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THE FACULTY OF MEDICINE IN THE UNIVERSITY OF LONDON

CNS ACTIVE PRINCIPLES FROM SELECTED CHINESE MEDICINAL PLANTS

Thesis presented by

MIN ZHU (BSc., MSc.)

for the degree of

Doctor of Philosophy

Department of Pharmacognosy The School of Pharmacy University of London

1994 ProQuest Number: 10105154

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT In order to identify potential central nervous system (CNS) active principles from plants, 10 Chinese herbs have been selected from literature reports, namely Schejflera hodinieri, Schejflera delavayi, Celastrus angulatus, Celastrus orbiculatus, Clerodendrum mandarinorum, Clerodendrum bungei, Periploca callophylla, Periploca forrestii, Alangium plantanifolium and Uncaria rhynchophylla. These plants were extracted by 70% and biologically screened by binding assays which included a 1-adrenoceptor, a2-adrenoceptor, p-adrenoceptor, 5HT1, 5HT1A, 5HT1C, 5HT2, , , Ca^-(DHP), K^-ion channel, 1, dopamine 2, adenosine 1, muscarinic, 1, Na'^/K^ ATPase, GABA^ and GABAg receptors.

The results of extract screening showed that all these plants were able to inhibit the

specific binding of radioligands to at least one receptor at concentrations of 1 mg/ml. Four of the species were then selected according to their distinct biological activities for further investigation in order to isolate their active principles . A total of forty-two compounds have been obtained by combination of various chromatographic techniques and their structures were determined by spectroscopic methods. The complete interpretation of all the spectra has been achieved and twelve compounds were found to be of novel structure.

From Schejflera bodinieri leaves and roots, fourteen triterpenoids and oligosaccarides have been obtained and eleven of them are novel. The extract screening showed that the plant was able to bind to a l and a2 adrenoceptors, 5HT1,5HT2, opiate, adenosine 1, Ca^-ion channel (DHP), K^-ion channel, dopamine 1, dopamine 2, GABA^ and GABAq receptors. In further screening, two compounds isolated from the plant bound to the 5HT2 receptor, one compound bound to the Ca^-ion channel receptor, one

compound bound to the dopamine 2 receptor and three compounds bound to the

muscarinic receptor (IC5 0 0.9-8.0 pM). Five of the compounds had interactions with or antagonists of p-adrenoceptor, Ca^^-ion channel, 5HT1C, 5HT1A, 5HT2, histamine 1, K^-ion channel or adenosine 1 receptors decreasing their IC50 values and some of these compounds were able to influence the binding sites of the receptors. Fifteen compounds have been obtained from Clerodendrum mandarinorum root bark and one of them is novel. In ligand binding assays, the plant extract was able to bind to 5HT1, 5HT2, opiate, dopamine 2, adenosine 1, K^-ion channel, GABA^ and GABAg receptors. Three isolated compounds were able to bind to adenosine 1, muscarinic and K^-ion channel receptors (IC 5 0 3-7.5pM). Six compounds had the ability to lower the IC50 values of agonists or antagonists of P-adrenoceptor, 5HT1 A, 5HT2, histamine 1, adenosine 1, Ca^-ion channel and K"^-ion channel receptors and change the binding sites of some of the receptors.

The extract of Alangium plantanifolium root bark was able to bind 5HT1, 5HT1A,

5HT2, histamine 1, adenosine 1 , dopamine 1 , opiate, Ca^^-ion channel, GABA^ and GABAg receptors. Five compounds have been obtained, two of them bound to the muscarinic receptor (IC50 6.7-S.5 pM) and two compounds have the ability to decrease the IC50 value of agonists or antagonists of adenosine 1, 5HT1A and 5HT1C receptors and alter the characteristics of some of the receptors.

The extract of Uncaria rhynchophylla bound to 5HT1A, 5HT2 and opiate receptors and eight compounds were obtained. Three compounds were able to bind to the K^-ion channel, 5HT1 A, 5HT2, opiate, a l and p-adrenoceptors (IC50 0.14-6.7 pM) and three compounds decreased the IC50 values of agonists or antagonists of 5HT1C, opiate, histamine 1 and K^-ion channel receptors and influenced some of the binding site.

The present investigation has resulted in the isolation of different chemical types of CNS active principles including triterpenoids and their glucosides, oligosaccarides, , and . The majority of clinical medicines in use are nitrogen-containing compounds, therefore it is possible that new non-nitrogen containing drugs may be developed from a knowledge of the structures identified in the present investigation. Plant medicines contain a mixture of chemical substances and their clinical efficacy cannot always be correlated with a single chemical component. The present results show that individual compounds may react with a single receptor or with a number of different receptors or affect other compound binding to receptors. The application of receptor ligand binding assays to plant extracts and their individual principles will further our understanding of the action of plant medicines. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Professor J. David Phillipson, Head of the pharmacognosy, for his conscientious supervision on phytochemistry research, and his constant encouragement, kindness and friendship. I am deeply grateful to Doctor Pam M. Greengrass (Discovery Biology Department, Pfizer Limited) for her invaluable guidance on the radioligand receptor binding work and her interest and kindness. Appreciation is also expressed to Professor Norman G. Bowery (Head of Pharmacology, The School of Pharmacy) for capable supervision on the pharmacological study, especially on the G ABA receptors research. These three supervisors gave me great help and consideration during the course of this study, without their encouragement, this work would not be possible.

I acknowledge the colleagues in Discovery Biology Department, Pfizer Limited for their friendly cooperation, in particular I would like to mention Mr. Mike Russell for his patient guidance in the practical ligand binding work, and Dr. Mike William, Head of the Department, for his carefulness and guidance in the whole Ph.D course. I would also like to express my sincere thanks to Doctor David V. Bowen (Head of Spectroscopy Department, Pfizer) for his kindly running partial NMR spectra and MS spectra in his leisure time.

My thanks also go to Professor Zhengyu Liu (Head of Department of Botany, Institute of Medicinal Plant Cultivation, Chong Qing, China) and his colleagues for their help in collection and identification the plant material. I thank Mrs. J. Hawkes for running the NMR experiments ( University of London Intercollegiate Research Service at King’s College) and Dr. K. Welham and his colleagues for running the mass spectra at the Mass Spectrometry Unit at The School of Pharmacy, University of London.

I am grateful to my friends and colleagues in the Department of Pharmacognosy, The School of Pharmacy with whom I have collaborated in various aspects of this work, particularly Doctor Shiling Yang, Doctor Ya Cai, Mrs. Hong-Wen Yu, Mrs. Janice Hallsworth, Miss Maria Camacho, Miss Caroline Lang’at and Dr. Pablo Solis. Also, I would like to thank Mrs. M. Pickett and Mr. G. Ronngren for their willing technical assistance and Mrs A. Cavanagh for graphical work in the preparation on posters for conferences.

I would like to express my appreciation and thanks to Professor J. David Phillipson, Doctor Pam M. Greengrass and Doctor Ya Cai for their attentively consideration, constructive suggestions and valuable discussions on the manuscript, from which I learned a great deal.

Undoubtedly, I am eternally indebted to my family members, for their encouragement, comprehension, cooperation and patience during these three years.

Finally, I gratefully acknowledge Pfizer limited for providing a research scholarship and modem facilities for the radioligand receptor binding assays. CONTENTS

Page

Abstract 2 Acknowledgement 5 List of Abbreviations 11 List of Figures 14 List of Tables 16

CHAPTER 1 INTRODUCTION 18 1.1 Central nervous system (CNS) disorders 19 1.1.1 Pain and 20 1.1.2 Depression and 23 1.1.3 Anxiety, insomnia and related medicines 28 1.1.4 and drugs 33 1.2 Receptors related to the CNS 37 1.3 Receptor ligand binding assays 47 1.4 Other pharmacological approaches for the CNS drug development 51 1.5 Plants as medical resource for the treatment of CNS disorders 52 1.5.1 Plants used for CNS disorders 52 1.5.2 Traditional Chinese medicine used for the CNS disorders 54 1.5.2.1 History of traditional Chinese medicine 54 1.5.2.2 Traditional Chinese medicine used for CNS disorders 56 1.6 The purpose and strategy of the present study 60

CHAPTER 2 MATERIALS AND METHODS 61 2.1 Plant materials 62 2.2 Preparation of plant extracts 62 2.3 Chromatographic techniques used for isolation 62 2.4 Spectroscopy for structure identification 64 2.4.1 Ultraviolet spectroscopy 64 2.4.2 Mass spectrometry (MS) 64 2.4.3 Nuclear magnetic resonance spectroscopy (NMR) 64 2.5 Hydrolysis of glycosides 65

2 . 6 The protocol of ligand binding assays for extract screening 65

2.7 The protocol of ligand binding assays for fraction screening 6 8

2 . 8 The protocol of ligand binding assays for compounds

screening 6 8 2.9 The protocol of ligand binding assays for testing the interaction of the isolated compounds with agonists or

antagonists of various receptors 6 8

2 . 1 0 Analysis of ligand receptor binding data 6 8

CHAPTER 3 IN VITRO BIOLOGICAL SCREENINGS OF 10 SPECIES OF CHINESE PLANTS 72 3.1 Introduction 73 3.2 The screening results of plant extracts 73 3.3 Discussion 84 3.3.1 Results of extract screening 84 3.3.2 Extract screening method 87 3.3.3 Summary 89

CHAPTER 4 Schejflera bodinieri 90 4.1 Introduction 91 4.1.1 Araliaceae and Schefflera 91 4.1.2 Schejflera bodinieri 93 4.2 Biological screenings on extract fractions 93 4.3 Isolation and structure identification of the isolated compounds 98 4.3.1 Identification of bodinitin A, bodinitin B, bodinitin C and bodinitin D 98 4.3.2 Identification of bodirin A, bodirin B, bodirin C and bodirin D 102 4.3.3 Identification of bodinone, bidinone glycoside and bodinin 105

8 4.3.4 Identification of D-sorbitol 108 4.3.5 Identification of trisaccharides 109 4.3.6 Identification of stigmasterol-3-O-P-D-glycoside 109

4.3.7 Spectral data 1 1 0 4.4 Biological tests on isolated compounds and related compounds 118 4.5 Discussion 124

CHAPTER 5 Clerodendrum mandarinorum 128 5.1 Introduction 129 5.1.1 Verbenaceae and Clerodendrum 129 5.1.2 Clerodendrum mandarinorum 130 5.2 Biological screenings of extract fractions 133 5.3 Isolation and structure identification of the isolated compounds 133 5.3.1 Identification of clerodirine 136 5.3.2 Identification of friedelanone 137 5.3.3 Identification of lupeol and betulinic acid 138 5.3.4 Identification of 24S-stigmasta-5, 25 dien-3p-ol and 22E, 24S-stigmasta 5, 22, 25 trien-3P-ol 139 5.3.5 Identification of cirsimaritin, cirsimaritin-4’-glucose and -3-methyl-ether 140 5.3.6 Identification of a-tetrahydropyrone 141 5.3.7 Identification of a-D-GIucopyranose-1 -ethylether, Sucrose and p-D-fructofuranose-2-ethylether 142 5.3.8 Identified a-D-glucopyranose and P-D-glucopyranose 143 5.3.9 Spectral data 143 5.4 Biological tests on isolated compounds and related compounds 150 5.5 Discussion 156

CHAPTER 6 Alangium plantanifolium 159

6 . 1 Introduction 160

6 . 2 Biological screenings of extract fractions 160 6.3 Isolation and structure identification of the isolated compounds 163

9 6.3.1 Identification of p-sitosterol, stigmasterol and sitosterol-3-O-P-D-glucopyranoside 165 6.3.2 Identification of 5p, 6P-hydroxyl-2,3-ene-cyclohexanoI -1-p-D-glucoside 166 6.3.3 Identification of p-D-fructofuranosyl-(l-4)-a-D-fructopyranoside 167 6.3.4 Spectral data 167 6.4 Biological tests on isolated compounds 173 6.5 Discussion 176

CHAPTER 7 Uncaria rhynchophylla 177 7.1 Introduction 178 7.2 Biological screenings of extract fractions 180 7.3 Isolation and structure identification of the isolated compounds 180 7.3.1 Identification of ursolic acid 183 7.3.2 Identification of hirsutine and epiallocorynantheine 183 7.3.3 Identification of (+)-, (-)-epicatechin, (+)-gallocatechin (-)-epigallocatechin and quercetin 186 7.3.4 Spectral data 187 7.4 Biological tests on isolated compounds and two alkaloids 191 7.5 Discussion 197

CHAPTER 8 GENERAL DISCUSSION 200 8.1 Difference between traditional Chinese medicine (TCM)

and western medicine 2 0 1 8.2 The Chinese medicine theory 203 8.3 Traditional Chinese medicine used for the treatment of CNS disorders 206 8.4 New drug development and TCM research 215

APPENDIX SPECTRA 221 REFERENCES 260

10 List of Abbreviations

jiM Micromolar ‘^C Dept Distortionless enhancement by polarization transfer (differentiation

between CH, CH2 , and CH 3 using the improved sensitivity of polarisation transfer) Centigrade '^C Carbon thirteen ‘H Proton 2D Two dimensional 5HT Al Adenosine 1 receptor Ach ATP Adenosine 5-triphosphate Bq Becquerel (1 radioactive delay per second) br Broad BuOH n-Butanol CC Column chromatography

CD3 OD Deuteromethanol

CDCI3 Deuterochloroform

CHCI3 Ci Curies (lCi=2.22 x 10*^ d.p.m.) CIMS Chemical ionization mass spectrometry cm'^ Centimetre to the minus one CNS Central nervous system COSY-45 Two dimensional ‘H-^H correlation spectroscopy d Doublet D1 Dopamine 1 receptor D2 Dopamine 2 receptor dd Double doublet DHP Dihydropyridines potent Ca^^ channel modulators with the ability both to inhibit and to stimulate transmembrane Ca^^ current

11 DMSO-dg Deuterodimethylsulphoxide DPM Radioactive decays per minute EIMS Electron impact mass spectrometry EtOAc Ethylacetate EtOH Ethanol eV Electron volt = 94.487 kJ/mol = 23.06 kcal/mol FABMS Fast atom bombardment mass spectrometry g Gram G ABA y-Aminobutyric acid Glc Glucose HI Histamine 1 receptor HPLC High performance liquid chromatography HRMS High resolution mass spectrometry hrs Hours Hz Hertz

IC5 0 Inhibition concentration fifty J Coupling constant K Association (affinity) constant Kd Dissociation constant Kg Kilogram L Ligand m/z The mass of the ion in dalton divided by its charge m Multiplet M Molar max Maxima MeOH Methanol mg Milligram MHz Mega Hertz ml Millilitre MS Mass spectrometry/spectrum NA Noradrenaline nm Nanometre

12 nM Nanomolar NMR Nuclear magnetic resonance ppm Part per million R Receptor Rham Rhamnose s Singlet SEM Standard error or mean sh Shoulder t Triplet TCM Traditional Chinese medicine TLC Thin-layer chromatography TMS Tetramethylsilane UV Ultraviolet Xyl Xylose

13 LIST OF FIGURES Page

Figure 1 . 1 Agonists of opiate receptors 2 1

Figure 1 . 2 Mixed agonists and antagonists of opiate receptors 2 2 Figure 1.3 Structures of some synthetic opiate candidates 23 Figure 1.4 The structures of some MAO Inhibitors 25 Figure 1.5 Some drugs 26

Figure 1 . 6 Some second generation antidepressants 27 Figure 1.7 Clinically used 31

Figure 1 . 8 Clinically used 32 Figure 1.9 Neuroleptic agent 36

Figure 1 . 1 0 Structures of plant psychodepressants 53

Figure 1 . 1 1 Structures of plant psychostimulants 54

Figure 3.1 The % inhibition of radioligand specific binding of the plant extracts at different receptors 78

Figure 4.1 The diversity of compound structures in Schefflera 92 Figure 4.2 Isolation of compounds from Schefflera bodinieri leaves 94 Figure 4.3 Isolation of compounds from Schejflera bodinieri roots 95 Figure 4.4 Possible MS fragmentation patterns of the aglycone of compounds 5, 7,13,14 100 Figure 4.5 Possible MS fragmentation patterns of the aglycone of compounds 3, 4, 10,12 104

Figure 4.6 Possible MS fragmentation patterns of compounds 1, 2, 6 106 Figure 4.7 The compounds isolated from Schejflera bodinieri 117

Figure 4.8 IC 5 0 curves of control compounds and the control compounds with

added isolated compounds 1 2 1 Figure 5.1 The diversity of compound structures in Clerodendrum species 132 Figure 5.2 Isolation compounds from Clerodendrum mandarinorum 135 Figure 5.3 The compounds isolated from Clerodendrum mandarinorum 149 Figure 5.4 Some flavonoids tested by ligand receptor binding assays 151

Figure 5.5 IC5 0 curves of control compounds and the control compounds with

14 added isolated compounds 153

Figure 5.6 Structures of benzo[b]furan, compound 21 and sorbifolin-4’-glucose 157

Figure 6.1 The diversity of compound structures in Alangium species 162 Figure 6.2 Isolation of compounds from Alangium plantanifolium 163

Figure 6.3 IC5 0 curves of control compounds and the control compounds with added natural products 175

Figure 7.1 Some compounds isolated from Uncaria rhynchophylla 179 Figure 7.2 Isolation of compounds from Uncaria rhynchophylla 181 Figure 7.3 The possible MS fragmentation pattern of compounds 36, 37 185 Figure 7.4 Compounds isolated from Uncaria rhynchophylla in the present study 190 Figure 7.5 The structures of and hemandizine 192

Figure 7.6 IC5 0 curves of control compounds and the control compounds with added natural products 194

Figure 8.1 Traditional Yin-Yang Symbol 204

15 LIST OF TABLES Page

Table 1.1 Common Brain Disorders in the United States 19 Table 1.2 Agonists and blockers of several receptors and their therapeutic potential 42 Table 1.3 Calcium ion channel receptors 46 Table 1.4 Adenosine receptor subtypes 47 Table 1.5 Examples of some texts on Chinese medicine plants, animal and minerals 56 Table 1.6 Some CNS active principles isolated from Chinese herbs 58

Table 2.1 Plants for biological screenings 63 Table 2.2 Radioligand receptor binding methods 70 Table 2.3 Buffer preparation 71

Table 3.1 %Inhibition of radioligand specific binding of plant extract against different receptors 74 Table 3.2 Plants Extracts with >90% Inhibition of ligand’s specific binding to different receptors 77

Table 4.1 The fraction screening results of Schejflera bodinieri (leaves) 96 Table 4.2 The fraction screening results of Schefflera bodinieri (roots) 97

Table 4.3 ^^CNMR data of compound 2 1 1 1 Table 4.4 ^^CNMR and ^HNMR spectral data of compounds 4,5,6 113

Table 4.5 *^CNMR data of compound 1 1 115 Table 4.6 Results of compound screenings 118

Table 4.7 The IC 5 0 values of control compounds and the control compounds

with the added isolated compounds 1 2 0

Table 5.1 Compounds isolated from Clerodendrum species 131 Table 5.2 The fraction screening results of Clerodendrum mandarinorum 134 Table 5.3 ^^CNMR and ^HNMR data of compound 24 147 Table 5.4 ^^CNMR and 'HNMR data of compound 26 148

16 Table 5.5 The IC 50 values of tested compounds 150

Table 5.6 The IC 5 0 values of control compounds and the control compounds with added isolated compounds 152

Table 6.1 Principles isolated from Alangium species 161 Table 6.2 The fraction screening results of Alangium plantanifolium 164 Table 6.3 ^HNMR spectral data of compounds 30, 31, 32 169 Table 6.4 ^^CNMR spectral data of compounds 30. 31, 32 170 Table 6.5 ^HNMR and ‘^CNMR spectral data of compound 33 171

Table 6 . 6 ‘^CNMR spectral data of the compound 34 172 Table 6.7 The results of compounds screenings 173

Table 6 . 8 The IC 5 0 values of control compounds and the control compounds with added isolated compounds 174

Table 7.1 The results of fraction screening of Uncaria rhynchophylla 182 Table 7.2 ‘HNMR spectral data of the compounds 38, 39, 40, 41 189 Table 7.3 The results of compound screenings 191

Table 7.4 IC5 0 values of control compounds and the control compounds with added natural products 193

Table 8.1 Traditional Chinese medicine used for the treatment of the CNS disorders 210 Table 8.2 Emperor Cardiotonic Pill 214

17 Chapter 1

Introduction

18 1.1 Central Nervous System Disorders Disorders of the central nervous system (CNS) are one class of common diseases nowadays. An investigation undertaken in the United States of America (Regier, et al., 1988) showed that 32% of the U.S. population suffers from a disorder of the brain at least once in a lifetime and that 15% of the population greater than 18 years old suffered from a mental disorder during the period of any single month. These disorders included ethanol abuse, drug addiction, affective disorders, phobias, obsessive-compulsive disorders, antisocial behaviour, schizophrenia and impaired cognition. From this survey, an approximate estimate can be made as to the number of individuals in the United States who are afflicted with disorders of the brain (Table 1 .1).

Table 1.1 Common Brain Disorders in the United States (Regier, et al, 1988)

Disorder affected individuals (million) migraine headache 25 abuse 25 anxiety/phobia 25

sleep disorder 2 0

depression/mania 1 2

drug abuse 1 0 obsessive-compulsive disorder 4 Alzheimer’s disease 3

schizophrenia 2

stroke 2

epilepsy 2 HIV infection 1.5 Parkinson’s disease 0.5

The wide prevalence of brain disorders provides the challenge to neuroscience to develop new pharmaceuticals that are efficacious, not harmful, and can be administered in an outpatient or ambulatory setting that does not require hospitalization. In the following paragraphs, a brief review of the major central nervous system disorders and the medicines used are given.

19 1.1.1 Pain and Analgesics Pain is probably the earliest disease recognized by man and presumably by its evolutionary predecessors as well (Frederickson and Hynes, 1985). It is so common and almost everyone suffers from pain in his life time. During the last 20 years, a wealth of information on the complex mechanism involved in pain has led to a better understanding of how analgesic drugs work and the clinical conditions on which they should be used (Bession and Vickers, 1994).

Painful stimuli are detected by peripheral receptors (nociceptors) at the site of the stimulus and this information is transmitted via the spinal cord to various higher centres where it is perceived, processed and reacted (Melzack, 1973). The "reaction" consists of autonomic, somatic and psychological components. This description is largely simplified, but the mechanisms and neuronal circuitry of this array of phenomena in response to a painful stimulus are very complex (Shaw, et al,1982). Numerous sites in the central nervous system have been identified where opiate analgesics act to modulate perception of painful stimuli (Thompson, 1984). It is generally believed that the mechanism of a -like analgesic is to initiate descending supraspinal inhibition of the pain input at the spinal level (Duggan, et al, 1979a, 1979b). Thus, may induce this descending inhibition by direct action at any one or more of the sites. Opiates might also act to diminish the emotional- affective component of pain by some actions confined to the supraspinal sites and might also act directly at the spinal level to produce analgesia (Yaksh, 1981).

Substances acting on opiate receptors have proven efficacy as analgesics. The majority of available central analgesics can be classified as opiate analgesics or mixed agonist- antagonist analgesics (Jaffle and Matin, 1980). Some examples are given in Figure 1.1 and 1.2. The opiate agonists are the compounds that have affinity for one or more of the opiate receptor subtypes and have intrinsic activity (efficacy) at all the receptors for which they have affinity. They may differ in affinity and /or intrinsic activity at the different receptor subtypes, but they are not antagonists at any of these sites. Pure antagonists are the compounds that may have affinity for one or more of the opiate receptor subtypes but have no intrinsic activity. This property is

20 the basis for their antagonist activity, since by binding to a receptor they thereby prevent its activation. Mixed agonist-antagonist compounds are those which have affinity and intrinsic activity at one or more of the opiate receptor subtypes but at least also have antagonistic properties at one of the receptor subtypes (Frederickson and Hypes, 1985).

RO OH

R=H, morphine R=CH3 ,

Figure 1.1 Agonists of Opiate Receptors (Frederickson and Hynes, 1985)

In addition, a number of neuroleptic drugs commonly used for the treatment of have been used by themselves or in combination with for the treatment of pain. Antidepressant agents were also suggested to have analgesic properties and some of them can potentiate the analgesic activity of the narcotics in both experimental animal and man (Von Knorring, et al, 1983). The descending inhibitory pathways could become a target for analgesic drugs (Dubner and Max, 1992). The other widely studied agents which also have some analgesic effect are the serotonin-uptake inhibitors. They increase serotonin action on synaptic receptors by preventing its inactivation (Ho, et al, 1975; Herz, et al, 1980; Peroutka, 1990; Saxena and Ferrari, 1989; Tricklebank, 1989).

The classical analgesics which are still in use generally have a morphine-like structure. The structures for the newer synthetic opioid analgesic candidates vary to a large extent (Figure 1.3), and some of them are more potent and selective than morphine. , , , , and are mixed opioid agonist-

21 antagonists, , U-50488 and tifluadem are K-opiate receptor agonists (Frederickson and Hynes, 1985). It is of interest to note that these compounds have some structural characters in common. For instance, they all contain at least one nitrogen atom as tertiary or secondary amide; they all contain at least one ring; and they all contain at least one oxygen as either hydroxyl, carboxyl or ether group.

N— \ \ ----- ^ > CH] ^CH] ^ 3 ^CH] C—C—CH] O CH] \ \ OH CH] OH OCH]

OH"

OH' ‘OH

Figure 1.2 Mixed Agonists and Antagonists of Opiate Receptors (Frederickson and Hynes, 1985)

Over the last 100 years, a number of drugs available to treat pain have increased considerably, but few of them are found to have significant advantages over morphine (Twycross and Lack, 1983). Morphine remains the gold standard for the treatment of severe acute pain, but it is not used as effectively as it could be because of fears of the development of tolerance, addiction and abuse (Zenz, 1993). The substances acting on opiate receptors have various side-effects (Cowan, 1992). The opiate agonist analgesics as a class are characterized by respiratory depression and addicting properties. The mixed agonist-antagonist analgesics reduce or alter the addicting potential, but this comes at the expense of the introduction of potential. Opiate antagonists, such as , reverse the respiratory depression and precipitate withdrawal in opiate dependent subjects but generally they do not alter the

22 psychotomimetic phenomena. (Lorens, 1976, Way, 1983, Takemori, et al, 1982, Koob, 1992). Obviously there is still a great need to develop effective central analgesic drugs with less tolerance, less abuse liability (psychic and physical dependence), less respiratory depression and less psychotomimetic effects than the existing drugs.

CH2OCH3 II / \ CH2 OCH3 CH2CH2 \ H5—N CH2 —CH2—N V fl if o\

Alfentanil

OH N — (CH));

CH) OH

Ciramadol Meptazinol Bremazocine

CH3CH2CH2

Picenadol \ CH)

C=0

Figure 1.3 Structures of some synthetic opioid analgesic candidates (Frederickson and Hynes, 1985)

1.1.2 Depression and Antidepressants

Depression is one of the common mental illness diagnosed by psychiatrists. In 1976, it was estimated that up to 15% of the adult population of the United States suffered from a serious depressive disorder in any given year (Burrows, 1976). In the same

23 year in the United States, 400,000 depression cases were treated and 20,000-26,000 suicides were attributed to acute mental depression and depression is ranked as number 10 in the diseases that cause death (Hollisler, 1981). Other surveys showed that up to 23% of the population suffered from the depression which was severe enough to impair normal mental function (Greist, 1979). It has been estimated in the United Kingdom that approximately 5% of the general practitioners’ workload and 2% of the total bill of the National Health Service are directed toward the treatment of depression (Taylor, 1975).

Depression, which is both a normal and an abnormal phenomenon, most reflects the human condition (Horwell, 1985). It is regarded as normal if it follows a traumatic experience such as bereavement or redundancy, but it would be abnormal if it follows a great personal success, such as passing an examination or giving birth. Such depression can be controlled by the passing of time or by various activities such as counselling with family, friends, priest or doctor or by taking up new interests and relationships. However, depression can become so disabling that it leads to the inability to communicate or to think, or even to the act of suicide. Such abnormal depression certainly warrants treatment by drugs or hospitalization (Horwell, 1985; Gelder, et al, 1983).

Since 1950s, there have been a great number of drugs available for the treatment of depression. The first major class of drugs receiving widespread acclaim was the monoamine oxidase (MAO) inhibitors (Kline and Cooper, 1980; Tyrer, 1982). The general structure of MAO inhibitors (Figure 1.4) contains one aromatic ring and a side chain including an amide group. They are particularly effective in the treatment of reactive (or secondary) depression, but these drugs can precipitate a hypertensive crisis when ingested with certain other drugs or with a wide variety of common foods that contain sympathomimetic (amine), such as tyramine and phenylethylamine.

24 C0NHNH2 CONHNH (CH2) 2CONHCH2 . CH2NHNHCOn CH3 n o o N- nialamide isocarboxazide isoniazid

CH2ÇHNHNH2 CH2ÇHNH2 CH3 CH3 NH: o Q (CH3)2N CH3 pheniprazine tranycypromine FLA 33 6

ÇH2NCH2C —CH CH2CHNCH2C — CH 0(CH2)2NCH2C—CH CH3 CH3 CH3 CH3

o deprenyl o pargylxne (-isomer) Cl clorgyline

Figure 1.4 The Structures of some MAO Inhibitors (Horwell, 1985)

Another class of the drugs are tricyclic antidepressants (Coppen, 1979; Rogers, et al, 1981; Bickel, 1980) which are effective and currently available for the treatment of depression. There are 20-30 compounds belonging to this class which are the most widely prescribed. Some examples are given in Figure 1.5.

The most severe side effects of tricyclic antidepressants are those associated with their properties (e.g. dry mouth, sedation, blurred vision, constipation). They cause cardiovascular effects, particularly hypotension. Cardiotoxicity has proved fatal in the elderly and in patients with heart disease (Blackwell, 1981a; 1981b). However, N-oxide that is claimed to have less cardiovascular toxicity has been introduced into the clinic.

25 Figure 1.5 Some drugs (Horwell, 1985)

(CH3)3NHCH3' h

amitriptyline

R=(CH2)3N(CH3)2 X=CH2 , R=(CH2)2N(CH3)3 R=CH2CH (CH3) CH2N(CH3) 2 dothiepin X=S R= ( CH2 ) 3 ^ r ~ \ CH2CH2OH R= (CH 2) 2 N ( CH3 >2

X=0 , R=(CH2)2N(CH3)2

X=CH2 ,

R=(CH2 ) 2 NHCH3

X=CH2 , R=CH(CH3)N(CH3)2

During the 1970s, the "second generation" antidepressants were developed. They appear to have less pronounced cardiovascular and anticholinergic side effects than the . Some of them, such as , and , have anxiolytic properties in addition to their antidepressant effects (Al-Yassiri, et al 1981). They have various structures (Figure 1.6) and the only similarity is that all of them contain aromatic rings and amine groups. The action of these drugs varies depending on their structures. Several compounds in this group appear to offer no advantage over those classical drugs but, as with most psychotropic agents, individual patients may

26 tolerate one drug better than another closely related compound (Horwell, 1985).

Figure 1.6 Some second generation antidepressants (Horwell, 1985)

CH2 CONH2

NH2 Mianserin Nomifensine

N—H

CH—CH2N(CH3)2 a F3 C Ï Ï A " \ yO--- N — (CH2) 3—N N 0 4(CH2)2NHCH3 Cl Trazodone

Interest has focused on the influence of the brain amines on the hypothalamus- pituitary-adrenocortical axis as one of the current trends in research into depression. The amines, such as histamine, acetylcholine, noradrenaline, dopamine and 5- hydroxytryptamine (5HT), functioning in the limbic area of the brain, have been shown to influence peptide transduction mechanisms in the hypothalamus that regulate the secretion of hormones from the pituitary gland (Horwell, 1985). Peptide analogues of the neurosecretory hormone have been synthesized as potential antidepressants and adrenocorticotrophic hormone, substance P and pineal have been implicated in depression. Alternative drug therapies include treatment with vitamins, ^-blockers.

27 such as , and lithium carbonate (Tyrer and Shaw, 1982; Baldessarini, 1980). The specific 5HT inhibitors are unequivocally effective as antidepressants, equal to or possibly better than the tricyclic compounds in major depression, and clearly superior in severe depression where tricyclics have poor efficacy (Hollister and Claghom, 1993). The specific 5HT reuptake inhibitors provide effective prophylaxis when given chronically, and evidence is accumulating that they are also active on anxiety associated with depression and in obsessive compulsive disorders. In addition, benzodiazepines, partial agonists to the 5HT1A receptor, Heterocyclics and non-selective aminergic neurotransmitter uptake inhibitors are also involved in the treatment of depression (Hollister, 1994).

1.1.3 Anxiety. Insomnia and Related Medicines Since the organization of sleep and wakefulness is highly elaborate and their function much influenced by external events, it is not surprising that the disorders of these systems are common. Sleeping and waking mechanism operate together as a homogeneous functional unit, the final output of which determines the level of arousal. Thus, disorders of one mechanism inevitably tend to affect others: anxiety is accompanied by insomnia and poor night-time sleep is associated with daytime sleepiness (Ashton, 1987).

Anxiety is a normal adaptive response to certain types of stress. It occurs when the degree of stress to which an individual is exposed overcomes his ability to adapt to it efficiently (Gelder, et al, 1983). Vulnerability to stress appears to be linked to certain genetic factors, such as trait anxiety (Roth, 1984) and to environmental influences. In normal subjects, anxiety symptoms may be induced by a number of factor, including severe stress, taking certain drugs (such as , , psychotomimetic, lactate or infusions), withdrawal from central nervous system depressant medicines (eg. narcotics, , anxiolytics), hypoglycaemia, hypercarbia, starvation or prolonged sleep loss. Anxiety may accompany hormone- secreting tumours, such as phaeochromocytoma or carcinoid and psychiatric disorders, eg. schizophrenia and depression (Ashton, 1987). Lader and Petursson (1981) estimated that one in ten of all men and one in five of all women in the United

28 Kingdom took tranquilizers or hypnotics for some time each year, usually for several weeks and many patients had taken them for years.

By the 1970s, benzodiazepines became the most commonly prescribed drugs in the western world. They calmed the mind, relaxed the muscles, controlled insomnia, helped people tolerate life-stressed and were also useful as (Ashton, 1987; 1994). They were safe and their dependence-producing potential seemed to be much lower than that of barbiturates. However, it became clear that these drugs do have adverse effects (Ashton, 1986), especially with chronic use. The major side effects of benzodiazepines, particularly from higher doses are drowsiness or stupor with only a small decrease in blood pressure and respiration. Most acute reactions to benzodiazepines are dose-related and consist of sedation, lightheadedness, ataxia and lethargy (Baldessarini, 1980). Upon continued treatment, tolerance will develop to these effects but not to the antianxiety effects. Occasional reactions to doses include impaired mental and psychomotor function, dysathria, headache, confusion, euphoria, delayed reaction tame and xerostomia. It is currently believed that the anxiolytic, , and effects of benzodiazepines are mediated by the benzodiazepine receptor (Paul, 1979). Moreover, the binding affinity of individual benzodiazepines is highly correlated with their clinical and pharmacological potencies. The benzodiazepine receptor is thought to be part of a supermolecular complex consisting of the GABA receptor and chloride ionophore (Olsen, 1982). It is believed that there are specific interactions among the benzodiazepine receptor, GABA receptor and chloride ionophore (Doble and Martin, 1992). The distribution of GABA receptors is similar to that of benzodiazepine binding sites. Occupation of these sites by benzodiazepines enhances the activities of GABA and the actions of the benzodiazepines require the presence of GABA (Speth, 1980).

Insomnia is a symptom that may have many underlying causes. Kastman (1980) estimated that up to 30% of the population at some time complain of difficulty in sleeping. Pain, discomfort, anxiety, increased sensory stimulation, stimulant drugs, hyperactive states, sleep apnoea and nocturnal myoclonus can increase noradrenergic.

29 and cholinergic activity which leads to difficulty in falling asleep; endocrine abnormalities, metabolic abnormalities and normal ageing can decrease activity which leads to difficulty in remaining asleep. Several neurotransmitters, neuromodulators and hormones appear to interact in a highly complex manner in the sleep-wakefulness cycle. These include serotonin (and possibly melatonin), noradrenaline, dopamine, acetylcholine, y-aminobutyric acid and probably various polypeptides and hormones (Gaillard, 1983; Koella, 1981a, 1981b; Monnier and Gaillard, 1981). The benzodiazepines can be used as adjunct treatment for symptomatic relief. Once the decision to use hypnotic has been made, the benzodiazepines are preferable to the barbiturates.

Benzodiazepines and barbiturates are two groups of compounds for disorders of the sleep system and antianxiety. Some of the currently marketed compounds are displayed in Figure 1.7 and Figure 1.8. Briefly, the structure-activity relationship of benzodiazepines (Figure 1.7) is that substitution at Rl, R2 and ring C are important in modifying the potency of these compounds (Stembach, 1973). The introduction of triazolo-1,4-benzodiazepine analogues has yield a new generation of potent compounds and this class of compound may possess pharmacological action different from the 1,4-benzodiazepines (Feighner, 1981). Alterations in structure that result in changes of potency may be the result of one or more contributing factors, such as changes in the pharmacokinetic properties which result from an alteration in the lipid solubility of the compounds or their susceptibility to biotransformation. Structural changes also alter the dynamics of interaction with the site of action.

Barbiturates (Figure 1.8 ) are derivatives of barbituric acid. CNS-active barbiturates are those which have alkyl or aryl substituent at Rl or R2. Polar substituents at Cj (Rl, R2) diminish hypnotic activity whereas alkyl side chains of increasing length at Cj impart activity. A phenyl group at the C 5 or N positions will selectively confer anticonvulsant activity. Any modification of structure that alters lipid solubility changes the hypnotic potency, the onset and duration of action, and the rate of metabolic transformation (Gee and Yamamura, 1985; Widler and Bruni, 1981).

30 Despite the relative effectiveness of the compounds available today for the treatment of anxiety and insomnia, they are far from ideal. The major problems with the benzodiazepines and barbiturates are the lack of selectivity (barbiturates being less selective than benzodiazepines), rapid development of tolerance to some of the therapeutic effects, and the abuse liability (Ashton, 1994). Some derivatives represent as more selective antianxiety agents. The selectivity of these compounds may be related to their partial agonist properties (Gee, et al, 1983). In other words, these components have lower efficacy than the clinically useful benzodiazepines. Consequently, benzodiazepines that have full efficacy (ie., full agonists) will produce antianxiety, anticonvulsant and sedative hypnotic effects, whereas compounds that

Figure 1.7 Clinically used benzodiazepines (Gee and Yamemura, 1985)

benzodiazepines

N a m e R l R 2 R 3 R 4 R 5 R 6

Chlordiazepoxide H C l C l 0 H - N H C H 3

C lorazepate H C l H - COGH = 0

C l o n a z e p a m H N O 2 C l - H = 0 D i a z e p a m - C H 3 C l H - H = 0 F lunitrazepam - C H 3 N O 2 F - H = 0

F l u r a z e p a m -(C H 2)2-N (C zH g)2 C l F - H = 0

H a l a z e p a m - C H 2 C F 3 C l - - H = 0 L o r a z e p a m H C l C l - OH = 0

M e d a z e p a m - C H 3 C l H - H H N i t r a z e p a m H N 0 2 H - H = 0

O x a z e p a m - C H 2 - A C l H - OH = 0 T e m a z e p a m - C H 3 C l H OH = 0

31 have only partial efficacy only produce a limited spectrum of pharmacological effects. The design of partial agonist compounds may therefore yield a variety of highly selective drugs useful in the treatment of anxiety and related disorders (Gee, et al, 1983). Studies on the neurobiological mechanism underlying the anxiolytic action of benzodiazepines have led neuropharmacologists to focus on central monoamine cholecystokinin (CCK) and glutamate system, with the hope of developing novel anxiolytics acting directly on certain subtypes of receptors for 5HT, CCK and excitatory amino acids. Recent preclinical data support the idea that selective ligands for 5HT1 A, 5HT2A. 5HT2C, 5HT3, atypical benzodiazepine, CCK, NMD A receptors and atypical benzodiazepine receptor agonists are of potential interest for the development of such drugs (Hamon, 1994; Hollister, 1994; Haefely et al, 1990).

Figure 1.8 Clinical used barbiturates (Gee and Yamamura, 1985)

barbiturates

Name Rl R2 R3 X

Amobarbital Ethyl Isopentyl H 0

Barbital Ethyl Ethyl H 0

Butabarbital Ethyl Sec-butyl H 0

Mephobarbital Ethyl Phenyl CH3 0

Metharbital Ethyl Ethyl CH3 0

Pentobarbital Ethyl 1-Methylbutyl H 0

Phenbarbital Ethyl Phenyl H 0

Secobarbital Allyl 1-Methylbutyl H 0 Allyl 1-Methylbutyl H S Thiopental Ethyl 1 -Methylbutyl H S

32 1.1.4. Schizophrenia and Antipsychotic Drugs Schizophrenia is a common disorder likely to affect almost 1% of the population (Reynolds, 1992). The symptoms of schizophrenia are divided into positive symptoms (delusion, hallucinations and incongruous affect), which are characteristic of the acute schizophrenic syndrome, and negative symptoms (withdrawal, loss of drive and flatten affect), seen mainly in chronic schizophrenic states (Gelder, et al, 1983; Reynolds, 1992). It is possible that in schizophrenia, as in Parkinson’s disease and Alzheimer’s disease, the neurones of one transmitter system may be primarily or mainly affected. It has been observed that antipsychotic drugs which are effective in controlling some schizophrenic symptoms, have in common a blocking action, and that dopamine receptor agonists and drugs which release dopamine can precipitate schizoid psychotic reaction, and greatly aggravate the symptoms of existing schizophrenia (Carlsson, 1978; Bunney, 1984; Baldessarini, 1980). The dopamine receptors may consist of a single macromolecular complex in which the conformation (and therefore the effective subtype) varies depending upon the presence or absence of co-operatively linked serotonergic or noradrenergic subunits (Cools, 1982). Reynolds et al.(1981) suggested that patients with paranoid psychosis tend to show an increase in D2 receptor density while patients with non-paranoid psychosis show a significant decrease. Thus changes in D2 receptor density may be related to the type of schizophrenia. The release of dopamine at synapses appears to be controlled partly by complex feedback loops involving cholinergic and GABA-ergic neurone and partly by dopaminergic autoreceptors (D3 receptor). Stimulation of D3 receptor by dopaminergic agonists decrease dopamine release and hence reduces dopaminergic activity (Farlely, et al, 1980). It has been suggested that GABA deficiency in limbic pathways might be a characteristic of some forms of schizophrenia (Hollister, 1994). The possibility that noradrenergic activity may be altered in schizophrenia has been considered by a number of authors (Lake, et al., 1980; Van Kammen, et al, 1990) and the elevation of noradrenaline is most marked in patients with paranoid features. Whether such abnormalities represent overactivity or underactivity of noradrenergic systems is unclear. Opiate receptors are present on dopaminergic nerve terminals in the corpus striatum and mesolimbic regions (Reisine, et al., 1980). Endogenous opiates have been shown to stimulate dopaminergic activity in the mesolimbic system (Koob

33 and Bloom, 1983). Smith and Copolov (1979) reported that schizophrenia might be associated with excessive endogenous opiate activity in the brain. The strongest biochemical and pharmacological evidence suggests an involvement of dopaminergic mesolimbic and mesocortical pathways, perhaps mediated through an endogenous opiate-related polypeptide mechanisms, in schizophrenia (Hong et al, 1980).

The antipsychotic drugs are chemically a heterogenous group of compounds with the property of controlling certain psychotic symptoms in man. All the antipsychotic drugs in clinical use block dopamine receptors in the brain (Ashton, 1987). Animal and human studies show that chronic treatment with antipsychotic drugs can lead to adaptive increase in D2 receptor sensitivity. Both limbic and striatal dopaminergic neurones receive an inhibitory GAB A-ergic input (Amt and Scheel-Kruger, 1979). Some antipsychotic drugs have significant central and peripheral a- and 13- adrenoceptor antagonistic activity (Snyder, 1982), but this action does not appear to be necessary for an anti-schizophrenic effect since it is not shared by all antipsychotic agents and adrenergic blockade does not correlate with therapeutic potency over the range of drugs. Many of the antipsychotic agents have blocking effects on serotonergic receptors (Carlsson, 1978; Sloviter, et al. 1980; Reynolds, et al. 1983). It is possible that serotonergic receptor antagonism contributes to the effect of antipsychotic drugs in reversing the action of some psychotomimetic agents. Some of the agents also have fairly potent antagonistic actions at histamine-1 and histamine-2 receptors (Richelson, 1981). It does not seem likely that this is important for the antischizophrenic actions but it may contribute to sedative effects. New antipsychotic agents may include selective D2/D3 receptor antagonists^ partial D2 receptor agonists, 5HT3 receptor antagonists, glutamate/a-opiate receptor agonists, blockers and drugs blocking several receptors (eg. D2, 5HT1, 5HT2, a 1-adrenoceptor, muscarinic, etc.) (Hollister, 1994).

The clinically used neuroleptics include derivatives, compounds, derivatives, thioxanthine derivatives, piperazinyldibenzoxazepine derivatives and substituted drugs (Jenner and Marsden, 1985; Stanley, et al, 1980; Robert, et al, 1978; Figure 1.9). The activities of

34 neuroleptics to act at different receptor sites vary enormously (Leysen, 1982). Most neuroleptic drugs not only act on cerebral dopamine receptors, but also exert a range of effects on other neurotransmitter systems, eg. noradrenaline, 5HT, histamine, acetylcholine, GABA and peptides. This is either the result of their action on dopamine system causing secondary effects on other neurotransmitter pathways or because the neuroleptics also act directly on other neurotransmitter’s receptors (Jenner and Marsden, 1985).

Antipsychotic drugs do not cure schizophrenia, but merely alleviate symptoms. Some patients do not respond at all and a few of those who do respond revert to complete or permanent normality, and even the best drug responders tend to relapse when treatment is stopped (Ashton, 1987). Nevertheless, it is generally agreed that the antipsychotic drugs have unique and relatively specific effects on certain schizophrenic symptoms, and their effects are not due to general sedation (Rogers et al., 1981).

Although some non-drug treatments are available for CNS disorders, such as electroconvulsive therapy (ECT) for antidepression, acupuncture for the relieve of pain and even some brain surgeries, the main treatments are still by medicine. These substances have various chemical structures, but also possess some common characteristics. The following general consideration can be summarized based on the structures listed in Figure 1.1-1.9: - high electron-affinity atoms in the structure, such as N, S, O, for increasing affinity to receptor protein and enhancing activity - lipid groups, such as benzene rings or long side-chains, for increasing lipid solubility of the compounds and changing their susceptibility to bio-transformation. - small molecular size, the molecular weight is usually around 200-600.

35 ‘CF 'SCH-

CH2 ) (ÇHo);

y \ N — CH- C « 3 CH3

CH3

OH

Haloperidol

CH

CH CF: ‘Cl

CH2 ) CL CH Cl

(CH2 ) OH

Flupenthixol

Cl CH3 SO2 C,Hc NH- CONHCH2 CH2 N ^ CONHCH2CH2N C2 H5 C,H. OCH3 ^OCH,

NH2 SO2 C2 H5 SO2

CONHCH2 ONHCH2 ' O N' N I OCH3 \ 0 CH3 C2 H5 C2 H5

Sulpiride

Figure 1.9 Neuroleptic Agents (Jenner and Marsden, 1985)

36 1.2. Receptors related to CNS As described in section 1.1, the disorders of the CNS are closely related to various receptors. There are more than ten neurotransmitters which have been characterized, such as acetylcholine, , dopamine, 5-HT, GABA, , opioid, benzodiazepine, noradrenaline, histamine, etc. (Kruk and Pycock, 1991). It is becoming increasingly clear that there are several different receptor subtypes in each neurotransmitter which produce various pharmacological actions. The major neurotransmitter receptors in CNS, which have been involved in the present study, are summarized as below.

5HT is a neurotransmitter present in most organisms. A number of 5HT receptor subtypes have been identified and characterized, such as 5HT1 A, 5HTlBa, 5HTlBp, 5HT1C, 5HTlDa, 5HT1D|3, 5HT1E, 5HT1F, 5HT2, 5HT3, 5HT4, etc. (Gothert and Schlicker, 1990; Hen, 1992; Maroteau et al, 1992, Viogt, et al, 1991; Hartig, et al, 1992; Peroutka, et al, 1990; Dumuis, et al, 1988; Craig and Clarke, 1990;). There are functional second message correlations for each of these binding site. For instance, the 5-HTlA, 5-HTlB and 5-HTlD sites inhibit the adenylate cyclase system, while the 5-HT 1C site stimulates phosphatidyl inositol turnover as does the 5-HT2 (renamed 5HT2C) receptors. The 5-HT 1 A, 5-HTlB and 5-HTlD receptors have functions of inhibition of neurotransmitter release and contraction of some vascular smooth muscle, whilst the 5-HT 1C and 5-HT2C receptors have the function of gastrointestinal and vascular smooth muscle contraction and platelet aggregation. The 5-HT3 receptor subtype forms part of ion channels for sodium, potassium and calcium ions. Blockers of 5-HT3 receptor are highly effective anti-emetic agents which are able to control nausea and vomiting caused by nearly all chemotherapeutic agents and radiation (Kruk and Pycock, 1991). 5HT4 exhibits similar characteristics as 5-HT3. It is linked positively to adenylate cyclase. ( Bockaert, et al, 1992). In general, the central 5-HT mechanism is important in the control of mood and behaviours, motor activity, feeding, hunger, thermoregulation, sleep, certain hallucinatory states and possibly some neuro endocrine mechanism in the hypothalamus.

The amino acid neurotransmitter y-aminobutyric acid (GABA), which is coupled to

37 chloride ion channels in mammalian neurones, can be further differentiated on pharmacological grounds into GABA^ and GABAg subclasses (Bowery and Pratt, 1992, Desarmenien et al. 1984). GABA^ receptors are coupled to chloride ion channels and possess allosteric sites for benzodiazepines, barbiturates and neuroactive steroids mediating fast synaptic inhibition. The GABAg receptors are coupled through G- proteins to neuronal or Ca^ channels. Activation of the receptors increase or decrease Ca"^"^ conductances and mediate slow synaptic inhibition. Inhibition and potentiation of stimulated adenylyl cyclase activity can be attributed to GABAg site activation. Presynaptic GABAg receptors influence the release of neurotransmitters and neuropeptides, such as GABA, glutamate, noradrenaline, dopamine, 5HT, substance P, cholecystokinin and somatostatin (Bowery and Pratt, 1992). The potential therapeutic applications of GABAg may include in the treatment of epilepsy, depression and cognitive processes (Bittiger, et al, 1993).

There are two main types of cholinergic receptors: muscarinic receptors and nicotinic receptors. Both of them exist in the autonomic nervous system (ANS) and the central nervous system (Kruk and Pycock, 1991). Drugs which activate or inhibit cholinergic mechanisms in the CNS have vaiying effects on arousal and wakefulness in man and animals. Cholinergic mechanisms are involved in nausea, vomiting and possibly vertigo. Resting tremor and rigidity are associated with excess cholinergic activity in Parkinson’s disease (Bowen and Davison, 1980). Muscarinic receptor blockers are effective treatments for these symptoms and also inhibit short term memory (Kruk and Pycock, 1991). In the ANS, muscarinic-receptor stimulation occurs physiologically when parasympathetic nervous system is active during rest and sleep. Muscarinic receptors mediate a slowing in the rate of contraction of heart and a decrease in the force of contraction. was found to mimic the effect of parasympathetic nervous stimulation. On the basis of pharmacological studies five muscarinic receptor subtypes are recognized and have been designated as M1-M5 (Barnes, et al. 1988; Caulfield, 1993). The Ml and M3 receptors are linked to Gs protein and phospholipase C, and their activation leads to increasing synthesis of inositol trisphosphate and diacyl glycerol. The M2 receptor has two effector pathways: inhibition of adenylyl cyclase leading to decreased cyclic AMP synthesis and G

38 protein regulated opening of potassium ion channels.

There are three types of histamine receptors which differ in agonist and antagonist sensitivities. On the basis of experience with drugs which block histamine receptors, it has been suggested that histamine might have a role in arousal, in mechanisms related to nausea and vomiting and in the control of blood pressure and water metabolism (Kruk and Pycock, 1991). HI receptor is linked to phospholipase C by a Gs regulatory protein resulting in release of the secondary messengers diacyl glycerol and inositol triphosphate. The competitive H2 receptor blockers linked to adenylyl cyclase by a Gs regulatory protein. H3 receptors have been identified as inhibitory histamine autoreceptors on all cells which release histamine (Prell and Green, 1986; Van der Werf and Timmerman, 1989).

Potassium ion channels play a critical role in determining the time course of electrical changes in most types of cell and they can be distinguished as voltage-gated channel, calcium-activated K^ channel and ATP-dependent channel. To date, at least 13 major types of channel receptors are recognized and, within each type, several sub-groups exist (Rudy, 1988; Cook, 1988; Edwards and Weston, 1990). The activators or openers of channels appear to possess smooth muscle actions. The probable sequence of events from channel opening to smooth muscle relaxation is that channel opening can enhance efflux which causes hyperpolarisation of nerve terminals reducing both Ca^ influx and the release of Ca^^ from intracellular stores. The final effect of this sequence is smooth muscle relaxation (Longman and Hamilton, 1992). The therapeutic potential of these smooth muscle relaxants is for the treatments of hypertension, asthma and incontinence. Drugs that modulate the activity of different types of channel are also potential useful for the treatment of heart arrhythmias (Pongs, 1992).

Noradrenaline (NA) is a catecholamine neurotransmitter in postganglionic sympathetic nerves and within the CNS. NA can combine with two main types of receptor called a-adrenoceptors and p-adrenoceptors (Ahlquist, 1948; Lands et al. 1967). All a- adrenoceptors belong to the G-protein regulated super family. Activation of the

39 receptors leads to transduction by different effector systems. Activation of al- adrenoceptors leads to increase IP3 and diacyl glycerol synthesis via phospholipase

C activation. Activation of a 2 -adrenoceptors leads to inhibition of adenylyl cyclase or opening of a G protein regulated potassium ion channel, p-Adrenoceptors are all linked to adenylyl cyclase by a Gs regulatoiy protein. Activation of P-adrenoceptors leads to increased synthesis of C-AMP (Heal and Marsden, 1990).

Dopamine is a precursor of noradrenaline and adrenaline. It is also a catecholamine neurotransmitter in the CNS and the autonomic nervous system. Five subtypes of dopamine receptors, D1-D5, have been found continouslly (Kebabian and Caine, 1979; Sokoloff et al, 1990; Van Toi, et al, 1991; Sunahera, et al, 1991). D1 receptor belongs to the G-protein receptor super family and its agonists stimulate the production of cyclic AMP. Gene sequence studies for D2 receptors have revealed that there may be two forms of the D2 receptors (D2s and D21). One involves a Gi protein which inhibits adenylyl cyclase and leads to decreased cyclic AMP production, the other is linked by a G protein to a potassium ion channel (Sibley and Frederick, 1992). D3 receptor, which is within the D2 subfamily (Sokoloff, et al, 1990), is found mainly in the mesolimbic pathways associated with cognitive and emotional functions and also inhibits adenylyl cyclase activity. It is likely that antipsychotic drugs interact at this site as well as at D2 receptors. D4 receptor, a member in the D2 subfamily, displays similar or lower affinities for dopamine receptor antagonists and agonists compared with the D2 receptor. However, the alozapine and its congener exhibited about ten-fold higher affinity for the D4 receptor (Van Toi, et al, 1991). D5 receptor, the second member of D1 receptor subfamily is linked to stimulation of adenylyl cyclase activity with a D1 receptor-like pharmacology (Sunahara, et al, 1991). The various agonist and antagonist ligands exhibit similar affinities for the D1 and D5 receptors, with the notable exception of dopamine, which is about ten-fold more potent at D5 than at D1 receptors (Sibley and Frederick, 1992). Dopamine receptor blockers are effective in the treatment of schizophrenia. They are also referred to as neuroleptics or major tranquillizers (Hollister, 1994). Dopamine receptor blockers have anti-emetic activity, which can reduce nausea and vomiting caused by chemicals or radiation (Kruk and Pycock, 1991). They also have a common

40 property of causing sedation without inducing readily reversible loss of consciousness- "sleep", and they do not possess hypnotic activity. This sedative action is useful in the treatment of severe agitation or mania (Lees, 1986; McGeer et al, 1984).

The existence of multiple opiate receptors has been demonstrated by pharmacological bioassay techniques and by radiolabelled opiate binding studies. Within the CNS, four major opiate receptor subtypes are proposed, and they are assigned as p-, Ô-, K-, a- opiate receptors (Hughes and Kosterlitz, 1983). Stimulation of p-opiate receptor induces hyperpolarization by opening channel and closing Ca^ channel or by inhibition of presynaptic mechanisms as on primary afferent neurones in the dorsal horn of the spinal cord. In keeping with this general suppressant action, some p- receptor appear to be associated with the inhibition of the adenyl cyclase system resulting in reduced production of cyclic AMP (Millan, 1990). However at some sites, p-receptors are linked to excitatory actions. For example, in the hippocampus where p receptor mediated presynaptic inhibition on GABA-ergic nerve terminals results in inhibition of GABA release so evoking an overall excitatory response (Simon, 1991). Overall 0-opiate receptor stimulation mediates hyperpolarization similar to that of p- receptor. K-Opiate receptors differ from p- and ô-receptors which have a different spectrum of analgesic activity (Shearman and Herz, 1982; Bertalmino and Woods, 1987). They are believed to act predominantly at spinal sites, and thereby do not cause significant respiratory depression at doses which produce a similar degree of analgesia as other opioid agonists. They are however more liable to cause hallucinations and sedation than other opioid agonist drugs (Kruk and Pycock, 1991; Morley, 1983).

The therapeutic potential of adrenoceptors, dopamine, 5HT, histamine, benzodiazepine, muscarinic, GABA, K^-channel, opiate receptors is summarized in Table 1.2.

41 Table 1.2 Agonists and Blockers of Several Receptors and Their Therapeutic Potential

Receptors Therapeutic potential Drugs Adrenoceptors a l selective agonist nasal decongestant a l selective blocker antihypertensive a2 selective agonist hypotensive on central a- a-methyl-NA adrenoceptors, analgesia clonjidine a2 selective blocker depression

Non-selective a- hypertensive, vascularspasm 1 blocker pi selective agonist hypertensive,stimulant of heart pi selective blocker hypotensive, antidysrhythmias

P2 selective agonist bronchodilator,asthma, premature labour Non-selectiveP-agonist asthma Non-selectivep-blocker antihypertensive,antianginal, (-)-propranolol antidysrhythmias

Dopamine receptors D1 selective agonist fenoldipam D2 selective agonist jquinpirole

D 1 selective blocker experimental SCH23390 D2 selective blocker antipsychotic

Non-selective emetic, Parkinson’s disease, 1 -dopa dopamine prolactin-induced subfertility receptor agonist acromegaly j)iribedii Non-selective major tranquillizer, chlorpromazine dopamine antipsychotic,schizophrenia, receptor blocker mania,antiemetic pimozide

42 5-HT receptors 5HT1A selective anxiolytic, hypotensive agonist 5HT1C selective a-methyl-5HT agonist 5HT1C selective kentanserin blocker 5HT2 selective agonist a-methyl-5HT 5HT2 selective blocker 5HT3 selective agonist 2-methyl-5HT 5HT3 selective blocker antiemetic, antipsychotic 5HT4 agonist certain 5HT4 blocker ICS205930 5HTl-like selective antimigraine GR43175 agonist 5HTl-like non- methiothepin selective blocker 5HT non-selective LSO agonist analgesia, anxiety Histamine receptors

HI agonist 2 -methyl-histamine

HI blocker allergy, cause sedative, travel sickness

H2 agonist gastric acid secretion 4-methyl-histamine

H2 blocker gastric and duodenal ulcers rinatidine H3 agonist R-oc- methylhistamine H3 blocker thioperamine Benzodiazepine BZl anxiety BZ2 sedative BZ3 PK11195

Muscarinic receptor non-selective agonist senile dementia non-selective blocker reduce bronchial and gastro­ intestinal secretions hyosine motion sickness,anaesthetic Ml selective blocker for treatments of gastric and duodenal ulceration caused by excess acid secretion

43 GABA receptors GABA a agonist GABA a blocker bicuculine GABAg agonist treatment of spasticity GABAg blocker potential use for epilepsy, cognition deficits, anxiety, saclophen depression, and have neuroprotective properties

K^-ion channel agonist smooth muscle relaxation blocker

Opiate receptor p -opiate agonist supra-spinal(cerebral) analgesia p-endorphin respiratory depression, morphine tolerance, withdrawal physical met- dependence, euphoria, sedation, leu-enkephalin partial agonist associated with K^-ion channel buprenorphine adenylate cyclase system meptazinol blocker naloxone selective agonist sufentanyl selective blocker

K-opiate receptor spinal analgesia, hallucinations, bremazocine agonist tolerance, appetite suppression, pentazocine sedation, associate with C a t­ ethylketocyclazocine ion channel butorphanol partial agonist blocker naloxone selective agonist endogenous ligand 1-13 fragment of

Ô -opiate receptor spinal analgesia, affective leu-enkephalin agonist behaviour, tolerance, associate met-enkephalin blocker with Ca^-ion channel naloxone selective agonist inhibition of adenylate cyclase ketocyclazocine

Reference: Hughes and Kosterlitz (1983); Morley (1983); Atweh and Kuhar (1983), Jaffe and Martin (1980); Kruk and Pycock (1991); Reynolds (1992); Hen, (1992); Hartig, et al, (1992)

44 Calcium ions play a number of important roles in the function of nerve cells, including neurotransmission. Modulation of synaptic transmission and transmitter release associated with memory and learning are all profoundly influenced by Ca^^. Ca^^ also has a critical effect on the electrical excitability of the cell. Ca^^ channels have been classified into several subtypes based on their physiological properties (Tsien, et al,1991) and these subtypes are summarized in Table 1.3. Low-voltage activated or T- type Ca^^ channels are activated at relatively negative membrane potential, have a small single-charmel conductance, and mediate a transient Ca^^ current that is important in determining the frequency of action potential generation in neurones and cardiac muscle cells (Puceat, et al, 1992). High-voltage activated Ca^^ require a more positive membrane potential for activation and include L, N and P channel types (Catterall and Striessing, 1992). The T and L types are expressed primarily in neurones where they play an important role in neurotransmitter release. L-type Ca^^ channels are the primary type in muscle cells and are responsible for the inward movement of Ca^^ that initiates contraction in cardiac and smooth muscle cells (Tsien, et al, 1991). The Ca^^ channel antagonists inhibit voltage-gated channels in many different cell types. Inhibition of Ca^^ channels in smooth muscle and cardiac muscle cells by these drugs is valuable in the therapy of a wide range of cardiovascular disorders including hypertension, atrial arrhythmia and angina pectoris (Zemig, et al, 1990). The antagonists of Ca^^ channel receptors can also be used for anticonvulsant (Desmedt and Janssen, 1975; Overweg, et al, 1984), pain relief (Del Pozo, et al, 1987), treatment of alcoholism (Koppi, et al, 1987) and psychiatric disorders (Ross, et al, 1987).

Adenosine is a neuromodulator that plays a pivotal role in maintaining adequate oxygen energy supply throughout the body and it may be an endogenous protective agent in cerebral ischaemia. The actions of adenosine are mediated through specific cell-surface receptors, of which at present two subtypes are known (Galen, et al, 1992). Potential therapeutic applications for agonists include the prevention of reperfusion injury after cardiac ischemia or stroke and the treatment of hypertension and epilepsy. Adenosine antagonists might be effective as cognition enhancers. The function of adenosine subtypes are summarized in Table 1.4 (Rudolphi, et al, 1992).

45 Table 1.3 Calcium Ion Channel Receptors

(Tsien, et al, 1991)

Receptor Properties Function/Location

Ca^-ion channel Ca^-L High-voltage activated, Excitation-contraction voltage-dependent, long- coupling; central to cardiac lasting current responsive muscle contraction and to dihydropyridines(agonist involved in most forms of and antagonist), slow smooth muscle inactivation contraction. Excitation- secretion coupling in endocrine cells and some neurons

Ca^-N High-voltage activated, Identified only in neurons vol tage-dependent, participate in moderate rate of neurotransmitter release inactivation

Ca++-T Low-voltage activated, Influence on SA voltage-dependent transient pacemaker activity in heart current. Channels and repetitive spike deactivate slower than L or activity in neurons and N channel endocrine cells

Ca^-P Moderately high-voltage Identified in some CNS activated neurons

46 Table 1.4 Adenosine Receptor Subtypes (Galen et al. 1992)

System Action Tissue/cell Subtype

Adenylate cyclase inhibition adipocytes Al heart,smooth muscle Al stimulation platelet A2a striatum,smooth muscle. A2a brain, fibroblasts A2b Guanylate cyclase activation smooth muscle Al Low Km cAMP adipocytes Al phosphodiesterase brain Al Potassium efflux activation heart,hippocampus Al ATPsensitive K^- activation heart Al channels Calcium influx inhibition brain Al neuromuscular junction A3? Phosphoinositole inhibition GH3 cell Al metabolism stimulation kidney RCCT cells Al thyroid FRTL-5 cells Al

1.3 Receptor ligand binding assays The vast majority of biological phenomena involve molecular communication between cells. The molecules which mediate this communication require the presence of a target molecule or receptor, on the target cell which recognizes the signal and translates this, usually by a complex series of intracellular events, into a biological effect or response (Boeynaems and Dumont, 1980). The study of the distribution, concentration, structure and function of these receptors is thus fundamental to the understanding of the biological response. As a consequence, the development of drugs and the drug design without consideration of receptors involved in the action is no longer realistic (Ariens, 1986).

Radioligand receptor binding studies are now used routinely to measure and characterize the interactions of ligands with a variety of receptors for hormones, neurotransmitters and drugs and constitute an important technique in searching for

47 CNS active compounds (Bennett and Yamamura, 1985). The principle of such assays is that the amount of radioligand bound to the membrane receptor is quantitatively reduced by the amount of unlabelled displacing ligand present in the incubation medium. These assays are quite sensitive, simple and rapid, and have provided a valuable molecular strategy for the drug development. A number of studies have indicated that these assays are a versatile procedure that can be used to examine many issues of neurobiological interest (Hulme and Birdsall, 1992).

The most logical way to study receptors is to use a labelled version of the specific molecule, and in the vast majority of cases this label is radioactive. The most commonly used radionuclides are tritium and iodine-125 (Hulme and Birdsall, 1992).

The general binding assay procedure is as follows (Hulme and Birdsall, 1992): a. Choose and make a tissue preparation containing the receptor. b. Select a suitable labelled ligand. c. Incubate the receptor preparation with an appropriate concentration of a labelled ligand for a defined time at a defined temperature. d. Separate the bound ligand from the free one, using an appropriate separation technique. e. Measure the bound and free ligand concentrations. f. Repeat steps (c)-(e) with the addition of unlabelled ligands or modulating agents as dictated by the aims of the experiment. g. Analyze the data to extract quantitative estimated of rate constants and/or affinity constants.

The mathematical analyses used to characterize a particular receptor in terms of its interactions with ligands are given in equations 1-14 as follows (Williams, 1991; Hulme and Birdsall, 1992).

The kinetics of the ligand binding is similar to those of classical -substrate interactions. The simplest case of ligand-receptor interaction deals with a homogeneous, univalent species of ligand (L) and a single non-interacting population

48 of binding sites (R). Thus, L+R RL (1) k ’^ and at equilibrium, ^ ^

T " W] (2)

k- 1 (3) = k where is the equilibrium dissociation constant. If the maximum number of specific receptor sites is Bmax, then

[LR] + [R] = Bmax ( 4) Multiplying by [L],

[LR] [L] + [L] [Rj = Bmax [L] ( 5)

Substituting (2) in (5) leads to Michaelis-Menten Equation ( 6 )

[RL] [L] Bmax [L] + Kd (6)

The ratio RL/Bmax represents the fraction of total receptor sites occupied by ligand. At half maximal occupancy of the receptor, RL/Bmax = 1/2, and = [L]. Hence the concentration of L required for half maximal occupancy of the receptor sites is equal to Kq . a number of useful equations can be derived from this relationship, such as Scatchard Equation:

[RL] [L] + [RL] Kd . Bmax |L] ( 7 ) Divided through by [L],

[RL]Kd - Bmax - [RL] [L] (8 )

Rearrange ( 6 ) gives Scatchard equation:

[RL] _ Bmax - [RL] [L] ' Kd (9)

49 A plot of [RL]/[L] versus [RL] has a slope equal to the negative reciprocal of the dissociation constant, -I/Kq and the intercept on the abscissa provides a measure of the concentration of binding sites (Bmax). The main advantage of the Scatchard plot is that it linearizes the data, being particularly valuable in binding systems that have a high level of non specific binding. A curved Scatchard plot may indicate cooperative interactions among receptors or if the ligand is binding to two or more sites with different affinities.

In investigations of the specificity of receptor binding sites, the ability of an unlabelled agent I to compete with radioligand L for the binding site is often measured. If the competing agent I is assumed to be a competitive inhibitor of the binding of R, then the receptor is in equilibrium with both I and L. Thus,

l + R Rl L+R RL Hence,

K, -[RI[I]/[RI]

Kd -IRI[L]/[RL] (10) and solving for [RL] gives,

(11) [RL] . Kd (1 + [I]/K ,) + [L1 where [I] is concentration of free unlabelled ligand, Kj is equilibrium dissociation constant for the interaction of I with the receptor. When there is no competing ligand present, i.e, 1 = 0, then the amount of binding [RL]o is given by,

From equation (1 1 ), adding a concentration of competing ligand, I^g, which reduces binding RL 5 0 by 50%, gives:

[ R L l ____ 'so Kod+W K,) +[L]

50 (13)

Since when I = I 5 0 , [RLj^g = [RL]q/2, equations (12) and (13) give,

2 [L] Bmax [L] Bmax

Kd ( 1 +I«/Ki) + [L] Kd +[L] which is simplified to

K ,- — ------(14) ' 1 +L/Ko

Thus using equation (14), the equilibrium dissociation constant of an unlabelled competing ligand I can be determined by measuring the concentration (I 5 0 ) of I which half-maximally inhibits the binding of the radioligand present at the concentration [L].

Competition data were routinely analyzed using the non-linear least squares programme which is specifically designed for the interpretation of sigmoidal concentration-response curves in terms of non-specific binding and total binding as well as inhibition constants and curve steepness.

1.4 Other pharmacological approaches for CNS dru^ development Although the radioligand binding assays are quick and simple, and can provide a valuable molecular strategy for drug development, they are only in-vitro tests and can not explain all the effects of CNS active drugs. Once a particular activity of a compound has been determined, its binding activity must be confirmed in a sequential and ascending series of functional assays comparing newly generated data with that derived in vitro. This places great and necessary emphasis on the need for integration of data from a variety of sources before a compound can properly be assessed for its therapeutic potential. A number of methods have been developed for different targets. For instance, to investigate the functional consequences of channel modulators in rat brain synaptosome, the effects of various agents known either to block channels or to open channels can be studied on the synaptosomal function of Ca^^ uptake, membrane potential and oxygen consumption (Smith, 1989).

51 For screening effects of central inhibition, the following animal behavioural tests are usually used to measure (Kasahara and Hikino, 1987): - revolution activity in wheel cages; - locomotor activity by using a counting box; -analgesic activity by thermal method (hotplatejtailflick), chemical method (acetic acid induced writhing),or mechanical method (tail clip); - effect on caffeine-induced convulsion and death by determining convulsion and lethal time; - muscle relaxant activity by use of a inclined board; - effect on -induced sleeping time. After various in-vivo animal tests for activity and toxicity, the different stages of clinic trials will be undertaken using human subjects.

1.5 Plants as a source of medicines for CNS disorders 1.5.1 Plants used for CNS disorders Plants have been used in many countries throughout the world for centuries including the treatment of disorders of the central nervous system. Separation and identification of the active constituents from medicinal plants began to attract scientists’ interests in the early 19th century. Sertumier isolated morphine from in 1806, Pelletier and Caventou obtained in 1818 and Runge extracted caffeine in 1820. Heffter isolated and other Cactus alkaloids in 1896. In 20th century, the active principles of Rauwolfia serpentina and indica have been studied. These compounds together with , which was isolated in 1860 and shown to possess both central and local activities, are the most important substances acting on the CNS. Early progress in this field made it possible to speculate on the relationship between

structure and biological activity and, in the last 2 0 years, on target tissues, receptor sites and mechanism of binding. These results have disclosed avenues of the greatest importance for neurochemistry and neurobiology, and many possibilities for the synthesis of new highly specific drugs have been generated (Bettolo, 1986).

When plant-based traditional medicines are used, they mainly consist of mixtures of either the plant material or its crude extract. A great number of plants have been

52 subjected to the isolation and structure elucidation of major active constituents, and some of them also to the pharmacological studies. In some cases, the pharmacological effects of the active constituents can explain the effects of the plant itself, but in other cases, it is not easy to find the constituents which can present significantly the total effects of the herb. There is only a little knowledge about the mechanism of plants at the cellular and molecular level underlying these effects. Many of the chemical substances isolated are thought to inhibit or mimic neurotransmitters in the CNS, but since there is still much to learn about these, it is not surprising that our understanding of the activities of various plants in the CNS is still in its infancy (Houghton and Bisset, 1985). The CNS active chemical substances isolated from plants possess various structures, such as indole and isoquinoline alkaloids, purines, phenethyl amines, monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoid. Several examples are given in Figure 1.10 and Figure 1.11.

0 CH3 RO. OCH3

•OCH3

R=R1=H, AV, Kawain HO R=R1=H, Dihydrokawain

R,R1=0Œ20, A7, Methysticin r =h , Morphine Mesembrine R=CH), Codeine R=H, R1=0CH3, A s , Yanyonin O OH

‘OH

CH3' Kessyl alcohol Jataitiansone Valtrate

CH3O CH. OHC ,CH200CCH2CH(CH3)2

CH3OOC

Reserpine Homobaldrinal

Figure 1.10 Structures of plant psychodepressants (Houghton and Bisset, 1985)

53 CHav^NHa OCH3 CH3. = R N CHa CH3 AT ,N CH3

R=CH3, caffeine R=H,OH, d-nor- R=H, th e o b ro m in e ad ch o m ein e R=0, cathinone

ÇH3 O'COOCH3 I CH3OOC

n i c o t i n e a r e c o l i n e

Figure 1.11 Structures of plant psychostimulants (Houghton and Bisset, 1985)

1.5.2 Traditional Chinese medicines used for CNS disorders 1.5.2.1 History of Traditional Chinese medicine The traditional Chinese medicine (TCM) has been used for thousands of years and still plays an important role in the health service in China. TCM has accumulated vast experience and has resulted in some specific beneficial effects on certain diseases. The theories of TCM have been developed and continually enriched through practice.

More than 2000 years ago, "Canon of Medicine", the earliest of the extant medical classics in China, was produced. The book summarized the previous experience of treatment and theories of herb medicine, dealt at length with anatomy, physiology and pathology of the human body, and diagnosis, treatment and prevention of diseases (Zhang, 1988). Since then, a large number of books have been published on Chinese medicines. Several examples are given in Table 1.5. From the Qin and Han dynasties

54 (221 B.C.-220 A.D.), doctors in the interior of China began to prescribe more and more herbs from the minority nationalities in remote region of the country and the southeast of Asia further enriching the knowledge of TCM. "The Herbal" (Sheng Nong Ben Cao Jing) is the earliest extant classic on materia medica and summarizes pharmaceutical knowledge which was known before the Han dynasty. It discusses the use of 365 herbs in detail and raises the pharmacological theories of "Jun, Chen, Zuo and Shi" (monarch, minister, assistant and guide), indicating the degree of the importance of the individual herbs in a prescription. It also describes "Qi Qing He He" (seven conditions in making up prescriptions), "Si Qi" (four properties of drugs), "Wu Wei" (five kinds of tastes of drugs, ie, sour, bitter, sweet, acrid and salty) and other tenets of TCM. Long-term clinical practice and modem scientific research have proved that most effects of the herbs recorded in the book are correct (Zhang, 1988).

In 659, the first Chinese pharmacopoeia was produced (Tang Ben Cao) which included many original drawings of herbs for the first time. In the Ming Dynasty, the famous herbalist Li Shizhen wrote his book "Ben Cao Gen Mu" (compendium of materia medica). He personally collected a large number of herbs, made conscientious investigations, dissected some medicinal ingredients from animals and compared and refined some medicinal minerals. He consulted more than 800 references and took 27 years to accomplish the book which lists 1892 medicines and more than 10,000 prescriptions. His work is a great contribution to Chinese traditional medicine (Xiao, 1981a, Zhang, 1988).

"Chinese Materia Medica" is regarded as a modem scientific works of traditional Chinese medicine. This series works clarify many previous confusion in Chinese herbs on the basis of latest experimental results.

The Chinese Pharmacopoeia (1990) lists 782 dmgs or prescriptions which are being used in China nowadays and 45% of these dmgs are medicinal herbs. There are 35,000 species of plants in China and 20% of them are recorded as medicinal plants (Anon, 1977; Xiao, 1981b).

55 Table 1.5 Examples of some texts on Chinese medicinal plants, animals and minerals

(Xiao, 1981b)

Date Title Number of volume and item contained

221B.C.- Sheng Nong Ben Cao Jing 1 vol., 365 items 220A.D. 659 Tang Ben Cao 53 vol., 844 items

1086-1106 Zheng Lei Ben Cao 31 vol., 1748 items

1590-1596 Ben Cao Gang Mu 52 vol., 1894 items

1961-1963 Chinese Meteria Medica 4 vol., 994 items (first edition) 1979-1993 (second edition) 6 vol., 665 items 1990 Pharmacopoeia of People’s 1 vol., 784 items (lastest edition) Republic of China 1992 (English edition)

1.5.2.2 Traditional Chinese medicine used in the treatment of CNS disorders The traditional Chinese medicine has been used for the treatment of various disorders of the CNS. About 50 herbs listed in the Chinese Pharmacopoeia show effects in the CNS (Xiao, 1989;). Some examples are given in Table 1.6 (p.58). Traditional Chinese medicine is a coherent and independent system of thought and practice. TCM believes that all the higher nervous activities, such as mental activity, consciousness and thinking mainly come from the functions of Heart and Liver (here the term Heart and Liver differ from the heart and liver in normal English, being capitalized). Because

56 the Chinese doctor prescribs medicine based on this theory, there are a number of differences in the medical treatment between the east and the west, which also influence the drug development.

The synthetic drugs used in clinical practice for the treatment of the CNS disorders are mainly nitrogen-containing, small molecular weight (MW 200-600) and lipid soluble compounds, whereas the CNS active constituents isolated from plants possess a wide range of structural types. They may be either nitrogen containing compounds such as berberine-, morphine-, aporphine-, diterpene-, - and some other types of or non-nitrogen containing such as sesquiterpene lactones, essential oils, benzyl alcohol-glucosides, coumarins, flavonoids, triterpenoids, irodoids or other types of compound. Some of them have larger molecular weight (MW 700-1200) and are water soluble. These different characteristics of plant principles exhibit the possibility to develop new types of drugs by modem technology. Traditional Chinese medicines have resulted from millennia of trials and errors and their use is based on TCM theory. It need to be carefully investigated if new drugs, which meet the criteria of modem treatment, can be developed from these herbal medicines. In general, new drugs should be based on the following characteristics: - greater therapeutic efficacy and selectivity than existing dmgs - lower toxicity - fewer or less disturbing side effects - more desirable pharmacokinetic features - different mechanisms of action - a basically different chemical stmcture

57 Table 1.6 Some CNS active principles isolated from Chinese herbs (Xiao, 1981b)

Plant Name Active principles Effect C o i y d a l i s analgesic tarschanlnovii PCH, sedative

C H 3 1

Sinoihenium acutum analgesic C H î O v / ^ sedative

S te p h a n ia 1-dicentrine analgesic dicentrinifera sedative

C H 5 0 .

CH, A c o n itu m delavaconitine surface anesthesia e p is c o p a le

CH.O

Datura met el used in Chinese traditional anesthesia

rcH, 00c—c- .CH,OH'

L o r a n th u s coriairrytin (a for the treatment parasiticus (b) of schizophrenia

R OH b; R=OH

58 Table 1.6 continued

Acorus gramineus (a), CNS depressant, (3-asarone (b) for the treatment of epilesy CCHj

a b 1 Asarum forbesil elemicin strong sedative, anesthesia _

CHjO— '^CH,CH=CH;

CH.O-^

P a t r i n i a patrinene sedative,used in scabiosaefolia neurasthenia with insomnia as its main symptom ! 1

Gastrodia elata gastrodin sedative, hypnotic for the treatment of vertigo

/f H / HoTH °"

Daphne oleoides daphnetin analgesic sedative

HO

59 1.6 The Purpose and Strategy of This Study Ten Chinese plants have been subjected to the detail in the present study. The strategy for the investigation includes: - pharmacological screening of the plant extracts to ensure they are effective - bio-assay guided fractionation of the active extracts - isolation of major principles from the active fractions - identification of structures of these principles - pharmacological studies on these principles The purposes of this study are: - to search for potential CNS active compounds which have novel structures - to screen the plant extracts for the particular biological activities in order to prove the rationale of using these plants - to understand the mechanism of the herb action

60 Chapter 2

Materials and Methods

61 2.1 Plant material Nine Chinese plants were collected from the Jinfo Mountain, southwest of China on November 30, 1990 and identified by Professor Z.Y. Liu, the head of the Department of Botany, Institute of Medicinal Plant Cultivation, Chong Qing, Si Chuan Province, P. R. China. The plants were identified as Schefflera bodinieri (Levi.) Rend, S. delavayi (Fr.) Harms, Celastrus angulatus Maxim, C. orbiculatus Thunb., Periploca callophylla (Wight) Falc., P.forrestii Schlechter, Clerodendrum mandarinorum Diels, C. bungei Steud, Alangium plantanifolium (Sieb. et Zucc.) Harms. The voucher specimens are kept in the herbarium of the Institute of Medicinal Plant Cultivation, Si Chuan, P.R. China. Another species, Uncaria rhynchophylla (Miq.) Jacks, purchased from a herb shop in London and identified by Ms. H.W. Yu (an expert of identifying crude drug, from Shanghai College of Traditional Chinese Medicine, P.R. China), was also studied in the present work.

2.2 Preparation of plant extracts The air-dried plant materials were ground, then soaked and percolated repeatly by 70% aqueous ethanol at room temperature until no more coloured material could be extracted. Removal of the solvent under vacuum at 50°C yielded reddish brown or dark green semi-solid extracts. The yields of each extract are given in Table 2.1.

2.3 Chromatographic techniques used for the isolation Column chromatography was used routinely for fractionation of the cmde extracts. The plant extract was dissolved in MeOH, then mixed with silica gel (35-70 mesh, Merck, Art 7733) at a ratio 1:1 (w/w). The mixture was dried and added to the top of a column filled with silica gel (70-230 mesh, Merck, Art.7734). The column was

eluted successively with petroleum spirit, CHCI 3 , CHCl^-MeOH mixture (with increasing the ratio of MeOH) and aqueous MeOH (with increasing the ratio of water). The fractions were collected and monitored by thin-layer chromatography (TLC) on

silica gel 6 OF2 5 4 precoated plates (Merck, Art.5554). The similar fractions were combined. Purification of compounds from the fractions was conducted on another column filled with silica gel (Sorbsil™ 60, M.P.D. 60A, 40-60H microns, Rhone Poulenc, SL 1330) and the column was eluted with solvent systems selected by TLC.

62 Table 2.1 Plants for Biological Screenings

Name of plant Part Extract (%) Schefflera bodinieri (Levi.) Rend leaf 16 root 7

S. delavayi (Fr.) Hams leaf 1 2 root 4

(Araliaceae) stem 1 Celastrus angulatus Maxim stem 3 C. orbiculatus Thunb. stem 3 (Celastraceae)

Clerodendrum mandarinorum Diels root bark 1 0 C. bungei Steud root bark 5 (Verbenaceae)

Periploca callophylla (Wight) Falc. stem 6

P. forrestii Schlechter stem 2 (Asclepiadaceae)

Alangium plantanifolium (Sieb. et Zucc.) root bark 6 Harms (Alangiaceae)

Uncaria rhynchophylla (Miq.) Jacks. stem hook 6 (Rubiaceae)

Several compounds were obtained pure by means of a second chromatographic column. However, for some compounds, repurification was needed in order to obtain the single entity.

Sephadex columns (LH-20,Sigam, UK) were used for purification of compounds from polar fractions of the plant extracts. Polyamide columns (polyamide 6 6 , MW 16,000- 20,000, Shanghai Regent Factory, China) were used to purify flavonoids. HPLC (Waters 991, photodiode array detector, Millipore Ltd. Column: normal phase. Si dp, 5p, 4.6mm X 25cm, Beckman, USA) was employed to separate the compounds whose structure closely related. The reverse phase HPLC (Column: ODS dp, 5p, 4.6mm X 25cm and 10.0mm 25cm, Beckman, USA) was used to separate ethanol soluble or water soluble compounds.

63 The fractions and compounds were examined on TLC plates observed under UV lamp (254nm or 366nm). Spray reagents were also employed. Dragendorff reagent was used to detect alkaloids; vanillin-sulphuric acid reagent for detecting and saccharides, and NH 3 atmosphere for detecting flavonoids and coumarins.

2.4 Spectroscopy for structure identification 2.4.1 Ultraviolet spectrophotometry (UV) UV spectra were recorded on a double beam Perkin-Elmer 402 ultraviolet-visible spectrophotometer. The shift reagents, including AICI 3 , HCl, NaBO^, NaOAc and NaOH were prepared according to Mabry, et al (1970). The samples were made in methanol and the concentration of sample was 1 mg/100ml. The measured region of wavelength was 200-400 nm.

2.4.2 Mass spectrometry (MS) Electron impact mass spectrometry (EIMS) and accurate mass were recorded on an Analytical ZAB-2F (VG, Micromass Ltd.) mass spectrometer operating at 70 ev, at inlet temperatures of 170-240°C. Fast atomic bombardment mass spectrometry

(FABMS) using 8 K ev atoms with an ion current of 0.5mA was conducted on an Analytical ZAB-2F (VG Micromass Ltd.) mass spectrometer. Samples for FABMS were prepared using the matrix indicated on each spectrum. Chemical ionization mass spectra (CIMS) were measured on a VG masslab 12-250 quadrupole instrument using ammonia as reactant gas unless otherwise stated.

2.4.3 Nuclear magnetic resonance spectroscopy (NMR) Proton magnetic resonance (PMR) spectra were recorded on Bruker WM-250, Bruker AMX-300, Bruker AMX-400 or Bruker AMX-500 spectrometers, operating at 250 MHz, 300 MHz, 400 MHz and 500 MHz respectively. Chemical shifts are reported in parts per million (ppm) on the Ô scale and are relative to tetramethylsilane ( T M S ) .

Solvents used were spectroscopic grade deuterochloroform (CDCI 3 ), methanol-d^

(CD 3 O D ) and D M S O -d g . All two-dimensional experiments were performed either on the 400 MHz or 500 MHz spectrometers.

64 Carbon-13 magnetic resonance (CMR) spectra were recorded on Bruker AMX-500 operating at 125.7, Bruker AMX-400 operating at 100.6 MHz, Bruker AMX-300 operating at 75.2, or Bruker WM-250 operating at 62.9 MHz. Chemical shifts are reported in parts per million (ppm) on the 5 scale relative to TMS. The solvents used were the same as for PMR analysis.

2.5 Hydrolysis of glycosides 2mg of a glycoside was dissolved in 10 ml MeOH with 10% HCl and heated at 80°C for one hour. The reaction solution was diluted with water, and extracted with CHCI 3 .

The extract was dried over anhydrous Na 2 S0 4 and filtered. Removal of the organic solvent resulted in the aglycone. The water layer was neutralized by NaOH and dried in vacuo to give the sugar moieties of the glycoside. The dry materai was dissolved in MeOH and identified by co-TLC with authentic samples. The solvent systems used for co-TLC to identify glucose and rhamnose included (1) CHCl^-MeOH-HAc 2:2:1

(glucose Rf 0.51, rhamnose Rf 0.68); (2) BUOH-HAC-H 2 O 5:1:2 (glucose Rf 0.17, rhamnose Rf 0.46); (3) EtOAc-MeOH-HAc 3:2:1 (glucose Rf 0.35, rhamnose Rf 0.58).

2.6 The protocol of ligand-binding assays for plant extract screening Each extract was tested by radio-ligand receptor binding assays. The receptors tested included a l, a2, p-adrenoceptors, 5HT1,5HT1A, 5HT2, opiate, benzodiazepine. C a t­ ion channel, K^-ion channel, dopamine 1, dopamine 2, adenosine 1, muscarinic, histamine 1, Na^/K"^ ATPase, GABA^ and GABAg.

The concentration range of the extracts was 0.1-1.0 mg/ml. Each extract was dissolved in absolute ethanol, 70% aqueous ethanol and the buffer used in the experiment, respectively (Table 2.2, page 70).

The details of each receptor binding assay are given in Table.2.2. The general procedure, except for GAB A tests, is given below: Each test plant extract (50 pi, at certain concentration) and required [^H]-ligand (50pl, at certain concentration) mixed with target tissue (400pl, final protein concentration at 0.4 mg/ml). The mixture was incubated for a specified time at specified temperature

65 (details in Table 2.2). Filtration was carried out on a pressure reduced Brandel Cell Harvester, (a filtration machine, Brandel, Gaithorsberg, MD). Samples were filtered on GF/B filters, which were presoaked in ice-cold 0.5% PEI for at least 15 minutes, and washed three times by ice-cold working buffer (see Table 2.2). The filter covered with samples was either punched into vials and soaked in scintillation solution for about one hour before counting or the whole filter was prepared for P-plate counting. The scintillation counters (LS-6000TA, beta plate or big spot counters, Beckman Ltd.) gave sample counts in either DPM or CPM.

For the GABA^ test,(-)-baclofen (62.5pl, 1.6mM, lOOpM final concentration) was added to SOOpl rat synaptic membranes for binding all the GABAg receptor on the tissue. The ligand ^H-GABA (lOOpl, lOnM final concentration) and test compound (lOOpl, lO^M-lO^M final concentrations) were then added. The non-specific binding was determined by adding isoquavacine HCl (lOOpl, lOOpM final concentration).

In the GABAg test, isoquavacine HCl (62.5pl, 640pM ,40pM final concentration) was added to SOOpl rat synaptic membranes for binding all the GABA^ receptor on the tissue. CaClz (62.5pl. 40mM, 2.5mM final concentration), ^H-GABA (lOOpl, lOnM final concentration) and test compound (lOOpL lO^M-lO^M final concentrations) were then added. The non-specific binding was determined by adding (-)-baclofen(lOOpl, lOOpM final concentration).

In both GABA;^ and GABAg experiments, the synaptic membranes with added compounds and radio ligand were incubated for 15 minutes at room temperature, then were washed by ice-cold 50mM Tris buffer with Brandel Cell Harvester. Samples were filtered on GF/B filters, then punched into vials and soaked in scintillation solution for one hour before counting. The counting procedure was the same as other receptor binding assays.

The membranes were prepared as follows: (lIFromi rat brain, pig brain and pig striatum Frozen whole rat brains were obtained from Charles River Ltd. and frozen pig brains

66 were obtained from Serotec Ltd.. The pig striatum was dissected out of the brain. The tissues were homogenised in 10 volumes of ice cold Tris HCl buffer (50 mM, pH 7.4) using a polytron, setting 5 for 20 seconds (Mechanically driven Potter-S loose-fitting glass teflon homogeniser). The homogenates were pooled and then centrifuged for 20 minutes at 50,000g in a Sorvall centrifuge (Sorvall RC5C Centrifuge SS-34 rotor). After spinning the supernatant was discarded and the pellet reconstituted in fresh ice- cold buffer (10 volumes), using the polytron at setting 5. The suspension was recentrifuged under the same conditions and the supernatant was discarded. The last stage was repeated once more. The final pellet was homogenised using the Polytron (setting 5 for 20 seconds) in 10 volumes of ice-cold Tris HCl (50 mM) and the homogenate was stored in aliquots at -70°C for subsequent study.

(2). From Guinea pig brain A similar procedure was used for frozen guinea pig brain obtained from Charles River Ltd.. The differences included that the suspension} need to be incubate at 37°C for 10 minutes before the final spinning and the storage buffer (Tris HCl, 50 mM) | ( need containing 4.0mM CaClj, l.Og/1 ascorbic acid and lOpM pargiline.

(3). Rat brain synaptic membrane This procedure was based on the method of Bowery et al (1983). A freshly dissected whole brain from male Wistar rat (200-250g) was homogenised in 14 volumes (approximately 25ml/brain) ice-cold sucrose (0.32M) solution using a Potter-Elvehjem homogeniser. The homogenate was centrifuged at lOOOg for 10 minutes, the resulting PI nuclear pellet discarded and the supernatant recentrifuged at 20,000g for 20 minutes forming a P2 pellet. This was lysed by resuspension in 20ml ice-cold distilled water followed by centrifugation at SOOOg. The "buffy coat" was retrieved by washing with the supernatant and the combined suspension was then centrifuged for a further 20 minutes at 48,000g. The resultant crude synaptic membranes were stored at -20°C for a minimum of 24 hours. The frozen membrane was then thawed and resuspended

in 25ml Tris-HCl buffer (50mM, pH7.4) containing 2.5mM CaCl 2 , standing for 45 minutes at ambient temperature prior to centrifugation at 9000g for 10 minutes. After spinning the supernatant was discarded, the pellet reconstituted in fresh ice-cold

67 buffer and incubated for 15 minutes in ice. The suspension was recentrifuged at 9000g for 10 minutes and this washing procedure was repeated further two times with 15 minutes incubation periods in ice between each centrifugation. All centrifugation steps were performed at 4°C. The final pellet was resuspended in 35 ml buffer and was ready for use .

2.7 The protocol of ligand binding assays for fraction screening The fractions were obtained from the separation of the crude extracts as described in Section 2.3. They were tested by ligand-receptor binding assays following the procedure given in Section 2.6. The concentrations of the test samples were 0.1 mg/ml and 1 mg/ml. The crude extracts were also tested at the same time as references.

2.8 The protocol of ligand binding assays for compound screening The isolated compounds were dissolved in the appropriate solvent to make a stock solution of lO'^M, then serially diluted to lO^M - lO'^M using an automated liquid handling system (Tecan, UK Ltd). The same procedure as listed in Section 2.6 was used for the screening.

2.9 The protocol of ligand binding assays for testing interaction between the isolated compounds and agonists or antagonists of receptors In order to investigate interaction between the isolated compounds with either agonists or antagonists (here especially the control compounds used in assays) of various receptors, the isolated compounds were added to control compounds in the tests. The analysis control compounds was followed the similar japproachdescribed in Table 2.2 (p.70) except containing 10 ^ M (final concentration) of isolated compounds in each sample. The IC50 curves and were established for both the control compounds alone and controls plus isolated compound.

2.10 Analysis of ligand-receptor binding data Non-specific binding was estimated in the presence of a high concentration of a

68 receptor specific non-radioactive compound (the compounds were listed in Table 2.2 as NSB). The amount of ligand non-specifically bound was subtracted from the total amount of radioactivity bound in the absence of any compound to yield the amount of ligand specifically bound. The specific portion of bound radioactivity was then expressed as a percentage of the total radioactivity bound. Alternatively, the amount of radioactivity specifically bound at a given concentration of non-radioactive compound was expressed as a percentage of the total ligand specifically bound.

69 Table 2.2 Radioligand Receptor Binding Methods

Receptor Ligand (Conc.nM)W. Control (Conc.M) NSB * (conc.M) Inc.* (min)/°C Buffer* Tissue al-adrenocetor prazosin (0.2) prazosin (10 ’-10 “) (10’^) 30/25 50mM Tris rat brain a2-adrenoceptor UK14304 (0.5) phentolamine (10^-10 '^ phentolamine (10^) 30/25 50mM Tris rat brain P-adrenoceptor DHA (2.0) propranolol (10^-10 '") propranolol (10’°) 30/25 50mM pig brain Tris+lOmMMgCl, adenosine 1 CHA (2.0) CHA (lO^-lO"*) CHA (10’°) 60/25 Krebs 50mM Tris rat brain dopamine 1 SCH-23390 (0.5) SCH-23390 (10-®-10 ‘") (+)- (10’°) 30/25 50mM Tris+2mM pig brain MgCh striatum dopamine 2 (0.2) butaclamol (lO'^-lO ") butaclamol (10’°) 30/25 50mM Tris pig brain striatum muscarinic QNB (0.2) atropine (10"*-10 '®) atropine (10’°) 60/25 Krebs Tris pig brain Ca^+ DHP (0.2) nitrendipine (lO'^-lO ") (10’’) 30/25 Krebs Tris pig brain ATP (l.O) (lO’-lO"") glibenclamide (10’°) 30/25 Krebs Tris pig brain 5HT1 5HT (2.0) 5HT (lO'^-lO’) 5HT (10’°) 30/25 5HT buffer guinea pig bram 5HT1A 8-OH DPAT (1.0) 8-OH DPAT (10“*-10 ‘®) 8-OH DPAT (10’°) 30/25 5HT buffer guinea pig brain 5HT1C mesulergin (2.0) mesulergin (10^-10^) mesulergin (10“*) 30/25 5HT buffer guinea pig brain 5HT2 (2.0) spiperone (10^-10 '°) spiperone (10’°) 30/25 50mM Tris rat brain histamine 1 pyrilamine (1.0) pyrilamine (10^-10 '") promethazine (10’°) 60/25 50mM Tris pig brain benzodiazepine (1.0) flunitrazepam( 10"*-1 O’ '“) diazepam (10 °) 60/5 50mM Tris pig brain opiate naloxone (1.0) naloxone (10"^-10’'°) naloxone (10’°) 30/25 50mM Tris pig brain

GABA a G ABA (10) G ABA (10’’-10’°) isoguavacine (10“*) 15/25 50mM Tris rat brain synap.membr. GABAg G ABA (10) G ABA (10^-10’°) (-)baclofen (10“*) 15/25 50mM Tris rat brain synap. membr. Na+/K+ ATPase ouabain (5.0) ouabain (lO’^-KT’) ATP buffer 2 60/37 ATP buffer 1 rat brain

*: buffer preparation see Table 2.3; NSB: the compound for determining non specific binding; Inc.: incubation time. * Kd: the Kd values see the page back

70 Range of the Kd value (nM) of radioligands in the assays

al-adrenocepter 0.2-0.3

a 2 -adrenocepter 0.4-0.8

P-adrenoceptor L5-4.5

adenosine 1 1.2-3.8

dopamine 1 0 .2 -0 . 6

dopamine 2 0.1-0.3

muscarinic 0.1-0.3

Ca^+ DHP 0.1-0.4

K+ ATP 0.7-1.5

5HT1 1.5-4.5

5HT1A 0 .8 - 1 . 8

5HT1C 1.2-3.0

5HT2 1.5-4.0

histamine 1 0.8-1.5

benzodiazepine 0.9-2.8

opiate 0 .8 -2 . 0

NaVK^ ATPase 4.0-7.0

GABA^ 50-80

GABAg 50-8 Table 2.3 Buffer Preparation

Buffer Preparation

50 mM Tris 50mM Tris.HCl (MW 151) in distilled water, adjusted to pH 7.5 at 25°C

Krebs Tris 50mM Tris.HCl; 136mM NaCl (MW 58.44); 5mM KCl (MW 74.55); 2.5mM CaCl^ (MW 219); 1.2mM MgClz (MW 203) in distilled water, adjusted to pH 7.5 at 25°C

5HT buffer 50mM Tri.HCl, 2mg pargiline, 4ml CaCl; solution, and Ig Ascorbic acid in IL distilled water, adjusted to pH 7.5 at 25°C

ATP buffer 1 (assay buffer) 50mM Tris.HCl, 300mM NaCl, lOmM MgClz, lOmM Tris-ATP (vanadium free. Sigma A0520) in distilled water, adjusted to pH 7.7 at 25°C

ATP buffer 2 (NSB buffer) 50mM Tris.HCl, 300mM NaCl, lOmM MgClg in distilled water, adjusted to pH 7.7 at 25°C

71 Chapter 3

In vitro biological screenings of 10 species of Chinese plants

72 3.1 Introduction A number of Chinese herbs are used for the treatment of CNS disorders in China. Some in-vivo tests have confirmed their activities and the active principles have been identified. However, veiy little is known about their actions at the molecular level. Modem ligand-receptor binding assays enable plant extracts to be tested against the receptors associated with pharmacological activities. Such an approach allows for bioassay guided fractionation of extracts in order to isolate active compounds and provides scientific information to corroborate the activities offered in clinical practice.

There are 55 traditional herb medicines listed in the Chinese Pharmacopoeia (Anon 1990; 1992) which are generally used for the treatment of CNS disorders. Most of these herbs have been studied for their chemical constituents and for their major pharmacological functions (Anon, 1979-1993). There are also several hundreds of herbs which have various effects on the CNS, and they are to some extent used in different areas of China (Anon, 1977). Most of these herbs have not been subjected to a detailed study. In searching for new types of CNS active principles and for a better understanding of their actions, ten plants were selected for the present study. Apart from Uncaria, these plants have not been previously investigated chemically or biologically. The extraction of these plants was described in Section 2.2 and their biological screening method was described in Section 2.5.

3.2 The screening results of the plant extracts The screening results of the plant extracts against 18 receptors are listed in Table 3.1. The highest %inhibition of each extract against different receptors has been plotted as bar graphs (Figure 3.1, p.78) in order to visualize the major biological activities of the plants and to facilitate the choice of plant for further investigation. The plants with % inhibition values over 90% at concentration 1 mg/ml are listed in Table 3.2.

73 Table 3.1 % Inhibition of Radioligand Specific Binding of Plant Extracts Against Different Receptors

Ex.l* Ex.2 Ex.3 Ex.4 Ex.5 Ex.6 Ex.7 Ex.8 Ex.9 Ex.10 Ex.ll Ex. 12 Ex.13

R(D* Cone 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1

al* SI* 76 5 56 5 70 6 56 8 74 6 41 4 60 3 17 1 29 0 53 5 57 3 80 28 32 0 (prazosin) 2 93 13 92 9 94 13 87 10 93 7 72 19 60 6 19 0 22 0 52 2 46 3 78 3 80 51 3 48 8 9 4 9 0 13 0 14 0 12 3 0 0 0 7 0 0 0 0 33 4 37 3 23 16

oc2 SI 80 24 63 20 92 13 65 15 52 18 76 44 83 44 38 39 69 30 79 49 78 49 80 33 65 40 (UK14304) 2 86 32 72 11 100 20 77 25 71 10 72 39 74 61 47 44 44 37 73 44 61 44 64 31 80 51 3 62 34 9 0 43 20 22 15 24 7 49 14 66 47 37 33 37 53 25 33 47 48 28 18 67 40 P SI 35 4 56 5 8 0 30 0 12 0 60 21 17 22 2 1 0 0 0 25 0 4 0 11 12 0 (DHA) 2 38 6 40 13 0 0 40 4 0 1 63 21 0 19 0 8 0 2 3 25 0 8 0 11 11 0 3 60 16 24 14 9 2 15 0 18 1 10 17 0 2 0 24 0 17 0 22 0 6 0 0 5 0

5HT1 SI 100 28 69 46 82 0 81 0 54 0 78 35 98 67 0 0 8 35 61 45 40 29 90 44 - (5HT1) 2 100 38 100 41 100 0 76 26 35 0 87 40 98 96 0 0 74 54 50 61 0 37 92 33 -- 3 100 32 28 0 41 0 0 23 70 46 61 39 17 51 0 18 0 30 0 27 38 20 42 30 --

5HT1A SI 35 7 25 9 0 0 0 0 0 0 67 56 100 98 29 31 33 56 98 66 68 57 74 56 67 49 (DPAT) 2 63 4 42 0 11 0 8 0 0 0 65 64 100 98 29 30 61 68 64 54 43 50 69 67 92 64 3 40 13 12 10 5 0 12 3 0 0 63 31 84 98 35 33 87 63 48 58 38 40 54 60 90 62

5HT2 SI 81 16 73 34 54 0 41 0 51 2 76 17 85 25 0 0 0 0 37 6 0 10 89 13 46 17 (kentanserin) 2 98 18 62 50 18 3 48 0 32 9 95 70 81 33 0 6 3 7 40 5 1 4 72 11 87 62 3 75 20 37 11 17 0 3 0 11 0 33 13 0 13 0 7 0 0 0 0 6 2 50 2 43 2

74 Table 3.1 continued

Ex.l Ex.2 Ex.3 Ex.4 Ex.5 Ex.6 Ex.7 Ex.8 Ex.9 Ex. 10 Ex. 11 Ex. 12 Ex.13

R(L) Cone. 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1

opiate SI 100 82 100 87 100 82 100 79 100 84 100 60 100 55 0 14 100 58 100 50 94 31 92 39 68 42 (naloxone) 2 100 89 100 88 100 87 100 76 100 95 100 45 100 53 9 35 100 55 100 41 100 45 100 36 89 67

3 100 85 100 72 100 67 100 67 100 60 85 33 41 28 1 45 22 15 41 40 48 45 82 24 39 14

adenosine 1 SI 75 0 67 0 0 0 0 0 0 0 98 51 93 39 38 25 37 26 87 40 80 20 58 46 22 5 (CHA) 2 100 0 76 0 0 0 0 0 0 0 96 54 95 40 43 24 53 24 83 36 76 25 87 33 45 12 3 40 0 30 0 0 0 0 0 0 0 92 50 38 19 39 16 47 14 37 17 48 21 8 17 12 2 benzodiazepine SI 59 28 38 19 51 23 46 40 46 21 19 9 20 9 51 5 29 6 61 6 23 2 36 10 - (flunitrazepam) 2 63 11 37 0 51 0 35 30 32 20 29 11 35 8 54 7 36 9 17 1 24 0 33 3 -- 3 51 23 31 14 34 15 38 18 35 12 26 7 19 3 28 13 14 7 21 1 14 0 15 0 - - Ca^channel SI 94 33 87 75 63 17 58 21 60 33 59 26 100 59 5 17 4 17 74 37 34 23 47 24 46 21 (nitrendipine) 2 92 44 59 48 34 15 66 21 24 19 33 22 100 53 3 22 18 24 49 26 21 21 48 17 65 17 3 88 32 66 35 15 14 43 16 43 12 27 23 27 22 14 20 17 24 21 23 42 24 21 22 33 14

K'^channel SI 95 24 65 14 79 25 58 10 81 23 39 4 60 16 39 7 4 11 60 14 52 7 54 2 43 18 (glibendamide) 2 100 27 61 16 99 20 56 16 77 27 40 7 66 9 37 9 21 9 35 6 34 7 57 7 47 21 3 78 28 32 11 33 6 31 10 38 10 18 10 9 3 7 3 11 6 9 5 43 9 17 1 65 23

Dopamine 1 SI 98 28 79 0 77 10 85 6 94 16 63 13 70 11 15 10 49 18 65 14 63 12 77 15 64 37 (SCH-23390) 2 100 27 95 0 81 10 88 4 59 11 69 15 59 12 18 8 60 20 47 13 64 14 69 14 47 21 3 88 32 17 0 26 0 24 1 22 0 16 11 6 11 4 5 3 13 1 9 33 10 6 12 12 2

75 Table 3.1 continued

Ex.l Ex.2 Ex.3 Ex.4 Ex.5 Ex.6 Ex.7 Ex.8 Ex.9 Ex.10 Ex. 11 Ex.l2 Ex.13

R(L) Cone. 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.1

Dopamine 2 SI 100 33 79 27 74 28 89 14 77 18 78 22 88 30 0 0 15 18 75 20 63 46 85 13 -- (spiperone) 2 100 33 85 0 80 27 79 27 77 28 100 92 92 53 0 0 27 21 41 16 15 29 80 15 -- 3 100 39 37 10 53 0 48 8 38 8 57 28 27 22 0 0 0 14 0 17 20 23 17 13 --

muscarinic SI 32 2 2 1 18 3 0 0 3 3 7 6 8 7 36 5 24 2 44 9 44 15 6 11 -- (QNB) 2 40 4 5 3 43 1 2 4 8 0 16 5 14 5 22 4 23 6 13 5 12 7 7 9 - - 3 45 5 6 0 3 0 4 3 11 0 8 6 5 5 6 5 10 7 10 6 24 7 8 25 --

histamine 1 SI 79 26 65 13 45 16 62 15 56 24 74 27 93 54 0 0 0 21 82 22 61 9 93 30 33 18 (pyrilamine) 2 87 55 39 4 66 10 89 6 34 9 73 23 95 42 0 0 50 23 76 33 89 12 92 35 45 27 3 53 9 9 2 26 17 5 17 23 14 13 13 10 21 0 0 0 21 0 18 13 8 14 27 70 46

Na+/K+ATPase SI 36 10 10 10 28 8 8 8 2 2 22 0 7 0 23 0 0 0 30 4 47 0 27 2 -- (ouabain) 2 53 6 3 3 26 7 3 3 23 0 0 0 0 0 56 0 0 0 0 16 41 0 0 0 -- 3 33 20 10 10 28 9 10 10 36 10 8 0 7 3 29 0 0 0 25 2 51 0 30 3 - -

GABA* SI 37 26 100 71 79 64 88 55 91 88 100 100 96 93 0 81 73 91 0 81 0 83 95 95 79 41 (GABA)

GABA b SI 67 24 84 68 100 75 100 62 100 95 100 100 99 87 0 53 79 87 0 90 0 45 100 100 82 55 (GABA)

Note; *. 'ExA-Schefflera bodinieri (leaf), Ex.2-S. bodinieri (root), Ex.3-S. delavayi (leaf), Ex.4-S. delavayi (root), Ex.5-S. delavayi (stem), Ex.6-Clerodendrum mandarinorum (root bark), Ex.l-Alangiwn plantanifolium (root bark), Ex.%-Periploca callophylla (stem), Ex.9-Celastrus orbiculatus (stem), ExAO-Periploca forrestii (stem), ExAl-Celastrus angulatus (stem), ExAl-Clerodendrum bungei (root bark), Ex.l3-f/ncar/a rhynchophylla (stem with hooks), all extracts were tested in concentrations of 0.1 mg/ml and 1.0 mg/ml; R-receptor, L-ligand ; S-solvent: 1-70% ethanol, 2-100% ethanol, 3-working buffer (see Table 2.2); a l- a l adrenoceptor, o2-a2 adrenoceptor, p-p adrenoceptor^ each experiment was conducted three times and the SEM range o f the extract screening was around ±15.

**. - not tested

76 Table 3.2 Plant Extracts with >90 % Inhibition of Radioligand

Specific Binding to Different Receptors

Receptor Plant

a 2 -adrenoceptor Schejflera delavayi

5HT1 S. bodinieri, S. delavayi, Alangium plantanifolium

5HT2 S. bodinieri opiate S. bodinieri, S. delavayi, Clerodendrum mandarinorum,

Alangium plantanifolium, Celastrus orbiculatus, Periploca

forrestii, Celastrus angulatus, Clerodendrum bungei, Uncaria

rhynchophylla

adenosine 1 Clerodendrum mandarinorum, Alangium plantanifolium

GABA a S. bodinieri, Clerodendrum mandarinorum, Alangium

plantanifolium, Clerodendrum bungei

GABA b S. bodinieri, S. delavayi, Clerodendrum mandarinorum,

Alangium plantanifolium, Clerodendrum mandarinorum

Ca^channel S. bodinieri, Alangium plantanifolium

K^channel S. bodinieri

dopamine 1 S. bodinieri

dopamine 2 S. bodinieri, Clerodendrum mandarinorum, Alangium

plantanifolium

histamine 1 Alangium plantanifolium, Clerodendrum bungei

77 Figure 3.1 The % Inhibition Radioligand Specific Binding at Different

Receptors of Each Plant Extract ( Data from Table 3.1)

Schefflera bodinieri (le a v e s)

% Inhibition

Receptor*

Schefflera bodinieri (Roots)

% Inhibition

100 —

H I J Receptor *:Receptor: A -al adrenoceptor, B-a2 adrenoceptor, C-p adrenoceptor, D- 5HT1, E-5HTIA, F-5HT2, G- opiate, H-benzodiazepine, I-Ca^* channel, J-K^channel, K-dopamine 1, L-dopamine 2, M-adenosine 1, N- muscarinic, 0-histamine 1, P-Na*/K^ ATPase, Q-GABA^, R-GABAg

78 Schefflera delavayi (leaves) % Inhibition

80 — i

Receptor CDEFGHl JKLMN Schefflera delavayi (roots) % Inhibition

I p

g 1 ! __ H

ABCDEFGHI JKLMNOPQR

Schefflera delavayi (stems) % Inhibition

I I I I I J K M N 0 P Q R Receptor

^:Receptor; A -al adrenoceptor, B-o2 adrenoceptor. C-P adrenoceptor. D- 5HT1. E-5HT1A. F-5HT2. G-opiate. H-benzodiazepine, I-Ca^’ channel. J-K*channel. K-dopamine 1. L-dopamine 2. M-adenosine 1. .N-muscarinic, 0-histamine 1. P-Na*/K" ATPase, Q-GABA^, R-GABAg

79 Clerodendrum mandarinorum

% Inhibition

100 — I

I I I I I I I I Receptor ABCDEFGH JKLMNOPQR

Clerodendrum bungei

% Inhibition

Receptor ABCDEFGH I JKLMNOPQR

*:R eceptor: A -al adrenoceptor, B -a2 adrenoceptor, C-(3 adrenoceptor, D - 5H T 1, E -5H T 1A , F-5H T 2, G -opiate, H -benzodiazepine, I-C a^^ channel, J-K ^channel, K -dopam ine 1, L -dopam ine 2, M -adenosine 1, N -m uscarinic, -histam ine 1, P-N aW ATPase, Q-GABA^, R-GABAg

8 0 Celastrus orbiculatus

% Inhibition

Receptor F G H 1 J K L

Celastrus angulatus

% Inhibition

100 -

90 —

80 -4

70

60 —'

50 -j - 40 -g

30 -j 20 A 10 —1 \ IB > n 1

ABC Receptor

*:R eceptor: A -al adrenoceptor, B -a2 adrenoceptor, C -p adrenoceptor, D - 5H T 1, E -5H T 1A , F -5H T 2, G -opiate, H -benzodiazepine, I-C a^^ channel, J-K ^channel, K -dopam ine 1, L -dopam ine 2, M -adenosine 1, N -m uscarinic, -histam ine 1, P-N aW ATPase, Q-GABA^, R-GABAg

8 1 Periploca forrestii

Vo Inhibition

I I I I ABCDEFGHI JKLMNOPQR Receptor

Periploca callophylla

% Inhibition

EFGHI JKLMN 0 P Q R Receptor

*:R eceptor: A -al adrenoceptor, B -a2 adrenoceptor, C -p adrenoceptor, D - 5H T 1, E -5H T 1A , F-5H T 2, G -opiate, H -benzodiazepine, I-Ca^^ channel, J-K ^channel, K - dopam ine 1, L -dopam ine 2, M -adenosine 1, N -m uscarinic 0-histam ine 1, P-N a^/K "^ A TPase, Q -G A BA ^, R-GABAg

82 Alangium plantanifolium

% Inhibit! on

100 1 90

80

70 1 60 J J 50 1 1 40 n

30 ! 20 -J 10 H

0 I I I I I I Receptor JKLMNOPQR

Uncaria rhynchophylla

% Inhibition 100

ABCEFGI JKMOQR Receptor

*:R eceptor: A -al adrenoceptor, B -a2 adrenoceptor, C -|3 adrenoceptor, D - 5H T 1, E -5H T 1A , F-5H T 2, G -opiate, H -benzodiazepine, I-Ca^^ channel, J-K ^channel, K - dopam ine 1, L -dopam ine 2, M -adenosine 1, N -m uscarinic O -histam ine 1, P-N a^/K ^ A T P a s e , Q-GABA^, R-GABAg

83 3.3 Discussion 3.3.1 Discussion on the screening results of the extracts 1. Several species of Schejflera are used as folk medicines for the treatment of pain, rheumatism, arthritis, fracture, sprains, lumbago and stomachache in China. Previous animal tests showed that an ethanol leaf extract of Schejflera arboricola had sedative, hypnotic, analgesic, anticonvulsant, muscle smooth and brochodilatory effects and these results supported it clinical uses in the treatment of trigeminal, neuralgia, migraine, sciatica neuralgia, bronchitis and asthma (Liao, 1986). In the present receptor-1 igand binding assays, it was found that the extracts of two species of Schejflera, namely S. bodinieri and S. delavayi, were able to bind with a l-, a2- adrenoceptors, 5HT1, 5HT2, opiate, Ca^-ion channel, K^-ion channel, dopamine 1, dopamine 2, adenosine 1, histamine 1, GABA^ and GABAg receptors. (Figure 3.1, p.78-79). These results correspond to the use of Schejflera species. For instance, binding to opiate , a2-adrenoceptor and 5HT1 receptors may be related with analgesic effects; binding to histamine 1 and opiate receptors may produce the sedative effects; binding to K^-ion channel receptor conformed to the effect of smooth muscle relaxation; binding to histamine 1 receptor supported the use for the treatment of bronchitis and asthma; binding with GABA receptors may be related to the anticonvulsant and sedative effects of the herb. The activities of Schejflera bodinieri leaves were generally stronger than those of roots with the exception of the GABA tests and this is in agreement with the clinical situation that the leaves are normally used in Chinese medicine. The leaf and root extracts of Schejflera delavayi gave similar screening results as those of Schejflera bodinieri.

2. Clerodendrum trichotomum is a medicinal herb which has effects as antirheumatic, analgesic and hypotensive. It is chiefly used in the treatment of rheumatism, hemiplegia, migraine, severe intermittent headache and hypertension (Xue, 1987). Its hypotensive, sedative, analgesic and antiinflammatory effects have been substantiated by animal tests (Xu, 1962; Ding, 1957). Two related species, C. mandarinorum and C. bungei, have been selected for the present studies. In initial screening (Figure 3.1, p.80), the extracts were able to bind with 5HT1, 5HT2, opiate, Dl, Al, HI, al-, a2- adrenoceptors, GABA^ and GABAg receptors which may explain their clinical use and

84 also can correspond to the results of the animal tests. Two species showed similar results and their chemical constituents were closely related as substantiated by TLC.

3. The root bark of Alangium chinense is used clinically as an antirheumatic, muscle- relaxant, channel stimulant and analgesic for the treatment of rheumatism, numbness of limbs and injuries due to impact, fractures, contusions and strains (Zheng, 1986). Previous animal tests on an ethanol extract of A. chinense showed the presence of muscle relaxant effect in dogs, rats and rabbits (Anon, 1971; 1972a). The extract was able to potentiate the effect of hypnotics in dogs, but did not produce hypnosis by itself (Anon, 1980). In clinical studies, dl-, an alkaloid isolated from this plant, was used as an adjuvant in acupuncture anaesthesia and Chinese herbal anaesthesia, with good results being obtained for 745 cases of various surgical operations (Anon, 1978). The ethanol extract of the fibrous roots was used for the treatment of chronic rheumatoid arthritis, various degrees of improvement or remission of clinical symptoms were obtained (Anon, 1972b). In the present study, an other species of Alangium, A. plantanifolium, was selected for the investigation. It showed that the 70% ethanol extract was able to bind with a2-adrenoceptor, 5HT1A, 5HT2, opiate, GABA^, GABAg and Ca^^-ion channel receptors (Figure 3.1, p.83) which was in agreement with the clinical uses of A. chinense. An analysis on TLC indicated that alkaloids, the major active constituents in A. chinense are hardly present in A. plantanifolium and its major principles were found to be terpenoids and their glycosides. Hence it is necessary to clarify the active components in A. plantanifolium.

4. Uncaria rhynchophylla is a traditional medicine listed in the Chinese Pharmacopoeia for treatment of stroke causing paralysis, facial paralysis, general numbness, hemiplegia, abnormal fetal movement, high fever, vertigo, dizziness, hypertensive, infantile convulsion and colic (Anon, 1992). Pharmacological studies (in- vivo) have shown that the aqueous decoctions and ethanol extracts have hypotensive, sedative, anticonvulsant and smooth muscle effects (Du, 1987). In the present ligand binding assays, the ethanol extract was found to bind to 5HT2, opiate, D l, a2- adrenoceptor, K^^-ion channel, GABA^ and GABAg receptors (Figure 3.1, p.83). These results were closely related to the finding observed from the animal tests and

85 suggested the presence of correlations between in-vitro ligand binding assays and in- vivo animal tests. For instance, this is especially used as an anticonvulsant for the treatment of epilepsy and it was found that the extract bound to GABA receptors which was related to this effect of the crude drug.

5. The root bark of Periploca sepium is used for the treatment of rheumatism, lumbago and arthritis (Hi, 1987). Since it is toxic, the herb is usually used in prescriptions with other herbs instead of being used alone. Two species of Periploca, P. callophylla and P. forrestii, were selected for this investigation. The results of ligand-receptor binding assays showed that the ethanol extract of P. callophylla inhibited specific binding of the ligand to the benzodiazepine receptor (54%,at 1 mg/ml), Na'^/K'^ ATPase receptor (56%,at 1 mg/ml), GABA^ receptor (81%, at 0.1 mg/ml) and GABAg receptor (53%, at 0.1 mg/ml) (Figure 3.1, p.82). These results were consistent with the finding in previous biological tests on P. sepium. P. sepium was found to contain some cardiotonic glycoside and these principles were involved in the inhibition of Na^/K^ ATPase of myocardial cellular membrane. 44% of Na^/K^ ATPase activity in guinea pigs could be inhibited by 1.5 mg of crude glycosides of the herb and nearly 90% inhibition by 2.0mg crude glycosides (Anon, 1974). However, the other species, P. forrestii tested in the present study, showed binding to the benzodiazepine receptor, Ca^^ and K^-ion channel receptors, D l, D2, HI, a l and a2 adrenoceptor, 5HT1A, Al, opiate, GABAy^ and GABAg receptors (Figure 3.1, p.82). TLC of the two species showed that the constituents in these plants are different which may result in the difference of their biological activities. It is realized that the active principles present in a particular genus may differ depending upon species and they are not necessarilly the same compounds. Hence it is important to investigate different species of herbal medicine in the same genus to provent their misuse. It is also essential that plants are correctly identified, particularly if toxicity is associated with the species.

6 . Several species of Celastrus are used in specific areas of China. The seeds have been found to possess sedative and hypnotic effects (Anon, 1977). C. angulatus and C. orbiculatus were selected for the present study. The results of binding assays

86 showed that the former was able to bind to opiate, A l, HI and GABA^ receptors and the latter bound to opiate, 5HT2, GABA^ and GABAg receptors (Figure 3.1, p.81). These receptors are related to the sedative effect of the crude drug.

7. A root extract {Panax ginseng, purchased from Sigma Ltd.) was also prepared in the present study in order to examine the consistency between the results from ligand-receptor binding assays and those obtained from in vivo tests since ginseng has been studied extensively. The 70% ethanol extract was subjected to eight receptor screenings. The results showed that the extract was able to bind to 5HT1A, 5HT1C, Dl, D2, HI and Ca^^ ion channel receptors and it did not bind to 5HT2 and ion channel receptors. The ginseng roots of are well known as a tonic herb in Chinese medicine. It is believed that they can replenish the vital energy of the body, increase the production of body fluid (secretory), act as a cardiotonic and tranquillizer and have various activities in cardiovascular system, centrol nervous system and immune system (Yang, 1986; Nagai, et al, 1971; Ganenko, 1968; Jie, et al, 1984; Yuan, et al, 1986; Choi, 1972; Wang, 1985; Abe, et al, 1979). The results of the present study showed the activities of ginseng which were related to the receptors tested, such as to be used as tranquillizer (bound to 5HT and dopamine receptors), in the treatment of asthma (bound to HI receptor) and heart failure (bound to Ca^^ ion channel receptors). It is well known that major principles in genseng are triterpenoids and their glycosides. However, triterpenoids and various saponins have not been used medically nowadays for the treatment of the CNS disorders. The finding in the present study exhibited the interest to further investigate this group of compounds.

3.3.2 Discussion on extract screening method It has been shown by the results of extract screening that radioligand receptor binding assays, which are normally used for the pure compounds, can also be used for plant extracts. This approach may provide reasonable explanations of the clinical effects associated with the medicinal plant and have consistency with the results obtained from in-vivo tests. The methods are rapid, simple and convenient for searching for active principles from plants. Since plant extract is normally a mixture of a large number of ingredients, several factors may influence the screening results:

87 1. The solvent used in ligand binding assays should be chosen carefully. Three different solvents were used for dissolving the plant extracts (absolute ethanol, 70% ethanol and working buffer). Working buffer is water based and is not a good solvent for all the compounds in the extracts at room temperature. 1 0 0 % ethanol can dissolve the most lipid molecules. However, it has influence on receptor protein and its use should be avoid. The best solvent in the assays is found to be 70% ethanol in which the majority of extracts were soluble in the tests and its influence on protein is not significant (proved by blank tests). It is essential that solvent should be tested as a blank for control purposes.

2. The concentration range of plant extract is crucial for obtaining reasonable results in the screening. It was found if the concentration is too high, the extract may not be dissolved completely; if the concentration is too low, activity may not be observed. In in-vivo tests, e.g. in a hot plate experiment, the ethanol extract of Schejflera arboricola (30g/]kg) increased the pain threshold of mice to heat and this effect was equivalent to that of 15mg/kg morphine (Anon, 1976). It can be calculated from this case that the concentration of the extract is 2 0 0 0 times higher than that of the single potent compound for producing the same effect. In ligand binding assays, the concentration of control compound, at which complete inhibition of specific binding of radioligand can be obtained, is around 1 0 (by taking the average molecule weight of control compounds as 400, the concentration is about 0.004mg/ml). It was found that as the concentration of extracts was lOmg/ml (over 2 0 0 0 times higher than that of control compounds), the extracts were not dissolved completely and it needed to be dilute to 1 mg/ml. It was found that the ideal concentration range of extract for preliminary screening was between 0 . 1 - 1 . 0 mg/ml.

3. In the pure compounds tested, a higher percentage of specific binding was usually obtained at a higher concentration. However, in the extract screening, this phenomenon was reversed sometimes, i.e., a lower specific binding was obtained at a higher concentration of an extract. This is due to the presence of particulate matter in not well dissolved sample which might entrap some radioligand and thus cause higher radio-activity counts. This problem can be overcome when the sample is diluted.

88 3.3.3 Summary The results of ligand receptor binding assays are consistent with the use of the herbal medicine in clinical practice and the finding in animal tests. Most of the extracts were active on GABA and opiate receptors at concentrations of 1 mg/ml, some of the extracts were also active at 5HT, dopamine, noradrenaline, adenosine, histamine and ion channel receptors. These findings indicate that the plants possess the activities in the central nervous system and are thus worthy of further investigation in order to isolate their active principles. However, none of the extracts were particularly active on the Na^/K^ ATPase, muscarinic or benzodiazepine receptors. As shown in Table 3.2 (p.77), Schejflera bodinieri, Clerodendrum mandarinorum and Alangium plantanifolium are the most active species in the original 1 0 plants tested and therefore, they as well as Uncaria rhynchophylla, a classical herb acting on the CNS, were selected for further chemical investigation.

Although the extracts tested showed various activities in the ligand binding assays, the plants are rarely used on their own in Chinese medicine and prescriptions frequently include some eight to fifteen herbs. It is likely that there are interactions between different herbs and between the active principles in a single prescription. It is not easy to clarify the function of the individual components in a prescription and it is necessary to obtain the major principles from plants in order to investigate their biological activities and interaction between them.

89 Chapter 4

Schefflera bodinieri

90 4.1 Introduction 4.1.1 Araliaceae and Schefflera Araliaceae belongs to the dicotyledons and contains about 80 genera and 900 species worldwide (How, 1984). There are some 23 genera and 160 species in China, being widespread in the southwest (most genera) and northeast {Panax) of the country (How, 1984). There are a number of Chinese herbs in the family and the most important medicinal genera include Panax, Acanthopanax and Tetrapanax which are generally used for providing energy, increasing the production of body fluids (secretory), improving blood supply and acting as a cardiotonic and tranquillizer. The major principles of these plants are triterpenoids, triterpenoid glucosides and polysaccharides.

Schejflera contains about 200 species worldwide with some 37 species in the southwest of China (How, 1984). S. arboricola Hayata, S. kwangsiensis Merr.ex H.L.Li and S. venulosa (Wight et Am.) Harms, are used as folk remedies for the treatment of pain, rheumatic arthritis, fractures, sprains, lumbago and stomachache in the southwest China. Several other species are also used medically in certain Asian countries such as Vietnam and India (Do, 1977; Jain, et al., 1982). Two species, S. octophylla and S. capitata, have been chemically investigated in detail resulting to the isolation of some twenty triterpenoids, triterpenoid glucosides and some oligosaccharides (Jain, et al. 1982; Strigina, et al 1975; Ty, et al 1984; Ikuta, et al. 1988; Sung, et al 1991a; 1991b; 1992; Sung and Adam, 1991; Kitajima, et al 1990; Adam et al. 1982; Lischewski, et al 1984). Several compounds which represent the different skeleton structures are shown in Figure 4.1.

91 'COOH COO Glc HO' Glc HO' CH2OH 'COOH Rham 28-0-[a-l-rhamnopyranosyl( 1-4) R= OH capitogenic acid -0-(3-D-glucopyranosyl(l-6)-] R= H cauloside A p-D-glucopyranoside of 3a-hydroxy- lup-20(29)-€ne-23^8-dioic acid

COO 'COOH Glc \ Glc HO'' Rham ho' COOH COOH

asiaticoside 3a,lla-dihydroxy-lup- 20(29)-€ne-23,28-dioic acid

t)H- OH 'COOH OHOH OH OH- OH' OH a-l-rhamnopyranosyl(l-4) oleanonic acid -0-P-D-glucopyranosy 1(1-6)- P-D-glucopyranose and its la-epimer

Figure 4.1 The diversity of compound structures inSchefflera (Sung, et al, 1991a; 1992; Sung and Adam, 1991; Kitajima, et al 1990; Lischewski, et al, 1984)

92 4.1.2 Schefflera bodinieri (Levi.) Rend Schejflera bodinieri (Levi.) Rend is a shrub which has not been recorded as a herb medicine in the Chinese literature, but it grows in the same environment as Schefflera herb species, and in some cases, it is collected mixing with the herb species for medicinal use. In the present biological screening by ligand-receptor binding assay, the leaf extract (70% ethanol) had| binding with Ca^^-ion channel, K^-ion channel, histamine 1, opiate, al, a2-adrenoceptors, 5HT1, 5HT2, adenosine 1, dopamine 1 and dopamine 2 receptors, and the root extract (70% ethanol) had binding with Ca^^-ion channel, opiate, a 1-adrenoceptor, 5HT1, dopamine 1 and dopamine 2 receptors. These observations prompted a further examination of the extracts in order to identify the active principles involved. There is no previous phytochemical report on the species, therefore, the present chemical investigation was undertaken for the roots and leaves.

4.2 Biological screenings on extract fractions Based on the observed activities of ethanol extracts of S. bodinieri , some of these receptor binding assays were selected for the fractional screening which included a l, a2-adrenoceptors, Ca^^, K^-channel, histamine 1, 5HT2, dopamine 1 and opiate receptors for the leaf extract, and a 1-adrenoceptor, Ca^^-channel, dopamine 1 and opiate receptors for the root extract. The fractions were obtained from column chromatography (Si 0 2 ) in order of their polarity and collected every ICX) ml. The detail of the fractions are shown in Figure 4.2 and Figure 4.3. The screening approach used was according to the Section 2.7 and the results of the screening are shown in Table 4.1 and Table 4.2.

93 Leaf extract (20g)

C C (S102)

Fr.1-3 Fr.4-10 pr.11-23 Fr.24-60 Fr.61-100 Fr.101-140Fr.141- (0.05g) (2g) (2g) (l9) Og) (Ig) 170(5g)

HPLC HPLC sephadex brown ma j or compd. 1 compd.3 syrup chlorophyll (lOOmg) (4mg) compd.2 compd.8 compd.9&9a (5mg) (20mg) (100 mg)

con^d.4 compd. 5 compd. 6 ccxnpd.7 (15mg> (20mg) (lOmg) (Img)

* solvent system: 1- petroleum, 2- CHCI 3 , 3- CHClg-MeOH (9:1),

4 - CHCVMeOH (8:2), 5 - CHClj-MeOH (7:3), 6 - MeOH

7- MeOH-water (9:1)

Figure 4.2 Isolation of compounds from Schefflera bodinieri leaves

94 Root extract (20g)

CC (S i0 2 )

F r .1 - 4 0 (ig ) Fr. 41-60 Fr. 61-100 Fr.101-180 (Ig) (3g) (lOg)

CC HPLC HPLC

compd. 4 (lOOmg) brown chlorophyll compd.3 (3mg) compd. 5 (lOOmg) syrup compd.10 (4mg) compd. 6 (20mg) compd.11 (5mg) compd. 7 (5mg) compd.12 (3mg) compd.13 (2mg) compd.14 (4mg)

*: solvent system 1- CHCI3, then CHClg-MeOH (9:1), 2- CHCl^-MeOH (8:2)

3- CHClj-MeOH (7:3), 4- MeOH, then MeOH-water (9:1)

Figure 4.3 Isolation of compounds fromSchefflera bodinieri roots

95 Table 4.1 The fraction screening of Schefflera bodinieri (leaves)

Fr.\ Reo. Ca^*-channel K*-channel HI al-adreno. (x2-adreno. 5HT2 Dl opiate Total ExL +• + -F -F-F -F -F-F -F+ -F Fr.1-3 ** -H- + -F-F Fr.4-10 -F-F Fr. 10-12 -F-F Fr.13-15 -H- Fr. 16-23 •f Fr.24-31 ++ -F-F -F Fr.32-33 + Fr.34-39 -H- -F -F Fr.40-43 -F + -F Fr.43-49 -F -F Fr.50-65 -H- + -F -F+ -F-F Fr.66-78 -H- ++ -F -F Fr.79-86 -F -H- -F-F Fr.87-90 -H- + -F-F -F-F Fr.91-94 -H- Fr.95-98 -F -F-F -F+ Fr.99-107 -F-F -F Fr. 108-111 ++ -F-F 4-F Fr.111-119 ++ -F -F + Fr.l20 + -F -F -F-F Fr.121-126 -H- -F -F-F -F-F -F-F -F Fr.l27 -F -F -F Fr.128-130 Fr.131-137 -F -F Fr.138-144 Fr. 145-147 + + Fr.148-156 + -F Fr.157-160 -F -F -F-F -F -F-F -F+ Fr.161-170 -F-F -F -F-F

*: %Inhibitioa between 50-79%, %Inhibition between 80-100%. ** the fractions see also Figure 4.2.

96 Table 4.2 The fraction screening of Schefflera bodinieri (roots)

Fr. \ Rep. Ca^+ al-adren. D1 opiate Total Ext. ++* 4-4- 4- 4- Fr. 1 -2 ** 4 - + 4-4- Fr.3-10 Fr.11-14 4- Fr.15-16 Fr. 17-20 4- Fr.21-24 + 4-4- Fr.25-27 Fr.28-30 4- Fr.31 + Fr.32 Fr.33-35 + Fr.36-38 4- 4-4- Fr.39-41 4- Fr.42-43 4- 4-4- Fr.44-46 4 - + Fr.47-50 4-4- 4- 4-4- Fr.51-55 4-4- Fr.56-58 4-4- 4- Fr.59-61 4-4- Fr.62-63 4- Fr.64-67 ++ 4- Fr.68-79 4-4- Fr.80-89 4-4- 4- Fr.90-95 4-4- Fr.96-100 4-4- Fr.101-110 Fr.l 11-120 4-4- Fr.121-131 Fr.132-138 4-4- 4- Fr.139-150 4-4- Fr.151-161 Fr.162-168 4-4- 4- Fr.169-175 4-4- Fr.176-180 4-4-

* : %Inhibition between 50-79%, %Inhibition between 80-100% **: the fractions see also Figure 4.3

97 4.3 Isolation and structure identification of the isolated compounds The general separation procedure have been outline in Section 2.3. For this species, the crude extracts were fractionated by repeated flash chromatography (SiOz). Further purification of different fractions were carried out with sephadex columns and HPLC (Figure 4.2, Figure 4.3). Of the 14 compounds isolated from leaves and roots of S. bodinieri, 11 triterpenoids and triterpenoid glucosides (compounds 1, 2, 3, 4, 5, 6 , 7, 10, 12, 13, 14 ) were found to be novel and each of them has been given a trivial name. The structure elucidation of these compounds is described in the following sections. The spectra of these novel compounds are included in Appendix.

4.3.1 Identification of Bodinitin A (5), Bodinitin B (7), Bodinitin C (13), and Bodinitin D (14)

18

Rham

, R2=H

, R2=CH3

13 R1=0, / R2=H 14 R1=0, , R2=CH3

Compounds 5, 7, 13, 14 are a group of structurally closely related compounds as indicated by EIMS, FABMS, ^HNMR. Detailed examination by ^^CNMR-DEPT, *H-*H COSY, and '^C-'H COSY spectra was carried out on compound 5. They were all the triterpenoid glucoside with the same sugar moieties as shown in their 'HNMR spectra.

The anomeric proton signals in each of the four compounds ) were around (±0.02) (IH, d, J=7Hz), 4.40 (IH, d, J=7Hz), 4.85 (IH, br.s), respectively and a methyl group

98 signal these compounds appeared at Ô 1.25 (3H, d, J= 6 Hz), indicating the presence of two glucoses and one rhamnose as sugar residues. The hydrolysis of the glycosides gave glucose and rhamnose as sugar components as indicated by TLC. In the further examined compound 5, its *^CNMR spectrum data confirmed the presence of the three sugar moieties (signals for anomeric C-atoms: Ô95.7, 102.9, 104.2) and the COSY spectrum indicated that one of glucoses was linked to the genin via carboxylic group since there was a cross peak between proton at ô5.35ppm and carbon at Ô95.7 (Spectrum 5.6). The 'H-‘H COSY (Spectrum 5.5), *^C-’H COSY and ‘^CNMR-DEPT (Spectrum 5.4) data of compound 5 further established that the structure of sugar moieties was a-L-rhamopyranosyl(l-4)-6-D-glucopyranosyl(l - 6 )-6 -D-glucopyranoside since the chemical shift of the carbon at C - 6 on the glucose linked with genin and the carbon at C-4 of second glucose were both 7 ppm downfield. Moreover, these spectral data of sugar moieties had a close agreement with those of triterpenoid glucosides isolated from Schejflera (Sung, et al, 1991a; 1991b; 1992).

The major difference in the structures of these compounds lies in their aglycones

Compound 5 (p.l 12 for data) The molecular weight of compound 5 was found to be 912.5085 (required 912.5083) and the molecular formula was established as by high resolution FAB mass spectrometry (Spectrum 5.1). The El mass spectrum showed the of aglycone at miz 442 (Spectrum 5.2). There were no characteristic ion peaks due to retro-Diels-Alder (RDA) type fragmentation of an olean-12-en or urs- 12-en-oci acid bearing substituent groups on rings C, D and E (Bombardell, et al, 1974) in EIMS, but there was a peak arising out of RDA fragmentation involving the 14:15 double bond in the ring D at mIz 275 (ion a. Figure 4.4, p. 100). The fragments formed by collapse of ring C were also observed at miz 207(ion b) and 234(ion c), and their further decomposition product at miz 189 and 190. Comparison was made with the related compound 36-acetoxy-D-friedoolean-14-en-28-oic acid (Aiyar, et al, 1971), suggesting that 5 contained a C,g-Me, and no methyl group was at C@ or C^^. The *^CNMR-DEPT spectrum substantiated the presence of twenty-nine carbon atoms of aglycone including a carboxylic carbon ( 6 178.1, C^^-COOH), a carbon linked with a hydroxyl group (Ô 76.7, 3p-0H) and two double bond carbons (Ô 137.5, 127.9).

99 ‘coo H RDA

HO'

5: m/z 275 5: R1=0H,H R2=H, M+ 442 7 : m/z 289 7: R1=0H,H R2=CH3 M+ 456 13: m/z 273 13: R1=0 R2=H M+ 440 14: m/z 287 14: R1=0 R2=CH3 M+ 454 m/z 219

-CH3>

+ COOH

m/z 189 ion b: 5: m/z 207 7: m/z 221 ion c : m/z 234 13 : m/z 205 14 : m/z 219 m/z 189 m/z 190

Figure 4.4 Possible MS Fragmentation Patterns of the Aglycones of Compounds 5, 7, 13, 14

The ‘HNMR spectrum (Spectrum 5.3) exhibited the presence of one olefmic proton (Ô 5.65), a proton linked with a hydroxyl group (ô 3.32, H-3a) and six tertiary methyl groups on the aglycone. From biogenetic point of view, the hydroxyl group should be assigned to C 3 position and this was further substantiated by the 'H-H COSY spectrum. The C 3 -OH of compound 5 was p-equatorial oriented which was mainly determined by chemical shift of C 3 ( 6 76.7) and H 3 (5 3.32) and in comparison with the related compounds (Chakravarty, et al. 1991). Hydrolysis of compound 5 gave its aglycone which had an identical El mass spectrum to that of compound 5 (spectrum 5.7). Based on these results and compared with the related data in references (Woo, et al, 1977, Sakurai, et al, 1987), the assignment of protons and carbons of the compound has been achieved (Table 4.4, p.l 13). The structure of 5 is found to novel and established as 28-0-[a-L-rhamopyranosyl(l-4)-B-D-glucopyranosyl(l-6)-]-B-D- glucopyranoside of 3p-hydroxyl-8-demethylisoaleuritolic-14( 15)-ene-28-acid, which is named bodinitin A.

100 Compound 7 (p.l 12 for data) The aglycone structure of compound 7 is closely related to compound 5 except the presence of an additional tertiary methyl group as shown in the ‘HNMR (Spectrum 7.3). This was confirmed by the EIMS showing a peak for the aglycone at m/z 456 (Spectrum 7.1) and the CIMS showing the M^ at m/z 926 (Spectrum 7.2). The additional tertiary methyl group was located at Cg as indicated by the fragment m/z 289 (ion a, Figure 4.4). The peak at m/z 275 which was formed by loss of allylic methyl group at Cg was also observed. The analysis of the 'HNMR spectrum indicated that compound 7 had the same sugar residues as found in compound 5 (the same signals for sugar’ proton). Therefore the structure of 7 is represented by 28-0-[a-L-rhamopyranosyl(l -4)-6-D-glucopyranosyl( 1 - 6 )-]-6 -D- glucopyranoside of 3p-hydroxyl-isoaleuritolic-14(15)-ene-28-acid which is named bodinitin B.

Compound 13 (p.l 15 for data) The difference between the aglycones of compounds 13 and 5 was the presence of a carbonyl group at C-3 instead of the hydroxyl group at C-3. In the EIMS (Spectrum 13.2), the M^ of the aglycone was at m/z 440 and there was a peak at m/z 273 due to the RDA cleavage (ion a, Figure 4.4) which substantiated the presence of the carbonyl group of C 3 . The HRFAB spectrum established the molecular formula as C 4 7 H7 4 O 1 7 (Spectrum 13.1). The ‘HNMR (Spectrum 13.3) showed that the compound had a similar spectral patten as compound 5 and the same signals of the sugars, indicating the presence of the similar aglycone and the same sugar moieties. Hydrolysis of compound 13 gave glucose and rhamnose as sugar components as indicated by TLC. The structure of this new glycoside is then determined as 28-0- [a-L-rhamnopyranosyl (l-4)-p-D-glucopyranosyl(l-6)-p-D- glucopyranosides of 3-oxo-8-demethylisoaleuritolic-14( 15)-ene-28-acid which is named bodinitin C.

Compound 14 (p.l 16 for data) The difference between compounds 14 and 5 included the presence of a carbonyl group at C-3 in 14 instead of a hydroxyl group in 5 and an additional tertiary methyl group. The HRFAB spectrum established the molecular formula as C4 gH 7 6 0 , 7 (Spectrum 14.1). In the EIMS (Spectrum 14.2), the M^ of the aglycone was at m/z 454 and there was a diagnostic peak at m/z 287 (ion a. Figure

101 4.4), indicating the presence of the additional tertiary methyl group at Cg and the carbonyl group of C 3 . The ‘HNMR spectrum showed the presence of seven tertiary methyl groups and the same signals for the sugar’ protons. Hydrolysis products also indicated that the sugar moieties were the same as those of compound 5. Therefore, the compound is 28-0-[a-L-rhamnopyranosyl (l-4)-0-p-D-glucopyranosyl(l-6)-P-D- glucopyranosides of 3-oxo-isoaleuritolic-14(15)-ene-28-acid which is named bodinitin D.

4.3.2 Identification of Bodirin A (4), Bodirin B (3), Bodirin C (10) and Bodirin D (12) The compounds 3, 4,10,12 are a group of structurally related triterpenoid glucosides as identified by EIMS, FABMS and 'HNMR. Detailed examination by ‘^CNMR- DEPT, 'H-‘H c o sy and *^C-‘H COSY spectra was carried out on compound 4. Compounds 3, 4, 12 have the same sugar moieties as compound 5 (ie., two glucoses and one rhamnose), but compound 1 0 has two glucoses as sugar components. Hydrolysis of these compounds substantiated the presence of the sugars as indicated by TLC. Structures of these four compounds are displayed as below.

\ 6 — *-l 14 Glc —^1 R Rham

compound 3 Rl=0, R2=H

,0H compound 4 Rl= R2=H H ,0H compound 10 Rl= R2=H

,0H compound 12 Rl= , R2=CH3

102 Compound 4 (p.l 12 for data) The HRFAB mass spectrum established the molecular formula of the compounds as (Spectrum 4.1) and the EIMS showed the of the aglycone at m/z 442. The ‘HNMR spectrum of 4 (Spectrum 4.3) indicated that it was a triterpenoid glycoside including three sugar moieties, ie. two glucoses and one rhamnose (signals for anomeric protons: 55.35, 4.40, 4.85), and six tertiary methyl groups (5 0.75, 0.80, 0.83, 0.87, 0.91, 0.99). The sugar units involved and their structures were the same as compound 5 as indicated by the data of ‘HNMR and ‘^CNMR, and the results of the hydrolysis. The ‘^CNMR-DEPT spectrum showed that there were two double bond quaternary carbons, one carboxyl carbon and a total of twenty-nine aglycone carbon atoms including eleven methylenes, three methines and five quaternary carbons (in addition of -C=C, -COOH) (Spectrum 4.4). In EIMS spectrum, the major fragments came from allyl cleavage showing peaks at m/z 275,

207 and 234, which indicated that the double bond position was at C 1 3 -C 1 4 and that there was a methyl group on C,g (Figure 4.5). The proton signal at 5 3.33 was assigned to C 3 as revealed by the ‘H-‘H COSY (Spectrum 4.5) and the C 3 -OH was p- equatorially oriented as indicated by the chemical shift of H-3 (5 3.33) and in comparison with the corresponding signal of the a-axial isomer (5 3.67 for Hgq-3) (Chakravarty, et al, 1991). The assignment of protons and carbons was made from the ‘HNMR, 'H-‘H COSY, ‘^C-'H COSY and '^CNMR-DEPT spectral data and by comparison with the related data in references (Pelletier, et al., 1964; Dugan, et al., 1964). The structure of compound 4 is then represented by 28-0-[a-L- rhamnopyranosyl (l-4)-P-D-glucopyranosyl(l-6)-]-p-D-glucopyranoside of 3P- hydroxyl-isopolygalic-13(14)-ene-28-acid which is named bodirin A.

Compound 3 (p.l 11 for data) The HRFAB spectrum exhibited the molecular formula as C4 7 H7 4 O 1 7 (Spectrum 3.1). The ‘HNMR (Spectrum 3.3) showed that compound 3 had a similar structure to compound 4 except for a carbonyl group substituted at C-3 as indicated by ‘^CNMR spectrum (absence of one C-OH carbon and presence of a carbonyl carbon signal at 5 220.96 compared with 4; Spectrum 3.4). In EIMS, the M"^ of the aglycone was at m/z 440 and a series of peaks which had 2 mass units less than compound 4 showed clearly (Spectrum 3.2). The major fragments of 3 were shown in Figure 4.5. The sugar moieties presented the signals of anomeric protons at 55.35,

103 4.85, 4.40 in ‘HNMR and the signals of anomeric carbons at Ô 95.5, 104.1, 102.8 in ‘^CNMR, suggesting the presence of the same sugar moieties as compound 4. Hydrolysis products of 3 were glucose and rhamnose as indicated by co-TLC. From these data, the structure of this new glycoside is established as 28-0-[a-L- rhamnopyranosyl (l-4)-0-P-D-glucopyranosyl(l-6)-p-D-glucopyranoside of 3-oxo- isopolygalic-13(14)-ene-28-acid which is named bodirin B.

■COOR + HO' "COOH

3: m/z 205 m/z 23 I 4: m/z 207 3: R1=0, R2=H M^440 10: m/z 207 COOH 4: Rl = R2=H M"" 442 12: m/z 221 / \ 10: Rl= R2=H M+442 ^OH m/z 219 m/z 190 12: R1 ”^OH R2=CH3 M+ 456

m/z 189 m/z 190 KG'

ion a 3: m/z 273 4: m/z 275 10: m/z 275 12: m/z 289

Figure 4.5 Possible MS Fragmentation Patterns of the Aglycones

of Compounds 3, 4,10 , 1 2

Compound 10 (p.l 14 for data) The ‘HNMR (Spectrum 10.3) indicated that compound 10 had a similar structure to compound 4, but no rhamnose unit was found (signals for anomeric protons: 5 5.35, 4.40 and no methyl group at Ô 1.25). The chemical shift of the aglycone protons were the same as compound 4 (eg. H-12 at Ô 2.48, H-3a at 5 3.33). The FABMS gave the M^ at m/z 766 (Spectrum 10.1) and

104 EIMS showed the of the aglycone at m/z 442 (Spectrum 10.2), and the fragments were the same as compound 4 (Figure 4.5). Based on these data, the structure of 10 is determined as 28-0-|3-D-glucopyranosyl(l-6)-P-D-glucopyranoside of 3P-hydroxyl- isopolygalic-13(14)-ene-28-acid which is named bodirin C.

Compound 12 (p.l 15 for data) Compare with compound 4, 12 had the same sugar moieties as indicated by ’HNMR spectrum (Spectrum 12.2), showing the anomeric proton signals at 5 5.35 (IH, d, J=7Hz, H-1 of Glc-1), 4.40 (IH, d, J=7Hz, H-1 of Glc-2), 4.85 (IH, br.s, H-1 of Rham) and hydrolysis of the compound gave glucose and rhamnose as sugar residues as revealed by co-TLC. The EIMS gave the M"^ of the aglycone at m/z 456 (Spectrum 12.1) and the CIMS spectrum gave the molecular weight at m/z 926 (Spectrum 12.3). The compound had a similar parent skeleton of aglycone to compound 4 except for having one extra methyl group exhibiting in the HNMR spectrum. The extra methyl group was assigned to Cg due to a diagnostic peak at m/z 289 (ion a) in EIMS (Figure 4.5). The compound had a lower Rf value than that of compound 4 on TLC and showed different colour after spraying with the vanillin-sulphuric acid reagent. Based on these data, the structure of compound 12 is represented by 28-0-[a-L-rhamnopyranosyl (l-4)-p-D-glucopyranosyl(l-6)-]-p-D- glucopyranoside of 3 p-hydroxyl-18-methyl-polygalic-13(14)-ene-28-acid which is named bodirin D.

4.3.3 Identification of Bodinone(l), Bodinone-glycoside (6) and Bodinin (2)

HOOC

'COOR COOH

15

HO''''

compound 1 R=H compound 6 R= -Glc-Glc-Rham compound 2 6"* 1 4-*"l

105 The compound 1 and 6 are two structurally related triterpenoids and 1 is the aglycone of 5 as indicated by 'HNMR, '^CNMR and EIMS.

Compound 1 (p.l 10 for data) was obtained as a white amorphous powder. The El mass spectrum gave the molecular weight at m/z 484 (Spectrum 1.2) which was further substantiated by the high resolution mass spectrum as the formula C 3 0 H4 4 O5 (Spectrum 1.1). The 'HNMR data (Spectrum 1.3) showed the presence of one olefinic proton (Ô 5.73) and six tertiary methyl groups. The '^CNMR-DEPT spectrum indicated the presence of two carboxyl groups (ô 178.7 and 181.0), one carbonyl group (Ô

2 2 0 .2 ) and a total of thirty carbon atoms including ten methylenes, three methines in addition to CH=, and six quaternary carbons in addition to C=, C=0, COOH (Spectrum 1.4). In the EIMS, there was a peak due to the RDA fragmentation involving 14:15 double bond in the ring D at m/z 273. The fragments formed by collapse of ring C were also observed, containing rings A and B part and rings D and E part, respectively, indicating the functional groups on the two part (Figure 4.6). The signal of one carboxylic group at 6178.1 in '^CNMR was assigned to C^^-COOH and the other -COOH signal at 6181.0 was assigned to C 2 9 due to the fragment of ion b

.COOH

RDA

iona l:m/z 287

compound 1 R1=0, R2=H; MW 484 6;m/z 287 compound 2 R1=0H, H, R2=H; MW 486 compound 6 R1=0, R2=-Glc-Glc-Rham; MW 956 COOH

-2 COOH

‘COOH

m/z 175 ion b: m/z 265 ion c l:m/z 205 2;m/z 207 6:m/z 205

Figure 4.6 Possible MS Fragmentation Patterns of Compounds 1, 2 and 6

106 in the EIMS. The carboxylic group of C 2 9 was a-equatorial oriented in comparison to the related compounds (Miyakoshi, et al, 1993). From these results and compared with related data in references (Woo, 1977, Djerassi, et al., 1962, Paul, et al., 1974), compound 1 is identified as 3-oxo-20-demethylisoaleuritolic-14 (15)-ene-28, 29-dioic acid which is named bodinone.

Compound 6 (p.l 12 for data) The HRFAB spectrum established the molecular formula as (Spectrum 6.1). The ‘^CNMR and 'HNMR showed that the compound 6 was a triterpenoid glycoside with three sugar moieties. The anomeric carbon signals appeared at 5 95.6, 104.0, 102.5 (Spectrum 6.4) and the anomeric proton signals at Ô 5.35, 4.40, 4.85 (Spectrum 6.3), which were the same as those exhibited in compound 5. One of the anomeric proton signals at ô 5.40 (Glc) indicated the glucose was connected to genin via carboxylic acid. Hydrolysis of the compound gave glucose and rhamnose as sugar components (co-TLC). In the EIMS, it showed the similar fragments to those of compound 1 except for that the ion at m/z 484 did not appeared due to the loss of the C 2 9 -COOH (a-cleavage). Such loss readily occurred under the same condition as for loss of the sugar moieties, and there was a clear peak at m/z 440 corresponding to the fragment of [aglycone-COOH+1]^ (Figure 4.6). Apart from the sugar signals, the ‘HNMR of 6 showed the similar spectral patten as 1, indicating the skeleton of the aglycone was the same as compound 1. From the ‘HNMR, H- H COSY, ‘^CNMR data and compared with related data in references (Sung, T.V., et al, 1991a, 1991b, 1992), the assignment of protons and carbons in compound 6 had been achieved in (Table 4.4). The structure of compound 6 is then represented as 28-0-[a-L-rhamnopyranosyl (l-4)-0-|3-D-glucopyranosyl(l-6)-]-p-D- glucopyranoside of 3-oxo-20-demethylisoaleuritic-14(15)-ene-28,29-dioic acid which is named bodinone-glycoside.

Compound 2 (p.l 10 for data) is a white amorphous powder. ‘HNMR (Spectrum 2.3) indicated that the compound was a triterpenoid with six tertiary methyl groups, one olefinic proton (5 5.55), one proton adjacent to a hydroxyl group (Ô 4.18) and two carboxylic acids (Ô 12.15, 2H, br.s, W 1 / 2 = 70Hz). ‘^CNMR exhibited the presence of two carboxylic carbons (5 176.8, 178.7), two double bond carbons ( 6 136.8, 125.4),

107 one carbon linked with a hydroxyl group (ô 73.76), three methines, ten methylenes and six quaternary carbons (Spectrum 2.4), corresponding to the formula which was substantiated by high resolution mass spectrometry (Spectrum 2.1). The chemical shift of C-3 was at ô 73.8, suggesting the hydroxyl group at C 3 was a-axial orientated. In the EIMS spectrum, the fragments arising from RDA cleavage were similar to those of compound 1 , except for the peaks due to the presence of hydroxyl group at C-3 instead of a carbonyl group as 1, showing the characteristic peaks of 14- ene at m/z 289, 275, 265, 207 and 189 (Budzikiewicz, et al, 1963, Razdan, et al, 1982;

Spectrum 2.2). These fragments suggested the double bond position at C, 4 , a methyl group at Cg, a hydroxyl group at C 3 and a carboxylic group at either C 2 9 or C3 0 (Figure 4.6). Comparing its *^CNMR data with those of compound 1, there was a 3a-0H carbon (Ô 73.76) in compound 2 instead of 3-ketonic carbon (Ô 220.2) in compound

1. The carboxylic carbon signal at ô 178.7 was typical for C 1 7 -COOH, therefore, the other carboxylic group (signal at Ô 176.8) was then assigned to C 3 0 due to this -COOH group being p-axial orientated. Based on these results, the structure of compound 2 is determined as 3a-hydroxyl-20-demethylisoaleuritolic-14( 15)-ene-28,30-dioic acid which is named bodinin.

4.3.4 Identification of D-sorbito! ( 8 )

^CHzOH

H "ohh" OH

In EIMS, M^ of compound 8 appeared at m/z 182 and M72 at m/2z 91. There was also a clear signal for M^-CH 3 0 H at m/z 150. The ‘HNMR spectrum showed the presence of only eight protons linked to the carbons to which hydroxyl groups were connected (Ô 2.90-4.65). These data suggested that the compound was a six carbon straight chain sugar. Comparing with an authentic sample of D-sorbitol by co-TLC with three different solvent systems, the compound had identical Rf values with those of D-sorbitol. Therefore, it is determined to be D-sorbitol (p.l 14 for data).

108 4.3.5 Identification of trisaccharides (9 & 9a) Compounds 9 and 9a (p.l 14 for data) were a mixture of epimeric trisaccharides including two glucoses and one rhamnose in an approximate ratio of 0 .8 (a): 1 .0 (P) as indicated by the '^CNMR spectrum. Hydrolysis of this mixture produced glucose and rhamnose (co-TLC). Their *^CNMR and ‘HNMR spectral data were identical to those of the sugar moieties of the triterpenoid glycosides isolated from this plant. The Cj-pH

CH OR OR R OH OH OH OH OH OR

compound 9 R = p OH compound 9a R = a OH and C,-aH of glucose-1 appeared at 8 5.12 (IH, d, J=3.1) and 8 4.50 (IH, d, J=7.6), respectively in 'HNMR, and C -la and C-lp appeared at 594.1, and 98.3, respectively in ‘^CNMR. The anomeric protons of glucose-2 and rhamnose appeared at 5 4.36 and 4.87 and the C-1 carbons of glucose-2 and rhamnose appeared at 5 104.5 and 102.9, as indicated by ‘H-'H COSY and '^C-'H COSY spectral data. The FAB mass spectrum of this mixture showed the M^ at m/z 511 (M^+Na^). Based on these results, compound 9 is determined as a-L-rhamnopyranosyl (l-4)-0-p-D-glucopyranosyl(l-6)- p-D-glucopyranoside and 9a is its la-epimer. These epimers have also been isolated from Schejflera octophylla (Sung, et al, 1991a).

4.3.6 Identification of Stigmasterol-3-O-p-D-glucoside (11)

28 27 la 20 23

17 26 13

10

GlcO' 109 Compound 11 (p.l 14 for data) The EIMS showed the of the compound at m/z 574 and [M-Glc]^ at m/z 412. ’HNMR spectrum showed the compound was a sterol glycoside with one glucose as the sugar moiety (anomeric proton signal at Ô 4.20) which was substantiated by hydrolysis (co-TLC). There were two tertiary methyl groups, three secondary methyl groups and one primary methyl group showing in the ’HNMR spectrum, which suggested that the compound might have a steroid structure with a side chain. There were three olefinic protons appeared at Ô 5.31 (IH, d, J=2), 5.15 (IH, dd, J=15, J=9) and 5.02 (IH, dd, J=15, J=9), respectively. The anomeric proton signal of the glucose was at Ô 4.20 (IH, d, J= 8 ) indicating that the glucose was linked to the genin via the hydroxy group at C-3. The signal of C-3 proton appeared at Ô 3.30 suggesting the glucose was p-equatorial oriented. The ’^CNMR-DEPT spectrum exhibited the presence of nine methylene carbons, three quaternary carbons including one double bond carbon, and ten methines including three double bond carbon and one carbon linked to a hydroxy group on the aglycone. The intensive peak in the EIMS at m/z 138 were due to the retro Diels-Alder cleavage of ring B, a typical cleavage of triterpenoids and steroids with 5(6)-unsaturated structure. The fragment at m/z 273 suggested that there was only one double bond in the parent nucleus and that the other double bond was on the side chain (Cong, 1987). Comparing the ’^CNMR and ‘HNMR data with those of stigmasterol, a closely agreement has been obtained (Kojima, et al, 1990). The compound is then determined as stigmasterol-3-0- P-D-glucoside.

4.3.7 Spectral data

Bodinin (1): white amorphous powder, soluble in CHCl^-MeOH (4:1). TLC (Si 0 2 ): Rf

0.67 (CHClg-MeOH 9:1). Found: [M]+ 484.3185, C 3 0 H4 4 O5 , requires 484.3189. ’HNMR

(DMSO-d^): 65.73 (t, J=l, H-15), 6 0.84, 0.89, 0.90, 1.00, 1.01, 1.05,(3H, each, s, tert-Me). ’^CNMR: 6 6.27, 18.19, 21.45, 23.30, 26.96, 33.15 ( 6 C, tert-Me); 6178.7

(C,7 -C0 0 H); 6181.0 (C 2 0 -COOH); 6220.2 (C-3); 137.1 (C-14); 126.3 (C-15). CH2 : 19.04, 23.30,24.17,24.69, 32.38, 33.94, 34.36,36.08, 39.49,43.56; CH: 43.63,46.41, 55.04, quaternary carbon: 30.95, 37.(X), 39.51, 47.61, 56.03. EIMS m/z (rel. int): 484 (5), 440 (27), 425 (55), 394 (7), 379 (23), 273 (15), 265(13), 234 (18), 205 (47),

110 189(19), 177 (29).

Bodinin (2): white amorphous powder, soluble in DMSO. TLC (Si 0 2 ) Rf 0.55 (CHCI3 - MeOH 9:1). Found: [M]^ 486.3342, requires 486.3345. 'HNMR (DMSO-d^): tert. Me (s): 5 0.75, 0.80, 0.81, 0.82, 0.86, 0.87. Ô 5.55 (IH, br.s, H-15), 4.18 (IH, br.s, H-3p), 3.15 (IH, br.s, H-13), 12.15 (2H, br.s, Wi/2=70 Hz, COOH-28, 30). ‘^CNMR see Table 4.3. EIMS m/z (rel. int.): 486 (M^, absent), 442 (M^-COOH, 10), 427 (26), 409 (51), 381 (15), 363(48), 317(2), 287(24), 275(29), 273(27), 243(13), 241(30), 227(45), 219(11), 201(42), 190(62), 189(65), 175(58), 135(95). FABMS m/z: 487 (M++1, 7), 469 (18), 441(10), 307(97), 289(100), 275(10), 273(24).

Table 4.3 "CNMR data of compound 2

No. Ô No. 6 No. Ô

1 33.11 1 1 18.00 2 1 36,17

2 23.98 1 2 40.18 2 2 22.58 3 73.76 13 46.42 23 33.25 4 37.11 14 136.8 24 18.20 5 46.40 15 125.4 25 16.10

6 25.45 16 23.90 26 22.50 7 33.54 17 55.35 27 23.60

8 36.90 18 43.01 28 178.6 9 48.40 19 31.85 29 28.80

1 0 36.90 2 0 30.57 30 176.8

The data were obtained from 400MHz NMR in DMSO.

Bodirin D (3): white amorphous powder, soluble in MeOH. TLC (SiOg): Rf 0.55 (CHClg-MeOH-HgO 8:2:0.1). Found: [M]+ 910.4929, C^yH^^O^^, requires 910.4926. 'HNMR (CD^OD-d^): Ô 0.90, 0.95, 0.95, 1.00, 1.05, 1.10 (3H, each, s, tert-Me), 5 2.48 (IH, dd, J=10, J=2, H-12), Ô 5.35 (IH, d, J=7, H-1 of Glc-1), 4.40 (IH, d, J=7,

H-1 of Glc-2), 4.85 (IH, br.s, H-1 of Rham) 1.25 (3H, d, J= 6 , H- 6 of Rham) 3.28- 4.15 (16H, sugar protons). ‘^CNMR (DMSO-dg): CHg: Ô 16.81, 20.73, 21.35, 25.13, 27.17, 33.01; CH^: 19.25, 20.84, 21.52, 32.00, 32.57, 34.92, 35.00, 39.55, 40.07, 42.29; CH: 40.05, 55.73, 56.67; quaternary carbon: 131.36 (C-13), 137.34(C-14), 178.12(C-17), 220.96(C-3); Glc-1: 95.56(C-1), 73.61 (C-2), 78.73(C-3), 70.85(C-4),

111 76.66(C-5), 69.42(C-6); Glc-2: 104.24(C-1), 75.11(C-2), 76.57(C-3), 79.60(C-4), 77.77(C-5), 61.80(C-6); Rham: 102.86(C-1), 72.27(C-2), 72.07(C-3), 73.78(C-4), 70.58(C-5), 17.82(C-6). EIMS m/z (rel. int.): 440 (3), 425 (92), 394 (17), 379 (78),

273 (29), 261 (9), 248 (12), 234(5), 227 (26), 219 ( 8 ), 205 (30), 201 (32), 189 (40),

190(35), 177 ( 6 8 ). FABMS m/z: 933 [M+Na]\ 787 [M+Na-Rham]\ 603 [aglycone+Glc+l], 441 [aglycone+1].

bodirin A (4): white amorphous powder, soluble in MeOH. TLC (Si 0 2 ): Rf 0.53

(CHCl3 -Me0 H-H2 0 8:2:0.1). Found: [M]^ 912.5085, requires 912.5083. 'HNMR and '^CNMR: see Table 4.4. EIMS m/z (rel. int): 442 (20), 427 (40), 409

(17), 381 (15), 363 (33), 275 (31), 261 (13), 234 ( 6 ), 227 (28), 219(11), 207 (48), 189(46), 191 (73), 190 (95), 175 (45), 161 (21), 147 (27), 135 (85). FABMS m/z: 934 [M+Na-1]\ 788 [M+Na-1-rhamnose]^, 604 [M-rhamnose-glucose]^.

Bodinitin A (5): white amorphous powder, soluble in MeOH. TLC (Si 0 2 ): Rf 0.45

(CHCl3 -Me0 H-H2 0 8:2:0.1). Found [M]+ 912.5084, C^^H^^O^^, requires 912.5083. HNMR and '^CNMR: Table 4.4. EIMS m/z (rel. int.): 442, aglycone (17), 427(35), 409(15), 381(9), 363(36), 275(27), 273(21), 234 (11), 219 (7), 229(32), 207(29), 190(81), 189(45), 175(43). FABMS m/z 934 [M+Na-lf, 788[M+Na-1-Rham]+, 604[M- Rham-Glu]^, 442[M-Rham-Glu-Glu]^. Aglycone of Bodinitin A (hydrolysis product) (5a): white amorphous powder, soluble in DMSO. TLC (SiO^): Rf 0.70 (CHCl3 -MeOH 9:1). EIMS m/z (rel. int.): 442 (M+, 25), 427 (49), 409 (23), 396(5), 382(11), 363(28), 275(24), 229(22), 207(28), 190(100), 175(52), 135(55).

Bodinone-glycoside (6 ): white amorphous powder, soluble in MeOH. TLC (Si 0 2 ): Rf

0.48 (CHCl 3 -Me0 H-H2 0 8:2:0.1). Found: [M]^ 954.4828, C^gH^^O^g, requires 954.4824. 'HNMR and '^CNMR: see Table 4.4. EIMS m/z (rel. int.): 440 (20), 425 (44), 394 (5), 379 (42), 273 (18), 265 (7), 220(5), 205 (17), 201 (19), 189 (23), 177 (36). FABMS m/z: 977 [M+Na]\

112 Table 4.4 "CNMR and HNMR Spectrum Data of Compounds 4, 5, 6

4 : R= OH R1=CH3 5 : R= OH R1=CH3

25 6; R=0 R1=C00H

R

A* œmpoimd 4 œmpoimd 5 ^omjgund 6 S* No. No.

1 32.7; 1.35,1.79 34.4 1.39,1.41 35.1; 1.98,1.50 o r

2 23.1; 1.98,1.55 25.2 1.98,1.55 35.2; 2.47,2.51 1 95.7; 5.35 3 76.6; 3.33 76.7 3.32 218.8; 2 73.7; 3.32 4 38.3; 38.3 44.7; 3 79.5; 3.65 5 48.8; 1.50 49.2 2.20 48.5; 2.10 4 71.0; 3.43 6 25.0; 1.60,1.90 25.2 1.95,2.00 20.7; 1.73,2.08 5 76.8; 3.55 7 33.8; 1.27,1.18 34.7 1.21,1.23 35.0; 1.58,1.47 6 69.4;3.83,4.12 8 38.1; 1.48 48.1 1.19 47.6; 1.20 G2

9 56.0; 1.28 50.0 1.30 56.4; 1.37 1 104.1; 4.40

1 0 38.6; 38.3 40.6; 2 75.3; 3.28

1 1 17.4; 1.55,1.50 19.3 1.30,1.32 24.2; 1.29,1.31 3 76.7; 3.50

1 2 41.0; 2.48,1.20 44.7 1.45,1.18 44.7; 1.42,1.18 4 78.6; 3.42 13 130.0; 44.5 2.92 44.7; 2.92 5 78.1; 3.30 14 137.0; 137.5; 137.5; 6 61.9;3.82,3.67 15 30.7; 2.18,2.07 127.9; 5.65 127.6; 5.70 R

16 39,3; 1.90.1.62 23.9; 1.98,1.95 25.7; 1.98,2.00 1 102.9; 4.85 17 53.7; 57.3; 57.1; 2 72.4; 3.87 18 46.0; 40.9; 39.4; 3 72.2; 3.67 19 40.9; 1.58,1.30 32.8; 1.58,1.30 40.6; 1.57,1.31 4 73.8; 3.43 20 30.2 31.5; 31.6; 5 70.6; 4.00

2 1 31.5; 1.52,1.32 37.7; 1.73,1.63 32.9; 1.58,1.45 6 17.9; 1.30 22 20.2; 2.00,1.78 25.7; 2.08,2.06 25.7; 2.08,1.73 23 31.6; 0.87 33.0; 0.90 33.7; 0.94 24 15.7; 0.83 18.3; 0.91 19.0; 0.93 25 16.5; 0.99 16.5; 1.01 17.9; 1.08

26 -- 21.8; 0.89 27 25.0; 0.91 23.6; 0.96 24.1; 1.06 28 178.0; 178.1; 178.0; 29 27.6; 0.80 28.5; 0.92 180.0; 30 19,9; 0.75 22.3; 0.85 27.3; 1.04

*: A-aglycone, S-sugar. G1-glucose 1, G2-glucose 2, R-rhamnose. The data were obtained from 500 MHz NMR in CD3OD. The assignment based on 'HNMR, '^CNMR, 'H-'H COSY, '^C-'H COSY and EIMS spectral data

113 Bodinitin B (7): white amorphous powder, soluble in MeOH. TLC (Si 0 2 ): Rf 0.40

(CHClj-MeOH-HzO 8:2:0.1). ‘HNMR (CD^OD-d^): Ô 0.75 (3H, s, tert-Me), 0.87 ( 6 H, s, tert-Me), 0.90 (9H, s, tert-Me), 0.95 (3H, s, tert-Me), 5.65 (IH, br.s, H-15), 5.35

(IH, d, J= 8 , H-1 of Glcl), 4.82 (IH, br.s, H-1 of Rham), 4.40 (IH, d, J= 8 , H-1 of Glc2), 3.32 (IH, br.s, H-3a), ô 3.28-4.15 (16H, sugar proton). EIMS m/z (rel. int.): 457, aglycone+1, (7), 443(15), 428(53), 410(25), 382(13), 364(18), 289(3), 275(37), 273(45), 234(8), 219(53), 207(43), 190(34), 189(62), 175(53), CIMS m/z 926[M]\ 780[M-Rha]\ 618[M-Rha-Glu]^, 456 [aglycone]^.

D-Sorbitol (8 ): white amorphous powder, soluble in MeOH and DMSO. TLC (SiOz): Rf 0.35 (CHCl^-MeOH-HzO 6:4:0.2). ‘HNMR (DMSO-d^): 54.65 (IH, d, J=2), 4.52

(2H, dd, J=14, J=3), 4.48 (IH, d, J=4), 4.35(1H, d, J= 6 ), 3.70 (IH, d, J=2), 3.14 (2H, dd, J=14, J=3). EIMS m/z (rel. int.): M+ 182 (4), 150 (72), 119(52), m/2z 91 (57).

Trisaccharides (9&9a): white amorphous powder, soluble in MeOH. TLC (SiOj): Rf 0.41 (CHClg-MeOH-HzO 6:4:0.1). FABMS (m/z) 511 [M+Na]\ 326[M-Glc]\ 342 [M-

Rham]+. ‘HNMR (CD 3 OD): 51.25 (6 H, d, J= 6 , Me-Rham), 4.50 (IH, d, J=7.6, a- anomeric proton on Glcl), 5.12 (IH, d, J=3, p-anomeric proton on Glcl), 4.87 (2H, br. s, H-1 of Rham.), 4.34 and 4.36 (2H, d, J=3, H-1 of Glc2). ‘^CNMR(CD 3 0 D): 5104.5 (1.8C, Cl-Glc2), 102.9 (1.8C, Cl-Rham), 98.3 (1C, P-D-Glcl), 94.1 (1C, a-D- Glcl), The chemical shift values of other signal were the same as those of sugar moieties listed in Table 4.4.

Bodirin C (10): white amorphous powder, soluble in MeOH. TLC (SiOj): Rf 0.70 (CHCl3-Me0H-H20 8:2:0.1). ‘HNMR (CD30D-d4): 5 0.80,0.85,0.93,0.94,0.97,0.98 (3H, each, s, tert-Me); 5 3.33(1H, br.s, H-3a), 52.48 (IH, dd, J=10, J=2, H-12), 5 5.35 (IH, d, J=7, H-1 of Glc-1), 4.40 (IH, d, J=7, H-1 of Glc-2), 5 3.28-4.12 (12H, sugar protons). EIMS m/z (rel. int.): 443 (12), 428(23), 410 (13), 397 (5), 382 ( 8 ), 364 (25), 289 (3), 275 (32), 273 (28), 234(8), 229 (38), 207(13), 190 (37), 189 (39). FABMS m/z: 780 [M]^, 618 [M-glucose]^.

114 Stigmasterol-3-O-^-D-glucose (11): white amorphous powder, soluble in DMSO. TLC

(SiOz): Rf 0.30 (CHClg-MeOH 9:1).‘HNMR (DMSO-d^): 8 0.67 (3H, s, CH3 -I 8 ), 0.93

(3H, s, CH3 -I 8 ), 1.02 (3H, br, s, CH3 -2 I), 0.82 (3H, d, J= 6 , CH3 -2 7 ), 0.84 (3H, d, J= 6 ,

CH3 -2 6 ), 0.87 (3H, t, J=4, CH 3 -2 9 ), 3.55 (IH, m, H-3), 5.35 (IH, d, J=3, H- 6 ), 5.15

(IH, dd, J=15, J=9, H-22), 5.02 (IH, d, J=15, J=9, H-23), 4.20 (IH, d, J= 8 , Glc H-1). '^CNMR (DMSO-de): see Table 4.5. EIMS mtz (rel. int): 574(M+, 2), 412 (5), 397(4), 394(8), 379(3), 369(3), 273(8), 271(6), 255(19), 231(12), 246(12), 229(13), 213(25), 138(65), 111(74), 97(94).

Table 4.5 *'^CNMR Data of Compound 11

Aglycone No. C Aglycone No. C Sugar No. C

1 37.0 17 55.6 1 1 0 1 . 0

2 29.5 18 11.9 2 76.3 3 77.1 19 19.3 3 77.0

4 37.0 2 0 40.3 4 70.3

5 140.6 2 1 21.3 5 77.1

6 121.4 2 2 138.0 6 61.3 7 31.6 23 129.0

8 31.6 24 50.8 9 49.8 25 35.7

1 0 36.4 26 2 0 . 8

1 1 2 0 . 8 27 19.9

1 2 38.5 28 25.7

13 42.1 29 1 2 . 0 14 56.4 15 24.1 16 28.0

The data were obtained from 400 MHz NMR in DMSO-dg.

Bodirin B (12): white amorphous powder, soluble in MeOH. TLC (SiOz): Rf 0.50

(CHCl3-Me0H-H20 8:2:0.1). *HNMR(CD30D-d4): 8 0.75,0.83,0.87,0.91,0.93,0.95,

0.95 (3H, each, s, tert-Me); 8 3.33 (IH, br.s, H-3a); 82.48 (IH, dd, J=10, J=2, H-12),

8 5.35 (IH, d, J=7, H-1 of Glc-1), 84.40 (IH, d, J=7, H-1 of Glc-2), 8 4.85 (IH, br.s,

H-1 of Rham), 8 1.25 (3H, s, H - 6 of Rham), 8 3.28-4.15 (16H, sugar protons). EIMS m/z (rel. int): 456 (5), 442 (5), 428 (10), 409 (7), 396 (7), 382 ( 6 ), 289 (3), 275 ( 8 ),

115 273 (10), 234(8), 229 (9), 227(6), 221(5), 207(21), 190 (7). FABMS m/z: 949 [M+Na]^, 780 [M-rhamnose]^, 618 [M+glucose]^

Bodinitin C (13): white amorphous powder, soluble in MeOH, TLC (Si 0 2 ): Rf 0.52 (CHCl^-MeOH-HzO 8:2:0.1). Found: [M]+ 910.4929, requires 910.4926. 'HNMR (CDgOD-d^): ô 0.90, 0.92, 0.94, 1.02, 1.07, 1.07 (3H, each, s, tert-Me), ô 5.65 (IH, t, J=2, H-15), ô 5.35 (IH, d, J=7, H-1 of Glc-1), ô 4.40 (IH, d, J=7, H-1,

Glc-2), 6 4.85 (IH, br.s, Rham-1), Ô 3.28-4.15 (16H, sugar protons); EIMS m/z (rel. int.): 440 (24), 425 (53), 394 (11), 379 (41), 273 (20), 261 (5), 227 (19), 207 (25), 190(27), 189 (25), 177 (47).

Bodinitin D (14): white amorphous powder, soluble in MeOH. TLC (Si 0 2 ): Rf 0.51

(CHCl3 -MeOH-H2 0 8:2:0.1). Found: [M]^ 924.5086, C^gH^^O;^, requires 924.5083. 'HNMR (CDgOD-d^): ô 0.87, 0.90, 0.97, 1.00, 1.02, 1.04, 1.10 (3H, each, s, tert-Me), ô 5.75 (IH, t, J=2, H-15), 65.35 (IH, d, J=7, H-1 of Glc-1), ô 4.40 (IH, d, J=7, H-1, Glc-2), ô 4.85 (IH, br.s, H-1 of Rham), Ô 3.28-4.15 (16H, sugar protons); EIMS m/z (rel. int.): 454 (14), 440 (2), 426 (3), 408 (15), 379 (3), 335 (10), 287 (13), 239 (21),

234(7), 219(8), 207 (46), 190 (9), 189 ( 8 ), 169 (90).

The compounds isolated from Schejflera bodinieri are summarized in Figure 4.7.

116 Figure 4.7 The compounds isolated from Schefflera bodinieri

Ic

‘Rham

5 Rl= R2=H 3 Rl= 0 , R2=H, R3=-Glc-Glc-Rham

7 Rl= < ■ R2=CH3 4 , R2=H, R3=-Glc-Glc-Rham

1 0 , R2=H, R3=-Glc-Glc 13 R1=0, ' R2=H 14 R1=0, R2=CH3 12 Rl= R2=CH3, R3=-Glc-Glc-Rham

'COOR. OH OH OH

iTm o h conpound 9 R=alpha H ccîtpound 9a R=beta H

coitpoiand 1 R1=0, R2=H, OH R3=C00H, R4=CH3 VDH coirpound 2 Rl= , R2=H, R3=CH3, R4=C00H

coitpound 6 R1=0, R2=-Glc-Glc-Rham R3=C00H, R4=CH3

K ,0H CHgOH Gb0‘ CHgOH " ÔHH OH

compound 1 1 corrpound 8

117 4.4 Biological tests on isolated compounds and related compounds

Compounds 1, 2, 3, 4, 5, 5a (aglycone of 5), 6 , 8 , 9 & 9a, 11 isolated from S. schefflera as well as ginsenoside-Rbl, ginsenoside-Rc and ginsenoside-Re (purchased from Sigma) were tested by ligand receptor binding assays following the approach described in Section 2.8. The receptors tested included a l, a2, p-adrenoceptors, 5HT1A, 5HT1C, 5HT2, dopamine 1, dopamine 2, muscarinic, adenosine 1, histamine 1, Na^/K^ATPase, Ca^^-channel (DHP), K^-channel, opiate (for all the compounds above) and GABA^ and GABAg (for isolated compounds only). Of the 13 compounds tested, 8 compounds were found to bind to the different receptors. The ICjq values of these compounds are listed in Table 4.6.

Table 4.6 Results of compound screenings

Compound Receptor IC5 0 MM* ± SEM N

compound 1 muscarinic 0.91 ± 0.15 3

compound 4 dopamine 2 1.83 ± 0.36 3

compound 6 muscarinic 3.57 ± 1.41 3

compound 8 muscarinic 3.24 ± 1.22 3 compound 9&9a Ca^^-channel 8.03 ± 2.01 3 5HT2 3.81 ± 1.34 3

compound 1 1 5HT2 8.04 ± 2.31 3 ginsenoside Rbl Ca^^-channel 4.23 ± 1.64 3 ginsenoside Rc Ca^^-channel 4.46 ± 0.62 3

*: The IC 5 0 of control compounds in the present study were 0.16nM for atropine (muscarinic receptor), 5.62 nM for spiperone (5HT2 receptor), 2.00nM for nitrendipine (Ca^^-channel receptor) and 2.08 nM for butaclamol (dopamine 2 receptor).

Compounds 1, 4, 5, 5a, 6 , 8 , 9&9a were further investigate to see whether they interacted with agonists or antagonists (here especially the control compounds used in each ligand binding assay) at various receptors following the approach described in Section 2.9. The binding assays involving a l, a2, p-adrenoceptors, Ca^^-channel, K^-channel, dopamine 1, dopamine 2, adenosine 1, histamine 1, opiate, 5HT1A,

118 5HT1C, 5HT2 receptors were selected to this study. The IC 5 0 values of the control compounds which has been changed by isolated compounds are listed in Table 4.7.

The IC 5 0 curves have also been established for the control compounds of which the binding sites have been affected by the isolated compounds (Figure 4.8).

119 Table 4.7 The IC 50 Values of the Control Compounds and the Control compounds with Added Isolated Compounds

Compound Receptor' IC5 o(nM)±SEM IC5 o(nM)±SEM IC5o(nM)±SEM N

(Control) (Iso. Compd)^ (Ctr.+Iso)^

1 P 1.41 ± 0.10 4 0.56 ± 0.10 3 opiate 1.02 ± 0.17 - 0.63 ± 0.08 3

5HT2 5.62 ± 0.47 - 1.77 ± 0.19 3

4 opiate 1.02 ± 0.17 - 0.14 ± 0.02 3

5HT1A 1 . 0 0 ± 0 . 1 2 - 0.08 ± 0 . 0 2 3

5HT1C 70.8 ± 5.26 - 1.58 ± 0.47 3

5HT2 5.62 ± 0.47 - 0.76 ± 0.08 3

HI 7.41 ± 0.77 - 0.40 ± 0.06 3

5 opiate 1.02 ± 0.17 - 0.35 ± 0.06 3

K+ 0.79 ± 0.05 - 0.79 ± 0.08* 3

5HT1C 70.8 ± 5.26 - 7.94 ± 0.87 3

5HT2 5.62 ± 0.47 - 0.63 ± 0.09 3

HI 7.41 ± 0.77 - 0.56 ± 0.08 3

5a opiate 1.02 ± 0.17 - 0.70 ± 0.05 3

5HT1C 70.8 ± 5.26 - 3.98 ± 0.53 3

5HT2 5.62 ± 0.47 - 0.45 ± 0.05 3

6 opiate 1.02 ±0.17 - 0.40 ± 0.05 3

5HT2 5.62 ± 0.47 - 0.40 ±0.17 3

A l 14.1 ± 0 .7 2 - 1 0 . 0 ± 1 . 1 2 * 3 9&9a P 1.41 ± 0.10 - 10.0 ± 0.54* 3 opiate 1.02 ± 0.17 - 0.13 ± 0.07 3

HI 7.41 ± 0.77 0.16 ±0.03* 3

1: receptor P- P adrenoceptor, K^- K^-ion channel receptor, A l- adenosine 1, HI- histamine 1. 2: isolated compound 3: control compound + isolated compound 4: inactive *: the binding site was also affected, see also Figure 4.8.

120 Figure 4.8 IC 50 Curves of the Control Compounds and the Control Compounds with Added Isolated Compound

GlibencUmide Displacement (K->—channel Receptor) 100 w

» Glibenciaaiide

* G lib .-C om p ou n d 5

u u a cn §

c

Pyrilamine Displacement (Histamine 1 R e c p to r ) 100

P y r ila m in e o>c Pyri.+Compound 1m

u a. cn

1 0 1 2 3 4 [Displacer] nM

121 Ketanserin Displacement (5HT2 Receptor) ICC

spiperone [Spip.+Compound 6

SC

[Displacer] nM

CHA Displacement (Adenosine 1 R e c e p to r ) 100

■ CHA oi c CHA+Compound 6 c m ■H u Q. 50 V) §

Xi I

122 DHA Displacement (S-adrenocepCor) 100

propranolol prCi+Compound 9

§

[Displacer] nM

Pyrilamine Displacement (Histamine 1 R e c p to r ) 100

P y r ila m in e

Pyri.+Compound 9

§

I

1 0 2 3 4 [ D is p la c e r ] nM

123 4.5 Discussion 1. Although the major principles of Schefflera bodinieri are triterpenoids which were similar to those obtained from other species of Schefflera and Araliaceae, they have their own characteristics, ie. the typical types of the compounds are pentacyclic triterpenoids with unsaturation at either C 1 3 -C 1 4 , or C 1 4 -C 1 5 . Comparing the constituents of the leaves with those of the roots, the leaves contain mainly aglycone and free sugars, whist the roots contain mainly glycosides. For instance, as shown in Figure 4.2 (p.94), the major constituents of the leaves are compound 1 (triterpenoid aglycone), compound 8 (six carbon sugar alcohol) and compound 9&9a (trisaccharides, the basic sugar moiety of the saponins in the plant) which can be calculated as 82% of the total weight of the isolated compounds. In contrast, all the compounds isolated from the roots are triterpenoid glycosides, suggesting that most of the glycosides are either synthesed or stored in the roots.

2. The majority of the isolated compounds are either structurally related or biosynthetically related and it is of interest to note the influence of the structure changes on their biological activities.

Compound 1 is the aglycone of compound 6 and compound 9 is the sugar moiety of compound 6 , both 1 and 5 were able to bind to muscarinic receptor (Table 4.6, p.l 18) and the aglycone (IC 5 0 0.9 pM) was more active than its glycoside (IC 5 0 3.5 pM). However, the sugar moiety was inactive at muscarinic receptor, but bound to Ca^^- channel receptor (IC 5 0 8.0 pM) and 5HT2 receptor (IC 5 0 3.8 pM). In interaction experiments (Table 4.7, p. 120), the aglycone (1) and its glycoside ( 6 ) had the same degree of influence on the 5HT2 receptor and the aglycone (1) and the sugar residue (9) had an effect on the p-adrenoceptor. The influence of 1 and 9 on the P- adrenoceptor were different. The aglycone changed the affinity of propranolol (the control compound) binding to the receptor, reducing its IC 5 0 value, whilst the sugar altered the binding site of the receptor (Figure 4.8, p. 121). Compound 6 , which was composed by 1 and 9, did not show any interaction with the control compound at this receptor.

124 CH; CH2OH o c 'COOR

compound 1 R=H compound 6 R= -Glc-Glc-Rham compound 9&9a

The compounds 4 and 5 are isomers, the only difference in the structure is the double bond position, 4 having a CJ 3 -C 1 4 double bond and 5 having a C 1 4 -C 1 5 double bond.

Compound 4 was able to bind to the dopamine 2 receptor (IC 5 0 1.8 pM, Table 4.6, p. 118), whilst compound 5 was inactive to all the receptors tested. In interaction experiments. Compounds 4, 5 and 5a (aglycone of 5) all had effects on the 5HT receptors. However, compound 4 acted on all the 5HT receptors tested non-selectively, while 5 and 5a acted on the 5HT1C and the 5HT2 receptors without influencing the 5HT1A receptor (Table 4.7, p. 120). As reviewed in Section 1.2 (p.37), the 5HT1A receptor belongs to different subfamily from the 5HT1C and 5HT2 receptor. The 5HT1A receptor has functions of inhibition of neurotransmitter release and contraction of some vascular smooth muscle, whilst the 5HT1C and 5HT2 receptors have the functions of gastrointestinal and vascular smooth muscle contraction and platelet aggregation. The finding in this study indicated that the influence of the isolated compounds to the control compounds are selective, which may be related to the structures or functions of receptors.

Glc Glc HO Rham HO Rham compound 5 R=CH3 compound 4 compound 6 R=COOH

125 Small structure difference between the compounds may result in marked differences in their biological activities. For instance, compound 5 affected the binding site of K^- channel receptors for glibenclamide (the control compound), whilst compound 6 , whose structure related to 5, did not show this activity, but showed an effect on the adenosine 1 receptor, on which compound 5 was inactive. Similarly, Compound 9 (the sugar moieties of 4 and 5), compounds 4 and 5 had an interaction with the control compound at the histamine 1 receptor, whilst compound 6 was inactive at this receptor. Therefore, to investigate the structure-activity relationship of these natural products will further our understanding to receptors and to drug-receptor interaction, and it will be beneficial to new drug development.

3. Triterpenoids and their glycosides showed various biological activities in the central nervous system. Previous studies on the biological screening of ginseng extracts and showed that ginsenosides were the active principles responsible for the physiological and pharmacological effects in brain, such as the abilities to affect the cyclic AMP and monoaminergic systems (Soe, 1980), to incorporate amino acids into proteins (Kim, 1979), to incorporate glucose into brain lipids (Shibata, et al, 1978), and to decrease sleeping time (Lee, 1974). These results suggested that ginseng saponins might play a role in the regulation of neurotransmission. In the present study, three ginseng glucosides were tested by ligand receptor binding assays and the results showed that ginsenoside Rbl and Rc were active at Ca^^-channel receptor (IC 5 0 4.2 and 4.4 pM, respectively. Table 4.6, p.l 18). It was reported (Tsang, 1985) that an extract fraction containing 40% ginsenoside Rc and 45% Rbl+Rb2 was able to inhibit the neurotransmitter uptake by 13-25% in the order of GAB A > noradrenaline > dopamine > 5HT > glutamate in ligand-receptor binding assays and it was then postulated that ginseng many exert its actions in the central nervous system by affecting the removal of neurotransmitter substances in synaptic regions. In the present study, it was also found the isolated compounds had ability to influence the agonists or antagonists binding to specific receptors. These results suggested that natural products may participate the regulation of neurotrasmission. The finding in this study and in the previous studies on ginseng saponins substantiated the activities of triterpenoid glycosides in the central nervous system.

126 4. The synergistic action of plant extract has been reported in some previous in-vivo studies, eg. an ethanol leaf extract of Schefflera arboricola produced a significant analgesic effect when lOOg/Kg of extract was given subcutaneously along with 5mg/kg of morphine hydrochloride (Anon., 1976). In the present study, several isolated compounds were found to interact with the agonists or antagonists of the various receptors, either decreased the IC 5 0 values of these compounds or affected the binding sites of the receptors. These facts indicated that the synergistic effect of the extract might be produced by the constituents of the plant interacting with either the endogenous or exogenous substances which were able to bind to the receptors.

5. Receptor ligand binding assays can be used as a guidance to isolated active principles from plant extracts. For instance, the extracts of Schefflera bodinieri were found to bind to the 5HT receptors, then compounds 9&9a, 11 were obtained which were active at 5HT2 receptor and compounds 1, 4, 5 and 6 were found to be able to affect the control compounds binding to 5HT1A, 5HT1C and 5HT2 receptors. However, it is necessary to mention that there are a large number of constituents in plant extract, they may exert their actions by a complex mechanism. Therefore, it is not surprising that the isolated single compounds do not represent the full effects of the plant itself, and similarly, the individual compounds can have certain effect which the extract does not show markedly.

127 Chapter 5

Clerodendrum mandarinorum

128 5.1 Introduction 5.1.1 Verbenaceae and Clerodendrum Verbenaceae belongs to the dicotyledons and contains about 75 genera and 3000 species worldwide (How, 1984). Some 20 genera and 174 species have been found in China and they mainly grow in the south of the country (How, 1984). A number of Chinese medicinal herbs are in the family which are mainly in the genera of Callicarpa, Premna, Vitex, Clerodendrum, Lantana and Verbena.

Several Callicarpa species, eg. C. pedunculata, C dichotoma, C. nudiflora, C. japonica, C. macrophylla and C. cathayans, show hemostatic effects and are used to treat hematemesis, hemoptysis, epistaxis, bloody stool, metrorrhagia, wound bleeding and are used as antibacterials (Hou, 1987). The major constituents in the genus are flavonoids such as , , luteolin-7-glucoside; phenols, ketone, tannins, and polysaccharides (Liu, 1979).

Premna microphylla is used to dispel swelling, hemostatic and has antipyretic effect (How, 1984). There has been no report on its chemical study.

Vitex cannabifolia and V. trifolia are the activators of the pituitary in the adrenocortical system and also have antibacterial, hypotensive, immunosuppressant, sedative and hypnotic effects (Anon, 1977). V. negundo var. cannabifolia is credited with having expectorant, antitussive and antiasthmatic actions and it is mainly used in chronic bronchitis (Bao, 1986). The fresh leaf and seed of the species contain volatile oil which is composed of p-caryophyllene, caryophyllene oxide, p-elemene, a-pinene, 1 ,8 -cinede (eucalyptol), limonene, p-cymene and ; glucosides, cardiac glycosides and alkaloids (refs, in Bao, 1986). Vitex negundo is reputed to be antirheumatic, stomachic, antitussive, fever-clearing, deobstruent, expectorant and sialagogue. It is used to treat common cold, gastric diseases, carbuncle, furuncle, cough and asthma. The fruit and root of the plant contain flavonoid glucosides, cardiac glucosides, alkaloids, amino acid and volatile oils (Bao, 1987).

129 The leaf decoction of Lantana camara is used as a bath agent to treat skin disease in the south of China (How, 1984).

Verbena officinalis is used to promote the blood circulation and relieve blood stasis, to counteract toxicity, and to cause diuresis. It is used for the treatment of amenorrhea and dysmenorrhoea, inflammation of the throat, carbuncles, boils, acute infection of the urinary tract and edema(Anon., 1992). The major components of its aerial part are iridoid glycosides; comin; hastatoside; flavones, eg. artemetin; triterpenoids, eg. lupeol, ursolic acid, p-sitosterol; and phenylpropane glucosides, eg. verbascoside and eukovoside (Jensen, et al., 1973; Buchi, et al.,1960; Makboul, 1986; Kui and Tang, 1985; Bianco, et al, 1984; Tang and Eisenbrand, 1992).

The plants of Clerodendrum grow in tropical or subtropical area including 400 species worldwide and 30 species in China (How, 1984). C. fortunatum, C. inerme, C. japonicum and C. trichotomum are recorded as medicinal species. The leaf of C. trichotomum is used as antirheumatic, analgesic, hypotensive and for the treatment of bloody stool (Xue, 1987; Li, 1992). There are several phytochemical reports on the plants of this genus. The major constituents isolated from the plants are listed in Table 5.1 and several examples which showed different type of structures are given in Figure 5.1.

5.1.2 Clerodendrum mandarinorum Clerodendrum mandarinorum Diels grows in the southwest of China and has not been recorded as a medicinal herb but it is often confused with C. trichotomum in certain areas since they have similar morphology. There is no previous chemical or pharmacological report on the species. In order to investigate the medicinal resource of Clerodendrum, this plant was selected in this study to search for the CNS active principles.

130 Table 5.1 Compounds isolated from Clerodendrum species

Species Constituent Reference C. hrachyanthum clerodinin C, clerodinin D, Lin,et al,1989 clerodiol

C. calamitosum 3-epicaryoptin Harada, et al, 1978

C. inerme clerodermic acid, neokadsuranin, Achuri,et al, 1990; 3,16,19,-tri Ac, 15-methoxy, 14,15 1992, Spencer,et al, -dihydro-3-epicaryopitin, 1981; Li, et al, 3,13-clerodadien-15,16,olid-18- 1988; Zdero,et al, oic acid 1991;Raha,et al, 1991

C. infortunatum 1 2 -lupanon, clerodin Manzoor-i-Khuda,et stigmasta-5,25,dien-3-ol and al, 1966; Rogers, et its 3 p O-D-glucopyranoside al 1979; Wagner et 7-0-P-D-glucoside-5-hydroxy- al, 1976 4-methoxy-fiavone

C. nerifolium 4 ’,5,6,7-tetrahydroxyflavone Prakach,et al, 1982

C. phlomides 4’ ,5,6,7-tetrahydroxyflavone Markham, 1983

C. serratum serratagenic acid Alvarado, et al, 1981 4’ ,5,6,7-tetrahydroxyflavone

C. scandens a serial stigmasta-3-ol Akihisa, et al, 1990

C. trichotomum clerodendrin A, trichotomine G l, Kato, et al, 1973 2,3,5,6,1 lb-hexahydro-3-oxo-lH- Toyoda, et al, 1982 indolizino(8,7-b)indole-5- Irikawa,et al, 1989 carboxylic acid-(5s,llbS)form and Harbome, et al, -(5s,llbR)form; trichotomine, 1971 N,N ’ -Di-(D-glucopy ranosyl)- Shelyuto, et al, trichotomine, clerodendroside, 1972 2,3,6,11 -tetrahydro-3-oxo-1H- indolizino-(8,7-b)indole-5,llb(5H) -dicarboxylic acid-(5s,l lbR)-form and -(5s, 1 lbS)-form 7-0-[p-D-glucuronosyl-(l-2)-P-D- glucuronoside-5-hydroxy-4 ’ - methoxy-flavone

131 Figure 5.1 The diversity of compound structures isolated from

Clerodendrum species

CH2OH COOH 18-hydro-3,13-clerodadien Clerodin -16,15-olide clerodermic acid

HOOC

OH

OH, 'COOH

OH HO' OH

serratagenic acid 3 , 3 ' ,4 ' ,5'-tetramethoxy 4',5,6,7-tetra-

- 4 ,5-methylene-dioxy-2 , hydroxy-flavone

2 ' -cyclo-7,7,-epoxylignan

As, A 7 ,or^ll

1 2 -lupanone /

COOH R=

2,3,5,6,11,llb-hexahydro-3-

oxo-lH-indolizino[8 ,7,b]indole- 5-carboxylic acid

132 5.2 Biological screenings of extract fractions The 70% ethanol extract of the C. mandarinorum root bark strongly bound to opiate, adenosine 1, 5HT1, 5HT2, dopamine 2, GABA^ and GABAg receptors (Figure 3.1, p.80), and therefore, the binding assays of the first four of these receptors were selected for fractional screenings as the test tissue was readily available. The fractions were prepared by column chromatography in order of polarity and the screening approach used was the same as described in the Section 2.7. The results are shown in Table 5.2.

5.3 Isolation and Structure identification of the isolated compounds Experimental details have been given in Sections 2.1-2.5 of Chapter 2. In brief, flash chromatography (Si 0 2 ) was repeatedly used for separation of 2 0 g of the root bark extract and the fractions were collected every 1 0 0 ml eluate in the order of polarity. The further purification of a number of compounds was carried out on sephadex column and HPLC. The procedure of isolation are summarized in Figure 5.2. From the root bark of C. mandarinorum, 15 compounds (15-29) have been isolated and identified. They include triterpenoids, oligosaccharides, steroids, flavonoids, a coumarin, and a lactone. A coumarin glycoside has found to be a novel compound.

133 Table 5.2 The fraction screening results of Clerodendrum mandarinorum

Fr.\ Rep. 5HT1 5HT2 adenosine 1 opiate

Total Ext. ++ * 4- 4-4- 4-

Fr.1-5 ** 4-

Fr.6 - 8 + 4-4- 4-4-

Fr.9-10 4- ++

Fr.ll + 4-4- 4-4-

Fr.l2 4- 4-4-

Fr.l3 + 4-4-

Fr.l4 4-4-

Fr.l5 4- 4-4-

Fr.l 6 4-4-

Fr.l7 4- 4-4-

Fr.l 8 4-

Fr.l9 4-4-

Fr.20 4-

Fr.21 4- 4- Fr.22

Fr.23 4- Fr.24-30 Fr.31 +

Fr.32-34 4- Fr.35-44

Fr.45 4-4- 4-4-

*: % Inhibition of radioligand’s specific binding between 50-79%

% Inhibition of radioligand’s specific binding between 80-100%

**: fractions see also Figure 5.2.

134 Figure 5.2 Isolation compounds from Clerodendrum mandarinorum

70% EtOH Extract root bark

CC (Si02)

Fr. 1-5 Fr . 6-17 Fr. 18-22 Fr. 23-35 Fr. 36-45 (0.5 g) (5g) (2g) (2g) (8g) brown syrup CC sephadex CC chlorophyll HPLC compound 15(5mg) CC HPLC compound 2 6 compound 2 7 (2 0mg) (100 mg) compounds 2B&29 compound 16 (2Omg) (2mg) compound 17 (2Omg) compound 18 (3mg) compound 19 (lOmg) compound 23(20mg) compound 20 (5 Omg) compound 24(2 Omg) compound 21 (200mg) compound 2 5 (15mg) compound 22 (5mg)

l-CHClj, 2-CHCl3-MeOH (9:1), S-CHClg-MeOH (8:2), 4-MeOH, S-MeOH-HzO (9:1)

135 5.3.1 Identification of Clerodirine (24) (p. 146 for data)

ORi 24a: RI = H

R 2

3 - * - l C H2C H 2O—Xy I— Rham 7* 8< OH 24b: RI =

, 0 1 R2 = H CH2CH2O—Xyl— Rham

Compound 24 is a trans-cmndimxc acid derivative with two sugar units as indicated by HNMR (Spectrum 24.3 in appendix). The spectrum revealed a pair of olefinic protons with trans- configuration at 5 6.3 and 7.6 (J=15 Hz) which can be assign to Cg and C 3. The ‘^CNMR-DEPT spectrum (Spectrum 24.4 in Appendix) exhibited eleven sugar carbons, fourteen aromatic carbons, one carbonyl carbon ( 6 168.3) and an ethanolic group, suggesting the presence of a structure as a phenyl substituted cinnamic acid derivative. There are two sets of three proton signals appeared as ABC system on both aromatic rings, respectively, and two sets of ortho- hydroxyl groups on both rings (four carbons at Ô 144.7, 146.6,146.9,148.1). The 'H-’H COSY spectrum established the linkage of the protons. The two coupling methylene signals (Ô 2.78, 2H, t, J=5, H-

7’; Ô 3.70, IH, m, H-8 ’; Ô 4.05, IH, m, H-8 ” ) suggested that there was a -CH 2 CH2 -O- group. The position of this group at C-4’ was determined by 'H-^H COSY, because

H-5’ (5 6.55, IH, dd, J= 8 , J=1.5) was weakly coupling to one of H-7’, and H- 6 ’ (Ô

6 .6 8 , IH, d, J= 8 ) was weak coupling with H-5 (Ô 6.95, IH, dd, J= 8 , J=1.5) (Spectrum 24.5 in Appendix). Although there are two possibilities to link the phenyl moiety to the parent structure (structures were supposed to be 24a and 24b), 24a was preferable structure based on the *H-‘H COSY spectrum. The sugar units were linked to the Cg -

OH since the chemical shift of C- 8 ’ (5 70.4) was 8 pm downfield comparing with the

value of that with free OH ( 8 62). The C 2 -OH was free because of the presence of a

136 hydroxy proton signal at 6 5.18 (IH, s, C 2 -OH). In ‘HNMR spectrum, two sugar anomeric proton signals were at 5 4.42 (IH, d, J=6.5) and 4.90, respectively, and one methyl signal was at Ô 1.18 (3H, d, J= 6 ). Hydrolysis of the compound afforded xylose and rhamnose (by co-TLC, arabinose, galactose, mannose and glucose were also spotted on the same TLC plate as additional control compounds). The C-1 of the rhamnose linked to the xylose via the C-3 of xylose since the chemical shift of the C- 3 was shifted 5ppm downfield. The ‘^CNMR data of the sugar units were identical to those of P-D-xylopyranoside and a-L-rhamnopyranoside in the references (Hughes, et al, 1976; Agrawal, 1992).The ElMS showed the ion peak of the aglycone at miz 316 (Spectrum 24.2 in Appendix). From these results, and compared with the related compound in reference (Nyandat, et al, 1993), the structure of compound 24 is 18-0- [a-L-rhamnopyranosyl ( 1 -3)-p-D-xylopyranoside]-4’-ethyl-ol-2’-phenolic-7-0-phenyl- tra/15-cinnamic acid. It is supposed to be a novel compound and is named clerodirine. To conform this structure, the long range C-H coupling NMR technique is needed.

5.3.2 Identification of friedelanone (15)

Compound 15 (p. 143 for data) was obtained as white needle crystals. The ‘HNMR spectrum showed the presence of seven tertiary methyl groups, one secondary methyl group and gave a typical spectral profile of a triterpenoid without a double bond in the structure. The ElMS indicated it molecular weight at m/z 426 and the characteristic ions of cleavage of friedelane-type compounds at mIz 273, 205 and 123 (Cong, 1987). The ‘^CNMR spectrum exhibited that the compound contained eight

137 methyl carbons, eleven methylenes, four methines and six quaternary carbons in addition to a ketone carbon corresponding to the formula C 3 0 H5 2 O. These spectral data suggest that the compound 15 is friedelanone. It is further substantiated by comparison of the mass spectrum of friedelanone (Cong, 1987) and the spectral data in the references (Sengupta, et al. 1968; Majumdar and Thakui, 1968).

5.3.3 Identification of Lupeol (16) and Betulinic acid (17)

HO'

compound 16: R=CH3 compound 17 : R=COOH

Compound 16 (p. 144 for data) was obtained as white needle crystals. ‘HNMR spectrum exhibited that it was a triterpenoid having the signals of five tertiary methyls, and one methyl on a double bond ( 6 1.68). Two coupling protons at ô 4.57 and 4.69 suggested the presence of a terminal methylene of an isopropenyl group on the side chain and the signal at 6 3.18 (H-3) indicated the presence of a 3p-0H. The '^CNMR spectrum exhibited that there were seven methyls, ten methylenes including one with a double bond (6109.3), six methines including one linked with a hydroxyl group (679.0), six quaternary carbons including one double bond carbon (6151.0)

corresponding to the formula C 3 0 H5 0 O. The ElMS data showed the molecular weight of the compound at miz 426 and the characteristic ions of lupane-type cleavage at miz 218 (100), 207(60) and 189(64) (Cong, 1987). Based on these spectral data and compared with the data in reference (Sholichin, et al., 1980), the compound 16 is identified as lupeol.

Compound 17 (p. 144 for data) was obtained as white needle crystals. The ‘HNMR

138 spectrum exhibited a similar pattern to compound 16, except for one methyl group less. The ‘^CNMR spectrum also showed similar chemical shift values as compound

16 except for the presence of one carboxylic carbon ( 6 179.3) instead of one methyl carbon. The ElMS further substantiated that C 1 7 -CH3 in compound 16 was replaced by a carboxylic group with diagnostic peaks at m/z 248, 234 and 220. Therefore, compound 17 is determined as betulinic acid (Sholichin, et al., 1980).

5.3.4 Identification of 24S-stigmasta>5, 25 dien-3|3-ol (18) and 22E, 24S-stigmasta 5,22,25 trien-3p-o! (19)

HO' MG'

compound 18 compound 19

Compound 18 (p. 144 for data) was obtained as white needle crystals. The 'HNMR data indicated that it had a stigmasterol-type structure ( 6 3.52, IH, m, H-3a; Ô 5.35

IH, d, J= 6 , H-6 ) with a terminal methylene (Ô 4.73, 4.63, C=CH2, H-26) of an isopropenyl group (Ô 1.59, 3H, s, C 2 5 -CH3 ) on the side chain was involved (Akihisa, et al., 1989). The ElMS showed the M^ at m/z 412 and the fragmentation of the compound was identical with those of stigmasta-5,25,-dien-3p-ol (Massey and Djerassi, 1979). The '^CNMR data further substantiated that the compound had a structure of 24S-stigmasta-5,25 dien-3P-ol which was in a good agreement with the data in the references (Kojima, et al. 1990; Akihisa, et al., 1990).

Compound 19 (p. 144 for data) has a similar structure as compound 18 except for an extra double bond between C 2 2 -C2 3 ( 6 5.20, IH, t, J=6 , H-22; 5 5.17, IH, t, J=6 , H-

23). The MS spectrum showed the M^ at m/z 410 and a characteristic peak of C 2 2 - unsaturated stigmasterol at m/z 271 (Pakrashi and Achari, 1971; Bolger, et al. 1970).

139 The ‘^CNMR also gave the identical data to 22E, 24S-stigmasta-5,22,25 trien-3p-ol reported in the references (Kojima, et al., 1990; Akihisa, et al., 1990).

5.3.5 Identification of Cirsimaritin (20), Cirsimaritin-4-glucose (21) and Quercetin-3-methyl-ether (22)

Rs

HO 0

compound 20 Rl=R2=OCH3, R3=R4=H, R5=0H

compound 21 Rl=R2=OCH3, R3=R4=H, R5=0Glc

compound 22 Rl=R4=R5=OH, R2=H, R3=OCH3

Compound 20 (p. 145 for data) was obtained as yellow crystals and had a positive reaction to ammonia, indicating that it may be a flavonoid. The UV spectrum of the compound showed it as a flavone 284, 338nm). In ElMS, the basic peak was at m/z 314 suggesting that there were two methoxyls and two hydroxyls in the molecule. The fragment m/z 299 [M-15]^ indicated that there was a methoxyl at either

C - 6 or C- 8 (Kingston, 1971). The fragments at m/z 181,167 and 153 showed that two methoxyls and one hydroxyl were located on A ring, while the fragment at m/z 121 showed the presence of a hydroxyl group on the B ring. The ‘HNMR (DMSO)

spectrum exhibited a set pair of two proton doublets (Ô7.97, 6.94, 2H each, d, J= 8 Hz)

and an aromatic singlet at Ô 6.92 and they were assigned to H-3’, H-5’, H-2’, H- 6 ’,

H-3, respectively. Since the chemical shift of H - 6 usually appears around Ô 6.14-6.46

and H - 8 appears around 8 6.35-6.92 (Yang, 1992), another aromatic singlet at 8 6.80

140 was assigned to H- 8 . Consequently, the compound is 5,4’-dihydroxyl- 6 ,7 dimethoxyl flavone (cirsimaritin). This was confirmed by comparison with the authentic sample (co-TLC).

Compound 21 (p. 145 for data) was a pale yellow amorphous powder and was positive to ammonia. The ’HNMR(DMSO) spectrum of the compound was similar to that of

20 except for the presence of a glucose ( 6 5.40, IH, d, J=2Hz, glucose C,-H and Ô

3.70-5.15, 5H, glucose C2 .5 -H). In the CIMS, a [M]"^ +1 peak appeared at m/z 477 and [M]^-glucoside+l peak appeared at m/z 315. The UV spectrum showed no bathochromic shift and an increase in the intensity with MeONa, indicating the presence of a 4’-glucose in B ring. Hydrolysis of the compound gave cirsimaritin and glucose as products. Therefore, the compound was identified as cirsimaritin-4’- glucoside.

Compound 22 (p. 145 for data) is also a known flavonoid and the structure was determined as quercetin-3-methylether by means of spectral analysis and comparison with authentic sample (co-TLC).

5.3.6 Identification of a-tetrahydropyrone (23)

O

O

Compound 23 (p. 145 for data) was obtained as a light yellow oil-like substance. The ElMS showed the M^+1 at m/z 101. ^^CNMR-DEPT spectrum displayed four methylenes, one of which linked to an oxygen atom in the ring (Ô 59.0), and one ketone carbon (Ô 214.8), corresponding to the formula C^HgOg. The ‘HNMR exhibited eight protons at Ô 3.80 (2H, t, J=6.5, H-5) and 5 1.8-2.7 ( 6 H), respectively, suggesting that the compound was tetrahydropyrone. The ‘H-'H COSY spectrum indicated the linkage of protons, locating the ketone at the a-position. The compound was then identified as a-tetrahydropyrone.

141 5.3.7 Identification of a-D-GIucopyranose-l-ethylether (25), Sucrose (26) and p-D- fructofuranose-2-ethylether (27)

CH^OH

H OH CHoOH CH3CH26 OH

compound 2 5 HOHoC

OH HOHoC OH

compound 2 6 OH

compound 27

Compound 25 (p. 146 for data) was obtained as white crystals and was soluble in EtOAc and MeOH. The CIMS gave the molecular ion peak at m/z 208. The ’HNMR and '^CNMR spectra indicated that the compound was an a-D-glucose (Ô 4.7, IH, d,

J=3, glc H-1) with an ethyl group at C-1 (5 1.24, 3H, t, J=7, CH 3 -I, Ô 3.76, 2H, q,

J=2, CH2 -I). The ’H-’H c o s y spectrum exhibited the linking order of protons. Comparing with the data in literature (Berlin, 1970), the structure was identified as a- D-glucopyranose-1 -ethylether.

Compound 26 (p. 148 for data) was obtained as a white amorphous powder and was soluble in DMSO and HjO. The ’HNMR spectrum indicated the presence of two sugar units with twenty-two protons between 5 3.10-5.15. The ’H-’H COSY spectrum displayed the linking order of the protons. The ’^CNMR spectrum showed the presence of twelve carbons including three methylenes between Ô 60.8-62.5 and a C-2 carbon of a fructofuranose at Ô 104. From these data, the compound was identified as a-D- glucopyranosyl-(l-2)-p-D-fructofuranose, ie., sucrose (Dorman and Roberts, 1971). The structure was further substantiated by co-TLC.

142 Compound 27 (p. 148 for data) was obtained as white powder and was soluble in MeOH. The ‘HNMR spectrum indicated that the compound was a sugar with nine proton signals at 6 3.50-4.12 and a methyl group signal at 5 1.30 (3H, t, J= 6 ). The ‘^CNMR showed that it was a fructose derivative (Ô 105.1 C-2) with an ethyl group on the C-2 (chemical shift of C-2 was 2ppm downfield). Comparing with the data in the reference (Dorman and Roberts, 1971), the compound was determined as p-D- fructofuranose-2 -ethylether.

5.3.8 Identified a-D-glucopyranose (28) and p-D-glucopyranose (29) Compounds 28 and 29 (p. 148 for data) were obtained as a mixture. The ElMS spectrum gave the M^-1 peak at m/z 181, and a series of peaks of loss 18 amu. The ‘HNMR spectrum showed signals of two sugar units and the anomeric proton signals at 5 5.10 (IH, d, J=3) and Ô 4.48 (IH, d J=7), respectively, suggesting that they might be a-D- and p-D- forms of glucopyranose. Examination the mixture on TLC with an authentic glucose sample showed that they were glucose rather than one compound composed of the two glucose units. Therefore, the compounds were identified as a-D- glucopyranose and p-D-glucopyranose.

All these compounds isolated from Clerodendrum mandarinorum were shown by TLC to be present in a crude extract of the plant and they were not artifacts formed during the isolation procedure. Their structures are summarized in Figure 5.3.

5.3.9 Spectral data

Friedelanone (15): white needle crystals, soluble in ether, CH2CI2, CHCI3. TLC (Si0 2 ): Rf 0.75 (CHCI3). ‘HNM R (CDCl^): tert. Me (s) Ô 0.73, 0.88, 0.95, 1.02, 1.04, 1.07,

1.20; secondary methyl, 50.90 (3H , d,J= 6 ). ‘^CNMR (CDCI3): methyl 5 6.83, 14.65, 17.94,18.67,20.26,31.78,32.09,35.02; methylene: 18.23,22.28,30.50, 32.41, 32.76, 35.33, 35.61, 36.00, 39.24, 41.28, 41.53; methines: 42.78, 53.10, 58.22, 59.47; quaternary carbons: 28.19, 30.02, 37.46, 38.31, 39.72, 42.16, 213.2 (3-ketone). ElMS m/z (rel. int.): 426 (M+, 7), 411(9), 341(4), 302(10), 287(7), 273(24), 231(18), 218(22), 205(40), 163(37), 137(49), 123(94), 109(97), 95(100).

143 Lupeol (16): white needle crystals, soluble in CHCI 3 . TLC (Si0 2 ): Rf 0.60 (CHCI3 -

MeOH 9:1). ’HNMR (CDCI 3 ): tert. Me (s) Ô 0.75, 0.80, 0.85, 0.93, 0.96, 1.00, 1.68;

Ô 4.57, 4.67 (2H, d, J=2, C=CH 2 , H-29), Ô 3.18(1H, m, H-3a). ’^CNMR (CDCI 3 ): methyls 6 14.55, 15.38, 15.98, 16.12, 18.00, 19.31, 28.00; methylenes: 18.32, 20.93, 25.14, 27.42,29.85, 34.28, 35.59, 38.72,40.01,109.33 (C-29); methines: 38.06,47.99, 48.30, 50.44, 55.30, 79.00 (C-3); quaternary carbons: 37.19, 38.33, 40.86, 42.86, 43.02, 150.99 (C-20). ElMS miz (rel. int.): 426(M\ 38), 411(10), 315(10), 218(100), 207(63), 203(50), 189(64), 135(67), 123(72), 95(78).

Betulinic acid (17): white crystals, soluble in CHCI 3 . TLC (Si0 2 ): Rf 0.50 (CHCI3 -

MeOH 9.5:0.5). ’HNMR (CDCI 3 ): tert. Me (5) 0.79, 0.84, 0.94, 0.96, 0.97, 1.69; Ô

4.72, 4.59 (2H, d, J=2, C=CH 2 , H-29), Ô 3.20, (IH, t, J= 8 , H-3a), Ô 3.04 (dt, J= ll,

J=6 , H-19). ’^CNMR (CDCI 3 ): methyls, 614.8, 15.5, 16.1, 16,3, 19.4, 28.0; methylenes, 18.5, 21.2, 25.8, 27.1, 29.9, 30.8, 32.5, 34.6, 37.4, 39.0, 109.6 (C-29); methines, 38.6, 47.3, 49.5, 50.8, 55.6, 79.0(C-3); quaternary carbons, 37.4, 39.0,40.9, 42.7, 56.5, 151.0(C-20), 179.3 (COOH). ElMS miz (rel. int.): 456(M+, 17), 438(10), 423(6), 395(6), 248(24), 234(17), 220(19), 207(47), 203(30), 189(100), 175(30), 135(54), 121(44), 95(55).

24S-Stigmasta-5,25-dien-3^-ol{\%): white needle crystals, soluble in CHCI 3 . TLC

(SiOz): Rf 0.45 (CHCl3 -MeOH 9.5:0.5). ’HNMR (CDCI 3 ): tert. Me (6 ) 0.68, 1.01,

1.59, 6 0.92 (3H, d, J=9, C 2 0 -CH3 ), 6 1.00(3H, t, J=3, C2 8 -CH3 ); 6 4.73, 4.63 (2H, d,

J=3, C=CH2 , H-26), 6 3.52, (IH, m, H-3a), 6 5.35 (IH, d, J= 6 , H-6 ). ’^CNMR

(CDCI3 ): methyls: 611.9, 12.0, 18.7, 19.4, 20.8; methylenes: 21.1, 24.3, 25.7, 28.2, 29.4, 31.7, 31.9, 33.7, 37.3, 39.7, 43.3, 109.5; methines: 31.9, 35.6, 49.5, 50.1, 56.1,

57.8, 71.8(C-3), 121.7 (C- 6 ); quaternary carbons: 36.5,42.3,140.7(C-5), 147.6(C-25). ElMS m/z (rel. int.): 412(M+, 32), 397(7), 394(12), 379(17), 314(13), 300(18), 299(20), 273(12), 255(33), 231(12), 229(13), 213(36).

22E,24S-Stigmasta-5,22,25-trien-3^-ol (19): white needle crystals, soluble in CHCI 3 .

TLC (Si0 2 ): Rf 0.42 (CHClj-MeOH 9.5:0.5). ’HNMR (CDCI 3 ): tert. Me (6 ) 0.68,

1.01, 1.65, 6 0.83 (3H, d, J=9, C 2 0 -CH3 ), 6 1.03(3H, t, J=3, C2 8 -CH3 ); 62.42(1H, q.

144 J=7, H-24); 5 4.69 (2H, m, C=C% H-26), 5 3.52, (IH, m, H-3a), Ô 5.35 (IH, d, J= 6 ,

H-6 ), 5.20 (IH, t, J=6 , H-22), 5.17 (IH, t, J=6 , H-23). ”CNMR (CDCI3 ): methyls: 511.9,12.1,17.8,19.4,20.2; methylenes: 21.1, 24.3,26.5,28.7,31.7,33.7, 37.3,39.7,

43.3, 111.4; methines: 31.9, 36.1, 40.2, 50.1, 55.9, 56.9, 71.8(C-3), 121.7 (C- 6 ), 130.6(C-23), 137.2(C-22); quaternary carbons: 31.9, 42.3, 140.7(C-5), 148.6(C-25). ElMS m/z (rel. int.): 410(M+, 10), 395(5), 392(7),300(14), 271(47), 255(32), 231(30), 229(14).

Cirsimaritin (20): yellow crystals, soluble in DMSO and slightly soluble in CHCI 3 .

TLC (SiOz): Rf 0.65 (CHCl^-MeOH 9:1). *HNMR (DMSO): 5 7.97, 6.94 (4H, d, J= 8 ,

H-2’, 3’, 5’, 6 ’), 3.92 (3H, s, OMe-7), 3.74(3H, s, OMe- 6 ). ElMS m/z (rel. int.): 314(M\ 100), 299(87), 285(20), 271(22), 181(27), 167(5), 153(40), 121(12).

Cirsimaritin-4' -glucoside (21): pale amorphous powder, soluble in DMSO, slightly soluble in MeOH. TLC (SiO^): Rf 0.35 (CHClg-MeOH 9:1). UV (nm) 284,358;

+MeOAc:300,330sh; +AICI 3 : 306, 358; +A1C13+HC1: 304, 352; +NaOAc: 286,384: fNaOAc+HgBO): 286,340. 'HNMR (d 6 -DMSO): 6 3.70-5.51 (5H, glucose C^^-H),

3.74(3H, s, OMe-6 ), 3.90(3H, s, OMe-7), 5.40 (IH, d, J=5Hz, glucose H-1), 6.96 (IH, s, H-8 ), 6.99 (IH, s, H-3), 7.20 (2H, d, J=7Hz, H-3’,5’), 8.06 (2H, d, J=7Hz, H-2’6 ). ElMS m/z (rel. int.): 314(M+-glucose, 98), 299 (100), 285 (31), 283(11), 271(44), 268(33), 181(33), 167(7), 153(76), 121(16), 119(32). CIMS m/z (rel. int.): 477 (M++1, 9), 315(20).

Quercetin-3-methylether (22): pale amorphous powder, soluble in MeOH. TLC (SiOg):

Rf 0.45 (CHClj-MeOH 9:1). UV (nm): 273, 350; +MeONa: 279,398; +AICI 3 :

280,300sh, 420; +AICI 3 +HCI: 280, 300sh, 350. ‘HNMR(d4-MeOH): Ô: 3.78 (3H, s,

OMe-3), 6.18 (IH, d, J=2Hz, H- 6 ), 6.40 (IH, d, J=2Hz, H- 8 ), 6.90 (IH, d, J=9Hz, H-

5’), 7.51 (IH, dd, J=9, 2 Hz, H- 6 ’), 7.65 (Ih, d, J=2Hz, H-2’). ElMS m/z (rel. int.): 316(M+,5), 273(4), 153(50), 137(10), 136(100).

Tetrahydropyrone (23): light yellow oil-like substance, soluble in MeOH. TLC (Si0 2 ):

Rf 0.70 (CHCl3 -MeOH 8:2). 'HNMR (CD 3 OD): Ô 3.80 (2H, t, J=6.5, H- 6 ), 1.80 (2H,

145 (IH, m, H-3’), 1.86 (IH, m, H-3” ). ‘^CNMR (CD 3 CD): Ô 214.8 (C-2), 37.8 (C-3),

44.5 (C-4), 37.7 (C-5), 59.0 (C- 6 ). ElMS m/z (rel. int.): 101 (M++1, 29), 83(24), 67(21), 55(64).

Clerodirine (24): yellow amorphous powder, soluble in MeOH. TLC (SiOj): Rf 0.32 (CHClj-MeOH 8:2). UV X.„„(MeOH, nm): 207, 222(sh), 340; +MeONa: 224, 410. ‘HNMR and ‘^CNMR data see Table 5.3. ElMS m/z (rel. int.) : 316 (aglycone, 3), 270

(5), 299 ( 8 ), 254 (20), 179 ( 8 ), 163 (5), 146 (10), 137(45), 132 (35), 131 (53), 110 (50), 106 (15), 91 (42), 55 (100).

a-D-glucopyranose-l-O-ethylether (25): white crystals, soluble in EtOAc and MeOH.

TLC (SiOz): Rf 0.30 (CHCl^-MeOH 8:2). ‘HNMR (CD 3 OD): Ô 4.78 (IH, d, J=3, H-1),

6 1.24(3H, t, J=7, CH3 -I), 3.76 (2H, q, J=2, CH^-l), 3.75 (m, H-2), 3.65-3.90 (5H,

overlapped, H-3, H-4, H-5, H- 6 ). ‘^CNMR (CD3 OD): Ô 15.3 (CH3 -I), 64.4 (CH^-l),

100.1 (C-1), 71.5 (C-2), 72.3 (C-3), 70.2 (C-4), 71.0 (C-5), 62.5 (C-6 ). CIMS m/z (rel. int.): 226(M++NH/, 100), 209 (7, M++1), 180 (12).

146 Table 5.3 "CNMR and 'HNMR data of Compound 24

.OH CH2CH2O—Xyl— Rham HO

OH

No. C H No. C H aglycone sugar

1 168.3 - xylose

2 117.4 6.30 (d, J=15) 1 103.0 4.42 (d, J=6.5)

3 146.6 7.60 (d, J=15) 2 74.4 3.42 *

4 127.4 - 3 80.3 3.28 * 5 114.7 7.05 (d, J=1.5) 4 70.6 3.85 *

6 144.7 - 5 60.9 3.40, 3.20 *

7 149.8 -

8 116.5 6.95 (dd, J=8,1.5) rhamnose

9 123.1 6.75 (dd, J= 8 ) 1 1 0 1 . 8 4.90 **

r 148.1 - 2 71.0 3.65 *

2 ’ 146.9 - 3 72.4 3.80 * 3’ 116.2 6.70 (s) 4 74.6 3.55 *

4’ 131.4 - 5 69.0 3.75 *

5’ 123.3 6.55 (dd, J=8,1.5) 6 17.1 1.18 (d, J= 6 )

6 ’ 115.2 6 . 6 8 (d, J= 8 ) V 35.3 2.78 (t, J=5)

8 ’ 70.4 3.70, 4.05 (m)

The data were obtained by 300 MHz NMR in CD3OD and assignment based on 'HNMR, '^CNMR- DEPT and 'H-'H COSY. *: J values could not be measured because the signals were overlapped. **: the signal overlapped with H^O peak in 'HNMR spectrum, but could be observed in H-'H COSY spectrum

Ref: Nyandat, E., Rwekika, E., Galeffi, C., Palazzino, G. and Nicoletti, M. (1993): Olinioside, 5-(4’-0-p-D-glucopyranosyl)-caffeoyloxy-5,6-dihydro-4-methyl-(2H)-pyran- 2-one from Olinia usamharensis. Phytochemistry, 33: 1493-1496

147 Sucrose (26): white amorphous powder, soluble in DMSO and H^O. TLC (SiOj): Rf 0.34 (CHClj-MeOH 7:3). ‘HNMR (DMSG-de) and ‘’CNMR (DMSO-d«) see Table 5.4.

ElMS m/z (rel. int.): 342 (M \ 2), 335 (4), 314 (6 ), 163 (10), 145 (18), 127 (22).

Table 5.4 "CNMR and 'HNMR data of compound 26

No. C H No. C H fructose glucose

1 62.5 3.41, 3.43 1 92.1 5.15

2 104.0 2 73.2 3.15 3 82.7 4.70 3 73.3 3.72 4 77.5 3.10 4 70.1 3.50 5 74.5 4.72 5 71.8 4.34

6 62.2 3.38, 3.42 6 60.8 4.12, 3.83

The data were obtained by 300 MHz NMR in DMSO and the assignment according to 'HNMR, '’CNMR-DEPT and 'H-'H COSY spectra.

P-D-fructofuranose-2-ethylether (27): white amorphous powder, soluble in MeOH. TLC (SiOî): Rf 0.25 (CHClj-MeOH 7:3). 'HNMR (CD^OD-d^): 5 1.17 (3H, t, J=7,

CH3 -2 ), 4.12 (IH, d, J= 6 , H-3), 3.95 (IH, t, J= 6 , H-4), 3.55 (2H, q, J=$, CH2 -2 ), 3.60-

3-80 (5H, overlapped, H-5, H- 6 , H-1). '’CNMR (CD3 OD): S 16.0 (CHj-2), 57.8 (CHj-

2), fructose: 64.8 (C-1), 105.2 (C-2), 83.2 (C-3), 78.2 (C-4), 77.1 (C-5), 61.8 (C- 6 ). ElMS m/z (rel. int.): 179 (2), 105 (36), 85 (5), 79 (100), 57 (87), 45 (51), 29 (73).

methanol. TLC (SiO;): Rf 0.51 (CHCl 3 -MeOH-HAc 2:2:1). 'HNMR (CDaOD-dJ:

55.10 (IH, d, J=4, H-1 a), 4.48 (IH, d, J= 8 , H-IP), 3.15-3.85 (sugar’s protons, overlapped). ElMS m/z (lel.int): 181 (M*-l, 2), 161(3), 144(3), 126 (14), 109(13), 91(13), 73(100), 57(90).

148 Figure 5.3 The compounds isolated from Clerodendrum mandarinorum

HO HO

compound 15 compound 18 compound 19

compound 23 HO HO

compound 16 R=CH3 compound 20 :Rl=R2 = 0CH3 compound 17 R=COOH R3=R4=H, R5=0H

compound 21:Rl=R2=OCH3 R3=R4=H, R5=0Glc

compound 22:R1=0H, R2=H CH2CH2O—Xyl—Rham

compound 24

HOH2Ç CH 2 OH OH HOH 2 Ç OH CH2 OH OH C2H5O •Ho h r I CH2OH compound 25 C 2 H 5 OH OH OH OH compound 27 R,, compound 26 OH

H compound 28 R= ""OH

compound 29 R= OH

149 5.4 Biological tests on isolated compounds and related compounds The isolated compounds except 15 ,28 and 29, and seven structural related flavonoids, namely luteolin, chrysoeriol, chrysosplenol-D, chrysoplenetin, casticin, sorbifilin-4’- glucose and diosmetin-7-rhamnosyl-glucose (structures see Figure 5.4, p.l51), were screened by ligand receptor binding assays following the approach described in Section 2.8 and 2.9. The receptors tested included a l, a2, p-adrenoceptors, 5HT1A, 5HT1C, 5HT2, dopamine 1, dopamine 2, adenosine 1, histamine 1, Na^/K^ATPase, Ca^^-channel, K^-channel, opiate, muscarinic (for all the compounds above), and GABA^ and GABAg (for compounds isolated in this study). Of the nineteen compounds tested, compounds 16, 21 and 25 were found to bind to the K^-channel, adenosine 1 and muscarinic receptors, respectively. The IC 5 0 values of these three compounds are listed in Table 5.5.

Table 5.5 The IC 5 0 values of tested compound

Compound Receptor IC 5 0 * (pM) ± SEM N

compound 16 K^-channel 7.5 ± 1.4 3

compound 2 1 adenosine 1 3.0 ± 0.8 6 compound 25 muscarinic 5.5 ± 1.4 3

* The IC 5 0 values of control compounds in the present study were 0.79 nM for glibenclamide (K^-channel receptor), 0.16 nM for atropine (muscarinic receptor) and 14.1 nM for CHA (adenosine 1 receptor).

Seven compounds (16, 17,19, 20, 21, 25, 26, structures see Figure 5.3, p. 149) were further tested to investigate if they interacted with the control compounds used in each ligand binding assay against various receptors. The binding assays of a l, a2, p- adrenoceptors, Ca^^-channel, K^-channel, dopamine 1, dopamine 2, adenosine 1, histamine 1, opiate, 5HT1A, 5HT1C, 5HT2 receptors were selected for this study. The experimental details were described in Section 2.9, Chapter 2. The IC 5 0 values of those control compounds (whose IC 5 0 values were changed) and the control compounds with added the individual isolated compounds are listed in Table 5.6 (p. 152). The IC 5 0 curves of these compounds are shown in Figure 5.5(p.l53).

150 Figure 5.4 Some flavonoids tested by ligand-receptor binding assays

HO HO .OCH HO

HO. R h a m — G l c

H O O OH

luteolin diosmetin-7-Rhamnosylglucoside

. 0 0 Ic .OCH

HO' OH OH

sorbifolin-4'-Glucose casticin

HO

,H0

HO.

HO OH

chrysoeriol chrysoplenetin

,H0

OCH

HO

chrysosplenol-D

151 Table 5.6 The IC 50 Values of Control Compounds and the Control Compounds with Added Isolated Compounds

Compound Receptor' IC5o(nM)±SEM IC5o(nM)±SEM IC%)(nM)±SEM N

(Control) (Iso.Compd.)^ (Ctr.+Iso)^

16 opiate 1.02 ± 0.17 4 0.10 ± 0.02 3

- 17 K+ 0.79 ± 0.05 1.00 ± 0.07* 3

A l 14.1 ± 2.31 3.98 ± 0.29* 3

- 19 K+ 0.79 ± 0.05 0.70 ± 0.08* 3

- 20 Ca'+ 2.00 ± 0.32 1.00 ± 0.09* 3

K+ 0.79 ± 0.05 0.40 ± 0.02* 3

A l 14.1 ± 2.31 10.0 ± 1.74* 3

- 21 opiate 1.02 ± 0.17 0.11 ± 0.03 3

A l 14.1 ± 2.31 ■ 10.0 ±1.17* 3

HI 7.41 ± 0.77 1.29 ± 0.30 3

- 25 a2 5.67 ± 0.47 1.00 ± 0.12 3 P 1.41 ± 0.10 10.0 ±1.16* 3

5HT1A 1.00 ±0.12 ■ 0.14 ± 0.09 3

HI 7.41 ± 0.77 0.13 ± 0.05 3

- 26 «2 5.67 ± 0.47 1.00 ± 0.08 3 P 1.41 ± 0.10 10.0 ± 1.28* 3 opiate 1.02 ± 0.17 0.14 ± 0.06 3

HI 7.41 ± 0.77 1.00 ± 0.45 3

1: receptor a2, p- a2, p adrenoceptor subtypes, - Ca^^, ion channel receptors, Al- adenosine 1 receptor, HI- histamine 1 receptor. 2: isolated compound 3: control compound + isolated compound 4: inactive *: the binding site was also influenced, see also Figure 5.5

152 Figure 5.5 IC50 Curves of control compounds and the control compounds with added natural products

CHA Displacement (Adenosine 1 Receptor) 100

■ CHA

* CHA* Compound 1 7

50 g

[Displacer] nM

Glibenclamide Displacement (K+-channel Receptor) 100

■ Glibenclamide

g

2

-2 [Displacer] nM

Glibenclamide Displacement (K+-channel Receptor) 100

■ Glibenclamide

*• Glib + Compound 19

I 5 0 g I X

-2 [Displacer] nM

153 Glibenclamide Displacement (K+-channei Receptor) 100

■ Glibenclamide

Glib + Compound 20

m

50 §

-2 0 1 2 3 [Displacer] nM

Nitrendipine Displacement (Ca++-channel receptor) 100

■ Nitrendipine

A Nitre.+Compound 20

œ

50

-2 [Displacer] nM

CHA Displacement (Adenosine 1 Receptor) 100

- CHA I A CHA+Compound 2 0 m

ina 5 0 g I

[Displacer] nM

154 CHA Displacement (Adenosine 1 Receptor) 100

• CHA

CHA+Compound 21 m

50 g

[Displacer] nM

DMA Displacement (B-adrenoceptor) 100

propranolol pro.*-Compound 25 m

50

§

[Displacer] nM

DMA Displacement (0-adrenoceptor) 100

propranolol prOi+Compound 26

50 §

[Displacer] nM

155 5.5 Discussion 1. The constituents isolated from Clerodendrum mandarinorum are all non-nitrogen containing. Their structures differ from the nitrogen-containing synthetic compounds used clinically for the treatment of the CNS disorders. Compounds 16, 21, 25 were mildly active against K^-channel, adenosine 1 and muscarinic receptors (IC 5 0 3.0-7.5 pM, Table 5.5, p. 150). In interaction experiments, 17,19, 20 had marked influence on ion channel receptors (Ca^^, K^-channel receptors), reducing the binding sites of the receptors (Figure 5.5, p. 153). These findings suggested that the principles isolated from this plant may exert their effects either by the activities at high concentration or by influence on the binding of other compounds (endogenous or exogenous) to the receptors. The further function assays are needed in order to understand the mechanism of action of these natural products.

2. The root bark extract of C. mandarinorum showed a strong binding to adenosine 1 receptor. Following the separation of active fractions, compound 21, a flavonoid glucoside, was found to be the active constituent (IC 5 0 3.0 pM, Table 5.5, p. 150). It is of interest that all known adenosine agonists are closely related to adenosine itself, and very few modifications to its basic structure are allowed. (Galen, et al, 1992). For the adenosine antagonists, they are generally planar, aromatic (or having a high n- electron density), nitrogen-containing heterocycles. The most potent receptor sensitives are 6:5-fused bicyclic or 6:6:5-fused tricyclic heterocycles. The naturally occurring benzo[b]furan is an only exception of this general rule which contains an O- rather than a N- 6:5-fused heterocycle, yet maintains considerable potency (Ki 17nM) in binding to the adenosine 1 receptor (Galen et al, 1992). The structure of compound 21 has some similarity to benzo[b]furan (Figure 5.6). It has not been shown previously

that flavonoids are able to bind with adenosine 1 receptor and compound 2 1 is first one to be observed with this activity. The structure of compound 21 is specific to Al receptor since none of the other eight flavonoids tested were active, even for sorbifolin-4’-glucose whose structure is very close to compound 21. In interaction experiments, compound 21 showed an effect on the binding of CHA (an agonist of Al receptor) to the receptor, and this fact may be related to its activity at Al receptor at high concentration.

156 OMe .OGlc OMe

RO' OH O compound 21 R=CH3 sorbifolin-4'-Glucose R=H benzo[b]furan

Figure 5.6 Structures of benzo[b]furan, compound 21 and sorbifolin-4-glucose

3. The flavonoids are one of the largest groups of natural products. Several flavonoids are obtained in the present study and they are also present in many species of Chinese herbs. The diverse pharmacological activities of flavonoids have been described (Havsteen, 1983). Activities include their effects on the synthesis of , on various enzyme systems and effects as anti allergic, anti-inflammatory, and anti­ asthmatics. In the present study, two flavonoids (compound 20, 21) have been tested for their interaction effect on the control compounds, and both of them showed the influence on specific receptors, such as histamine 1, adenosine 1, Ca^^-channel and K^- channel receptors. The synergistic effects of flavonoids have been found in a number of previous studies. For instance, it was reported that some flavonoids (eg. quercetin) were able to potentiate or to inhibit the release of histamine by rat peritoneal mast cell induced by a compound (called compound 48/80) according to the concentration used. (Amellal, et al, 1985). It was also found that the antimalarial activity of (qing haosu) was markedly enhanced by the presence of methylated flavones such as artemetin and casticin from Artemsia annua L. (Yang, et al, 1988). It is not inconceivable that flavonoids may act through a variety of different mechanisms (eg. on membrane ATPase, or on prostaglandins synthetase). Low activity and large doses needed are common disadvantages of the flavonoids, on the other hand, they have generally lower toxicity and offer a wide variety of chemical structures. The rational utilization of this group of compounds, especially for their synergistic effects, may be beneficial to new drug development. Moreover, the investigation into the functions of

157 flavonoids at the molecular level will further increase our understanding of their actions in herbal medicines.

4. Five oligosaccharides have been isolated from this plant and they are the major group of compounds present in the highly polar fractions. Meanwhile, the saccharides are also the main constituents of the herb decoction. Compound 25 showed weak activity against the muscarinic receptor (IC 5 0 5.5 pM, Table 5.5, p. 153) and compounds 25 and 26 exhibited interactions on the control compounds binding to histamine 1, 5HT1A and a2-adrenoceptor(Table 5.6, p. 152). Glucose is the basic unit of these compounds which is almost the sole substance for cerebral oxidative metabolism (Bachelard, 1978; Sokoloff, 1977). Therefore, further investigation of the functions of saccharides may help understand the action mechanism of the plant decoction.

158 Chapter 6

Alangium plantanifolium

159 6.1 Introduction Alangiaceae belongs to the dicotyledons and contains only one genus, Alangium, in the family. There are about 30 species spreading from Africa to Japan and Fiji islands

(How, 1984). There are 8 species in China mainly growing in the south of the country (How, 1984). The most common species in China is Alangium chinense and it has been used as a medicinal herb for the treatment of rheumatism, numbness of the limbs and injuries due to impact, fractures, contusions and strains (Zheng, 1986). The fibrous roots and root barks of A. chinense contain alkaloids, glycosides, phenolic compounds, amino acid, cardiac glycosides and resin (Zheng, 1986). There are several phytochemical reports on Alangium species, a summary is listed in Table 6.1 and the major types of the compounds isolated are given in Figure 6.1. There are several pharmacological studies on A. chinense showing that the decoction of the fibrous roots of A. chinense had the effects of muscle relaxant, hypnotic, stimulating respiration, adjusting blood pressure (lowing blood pressure of normal rabbits and raising that of anaesthetized dogs) and heart contractility (increasing the cardiac contractility of isolated rabbit heart, but reducing the effect by increased dosage),( references cited in Zheng, 1986). A. plantanifolium (Sieb. et. Zucc.) Harms, is a species growing in the southwest of China. There are no previous pharmacological or phytochemical reports on this species. In order to investigate the medicinal resource of Alangium, this species was selected for the present study. The biological screening of the plant extract was undertaken before the chemical study to search for CNS active compounds.

6.2 Biological screenings of extract fractions The 70% ethanol root bark extract of A. plantanifolium showed strongly binding to 5HT1,5HT2, opiate, adenosine 1, Ca^-channel, histamine 1, dopamine 2, GABA^ and GABAg receptors (Figure 3.1, p.83). The first six binding assays were adopted for the sreenings in its fractions, since the test tissue was easy to obtain. The fractions were prepared by column chromatography in order of polarity and the screening approaches used were the same as described in Section 2.7. The results of these screenings are shown in Table 6.2.

160 Table 6.1 Principles isolated from Alangium species species constituent reference A. lamarckii alamarckin, (-)alamarine, alagamide Pakrashi, et al, (-)alancine, (+)alangicine, alangiside 1967; 1971; 1985 (-)alangimarckine, (R)alagimaridine, Bhattacharjya, et al, alangimarine, alangimarinone, 1988; alangine A, alangine B, alangium Chattopadhyay, et alkaloid AL64, (-)ankorine, benzoyl- al, 1984; phenylalaninol, bharatamine, Fujii, et al, 1976; (-)cephaeline, isoalamarine, (-) 1977; 1983; 1987; demethylprotoemetinol, (-)emetine, Kapil, et al, 1971; demethylcephaeline, psychotrine, Shoeb, et al, 1975; isoalangimarine, isocephaeline, Sawayama, et al, 9-demethylprotoemetinol, 1989;

1 0 -demethy Iprotoemetinol, (+)-9-demethylpsychotrine,

1 0 -demethyltubulosine, alangidinol, (-)deoxytubulosine, isoalangidiol, dihydroalamarine, (-)tubulosine, isoalangimarine, isotubulosine, protoemetinol, (+,-)-1,2,3,4- tetrahydro-6-hydroxy-7-methoxy-1 - methylisoquinoline, stigmasta- 5,22,25-trien-3P-ol

A. salviifolium (-)ankorine, cephaeline, psychotrine Pakrashi, et al, 1967

A. vitiense 9-demethyltubulosine Ohba, et al, 1980

A. villosum deoxyemmolactone, Burbage, et al, 1970 isodeoxyemmolactone

A. plantinifolium. alagifolioside Otsuka, et al, 1989 var trilobum

A. chinense di-anabasine Zheng, 1986

161 Figure 6.1 The diversity of compound structures in Alangium species

OH

HO' HO' isoalangidiol stigm asta-5,22,2 5- t r i e n - 3 - o l

.....OH OGlc COOH

OH

alanggifolioside deoxyemmolactone

OH MeO. MeO'

CH3 HO' MeO'

‘COOH

alamaridine a la n c in e MeO.

HO'

H ÇH2OH

H

OGlc CH2Ph

a la n g is id e benzoylphenylalaninol

162 6.3 Isolation and Structure Identification of the Isolated Compounds Experimental detail is given in Sections 2.1-2.5 of Chapter 2. The separation procedure is summarized in Figure 6.2. From the root bark of A. plantanifolium, five compounds have been isolated and identified (30-34). They are two steroids, a steroid glucoside, a cyclohexenol glucoside and a bisaccharide. The structure identifications of these compounds are described as following sections.

Figure 6.2 Isolation of compounds from Alangium plantanifolium

70% EtOH Root Bark Extract ( 8g )

CC (Si0 2 )

Fr. 1-2 Fr. 3-5 Fr. 6-7 Fr. 8-11 Fr. 11-18 (0.5g) (O.Sg) (0.5g) (0.5g) brown syrup (4g) Chlorophyllcompound 3 0 compound 32 compound 33 (10 mg) (20 mg) (2 0mg) compound 31 HPLC (10 mg) compound 3 4 (20 mg)

1. petroleum spirit, 2. C H C I3 , 3. CH3OH-CHCI3 1:9 4. C H 3 O H -C H C I3 2:8, 5. C H 3 O H

163 Table 6.2 The fraction screening results ofAlangium plantanifolium

Fr.NRep. Ca++ * H1 * 5HT1 5HT1A 5HT2 A1 * opiate

Total Ext. 4- 4- 4-4- 4- 4-4- 4-

Fr.l *** ++ 4-

Fr.2 ++ 4- 4-

Fr.3 + 4- Fr.4

Fr.5 4- 4-

Fr. 6

Fr.7 4-4-

Fr. 8 ++ 4-4- 4-

Fr.9 ++ 4-4- 4- 4- 4-4-

Fr.lO 4- 4-4- Fr.ll

Fr.l 2 ++ 4- Fr.l 3 ++

Fr.l4 4-4- 4-

Fr.l 5 4-4- 4- 4-4-

Fr.l 6

Fr.l 7 4- 4-4-

Fr.l 8 ++

*: Ca^^-Ca^ ion channel receptor, HI-histamine 1 receptor, A1-adenosine 1 receptor

**: "+"- %Inhibition of ligand’s specific binding between 50-79%

"++"- %Inhibition of ligand’s specific binding between 80-100%

***: fractions see also Figure 6.2

164 6.3.1 Identification of P-sitosterol (30), stigmasterol (31) and sitosterol-3-O-P-D- glucopyranoside (32) III,, III,,

HO’

compound 30 R=H compound 31

compound 32 R=Glc

Compound 30 (p. 167 for data) was obtained as a white crystalline solid. The EIMS spectrum showed the molecular ion peak at m/z 414 and the fragmentation was identical with that of P-sitosterol. ‘HNMR indicated the presence of one olefinic proton (Ô 5.35, d, J=4), two tertiary methyl groups (Ô 0.68, 1.02), four overlapped methyl signals between Ô 0.80-0.95 and a characteristic peak of H-3 proton at 5 3.55 (IH, m). These signals were identical to those of p-sitosterol. The *^CNMR data were consistent with those given in reference (Kojima et al, 1990) for p-sitosterol and the compound was further substantiated by co-TLC with the authentic sample.

Compound 32 (p. 168 for data) The ‘HNMR spectrum of the compound exhibited that

it was a sterol glucoside with an anomeric proton at 6 4.42 (IH, d, J=7). Except for the sugar’s signals the spectrum was similar to that of compound 30, suggesting that the compound was sitosterol-3-O-p-D-glucoside. Hydrolysis of 32 gave glucose as the sugar moiety and the EIMS spectrum showed a peak of M^-glucose at m/z 414. The ’^CNMR spectral data were identical with those given in the reference (Kojima, et al, 1990), further substantiating that the glucose was p-equatorially oriented (5 79.0, C-3). Therefore the compound is sitosterol-3-O-p-D-glucopyranoside.

Compound 31 (p. 167 for data) The ^HNMR and ‘^CNMR spectra of the compound indicated that it was also a sterol, but had three olefinic protons (Ô 5.35, IH, d, J=4;

165 ô 5.15, IH, dd, J=15, J=9; ô 5.02, dd, J=15, J=9) and four double bond carbons at ô 140.7, 121.7, 138.3 and 129.2. The EIMS give the molecular weight at m/z 412 and a fragment at m/z 273 suggested that one double bond was in the parent nucleus and the other one was on the side chain (Cong, 1987). Based on these results and compared with the references (Kojima et al, 1990; Wright, 1978), the compound is stigmasterol which was further substantiated by comparison with the authentic sample (co-TLC).

6.3.2 Identification of 5|3, 6P-hydroxyI-2,3-ene-cycIohexanol-l-p-D-gIucoside (33)

GlcO 5^H0 HO

Compound 33 (p. 171 for data) was a white amorphous powder, and soluble in DMSO. The ‘HNMR exhibited the presence of two olefinic protons at Ô 5.55 (IH, | m) and

6 5.49 (IH, m), one CH 2 group (5 2.05, IH, m, 5 2.47, dd, J=14, J=2), one anomeric proton of sugar (Ô 4.30, IH, d, J= 8 ) and nine proton signals of CHOH. In '^CNMR spectrum, there were two double bond methine carbons (Ô 133.0, 126.5), one methylene carbon (Ô 33.59), three CHOH carbons and a set of glucose signals. The FARMS indicated the molecular weight at m/z 292 and M^-glucose at m/z 130. The high resolution MS gave the accurate mass at m/z 315.1052 for the formula (^izHzoOgNa (required 315.1056). All these results suggested that the compound was a cyclohexanol with a double bond, two free hydroxyl groups and one glucosylated hydroxyl group. In *H-‘H COSY spectrum, the methylene protons (H-4) were coupled

with one olefinic protons at 6 5.65 (H-3) and a proton at Ô 3.70 (H-5), the other olefinic proton (5 5.49, H-2) was coupled with the protons at Ô 5.65 (H-3) and 3.10 (H-1). The glucose was linked to the aglycone via the hydroxyl group at C-1 since the glucose protons at C’-2 and C -5 had weak coupling with the proton at C-2 (Ô 5.49). The glucose and two hydroxyl groups were all equatorially oriented as suggested by their chemical shifts of the carbons (Ô 79.2, 79.4, 79.7). The compound is then determined as 5p, 6p-hydroxy-2,3,-ene-cyclohexanol-1 -p-D-glucoside.

166 6.3.3 Identification of p-D-fructofuranosyl-(l-4)-a-D-fructopyranoside (34)

0 HO

HO CH2OH

Compound 34 (p. 172 for data) The 'HNMR spectrum indicated that the compound was composed of sugar units without an anomeric proton and all signals were overlapped between Ô 3.45-4.10. The '^CNMR spectrum exhibited the presence of two quaternary carbons at 8 103.4, 99.5, six CHOH carbons 8 83.4, 77.4, 76.9, 72.1, 71.5,

69.6 and four CH 2 OH carbons at 8 65.6, 64.4, 64.4, 62.9 indicating that two six- carbon sugar joined together. In CIMS, the clear peaks at m/z 180 and 149

(I 8 O-CH2 OH) suggested the presence of the fructose-like structure. Comparing with the '^CNMR data of p-D-fructofuranose, a-D-fructofuranose, a-D-fructopyranose and p-D-fructopyranose (Bock, et al, 1983), one of the sugar residuals of 34 is identical to that of p-D-fructofuranose except for the chemical shift of C-1 being 2.5 ppm downfield and the other one was consistent with that of a-D-fructopyranose with a 1.6 ppm downfield shift of C-4. These data suggested that the compound was composed of a-D-fructopyranose and p-D-fructofuranose linked via the C-1 of p-D- fructofuranose to the C-4 of a-D-fructopyranose. The hydrolysis of compound 34 gave fructose only as the product. Therefore, the compound is determined as P-D- fructofuranosyK 1 -4)-a-D-fructopyranoside.

6.3.4 Spectral data

p-sitosterol (30): w hite crystals, soluble in C H C I3. T L C (Si0 2 ): Rf 0.75 (CH Clg-EtO A c

9:1). 'H N M R and '^C N M R (CD CI3) see Table 6.3 a n d T a b l e 6.4. EIMS m/z (rel. in t.) 414 (M+, 53), 399 (30), 396(63), 381(11), 329(22), 303(23), 275(6), 273(17), 271(6), 255(21), 231(14), 213(20, 246(5), 229(8).

Stigmastero/(31): w hite crystals, soluble in C H C I3 . TLC (SIO2 ): Rf 0.63 (C H C I3 -

167 EtOAc 9:1). ‘HNMR and ‘^CNMR (C D C I3 ): see Table 6.3 and Table 6.4. EIMS m/z 412 (M+ ,85), 397(5), 394(6), 379(7), 369(28), 273(22), 271(72), 255(100), 246(4), 231(18), 229(19), 213(41).

Sitosterol-3-0-^-D-glucopyranoside (32): white amorphous powder, soluble in DMSO. TLC (SiOz ): Rf 0.43 (CHClg -MeOH 9:1). ' HNMR and ‘^CNMR (C D C I3 ): see Table 6.3 and Table 6.4. EIMS m/z (rel. int.): 414 (M^ -glucose, 9), 399(13), 396(25), 381(10), 329(6), 303(6), 275(8), 273(7), 271(5), 255(7), 246(3), 231(8), 229(7), 213(10).

168 Table 6.3 HNMR spectral data of compounds 30, 31, 32

28

24 26

14

RO

compound 3 0 R=OH compound 31 R=OH, A 22 compound 3 2 R=OGlc

H No. 30 31 32 3 3.55, (m) 3.55, (m) 3.55, (m)

6 5.35, (d= 6 ) 5.35, (d, J=5) 5.39, (m)

Me-18 0 .6 8 , (s) 0.69, (s) 0.69, (s)

Me-19 1 .0 2 , (s) 1 .0 1 , (s) 1 .0 1 , (s)

Me-21 0.92, (d, J= 6 ) 1.02, (d, J= 6 ) 0.94, (d, J= 6 )

2 2 5.15, (dd, J=15, J=9) 23 5.01, (dd, J=15, J=9)

Me-26 0.83, (d, J= 6 ) 0.85, (d, J= 6 ) 0.84, (d, J= 6 )

Me-27 0.82, (d, J= 6 ) 0.80, (d, J= 6 ) 0.82, (d, J= 6 )

Me-29 0.85, (t, J=7) 0.81, (t, J=7) 0.85, (t, J= 8 ) Glc-1 4.42, (d, J=7) Glc-2 3.21, (t, J=9) Glc-3 3.40, * (m) Glc-4 3.30, * (m) Glc-5 3.35, * (m)

Glc- 6 3.72, (dd, J=10, J=4) 3.85, (dd, J=10, J=3)

The data was obtained from 400 MHz NMR in C D C I3 . *: the assignment interchangeable.

169 Table 6.4 "CNMR spectral data of compound 30, 31, 32

No. 30 31 32 32 sugar aglycone No. C

1 37.5 37.5 37.2 r 1 0 1 . 1

2 31.9 31.9 29.5 2 ’ 73.6 3 72.0 71.8 79.4 3’ 76.5 4 42.6 42.3 38.6 4’ 70.2 5 141.0 140.7 140.3 5’ 76.0

6 1 2 2 . 0 121.7 1 2 2 . 0 6 ’ 61.7 7 32.2 32.0 31.9

8 32.1 31.9 31.9 9 50.4 50.2 50.2

1 0 36.7 36.5 36.7

1 1 21.3 2 1 . 2 2 1 . 0

1 2 40.0 39.7 39.8 13 42.5 42.2 42.3 14 57.0 56.8 56.8 15 24.6 24.5 24.2 16 28.5 28.7 28.2 17 56.3 55.9 56.0

18 1 2 . 1 1 2 . 0 11.7 19 19.7 19.4 19.2

2 0 36.4 40.5 36.1

2 1 19.0 2 1 . 2 18.6

2 2 34.2 138.3 33.9 23 26.3 129.2 26.0 24 46.1 51.2 45.9 25 29.4 31.9 29.1

26 2 0 . 1 2 1 . 2 19.6 27 19.3 19.0 18.6 28 23.3 25.4 23.0

29 1 2 . 2 12.3 1 1 . 8

The data was obtained by 400 MHz NMR spectroscopy in CDCI 3

170 5^,6^-hydroxy 1-2,3-ene-cyclohexanol-l-^-D-glucoside (33): white amorphous powder soluble in DMSO. TLC (SiO^): Rf 0.45 (CHCl^-MeOH 8:2). ‘HNMR (C D 3 O D ) and

'^CNMR (CD3OD): see Table 6.5. HRFABMS m/z 315 (M^+Na, measured 315.1052, required 315.1056), 130 (M^-glucose).

Table 6.5 ‘HNMR and "CNMR spectral data of compound 33

HO HO

No aglycone No Glucose C HCH

1 79.7 3.10, (d, J= 8 ) r 103.7 4.30, (d, J= 8 )

2 133.1 5.49, (d, J=10) T 74.8 3.00, (t, J=9) 3 126.5 5.65, (t, J=3) 3’ 78.2 3.15, (m) 4 33.6 2.47, (dd, J=4, 2) 4’ 72.7 3.38, (t, J=10) 2.05, (m) 5 79.2 3.70, (m) 5’ 75.8 3.62, (m)

6 79.5 3.40, (m) 6 ’ 63.8 3.30, (t, J=10) 3.95, (br. s)

Data was obtained from 400 MHz NMR in DMSG-d^.

171 p-D-jructoJuranosyl(l-4)-OL-D-fructopyranoside (34): yellow pale amorphous powder,

soluble in MeOH. TLC (SiOj: Rf 0.37 (CHCl^-MeOH 7:3). ‘HNMR (CD3 0 D-d 4 ): Ô

3.45-4.10 (14H, overlapped. ‘^CNMR (CD 3 0 D-d 4 ): see Table 6.4. CIMS m/z (rel. int.):

M+(absent), 198(7), 180(30), 167(50), 162(16), 149(11), 145(14).

Table 6 . 6 CNMR data of compound 34

HO , HO HOH2 C

1 '

No. p-D-fructofuranose No. a-D-fructopyranose

1 6 6 . 1 r 64.8

2 103.4 2 ’ 99.5

3 77.8 3’ 69.4

4 76.9 4’ 72.1

5 83.4 5’ 71.5

6 62.6 6 ’ 64.8

172 6,4 Biological tests on isolated compounds The isolated compounds (30-34) were tested by the radio-ligand receptor binding assays following the approaches described in Sections 2.8 and 2.9. The receptors tested included al, a2, p-adrenoceptors, 5HT1A, 5HT1C, 5HT2, dopamine 1, dopamine 2, adenosine 1, histamine 1, Na^/K^ATPase, Ca^^-channel, K^-channel, opiate, muscarinic GABA^ and GABAg. Compounds 30 and 33 were found to bind to the muscarinic receptor and the IC 5 0 values were listed in Table 6.7.

Table 6.7 The results of compound screenings

Compound Receptor IC5 0 (pM) ± SEM N compound 31 muscarinic 8.52 ±2.13 3 compound 34 muscarinic 6.74 ± 0.75 3

* The IC 5 0 value of control compound is 0.16 nM for atropine..

Compounds 30-34 were further tested to investigate if they had any interactions with the control compounds against the various receptors. The binding assays including a l, a2, P-adrenoceptors, Ca^^-channel, K^-channel, dopamine 1, dopamine 2, adenosine 1, histamine 1, opiate, 5HT1A, 5HT1C, 5HT2 receptors were selected in this study.

The experimental details were the same as described in Section 2.9. The IC 5 0 values of these control compounds alone and the control compounds with the added individual isolated compounds are listed in Table 6 .8 . The IC 5 0 curves were also established for the control compounds whose binding sites had been changed by the the isolated compounds.

173 Table 6.8 The IC 50 Values (nM) of Control Compounds and the Control Compounds with Added Isolated Compounds

Compound Receptor' ICjoCnM) ± SEM IC5o(nM) ± SEM ICgo(nM) ± SEM N

(control) (iso. compd)^ (ctr. +iso)^

30 A l 14.1 ± 2.31 4 1.00 ± 0.38* 3

32 opiate 1.02 ± 0.17 - 0.14 ± 0.04 3

5HT1A 1.00 ± 0 .1 2 - 0.06 ± 0.01 3

5HT1C 70.8 ± 5.26 - 3.16 ± 0.43 3

A l 14.1 ± 2.31 - 1.00 ± 0.27* 3

33 D1 3.10 ± 0.23 - 0.75 ±0.18 3

1: Receptor D1 - dopamine 1, Al- adenosine 1

2: isolated compound

3: control compound plus isolated compound

4: inactive

*: the receptor binding sites were also changed (see also Figure 6.3)

174 Figure 6.3 IC5 0 Curves of Control Compound and the Control Compounds

with Added Natural Products

CHA Displacement (Adenosine 1 Receptor) 100

■ CHA

CHA+ compound 30 m

50

1 0 2 3 4 [Displacer] nM

CHA Displacement (Adenosine I Receptor) 100

• CHA

CHA+ compound 32

i zc

[Displacer] nM

175 6.5 Discussion 1. The compounds isolated from A. plantanifolium are two steroids, a steroid glucoside, a cyclohexenol glucoside and a bisaccharide. Alkaloids have been reported to be present in A. lamarckii and A. chinense and they were found to be the active principles (Pakrashi, et al, 1971; 1985; Zheng, 1986). However, in the present studies, an extraction procedure specific for alkaloids has been undertaken and it was found that the content of alkaloids in this plant was so low (less than 0 .0 0 1 %) that they could not be isolated following the separation procedure listed in Figure 6.2. This fact suggested that active constituents found in the bioassays of the extract may be non­ alkaloid compounds.

2. Of those five compounds isolated from the plant, compounds 30 and 33 were found to bind to the muscarinic receptor (IC 5 0 8 . 5 and 6.7 pM, respectively. Table 6.5). This was consistent with the finding that the decoction of the fibrous roots of A. chinense had significant muscle relaxant effect in dogs (Anno, 1971, 1972). In the interaction experiments, 30 and 32 showed the same effects on the adenosine 1 receptor reducing the IC 5 0 value of CHA by more than 10 times (calculated from the data in Table 6 .8 , p. 174). In addition, compound 32 had the effects on 5HT1 receptors reducing the IC 5 0 values of control compounds for 5HT1A and 5HT1C receptors by more than 20 times (the data calculated from Table 6 .8 ). These behaviours were consistent with the finding that the ethanol extract of A. chinense potentiated the effect of hypnotics, but did not induce hypnosis by itself (Anno, 1980). Compounds 30 and 32 are structurally related, ie. 30 is the aglycone of 32. It is of interest to note that 30 (P-stitosterol) and its glucoside (32) induce effects in the central nervous system. These finding also provide the explanation for the herb’s use as muscle relaxant and hypnotic.

176 Chapter 7

Uncaria rhynchophylla

177 7.1 Introduction Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) is one of the species listed in the Chinese Pharmacopoeia. It is used as an antipyretic and anticonvulsant for the treatment of headache, vertigo and epilepsy (Anon., 1992). The medicinal part of the plant is the branches bearing hooks. The related species in the genus, namely U. macrophylla Wall., U. hirsuta Havil., U. sinensis (Oliv.) Havil. and U. sessilifructus Roxb. also have the same medicinal uses.

There are a number of phytochemical studies on the plants in the genus. The species which have been investigated include U. attenuala, U. bernaysii, U. callophylla, U. canescens, U. ferrea, U. florida, U. formosana, U. gambier, U. kawakami, U. macrophylla, U. oritentalis, U. petreopoda, U. rhynchophylla, U. sinensis, U. thwaitesii, U. tomentosa, etc. ( Raymont-Harmet, 1936; Kondo, et al, 1941; 1942; Nozoye, 1957; 1958; Yeoh, et al, 1966; Beecham, 1967; Pousset, et al, 1967; Beecham et al, 1968; Chan, 1969; Aimi, et al, 1977; 1982; 1989; Phillipson et al, 1973; 1975; 1978; Phillipson and Hemingway, 1975; Herath, 1978; Iwu, et al, 1979; Nonaka, et al, 1980; Yamanaka, et al, 1983; Tanaka, et al, 1983; Goh, et al, 1985; Martin, et al, 1986; Amone, et al, 1987; Liang, et al, 1990; Aquino, 1990; Liu and Feng, 1993). More than a hundred compounds have been isolated, most of them are indole alkaloids, triterpenoids, flavonoids and phenols. The structures of some alkaloids isolated from U. rhynchophylla are listed in Figure 7.1.

The total alkaloid fraction from U. rhynchophylla was found to have a hypotensive and sedative effects in animal tests (Chang, et al, 1978; Che, et al, 1965). Hence, U. rhynchophylla has been selected in the present studies to investigate CNS active principles.

178 Figure 7.1 Some compounds isolated from Uncaria rhynchophylla

CH2CH3

O2 CH3

corynoxan isorhynchophylline rhynchophylline

•CH-CHz

O2CH3 CO2CH3

corynoxeine 1 socorynoxeine hirsuteine

'"CH— CH2 "CH2Chb CH; O2CH3 O2CH3

hirsutine corynantheine dihydrocorynantheine

HO,

COOCHi COOGH3

corynan OH geissoschizine OH rhynchophine methyl ether

(Kondo, et al, 1928; 1937; Nozoye, 1957; 1958; Yamanaka, et al, 1983; Aimi, et al, 1973; 1982; Iwu, et al, 1979; Damak, et al, 1976)

179 7.2 Biological screenings of extract fractions The 90% methanol extract of stem with hooks of U. rhynchophylla showed strong binding to the a l, a2-adrenoceptors, 5HT1A, 5HT2, opiate, GABA^ and GABAg receptors (Figure 3.1, p.83). These receptor ligand binding assays (except for GABA^ and GABAg assays) plus ion channel receptors (Ca^^ and K^) and histamine 1 receptor were adopted for the fractional screenings. The crude extract was fractionated successively with chloroform, ethylacetate, methanol, 50% methanol and water. The details of fractionation are presented in Figure 7.2. The biological screening approach used was the same as described in Section 2.7. The results of the fraction screening are shown in Table 7.1.

7.3 Isolation and Structure identification of the isolated compounds The isolation procedure is summarized in Figure 7.2. The compounds were mainly separated by repeated flash chromatography (Si 0 2 ) and the purification was carried out on sephadex (LH-20) columns and HPLC. From the stems with hooks of U. rhynchophylla, eight compounds were isolated and identified (35-42) including two indole alkaloids, one triterpenoid, one flavonol and four flavanols. The structure determinations of these compounds are described in the following sections.

180 Figure 7.2 Isolation of compounds fromUncaria rhynchophylla

Crude Drug (stem with hooks)

pulverized

120g boil water 90% MeOH E x t r a c t (1) (O.lg)

Marc MeOH Ext. (2) (7g) 5 0% MeOH

Marc MeOH Ext.(3) (2g) CHC13 EtOAc BuOH

CHC13 Ext. (4) EtOAc Ext. (6) BuOH Ext. (8) (1.5g) (2g) (2.5g) CHC13

CHC13 Ext.(5) EtOAC Ext.(7) (0.2g) (0.5g)

Fr.C86-C130 sephadex (0.5g) Fr.E1-E30 Fr.E31-E47 column (Ig) (ig) Fr.Cl-C28 Fr.C55-C85 (0.2g) HPLC (0.3g) sephadex compound column 42(2mg)

compound 35(30mg) Fr.C29-C54 (0.5g) compound 3 8 (lOmg) compound 36(5mg) compound 3 9 (5mg) compound 37(6mg) compound 40 (3mg) compound 41 (5mg)

181 Table 7.1 The results of fraction screenings of Uncaria rhynchophylla

Fr. ***\ Rep. a l* a2* 5HT1A 5HT2 opiate Ca"+ * K+* Hl*

H2 O Ext.(l) 4-4- 4 - 4 -

MeOH Ext.(2) 4- 4- 4 -

MeOH Ext.(3) + 4- 4 -

C H C I3 Ext.(4) + 4- 4 -

C H C I3 Ext.(5) ++ 4-4- 4 -4 - 4 -4 -

Fr. C1-C28 4 - 4 -

Fr. C29-C54 4-4- 4-4- 4 -4 - 4 -4 -

Fr. C55-C85 ++ 4- 4-4- 4 -4 -

Fr. C86-C130 ++ 4 -4 - EtOAc Ext.(6) 4- 4-

EtOAc Ext.(7) 4-4- 4-4- 4 -4 - 4- 4-

Fr. E1-E30 4- 4-4- 4 -4 - 4-4- Fr. E31-E47

BuOH Ext.(8) 4-4- 4 -

*: a l, a 2 -adrenoceptor subtypes, Ca^-Ca^ ion channel receptor, K^-K^ channel

receptor, HI-histamine 1 receptor

%Inhibition of ligand’s specific binding between 50-79%

"++"- %Inhibition of ligand’s specific binding between 80-100%

***: fractions were described in Figure 7.2

182 7.3.1 Identification of Ursolic acid (35)

COOH

HO'

Compound 35 (p. 187 for data) was obtained as a white amorphous powder. ^HNMR exhibited the presence of one olefinic proton (5 5.25, t, J=4), five tertiary methyl groups, two methyls adjacent to protons (5 0.85, 3H, d, J= 6 ; 5 0.97, 3H, d, J= 6 ) and one proton adjacent to a hydroxyl group (5 3.20, IH, t, J= 6 ), suggesting that the compound had a structure as urs-12-en. The *^CNMR spectrum presented the signals of one carboxyl group ( 6 181.5), one hydroxyl group (Ô 79.2) and one double bond (Ô 126.2, 139.1) in the molecule. The EIMS data gave the molecular ion peak at m/z 456 and the characteristic peak of an olen-12-en or urs-12-en-oic acid at mtz 248. The chemical shift of C 3 appeared at 6 79.2, suggesting that the hydroxyl group at C 3 was P-equatorial oriented. The ‘H-’H COSY spectrum established the linkage of the protons further substantiating the compound was ursolic acid.

7.3.2 Identification of Hirsutine (36) and Epiallocorynantheine (37)

compound 3 6 compound 3 7

Compound 36 (p. 187 for data) was positive to the Dragendroff reagent. The EIMS spectrum gave the molecular ion peak at mh 368 and characteristic peaks of P-

183 tetrahydrocarboline-type compounds at miz 156, 169, 170, 184 (Cong, 1987).The ‘HNMR spectrum exhibited the presence of four aromatic protons (Ô 7.10-7.45), one olefinic proton (Ô 7.30, s) and two methoxyl groups ( 6 3.65, 3.71). These data revealed a heteroyohimbine structure with the C-20 ethyl substituent. The signal of H-3 at ô 4.51 (IH, m) indicated the cis configuration of the C-3H to lone pair of electrons. The unsymmetric triplet signal of methyl group suggested the ethyl group was a-configuration (Supavita, 1982). The H-15 was also a-configuration since the tetracyclic heteroyohimbine type of alkaloids were derived from monoterpene secologanin (Stockigt, 1980). In EIMS, the intensity of the peak at m/z 239 (ion b. Figure 7.3) was lower than that of the peak at m/z 225 (ion c) also suggesting the presence of H-20p (Cong, 1987). The data of 36 in EIMS were in a good agreement with that of hirsutine (Cong, 1987), therefore, the compound is determined as hirsutine.

Compound 37 (p. 187 for data) was also positive to the Dragendroff reagent. It showed a similar El mass spectrum as that of compound 36 except for the molecular ion peak (m/z 366) and a series of peaks being two mass unit less than those corresponding peaks of compound 36 (Figure 7.3), indicating 37 had a similar structure to compound 36. The *HNMR spectrum exhibited the presence of four aromatic protons (Ô 7.10- 7.50), two methoxyl groups ( ô 3.67, 3.75), one olefinic proton (5 7.30, IH, s) and a vinyl group in the molecule (Ô4.97, IH, m; 4.83, IH, dd, J= 8 , J=2; 4.87, IH, dd

J= 8 , J=2), suggesting the presence of a similar structure to compound 36 and a double bond between C^g-C^g. The signal of C-3H at Ô 4.50 (IH, m) revealed the presence of H-3p configuration. In EIMS, the intensity of the peak at m/z 237 (ion b. Figure 7.3) was higher than that of the peak at m/z 223 (ion c) suggesting the presence of H-20a (Cong, 1987). The EIMS data were consistent with those of epiallocorynantheine (Phillipson and Hemingway, 1975). Hence, the compound is determined as epiallocorynantheine.

184 Figure 7.3 Possible MS Frafmentation Patterns of Compounds 36 and 37 (Cong, 1987)

-H 15

CH3 OOC

compound 3 6 M+ 3 68 compound 37 M+ 3 66

14/15

15/20

251 249 15/20

CH3 OOC

ion b 36: 239 37: 237 3/14

ion c 36:225 37:223

185 7.3.3 Identification of (+)-Catechin (38), (-)-Epicatechin (39), (+)-GaIlocatechin (40), (-)-Epigallocatechin (41) and Quercetin (42)

.OH OH

OH OH OH OH

OH OH OH OH

compound 3 8 R=H compound 3 9 R=H compound 4 0 R=OH compound 41 R=OH

Compounds 38, 39, 40, 41 were structurally related. Compounds 38 and 39 (p. 188 for data) had the same El mass spectrum which gave the molecular ion peak at m/z 290 and the characteristic peaks of flavonol at m/z 152, 139 and 123 indicating that both of them had the catechin-type structure. The 'HNMR spectrum of 38 showed five aromatic protons ( 8 5.87-6.86) and four protons at 8 2.52, 2.90, 3.99, 4.55, respectively. The 'HNMR spectrum of 39 also exhibited five aromatic protons ( 8 5.91-

7.00) and four protons at 8 2.73, 2.86, 4.20, 4.87, respectively. Comparing with the values in the literature (Cai, et al, 1991), compound 38 is (+)-catechin and compound 39 is (-)-epicatechin.

Compounds 40 and 41 (P. 188 for data) had the same El mass spectrum which showed the molecular ion peaks at m/z 306 and the peaks at m/z 168, 140, 139 indicating that they had gallocatechin structure. The 'HNMR exhibited that compound 40 had four

aromatic protons at 8 6.45-5.87 and four protons at 8 4.53-2.52. Compound 41 had four aromatic protons at 8 6.57-5.91 and four protons at 8 4.80-2.73. Since the signal of H-2 proton of compound 40 appeared at 8 4.53 (d, 1=7) and the signal of H-2

proton of compound 41 appeared at 8 4.80 (s), the former is determined as ( 4 -)- gallocatechin and the latter is determined as (-)-epigallocatechin (Cai, et al, 1991).

Compound 42 (p. 188 for data) was a flavonoid (positive to NH 3 vapour) and identified

186 as quercetin by EIMS and by comparison with authentic sample (co-TLC).

The structures of compounds isolated from U. rhynchophylla in the present study are summarized in Figure 7.4.

7 J.4 Spectral Data Ursolic acid (35): white amorphous powder, soluble in CHClj/MeOH (1:1). 'HNMR

(CD3 OD/CDCI3 ): 5 0.75, 0.81, 0.91, 0.98, 1.05 (3H, each, tert. Me); 8 0.85 (3H, d,

J=6 , Me-C„); 0.97 (3H, d, J= 6 , Me-Qg); 2.10, (IH, d, J=10, H-18); 3.20 (IH, t, J= 6 , H-3); 5.25 (IH, t, J=2, H-12); 1.32 (IH, br.t, 1=7, H-19); 1.68 (IH, dd, 1=10,1=2, H-

20). '^CNMR (CD3 OD/CDCI3 ): CH3 : 8 15.8, 16.1, 17.4, 17.4, 21.4, 24.0, 28.5; CH;: 19.0, 23.9, 24.8, 27.3, 28.7, 31.3, 33.8, 37.5, 39.4; CH: 39.7, 39.8, 48.3, 53.6, 56.1, 79.2 (C-3), 126.2(C-12); quaternary carbon: 37.6, 39.4,40.2,42.8, 50.0, 139.1(C-13), 181.5(COOH). EIMS m/z (rel. int.): 456 (M*, 3), 411(3), 438(2), 410(4), 248(100), 219(37), 207(41), 203(78), 189(41), 133(83). FABMS m/z (rel.int.): 456(M \ 5), 438(17), 410(12), 248(65), 165(100).

Hirsutine (36): white amorphous powder, soluble in C H C I3 . 'HNMR (C D C I3 ): 8 7.45 (IH, dd, 1=8,1=2, H-12); 7.37 (IH, dd, 1=8,1=2, H-9); 7.32 (m, H-10); 7.30 ( I H , s, H-17); 7.12 (IH, t,d, 1=10,1=2, H-11); 4.51 ( I H , m, H-3); 3.71(3H, s, CO;Me), 3.65 (3H, s, OMe); 3.30 ( I H , dd, 1=8,1=3, H-21a); 2.09 ( I H , dd, 1=10,1=2, H-21b); 2.25

(2H, t, 1=10, H-5); 2.45 ( I H , t, 1=12, H-6 a); 2.28 (IH, t, 1=8, H- 6 b); 2.85 (IH, d, 1=8, H-20); 2.55 ( I H , dd, 1=12,1=3, H-15), 1.60 (2H, m); 1.30 (2H, m); 0.75 (3H, t, 1=10, H-18). EIMS m/z (rel.int.): 368 (M* , 95), 367(62), 353(100), 339(9), 337(23), 311(29), 251(18), 239(13), 225(26), 184(62), 170(26), 169(38), 156(40), 143(15).

Epiallocorynantheine (37): white amorphous powder, soluble in CHCI 3 . 'HNMR

(CDCI3 ): 8 7.50 (IH, dd, 1=8,1=2, H-12); 7.37 (IH, dd, 1=8,1=2, H-9); 7.32 (IH, m, H-10); 7.30 (IH, s, H-17); 7.10 (IH, td, 1=10, 1=2, H-11); 4.97 (IH, m, H-19); 4.87(1H, dd, 1=8,1=2, H-18a); 4.83(1H, dd, 1=8,1=2, H-18b); 4.50 (IH, m, H-3); 3.75 (3H, s, CO;Me), 3.67 (3H, s, OMe), 3.30 (IH, dd, 1=8,1=3, H-21a); 2.09 (IH, dd, 1=12, 1=1, H-21b); 2.76 (IH, dd, 1=12,1=2, H-20); 2.39 (2H, dd, 1=12,1=2); 2.55

187 (2H, dd, J=12, J=2); 2.63 (IH, d, J=4, H-15); 1.55 (2H, d, J=10). EIMS mIz (rel. int.): 367 (M++1, 100), 353 (96), 339 (5), 249(17), 237(25), 223(21), 184(60), 170(25), 169(32), 156(29), 143(7).

(+)-Catechin (38): white powder, ‘HNMR data see Table 7.2. EIMS m h (rel.int.): 290 (M \ 38), 272(14), 255(6), 152 (85), 139(100), 123 (92).

(-)-Epicatechin (39): white powder, 'HNMR data see Table 7.2. EIMS m h (rel,int.): 290 (M \ 27), 272 (11), 255(8), 152(76), 139(100), 123 (87).

(+)-Gallocatechin (40): white powder, 'HNMR data see Table 7.2. EIMS m h (rel,int.):

306 (M+, 6 ), 168(12), 140(27), 139(100), 124(51), 123(90), 110(63).

(-)-Epigallocatechin (41): white powder, 'HNMR data see Table 7.2. EIMS m/z (rel,int.): 306 (M+, 4), 168(15), 140(23), 139(100), 124(41), 123(89), 110(53).

Quercetin (42): yellow powder, slightly soluble in MeOH. EIMS miz (rel.int.): 302(M\ 84), 301(30), 285(20), 274(15), 273(23), 251(9), 245(27), 228(23), 169(24), 153(26), 152(5), 150(14), 137(52), 124(11), 110(32), 109(19).

188 Table 7.2 HNMR spectral data of compounds 38, 39, 40, 41

OH OH

OH OH OH OH

OH ■OH OH OH

compound 3 8 R=H compound 3 9 R=H compound 4 0 R=OH compound 41 R=OH

No. compound 38 compound 39 compound 40 compound 41

2 ’ 6 .8 6 , d, J=2 7.00, d, J=2 6.45, s 6.57, s

5’ 6.25, d, J = 8 6.74, d, J = 8 --

6 ’ 6.73, d, J = 8 6.82, d, J = 8 6.45, s 6.57, s

6 5.87, d, J=2 5.91, d, J=2 5.87, d, J=2 5.91, d, J=2

8 5.98, d, J=2 5.98, d, J=2 5.97, d, J=2 5.97, d, J=7

2 4.55, d, J=7 4.87, s 4.53, d, J=7 4.80, s 3 3.99, m 4.20, m 3.95, m 4.19, m 4a 2.90, dd, J=16, 2.86, dd, J=16, 2.86, dd, J=16, 2.84, dd, J=16,

J= 6 J=3 J=5 J=3 4b 2.52, dd, J=16, 2.73, dd, J=16, 2.52, dd, J=16, 2.73, dd, J=16,

J= 8 J=3 J= 8 J=3

The data was obtained from 400 MHz NMR in CD^OD-d..

189 Figure 7.4 Compounds isolated from Uncaria rhynchophylla

in the present study

OH OH

OH. COOH

OH HO'

ursolic acid (35) quercetin (42)

CH—CH2 H CH30^...^ s?^^C 02C H 3 H

hirsutine (36) epiallocorynantheine(37)

OH .OH OH. OH , I OH OH "OH OH OH

catechin (38): R=H gallocatechin (40): R=OH epicatechin (39) : R=H epigallocatechin (41): R=H

190 7.4 Biological tests on isolated compounds and two alkaloids Compounds 35-42 (structures in Figure 7.4, p. 190) were screened by the radio-ligand receptor binding assays following the approaches described in Sections 2.8 and 2.9. Two bisbenzylisoquinoline alkaloids, namely berbamine and hemandizine (structures in Figure 7.5, p. 192) were also tested since the berbamine was reported to have hypotensive effect and hemandizine was structurally related to it. The receptors tested included a l, a2, P-adrenoceptors, 5HT1A, 5HT1C, 5HT2, dopamine 1, dopamine 2, adenosine 1, histamine 1, Na^/K'^ATPase, Ca^^-channel, K^-channel, opiate, muscarinic GABAa and GABAg. Compounds 35, 36, 37, berbamine and hemandizine bound to

several receptors and the IC 5 0 values of these compounds are listed in Table 7.3.

Table 7.3 The results of compound screenings

Compound Receptor IC5 0 ± SEM N compound 35 muscarinic 6.63 ± 1.58 3 K^-channel 2.83 ± 1.66 3

compound 36 K^-channel 0.14 ± 0.07 3

a 2 -adrenoceptor 0.15 ± 0.05 3 p-adrenoceptor 4.53 ± 1.20 3 5HT2 1.60 ± 0.43 3 5HT1A 3.47 ± 0.66 3 opiate 0.11 ±0.04 3

compound 37 p-adrenoceptor 6.73 ± 1.65 3 5HT2 3.54 ± 1.23 3 5HT1A 3.48 ± 0.47 3 opiate 0.65 ±0.12 3

berbamine dopamine 2 3.32 ± 0.45 3

hemandizine dopamine 1 4.12 ± 1.04 3 muscarinic 6.13 ± 1.27 3

* The IC 5 0 value of control compounds in the present study are atropine 0.16 nM (muscarinic receptor); glibenclamide 0.79 nM (K^-channel receptor); phentolamine 5.64 nM (a2-adrenoceptor); propranolol 1.41

nM (P-adrenoceptor); 8 -OH DPAT 1.00 nM (5HT1A receptor); spiperone 5.62 (5HT2 receptor); SCH-23390 3.10 nM (dopamine 1 receptor); butaclamol 2.08 nM (dopamine 2 receptor); naloxone 1.02 nM (opiate receptor).

191 Compounds 35, 38, 41, berbamine and hemandizine were further tested to investigate if they have any interactions with the control compound used in each binding assay at the various receptors. The binding assays of a l, a2, p-adrenoceptors, Ca^^-channel, K^-channel, dopamine 1, dopamine 2, adenosine 1, histamine 1, opiate, 5HT1A,

5HT1C, 5HT2 receptors were adopted in the study. The IC 5 0 values of the control compounds whose IC 5 0 value were changed are listed in Table 7.4. The IC 5 0 curves have also been established for the control compounds whose binding sites have been altered by isolated compounds (Figure 7.6, p. 194).

Figure 7.5 The structures of berbamine and hemandizine

OH

berbamine

OCht

CH3 0

CH3 ‘OCH b

hemandizine

192 Table 7.4 The IC 50 Values (nM) of Control Compounds and the Control Compounds with Added Natural Products

Compound Receptor' IC5o(nM)±SEM IC5o(nM)±SEM IC5o(nM)±SEM N

(Control) (Iso.Compd)^ (Ctr.+Iso)^

35 opiate 1.02 ± 0.17 - 0.25 ± 0.05 3

5HT1C 70.8 ± 5.26 - 1.66 ± 0.18 3

HI 1.66 ± 0,18 - 0.10 ±0.03 3

38 K+ 0.79 ± 0.05 - 0.63 ± 0.10* 3

41 D2 2.08 ± 0.26 - 1.00 ± 0.46* 3

0.50 ± 0.07 berbamine al 5.64 ± 0.47 3 0.16 ± 0.13 5HT1A 1.00 ±0.12 3 1.00 ± 0.24 A l 14.1 ± 2.31 - 3

0.10 ± 0.08* hemandizine Ca"+ 2.00 ± 0.32 3 12.6 ± 1.75* 5HT1C 70.8 ± 5.26 3 0.03 ± 0.01 5HT2 5.62 ± 0.47 3 0.27 ± 0.04 HI 7.41 ± 0.77 - 3

1 : receptor al- al adrenoceptor subtypes; D2- dopamine 2 receptor; Ca^^, - Ca^^, ion channel receptors; A l- adenosine 1 receptor; HI- histamine 1 receptor

2: isolated compound

3: control compound + isolated compound

4: inactive

*: the binding site was also influenced, see also Figure 7.6

193 Figure 7.6 IC5 0 Curves of Control Compounds and

the Control Compounds with Added Natural Products

Glibenclamide Displacement (K+-channel Receptor) 100

■ Glibenclamide

Glib.+compound 38 cS

(/Ia 50 g

- 2 1 0 I 2 3 [Displacer] nM

Spiperone Displacement (Dopamine 2 Receptor) 100

butaclamol CT butaclamol+compound 41 C m£

g

X

[Displacer] nM

194 Pyrilamine Displacement (Histamine 1 Recptor) 100

Pyrilamine Pyri.+Hernadizine

g

1 0 1 2 3 4 [Displacer] nM

Nitrendipine Displacement (Ca++-channel receptor) 100

Nitrendipine

*■ nitre.+Hernadizine c in

-2

195 (5HT1C Receptor)

cn 100 Mesulergine

M esu. +compound 35 m

g 5 0

[Displacer] nM

Pyrilamine Displacement (Histamine 1 Recptor) 100

P y r ila m in e D) C ■o Pyri.-fcompound 35 mc

5 0

10 1 2 3 4 [Displacer] nM

Naloxone Displacement (opiate receptor) 100

■ N aloxone Naio.+ compound 35

t/1CL 5 0 o

[Displacer] log nM

196 7.5 Discussion 1. Uncaria rhynchophylla is documented as an antipyretic and anticonvulsant for the treatment of headache, vertigo and epilepsy (Anon, 1992). The pharmacological studies on the aqueous decoction and the ethanol extract showed hypotensive, sedative, anticonvulsant and smooth muscle effects (Du, 1987). In the present radioligand receptor binding assays, the 70% ethanol extract was able to bind to a l, a2- adrenoceptors, 5HT1A, 5HT2, opiate, Ca^^-channel, K^-channel, histamine 1, GABA^ and GABAg receptors (Figure 3.1, p.83). These receptors are believed to be related to hypotensive, sedative, anticonvulsant and smooth muscle effects. The results of fraction screening (Table 7.1, p. 182) exhibited the active fractions and the most of the fractions tested were able to bind a2-adrenoceptor and 5HT1A receptor suggesting that the hypotensive and sedative effects of the plant mainly due to the effects on these receptors. Following the isolation of active fraction C29-C54, hirsutine (36) and epiallocorynantheine (37) were obtained. Hirsutine was reported to have antihypertensive effect (Horre, et al, 1992) in in vivo tests and it was found to bind to a2, P-adrenoceptors, 5HT1 A, 5HT2, K^-channel and opiate receptors in the present studies (Table 7.3, p. 191). These results were consistent with the finding of the in-vivo tests. The structurally related epiallocorynantheine (37) had ability to bind to p- adrenoceptor, 5HT1A, 5HT2 and opiate receptors which also related to hypotensive and sedative effects. From the EtOAc extract ( 6 ), four catechin-type compounds were obtained. These four compounds did not show any activity against the receptors tested, but (+)-catechin (38) was able to alter the binding site of glibenclamide (an antagonist of K^-channel receptor) at K^-channel receptor, while epigallocatechin (41) change the binding of butaclamol (an antagonist of dopamine 2 receptor) at D2 receptor. These two receptors (K^, D2) involved are related to sedative effect and antihypertensive. From fractions C1-C28, ursolic acid (35) was obtained as the major principle which showed the ability to bind with muscarinic receptor, K^-channel receptor (Table 7.3, p. 191) and to interact to the ligands binding to opiate, 5HT1C and histamine 1 receptors (Table 7.4, p. 193). The finding in the present study suggests that the plant extract include a variety of constituents which may not show the same extent of activities, but the individual components may contribute to overall effects. Some of them may act on receptors directly (eg. compounds 36, 37); some of them have

197 synergistic effect on the ligands of various receptors (eg. compounds 38, 41); and some of them can act on several receptors and also have synergistic effect to the ligands of other receptors (eg. compound 35). The symptoms of the CNS disorders are usually caused by several factors and involved different receptors. For examples, in the treatment of hypertensive, drugs are applied that interfere at various sites with the efferent sympathetic system, that occupy a- or p-adrenergic receptors, that may stimulate afferent parasympathetic activity, or that influence renal handling of water, sodium and potassium. Single compounds, such as a- or p-adrenergic antagonists, vasodilators, or diuretics, do not aim at completeness in overall antihypertensive therapy. The plant extract produces multiple effects by its different ingredients having the possibility to act on several targets (eg. a, p-adrenoceptor, 5HT1 A, K^-channel and muscarinic receptors) at the same time which may cause relief of a single symptom (eg. hypertensive).

2. In the fraction screenings, the C H C I3 extract ( 6 ) which was obtained from the 50% MeOH extract (3) was more active than the C H C I3 extract (4) which was derived from the 90% MeOH extract (Table 7.1, p. 182) suggesting that the constituents in these two C H C I3 fractions might be different. The same phenomenon was observed with the EtOAc extract (7) (made from the 50% MeOH extract) and EtOAc extract (5) (obtained from the 90% MeOH extract) suggesting the presence of some active components in the more polar fraction. The water extract (1) containing the same constituents as the decoction used in traditional medicine was able to bind with a 2 - adrenoceptor, 5HT1A, K^-channel and histamine receptors which were related to the hypotensive and sedative effects of the herb.

3. Two bisbenzylisoquinoline alkaloids, berbamine and hemandizine were tested by radioligand-receptor binding assays. Berbamine was reported to have an antihypertensive effect (Liu, et al, 1983). It was found in the present study that the compound was able to bind to the dopamine 2 receptor (Table 7.3, p. 191) and had interactions with the ligands of a2-adrenoceptor and 5HT1A receptor (Table 7.4, p. 193). The related compound hemandizine was able to bind to dopamine 1 and muscarinic receptors and had influence on the binding of the radioligands to Ca 2+

198 channel, 5HT1C, 5HT2, adenosine 1 and histamine 1 receptors (Table 7.4). These receptors are also related to hypotensive and sedative effects and the results obtained in the binding assays were consistent with those in vivo tests. The alkaloids are a major group of compounds present in plants which have various effects in the central nervous system. The medicines commonly used in clinics for the treatment of the CNS disease are either alkaloids or nitrogen-containing compounds. Therefore, to develop new leads from natural products, alkaloids are still the main source.

199 Chapter 8

General Discussion

200 In a search for CNS active principles from natural products, ten species of Chinese plants were selected and tested for their activities at biological receptors by radioligand receptor binding assays. Four species were subjected to chemical studies following bio-activity guided fractionation. Forty-two compounds have been obtained and identified. Thirty-four of the isolated compounds were examined for their activities in the central nervous system by radioligand receptor binding assays. The results showed that fourteen of these compounds were able to bind to specific receptors with the IC 5 0 values at the pM level. Eleven structural related compounds from other plant sources were also tested, and four of them bound to specific receptors with IC 5 0 values at the pM level. Twenty-four of these compounds were examined in order to determine whether they had any interactions with the control compounds at various receptors, eighteen of them were found to have the ability either reducing the

IC5 0 values of the control compounds or changing the receptor binding sites of these compounds.

The compounds isolated from the plants were not as active as the control compounds

(which have IC 5 0 values at the nM level), but 40% of the tested compounds showed a vaiying degree of activities and 75% of the tested compounds had the ability to affect the binding of the control compounds. These results lead to the question as to whether new drugs which have strong CNS activities can be developed from Chinese medicinal plants? To answer this question, it may be helpful to realize the difference between the traditional Chinese medicine (TCM) and western medicine, and to understand the basic theory of TCM.

8.1 Differences between the Chinese traditional medicine and western medicine Chinese medicine is a coherent and independent system of thought and practice that has been developed over two thousand years. Based on ancient texts , it is the result of a continuous process of critical thinking as well as extensive clinical observation and testing. It represents a thorough formulation and reformulation of material by respected clinicians. It is also, however, rooted in the philosophy, logic, sensibility and habits of a civilization and has developed its own perception of the body and of health and disease.

201 Both Chinese medicine and western medicine have their own ways of seeing and thinking, and the two different logical structures have pointed the two forms of medicine in different directions. Western medicine is concerned mainly with isolated disease categories. A western physician starts with a symptom, then searches for the underlying mechanism- a precise cause for a specific disease. The disease may affect various parts of the body, but it is a relatively well-defined, self-contained phenomenon. Precise diagnosis frames an exact, quantifiable description of a narrow area.

A Chinese physician, in contrast, directs his attention to the complete physiological and psychological individual. All relevant information, including the symptoms as well as the patient’s other general characteristics, is gathered and woven together to form what Chinese medicine called a "pattern of disharmony". This pattern of disharmony describes a situation of "imbalance" in a patient’s body. Oriental diagnostic technique does not turn up a specific disease entity or a precise cause, but renders a workable description of a whole person. The Chinese are interested in discerning the relationships among bodily events occurring at the same time. The logic of Chinese medicine is organismic or synthetic, attempting to organize symptoms and signs into an understandable configuration. The total configuration and the patterns of disharmony provide the framework for treatment, the therapy then attempts to bring the configuration into balance, to restore harmony to the individual. To western medicine understanding an illness means uncovering a distinct entity that is separated from patient’s being; to Chinese medicine, the understanding means perceiving the relationships between all the patient’s signs and symptoms. The Chinese method is thus holistic, based on the idea that no single part can be understood except in its relation to the whole. A symptom, therefore, is not traced back to a cause, but is looked at as a part of totality. If a person has a symptom, Chinese medicine wants to know how the symptom fits into the patient’s entire bodily pattern. A person who is well, or "in harmony" has no distressing symptoms and expresses mental, physical and spiritual balance. When that person is ill, the symptom is only one part of complete bodily imbalance that can be seen in other aspects of his or her life and behaviour. Understanding that overall pattern, with the symptom as part of it, is the challenge of

202 Chinese medicine. Hence the difference in the thought of "whole" and "part" leads to the Chinese medicine and western medicine going different ways in their treatment. On the other hand, Chinese medicine uses terminology that is strange to the western ear. For example, the Chinese refer to certain diseases as being generated by "Dampness", "Heat", or "Wind". (The words here should be accounted for by the special meaning with Chinese terminology rather than ordinary English terms). Modem western medicine does not recognize Dampness, yet can treat what Chinese medicine described as Dampness of the Spleen (eg. oedema, damp-phlegm, diarrhea, etc.) . Chinese medicine considers important certain aspects of the human body that are not significant to western medicine. At the same time, western medicine observes and can describe some aspects of the human body that are insignificant or not perceptible to Chinese medicine. For instance, Chinese medical theory does not have the concept of a nervous system. Nevertheless, it has been demonstrated that Chinese medicine can be used to treat neurological disorders. Therefore, the perceptions of the two traditions reflect two different worlds but both can heal the same body.

8.2 The Chinese Medicine Theory The logic underlying Chinese medical theory- a logic which assumes that a part can be understood only in its relation to the whole- can also be called synthetic or dialectical. This dialectical logic that explains relationships, patterns and changes is called Yin-Yang theory. Yin-Yang theory is based on the philosophical construct of two polar complements, called Yin and Yang. Yin originally meant the shady side of a slope. It is associated with such qualities as cold, rest, responsiveness, passivity, darkness, inferiority, downward, inwardness, and decrease. The original meaning of yang was the sunny side of a slope, the term implies brightness and is part of one common Chinese expression for the sun. Yang is associated with qualities such as heat, stimulation, movement, activity, excitement, vigour, light, exteriorly, upwardness, outwardness, and increase. In the Yin-Yang system of thought, all things are seen as parts of a whole. No entity can ever be isolated from its relationship to other entities; no thing can exist in and of itself. There are no absolutes. Yin and Yang must

Reference : Zhang, 1988; Kaptchuk, 1983

203 necessarily contain within themselves, the possibility of opposition and change. The Yin-Yang theory is well illustrated by the traditional Yin-Yang symbol (Figure 8.1). The circle representing the whole is divided into Yin (black) and Yang (white). The small circles of opposite shading illustrate that within the Yin there is Yang and vice versa. The dynamic curve dividing them indicates that Yin and Yang are continuously merging. Thus Yin and Yang create each other, control each other, and transform into each other.

Figure 8.1 Traditional Yin-Yang Symbol

TCM has many characteristics both in the understanding of the physiology and pathology of the human body and in the diagnosis and treatment of disease. These characteristics can be summarized in the following two aspects: (a). The concept of the organism as a whole; (b). Diagnosis and treatment based on an overall analysis of signs and symptoms.

"Organic whole" means entirety and unity. TCM attaches great importance to the unity of the human body itself and its relationship with nature, and considers that the human body itself is an organic whole and has very close and inseparable relations with the external natural surroundings. Such a concept penetrates through the entire theory and is of great significance in guiding diagnosis and treatment. For example, TCM believes that "the Heart has its specific opening in the tongue", so the physiological functions and pathological changes of the Heart can be known by observing the tongue. A pale tongue indicates blood deficiency of the Heart; purple tongue with

204 petechiae suggests that the circulation of the Heart-blood is not well. To cure heart diseases, the most important thing is to find out the pathogenesis according to the relationship between the heart and the tongue, by taking into consideration the organism as a whole, and by making a comprehensive analysis of the case.

"Diagnosis and treatment based on an overall analysis of signs and symptoms" means analysing the relevant information collected by diagnosis and determining the corresponding therapeutic method according to the conclusion of an overall differentiation of symptoms and signs. In clinical treatment, TCM physicians do not focus their main attention on the similarity and distinction between diseases but on the differences between the symptoms they have. Generally speaking, the same symptoms are treated in similar ways, and vice versa. Take cold for an example, if it manifests itself in more severe chillness and with slight fever, a tongue with thin and white fur then it belongs to the exterior symptoms caused by Wind and Cold, and should be treated with strong sudorifics which are pungent in taste and warm in property to dispel the Wind and Cold; if its manifestations are more severe fever, milder chilliness, a tongue with thin and yellow fur, then it belongs to the exterior symptom caused by Wind and Heat, and should be treated with diaphoretics which are pungent in taste and cool in property to dispel the Wind and Heat. Sometimes, different diseases have the same symptom in nature, then their treatments are basically the same.

In TCM, the internal organs of the human body are divided into "five viscera" (the Heart, Liver, Spleen, Lungs, Kidneys) and "six bowels" (the Gall bladder. Stomach, Large intestine, Small intestine. Urinary bladder, triple Burner). The former have common functions of production, transformation, regulation and store of vital substances, and the latter have the functions of receiving, breaking down and absorbing the part of food that will be transformed into the vital substances, and transporting and excreting the unused portion. It is necessary to mention that the name of the internal organs are basically the same as those used in western medicine, but all the concepts are different. The functions of an organ in TCM may contain the functions of many organs in western medicine; meanwhile, the function of an organ

205 in western medicine may be contained in the functions of several viscera and bowels in TCM. For instance, the Heart in TCM does refer to the same anatomic entity as in western medicine, in addition, it also contains some of the functions of the nervous system, especially some functions of the brain.

According to the TCM, the main physiological functions of the Heart include controlling blood circulation, taking charge of mental activities, and having relations with the tongue and face. It believes that the Heart stores and rules the spirit: if the spirit is nourished, the individual responds appropriately to the environment; if the spirit-storing of the Heart is impaired, the individual may show symptoms associated with the spirit, such as insomnia, excessive dreaming, or forgetfulness. More serious disorders of this type are hysteria, irrational behaviour, insanity, and delirium. TCM also believes that the mental activities of human beings are also associated with the Liver which is responsible for the smooth movement of bodily substances and for the regularity of body activities. The Liver harmonizes the emotions. Its gentle movement is responsible for creating a relaxed easygoing internal environment -an even disposition. Any sudden change in the normal pattern of emotions can affect the functions of the liver, and a disharmony of the Liver will directly affect the emotional state of the individual. Anger and emotional frustration have especially influence on the Liver.

8,3 Traditional Chinese medicine used for the treatment of the CNS disorders There are three types of traditional Chinese medicine which are used for the treatment of the CNS disorders, namely tranquilizers, drugs for calming the Liver, and drugs for inducing resuscitation. Some examples are given in Table 8.1 (p.210).

Drugs whose principal effects are tranquillizing the mind and relieving uneasiness are called tranquilizers. Tranquilizers exert their effects either through sedation or through nourishment of the Heart. They are chiefly used in the treatment of irritability, palpitation, insomnia and dreaminess due to deficiency of the Heart-Qi (Qi is a specific term in TCM which can be thought as matter on the verge of becoming energy, or energy at the point of materializing) and insufficiency of the Heart-blood,

206 or due to exuberant Fire* resulting from hyperactivity of the Heart. Tranquilizers are also used to treat such diseases as infantile convulsion, epilepsy and mania. Drugs of this class can be selected for use according to the etiology and pathogenesis of the disease and used together with suitable drugs of others classes. For symptoms due to deficiency of Yin and insufficiency of the blood, drugs for nourishing the blood and strengthening Yin should be added; for symptoms due to exuberant Fire caused by hyperactivity of the Heart, drugs for removing excessive Fire from the Heart should be given as well; as for epilepsy and infantile convulsion, drugs for resolving phlegm and inducing resuscitation or drugs for calming the Liver to stop endogenous Wind** should be used as main ingredients with the tranquilizers as the adjuvant.

The sedative tranquilizers are mainly minerals, such as Cinnabaris, which have the effects of tranquillizing the mind and clearing away heat and toxins. The tranquilizers for nourishment of the Heart are normally plant materials, such as Semen Ziziphi Spinosae, Semen Biotae, Radix Polygalae, for the treatment of insomnia, dreaminess, palpitation induced by terror, and severe palpitation due to blood insufficiency in the Heart and Liver. The constituents of this class of herbal medicine include alkaloids, terpenoid saponins and polyssacharides.

Drugs whose principal effects are calming the Liver to check endogenous Wind and to relieve convulsion and calming the Liver to suppress hyperactivity of Liver-Yang are called drugs for calming the Liver. These drugs are chiefly indicated for dizziness and blurred vision due to hyperactivity of the Liver-Yang, and spasms and convulsions

* Fire: The characteristic of Fire is hot and active. The signs of Fire are high fever, a red face, red eyes, dark and reddish urine and symptoms as carbuncles, boils, reddish ulcers or other skin lesions that are red, swollen, raised, and painful (those in the west would be called inflammation).

** Wind: Wind in the body resembles wind in nature. It is both movement and that which generates movement in what would otherwise be still. It produces change and urgency in what would otherwise be slow and even, and it causes things to appear and disappear rapidly. Because Wind is associated with movement, it is often recognized by such signs as pains that move from place to place, itching or skin eruptions that change locations, spasms, tremors of the limbs, twitching, dizziness, or tetany. Wind can be divided to External Wind which is accompanied by fever, fear of drafts, sweating, sudden headaches, stuffed nasal passages, itchy or sore throat, and Internal Wind which usually accompanies a chronic disharmony, frequently, though not exclusively, of Liver. Signs of Internal Wind may include dizziness, tinnitus, numbness of the limbs, tremors, convulsions and apoplexy.

207 due to up-stirring of endogenous Wind resulting from disorders of the Liver. Some drugs of this class also have the effects of clearing away heat from the Liver and are therefore effective for blood-shot, swollen and painful eyes and headache due to intense heat in the Liver. Plants used for this type of medicine generally contain alkaloids and various saponins with phenolic compounds (quinone, flavonoid, coumarin) and terpenoids as aglycones. Drugs for calming the Liver should be used in combination with drugs of other classes according to the causes of the disease and they are often used in combination with tranquilizers for the treatment of mental disorders.

Plants which are mainly used for restoring consciousness are referred to as drugs for inducing resuscitation, they have strong aromatic odours and remarkable action of stimulating the sense organs and restoring consciousness (essential oils exist in the most of these plants). Such plants are chiefly used for treating loss of consciousness and delirium due to invasion of heat into the pericardium or obstruction of the Heart by pathogenic phlegm. They are also indicated for unconsciousness of excess type like abrupt faint away in the course of terror-induced epilepsy or apoplexy.

In the application of drugs, sometimes a single drug is used and sometimes two or more drugs are used together. The combined application of two or more drugs has the effect of compatibility as "mutual reinforcement", "mutual assistance", "mutual restraint", "mutual detoxication", "mutual inhibition", or "incompatibility" which can be explained as follows: Mutual reinforcement - Drugs of similar characters and functions are used in coordination to strengthen their effects. Mutual assistance - Drugs similar in certain aspects of their characters and functions can be used together, with one as the principal and the other or others as subsidiary, to help increase the effects of the principal. Mutual restraint - When drugs are used in combination, the toxicity and side effects of one drug can be reduced or eliminated by the other. Mutual detoxication - One drug can remove the toxicity and side effects of the other. Mutual inhibition - When two drugs are used together, they inhibit or check each other

208 to weaken or even lose their original efficacy. Incompatibility - When two drugs are used in combination, toxicity or side effects may result. In a word, when two or more drugs are used in one prescription, they will give rise to interactions. They may coordinate each other to increase their effects; counteract each other to reduce or remove their toxicity or side effects; or contradict each other to weaken or lose their effects, or even result in toxicity and side effects. For this reason, when two or more drugs are needed, they should be selected carefully according to the conditions of the patient, and the characters and functions of the drugs. "Mutual reinforcement" and "mutual assistance" drugs should be employed whenever possible. "Mutual restraint" and "mutual detoxication" drugs can be considered when using poisonous drugs. "Mutual inhibition" and "incompatibility" drugs should be avoided to use.

It is shown in Table 8.1 that the traditional Chinese medicines used for the CNS diseases are mainly via Heart and Liver channels which influence the actions of the Heart and Liver controlling the mental activities. Some drugs are also related to the Kidney, Spleen and Lung which further reflect the treatment of "organism as a whole". The constituents of these plants are diverse including essential oils, monoterpenes, sesquiterpenes, diterpenes, triterpenes, alkaloids, flavonoids, polysaccharides, phenolic compounds, and various saponins with different aglycone structures. In western medicine, the majority of drugs used clinically for the treatment of the CNS disorders are nitrogen-containing compounds, whilst, of the 55 Chinese plant medicines with CNS activities in the Chinese pharmacopoeia, only 9 drugs are reported containing alkaloids and the most drugs have various saponins as major constituents. Some of these saponins have sedative and tonic effects and some of them show immunomodulatory action. In the drug applications, each drug has its individual functions and few drugs can substitute each other. For instance, both Semen Ziziphi Spinosane and Semen Biotae have the effect of "nourishing the Heart to tranquillize the mind", but the former is used for insomnia due to blood insufficiency in the Heart and Liver, while the latter is used for insomnia due to anxiety. These two drugs can

209 Table 8.1 Traditional Chinese Medicines Used for the Treatment of the CNS Disorders

Plant (part) channel Therapeutic applications Chemical constituents tropism

Acanthopanax senticosus Liver sedative, anticonvulsion, antifatigue, isofraxidin, sesamin, p-sitosterol, ffiedelin, polysaccharides, (root) Kidney detoxicant, hypotensive, increase blood cell coumarin glycoside: eleutheroside Bl; hydrobenzene glycosides: reproduction and the functions of the Liver. eleutherosides A, B, C, D, E, I, K, L, and M; triterpenoid The polysaccharide fractions are glycosides: ciwujianosides A l, A2, A3, A4, B, Cl, C2, C3, C4, D l, immunostimulating agents, they potentiate D2, D3, and E. the antibody response against sheep red blood cells and stimulate phagocytosis by peritoneal macrophages of mice and decreased toxic effects of thioacetamine and phytohemagglutinin in mice, and to enhance resistance to X-ray irradiation. The ethanol extract had immunomodulatory activity showing a drastic increase in the absolute number of immune competent cells, especially T lymphocytes.

Albizzia julibrissin (flower) Heart sedative, for the treatment of distractibility triterpenoids: machaerinic acid lactone, acacic acid lactone, Spleen and insomnia due to anxiety machaerinic acid, methylseter, acacigenin B, 16-deoxyacecigenin B, glycoside: cyanidin-3-P-D-glucoside; flavonoid: 7, 3’, 4’- trihydroxyflavone

Acorus calamus (rhizome) Heart for impairment of consciousness in epilepsy; essential oils: a, (3, y- asarone, methyl egenol, bisasaricin Liver as sedative and expectorant

Cornus officinalis (ripe fruits Liver for dizziness and lumbago saponins: comin, morroniside, 7-0-methyl-morroniside, sweroside; without seeds) Kidney ursolic acid, gallic acid, loniceroside, betulic acid, coumarins, amino acids, flavonoids

210 Corydalis yanhusuo, Heart for insomnia, general pain and hypertension Benzylisoquinoline alkaloids: corydaline, dl-tetrahydropalmatine, C.turtschaninovii. f yanhusuo Liver protopine, 1-tetrahydrocoptisine, dl- tetrahydrocolumbamine, a- C. ambigua Kidney allocryptopine, ambinine, , corybulbine, adlumine, etc. C. decumbens (tubers)

Eucalyptus globulus Heart neuralgia essential oils: a-pinene, camphor, thymol, a-phellandrene

Gastrodia elata Liver for infantile convulsions, epilepsy, dizziness, benzyl alcohol glycoside: gastrodin, gastrodioside (tuber) numbness of limbs, hypertensive, insomnia, others: vanilyl alcohol, vanillin, 4-hydrobenzaldehyde, succinic acid, neurasthenia, general paralysis, vertigo citric acid, palmitic acid, sucrose, p-sitosterol, daucosterol, and more than twenty benzyl alcohol derivatives

Lycium barbarum Liver for dizziness, hypertension sesquiterpenes: solavetivone, dehydro-occyperone, zeaxanthine, (ripe fruit) Kidney physalien; 36 volatile substances including alcohols, aldehydes, ketones, esters, tetols, lactone, pyriones, and pyridines

Morus alba Heart tonic, sedative, analgesic lipids (63%) which are mainly fatty acids: heptanoic acid, caprylic (fruit) Kidney acid, pelargonic acid, capric acid, myristric acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid and honadecanoic acid; organic acids (27%); others: alcohols; rutin and some flavonoids; essential oils: cinenol, linalool, camphor, a- pinene, limonene

Polygala tenuifolis Heart for neurasthenia, insomnia, epilepsy, triterpene saponins: tenuifolin, senegenin, hydroxysesegenin, (root) Kidney forgetfulness, palpation, apoplexy; saponin onjisaponins A, B, E, F, G; Xanthone derivatives: wubengzisides Lung fractions showed inhibitory activity against A, B, C, 6-OH-1,2,3,7-tetramethoxy-xanthone, 3,4,5-trimethoxy- cAMP phosphodiesterase. cinnamic acid, polygalite

211 Polygonum multiflorum Heart for neurasthenia, insomnia, nightmare; used anthraquinones: emodin, physcion; hydroxylated stibene glycosides: (stem) Liver as sedative and antirheumatic 2,3,5,4’-tetrahydroxystibene-2-0-P-D-glucoside, polygoacetophenoside

Salvia miltiorrhiza Heart for insomnia, menstrual disorders, blood phenanthrofurane quinone derivatives: tanshinones I, II, HE, (root) Liver circulation diseases cryptotanshinone, methyl tanshinonate, isotanshinones I, H, tanshindiols A, B, C, Daushenxinkuns A, B, C, salviol, miltirone, mitiodiol

Valeriana officinalis (root and Heart for neurasthenia, insomnia, epilepsy and essential oils: bomyl isovalerate, pinene, limonene, bomeol, rhizome) Lung hysteria terpineol, myrcene, phellandrene, 1-caiyophyllene, y-terpinene; alkaloids: valerianine, actinidine, jatamansine; caffeic acid, valeroside, isoferulic acid, chlorogenic acid; others: valerianol, valerenal, , cryptofauronol, faurony acetate

Ziziphus spinosa Heart for insomnia, fidgeting excessive sweating triterpenoid saponins: jujubosides A, B, ziziphin; triterpenoids: (ride seed) Liver due to debility; used as sedative and tonic alphitolic acid, betulonic acid, oleanonic acid, maslimic acid, Spleen drug unsolic acid; benzylisoquinoline alkaloids: norisoboldine, asimilobine, yuziphine, zizphusine; cyclopeptide alkaloids mauritines A, D, nummularines A, B, jubanines A, B, frangufoline, daechucyclopeptide-1; flavonoid glycosides: swertisin, spinosin, sinapoylspinosin, zivalgavin

References: Xiao, 1989; Wang, 1985; Tang and Eisenbrand, 1992; Li, 1992.

212 also be used together for insomnia, palpitation, dreaminess and dryness in the mouth due to *' deficiency of the Heart and kidney" as well as hyperactivity of Yang due to the Yin deficiency. "Emperor Cardiotonic Pill" is an effective drug for sleeping disorder especially for insomnia, palpitation, forgetfulness, dreaminess, and dryness in the mouth and throat. The prescription of this drug is composed of 15 plant medicines and one mineral, and it can be taken as an example to show how the individual drugs are combined (Table 8.2).

The 16 components in the "Emperor Cardiotonic Pill" can be divided into 4 groups based on their functions. Drugs 1-6 have the major effects of the anticonvulsion, hypotension and sedative. Drugs 7-11 have subsidiary effect which are used for increasing coronary and cerebral blood flow, lowering peripheral blood pressure, increasing blood reproduction in the liver, and cardiotonic. Drugs 12-15 exert their sedative effects from clearing the internal Fire and antianxiety. Drug 16 is used in the most TCM prescriptions since it is a detoxicant and mediator to make the individual drugs in the prescription in harmony. It is obvious that for a single symptom (eg. sleeping disorder), TCM not only acts on the effectors (eg, receptors in the brain, like most western medicines do), but also improves the whole internal environment in the patient’s body to eliminate the cause of the disease and to bring the whole back to a harmonious state. The actions of mutual reinforcement, mutual assistance and mutual detoxication of these drugs have been reflected in the prescription which make the drug more effective than a single component. For the treatment of the CNS disorders, the clinical effects of most TCM prescriptions start to be observed after the patient takes the medicine for one course (7-10 days). This fact implies that TCM may have a different mechanism of action from western medicine. In contract, the effects of western medicine for the same uses can usually be observed in a very short time (30- 60 minutes for pain killers and sleeping pills) indicating that the drugs act directly on the effectors, whilst the patients have to depend on the use of medicine to relieve the symptoms continuously. Rapid, effective, alleviation symptoms (especially for the CNS disorders) are the features of the western medicine, whilst, slow but effective elimination of the causes of disease are the features of the Chinese medicine.

213 Table 8.2 Emperor Cardiotonic Pill

Actions: for the syndrome of deficiency of blood*, deficiency of Yin** and the dysfunction of Heart***.

*: A morbid condition marked by pallor, dizziness, palpitation, insomnia and numbness of the limbs. **: A general term for lack of body fluid, vital essence and blood, often resulting endogenous Heat or Fire. ***: A viscus that maintains the blood circulation and controls the mental activities. Thus not only the disturbance of blood circulation and irregularity in the sequence of pulse beat but also various disorders of the central nervous system, such as insomnia, forgetfulness, impaired consciousness, are attributed to the Heart.

Usage and Dosage: take orally, 15 pills once, twice a day (1 Spills equal 5.625g crude drug).

Ingredients:

Plant Functions

1. Ziziphus jujuba anticonvulsant, antishock, hypotensive, sedative, hypnotic, tranquillizer 2. Biota orientalis sedative, moistening the bowels to relieve constipation

3. Scrophularia buergeriana anticonvulsant, antitoxin, cardiotonic, hypoglycaemic, hypotensive, sedative, hypnotic 4. Acorus gramineus anticonvulsant, sedative, hypnotic, tranquillizer

5. Poria cocos antineoplastic, cardiotonic, immunoadjuvant, liver protective agent, sedative, hypnotic 6. Cinnabaria sedative, clearing away Heat and toxins

7. Rehmanniia glutinosa coronary vasodilator, hypoglycaemic, liver protective agent, sedative, hypnotic 8. Angelica sinensis antiarrhythmic, antilipemic, haematinic, coronary vasodilator, hypotensive, immunosuppressant, liver protective agent, peripheral vasodilator 9. Schisandra chinensis cardiotonic, central stimulant, peripheral vasodilator, promoter of anabolism 10. Salvia miltorrhiza anticoagulant, antilipemic, antineoplastic, coronary and peripheral vasodilator, immunoadjuvant, hypotensive, hypoglycaemic 11. Codonopsis pilosula adaptogen, haematinic

12. Asparagus cochinchinensis for dry throat, cough with sticky phlegm, anxiety

13. Ophiopogon japonicus for insomnia, palpitation, hyperglycaemic

14. Polygala tenuifolia anticonvulsant, expectorant, sedative, hypnotic

15. Platycodon grandiflorus antiinflammatory, expectorant, hypoglycaemic, hypotensive

16. Glycyrrhiza uralensis analgesic, anticonvulsant, detoxicant, liver protective agent, antilipidemic

214 The Chinese people usually have their own way to choose the type of medical treatment. For most of the acute diseases, they choose western medicine, and sometimes, when the symptoms are alleviated, they switch to the Chinese medicine to seek complete recovery. For most of chronic diseases, they simply choose the Chinese medicine. These features of the two types of medicine lead to different approaches for the development of drugs.

8.4 New drug development and TCM research Up to now, a huge number of studies have been undertaken to isolate the active components from medicinal herbs in attempts to introduce them into the framework of modem western medicine. In China, most herbal medicines listed in the Chinese Pharmacopoeia have been phytochemically studied for their active principles and a number of medicines have been developed from them. For instance, an anti-malarial constituent, artemisinin (qing haosu) is obtained from Artemsia annua L, a anti­ bacterial principle berberine from Coptis chinensis and a anti-tumour ingredient camptothecin from Camptotheca acuminata. For the drugs which act on the central nervous system, there are schizandrin, a CNS depressant, from Schisandra chinensis; tetrahydropalmatine, an analgesic, from Corydalis yuanhusuo; and sinomenine, an analgesic and sedative, from Sinomenium acutum (more examples in Table 1.6, p.58). Along with the applications of the more and more modem techniques, great progress has been made in developing modem medicine from the traditional Chinese medicine.

As reviewed in Chapter one, new dmgs should be based on the following characteristics: greater therapeutic efficacy and selectivity than existing dmgs; lower toxicity; fewer or less disturbing side effect; more desirable pharmacokinetic features; different mechanism of action and a basically different chemical stmcture. The following section indicates whether or not the constituents isolated from Chinese plants can meet these criteria.

Therapeutic efficacy Some compounds from TCM have greater therapeutic efficacy than existing dmgs, for instance, lappaconitine, a diterpene alkaloid from Aconitum spp., exhibited a strong analgesic activity in animal test (Tang, et al, 1983). An

215 anaesthesia test on rabbit cornea revealed that the surface anaesthetic potency of lappaconitine was eight times stronger than that of cocaine. Local anaesthetic effects on sciatic nerve block in mice were five times stronger than those of cocaine (Jiang, et al, 1983). In the present study, although no highly active compounds have been found, there are fourteen compounds which showed some activities at the pM level and they may become starting material for structure modification ( compounds which have IC 5 0 values less than 10 pM can be used to modify the structure).

Selectivity In plants, some structurally related compounds may have very different effects depending on their substitution, configuration and conformation. For instance, ginsenoside Rbl [(20s)-protopanaxadiol glycoside] is a CNS depressant and ginsenoside Rgl [(20s)-protopanaxatriol glycoside] is a stimulant. The difference between their structure was that the former had a proton at C-5 and the latter had a hydroxyl group at C-5. In the present study, compound 4 and its isomer 5 (4 has a

C,3 -Ci4 double bond and 5 has C, 4 -C ,5 double bond, structures in Figure 4.7, p. 117) showed the different activities. Compound 4 bound to dopamine 2 receptor, whilst 5 was inactive to all the receptors tested. In the interaction 4 did not show any activity at ion channel receptors, but 5 markedly affected the binding site of glibenclamide (antagonist) at K^-channel receptors. To 5HT receptors, 4 interacted with the control compounds binding to all the 5HT receptor subtypes tested non-selectively, while 5 interacted with the control compounds binding to the 5HT1C and 5HT2 receptors without influencing the 5HT1A receptor.

Lower toxicity Ginseng glycosides which have sedative effect in mice showed LD 5 0 as Rbl lOmg/Kg, Rb2 305mg/Kg, Rc 410 mg/Kg, Rd 324 mg/Kg, Re 405 mg/Kg, Rf 1304 mg/Kg, Rgl 1250mg/Kg, and it was found that the compounds having glucose as sugar moiety have lower toxicity (Li, 1992). Gastrodin, a 4-hydroxybenzyl alcohol glucoside from Gastrodia elata, was not toxic to mice when given orally or intravenously at doses below 5g/Kg, and this compound showed sedative effects in both animal and human subjects (Deng and Mo, 1979). In the present study, the glycosides from Schejflera bodinieri have been tested for cytotocity (by method in Anderson, et al, 1991) and none of them were found to be toxic.

216 Different mechanisms of action A number of compounds isolated from Chinese plants have various mechanisms of actions for the treatment of the CNS disorders. For instance, gastrodin, a sedative and an anticonvulsant, was found to increase the content of the DNA, RNA and glycogen at the heart cell of neonatal rats and the activities of their succinate dehydrogenase, lactate dehydrogenase, and ATPase, indicating that gastrodin can promote the energy metabolism of the heart especially under hypoxia conditions (Huang, et al, 1986). A mechanism in the tranquillizing action of 1- tetrahydropalmatine (from Corydalis spp.) was found to block the dopaminergic receptors without depletion of monoamine stores or to increase the functions of GAB A (Jin, et al, 1983). In the present study, some compounds were able to bind to several receptors, such as hirsutine (36) and epiallocorynantheine (37) were active at ion channel receptors, noradrenaline receptors, 5HT receptors and opiate receptors. As reviewed by Hollister (1994), one type of new antipsychotic agents is drugs blocking several receptors which may be related to 5HT, noradrenaline, dopamine and muscarinic receptors. Moreover, a number of compounds tested in the present work exhibited the ability to interact with the control compounds at two or more receptors. To further understand this type of mechanism of action may be beneficial to the new drug development.

The actions of the constituents in plants are diverse. As showing in this study, they may bind to a specific receptor, or be active at several receptor or affect other compounds binding to specific receptors. The interaction of these compounds have been revealed at the receptor level in this work. The preliminary studies suggested that the isolated compounds may modify the sensitivity of receptors by up- or down- regulating receptor numbers or alter the affinity of agonists and antagonists at specific receptors. Receptor modulation is one of the current trends of new drug development. For instance, antidepressants of the noradrenaline/5HT uptake inhibitor class cause changes in receptor numbers (Hollister, 1993). In schzophrenia, the D2 and D3 receptor density is elevated by 10%, and the D4 receptor density is elevated by 600%. Therefore, to reduce the D4 receptor density may be a target for future antipsychotic drugs (Seeman and Van Toi, 1994). The finding in this work indicated that it is possible to find some compounds from natural products which have the ability to

217 regulate the receptor numbers.

The combination of the effects of several compounds may be the mechanism of action of natural products present in crude extract. Although a single compound may not show very strong effects, the combination of several compounds may exhibit synergistic effects to increase the activity.

Different Chemical structures Natural products provided a rich diversity of chemicals and within each chemical grouping there are minor structural modifications due to the state oxidation or the to substituent present. There are a large number of natural products available and they provide many compounds for testing. The developing of a series of in-vitro tests, which are simple, sensitive and which may provide information at the molecular level (eg. radioligand receptor binding assays), have enabled more and more natural products to be screened for biological activities. As shown in Table 1.6 (p.58) and Table 8.1 (p.210), there is a great diversity of the structures which may be active in the CNS. These natural product molecules may well give excellent leads for the development of new synthetic drugs and in learning about specificity of action. In the present study, the compounds which showed some activities at the receptors screened all had different structures from the existing clinical drugs and some of them only acted on one receptor subtype (eg. compound 2 1 was active at Al receptor). It may well be possible to devise synthetic modifications or to produce completely synthetic compounds which will help in understanding structure- activity relationships in drug design.

Some compounds tested in the present study did not show any activities to the receptors screened. The reasons may be as follows: 1). The compound is inactive. 2). The compound is inactive at the receptors tested, but active at other receptors or receptor subtypes which have not been screened. 3). The compound exerts its effect by non-receptor mechanism (eg. acts on some ). 4). The compound is degraded on the receptor by some enzymes. For instance, from

218 past experience, it is known that the ligands most likely to be degraded in CNS receptor studies are natural products or derivatives of natural products for which brain tissue possesses catabolic pathway (ie.,catecholamine, peptide). Overcoming this catabolism usually involves inhibition of the catabolic enzymes by including various drugs, e.g., monoamine oxidase or peptidase inhibitors (Bennett and Yamamura, 1985).

Therefore, there is every possibility of making further discoveries of new drug from natural products.

The technique of radioligand binding has aided significantly in the process of drug discovery. In addition to being pivotal in the identification and characterization of many new receptor classes and subtypes, binding technology has also permitted an easier means by which to study the receptor-ligand interaction at the molecular level. They not only can be used to examine the activities of a single ligand, but also can be used to observe the interaction between the ligand and its potentiators for both binding activity and intrinsic efficacy. The next decade in receptor related research will see an increased focus on potential ligand interactions with components of the second-messenger systems and with intracellular drug recognition sites. Moreover, while the central nervous system will remain a major therapeutic focus and sources of receptor-rich tissue, the explosion in knowledge in the area of inflammation and immunomodulation will result in a greater dependence on radioligand binding to identify and better define the roles of the many new receptors being discovered in the vascular and immune system (William, 1991). The diversity of the technique of radioligand binding also provides the possibility to discover the multiple functions of the Chinese medicine in different systems in the human body.

It is necessary to mention that the results obtained from the present study are preliminary, since only ten plant species have been biologically screened, four plant species have been investigated in detail, and biological test approaches are limited in the receptor binding assays. To better understand the actions of natural products and develop new medicines from them, this study should be extended at least to include: -to select more plant samples

219 -to investigate more types of compounds -to test the compounds at more receptors -to do the receptor binding assays in more details -to undertake more biological tests which are related to the CNS in both in-vitro and in-vivo.

Chinese and western medicine are two medical sciences with different theoretical systems developed under different historical conditions. They are both the fruits of intelligence and wisdom of all mankind. It is predictable that Chinese medicine and western medicine will gradually merge into a single entity because of the rapid advancement of science and technology in the world and the steady development of Chinese medicine and western medicine in both practice and theory as well as their renewal and mutual influence. Since only 10-20% Chinese plants have been developed in various extent, there is a great possibility to develop new CNS active compounds from traditional Chinese medicine.

220 APPENDIX Page

Spectrum 1.1 HREIMS of compound 1 223 Spectrum 1.2 EIMS of compound 1 223 Spectrum 1.3 ’HNMR of compound 1 224

Spectrum 1.4 ‘^CNMR of compound 1 225 Spectrum 2.1 HRFABMS of compound 2 226 Spectrum 2.2 EIMS of compound 2 226 Spectrum 2.3 *HNMR of compound 2 227

Spectrum 2.4 ‘^CNMR of compound 2 228 Spectrum 3.1 HRFABMS of compound 3 229 Spectrum 3.2 EIMS of compound 3 229 Spectrum 3.3 ‘HNMR of compound 3 230 Spectrum 3.4 '^CNMR of compound 3 231 Spectrum 4.1 HRFABMS of compound 4 232 Spectrum 4.2 EIMS of compound 4 232 Spectrum 4.3 ‘HNMR of compound 4 233 Spectrum 4.4 *^CNMR of compound 4 234

Spectrum 4.5 ‘H-’H c o s y of compound 4 235 Spectrum 4.6 '^C-*H COSY of compound 4 236 Spectrum 5.1 HRFABMS of compound 5 237 Spectrum 5.2 EIMS of compound 5 237 Spectrum 5.3 'HNMR of compound 5 238 Spectrum 5.4 ‘^CNMR of compound 5 239

Spectrum 5.5 ’H-'H c o s y of compound 5 240 Spectrum 5.6 '^C-'H COSY of compound 5 241 Spectrum 5.7 EIMS of hydrolysis product of compound 5 241

Spectrum 6.1 HRFABMS of compound 6 242

Spectrum 6.2 EIMS of compound 6 242

Spectrum 6.3 'HNMR of compound 6 243

Spectrum 6.4 ‘^CNMR of compound 6 244

Spectrum 6.5 'H-'H cosy of compound 6 245

221 Spectrum 7.1 EIMS of compound 7 246 Spectrum 7.2 CIMS of compound 7 246 Spectrum 7.3 ^HNMR of compound 7 247 Spectrum 10.1 FABMS of compound 10 248 Spectrum 10.2 EIMS of compound 10 248 Spectrum 10.3 ’HNMR of compound 10 249 Spectrum 12.1 EIMS of compound 12 250 Spectrum 12.2 CIMS of compound 12 250 Spectrum 12.3 ’HNMR of compound 12 251 Spectrum 13.1 HRFABMS of compound 13 252 Spectrum 13.2 EIMS of compound 13 252 Spectrum 13.3 ’HNMR of compound 13 253 Spectrum 14.1 HRFABMS of compound 14 254 Spectrum 14.2 EIMS of compound 14 254 Spectrum 14.3 HNMR of compound 14 255 Spectrum 24.1 UV of compound 24 256 Spectrum 24.2 EIMS of compound 24 256 Spectrum 24.3 ’HNMR of compound 24 257 Spectrum 24.4 ’^CNMR of compound 24 258 Spectrum 24.5 ’H-’H c o s y of compound 24 259

222 :CM=::1 I=J.3v TIG=3i::%j48 EU rCrC'CL-CP SuStTrS m FAB ,'KEA m i x PT=çy) Cil^CAVa Iwl -35 Ù 1 <3 ACR'i^ -05, ;sc15-C# 33J

: 3:3 H45 C5

-M !

:3 J 467 :9J :3i 33J

i111 I .:::|IH!i.l!!liil! 4W41 ^5 453 455 i;3 475 453 485 458 4% 588

Spectrum 1.1 HREIM S of compound 1

!] :9 9 ::l (I 303=8 I9-40V-37 :3 4-8 88 80 12-359 :3f.=3 I= 7:5.u H,=2930 ::c=877409a8 A'.' Rent Sus ST[>08F R« 49:4888 ■33 RJ5BECH:3H TEMP] E !- PT= 3“ Sat ICRL 5RSS 41 122, 41 Q= t ;i 85 93 75 70 85 82 425 55 52 205 45 40 35 90 177 105 440 23 1« 373 233 273

I, 394 4P4 jjiiKi'iiig 'ii i''!: iili' 7LÙLH-' ■ ‘ i ' " i • • I ‘ i 1 .1 , 3 5 : ; J i | , 1 103 322 408 502 583

Spectrum 1.2 EIM S of compound 1

223 )

C f N M I < OE-JOO

Z H U S U O J f ) 2 b J A N 9 1 UIN hiO K2i CULL 3 uvu OPIRAÎOK OVtt

hJ

^ ------^— W 1___ -I 1---- 1---- 1---- 1---- 1---- 1----r T T 5 1 PPM -O.JSf'PM

Speclmm 1.3 'lINMK of compound 1 j Hsb. KHjUt Ww ÜCH, Lh UNul

JCL3. 13C DEPT CH3. CM *VE. CM2 -VE. C 101-0

h i I Hill llillÜM'l ill/ YJi'iu,

Î :

62 896 N) 6600000 K>Ln '6600 0 0 0

Spectrum 1.4 'CNMR of compound 1 15-11-34 lO:35*0:O9:pg ZFB-ÎE R* A7 3U îtv W %:LTr.3

491 493 495 9 , ?

Spectrum 2.1 H RFA BM S of com pound 2

iE r-5SM .1 :C0=4 2 5 - : : ? - : ; : : 3 2 -j 29 23 :?5£: C - 0,1=9 -!»=2Ea T;C--9c'':5:34 2U 2cr,i ;us LÎEI ■ y. ü 2:.1S ?T: •]'’ û ;fl';C.ÎL yii

Spectrum 2.2 EIMS of compound 2

2 2 6 D4t# P«r«Mters êuQ0693 5

.cquUition P#r##tt#r#

P rO C ItS lfl0 P4T è«8t t r t

4 piQt parABcterf

13 600 ppm y

ti-rr-prm T rT m rrrtrT ri r

Spectrum 2.3 IN MR of compound 2 HI5J 1 1 1 1 J l ^

HT53

‘i V V V s, X, ^ »; :i rj x, rj ij w L is ;; u s. ' '' ^

HI53 ; i- s 1- M r Î r: g ri a ^ Ü :; t. :•: k; S j I; V. !.. j ; xrt : ;• Î' V V? v v ;= ; F. g. IT g; g s, g g s: ii: in g g g g g g is i . g g K 00

iwwm wwwWw##!^ ^ #wWWw%mw»

Speciriini 2.4 'CNMR of compound 2 xl :r:=0 ij! --d 7 : " ;; i Cr 5::# I=l3v Ha=l^a t V J i w 4/ • wvW i 11 ci3-r m \ C^L-FrECrL 933 ?1 __J,3 -8/33339 :a rCra 932.4E24

;3j C47H/4 017K4

c3j ErculB S33.4827 59J

CO I '"I :üj

918 »i< ', :, I III ... ^ 965 919 915 929 925 939 935 940

Spectrum 3. HRFABMS of compound 3

3 ::'3 tf S ll xl 3od=0 9S-.HN-5E 15 23 5 80 08 Z=32F [ I - :;V-0 i=«.3v H.=8:8 115=654334015 5V flcnt 6LEGP Sus LKEIRS HPP 134;3038 5 2 3 -' • : VfiRISUS TEHPEKATQES P1= 8" C it OfiVCflL MASS 34 ■?p 34

<25

308

<41

325 LA 455 <58 4gg . 350 =80 450

Spectrum 3.2 EIMS of compound 3

229 spectrum 3.3 ‘HNMR of compound 3

230 B BO f I

B 8 0 F ^ uj J, r r%c.»-'^€U-'r^r»uia.r«.«t|^cTi<^«r\ir'—• o r - t f ' O ' ^ ^ o r ' r i A » » <^ry-* A<0tp«c>c.r iDir*-*r^(aruiMooiDeC'Oc.ir'0"*'u r>r-A(ri

w B 80 F |g5?SP'2!ï;3Ê?SSSî' u r:"' -:E \ I I II111 Ct il i f r ifiT^ » <•;•>>

ww#W# Ü %/ m iwiwrniW" -I ^rrm-rrrTTTTTTT» ttt ^ttt t

Spectrum 3.4 '^CNMR of compound 3 CCI

Spectrum 4.1 HRFABMS of compound 4

xi ;ao=a 0S-#-9i ;b 24 00 aa ;H-;E3 : . '= 0 i : 2 .;v M .xà'3 TIC=35 50F SusSTEüOOF 13743888 ?T= Cil. ICfiL nass 55

-

427

353

442 433

381

...i. Æ i. 358 488 458

Spectrum 4.2 EIMS of compound 4

232 Ill BLFOME COW f|'.-ilO

u g g ,

ZlllJj.iS 15'0 411 P-flJu < t5 411 04 re i c 13

se jou 1 J5 SY J1Ü >1 01 7 c iO 15 J sr ic7fiO ro 30 760 s o uOcM OOo iiz/oi btit)

no ,1 ) no 0 0 4 0 J 730 Hi. 10 IIS .‘i2 re c'j7 e .1 7i 00 0 3 0 to l j U) fiP .31 PI U) 1 11 l‘)0 GO 0 I) CX J4 nu r:Y 3 i 00 FI to ‘jOlH F 3 ■JOjH llZ/IH 9/ 100 PPM/CM StI 40 Jj 27

U ,a,'F v 4J I __

10 0 Q 0 8 5 8 0 7 . 5 7 0 o 5 6 0 5 5 5

Spectrum 4.3 'HNMR of compound 4 Gt: 'T[-

A Z l l D - t 1 0 IFLHj ZMii UIN dU) : ûhEK^fOH UvU ONt HUI j t i£ ‘j.. kW4«*W,# PULSt * lu lll -

ua Of AuO> • LInC BhO^uuu ■

f HI ÜiJt'Ji-f - iPEL V.IÜIH- U| lOUKI t H u f ' rHrOtlLKk.1 - WW#,!|lYrW

K) UJ Vu tW

spectrum 4.4 '^CNMR of compound 4 K-'-> \

"B 8 " 150 ‘jMv F1 pnn I PI(II.IH) 001 KirPHO PIIIIJ. nil 001 AU onuG uAFtZ c 7 All3-? aa ria I0J4 ______It___ L, SI 1 5ia SWT i f ' 7 7 a c SHI 70 9 GO NIlO I

HUH.' W tlHl SSPIssaa m M-I

tu ni 7-'0"( Ul 6 IIGA ( ip 0 0 I'H 0 !■ NJ UF 3 'P 0; U) NS Ln p ? PO ' 10.10 UE 1 . ’=' IN 00 Of

5 . 0 0 1 5 1 0

Spectrum 4.5 'll-'ll COSY of compound 4 C-H CORRELATION CD30D

/n il IJ 150 S lU FI RROI PRO.III! 00 1 F 2 PIUI J PuuJ>: 001 All PfUlb ZIJ All [lAlE 2 -2 02

S l 2 20 4 b GI 1 2 5 6 Ck2 1 2 0 2 0 5 1 4 5W1 171)*, li5o lino 2

WI1H2 0 HllWl r , t 02 15 ;uHi 1 111 15 01,0 C.I12 0 0 OUI 0 0 ML 2 M PI IN Inin F 1 104 'I 4 IP F 2 4 70 4P Atm LOi iinii FI to 'I'll,' F2 9'-. ,.P

in 2 ooOOuOO £ j on PI 11 5 ' no lin o,,0 40 N) F 6 9 no U) 02 noJoOOO P5 4 JO Ov 1,4 001 iioOi, 2r n ROA nil n 0 PW 0 0 OF 51 JO US 1 70 ns 2 P9 172 on NE 64 IN 0 0 0 1 -.O'J 160 150 140 1 2 0 n o 0 0 ‘JO HO r.O 50

Spectrum 4.6 COSY of compound 4 *2i;i9K :ç-'j Î3-AI--4 a - s f~ :.H i=iSu :-;!=!:C3 Ti'>!:3ôK40:3 S Sea'US? Sst7f3 B5 Fa::A"iX:KEn rT= 3'' CÂraCAL K5.3 51 1,3

IBcAKO

Lv :

935.4352

iJiiJiiiiliidi! I 929 905 310 315 320 %5 g 935

Spectrum 5.1 HRFABM S of compound 5

2 ::2 5 8 4 ll xl Bcd=3 22-3CT-9I 11 5-8 8 8 83 12-858 E i- 3:fl=3 M 2.5v it'-izi TIC=51157S888 ÎV Acnt ÜLSOP Sus STEHDE' m- 13/33388 352T Ei VR8I0US TEMFEZATUKES FT= 3 ' C il ;C,=L 3ASS 38

383

442

488

Spectrum 5.2 EIMS of compound 5

237 /

w 00

IftL.

Spectmni 5.3 IN MR of compound 5 H-23-7 IN C0300 -HEF . MM290 13C DEPT CH ONLY 1 Jll I

H-23-7 IN CD30D. 13C DEPT CM3. CH *VE. CH2 -VE. C (01-0 M

II]T ! ! il 1

Iji, mt'

H-J3-7 IN CO300 -REF . MH290 13C llHl bWji^ r . .rnX' 1 ^T.ij.11. C3842S 001 ÀU PROG; eaoEPxzs 4U w TIME S 2 8 SF 82 898 VO SY 93 6600000 01 93300 000 SI 32760 TO 32768 SW 16666 667 M2/PT 1 017 PW 3 0 RO 0 0

FM 20900 02 9200 000 OP 14H &a LB 2 000 68 450 CX 40 00 CY 8 00 F| 198 0Û7P F2 -I 999P hZ/CM 314 484. PPM/CH 9 000

spectrum 5.4 ‘^CNMI^ of compound 5 ZHU M 23 7 CD300 ÛV3

^

1 ^3t=>

1 1

! 0 î

1 1

g

è

- 1 -

1 ; 1 ! a ^ I

j i

! i

'CZ> 1 1 1 1 ! 1 1 ...... J , 1 3. 5 5 Ü 4 3 4 0

Spectrum 5.5 11-11 COSY of compoiintl 5 ;;V:

2 . 5 0 5

Spectrum 5.6 I3r-C- 1H C O SY of com pound 5

33:21411 xl 100=0 lB-SOV-97 14 H ?B 38 1 2 -1 :8 E l- .'=3 :=l:3,u T1[:443318B0 3V lent lys STEIOEF 118 1924888 55H ?!C5E [I- ?T= 3" Cat 1C2.L 3SS3 138

41 1

135 175 427

185 207 363 113 442 275 489 223

147 ISI 231 392 !■ < II i l :Lii: f 1 1 : 108 lie 180 408 4:0 189

Spectrum 5.7 EIMS of hydrolysis product of compound 5

241 ü;:i=v :=i^/ ;i'; Fcrc^rrr L/," i,r;L:n * Xn r! = CcUFFSC-l iOvl 32/ 1.3

%5 9:^ 9;:3

spectrum 6. HRFABMS of compound 6

3 ! W : l t <: 3co=9 îî-m-i\ !5 ; - i îi 33 !2-3 :3 [ ! • 3oM l--2.;« :iC=S43S23908 W Act L123P iu s STi^OEF 13314383 3123-2 21 .nî:;:S lE.APtxfllUKÎ ?T-' 3 ' û 1C.ÎL

M uaMhliuiiiiiiii uni

Spectrum 6,2 EIMS of compound 6

242 ZhU 350-J Z3300 3vE

ZHU205 133 AU =AOG <00 AU DATE 2 6 - 1 - 9 2

300 135 3 Y 210 0 01 7239 153 SI 3 2 7 5 3 :o 3 2 7 5 3 SK 60 2 4 ■795 6Z, •PÎ 363

FW 0 .4 30 0 7 AJ 3G 2Ô NS 32 Tc 297

'6 0 0 02 OP SOL 30

l 3 300 00 0 7 Z < 34 00 Cf 23 00 10 SO tP -à 495P HZ''CM 97 ICO 30M/CH 324 SO 4539 27

7 .0 5 56.0 5 . 5 5 0 4 5 4 0 3 .5 3 0 2 5 2.0 1 .5 1.0 5 0.0

Spectrum 6.3 'HNMR of compound 6

243 G E N M R Q E - : 5 0 0

A71IU44 044 0 2 E E B 9 1

ZHU UIN W5Û 4 CU30D DVB

OKEHAioH ova

owe PULSE SEÛUtNCe

PUISE WIDTH - iO^DtthtCS BI9 iO U^EC

NO Of ACOS - 409 b OAIA SIZE • J 276& LINE 8 HCADHC - 2 00 HZ S PIN RATE " 16 NPS

fHEOUCNCr - 7S 493)24 LIlZ

DECOUPLER SlANDARU-64 UOUULAllON fREOuENCT - 4 000 PPU nXT'* POWER • 3 ) 00/ 2900 PLOT SCALE S 1Û 72 HZ/CU 6 76SI PPU/CU fROW 22S 00 TO 4 99 PPU I

n I I I I I 1 I I p * I r ^ I I i i i I T I I I r~ T ' I I I 1 BO 160 140 120 100 80 60 40 20 0 P P M

Spectrum 6.4 '^CNMR of compound 6 ZHU B 5 0 - 4 COSY CD30D DVB "g% " -'H ll.'JlJll St IX F 1 PfIO 1 O lidlM l (10 1 F<; M id 1 PliCildl 00 1 Ad Odd,, Ic:) Ad DAIl; a ; 1 - da

SIJ 1 0 /4 SI 1 s i a ___•-- ——— J,—■" - SW2 16.Î3 d o / SHI BIB J31 ddO 1

------Hdrli s (= s HdWl s S'.!W 0 c . ,C3 5SÜ1 0 ----- i M dt3 KsSSr OL Id ddw P 1 I. ( I'll- a t- Fa S /tiP ------Add Ldi.tiMd F 1 1, 0 .,0'

ltd 0 0 CD OH 0 (, d t j u b 00 <3 & dS -1 Ln j p IIS a t r ^ \ (10 o OOOOao ME i .;b j j 0 Id 001,61 10 fS L ^ O

o

as'' ... c d S S ^

J b J O h'f'H

III I Spectrum 6.5 H- II C O S Y o f com p ou n d 6 ÎEE'SeîJl x! 8 :0=0 3S-.W-52 15 \i-i 03 êâ ZEi;.- [> =:3v H.=533 î ::= S2 :;2 3 5 ;5 : 3V a c :: VL:2= :y s L!E!'.S H3S J3353333 333 3: vR'IOjS lïP[;:Ti:?.EE ?r- a= Û. zavcîL rR-R ;33 . 3! :: :33 :31

, i | l '

349358 I

Spectrum 7.1 EIMS of compound 7

335883511 xl 8 qa =8 I P - J N - K 11 3*0 80 8 8 12-358 ’S- 3:1=8 I=S.3tf H,=1308 T!C=5/0B52332 AV Sent 'ÜLSOP Sus 'SPSC.M HHR 48785880 353 IHÎÎlOSPIWr ?T= 8 « Cat •8 SS 143 181, •XlO-4

833 1113

Spectrum 7.2 CIMS of compound 7

246 IM CUJOÜ «IMS .(MJiO IH

250.134

2 179

2 9 7

63L PO

_ ! .■■■■1. 7 50 7 OP T lip 5 50 5 00 ^ 4 50 4 00 1 *5p“ ' ' '"l" H

Speclmm 7.3 IN MR ol' compound 7 5:.1=] !=:.2u i,=|:â3 T::=::4/::23a 4V S:r,i ;us TS'O 3=2:3209 'F :3 ^ ^ :<3 •X 1 3 >0 U 3

32 I I

=2 .

■2

:?

18 3 /8

8 ■888

Spectrum 10. FABMS of compound 10

522=82311 x! 3cq=8 8B-3P.N-:2 15 :7 -9 8 8 89 3P.E2F [ I - 3cM:8 1=18" H.=850 TICx5E8S51212H AV Rent iJLSDP 3us LÎEÎSS PUR :2390888 398-15 U VrxiCUS lEflPExPTUPES ?T= 8 “ Û JfiVC.AL ■SSS 34 188, 34

105 135

364 428

418 443

<86 jlLiJ1 1 ^ 150 288 258 300 358 488 458

Spectrum 10.2 EIMS of compound 10

248 IN LU3UI1 «IMS IN

H3646? 001 ÜAIE 0-iï-ai t i m e 12 21

37S9 396

200

9 60IP

to

IIII

S p ectru m 10.3 INMK of compoiiiKl 10 .22? 2ys 2 :9 " ] 2 ! .F 24 ': 2ai ]SvC:L

2 9 .

- i . ,

(2 :

299 iS tail*

S p e c t r u m 1 2 . 1 EIM S of com pound 12

23:9922:: Xl 9o:=9 ;7-j=H-=2 !: 9*9 90 03 1 5 -H5 9 75* ■zr-i :=2.2v H,:!E90 ’IM073::9SS3 0V flcnt X2ÎP Sus !£?2CN ■i.'s 5 : 0 : 9 9 9 3 5630 THEJflQSrsnY • p ": j** jiL

100, M9

93

68 7;

65 60

50 45

35

10 25

20 15

10 5 0 idtillj ihAAJIIililLjl'm j l i t I j ukx.I 400 '8 8 838 1000 1280

Spectrum 12.2 CIMS of compound 12

250 eao-o IN cuipo *ihs. Mwaso in

OAIE 8-12-91

2S0 134

SI 16384 TO 16384 SM 3759 HZ/PT

297

MZ/CH 62 927

LnNJ

Spectrum 12.3 'UNMR of compound 12 ■ - M . . r . , /I

S p ectru m 13.1 HRFABMS of compound 13

::S3743I1 d 300=8 as-#-31 17 2-8 88 38 12-2S8 [ ! • =5.9v H.:638 TIC=ScaeB:380 3V flcnc 'JLSOP Sus STEMOEF HUB 17173398 5121 Cl ?T= 8 * Û 1C2L 18SS 55 :8 L CÇ : S3 j

63 J

189 387

388 508

Spectrum 13.2 EIMS of compound 13

252 ZllU. .} 'JIN

U)K

Spectrum 13.3 'HNMR of compound 13 xl -cc-.î VS.U -r i? ' LV -:: ::4=:] >lbi - - = ! ; : 3 :V nCTiUcC? SystTS 1 2 2 F:::;S .'f,?::: PI= Csl:FrKrl 1S9_ 3 4 ;.: 51 1.3 4c:m ■Cci

S p e c t r u m 14.1 H RFA BM S of com pound 14

3isa;47ii xl 8 0 0 = 8 8 5 -W -9 1 17 8 4 8 8 38 12-253 5 I- 9d /1:8 =5E2»v H«=b58 TIC=2<5S5888 RV flcni ULSOP Sus 5TLR05F HW 737800 3122 [I PT= 3" Ù ICHL MASS 53

233

58 188 158 IBG 358 488 580

Spectrum 14.2 EIMS of compound 14

254 u»«

00 o o I f ) o Cû00 o en eunj o 0 0 GO ai

PPM Spectrum 14.3 'HNMR of compound 14 MeOH

MeONA

200 250 300 350 400

Spectrum 24.1 L'V of compound 24

.1 :cs=a ;3-” 2-22 !c 25-i 23 23 :=E2r C - -«=253 ':c=?;/"2 2 dis av act .le:? Eus û[;.’s ■:î3 .«!::s ■■.v5.î.3T'jîE3 :T= i' :,ivc=L '.21 -

53. .

581 30g 431 -iIa , 4 8 8

Spectrum 24.2 EIMS of compound 24

256 + HOP suppress»'

K) ■-JLA

1 L 1 I. I. I c'o

Spectrum 24.3 'IINMR of compound 24 AZHU-i3. 043 0 2 F E 3 9 1

:h u u :h h 2t 4 C3200 o^a

3PERATCM ova

ONE »ULSE SEOUCHCE

^ULSE éflOTH • 4 83 JE E : 30 jEi^EES *cQ. ? : u c - 819 20 w s e: RECYCLE T:UE • 2 81 3EC

•4Û or 4C0S. - 4098 0KT4 SI:t - 32788 L:sE 990A0NC • 2. 30 HZ S riN * a TE . 19 9PE

'■^EOu Èh CY « 75 49512* yn: S r ç c iVIOTh - 20000 h 2 5AIH . 90 .8

OECOV'lER 5TAHO*RO-«4 wooi / l a t : on •EOUENCY - 4 300 • • « OWCR • 3100/ 2900

'LOT SCAAE *510 72 h Z/CW 8 785J ••y/CW «"ROW 225 30 TO -* 39 "V

120 100 30 50 •5-U Z J 0 P = V

Spectrum 24,4 ‘^CNMR of compound 24

258 ZHU H 21 4 COSY C D 3 0 0 Ü V B

to LA VO

7 0 b b b 0 b u 4 b J Ü 2 0 1 b

Spectrum 24.5 COSY ot compound 24 References

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