleum pratense L. 211 3' -Chloro-abscisic acid 353 /?-Chloromercuribenzoate 736 Chlorophyta 735

943 Cholecystokinin effect 799,800 Cholelithiasis 378 Cholelithiasis therapy 378,382 Chromosome exchange 304 Chromosome mutation test 303 Chronic experiment 313 Chronic toxicity studies 308 Chrysamin 551 Chrysanolide 559 Chrysanthemum parthenium 376 Chrysentunone 592 Cibarian 522 Cimicifuga racemosa 111 Cimifuga species 395 Cinerinl 588 Cinerinll 588 /ran5-Cinnamaldehyde 16,33 from Cinnamomum osmopholeum structure of 33 Cinnamomum osmopholeum 16 /ran^-cinnamaldehyde from 16 Cinnamomum sieboldii Meisner 18,35 CinnamtanninB-1 from 18,35 Cinnamtannin D-1 from 18,35 Cinnamonum camphora 377 Cinnamonum zeylanicum 377 Cinnamtannin 1 18,35,36 from Cinnamomum sieboldii Meisner 18,35 structure of 36 Cinnamtannin D-1 18,35,36 from Cinnamomum sieboldii 18,35 structure of 36 Cinnamyl alcohol dehydrogenase 763 Citrus auranhtium ssp. amara 372-375,377 petitgrain oil from 372-375,377 Citrus aurantium 11,311 neohesperidin dihydrochalcone from 17 Citrus Clementine 373,377 Citrus genus 377 Citrus latifolia 372,377 Citrus limon 375,377 Citrus oils 372 Citrus paradisi 375 Citrus paradisi Macfad. 17 naringin dihydrochalcone from 17

Citrus reticulata 375 Citrus sinensis 375 Class III peroxidase 737 Clavosine 878,879 ClavosinesA 878 from Myriastra clavosa sponge 879 Cloned mammalian adenylylcyclases 825 Coix lachryma }6bi 191-212 Combined effects of 5-fluorouracil 438 Complex mixture 466 of Astragalus sapogenins 466 Complogenin 453 from Astragalus complanatus R. 453 Computational studies 338 16 Configuration 471 of secondary hydroxy 471 of Astragalus saponins 471 Conformational property 337 of analogs 336 Conformational studies of (-)-a-isoparteine 258 of (4-)-.p-isosparteine 259 Conformations 335 of abscisic acid 335,337 Conformers of abscisic acid 340 cisodialas 259 of sparteine 259 transoidalas 259 Coniferylalcohol 60 Coniferyl aldehyde 763 Conjugated forms 354 of abscisic acid 354 Consolida orientalis 191 Constitutional isomers 362 ofmyrcene 363 ofocymene 363 structure of 363 Contents oftannins 413,416 Corallydictyals A and B 855,856 Coriandrum sativum 376 Coronilla 519 Corosolic acid 861,862

944 Corydalis caucasica 163 Corydalis flabellata 169 Corydalis rutifolia 170 Corydalis severtzowii 163 Corynocarpin 522 Costunolide 559 Coumarins 660,847 in Astragalus spp. 503 from Artemisia sp. 847 ll-(9-p-Couinaroylnepecticyn 477 structure of 477 Coumestrans 894 /7-Coumaryl-CoA 768 Crataegus pinnatifida 861 Crispolide 560 Crossandra pungens 191 Crystal structure 335 for abscisic acid 335 Crystallographic studies 821 CumambrinA 576 CumambrinB 576 Curculigo latifolia Dryand. 18 curculin from 18 Curculigo latifolia Dryand. 39 curculinfrom39 Curculin 18,38,39 from Curculigo latifolia Dryand. 18,39 Curcumin 852 from Curcuma aromatica 852 from Curcuma longa 852 from Curcuma xanthrorrhiza 852 Cushman study 925 Cyclic hydroxamic acid 199 a-hydroxylation of 199 Cyclic oligosaccharides 365 Cyclic polypeptide inhibitors 879 9,19-Cyclo-5a,9p-lanostane 447 Cycloalpigenin 452 from Astragalus alopecuros Pall. 452 Cycloartane 447 frora Astragalus sg. 447 Cycloartane derivatives 448 Cycloartane structure of genins 4

Cycloartane from Astragalus sieversienus 483 Cycloartane-type triterpene glycoside 473 Cycloartenol 447 Cycloasgenin A 449 from Astragalus taschkendicus Bimge 449 Cycloasgenin B from Astragalus taschkendicus Bunge 448 Cycloasgenin C 448 from Astragalus taschkendicus Bunge 448 Cycloastragenol 449,473 from Astragalus dissectus 449 Cyclocanthogenin 448 from Astragalus tragacantha Habl. 448 Cyclocanthoside A 448 from Astragalus cephalotes 455 Cyclocanthoside B 448 from Astragalus tragacantha Habl. 448 Cyclocarioside A 15,28,29 from Cyclocarya paliurus 15 structure of 29 Cyclocarioside I 15,28,29 from Cyclocarya paliurus 15 structure of 29 Cyclocarya paliurus 15,28 cyclocarioside A from 15 cyclocarioside I from 15 P-Cyclodextrin 368,369 for the separation of enantiomers 369 Cyclodextrin glucanosyltransferase 365 from. Bacillus macerans 365 from Bacillus megaterium 365 Cyclodextrins 365 a-Cyclodextrins 365 P-Cyclodextrins 365 y-Cyclodextrins 365 structure of 366 Cyclodextrins 368 chromotographic techniques for 368

945 Cyclodextrin-type compound 365 from (+)-a-D-glucose 365 Cyclogalegenin 450 horn Astragalus galegiformis L. 450 Cyclohemiacetals with 196 synthesis of 197 Cyclohex-2-enone 335 ofabscisicacid 335 1,4-Cyclohexadiene 342 Cyclohexanone derivatives 338 Cyclohexenone ring 344 of abscisic acid 344 9,19-Cyclolamost-24£-ene-la,3p, 16(3,27tetraol 448 from Astragalus mongholicus Bunge 448 9,19-Cyclolanost-24E-en-3 P ,6a, 16P-tril 448 fiom Astragalus trigonusT>C 448 9,11-Cyclolanostane 468 from Astragalus membranaceus 468 Cyclomaltodextrin glucanotransferase 8 from Bacillus macerans 8 Cycloorbigenin 452 from Astragalus orbiculatus Ledeb 452 Cyclopropane methylsteroid 470 Cyclopycnanthogenin 450 from Astragalus pycnanthus Boiss 450 P-Cyclopyrethrosin 554 Cyclooxygenase 660 Cymicilen 483 Cyperaceaesy^. 766 Cyprinus carpi 439 Cytidine 5'-triphosphate 820 Cytisine 276,280 (-)-Cytisine 281 Cytisine-rich fruit 280 ofLupinus anagyroides 283 Cytochrome CYP2D6 280 Cytokine receptors 825 Cytokines 82 HODEs induced genesis of 82 in mammalians 82

Cytosal ascorbate peroxidases 736 Cytotoxic activity 927 of flavonoids 927 Cytotoxic flavonoids 925 Cytotoxic value 930 of flavonoids 930 of vinblastine 782 Cytotoxicity 94,924 of flavonoids on cancer cell line 893 of catechins 924 Dactylicapnos torulosa 111 DAG (diacylglycerol) 827-829 Daphnetin 847 Daphnoretin (4) 847 Dasyanthogenin 451 from Astragalus dasyanthus Pall. 451 Daucus spp. 686 Davanone 592 Deacetyl-8-cyclotulipinolide 557 28-Deacetylbelamcandal 835 Deacetyl-P-cyclo-pyrethrosin 555 Decoction 309 of Stevia leaves 309 14-Dehydro-15-phenylsparteine 262 structure of 262 2-Dehydro-17P-isopropyl-2-phenylsparteine 264 2-Dehydro-2-phenyl-17 P-methylsparteine 264 2-Dehydro-2-phenylsparteine 260,261 mesomeric form of 261 2-phenyl-2-dehydrosparteine salts of 260,261 3-Dehydrocycloasgenin 448 f^om Astragalus taschkendicus 448 11,13-Dehydrodesacetyl matricarin 580 Dehydrodicatechin B4 778 from (+)-catechin 778 Dehydroleutodin 580 DentatinA551 5-Deoxy analogue 842 of quercetin 842

946 DeoxyPTX 25IH alkaloid 247-249 from Epipedobates tricolor 247 structure of 249 1 '-Deoxy-1 '-fluoro-ABA biological activity of 334 5-Deoxy-6,8-dihydroxyepicatechins 838 from Acacia melanoxyIon 838 7-Deoxy-(5-ep/-castanospennine 517 I'-Deoxy-abscisicacid 334 8-Deoxycumambrin B 577 12-Deoxyphorbol-13-O-phenylacetate-20acetate 830 Ca^^-Dependent protein kinase 858 Derris scandens 844 Derris sp. 844 Desangelchrysamin 551 Desacetoxymatricin 581 Desacetyltulipinolide-1P, 1 Oa-epoxide 572 Desacetyllaurenobiolide 564 Detection 671 of sulfate groups 671 DHBoA 191,199,201 from Zea mays 191 l,6-Di-0-3-Nitropropanoyl-P-D-glucopyransoide 522 2,6-Di-0-penthyl-3-0-trifluoroacetyl 368 3,4-Di-O-cafFeoylquinic acid 418 from Artemisia montana 418 3,5-Di-O-caffeoylquimc acid 418 4,5-Di-O-caffeoylquinic acid 418 5,8-Diacetoxynerolidol 587 Diarylalkanoids 852 Diarylheptanoids 853 from Pinusflexilis 853 Diastereomers 363,364 1,4-Diazabicyclo [2,2,2]-octane 850 Diazepam binding inhibitor 803 DIBoA 191,195,196,197,199,202, 203,212 from Acanthus mollis 191 from Agropyrinrepens 191 from Aphelandratetragona 191 from Consolida orientalis 191 from Hordeum vulgare 191 from Saccharum officinale 191 from Secale cereale 191

from Triticum aestivum 191 from Zea mays 191 DIBoA 215-217 effect on Amaranthus retroflexus L. 217 effect on Brassica napus h. 2X1 effect on Echinochloa crus-galli (L.)Beauv. 217 effect on Lactuca sativa 111 effect on Lepidium sativum 111 effect on Lolium perenne I.. Ill effect on Poa annua I.. Ill effect on Secale cereale 111 effect on Triticum aestivum 111 effects on plant organisms 215-217 Dicentra spectabilis 158,163 Dicoumarol 847 Dicranostigmafranchetianum162 Dicranostigma lactucoides 155,162 Dicranostigma leptopodum 162 16p,23; 16a,24-Diepoxycycloartane compoxmds 472 5,5'-Diferulic acid 759 5,8'-Diferulic acid 759 8-0-4'-Diferulic acid 759 Difemloylmethane 852 8',8'-Difluoro-abscisic acid 347 2 'a-3 'a-Dihydro-2 'a,3 'a-epoxy-ABA 331 (2/?,3i?)-2,3-Dihydro-5,7,3',4'-tetrahydroxy-6-methoxy flavonol 17,35 from Hymenoxys tumeri 17 structure of 35 (2R, 3/?)-2,3-Dihydro-5,7,3',4'-tetrahydroxy-6-methoxy-3 -0-acetylflavonol 17,35 from Hymenoxys tumeri 11 structure of 35 (2;?,3/?)-2,3-Dihydro-5,7,4'-trihydroxy-6methoxy-3-(9-acetyl flavonol 18,35 from Hymenoxys turneri 18,35 structure of 35 4p,6a-Dihydro-5,7aH,8,11 pH-eudesma8,12-olide 553 2',3'-Dihydro-ABA 353 /rfl/25-2-aryl-2,3-Dihydrobenzofuran moiety 767

947 Dihydrochelirubine 163 m Bocconia integrifolia 163 Dihydrocycloorbigenin 474 Dihydrodidemnin 804 biological activity of 804 from Aplidium albicans 804 lla-Dihydroflabellin 579 liP-Dihydroflabellin 579 Dihydroparthenolide 565 (2JR, 3/?)-Dihydroquercetin 3-O-acetate 17,34,35 from Tessaria dodoneifolia 17 from Hymenoxys tumeri K. Parker 17 structure of 35 {2R, 3i?)-Dihydroquercetin 3-Oacetate 34 from Tessaria dodoneifolia 34 Dihydroquercetin 3-O-acetate 4'-methyl ether 17,35 from Tessaria dodoneifolia 17 structure of 35 Dihydroridentin 567 Dihydrosanguinarine 163,173 in Papaver somniferum 163 structure of 173 Z?w(6-Dihydrosanguinarinyl) ether 172 bond length & angles in 175 crystal data of 175 structure of 172 X-ray analysis of 174 (lli?)-ll,13-DihydrotatridinA 571 (lli?)-ll,13-DihydrotatridinB 571 Dihydroxy indolizidine alkaloid 517 from Astragalus lentiginosus 517 from spotted locoweed 517 3 P,4a-Dihydroxy-13 P-methoxylupanine 269 from Acosmium panamense 269 7,3 '-Dihydroxy-2 '-methoxy-4 ',5 'methylenedioxyisoflavan 499 from Astragalus gummifer 499 6a,25-Dihydroxy-20(/?), 24(S)-epoxycycloartan-3,16-dione 450 from Astragalus adsurgens Pall. 450

2,4-Dihydroxy-2H-1,4-benzoxazin-3(4//)one-skeleton 194-196 4p,6a-Dihydroxy-5,7aH,8pH-eudesnian8,12-olide 552 3,10-Dihydroxy-5,8-diacetoxy-1 (2), ll(12)-dehydrofamesol 586 1P ,4a-Dihydroxy-6a-angeloyloxyeudesmll(13)-en-8,12-olide 552 1P ,4a-Dihydroxy-6a-isobutyloxyeudesmll(13)-en-8,12-olide 552 1P ,3 P-Dihydroxy-7a, 11 PH-germacra-4z10(14)-dien-12,6a-olide 562 1 p,3P-Dihydroxy-7a, 11 PH-germacra4Z,10(14)-dien.l2,6a-olide 562 l,3p-Dihydroxy-7a,l ipH-germacra4Z,9Z-dien-12,6a-olide 562 1 a,3 P-Dihydroxy-7a, 11 PH-germacra4Z,9Z-dien-12-6a-olide 561 5,11 -Dihydroxy-8,9-dihydro-9,10dehydronerolidol 587 1 a,3 P-Dihydroxy-9P, 1 Op-epoxy7a, 11 PH-germacra-4Z-en-12,6a-olide 562 2,5-Dihydroxybenzyl 870 for inhibitory activity 870 6,7-Dihydroxycoimiarin 847 7,8-Dihydroxycoumarin 847 3 P, 16P-Dihydroxycyclolanost-24-en-6,one 448 horn Astragalus trigonus DC 448 Dihydroxyindolizidine 525 from natural sources 525 la,6a-Dihydroxyisocostic acid methyl ester 557 I-3,4-Dihydroxyphenylalanine 754 from peroxidase 754 jD-(-)-3,4-Dihydroxyphenylglycine 754 from peroxidase 754 8a,9P-Dihydroxy-^fl«5,^ra«5-germacra1 (10),4(5)-dien-^fl«5-6,12-olide 563 Dihydro-P-cyclo-pyrethrosin 554 DIM2B0A 191 ftovciZeamays 191 DIMBoA 191,192,195,196,197,199,202, 203,209,212,213,214 QffQct onAvenafatua 111 effect on Avena sativa 111

948 from Agropyrom repens 191 from CoixlachymajobiL. 212 from Saccharum officinale 191 from Triticum aestivum 191 fvovci Zea mays 191 in tritrophic interaction 213,214 Dimeric coniferyl alcohol derivatives 592 6',8'-Dimethoxyfeselol 592 3,4-Dimethoxyphenyl-O-D-glucoside 410 (2S*,3S*,6R*)2,6-Dimethyl-3,6-epoxyocta-7-enoic acid 594 2,5-Dimethylfuran 850 Dinoflagellates 875 Dinophysis 875 Dioscoreophyllum cumminsii 19 monellinfrom 19 Diosmetin 844 1,3-Dioxo-7a, 11 PH-2,3-secogermacra4Z,9Z-dien-12,6a-olide 573 10,17-Dioxosparteine skeleton 272 1,2-Diphenylpropane derivatives 894 Diprianpini 384 Dipterocarpaceae spp. 766 Discorhabdin P (53) 881,882 of Batzella species 881 Disseminated intravascular coagulation 435 Distortionless Enhancement by Polarization Transfer 468 5,8-Disubstituted indolizidines 235,238 conformers of 237 massfragmentationpathway of 238 3,5-Disubstituted indolizidines 236,240 conformers of 237 from Myrmicaria eumenoides 240 from Solenopsis species 241 FTIR spectral analyses of 241 3-hexyl-5-methylindolizidines 241 massfragmentationpathway of 236 (-)-myrmicarin 237A 240 (+)-myrmicarin 237B 240 structure identification of 240 1,4-Disubstituted quinolizidine 235,238, 244

massfragmentationpathway of 238 NMR characterization of 244 quinolizidine 217A 244 quinolizidine 231A 245 quinolizidine 233A 245 quinolizidine 2071 245 quinolizidine 235E 245 quinolizidine 275A 246 4,6-Disubstituted quinolizidines 246 from Mantella basileo 246 in Solenopsis sp. 246 quinolizidine 246 stereochemistry of 246 structure of 247 Diterpene acid 13,23 from pine tree 13 structure of 23 Diterpenoids 857 4-Cl-DMBoA 193 from Zea mays 193 5.CI-DMB0A 193 from Zea mays 193 DMBoA 193 from Zea mays 193 (Ac-D-Nal(2)-D-Phe(4CL)2-Pal(3)3-DCit6,D-AlalO) 795 Dolastatin-15 806,804 Douglanin 551 Dried herbs 418 of Artemisia species 418 for inflammation 418 6-Oxo-drimenol-3a-isovalerate isofraxidin ether 593 DulcosideA 13,22 from Stevia rehaudiana 13,22 structure of 23 DulcosideA 300 from Stevia rebaudiana 300 DynorphinA 803 DPE blockers 283 Effector-receptor complexes 379 Electron transfer 749 4,4'-6w-jatrorrhizine (v) 749 Oxidative halogenation 750 Oxygen transfer 751

949 7V-dealkylation 754 O-dealkylation 754 Eledoisin 800 Elettaria cardamonum 374 Elicitors 61 EUagic acid 413,755 Emodoin 849 from Polygonum cuspidatum 849 Enantiomeric composition 374,377,385 homRutaceaefdirmly Yll in conifers 370 of monoterpenoids 370,374 ofa-pinene 385 Enantioselective chromatographic analysis 361 Enantioseparations 387 of monoterpenoids 387 Endecaphyllins 522 Endocrine function 314 Endogenous abscisic acid 322 P-Endorphin 802,803 Endothelial cells 902 Engelhardtia chrysolepis 18,35 huangqioside E from 18,35 neoastilbin from 18,35 Enzymatic degradation 365 of staren 365 Enzymatic mechanism 383 ofterpenes 383 Enzyme inhibitory activity 850 (-)-Epicatechin gallate 416 Epidemiological studies 430 Epidermal growth factor 824 Epidermal growth factor (EGF)-derived synthetic peptide 838 Epidermal growth factor receptor 847 Epididymal sperum 311 4£-l-Epidihydroridentin 567 4Z-l-Epidihydroridentin 567 (-)-Epigallocatechin 416 structure of 418 (-)-Epigallocatechin gallate 416,839 L-Epinephrine 754 Epoxidation 753 of styrene 753

Epoxides 79,80 generation of 80 of linoleic acid 80 1P, 1 Oa-Epoxy-1,1 OH-desacetyllaurenobiolide 565 Epoxy-3 P,4a, 1 Oa-trihydroxyguaian6a,12-olide 579 lp,10a-Epoxy-3p-hydroxy-7a,l laHgermacra-4Z-en-12,6P-olide 563 3,4-8a-Epoxy-8-deoxycumambrin 577 Epoxyaldehydes 82 generation of 82 hydrolysis products of 82 A\R)-y -Epoxyangeloyloxy-4' -acetoxy-3' 4'-dihydroseselin 429 20(S), 24(R)-Epoxycyclo artane-3p,6a, 16P,25-tetraol 470 from Astragalus galegiformis 470 20(R), 24(S)-Epoxycyclo artane-3p,6a, 16p,25-tetraol 470 from Astragalus membranaceus 470 20,24-Epoxycycloartane derivatives 449 20,25-Epoxycycloartane derivatives 450 20,25-Epoxycycloartane-3P,6a,16P,24atetraol from Astragalus microcephalus willd 450 1 a, 1 OP-Epoxydeacetyl laurenobiolide 564 Epoxyflabellin 578 Epoxysantamarine 556 Equatorial side-chain 341 Erivanin 51 Escherichia coli 305 Eschscholtzia califomica 159 Essential oils 377 from Hedychiumflavum 377 from Lantana camara (+)-terpinen4-ol 377 5'-7V-Ethylcarboxamidoadenosine 878 Eucalyptus genera, 755 Eucryphin 395 Eugenia genera. 755 Euglenophyta 735 Eukaryots 736 Euphorbia genera 755

950 Follicle-stimulating hormone 796 Formation 346,877 of (+)-abscisic acid 346 of(-)-abscisicacid 346 Fagara macrophylla 168 of (HOABA) 346 Fagara xanthoxyloides 159 of phaseic acid 346 Fagaridine 159 Formononetin 498,505 from Fagara xanthoxyloides from Astragalus membanaceus Lam. 159 505 from Zanthoxylum nitidum Forward mutation test 303 DC. 159 Fostreus 883 from Zanthoxylum tessmanii 159 as phosphatase inhibitor 883 Fagaronine 159 Fostriecin 883 from Fagara xanthoxyloides from Streptomyces pulveraceus sp. Lam. 159 883 Fagaropsis angolensis 164 Fragacanth gum 510 Falcarindiol 422 frora Astragalus gummifer from Falcaria vulgaris All Lab. 510 Falcarinol and diynene 687 Free bases 170 from Aegopodium podagraria 687 ofQBA 170 from Pituranthus tortuosus 687 preparation methods of 170 p-Famesene 586 pseudobase of 171 Famesol 586 Free-energy 340 Fenton reaction 65 of ABA 340 French paradox effect 430 generation of OH radicals in 65 Fridelin 590 Femlic acid dimers 757 e/?/-Friedelinol 590 structure of 757 Fmctose content 311 Femlic acid 746 Fmits 283 Flabellin 578 ofLupinus anagyroides 283 3,5,7,4'-Flavone 844 5-FU derivatives 436 Flavonoid complex 505 from Astragalus centralpinus 505 Fungicidal isoflavans 498 Fusarium culmorum 382 Flavonoids 483,660,841 from. Astragalus spp. 745 Galactomannane 511 Flavonones 893 a-D-Galactopyranose 511 Flavour materials 374 3-O-P-D-Galactopyranosyde 477 fromLabiatae 374 D-Galactose 506 from Rutaceae 374 8'-Fluorinated abscisic acid analogs 346 6-0-p-D-Galactosyl-D-glucose 521 Fluorination 333 D-Galacturonic acid 506 of biologically active Galanin effect 799,800 confounds 333 Galanin receptor antagonists 799 3'-Fluoro-abscisicacid 349 Gastric tubing 314 8'-Fluoro-abscisicacid 335 for gastrointestinal absorption 314 5-Fluorouracil 795,436 Gastrin releasing peptide 800 FMBoA 211 Gastrointestinal peptide-hormones 797 Foeniculum vulgare Mill 16 Gastrointestinal hormones 794 ^a/15-anethole from 16 ExtrsLCts of Geranii 414 effects on serum total cholesterol 414

951 Gastrointestinal toxicity 436 Gaudichandioside A 14,24,25 from Baccharis gaudichaudiana 14,24,25 GDHBOA 188 hovci Coix lachryma ]oh\ 188 from Zea mays 188 GDIBOA 188,209 hora Acanthus mollis 188 from Consolida orientalis 188 from Secale cereale 188 synthesis of 210 from Triticum aestivum 188 GDIM2B0A 188,209 from Coixlachrymajobi 188 from Secale cereale 188 from Triticum aestivum 188 from Zea mays 188 synthesis of 210 Genistein(13) 843,844 Genus Astragalus 443,446 abrisapogenol B from 443,453 astragenol from 449 complogenin from 453 9,19-cyclo-5a,9P-lanostane from 447 cyclocanthogenin from 448 cycloalpigenin from 452 cycloalpigenin B from 449 cycloalpigenin C from 449 cycloalpigenin D from 449 cycloasgenin A from 449 cycloasgenin B from 558 cycloasgenin C from 448 cycloastragenol from 449 cyclogalegenin from 450 cycloorbigenin A from 452 cycloorbigenin B from 452 cyclopycnanthogenin from 450 dasyanthogenin from 451 3-dehydrocycloasgenin from 448 dihydroycloorbigenin A from 452 glycyrrhetic acid from 453 medicinal uses of 447 oleanohc acid from 453 oxytrogenin from 453 quisvagenin from 450 sapogeninfrom 448

sapogenin II from 453 saponins from from 446 soyasapogenol B from 453 terpenoid sapogenins from 446 Genus Bupleurum L. 660 Genus Tanacetum 547 Geraniin 840 GerawnHerba 413 corilaginfrom 413 of Geranium thunbergii 413 Gerinimum genera 755 Germacranolide 560 GermacreneD 573 GHBOA 188 from Blepharis edulis 188 from Zea mays 188 GHDMBOA 188 from Coix lachryma johi 188 from Triticum aestivum 188 from Zea mays 188 GHMBOA 188 from Zea mays 188 Gradient heteromultibond correlation 470 Grevillea robusta leaves 839 extract of 839 GHMBOA 188,209 from Coix lachryma j obi 188 from Secale cereale 188 from Triticum aestivum 188 from Zea mays 188 Gibberellins 305 Ginsenoside Rbi (33) 859,860 Glabridin 846 Glial cell 806 Glioma cells 797 Glucocorticoid receptor 877 P-D-Glucopyranose monoester 522 Glucose ester 520 of nitropropanoic acid 520 Glucose-1-phosphate 821 P-Glucosidase activity 354 in barley leaves 354 3-0-p-D-Glucoside 517 I'-O-Glucoside 354 P-D-Glucosides 520 ofnitropropanol 520,521 from Astragalus sipp. 520

952 1-0-Glucosyl ester 354 (1 l/?)-6-0-P-D-Glucosyl-l 1,13-dihydrotatridinB 572 Glucosyltransferase 354,778 Glycan 506 Glycogen phosphorylase molecule 821 Glycopeptides aeruginosin 205 Glycoproteins 736 a-Glycosidase 256 Glycoside structural elucidation 484 Glycosides 311,775,924 from .S^ma extract 311 p-Glycosidic bonds 314 Glycosidic lupine alkaloid 268 Glycosilation effect 475 Glycosylation 737 of enzyme 737 Glycyphyllin 17,33,34 from Smilax glycyphylla Sm. 17 structure of 34 Glycyrrhetic acid 453 from Astragalus glycyphyllos L. 453 18a-Glycyrrhetinicacid 858,859 Glycyrrhiza glabra L. 6,15,29 glycyrrhizin from 6,15,29 Glycyrrhiza inflata Batal 15,29,30,425 apioglycyrrhizin from 15,29,30 araboglycyrrhizin from 15,29,30 Glycyrrhizin 5,6,15,29 from Glycyrrhiza glabra L. 6,15 structure of 6 3-O-D-glycoronide (MGGR,7) 6,29 Gnetaceae spp. 766 Gnidimacrin 831 from Stellera chamaejasme 831 Gonadotropin hormone 795 Gonadotropin-releasing harmone 795,796 Gonadotropins 796 Gossypol 855 Gossypium herbaceum 855 Thespesia populnea 855 G-protein coupled receptors 825 Gradient hetero single quantum correlation 470

Griceps 385 for Velocitermes velox 385 Gurmarin 42 as sweetness inhibitor from Gymnema sylvestre 42 Gymnema altemifolium 41,43,44 altemoside I-V from 41 Gymnema sylvestre R. Br. ex Schutt. 41, 42,48,49 gymnemasoponin III,IV,V from 41 gymnemic acid IIVI,VIII-XVIII from 41 Gymnemagenin 42,44 structure of 44 Gymnemasaponins III-V 41,43 as sweetness inhibitors 41 from Gymnema sylvestre 41 structures of 41,45 Gymnemic acid I-VI,VIII,XVIII 41,43,44 as sweetness inhibitors 41 from Gymnema sylvestre 41 structures of 44 Gymnestrogenin-type aglycone structure of 47 Gynostemma pentaphyllum 15,28 gypenoside XX from 15 GypenosideXX 15,28,29 from Gynostemma pentaphyllum 15 HA inhibitors 116-129 2P-chlori-peracetyl-Neu methyl ester 124,127 4-(8-morpholin) capriloyl-Neu5Aca2Me 126,127 4,7-di-0-acetyl-Neu5Ac-a2Me 123 4-capriloyl-Neu5Ac-a2Me 125-127 4-0-acetyl-Neu5Ac-a2Me 123 4-0-capriloyl-Neu5Ac-2Me 125 4-0-phenyl-2-propionyl-Neu5Aca2Me 123 4-t-butyl-dimethyl-silyl ether 127

953 5-azido-5-deamino-Neu5Ac-a2Me 119 5-N-benzyloxycarbonyl-Neu-a2Me 119 5-N-propionyl-Neu-a2Me 119 7-deoxy-Neu5Ac-a2Me 123,127 8-aimno-8-deoxy-Neu5Ac 121,122 8-azido-8-deoxy-Neu5Ac 121,122 8-tosyl-Neu5AcMe ester 121,122 9-aimno-9-deoxy-Neu5Ac 121,122 9-amino-9-deoxy-Neu5Ac-a2Me 119 9-azido-9-deoxy-Neu5Ac 121,22 9-azido-9-deoxy-Neu5Ac-a2Me 119 9-0-acetyl-Neu5Ac-a2Me 119 9-tosyl-Neu5AcMe ester 121,122 Neu 5Ac methyl ester 120-122 Neu5Ac-a2Me 119-121 Neu5 Ac-a2Me amide 119,120 Neu5Ac-a2Me benzyl ester 125-127 Neu5 Ac-a2Me methyl ester 125-127 Neu5Ac-P2Me methyl ester 3|3 125,127 peracetyl-Neu-a2Me acid methyl ester 124,127 Haemagglutinin (HA) 103,107 inhibitors of 117-129 structure of 109-112 for influenza infection 103 Haematoxylon campechianum L. 18,37 hematoxylin from 18 Hakomori's procedure 512 Halichondria genus 875 Halistanol trisulphate (34) 861 Halogenation 749,752 of olefins 749,752 Hanfillin 564 HBoA 191 from Aphelandra tetragona 191 from Blepharis edulis 191 from Scoporia dulcis 191 from Zea mays 191 HDMBoA 191,202 from Zea mays 191

Head-to-tail polymerization 774 Heimerlein 564 Helichrysum gymnocephalum 377 Helix pomatia 468 Helminthosporium carbonum 498,499 Hematologic toxicity 436 Hematoporetic stem cell 806 (+)-Hematoxylin 18,37,38 from Haematoxylon campechianum L. 18 structure of 38 Hemiacetal ethers 189 retrosynthetic analysis of 189 Hemicryptophyte plant 663 Hemsleya carnosiflora 14 camosifloside V from 14 camosifloside VI from 14 Hemsleya panacis-scandens 15,25 scandenoside R6 from 15,25 scandenoside Rl 1 from 15,25 Hepatoma cell 806 Hepatotoxic principle 879 of Anabaena genera 879 of Microcystis g^ntrdi 879 of Oscillatoria genera. 879 (8E)-Heptadeca-l,8,dien-4,6-diyn-3,10diol 422 structure of 422 (+)-Hemaudulcin 13,21 from Lippa dulcis Trev. 13,21 structure of 21 Heterosaccharides(AH-l, AH-2) 508 from Astragalus mongholicus Bunge 508 Hexadeuterated abscisic acid 353 Hexahydroazepine amide alkaloid 868 from Verticillium balanoides 868 4,4',5,5',6,6'-Hexahydroxydiphenic acid dilactone 755 3-Hexyl-5-methylindolizidine 241 diastereomers of 241 High sugar diet-treated group 410 High fat-diet-treated group 401 Hiptagin 522 Histidine 78 Histidine kinases 824 Histological examination 310 of consuming stevioside effects

954 310 of reproduction tissue 310 HMBoA 191,209 from Coix lachryma johi 191 from Triticum aestivum 191 from Zea mays 191 ^H-NMR spectra 339 of abscisic acid in acetone-d^ 339 ^H-NMR spectrum 176,350 of chelerythrinefreebase 176 of phaseic acid 350 ^H-NMR studies 338 of abscisic acid 338 Hodulosides VII-X 41,45,46 from Hovenia dulcis Thunb. var. Tomentella Makino 41 Homochelidonine 162 in Chelidonium majus 162 Homooligomers 768 of rrflW5-resveratrol 768 Hongshengma 395 Hordatines 765 Hordeum vulgare 191,765 Hovenia dulcis Thunb. 41,43 hoduloside I-V,VIII-X from 41 hovenoside I from 41 jujuboside B from 41 saponin C2from41 saponin E,H from 41 Hovenolactone 45,47 structure of 47 Hovenosdiel 41,45,46 from Hovenia dulcis Thunb. var. tomentella Makino 41 structure of 46 135-HPOTE 71 degradation of 71 5-HT3 receptor 286 5-HT4 receptor 286 Huangqioside E 18,35 from Engelhardtia chrysolepis Hance 18 structure of 35 2-Huoro-ABA 355 Hydrangea macrophylla Seringe var. thunbergii 17 Hydrocotile genera, 661 Hydrocotyloideae farrnly 661

Hydrolysis 354 of abscisic acid-GE 354 of fat 402 of phosphate 872 16-Hydroperoxy-10,12,14-octadecatrienoic acid 71 generation of 71 9-Hydroperoxy-10,12,15-octadecatrienoic acid 74 by Baeyer-Villiger reaction 94 transformation of 74 {6S,1S, 10i?)-2-Hydroperoxy-2,6,10trimethyl-7,10-epoxydodeca-3,11dien-5-one 594 2£-4-Hydroperoxy-2-nonenal (4-HNE) 74,75,83 generation of 75 13-Hydroperoxy-9Z-ll£-octadecaenoic acid 69-71 generation of 69,70 by different lipoxygenases 70 Hydrophobic metabolites 447,484 Hydrophobicity 333 3(3-Hydroxy costunolide 560 cw,cw-2a-Hydroxy costunolide 560 Hydroxydavanone 592 4,5-Cw-3P-Hydroxy germacranolide 561 ip-Hydroxy-10,14-dehydro-l,10Hparthenolide 566 6a-Hydroxy-l l,13-dihydro-5,7aH,8, 1 ipH-eudesm-4(15)-en-8,12olide 552 (-)-3 p-Hydroxy-13a-tigloyloxylupanine 272 from Genista cinerea 111 (+)-15 P-Hydroxy-17-oxolupanine 271 fromLupinus albus 111 1 a-Hydroxy-1 -desoxochrysanolide 559 1 P-Hydroxy-1-desoxochrysanolide 559 1 a-Hydroxy-1-desoxoarglamine 549 1 a-Hydroxy-1 -desoxo-tamirin 568 1 P-Hydroxy-1 -desoxo-tamirin 568 (65*,75*, 10i?*)-3-Hydroxy-2,10,10trimethyl-7,10-epoxy-dodeca-1,11dien-5-one 594 7-Hydroxy-2 '-methoxy-4' 5' -methylenedioxyisoflavan 256 499

955 structure 473 of 25-Hydroxy-20,24-epoxy 473 (+)-(4 '-Hydroxy-3 '-methoxyciimamoyl)epil-upinine 268 5-3-Hydroxy-3-methylglutaryl 383 3-Hydroxy-3-methylglutarylcoenzyme 386 r-Hydroxy-4'-oxo-a-ionone 330 3-Hydroxy-5-(3,4-dihy(iroxyphenyl) 838 from Phyllocladus trichomanoides 838 11 -Hydroxy-5,14-diacetoxy-9,10dehydrofamesol acetate 586 6a-Hydroxy-5,7aH,8pH-eudesm-4( 15)en-8,12-olide 552 1 P-Hydroxy-6a-angeloyl-oxyeudesm4(15), ll(13)-dien.8,12-olide 552 1 P-Hydroxy-6a-angeloyl-oxygermacra4(5), 10(14), ll(13)-trien-8,12olide 561 5-Hydroxy-9-acetoxynerolidol 586 8*-Hydroxy-abscisic acid 335 7'-Hydroxyabscisic acid 348 8a-Hydroxyachillin 574 12a-Hydroxyamorphigenin 8a-Hydroxyanhydroverlotorin 572 3P-Hydroxyanhydroverlotorin 573 (+)-13a-Hydroxyaphyllidine 273 from Lupinus hartwegii 273 (+)-2P-Hydroxyaphylline 273 ip-HydroxyarbusculinA 549 8a-Hydroxyarbusculin A 549 1 a-Hydroxy-desacetylirinol-4a,5 Pepoxide 565 6-Hydroxydihydrochelerythrine 177 from Toddalia aculeata 111 A^^^^-15-Hydroxydihydroflabellin 579 6-Hydroxydihydrosanguinarine 173 fromDactylicapnos torulosa 111 structure of 173 8a-Hydroxyestafiafin 578 4-Hydroxyfamesolacetate 586 4P-Hydroxyhemandulcin 13,21 from Lippadulcis 13,21 structure of 21 8'-Hydroxylating activity 346 in maize cell 346

Hydroxylation by cytochrome P450 346 7'-Hydroxylation of (r/?)-(-)-Abscisic acid 348 Hydroxylation 749,754 ofarenes 749 of indoles 754 (-)-6a-Hydroxylupanine 269 from Lygos raetam var. sarcocarpa 269 13P-Hydroxymamanine 267 X-ray analysis of 267 (-)-baptifoline 267 (-)-14P-Hydroxymatrine 275 from Sophora tonkinensis 275 (-)-13P-Hydroxymultiflorine 273 from Lupinus varius 273 3P-Hydroxy-oxo-7a,l ipH-germacra4Z,10(14)-diene-12,6a-olide 561 3P-hydroxyparthenolide 566 3a-Hydroxyreynosin 555 8'-Hydroxyabscisic acid 345 8a Hydroxysantamarine 556 P-Hydroxy-P-eudesmol 558 Hylobius abietis 385 Hymenoxys tumeriK. Parker 17,18,35 {2R, 3/?)-Dihydroquercetin 3-0acetate from 17 (2i?, 3/^)-2,3-Dihydro-5,7,3'-4'tetrahydroxy-6-methoxy-3-0acetylflavanol from 17 (2R, 3/?)-2,3-Dihydro-5,7,3',4'tetrahydroxy-6-methoxyflavanol from 17 (2R, 3i?)-2,3-Dihydro-5,7,4'trihydroxy-6-methoxy-3-(9acetylflavanol from 18 Hypecoum imberbe 167,168 Hypecoum leptocarpum 168 Hypericin 850,851 Hypericin inhibited protein kinase C 850 Hypericum sp. 850 Hypersensitive response 61 Hypoglottis Bungc 61 Hypolipidaemic effects 400

956 Hypotensive activity 481 of Astragalus membranaceus roots 481,483 Hypothalamic neuropeptide 794 Hypothalamus-pituitary 794 Ilex paraguariensis 299 Illicium verum Hook f. 16 /ran^-anethole from 16 Immimocompetent organic toxicity 436 Immimostimulant effect 513 of polysaccharide 513 of Astragalus membranaceus 513 Inactivation pathway 321,345 of ABA 345 Indicimiinone 593 Indigofera genera. 519 Indole alkaloids 862 Indole-3-acetic acid 747 Indolizidine alkaloid 235,236-238 1,4-disubstituted quinolizidines 238 3,5-disubstituted indolizidines 235,2365,8-disubstituted indolizidines 235,238 as non-competitive blockers 249 biological activity of 249,250 pumiliotoxin alkaloids 238,239 quinolizidine alkaloid 235, 236-238 spectral features of 236 structure identification of 240-247 structure identification of 247-249 Indolizidine 223A 242,243,287 GG-MSfragmentsof 242 Indolizidine 249H 243 from Dendrobates auratus 243 structure of 244 Indolizidine 25IM 242,243 mass spectrum of 243 Indolizidine 267J 242,243 5E,9E stereochemistry of 243 Indolizidine alkaloids 233 from amphibians 234 from ants 234

bioactivity of 233 structure of 233 Indomethacin 689,690 INEPT (Intensive Nucleus Enhancement by Polarization Transfer) 469 Influence on intracutaneous permeability 378,380 promotors of sorption 380 Influenza A 108 Influenzae 108 Influenza infection 103 Influenza virus 107,108 enzyme mechanism of 114 representation of 107 Inhibition of LTC4-release 691 Inhibition of PGEz-release 690 from peritoneal macrophages 690 Inhibitory action 402 of chitosan 402 Inhibitory effects 437 of budlejasaponin IV 689,690,691 of chitosan 437,440 of carp extract 440,689 of ginseng saponins 423 of sandrosaponin I 689,690,691, 693 Insect-plant relationship 384 Insulin-like substances 394 in Korean ginseng 394 Integrins 825 Intense sweetness 4,11 Interconversion 336 of ring of abscisic acid 336,338 International council of harmonisation 304 Interstitial cells 310,311 lodination of tyrosine 751 Ipstypographus 384,385 ER spectrum 177 ofsanguilutine free base 177 8a-Isobutycyloxyestafafin 578 Isocyanate intermediate 330 Isoenzyme patterns 757 of Vitis vinifera 151 Isofagaridine 159 6-methoxy adduct of 159 from Zanthoxylum nitidum 159

957 Isoflavanones 893 Isoflavans 484,500 fyom Astragalus spp. 500 from Astragalus alexandrinus 500 from Astragalus membranaceus 500 from Astragalus trigonus 500 Isoflavones 484,745 Isoflavonoids 841 Isofraxidin drimenylether 593 Isohumulen 574 Isomenthone 375 Isomerism 361 ofmonoterpenoids 361 Isomerization rates 350 of 8'-hydroxyabscisic acid 350 of phaseic acid (PA) 350 Isomucronulatol7,2*-di-0-P-D-glucoside 499 Isoprenylflavone cycloheterophyllin 842 from Artocarpus heterophyllus 842 Isoquinoline alkaloids 819,863 Isospiciformin 568 (-)-3-Isothujone 384 Isotope effect 348 Itodulosides I-V 41,45,46,47 from Hovenia dulcis Thunb. var. tomentellaM?ikmo 41 structures of 46,47 Janus kinases (JAKs) JasmolinI 588 Jasmolinll 588 Jasmonic acid 64,71-73 biological activities of 72,73 generation of 71,72 Jatrorrhiza palmata 749 Jujubasaponins II-VI 42,48-50 from Ziziphus jujuba P. Miller 42 structures of 49,50 JujubosideB 41,42,45,46 from Hovenia dulcis Thunb. var. tomentellaMdikmo 41 structure of 46 Juniperus 372 (+)-limonene in 372

Juriperus communis 370,372 essential oils from 370 K562 cells study 877 Kalihinanes terpenoids Karakin 522 Kassinin (KAS) 800 ent-Kaurenate 305 enr-Kaurene glycosides 14,22 from Rubus suavissimus 22 from Stevia rebaudiana 22 suaviosideA 14,22 suaviosideB 14,22 suavioside G 14,22 suaviosideH 14,22 suavioside I 14,22 suavioside J 14,22 KazinolB(5) 834 from Broussonetia papyrifera 834 3-Keto-4a-H-germacran-1 (10), 11 (13)dien-6,12-olide 563 2-Keto-8a-hydroxy-5a,6a,7pH-guaianl(10),3(4),ll(13)-trien-6,12olide 580 a-Ketols 72 Y-Ketols 72 Ketopelenolide 564 Kinetic characteristics 350 of isomerization process 350 Kinetics of peroxidase-catalyzed reactions 741 Kinetic parameters 351 Kinetic studies 857 of cell 857 Labdane glycoside 14,22,24 baiyunoside 14,24 gandichandioside A 14,25 phlomisoside I 14,24 Labiataepinaceae 361 Lamininai 806 Lanost-9(ll)-ene 468 Lantana camara 836 Lapachol 851 P-lapachone(21) 851 P-Lapachone 851 a-1,5-L-arabino-p-3,6-D-galactan 510

958 (+)-Lariciresinol 503 Lavandula angustifoUa 374 Lavendustin A (45) 869,870 from Streptomyces griseolavendus 869 Leguminosae spp. 443,766 Leguminous plants 523 of Astragalus genera. 523 of oxytropis geneid. 523 Lentiginoside from Astragalus lentiginosus 251 Leucine-enkephalin (L-ENK) 798 Leucinostatin A 805 from Acremonium sp. 805 Leucosceptoside 837 Leu-enkephalin 802,803 Lentiginoside alkaloid 251 from Astragalus lentiginosus 251 structure of 251 Licochalcones A and B 425 structure of 427,428 Lignin polymer 760 Lignans 504 from Astragalus mongholicus 504 Li-methylsulfynil carbanion 512 (+)-Limonene 372 cf-Limonene 386 (+)-Linalool 384 Linalool enantiomers 379 Linalyl acetate 374 structure of 374 Linolenic acid 70 generation of 165-hydroperoxy9Z, 12Z, 14£'-octa-decatrienoic acid (165-HPOTE) from 70 generation of 95-hydroperoxy10£, 12Z, 16Z-octadecatrienoic acid from 70,71 Lipases 66 Lipid peroxidation products 84-86 derivatization with pentafluorobenzyl hydroxylamine 85 identification of 84-86 in biological materials 84-86 relation with cell death 86-88 spectra of 85 Lipid peroxidation 59,68,85,504,505 oflinoleicacid 68

Lipid hydroperoxides (LOOHs) 59,67,68, 73,78 degradation products of 68 Lipogenesis 395 insulin-inducedfromglucose 397 Lipolytic action 394 activity of 394 Lipopolysaccharide (LPS) 856 Lipooxygenase metabolites 660 Lipoxygenases 59,67-69,88 from soybean 69,70 of tomatoes 70 Lippadulcis 13,21 (+)-hemandulcin from 13,21 4P-hydroxyheman dulcin from 13,21 Listeria monocytogenes 381 Lithocholic acid 858,859 LO* radicals 78,79 by a Fenton-like reaction 78 generation of 78 a-Lobeline 283 Locois 523 cw-Longipinane-2,7-dione 587 /rfl«5-Longipinane-2,7-dione 587 Io/t/5 families 519 Low density lipoprotein-cholesterol 405 Low-temperature NMR analysis 344 LudovicinA 553 LudovicinB 553 l-€?p/-Ludovicin C 554 Lupenone 478 from Astragalus adsurgens 478 Lupeol 478 Lupeyl acetate 590 Lupine alkaloids 258 biological activity of 258 structure of 258 Lupinyl moiety 285 Lutinizing hormone-releasing hormone 794,796 Lymphocyte proHferation 693 Z)-Lysine 795 Lysolecithines 66 a(l,3)-Lysosoma 252 Lysosomal a-mannosidase 524

959 Matrix metalloproteinases 805 MboA 193,212,217 effect on Avenafatua 111 effect on Avena sativa 217 Mabinlin 18,38 from Aphelandra tetragona 193 from Capparis masaikai Levi. 18 from Coix lachryma jobi 193,212 Macarpine 159 from Scoparia dulcis 193 from Chelidonium majus 159 from Triticum aestivum 193 fiGm Eschscholtzia s^QCiQS 159 from Zea mays 193 from Eschscholtzia califomica Mechanism 159 of absicisic acid reception 322 from Macleaya microcarpa 159 Mechanistic features from Macleaya cordata 159 of kinase 821 from Stylophorum diphyllum 159 Medicarpin 498 from Stylophorum lasiocarpum Mentha 375 159 1-Menthol 381 Macleaya cordata 155,159,162,864 Menthone 375 Macleaya microcarpa 155,159,162 Metabolic activation system 303 Macrocyclic lactone Metabolic inactivation 322 from Cylindrocarpon sp. 871 of abscisic acid 322 from Dendryphionsp. 871 Metabolic transformations 748 Metabolism 301 Macrotanacin 580 of Berberidaceae family 754 Magainins 807 of Ranunculaceae family 754 Magnificol 590 ofstevioside 301 Magnoflorine 162 of tetrahydroprotoberberine from Chelidonium majus 162 alkaloid 754 Magnograndiolide 580 Met-enkephalin 802,803 Magnolialide 554 12-Methoxydihydrochelerythrine 163 Magnolol 853 in Bocconia integrifolia 163 in Magnolia officinalis 853 l-Methoxy-2,3-methylenedioxybenzene Malonyl-CoA 768 499 Mammalian cell mutation test 304 (-)-(3 '-Methoxy-4 '-a-L-rhamnosyloxyMangifera genera 755 cinnamoyl)-epilupinine 267,268 y-Mangostin 902 from Lupinus hirsutus 161 from Garcinia mangostana 902 4'-Methoxy-abscisic acid 335 1,4-P-D-Mannopyranose 511 activity of 335 a(l,6)-Mannosidases 252 6-Methoxydihydrochelerythrine 168 Marsglobiferin 47,48 from Chelidonium majus 168 structure of 48 from Hypecoum imberbe 168 Mass spectral fragmentation from Hypecoum leptocarpum 168 pathway of 238 6-Methoxydihydrofagaridine 168 of indolizidines 235,236 from Zanthoxylum nitidum 168 of indolizidines 235,238 6-Methoxydihydronitidine 168 of quinolizidines 235,236 from Fagara macrophylla 168 of quinolizidines 235,238 13P-Methoxylupanine 269 Mast cell 806 3-Methoxytanapartholide 585 Metabolic activation system 303

Lytic enzymes 67 proteases 67 nucleases 67

960 r-Methyl ether 341 of ABA 341 Methyl jasmonate 60 1 -Methyl-4-phenyl-1,2,3,6-tetrahydropyridine 283 6-(4-Methyl-2-oxopenlyl) dihydrochelerythrine 167 6-Methyldihydrocheresythrine 167 in Zanthoxylum simulans 167 a-Methylene butyrolactone 856 Methylene-abscisic acid 347 8'-Methylenoxides 348 8'-Methyhdyne-abscisic acid 347 bioactivity of 347 4'-Methylluteolin 843 gew-Methyls 333 P-Methyl-styrenes 753 8-Methylvestitol 505 from Astragalus alexandrinus 505 from Astragalus trigonus 505 Michelenolide 565 Michellaniines A,B,C alkaloids 865 analogue glaucine 868 apomorphine 866,867 (+)-boldine 866 bulbocapnine 866 from Ancistrocladus korupensis 865 nitrogenated compounds 868 sanguinarine 866,867 Micromeriafruticosa Zll Micromorphological studies 876 Micronucleus test 303 Microsomal cytochrome 346 Microsomal proteinfraction331 of Arabidopsis thaliana 331 Microtus montanus 213 responses to MBoA 213 Milkevetches 444 Miosin light-chain kinase (MLCK) 822 Mioxinjection ofabscisicacid 322 Miraculin 39 from Richardella dulcificia 39 Miserotoxin 526 Mitochondrial yeast cytochrome A peroxidase 736

Mitogen activated protein kinase 858 Mitogen activated protein kinases (MAPKs) 828,829 Mitogenic effect study 798 Miosin light-chain kinase 858 Modulation 819 of protein phosphorylation 819 MogrosidelV 14,26,27 from Siraitia grosvenorii 14 structure of 27 MogrosideV 14,26 from Siraitia grosvenorii 14 Mogroside V(2) 6,7 from Siraitia grosvenorii 6,7 structure of 7 Mollusk 804 Bolabella auricularia 804 Monatin 18,38 from Schlerochiton ilicifolius A. 18 structure of 38 Monellin 18,38 from Dioscoreophyllum cumminsii 19 Monodesmosidic 10 675 from root of Bupleurum rigidum 675 Monodesmosidic saponins 664 Mono-electronic peroxidase-catalyzed 770 Monofluoro alkane 334 Monoterpenic hydrocarbons 370 Monoteipenoid indole alkaloids 780 Mono-teruloyl-tri-/?-carmaroyl-esters 837 Monoxygenase 322 Motuporin 880 from sponge Mucrine 565 Mucronulatol 366 498 Mukurozioside lib 13,21 from Sapindus mukurossi Gaertn 21 from Sapindus rarak DC 13,21 structure of 22 Multifimctional calmodulin-dependent kinases 822,825,827 Multipoint attachment model 11 Musca domestica 384

961 Mutagenic action 304 of human lymphocytes 304 Mutagenicity tests 303,304 Ames test 303 chromosome mutation test 303,304 for stevioside 303,304 forward mutation test 303 mammalian cell mutation test 304 micronucleus test 303 Myelotoxicity 436 Myricetin 746,844 Myristoylated alanine-rich C kinase substrate 860,861 (+)-Myrmicarin 237 240 from Myrmicaria eumenoides 240 (-)-Myrmicarin 237A 240 from Myrmicaria eumenoides 240 Myrrhis odorata Scop. 16 trans-dxiQiholQfrom16 5-N-acetylneuraminic acid 104 AZ-linked glycosylation 256 nAChRs 281 Nallanin 844,845 Naphthoquinones 849 NaphtyUsoquinolines 819 Naringin dihydrochalcone*^ 17,34 from Citrus paradisiMdicfdid 17 structure of 64 N-[3-hydroxy-3-methyl-glutaroyl]tryptophan 480 N'SLiyl glycine amides 285 N-[(quinolizidin-1 a-yl)-methyl]benzotriazol-2-yl 285 Nasutitermes ephratae 385 Nasutitermes nigriceps 385 Natural inhibitors 836 of protein kinases 836 Natural oligopeptides 793 with biological activities 793 Natural products 398 anti-obesity action of 398 biochemical studies of 393 from foodstuffs 393 from medicinal plants 393 pharmacological studies of 393 Naturally occiuxing protein kinase activators 829

Naufraga 661 NDGA compound 692 Necrosis 87 in mammalians 87 Neoastilbin 18,35 from Engelhardtia chrysolepis 18 structure of 35 Neohesperidin dihydrochalcone 10 from Citrus aurantium L. 10 Neohesperidin dihydrochalcone 17,34 from Citrus aurantium L. 17 structure of 34 Neoplastic activity 306 of stevioside 306 Nerohoil 373 Neuraminic acid 103-152 biological activity of 103-152 chemistry of 103-152 effects of 105,106 Neuraminidase 103,107,108,112-116 enzyme mechanism of 114 from Anthrobacter sialophilus 115 NA-sialic acid interactions 113,114 of influenza virus 114 structure of 112-114 target against influenza infection 103 Neuraminidase inhibitors 130 4-amino-3 -nitrobenzoic acid 138 4-(N-acetylamino)-3 guanidinobenzoic acid 138 4-amino-3-hydroxy benzoic acid 138,139 I-(4-carboxy-2-(3 -pentylamino)phenyl)-5,5-(hydroxymethyl) pyrrolidin-2-one 150-152 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid 130,136 2,3-didehydro-2,4-dideoxy-4guanidyl-Neu5Ac 131,135 Neurohormones 794 Neurokinin A 800 Neurokinin B 800 Neuron cell 806 Neuropeptide Y 800 Neuropeptide analogues 798

962 Neurotensin 799,800 Neutrophil cytosol 842 A^-formyl-catharinine 778 Nicotinamide adenine dinucleotide phosphate (NADPH) 405 (-)-Nicotine 281 Nicotine 284 Nitidine 159 biological activity of 179 in Rutaceae faimly 164 from Zanthoxylum nitidum 159 l-(2-Nitro)phenylethyl ester 332 3-Nitro-1 -propylallolactoside 521 3-Nitro-1 -propylcellobioside 521 3-Nitro-1 -propyl-p-D-gentiobioside 521 /?-Nitroaniline 520 3-Nitropropanoate ester 522 from Indigofera endecaphylla 522 A^-Nitroso 6w(2-oxopropyl) amine 795 Nitro-toxins fj[oxa Astragalus s^i^. 521 Nitrotyrasanguinarine 167 from Hypecoum imberbe 167 NMR analysis 343 of abscisic acid 343 Nodularia spumigena cyanobacteria 880 NOE experiment 338 of abscisic acid 338 Non-cicosanoid mediators 693 Non-peptide polyether toxins Norathyri(18) 902 from Tripterospermum lanceolatum 902 Norathyriol 902 Norectal 483 Norepinephrine-augmenting lipolytic activity 400 of oolong tea 400 Nomuciferine(51) 880,881 Nuclear Overhauser effect (NOE) 335 of abscisic acid 336 Nutritional significance 314 of Stevia 314 ofstevioside 314 Obesity 398 gold thioglucose (GTG) in 398 model of 398

Ocimum basilicum 374,376 1-Oxo-a-longipinane 587 13-Oxo-9-hydroxy-10-octadecenoic acid 73 generation of 73 13-0x0-9,11-octadecadienoic acid 73 generation of 73 Odoratin 499 Odour of carvone enantiomers 379 ODS column 351 OECDandlCH 304 of mutagenesis test 304 Okadaicacid 875,876 Oleanolic acid 453 from Astragalus bungeanus Boiss 453 Oligomeric catechins 837 Oligomers 855 from Canagana sinica 855 Oligopeptides 793 antibacterial activities of 793-804 biological activities of 793-804 Oligosaccharidic moiety 524,673 of saponins 673 Olive oil 697 antiatherosclerotic effect of 714 antioxidant activity of 710 bioavailability of 727 biological activity of 714 cardiovascular effect of 723 composition of 698,699 effect on LDL oxidation 714,717, 723 effect on nitric oxide metabolism 721 effect on platelet aggregation 719 effect on reactive oxygen-induced toxicity 722 hydrocarbons from 700 influence of cultivar on 707 phenols from 702,703 prooxidant effects of 713 sterols from 702 tocopherols from 702,703 triterpenes from 702 volatile components of 700,701

963 4-en-6p-01-one 477 -3',5'oMe, 4'[(t-butyldimethylsilyl)oxi] 928 r-O-methyl-ABA 334 Optical isomers of carvone 378 Organoleptic properties 379 Orientation of cyclopropane 344 Origanum genus 376 Origanum syriacum 376 Origanum vulgare 376 D-Omithine 795 Osladin 16,31,32 from Polypodium vulgare L. 16,31 structure of 32 Osmorhiza longistylis DC 16 rraw5-anethole from 16 Oval glucose tolerance test 409 Oxidation of 749,774 (H-)-catechin 774 cytochrome P450 enzymes 348 terminal acetylene 348 xenobiotics 749 Oxidation 778 of vincristine 778 Oxidative burst 65,66 8-Oxo-2a,9-dihydroxy-/ran5, transgermacra-1(10),4(5)-dien-trfl«5-6,12olide 563 A^^^>-15-Oxo-flabellin 579 4-Oxoisophorone 337 10-Oxolupanine 271 from Dicraeopetalum stipulare 111 17-Oxolupanine 271 from Dicraeopetalum stipulare 271 Oxo-cyclo tautomerism 3-Oxo-malabarica-14(26), 1 IE,21 -triene 591 11-Oxomogroside V 14,26,27 from Siraitia siamensis 14 structure of 27 Oxosanguinarine 173 structure of 173 5-Oxotetrahydroftiran-3-acetic acid 522

Oxyferryl porphyrin 7C 738 Oxy-ftmctionalization of aromatic amines 749 of phenols 749 Oxygenated monoterpenoids 373 Oxysanguinarine 163 m Papaver somniferum 163 Oxytropis sericea 516 Oxytrogenin 453 from Astragalus shikokianus 453 Oxytropis spcciQS 444,514 Panax ginseng 394,445 Panaxynol 422 Panaxynol compound 686 from Panax ginseng roots 686 Pancreatic spasmolytic polypeptide 805 Papaveraceae 163 Arctomecon genera 163 Argemone gQntrdi 163 Eschscholtzia generdi 163 Glaucium genera 163 Hunnemannia genera 163 Hylomecon genera 163 Hypecoum genera 163 Meconopsis gQuera, 163 Platystemon genera 163 Pteridophyllum genera 163 Romneya genera 163 Stylophorum genera 163 Papaver oreophilum 163 Papaver radcatum 163 Papaver somniferum 163 Paraguay 309 of Stevia 309 ParishinA 581 Parthenolide 565,856 Pelargonium graveolens 375 Pelargonium roseum 377 Penicillium oxalicum 849 Pentacyclic triterpenes 474 from Astragalus spp. 474 Pentadin 19,38 from Pentadiplandra brazzeana 19,38 Pentadiplandra brazzeana 18,19,38 brazzeinfrom 18 pentadin from 19,38

964 Perillartine 13,20 3,5,7,3',4'-Pentahydroxyflavone 841 from Perilla frutescens 13,20 Peptide hormones 794,798 Peptides 804 structure of 20 biological activity 804 Permethyl-0-rhamnose moiety 879 Peptides Peroxidases as catalysts 755 from animal tissues 802 in synthesis of bioactive plant from plant tissues 802 phenols 755 Periandra dulcis Mart 15,16,30 in synthesis bioactive periandrin I from 16,30 alkaloids 788 periandrin n from 16,30 Peroxidase-catalyzed dimerization 765 periandrin III from 16,30 ofp-coumaryl-hydroxy periandrin IV from 16,30 oxyagmatine 765 periandrin V from 16,30 ofp-coumaryl-hydroxyagmatine Periandra mediterranea (Veil.) Taub 765 15,16,30 Peroxidase-mediated oxidation 777 periandrin I from 15,30 of (+)-catechin 777 periandrin II from 16,30 Peroxidases 735 periandrin III from 16,30 from grapevine 756 periandrin IV from 16,30 from Vitis vinifera 756,757 Periandrin II 16,30,31 heme-containing enzymes from Periandra dulcis 16,30 731,736 from Periandra mediterranea for synthesis of natural bioactive alkaloids 778 16,30 Peroxidized oil-treated group 408 structure of 31 3a-Peroxyarmefolin 550 Periandrin III 16,30,31 Peroxyparthenolide 566 from Periandra dulcis 16,30 Perpenthylated-P-cyclodextrin 368 from Periandra mediterranea Petitgrain oil 373 16,30 structure of 31 from leaves 373 Periandrin IV 16,30,31 from twigs 373 from Periandra mediterranea Phaca(L,) 444 16,30 Pharmacological actions 393,431 from Periandra dulcis 16,30 of Baicalein 431 structure of 31 Pharmacological properties 767,844 Periandrin V 16,30,31 P-Phellandrene 372 ftom Periandra dulcis 16,30 (-)-a-Phellandrene 379 structure of 31 (+)-a-Phellandrene 379 Periandrin I 15,30,31 Phenobarbital 5,6-benzoflavone 304 from Periaudra dulcis Mart 15,30 ofStevia 304 from Periandra mediterranea Phenolic compounds 483,836,853 (Veil.) Taub 15,30 Phenolic glycosides 687 structure of 31 3-Phenyl derivatives 841 Perilla frutescens 10,13,20 Phenyl propanoid 768 Perillartine 10,13,20 Penstemon linarioi 837 as sweetener 10 from phenylethanoid from Perilla frutescens 10,13,20 glycoside 837

965 Phenylpropanoid glycosides 836 from Digitalis purpurea 837 from Penstemon linarioi 837 Phenylpropanoid moieties 853 Phlomis betonicoides 14,24 baiyunoside from 14,25 phlomisoside I from 14,25 Phlomisoside I 14,25 fram Phlomis betonicoides 14 structure of 25 Phlorizin 17,33,34 from Symplocos lancifolia Sieb et. Zucc 17 structure of 34 Phoenix canariensis 840 Phorbol esters 830 Phorbol-12,13-dibutyrate 831 Phosphate acceptor 822 Phosphatidylinositol-3,4,5-trisphosphate (PIP3) 828 Phosphatidylinositol 3-kinase 843 Phosphatidylinositol kinases 842 orr/zo-Phosphoric acid 820 Phosphorylation 823 of histones 876 ofoxysterol 872 of serine 821 Photoaffinity probes 330 Photo-irradiation activity 850 Photoisomerization 354 of ABA 354 cis, ^a«5-Photoisomerization 355 Phyllodulcin 7,5,17 from Hydrangea macrophylla 1,\1 of stevioside 5 structure of 7 thaumatin 5 Phyllomedusin 800 Physalaemin 800 Physicochemical properties 363,365 of cyclodextrins 365 Phytoalexins 60-62,498 from ethylene 60 from glycoproteins 60 fromjasmonic acid 60 by liberation of enzymes 61 oligopeptides as 60 oligosaccharides as 60

reactive oxygen compounds (ROS) as 61 salicylic acid as 60 generation of 60,61 Phytolysis 330 of bovine serum albumin 330 Picea abies (L.) Karl. 370,385 Picea species 372 Pilocarpus jaborandi 522 (+)-a-Pinene 373 (-)-a-Pinene 385 structure of 385 (-)-P-Pinene 372 (+)-p-Pinene 372 Pinuspeuce 370,372 Pinus species 372 Pinus sylvestris 370 Piper marginatum Jacq. 16 /raw5-anethole from 16 Pissodes 384 Plant injury 59-88 chemical responses to 59-88 Plant aging 59-88 Plant peroxidases as versatile catalysts 737 insect-plant relationship 378 Plasminogen activator inhibitor-l(PAI-l) 433,435 structure of 435 Plasmopara viticola 768 Platelet arachidonate metabolism 422 of polyacetylene compoimds 422 from Saposhnikoviae radix 422 Platelet-derived growth factor 847 Platycarya genera 755 Polyacetylene alcohols 686 structure of 686 Polyacetylene compounds 422, 660,686, 870 from acetonic extract 686 frompanaxynol 870,871 from Bupleurum spp. 686 from roots Polycanthisine alkaloid 251 from genus Astragalus 251 NMR studies of 251,252 Polychlorinated biphenyl 304

966 Polygnum cuspidatum 405 for hyperlipidaemia 405 Polygraphus poligraphus 370 Polyhydroxyindolizidines 252 biological activity of 252 fi^om Astragalus spip. 523 Polymerization 760 of dihydrodiconiferyl alcohol 760 of guaiacylglycerol-P-O-coniferyl alcohol 760 of ciimamyl alcohols 760 of coniferyl alcohol 760 of/7-coumaryl alcohol 760 of sinapyl alcohol 760 ofpinoresinol 760 Polypodium glycyrrhiza 16,32 polypodoside A from 16,32 polypodoside B from 16,32 Polypodium vulgare L. 16 osladinfrom 16 Polypodoside A 16,32 from Polypodium glycyrrhiza DC 16 structure of 32 Polypodoside B 16,32 from Polypodium glycyrrhiza 16 structure of 32 Polysaccharides 506 from Astragalus membranaceus 506 Polyunsaturated fatty acids 59,62,67 Porphyrins 745 Praeterienolide 554 Prenyl substituents 929 Prenylated chalcone 928 from hops 928 Prenylnaphto-l,2-quinone 851 Prenylphloroglucinol 853 from Mallotus philippinensis 853 Prevention by carp extract 439 of gastrointestinal toxicity 439 of myelotoxicity 439 Prevention by chitosan 436 of myelotoxicity 436 Prexylisoflavones 844 Prickles 59 as plant protecting organs 59 Primary hormone 321

Proanthocyanidins 35,37,893 from Archniodes sporadosora 35 from Arachiniodes exilis 35 structure of 37 Pro-apoptopic 851 Probes 332 activity of 346 for 7',7'-difluoro-ABA 348 for 7'-hydroxylation 348 for8'-hydroxylation 346 for ABA receptors 332 for abscisic acid receptors 330 for determining the abscisic acid reception 331 for isomerization to phaseic acid 348 for structure activity relationship 332 of abscisic acid 332 Procyanidins 837 from Pseudotsuga menziesii 837,838 Prokaryots 736 9'-Propargyl-abscisic acid 348 Propranol 394 from glucose 394 5-Pro^ proton 338 Prorocentrum 875 Protein binding 377 Protein kinase C 822 classical forms of 822 Protein kinase classical form 822 of glycoprotein 737 Protein kinase C (PKC) 827,828,830,834 Protein phosphatase inhibitors 874 Protein phosphate 873,872 Protein phosphorylation 819 by natural products 819 biochemical basis of 820 Proteinaceous polysaccharide 511 G-Protein-coupled receptor 821 Protein-tyrosine kinase activity 842 offlavonoidaglycones842 of glycosides 842 of Koelreuteria henryi 842 Protoberberine alkaloids 749

967 Protopine 162 from Chelidonium majus 162 Proxyelocytic leukaemia (HL-60) cells 842 Pseudobases 171 disproportionation of 174 of sanguinarine 174 Psudobaptigenin 498 Pteridophyta 735 Pterocarpans 498 fiom Astragalus s^i^. 498 Pterocarpenoids 894 Pterocarya paliurus Batal 16 Pterocaryoside A from 16 Pterocaryoside B from 16 Pterocaryoside A 16,31,32 from Pterocarya paliurus Batal 16 structure of 32 Pterocaryoside B 16,31,32 from Pterocarya paliurus 16 structure of 32 Protein-tyrosine kinase inhibitory activities 839 PTX341A 247-249 from Epipedobates tricolor 247 structure of 249 PTX-A alkaloids 247-249 PTX B alkaloid 249,250 PTX 251 D alkaloid 250 (+)-Pulegone 375 from Mentha piperita 375 from Mentha longifolia 375 from Mentha pulegium 375 from Mentha sylvestris 375 Pulegone 375 structure of 375 Pumiliotoxin alkaloids 238,239 allO'?TX 323 B alkaloid 249,250 biological activity of 249,250 fragmentation pathway of 239 Punctatine 159 from Zanthoxylum punctatum 159 Pyranocoumarin decursin 835 Pyrethrin 581 PyrethrinI 589 Pyrethrin II 589 Pyrethroidinin 582 Pyrethrosin 566

Pyroglutamic acid 394 from red Ginseng root 394 structure of 394,395 Quaternary benzo [c]phenanthridine alkaloids chelerythrine 155,156, 161,162 chemistry of 155 fagaridine 156 formation offree bases in 155 from Bocconiasptcits 155 from Chelidonium majus 15 5,162 from Dicranostigma lactucoides 155,162 from Macleaya cordata 155 from Macleaya microcarpa 155 from Zanthoxylum species 155 in Fumariaceae family 155,157, 161 in Papaveraceae family 155,157, 161 in Rutaceae family 155,161 isofagaridine 156 nitidine 156 punctatine 156 sanguinarine 155,156,161,162 Quercetin 895,746 Quercus faginea 663 Quercus genera 755 Quercus ilex subsp. ballota 663 Quinolizidine 244,246 from Mantella species 244 structure of 244,246 Quinolizidine alkaloid 233,276,287 bioactivity of 233 for Lupinus albus 276 for Lupinus angustifolius 276 fromamphibans 234 from Anagyris foetida 278 from ants 234 from Lupinus mutabilis 278 structure of 233 Quinolizidine derivatives 284,285 testing against Mycobacterium tuberculosis 284 orr/io-Quinones 849 Quinones 849

968 Quisvagenin 450 I-Rhamnose 506 from Astragalus quisqualis Bunge L-Rhamno-a-l,4-D-galacturonan 506 450 Rhizoctonia leguminicola 515 Rhizomes 277 Radical oxidation 346 of Caulophyllum thalictroides 111 Ravensara aromatica 311 Rhodophyta 735 Reactivity Rhubarb (i?/iet/m/7fl/marMm) 849,850 of enzyme intermediates 740 Ribesrubrum 839,840 Rebaudioside A 5 extracts of 839 Rebaudioside 314 Ribes sanguineum 839 in Sprague-Dawley 314 extracts of 839 Rebaudioside 8,9,13,22 Richardella dulcifica 39 Rebaudioside A 300 miraculinfrom 39 from Stevia rebaudiana 8,300 Ridentin 566 Rebaudioside B 13,23,300 Rimantidine 108 ftom. Stevia rebaudiana 13,300 for treatment of influenza structure of 23 in infections 108 Rebaudioside C 13,22,23,300 Robinia pseudoacacia 374 from Stevia rebaudiana 13,300 Robustic acid 844 structure of 23 RotatingframeNuclear Overhauser Effect Rebaudioside D 13,23,300 spectroscopy 669 from Stevia rebaudiana 13,300 Role of structure of 23 cholecystokinin Rebaudioside E 13,23,300 gastrin 797 from Stevia rebaudiana 13,300 protein phosphorylation 821 structure of 23 Roosters 302 Receptor binding 377 ROS (Reactive Oxygen Spp.) 504,505 Receptor-like protein tyrosine Rotational isomers 340 phosphatases 874 structure of 340 a3p2-Receptors 282 Rotenoid 893 a4p2-Receptors 282 Rottlerin 853,854 Receptors 363 Rubus suavissimus 13,14,22-24 of chiral proteins 363 e«/-Kaurene from 22 with enzymatic activity 824 labaneglycosides from 22 without enzymatic activity 825 rubusodiefrom 13 4'-Reduced- abscisic acid 334 steviol 13-(9-p-D-glucoside Reduction from 13 from Croton genera 830 suavioside A from 14,22 from Euphorbia genera 830 suavioside B from 14,22,23 of blood leukocyte 437 suavioside G from 14,22,24 suavioside H from 14,22,24 of lymphocytes 437 suaviosdie I from 14,22,24 of diterpenes 830 suaviosdie J from 14,22,24 Sapium genera 830 Responsive element binding protein 825 Rubusoside 13,22,23 Resveratrol 854,855 from Rubus suavissimus S. Lee 13 tranS'Res\QT3tio\ oligomers 768 structure of 23 Reynosin 555 RupicolinA 582

969 RupicolinB 582 Rutaceae 361,372,375 (-)-Sabinene 372 (+)-Sabinene 372 Saccharum officinale 191 Safety evaluation 299 of stevin 299 of stevioside 299 SaikogeninF 671 Saikosaponin 6 674 structure of 675 Saikosaponin 8 674 structure of 675 Saikosaponin F 693 from Bupleurum kaoi 693 Saikosaponins 660,665,666,667 Bupleurum rigidum 665 from Aerial parts 665 Saikosaponins 671 from aerial parts of Bupleurum rigidum 671 Saikosaponins from Bupleurum falcatum 688 Salicylic acid 63 elicitor of pathogen related genes 63 generation of 63 Salmonella typhimurium TA 305 Salmonella typhimurium TM677 305 Salvia dominica 377 Salviafruticosa2>16^311 Salvia officinalis 2>16,2>11 Salvia sclarea 2>16,2>11 Sandrosaponin I-X 677,688 Sanguidimerine 138,169 from Sanguinaria canadensis 158,169 structure of 169 Sanguilutine 159 in Chelidonium majus 159 from Sanguinaria canadensis 159 Sanguilutine 172 free bases of 177 pK constant of 172 Sanguinaria canadensis L. 155,158,159, 162,169

Sanguinarine 158,164,172,173,177,178 biological activities of 178,179 derivatives of 172,173 from Bocconia pearcei 163 from Chelidonium majus L. 158 from Macleaya cordata 162 from Macleaya microcarpa 162 from Papaver oreophilum Rupr. 163 from Papaver radicatum Rottb. 163 from Papaver somniferum 163 from Sanguinaria canadensis L. 158 in Corydalis caucasica DC. 163 in Corydalis severtzowii Rgl. 163 in Fagaropsis angolensis 164 in Zanthoxylum conspersipunctatum 164 pK constant of 172 Sanguirubine 156-158,159,172 chelirubine 156-158,161,162 from Sanguinaria canadensis 159 pK constant of 172 Saniculoideae subfamily 661 Santamarine 555 Sapindus rarak DC 13 mukurozioside from 13 SAPK/JNK (Stress-activated PK/cJun Nterminal kinase 829 Sapogenin 448,473 from Astragalus oleifolius DC 448 from Astragalus microcephalus 473 Sapogenin II 453 from Astragalus glycyphyllos L. 453 Saponin C2 41,45,46 from Hovenia dulcis Thunb. var. tomentella Mdkino 41 structure of 46 Saponin E 41,45,47 from Hovenia dulcis Thunb. var. tomentella Mdkino 41 structure of 47 Saponin H 41,45,47 from Hovenia dulcis Thunb. var.

970 tomentellaMakino 41 Sesquiterpene lactone 856 structure of 47 from Astragalus spi^. 478 Saponin mixture 466 from Tanacetum parthenium 856 from Astragalus glycyphyllos 466 structure of 479 Saponins 446,671 Sialic acid analogues 116-129 of cycloartane series 446 aromatic sialic acid analogues Saponins 481 134-140 from Astragalus membranaceus carbocyclic sialic acid analogues 481 140-141 Saposhnikovia divaricata All long chain ester analogues Scandenoside Rl 1 15,25,26,27 122-129 from Hemsleya panacispolar analogues 118-122 scandens 15,25 Sialic acids 103 structure of 27 biological ftinction of 103 Scandenoside R6 15,25,26 effect on viscosity of glycoprotein from Hemsleya panacis105 scandens 15,25 glycosidic linkage of 105 structure of 26 in transport of molecules 105 Scanning electron microphotographs 399 in transport of viruses 105,106 structure of 399 model for 111 Schlerochiton ilicifolius A 18,38 structure of HAl binding site 110 monatinfrom 18 Siamenosidel 15,26,27 Scoporia dulcis 191 from Siraitia grosvenorii 15 Scutellaria baicalensis 431 from Siraitia siamensis 15 Secale cereale 191,193,125,216,217 structure of 27 Secalonic acid 849 Simulanoquinoline 167 Secophenanthoindolizidine alkaloid 517 in Zanthoxylum simulans 167 Secretoryfrmgalperoxidases 736 Sinapyl alcohol 743 Sedative 660 Siraitia grosvenorii 6,7,14,15 Seleniferous Astragalus 520 mogroside IV from 14 Selliguea feei Bovy 18,35 mogrosideV 6,7,14 selligueain A from 18,35 siamenoside I from 15 SelligueainA 18,35,36,37 Siraitia siamensis 14,15 from Selliguea feei Bory 18,35 siamenoside I from 15 structure of 36 (-)-Siringaresinol 503 Senescent plant 86 from Astragalus mongholicus 503, from Astragalus angustifolius 466 504,508 lipid peroxidation products of 86 structure of 504 sapogenins 466 Sitakisogenin 45,48 Separation scheme 507 structure of 48 of polysaccharide 507 Sitakisosides I-IX,XI-XIII,XVI,XVIII 41, ftom Astragali radix 507 45,47,48 Sepration method 507 from Stephanotis lutchuensis Sequiterpenoids 855 Koidz. ydx.japonica 41 Serine receptor 824 structures of 48 Serpentine 782 P-Sitosterol 477 (+)-Sesamin 593 SivasinoHde 556 Sesquiphellandren structure 587

971 Slaframine 252 biological activity of 252 from Rhizoctonia leguminicula 255 Slaframine alkaloids 250,255 as muscarinic agonist 255 from Rhizoctonia leguminicula 255 Slaframine type alkaloids 250 HPLC determination of 251 from Astragalus 250 from Eleocarpus 250 from Iponuga 250 Small cell lung cancer 799,802 Smilax glycyphylla Sm. 17 glycyphyllin from 17 Smimovia turkestana Bymge 517 Somatostatin analog 795,796 Sotmatal opening 345 for abscisic acid 345 Soyasapogenol B 453 from Astragalus complanatus 481 Soyasaponins 398 from soybeans 398 structure of 398 Sparteine 279,280 Sphaerotheca pannosa 178 Spiciformin 567 Src homology domain 2 824 Staurogyne merguensis Wall. 39,40 strogin 1 from 39,40 strogin 2 from 39,40 strogin 4 from 39,40 Staurosporeus 862,863 Stellattamide B 251 from Korean sponge 251 Stephanotis lutchuensis Koidz. var. japonica 41 sitakisoside I-IV, XI-XIII from 41 sitakisoside XVI from 41 sitakisoside XVIII from 41 Steric effect 355 of benzene ring 355 Steroid hormones 823 Steroidal saponins 477 from Astragalus alexandrinus All Stevia 299-317 acute toxicity tests of 307

assweetner 299-317 chronic toxicity studies of 306-308 consequences for bioavailability of nutrients 312-314 effect on fertility 309,310 nutritional significance of 314,315 safety evaluation of 299-317 subacute toxicity studies of 307,308 5/evw extract 307,310,314 for diabetics 314 for stevioside 307,310 Stevia leaves 315 immunological activity of 315 5^ev/a plant 315 allergenic activity of 315 Stevia rebaudianaBcrtord 13,22,299 Stevia rebaudiana 8,9,13,22,299,311 e«^kaurene from 22 labdane glycosides from 22 of asteraceae 299 rebaudioside A from 8,9,13 rebaudioside B from 13 rebaudioside C from 13 rebaudioside D from 13 rebaudioside E from 13 stevia extract from 8 steviolbioside from 13 stevioside from 8 sugar-transferred Stevia extract 8 Stevia water extracts 311 Steviol 304,305,307,312 acute toxicity tests of 307 effect on fertility 312 in Salmonella typhimurium TM677 305 mutagenicity tests with 304 Steviol 13-0-p-D-glucoside 13,22,23 from Rubus suavissimus 13,22 structure of 23 Steviol epoxide 302 Steviolbioside 13,23,300 from Stevia rebaudiana 13,300 strucutre of 23 Stevioside 8,9,13,22,299-317 acute toxicity tests of 307 assweetner 299-317 chronic toxicity studies of

972 306,307,308 effect on fertility 309,310 for phenylketonuria 315 from Stevia rebaudiana 8,9,13 from Stevia rebaudiana Bertoni 299 metabolism of 301,302,314 mutagenicity tests for 303,304 nutritional significance of 314,315 purity of 316,317 structure of 300 subacute toxicity of studies of 307,308 Stigmasterol 477 from Astragalus glycyphyllos All Stilbene oligomers 767 Stilbenes 745,766,854 from Polygonum cuspidatum 854 Streptococcus mutants IIS Streptococcus sobrinus 316,778 Strictosidine synthase 781 from secologanin 781 from tryptamine 781 from strictosidine 781 Stroginl 2,39,40 from Staurogyne merguensis Wall. 39,40 structure of 40 Structure elucidation 467,667 of saponins 667 by MS, IR, NMR spectroscopy 659 Structure of 323,438,423,501,677,758, 761,762,823 of achillamide 480 of acylated flavonoid glycosides 501 of astragaluquinone 500 of black sugar-1 412 ofsandrosaponinlll 674 of sandrosaponin 674 of eucryphin 397 of flavonoid 484 of saponins 677 of monoterpenoid indole alkaloids 781 of nitrocompounds 532 of 2-phenyl-4H-1 -benzopyran-4-

one 841 of tea saponins 402 Structure-activity relationship 893,923 offlavanoids 923 Structure identification 240-247 of (-)-myrmicarin 240-247 of (+)-myrmicarin 240-247 Structure-odour relationship 378 Structures ofconiferylalchol 746 Strychnine from Strychnos nux-vomica 780 Stylophorum diphyllum 159 Stylophorum lasiocarpum 159 SuaviosideA 14,22 from Rubus suavissimus 14,22 strucutre of 24 SuaviosideB 14,22,23 from Rubus suavissimus 14,22 structure of 23 SuaviosideG 14,22,24 from Rubus suavissimus 14,22 structure of 24 SuaviosideH 14,22,24 from Rubus suavissimus 14,22 structure of 24 Suaviosidel 14,22,24 from Rubus suavissimus 14,22,24 structure of 24 SuaviosideJ 14,22,24 from Rubus suavissimus 14,22 structure of 24 Subacute toxicity 307 from animals 307 of stevioside 308 Substrate specificity 745 of peroxidases Sucrose 3,4,5 as sweetener 3 Sucrose substitutes 4,5 acesulfame-kas 4 sweetness of 4,5 aspartame as 4 cyclamate as 4 glycyrrhizin as 5 mogrosideVas 5 neotame as 5 saccharin as 4

973 sucralose as 4 sucronic acid as 4 super aspartame as 5 Sugar sweetner mechanism 4,12 non-sugar sweetner system 11,12 Suicide inhibition 348 of 8'-hydroxylase 348 Suicide inhibitors 347 for abscisic acid 8'-hydroxylase 347 Sulfated spectra of trisaccharide 669 ^om. Bupleurum rigidum 659,669 Sulfate olefaction 678 of saponinfromBupleurum rigidum 678 of sandrosaponinl-X 678 Sulfated trisaccharide moiety 673 Sulfoxidation 752 of methyl p-tolyl 752 Swainsona canescens 515 Swainsona sipQCiQS 514 Swainsonine 252,253,255,524,525 biological activity of 252 effect on Lupinus species 254 from Astragalus s^t. 253 from gastrointestinal tract 525 from Metarhizium anisoplilae 255 from Oxytropis sp. 253 from Rhizoctonia leguminicula 254,255 from Swainsona galegifolia 255 gastanospermine 525 in Astragallus species 253 in Oxytropis species 253 inhibition (-i-)-ramannosidase by 255 inhibition cellular mannosidases by 252 inhibits naranginase 255 Sweet taste 10,11 activation by receptor-independent mechanism 12 receptor for 11 Sweetness inducers 39,40 arabinogalactin as 39 caffeic acid conjugates cynarin as 39 chlorogenic acid as 39

curculinas 39 miraculinas 39 strogin 1 as 39,40 strogin 2 as 39,40 strogin 4 as 39,40 Sweetness inhibitors 40 altemoside 141 altemosidell 41 altemoside III 41 altemoside IV 41 altemoside V 41 from Gymnema alternifolium 40 from Gymnema sylvestre 40-43 from HoveniadulcisThyxrrAy. 40 from Stephanotis lutchuensis Koidz. yar.japonica 40 from Ziziphosjujuta P. Miller 40 gymaemasaponin III 41,43 gymaemasaponin IV 41,43 gymaemasaponin V 41,43 gymnemic acid I 41,42 gymnemic acid II 41,42 gymnemic acid III 41,42 gymnemic acid IV 41,42 gymnemic acid IX 41,42 gymnemic acid V 41,42 gymnemic acid VI 41,42 gymnemic acid VIII 41,42 gymnemic acid X 41,42 gymnemic acid XI 41,42 gymnemic acid XII 41,42 gymnemic acid XIII 42,42 gymnemic acid XIV 41,42 gymnemic acid XV 41,42,45 gymnemic acid XVI 41,42,45 gymnemic acid XVII 41,42,45 gymnemic acid XVIII 41,42,45 hoduloside 141 hodulosidell 41 hoduloside III 41 hoduloside IV 41 hoduloside IX 41 hoduloside V 41 hoduloside VII 41 hoduloside VIII 41 hoduloside X 41 hovenosidel 41 jujubasaponin II 41

974 jujubasaponin III 41 jujubasaponin IV 41 jujubasaponin V 41 jujubasaponin VI 41 jujubasaponin zizyphus saponin III 41 jujubosideB 41 jujubosideB 41 saponin C2 41 saponin E 41 saponin H 41 sitakisoside XIII 41 sitakisoside XVI 41 sitakisoside XVIII 41 sitakisoside I 41 sitakisoside II 41 sitakisoside III 41 sitakisoside IV 41 sitakisoside IX 41 sitakisoside V 41 sitakisoside VI 41 sitakisoside VII 41 sitakisoside VIII 41 sitakisoside X 41 sitakisoside XI 41 sitakisoside XII 41 Ziziphin 41 Zizyphus saponin 141 Zizyphus saponin II 41 Symplocos Helminthosporium carbonum UUstrup 498 Symplocos lancifolia Sieb. et Zucc 17,33, 34 phlorizin from 17,33,34 Symplocos microcalyx Hayata 17,34 trilobatin from 17 2-(p-Tolyl)-sparteine 263 monoperchlorate salt 263 Tachykinins 798 of substances? 798 of substances K 798 of neuropeptides 798 TagetesfilicifoliaLag. 16 /ra«5-anethole from 16 Talin protein 9,10 as sweetner 9,10 Tamirin 568

Tanacetin 556 TanacetolA 574 TanacetolB 574 Tanacetum albipannosum 570,590 Tanacetum annuum 575 Tanacetum argenteum 551,565,579 Tanacetum argyrophyllum 555 Tanacetum aucheranum 586,587 Tanacetum balsamita 551,591 Tanacetum chiliophyllum 551,556 Tanacetum cilicium 575 Tanacetum cinerariaefolium 551,571 Tanacetum corymbosum 586 Tanacetum densum 551 Tanacetum ferulaceum 549 Tanacetum heterotomum 590 Tanacetum indicum 575 Tanacetum macrophillum 564,574 Tanacetum odessanum 586 Tanacetum parthenifolium 581 Tanacetum parthenium 548,566 Tanacetum polycephalum 5 5 9,5 65 Tanacetum poteriifolium 560 Tanacetum praeteritum 549 Tanacetum pseudoachillea 557 Tanacetum ptarmicaeflorum 558,567 Tanacetum santolina 550,555,566 Tanacetum santolinoide 551,567 Tanacetum serotinum 550 Tanacetum sinaicum 553 Tanacetum species 634,547 allergent activity of 634 anticoagulant and antifibrinolytic activity of 635 antihelmintic activity of 636 antiinflammatory activity of 637 antiulcer activity of 639 biological activities of 547 cytotoxic effects of 639 insecticidal activity of 641 phytochemical studies of 548-594 phytotoxic activity of 643 prophylactic activity against migraine 643 Tanacetum tanacetioides 555 Tanacetum vulgare 548,549 Tanachin 569 TanalbinA 570

975 TanalbinB 570 5^co-Tanapartholide 585 Tanaparthin-a-peroxide 582 Tanaparthin-p-peroxide 582 Tanaphillin 586 Tanapraetinolide 556 Tanapsin 557 Tanargyrolide 570 wo-Tanargyrolide 570 Tanavulgarol 593 Tanciloide 583 wo-Tanciloide 583 Tanciloide 8a-methylbutyrate 583 Tannin ,837,840 from Rhus species 840 TannunolideA 583 TannunolideB 584 Tannunolide C 584 TannunolideD 584 Tannunolide E 584 Tansanin 570 Taraxasterol 591 TatridinA 571 TatridinB 571 Tautomerization 771 ofM04 771 Tautomycin 879 from Streptomyces spiroverticillatus 879 Taxol 857 Terihanine 160 from Zanthoxylum nitidum DC. 160 6-oxoderivative of 160 Terminalia genera 755 Terpenoid 471 Terpenoid sapogenins 446 Terpenoids 20,855 (-)-Terpinen-4-ol 370,376 structure of 376 (-)-Terpinen-4-ol 377 (-)-a-Terpinol 364 (+)-a-Terpinol 364 Tessaria dodoneifolia 17,34,35 dihydroquercetin 3-0-acetate 4'methyl ether from 17,34

{2R, 3/?)-dihydroquercetin 3-0acetate from 17,34 Tetracyclic lupine alkaloid 268,273 frovaLupinus sp. 273,268 12-0-Tetradecanoylphorbol-13-acetate 482,827,830 Tetradium glabrifolium 164 benzophenanthridine alkaloids in 164 ^w-Tetrahydroxanthone 849 2,3,5,4'-Tetrahydroxy stilbenez-2-O-Dglucoside 407,408 24(^-3p, 16P,24,25-Tetrahydroxycycloartan-6-one 448 hora Astragalus membranaceus Bunge 448 5,7,3',4'-Tetrahydroxyflavone 843 1,6,7,8-Tetrahydroxyindolizidine 514 3p,22p,24,29-Tetrahydroxyolean-12-ene 453 from Astragalus trojanus Stev. 453 3 p, 16P,21 p,28-Tetrahydroxyoleanan-12en-22-one 47,48 structure of 48 2',3,4',5-Tetrahydroxystilbene 768 3,3',4',5-Tetrahydroxystilbene 768 Tetratogenicity 309 Thaumatin 19,38 from Thaumatococcus daniellii 19 Thaumatin 9,10 analogs of 9,10 from Thaumatococcus daniellii 9,10 Thaumatococcus daniellii 19 thaumatin from 19 Therapeutic potential 893 offlavonoids 893 Therapeutic effects 379 of aroma 379 Thermodynamic characteristics 350 3'-Thioether 331 Thiol reagents 736 Threonine kinases 824 Thuja plicata 384 (+)-3-Thujone 384 Thymic himioral factor-gamma 2 804

976 a-Thymosins 804,806 p-Thymosins 804 Thymus vulgaris 374 Thyroid hormone 314 Thyroxine 314 Titerpenoid sapogenin and saponin 470 from Astragalus genus 470 T-Lymphocytes 799 Tobacco syntaxin 330 Toddaliaaculeata?QTS. 164,158,177 Toddaline 158 from Toddalia aculeata TQTS 158 Tomentoside 473 from Astragalus tomentosus 473 Topoisomerase 1-inhibitory properties 851 from Tabebuia avellanadae tree 851 Topsentia sponge 861 Toxicity effects on animals 444 Toxicity study 306 ofstevioside 306 Toxicity to reproductive organs 311 Toxicological studies 301 Tragacantha Bunge 444 Tragacanthin polysaccharides 514 from Astragalus brachycentrus 514 from Astragalus echidnaeformis 514 Traumatic acid 75 generation of 75 Trigonella foenumgraecum 277 1,2,8-Triacetate 516 TRIBoA 191,199,201 fromZeama>;5 191 from Crossandra pungens 191 seco-{ 11,12)-12,13-Tricyclic quinolizidines 265 dehydromultiflorine 265 seco-(l l,12)-5,6-dehydromultiflorine 265,266 Tricyclic resin acid 22 Tridecanonchelarythrine 167 in Zanthoxylum integrifolium 167

8',8',8'-Trideutero-ABA 348 Trifluorinate p-ionylideneacelic acid 346 of 16,16,16-trifluororetinal 346 8'-Trifluorinated analog 347 8',8',8'-Trifluoro-ABA 346 from methyl ester 346 Trihydroxy fatty acids 77 enzymic generation of 77 la,3p,4p-Trihydroxy-(5a,7a,l lpH,10a methyl)-eudesman-12,6a-ohde 553 3,16,25-Trihydroxy-2(i?), 24(5)epoxycycloartan-6,11 -diene 450 from Astragalus aitosensis M.B. 450 3P,16P,25-Trihydroxy-20(/?), 24(5)epoxy-9,19-cyclolanostan-6-one 450 from Astragalus membranaceus Bunge 450 la,3p,10a-Trihydroxy-7a,l IPHgermacra-4Z-en-12,6a-olide 563 3 p,6a, 16p-Trihydroxy-9,19-cyclolanost25-en-24-one 448 from Astragalus trigonus DC 448 ip,6a,8a-Trihydroxycostic acid methyl ester 558 1 P,4a,6a-Trihydroxy-eudesm-l 1-en6a,12-olide 553 5,7,4'-Trihydroxyflavone 842 1,7,8-Trihydroxyindolizidine 514 3,4',5-Trihydroxystilbene 767 Triiodoth)n:onine 314 Trilobatin 17,33,34 from Symplocos microcalyx Hayota 17 structure of 34 3,4,6-Trimethoxy phenyl-0-glucoside 410 2,6,10-Trimethyl-2,5-epidi-oxy-7,10epoxydodeca-11-en 594 (5S,6S,7S,10R)-2,6,10-Trimethyl.2,5epidioxy-7,10-epoxydodeca-3,11 -dien5-ol 594 2,3,6-Tri-(9-methyl 367 Tripterpenoid sapogenins 448 fiovci Astragalus sipp. 448 Triterpenoid saponins 454-465 from Astragalus spp 454-465

977 Trisaccharide moiety 675 5,6,8-Trisubstituted indolizidines 242,243 from Dendrobates pumilio 242 Triterpenoid glycosides 3 as sweetaess-inhibitory principles 3 from Asclepidaceae 3 from Rhamnaceae 3 Triterpenoid saponins 454 acetylastragaloside 456 agroastragaloside I 454 agroastragaloside II 454 agroastragaloside III 456 agroastragaloside IV 456 alexandroside I 454 asemestioside A 456 asemestioside B 456 asemestioside C 456 askendosideB 456 askendosideD 456 askenodoside A 454 askenodoside C 454 askenodoside F 454 askenodoside G 454 astrachrysoside A 456 astragaloside I 456 astragaloside II 457 astragaloside III 457 astragaloside IV 457 astragaloside V 457 astragaloside VI 457 astragaloside VII 457 astragaloside VIII 457 astragaloside VIII Me ester 464 astramembrannin II 457 astrasieversianin I 457 astrasieversianin II 458 astrasieversianin III 458 astrasieversianin IV 458 astrasieversianin IX 458 astrasieversianin V 458 astrasieversianin VI 458 astrasieversianin VII 458 astrasieversianin VIII 458 astrasieversianin X 458 astrasieversianin XI 458 astrasieversianin XII 458 astrasieversianin XIII 458

astrasieversianin XIV 458 astrasieversianin XV 458 astraverrucin I 459 astravermcin II 459 astraverrucin III 459 astraverrucin IV 459 astraverrucin V 459 astraverrucin VI 459 astresieversianin XVI 459 astrojanoside A 464 azukisaponin V 464 brachyosideA 454 brachyosideB 459 brachyoside C 454 cephalotoside A 454 comploside II 464 cycloalpioside 463 cycloalpioside A 450 cycloalpioside B 459 cycloalpioside C 459 cycloalpioside D 459 cycloaraloside E 459 cycloaraloside F 459 cyclocanthoside A 454 cyclocanthoside B 454 cyclocanthoside C 454 cyclocanthoside D 455 cyclocanthoside E 455 cyclocanthoside G 455 cyclocarposide 459 cyclocephaloside I 461 cyclocephaloside II459 cyclogaleginoside A 460 cyclogaleginoside B 460 cycloglobiceposide A 460 cycloorbicoside A 463 cycloorbicoside G 463 cyclopycnanthoside 455 cyclorbicoside B 463 cyclosieversiosdie F 460 cyclosieversiosdie G 460 cyclosieversioside D 460 cyclosieversioside E 460 cyclosieversioside H 460 dasyanthoside A 462 from Astragalus species 454 giganteoside D 464 huangqiuenin 460

978 huangqiyenin B 455 huangqiyenin D 460 isostragaloside I 460 isostragaloside II 460 isostragaloside IV 460 macrophyllosaponin D 455 macrophyllosaponin C 455 macrophyllosaponin B 455 macrophyllosaponin A 455 monghlicoside II 455 mongholicoside I 455 robinioside 464 sieberosidel 460 sieberoside II 461 sophoraflavoside II 464 soyasaponini 464 soyasaponin I Me ester 464 soyasaponin II Me ester 464 soyasaponin III Me ester 464 soyasaponin IV 464 tomentoside I 462 tomentoside II 462 trigonosidel 461 trigonoside II 461 trigonoside III 461 trojanosideA 461 trojanosideB 461 trojanoside C 455 trojanosideD 455 trojanoside F 455 trojanoside H 461 trojanosieE 455 Triterpenoid saponins 454,671 from Astragalus spp. 454 of saikosaponin 671 Triterpenoids 858 Triticum aestivum 191,193,215,217,218 Tritrophic interaction 213,214 with between Sitobion avenae 213 vnUti Aphidius rhopalosiphi 213 DIMBoAin 213,214 Tryptophan derivative 479 from Astragalus trojanus 479 Tulirinol 572 Timior necrosis factor 851 Tunefulin 585 Tyrosine kinase activity 854 Tyrosine kinases receptor 824,825,827

Tyrosine phosphorylation 856 Tyrosine-specific phosphatases 873,874 Umbelliferae gQiiQXdi 361,686 Unnatural enantiomer 346 a,P-Unsaturated carboxyl system 349 Unsaturated epoxy hydroxy acids 81,82 by nonenzymic induced oxidation oflinoleicacid 81 Uperolein 754 Ureterolithiasis-drug-therapy 382 Uridine 5'-triphosphate 820 Usambanoline 160 from Zanthoxylum chalybaeum 160 from Zanthoxylum usambarense 160 Vaccinia virus HI (VHl) 873 Vanicosides A and B 837 from Polygonum pensylvanicum 837 Vasoactive intestinal peptide 800,801 Vasopressin 800 Velocitermes velox 385 from Peru 385 Vetiveria zizanioides 377 Vinblastine 744,781,782 Vincristine 744,780,781 from Catharanthus roseus 744,780 Vindoline 747,782 1-2-Viniferin 767 E-Viniferin 767 (+)-a-Viniferin 855 Viniferins 11Q,166 in spermatophytic plants species 770,766 Viral glycoprotein 109 haemagglutinin 109-112 neuraminidase 112-114 Viscera 309 of fetus 309 Vitaceae 766 Vulgarolide 594 VulgaroneA 588 VulgaroneB 588

979 Warangalone 844,845 Weolignans 765 biological activity of 765 Wortmaimin (30) from Talaromyces wortmannii 858 Xanthium strumarium 346 Xanthohumol 928 Xanthones 902 Xenobiotics 744 Xenopus laevis 805 Xenopus-derived 807 Yama-Ninjin 429 Yeast glycogen phosphorylase 821 Zanthoxylum ailanthoides 167 Zanthoxylum avicennae 159 Zanthoxylum chalybaeum 160 Zanthoxylum conspersipunctatum 164 Zanthoxylum integrifolium 167 Zanthoxylum nitidum 159,160,168 Zanthoxylum punctatum 159 Zanthoxylum simulans 167 Zanthoxylum spinosum 170 Zanthoxylum tessmanii 159 Zanthoxylum usambarense 160 Zeamays 191,193,215 Zinnia elegans 765 Ziziphin 42,47-49 structure of 49 Ziziphus jujuba P. Miller 42,47,48 jujuba saponin II from 41 jujuboside B from 41 ziziphin from 41 zizyphus saponin I-III from 42 Zizyphus saponin I-III 42,49 structures of 49

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