THE SEARCH FOR ANALGESIC COMPOUNDS FROM HIGHER PLANTS
Thesis presented by
JULIA H. SAMPSON
(B.Sc.)
for the degree of
Doctor of Philosophy
Department of Pharmacognosy The School of Pharmacy University of London 1995 ProQuest Number: 10104867
All rights reserved
INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest.
ProQuest 10104867
Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author.
All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC.
ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 TABLE OF CONTENTS PAGE NUMBER
Acknowledgements 1 Abstract 2 List of Abbreviations 5 List of Figures 6 List of Tables 9
CHAPTER ONE ; INTRODUCTION
1.1 Introduction 11 1.2 Uses of Plants 12 1.3 Initial Separation of Phytochemicals 13 1.4 Medicinal Plants for Use as Potential Analgesic Drugs 16 1.5 Alternative Health Care 19 1.5.1 Plants in Alternative Systems of Medicine 19 1.5.2 African Medicine 20 1.5.3 Ayurvedic Medicine 20 1.5.4 Traditional Chinese Medicine 21 1.6 Documenting and Evaluating Herbal Remedies 21 1.6.1 Intraspecific Variation 21 1.7 Secondary Metabolites Derived from Plant Metabolism 21 1.7.1 Alkaloids 28 1.7.2 Phenolics 31 1.7.3 Terpenoids 35 1.8 Nociception in the Mammalian Central Nervous System 37 1.9 The First Opioid Agonists and Antagonists 38 1.10 Non Opioid Analgesic Drugs 38 1.11 Mediation of Pain in the Mammalian CNS 40 1.12 Inflammation and Nociception 41 1.13 Discovery of Bradykinin and its Physiological Effects 43 1.14 Kininogens, Kallikreins and Kinins 45 1.15 The Mechanisms of Bradykinin Sensory Neurone Excitation 45 and Sensitisation 1.16 Bradykinin Receptors 48 1.17 Characterisation of Bradykinin Receptors 50 1.18 The Rationale for New Bradykinin Antagonists 52 1.19 Signal Transduction Mechanisms of Bradykinin Via 53 BK H Receptor Subtypes 1.20 Seven Transmembrane Receptors 54 1.21 The Structure of G-Protein Coupled Receptors 55 1.22 Signalling by Seven Transmembrane Receptors 56 1.23 Regulation of Bradykinin Activity 57 1.24 CGRP and Mediation of Nociception in the Mammalian CNS 59 1.25 CGRP Immunoreactivity 59 1.26 Physiological Effects Mediated by CGRP 60 1.27 CNS Actions of CGRP 61 1.28 The Rationale for the Use of a CGRP Antagonist in Migraine 62 CHAPTER ONE : INTRODUCTION
TABLE OF CONTENTS PAGE NUMBER
1.29 Co-localisation of CGRP with Other Neuropeptides 63 1.30 CGRP and Calcitonin Receptors 64 1.31 CGRP Receptor Agonists and Antagonists 65 1.31.1 CGRP 8-37 65 1.31.2 [Tyr]CGRP28-37 66 1.31.3 Human Calcitonin 66 1.32 CGRP Receptors Expressed in the SK-N-MC Cell line 66 1.33 Cleavage of CGRP 66 1.34 Substance P and the Neurokinin 1 Receptor 67 1.35 Aims 68
CHAPTER TWO: MATERIALS AND METHODS PAGE NUMBER
2.1 Considerations for Binding Assays 72 2.2 The Initial Development of the Bradykinin Radioligand 73 Binding Assay using a Rat Uterus Membrane Preparation 2.3 Cloning of the BK H Receptor in Chinese Hamster 74 Ovary Cells 2.4 Expression of the BK H Receptor in Chinese 75 Hamster Ovary Cells 2.5 Assay Conditions for the BK H Screen 77 2.5.1 Radiochemicals 77 2.5.2 Stock Radioligands 77 2.5.3 Non-Radioactive Ligands 78 2.5.4 Determination of Non Specific Binding 78 2.6 Separation of Bound from Free Ligand 77 2.7 Measurement of Radioactivity for Bradykinin 78 2.8 Data Analysis and Capture 78 2.9 The Preparation and Use of CGRP Membranes 79 2.10 Assay Conditions for the CGRP Screen 79 2.10.1 Non-Radioactive Ligands 79 2.10.2 Measurement of Radioactivity and Data Analysis 79 2.11 Determination of the Protein Content for BK H and CGRP 80 2.12 Time Course Studies for the BK H and CGRP Assays 81 2.12.1 Calcitonin G^ne Related Peptide 81 2.13 Saturation Analysis of the BK H and CGRP Receptors 83 2.13.1 Scatchard Analysis 83 2.14 Association and Dissociation Studies 83 2.15 Characterisation of the BK H Receptor 83 2.16 The Neurokinin 1 Receptor Radioligand Binding Assay 84 2.17 Composition of Solutions 86 2.18 Assay Protocols for Bradykinin BK H and CGRP 87 2.19 Assay Protocol for the CGRP Binding Assay 87 CHAPTER TWO ; MATERIALS AND METHODS______PAGE NUMBER
2.20 Assay Protocol for the BK H Binding Assay Using Rat Uterus 88 Membranes 2.21 Thin Layer Chromatography for Detection of Terpenoids 88 2.22 Preparation of Plant Extracts for Screening in in vitro 89 Binding Assays 2.23 Extraction and Isolation of Bioactive Entities 91 2.24 Plant Sample Preparation for Extraction and Isolation 91 2.25 Sample Processing on LH20 Columns 91 2.26 Automated Multiple Development-High Pressure Liquid 92 Thin Layer Chromatography 2.27 Preparation of Plant Extracts for in vivo Tests 92 2.28 Preparation of Mice for the in vivo Screening of Plant Extracts 94 in the Acetic Acid Writhing Test.
CHAPTER THREE : RESULTS______PAGE NUMBER
3.1 The Results of Screening Methanol Plant Extracts in the in vitro 95 Bradykinin BK H Rat Uterus Membrane Preparation 3.2 Results of the Development Experiments for the Bradykinin 96 BK H Assay. 3.3 Time Course Studies for the Bradykinin BK H Assay 96 3.4 Dissociation Studies of the Bradykinin BK H Receptor 96 3.5 Scatchard Analysis of the Bradykinin BK H Receptor 103 3.6 Displacement Curves of Bradykinin Analogues in the Bradykinin 109 BK H Assay. 3.7 Results from the CGRP Radioligand Binding Assay : Time Course Studies 112 3.8 Scatchard Analysis of the CGRP Receptor 114 3.9 Displacement Curve of CGRP in the CGRP Assay 116 3.10 Results of Screening 300 Ethnomedically Selected and 335 140 Non-Selected Plant Extracts in the Bradykinin BK H Assay 3.11 The In vitro and In vivo Results of the Methanol Extracts of Panax 143 ginseng and Ipomea pes-caprae 3.11.1 The in vivo Results of Panax ginseng and Ipomea pes-caprae 153 3.13 The Extraction of Bioactive Entities from Symplocos leptophylla 154 3.14 The Procedure Followed for the Extraction of Bioactive Entities 155 from Symplocos leptophylla 3.14.1 Results of Symplocos leptophylla using AMD 158 3.15 Displacement Curves of Fractions 1-8 from Symplocos leptophylla 174 in the Bradykinin BK H Assay CHAPTER FOUR ; DISCUSSION______PAGE NUMBER
4.1 A Discussion of Methods Available for Selecting Plants 176 for Analgesic Bioactivity 4.2 Discussion of Results from Screening Non-Selected and 181 Ethnomedically Selected Plants for Novel Leads to Analgesics 4.3 Non Peptide Bradykinin Antagonists 182 4.4 The Partial Purification of Symplocos leptophylla 188 4.5 Non Selective Analgesic Effects of Plants Used in Traditional 190 Medicine : Discussion of In vivo Results 4.6 Considerations of the Difficulties Encountered in Bioassay Guided 194 Fractionation of Plant Extracts 4.7 Final Conclusions 195 4.8 Future Work 196
APPENDIXES 217
REFERENCES 234 ACKNOWLEDGEMENTS
I very gratefully acknowledge and thank my supervisor Professor J.David Phillipson for his help, guidance and friendship throughout the three years spent studying for this Ph.D., which has contributed to my interest and enthusiasm for this work. I am also indebted to him for his help in editing this thesis.
My sincere thanks are also expressed to Professor Norman G.Bowery for his help and support whilst studying in the department of Pharmacology at the University of London, and for his help in editing this thesis.
I am indebted to Dr. M.J.O’Neill, Dr. RM.Tait and Dr. J.G.Houston at Glaxo Group Research for their support and unlimited use of facilities during this research. My thanks also to Dr .J.A. Lewis in the division of New Lead Discovery for the supply of plant samples and to Mr. J.G. Farthing for his help and supervision whilst carrying out the chromatographic techniques.
My thanks are also expressed to Mr. S. Fogarty in the department of Biomolecular Screening at Glaxo Group Research for help with the radio - ligand binding techniques used in this project, to Ms. T. Shaw-Hamilton for screening plant extracts in the Neurokinin 1 assay, to Mr. J.Coote for the preparation of CGRP membranes and to Dr. G.Vassart at the University of Belgium for the Chinese Hamster Ovary cells which express the bradykinin BK II receptor. I am also grateful to Ms. Marzia Malkangio from the department of Pharmacology, University of London for her help with the in vivo animal studies, and to Dr. Ian Bates in the department of Pharmacy Practice at the University of London for his kind help with the statistical analyses used for the in vivo research.
I am very grateful to Ms. P. J.Harrington for the preparation of the results tables.
Thanks are given to my parents for their encouragement and support, and I am indebted to my partner Shaun for his patience, help and encouragement throughout these three years.
I would like to thank the BBSRC (formerly SERC) for the provision of a grant enabling me to study for this Ph D and to Glaxo Research and Development for their sponsorship.
I would also like to express my appreciation both to Glaxo-Wellcome Medicines and the Department of Pharmacognosy, University of London, who enabled me to present a paper at the Third International Congress of Ethnopharmacology, held in Beijing, China in September 1994. ABSTRACT
Many important modem day plant dmgs have been discovered by following leads from traditional folk use. The aim of this work was to select plants from the medicinal and scientific literature of China, the West Indies, South America and West Afiica, which have been used traditionally for the treatment of pain and to screen them in the bradykinin BK II and calcitonin gene-related peptide (CGRP) binding assays in an attempt to find new leads to analgesic compounds. More than twenty endogenous neuropeptides have been imphcated in the mediation of pain within the mammalian central nervous system including bradykinin, CGRP and Substance P. The bradykinin BK II and CGRP in vitro radioligand binding assays were developed in order to screen plant extracts for new leads to analgesic compounds. Bradykinin is involved in the mediation of acute pain and a number of bradykinin peptide antagonists are available for in vivo and in vitro use, but there is a need for antagonists which are selective and stable in vivo. CGRP is thought to be a mediator of migraine and other vascular headaches and there is no single drug to prevent the development of migraines after the initial onset. Thus new non-peptide lead molecules to CGRP antagonists are required. The plant extracts which produced positive activity in the assay were treated with polyvinylpyrrolidone (PVP) to remove tannins, thus reducing the possibility of obtaining false positive results due to the non-specific binding of tannins to proteins. Three hundred ethnomedically selected plants and 335 randomly collected plants yielded 21 plants with apparent specific activity in the bradykinin BK II assay, of which 19 were ethnomedically selected. Five plants had selective activity in the CGRP assay. The extract of Symplocos leptophylla produced the most potent and selective inhibition of the BK II receptor and was chosen for further investigation in attempts to determine the active principle(s). The extracts of Ipomea pes-caprae and Panax ginseng inhibited binding of bradykinin to the BK II receptor and Substance P to the Neurokinin 1 receptor, and were thus deemed to be non-selective in the BK II assay. The extracts were tested in mice in the in vivo acetic acid writhing test and /. pes-caprae was found to significantly decrease the number of writhes when compared to control values, thus /. pes-caprae may have some broad based analgesic effects. Low throughput screens such as the bradykinin BK II and CGRP assays may be used in conjunction with ethnobotanical literature as a means of identifying plant derived natural products as leads to new analgesic compounds. LIST OF ABBREVIATIONS
a Alpha
ACE Angiotensin Converting Enzyme
ANS Autonomic Nervous System
AFP Aminopeptidase
ATP Adenosine Triphosphate
AMD Automated Multiple Development
AMP Adenosine Monophosphate
p Beta
BK Bradykinin
cDNA Complementary DNA
CGRP Calcitonin Gene Related Peptide
CHO Chinese Hamster Ovary
CNS Central Nervous System
CPN Carhoxypeptidase N
CPM’s Counts Per Minute
CT Calcitonin
Da Daltons
DNA Deoxyribosenucleic Acid
DPM s Disintegrations Per Minute
EDRF Epidermal Derived Growth Factor
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylenehis(oxyethylenenitrileo)tetraacetic acid
E.L Electron Impact
FAB Fast Atom Bombardment
FAG Food and Agricultural Organisation of the United Nations
GDP Guanosine Diphosphate
3 GTP Guanosine Triphosphate
Proton
HBSS Hencks Buffered Salt Solution
HEXES Hydroxyeicosatetraenoic
HPLC High Pressure Liquid Chromatography
HPTLC High Pressure Thin Layer Chromatography hCGRP Human Calcitonin Gene Related Peptide
5-HT S-Hydroxytryptamine
IL Interleukin
L P. Inter-peritoneally
L Litre
MeCN Acetonitrile
MeOH Methanol
mRNA Messenger Ribosenucleic Acid
MW Molecular Weight
MS Mass Spectrometry
NEP Neutral Endopeptidase
NKA Neurokinin A
NMR Nuclear Magnetic Resonance
NSAID Non Steroidal Antiinflammatory Drug
NSB Non Specific Binding
PA2 Phospholipase A2
PAF Platelet Activating Factor
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PGI2 Prostaglandin 12
PVP Polyvinylpyrrolidine
rCGRP Rat Calcitonin Gene Related Peptide SPE Solid Phase Extraction
TCM Traditional Chinese Medicine
TNF Tumour Necrosis Factor
TOF Time Of Flight ugs Microgrammes mgs Milligrammes g Grammes pM Picomolar nM Nanomolar uM Micromolar mgs/ml Milligrames per Millilitre
NAPRAleit Natural Products Alert
TLC Thin Layer Chromatography
WHO World Health Organisation LIST OF FIGURES
CHAPTER ONE
Figure 1 Narwedine
Figure .2 Reserpine
Figure .3 Vincristine and Vinblastine
Figure .4 Strychnine
Figure .5 Physostigmine
Figure .6 Morphine and Papaverine
Figure .7 Tetrandrine
Figure .8 Dendrobine
Figure .9 Cassinine and Deoxynupharidine
Figure . 10 Thionupharidine
Figure .11 Jacobine and Senecionine
Figure .12 Protoverine, Conessine and Funtamine
Figure .13 Kurchessine and Irehine
Figure .14 Cocaine and Hyoscyamine
Figure .15 Agrimonin, Rugosin D and Corilagin
Figure .16 Formononetin and Genistein
Figure .17 Kaempferol, Quercetin and Myricetin
Figure .18 Acacetin and Isorhamnetin
Figure .19 Apigenin and Luteolin
Figure .2 0 Naringenin, Catechin and Hesperetin
Figure .2 1 Camphor
Figure .2 2 Menthol
Figure .23 Abcisic acid, Farnesene and Gossypol
Figure .24 Giberellic acid
Figure .25 Aescin and Glycyrrhizin Figure 1.26 Digitogenin, Digitonin and Diosgenin
Figure 1.27 Simplified Diagrammatic Representation of the Main Neuronal Pathways Involved in the Transmission of Pain.
Figure 1.28 Kinin Formation.
Figure 1.29 Physiological Stimuli Required for the Synthesis of Bradykinin
Figure 1.30 Model of the Structural Domains of G-Protein Coupled Receptors.
CHAPTER TWO
Figure 2.1 Plant Sample Preparation for Screening in in vitro Radioligand Binding Assays.
CHAPTER THREE
Figure 3.1 Time Course Study for the Bradykinin BK H Assay
Figure 3.2 Association and Dissociation Studies for the Bradykinin BK H Assay
Figure 3.3 Scatchard Analysis of the Bradykinin BK H Receptor
Figure 3.4 Displacement Curves of Bradykinin and Hoe - 140 in the Bradykinin
Assay
Figure 3.5 Displacement Curves of Bradykinin Analogues in the Bradykinin
Assay
Figure 3.6 Displacement Curves of Bradykinin Analogues in the Bradykinin
Assay
Figure 3.7 Time Course Study for the CGRP Assay
Figure 3.8 Scatchard Analysis of the CGRP Receptor
Figure 3.9 Displacement Curve of CGRP in the CGRP Assay
Figures 3.10 - 3.17 Displacement Curves of Fractions 1-8 from Symplocos leptophylla in the Bradykinin BK H Assay
Figure 3.18 A Chromatogram of Fractions 1 - 8 from Symplocos leptophylla
Figures 3.19 - 3.26 ^H NMR Spectra of Fractions 1 - 8 from Symplocos leptophylla
1 LIST OF TABLES
CHAPTER THREE
3.1 The Results of Screening Methanol Plant Extracts in the in vitro Bradykinin BK H Rat Uterus Membrane Preparation.
3.2 IC50 and Ki values of Bradykinin Analogues
3.3 The Positive Results of Screening 635 Methanol Plant Extracts in the Bradykinin BK H Assay.
3.4 The Influence of PVP on the Activity of Plant Extracts in the Bradykinin BK Assay.
3.5 Serial Dilutions of the Most Potent Methanol Plant Extracts in the Bradykinin BK H Assay.
3.6 Plant Extracts with Selective Activity to the Bradykinin BK H Receptor.
3.7 Isolation of the Bioactive Fractions from the Stem and Bark of Symplocos leptophylla in the Bradykinin BK H Assay.
3.8 The Results of Screening the Methanol Extracts of Panax ginseng and Ipomea pes-caprae in the Neurokinin 1 Assay (NKl) Receptor Ligand Binding Assay.
3.9 The Results of Panax ginseng and Ipomea pes-caprae in the in vivo Acetic Acid Writhing Test.
3.10 The Results of Mass Spectral Analysis of Fractions 1-8
3.11 The Results of Screening 635 Plant Extracts in the Calcitonin Gene Related Peptide Assay
3.12 The Results of Treating the Methanol Plant Extracts Producing a Positive Result in the CGRP Assay with PVP.
3.13 Displacement Curves of the Methanol Plant Extracts of Physostigma venenosum and Typhonium giganteum in the CGRP Assay After Treatment with PVP.
3.14 The Displacement Curve of Physostigmine in the CGRP Assay.
3.15 Plants with Selective Activity to the CGRP Receptor.
8 3.16 The Bioactivity of Symplocos leptophylla in the Bradykinin BK H Assay Prior to Treating with PVP.
3.17 The Bioactivity of Symplocos leptophylla in the Bradykinin BK II Assay Following Treatment with PVP
3.18 The Dose Dependant Relationship of Symplocos leptophylla in the Bradykinin BK II Assay
3.19 IC50 Values for Fractions 1 -8 from Symplocos leptophylla in the Bradykinin BK II Assay
CHAPTER FOUR
4.1 Documented Phytochemistry of the Symplocos Species CHAPTER ONE INTRODUCTION CHAPTER ONE 1.1.INTRODÜCTION
'Dark behind it rose the forest Rose the black and gloomy pine trees'
The Song of Hiawatha, iii Hiawatha's Childhood. H.W. Longfellow 1807-1882
Dark behind us rises the forest and within it lies possibly the greatest treasure chest of unidentified plant species containing potentially thousands of compounds with unknown biological activities. On a worldwide scale it is estimated that the plant kingdom contains approximately 250,000 species of higher plant (Farnsworth et al, 1988), with the tropical rain forests containing a high percentage of these. The number of seed plants occurring in the tropics is estimated to be around 155,000 (Prance, 1977) of which approximately
120,000 (including 30,000 undescribed species) occur solely in the tropical moist forests
(Myers, 1987) and of these three-fifths occur in tropical America and one-fifth in each of tropical Asia and Africa.
According to an estimate of the World Health Organisation, around 80% of the people in developing countries rely on traditional medicine for their primary health care needs, of which a major portion involves the use of plant extracts or their active principles
(Farnsworth et al, 1985). In spite of the extensive use of plants for medicines it is estimated that only 5% to 10% of all plants in the world have been systematically investigated for their pharmacological activity (Baerheim-Svendsen, 1984). Great differences exist between different geographical areas, where richer countries can afford research and pharmaceutical products that are not within reach of poorer individuals and countries. In well developed countries drugs are available through a system that requires
10 products to be profitable and some pharmaceutical companies believe that plant based research is not profitable. There are also many sceptics who regard the use of plants and the medicinals derived fi*om them as either of Uttle use when compared to synthetically derived drugs or see their use as 'old wives tales', with no real scientific value, hence they are brushed aside and are of no use or interest. However, it is a fact that many important modem day plant dmgs have been discovered by following leads fi^om traditional folk uses e.g. digoxin used for the treatment of congestive heart failiure, is derived fi'om the foxglove Digitalis lanata, the sedative and analgesics, morphine and codeine, are derived from the opium poppy, Papaver somniferum, and quinine from Cinchona sp. which is used in the treatment of malaria. It has been calculated that 74% of the 121 biologically active plant derived compounds presently in use worldwide, have been discovered through following up research to verify the authenticity of information concerning folk or ethnomedicinal uses of plants (Farnsworth et al, 1985). Penso, (1982), estimates that approximately 20,000 species of plants are used medicinally throughout the world, thus leaving a huge number of plants, for which the detailed biochemistry and pharmacological activities are unknown.
1.2. USES OF PLANTS
Some of the early uses of plants date back to the eleventh century to a Dogma, known as the Doctrine of Signatures which was almost the sole means by which humans attributed medicinal value to plants. This dogma held that the colour, shape, habitat or other physical characteristics of a plant were indicative of its medicinal value. The worm shaped embus of
Chenopodium suggested anthelmintic use and the yellow colour of saffron indicated its use in liver disorders. No rational justification for the use of drugs selected in this manner was made (Farnsworth, 1966).
By experimentation, and trial and error, plants were found to be useful for the treatment of illnesses. Many of the uses of these plants were eventually documented in various herbals, books on medicinal botany and ethnobotanical manuscripts. Other uses of medicinal plants
11 were not written down, but existed in the work and languages of shamans and witch doctors, information that is indigenous within certain groups of people in largely underdeveloped countries.
Before the nineteenth century, slow progress was made in phytochemistry and only a few compounds such as camphor, starch, cane sugar and benzoic acid were known (Trease and
Evans, 1978). Complex mixtures of fats, oils, tars and resins had been prepared and used although little was known of their composition. By 1803 the first alkaloid, narcotine, was isolated and following this were the isolations of morphine, strychnine and emetine. In the ten years between 1813 and 1823 the chemical nature of fats and fixed oils were elucidated.
By the middle of the Twentieth century the principal structural types most commonly found in plants had mostly been elucidated and natural product chemists were studying biosynthetic pathways found in plants which was being made possible by the introduction of new techniques of separation and analysis.
Different compounds isolated fi’om plants were found to be important for differing reasons.
Alkaloids were of medicinal use, carbohydrates, fats and proteins were of dietary importance and starch and gums used in drug preparations, although not having any pharmacological action, were of value in the preparation of pharmaceuticals.
12 1.3. INITIAL SEPARATION OF PHYTOCHEMICALS.
Thin layer chromatography (TLC) was developed as a method of analysis in 1958 by Stahl (Trease and Evans, 1978). This method of separation was based on the properties of thin powdered layers of silica gel, alumina, cellulose or kieselguhr which adhered to a glass plate. The mixtures of compounds to be resolved were dissolved in a suitable solvent and placed as spots on the powder at one end of the plate which was dipped into a suitable solvent mixture and enclosed in an airtight chamber. The solvent front travelled up the plate and after a given period (from a few minutes to several hours) the plate was removed, and the solvent front marked. The solvent was allowed to evaporate and the positions of separated compounds were determined as coloured spots, by ultra-violet light absorption or by spraying with a locating agent.
Thin layer chromatography was advantageous over paper chromatography as separations could be effected more rapidly with greater resolutions and more sensitivity. Separated spots were generally more compact and more clearly demarcated from one another, also, spray reagents such as sulphuric acid could be used which would have destroyed a paper chromatogram. Thus, the separation and isolation of compounds from plant extracts was greatly facilitated. With the development of more sophisticated separation techniques such as gas-liquid chromatography and high pressure liquid chromatography (HPLC), even greater numbers of separations would be achieved. The emergence of spectroscopic techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), meant that minute quantities of compounds could be isolated by chromatography in yields of microgrammes and milligrammes, and chemical structures determined as previously gramme quantities were required for the analysis of such compounds. The methods available upto the present day for the extraction of biological entities continue to develop, enabling the separation of singular components from extremely complex plant extracts. 13 1.4. MEDICINAL PLANTS FOR USE AS POTENTIAL ANALGESICS.
Medicinal plants are defined as 'higher plants that have been alleged to have medicinal properties' i.e. effects relating to health or which have proven to be useful as drugs by
Western standards or which contain constituents that are used as drugs (Farnsworth et al,
1976). Plants which have been used traditionally as analgesics or which have yielded compounds which are used in pain relief include Cannabis sativa, (Cannabinaceae),
Mandragora officinarum, (Solanaceae), Papaver somniferum, (Papaveraceae) and Conium maculatum, (Umbelliferae). Thousands of plants are used to treat all manner of wounds and diseases. Currently available analgesics still produce enough undesirable side effects that the search for new ones is justified. When referring to herbal and ethnobotanical literature for sources of plants used traditionally in the treatment of pain, information that refers to a plant species as being effective for only one particular pain may suggest the actual effect of the remedy is not analgesia. If the species indicated is used for a number of different pains, (i.e. headaches, backache, wounding) it is more likely that the plant is useful as an analgesic rather than for example, an anti-inflammatory (Elisabetsky, 1990). If the information regards only pain that might obviously be related to e.g. muscular spasm (pain in abdomen, indigestion) it suggests the remedy's effect is possibly muscle relaxation rather than analgesia. Farnsworth (1976) also states that the evaluation of medicinal practices may be carried out by comparing the similarity of a plant use by different groups (the same plant for treatment of a different or the same disorder). Parallel use of a plant by widely separated groups shows an independent origin of plant use and therefore gives evidence supporting its effective use.
Some plant species used as 'panaceas' have a large number of distinct pharmacological effects due to their highly complex chemical composition e.g. Panax ginseng, which has been documented as a plant with analgesic, anti-stress and anti-ageing effects, also promotes immune function and metabolism and is also documented for its use in the cardiovascular
14 system and endocrine secretion. The uses of a single plant that may seem unrelated may be due to the different expression of a similar use e.g. inflammation may be expressed as a localized pain, localized heat or localized swelling. Some chemical compounds may have more than one therapeutic use e.g. the opioids, which are used for the treatment of pain, coughs and diarrhoea, highlighting the number of different compounds in a plant which may have a diversity of pharmacological effects.
In analysing the research activity within American pharmaceutical industries, (Mattison et
al 1988) it was shown that analgesics are one of the four highest therapeutic categories on which research efforts were being concentrated. The international pharmaceutical market of analgesics is estimated to be worth several billion dollars and the search for new analgesics has remained a priority since 1963. Analgesic compounds still on the market present a wide range of undesired effects, therefore new and better compounds are required.
Opioid analgesics, which are extremely effective in relieving mild to moderate pain, severe to acute pain and chronic pain management, have their applicability limited by their unwanted side effects such as abuse, psychological and physical dependence, addiction, tolerance, respiratory depression, nausea, vomiting and constipation (Foote, et al 1988). As yet, all known opioid agonists produce in varying degrees, the same unwanted effects found with morphine (Jaff et al, 1985). This could be explained by the fact that most of the effects of the withdrawal syndrome caused by these compounds are mediated at or near the brain sites through which analgesia is achieved (Wise, 1987). Some selective agonists acting at opioid receptors e.g. nalorphine, present lower risks of physical dependence but are characterized by psychomimetic effects. Methadone and meperidine, which are chemically different from morphine also lead to physical dependence and induce sedation.
Non-opioid analgesics used in the management of mild and severe pains also produce serious side effects e.g. some salicylate drugs and other drugs which act by prostaglandin 15 inhibition through cyclooxygenase are potentially associated with ulcerogeneity, damage to blood coagulation and neuropathologies (Brinkworth et al, 1988). Allergic reactions have also been asociated with acetylsalicylic acid based drugs used in young people (Brinkworth et al, 1988). Thus, the need for safer analgesic drugs with fewer side effects are extremely desirable and by studying worldwide literature on plants traditionally used in this therapeutic area, novel leads to new analgesic compounds may be found.
1.5.ALTERNATIVE HEALTH CARE
1.5.1 .PLANTS IN ALTERNATIVE SYSTEMS OF MEDICINE
Plants which have been selected for use as medicinals, often over thousands of years, constitute the most obvious choice for examination in the current search for new therapeutically active drugs. The World Health Orgaisation (WHO) has a policy at the present time to secure health for all by the year 2000 and there is an official interest in indigenous systems of medicine, especially plant remedies, based on the fact that about 80% of the world's population use herbal medicines. By developing such systems their methods for facilitating health care for all are expected to be met. Many people who use western systems of health care are concerned with the potency and side effects of new synthetically derived drugs and there is increasing interest in systems of alternative medicine.
A herbal remedy is one in which the main therapeutic activity depends on the plant or fungal metabolites it contains, (Croom, 1983) but it is not definable in terms of the particular system of medicine in which it is employed. It appears to be a general notion that mostly, herbal remedies, being natural products, are safer than the potent synthetic drugs which often have far reaching undesirable side effects. However, some plants contain harmful compounds for example carcinogens, as found in Sassafras spp. and pyrrolizidine alkaloids, as found in
Symphytum officinale so care should still be taken when natural products such as these are used. 16 Ethnomedicine is defined as the beliefs and practices relating to diseases which are products of indigenous cultural development and are not explicitly derived from the conceptual framework of modem medicine (Foster, 1983). Different groups of people develop alternative beliefs and methods for treating illnesses. In the Western countries, an illness is largely treated according to the symptons presented to a physician. For the majority of people using traditional methods of medicine the whole individual is taken into account, and the method of preparation of plants to treat the person are modified accordingly. In this way a more specific method of treating a certain illness may be brought about.
Many systems of healthcare use plants as a source of medicines, although this is to a lesser extent in the richer countries of the West. Holistic medicine takes into account an individuals family, their social life, their hereditary and cultural background, and so therefore, their psychosocial and clinical factors are taken into consideration for the diagnosis and therapy. Traditional healers involved in this type of healthcare are often old and highly respected members of the population. The use of the plants in some instances are important as they have rituahstic value when treating the individual with ill health. Such is the case in
African and West Indian traditional medicine.
1.5.2. AFRICAN MEDICINE
The physical and mental states of an individual are treated together in Afiican traditional medicine and medications are directed towards the mind and body (Koumare,
1983). Incantations accompany the preparation and administration of a medicament, and a healer mediates between spirits and humans.
17 1.5.3. AYURVEDIC MEDICINE
Ayurvedic medicine which means 'Science of Life' is one of the oldest formulated systems of medicine, and is widely practised in South East Asia, especially in India, Bangladesh and
Pakistan. The Indian population utilises around 540 plant species in different formulations,
(Kapoor et al, 1979). There are around seventy books containing around 800 recipes using largely plants as sources of medicines. India was one of the countries in the pioneering development and practice of well documented indigenous systems of medicine, the most notable being Ayurveda and Unani. About 75% of the population consult mainly traditional physicians and the turnover of indigenous medicines is around 1.5 times that of modem drugs (Rustogi 1980).
1.5.4.TRADITIONAL CHINESE MEDICINE Chinese Traditional Herbal Drugs or 'Zhong Cao Yao' serve as the fundamental basis for drug research and for identifying new dmgs in modem China (Pei-gen, 1980). Chinese herb medicines are derived from natural origin and have an equally important position in comparison with synthetic dmgs and antibiotics. Due to the fact that China has a long history in the use of herb medicine and has accumulated a vast experience which has demonstrated conspicuous and unique effects on certain diseases, the Chinese government has placed an emphasis on systemizing traditional medicine and raising it to a higher level. Chinese herb medicine originated from natural products used in China and dmgs and plants are classified according to their different uses :
Traditional Chinese Medicine (TCM) or Chinese Materia Medica, 'Zhong Yao' includes the use of plants which are directed by a series of systematic and self contained theories (Pei- gen, 1980) which have spread throughout the country and are regarded as the main component of Chinese herb medicine. There are approximately five hundred species of commonly used Chinese traditional dmgs in this class, which includes Panax ginseng and Glycyrrhiza uralensis. National Minorities Dmgs or 'Min Zu Yao' are closely connected with Chinese traditional
18 medicine. The use of this class of drugs are limited to people in certain geographical areas as the growth of these plants is limited to certain districts, and their use is governed by local theories. It is estimated that there are between 500-600 plant species in this class (Pei-gen, 1980).
The use of national minorities drugs are most frequently encountered throughout China and their use is characterised by trial and error and are species ailment orientated, usually without theoretical direction. This class of drugs has the largest number of species and at least 80% of these Chinese herbal medicines are of botanical origin. A recent survey of these drugs identified 4877 plant species with some degree of therapeutic value (Peigen, 1980). Chnical observation of the original uses of these plants determines whether or not the effectiveness is real and if the drug can serve as a research subject.
1.6. DOCUMENTING AND EVALUATING HERBAL REMEDIES.
Over the previous thirty years methods for the evaluation of the quality of botanical pharmacopoeial drugs and their preparations have been devised and standards for their use have been set (Croom, 1983). Herbal remedies generally include a mixture of different plant extracts and there are a number of pitfalls which may occur during the isolation of the active principles, however, precautions can be taken to minimise these, e.g. plant materials which are to be used for in vitro screening and subsequent isolation of bioactive compounds must be botanically authenticated. If pharmacological examination of a plant extract produces bioactivity, one would expect the same degree of activity from subsequent samples, however, if on examination of a subsequent sample the effect is not repeated as a result of incorrect identification of the plant species, a conclusive result cannot be reached.
There may be a number of cofactors in a plant extract which modify the absorption, distribution and elimination of the bioactive compounds (Croom, 1983). Simple extractive tests can be performed to identify the presence of major metabolites present in an extract e.g. saponins or alkaloids, and some form of quality control assured. Differences within a
19 plant species may exist due to the growth of identical plants in widespread geographical areas and these would be identified while carrying out quality control assays, ensuring plants extracts which are to be used in herbal medicinals are of a safe, consistent and standard preparation.
1.6.1. INTRASPECIFIC VARIATION
The identification of the correct species of a plant does not preclude chemical variation in a living plant. Within a plant species, individual plants may vary chemically, therefore pharmacologically, due to genetic differences which do not show up morphologically.
Environmental differences due to factors such as soil composition and climate, the stage of the plants development, environmental nutrients or moisture, may also cause varying yields of compounds. For example, the peak morphine content of the opium poppy Papaver somnifenim, occurs 2-3 weeks after flowering, harvesting earlier or later yields significantly less morphine (Tyler et al, 1976).
A number of factors influence the growth of a plant and in turn its ability to produce chemical compounds, e.g. temperature, rainfall, day length and altitude. Depending on whether a plant is cultivated or wild, the biochemical composition may alter in different plant parts. Certain drugs are now obtained almost exclusively fi'om cultivated plants e.g. ginger, peppermint oil, fennel, cinchona and opium. In other cases, wild and cultivated plant sources are utilized. Some plants have been cultivated from the very beginning of records e.g. the opium poppy, Papaver somniferum, others are grown because supplies of wild plants are insufficient to meet the demand of the general public or collection of the plant may be difficult. Also, plants under cultivation generally produce a better quality of drug.
Fairly rapid drying of plants prevents flowers and leaves from losing colour and retains the aroma of aromatic drugs. The temperature used for drying plants should be governed by the
20 constituents and physical nature of the drug required.
The differences in the ways in which plants are utilized pharmacologically may fall into three categories (Principe, 1991):(l)Constituents isolated from plants may be used directly as therapeutic agents e.g. digitoxin, from Digitalis purpurea, morphine from Papaver somniferum and atropine prepared from Atropa belladonna. (2) Plant constituents used as starting materials for the synthesis of useful drugs e.g. adrenal cortex and other steroid hormones are normally synthesized from plant steroidal sapogenins. (3)Natural products may serve as models for pharmacologically active compounds in the field of drug synthesis.
Occasionally some plant constituents which are potentially useful as drugs cannot be employed directly either because plant material is unavailable, is only available in limited quantities or the plant may not lend itself to cultivation.
1.7.SECONDARY METABOLITES DERIVED FROM PLANT METABOLISM
All organisms possess similar metabolic pathways by which they synthesise and utilise essential compounds, e.g. sugars, amino acids, fatty acids, nucleotides and proteins. This is primary metabolism, and these compounds, essential for the survival of organisms are primary metabolites. TSiatural Products' are called 'secondary metabolites' since it is thought that synthesis of these compounds are not essential for a plant's survival and development.
The pathways of synthesis and subsequent utilisation of these compounds constitute plant secondary metabolism but the division between primary and secondary metabolism is not clear cut e.g. many obscure amino acids are produced which are secondary metabolites and the sterols have an essential role in most organisms, also, primary metabolism provides molecules for the synthesis of many secondary metabolites. The following classes of compounds occur throughout the plant kingdom in different families and many of them are utilised for their pharmacological effects including that of analgesia.
21 1.7.1 ALKALOIDS
Alkaloids, derived largely from plant sources, are basic in nature and generally contain a nitrogen atom in a heterocyclic ring. They possess an array of structural diversity and physiological activity unrivalled by any other group of natural products (Harbome et al 1993). One of the reasons why alkaloids are of particular importance to humans is because of their medicinal properties, which are due to their effects on the CNS, e.g. morphine for its use as an analgesic and vincristine and vinblastine for their anti-cancer uses. The classification of alkaloids is generally based on the type of ring system present e.g. isoquinoline, indole etc., and on the biosynthetic origin from a particular protein amino acid precursor e.g.ornithine or lysine. Some alkaloids are noted for their universal occurence in particular families, for example the Papaveraceae, the common occurrence in others, e.g. the Rutaceae and rarity in others, for example the Umbelliferae. A very brief account of the occurrence of some alkaloids, the family in which they occur and for which therapeutic disorder they are used, serve to illustrate their diversity and wide occurrence. Narwedine occurs in the bulbs of the snowdrop, Galanthus nivalis, family Amaryllidaceae, and is used to potentiate the analgesic effects of morphine. Narwedine also has hypotensive activity, and increases the amplitude and frequency of respiratory movements (Harbome, 1993).
CHjO,
Figure 1.1 Narwedine
Approximately 275 alkaloids have been described in the Amaryllidaceae which are all
2 2 restricted in their occurrence to this family. The indole alkaloids are derived biosynthetically from tryptophan and a CIO monoterpenoid precursor secologanin (Harbome et al, 1993). A number of the alkaloids in this family are of clinical value in medicine e.g. reserpine from Ratiwolfia serpentina (Apocynaceae), is used for the treatment of hypertension.
.OCH,
•OCH,
OCH,
Figure 1.2 Reserpine Vincristine and vinblastine from the leaves of the Madagaskar periwinkle, Catharanthus Apocynaceae) are used to treat certain types of leukaemia and solid tumours.
C H ,0 .
.CM,
.CM, MO ^— OCH, o
Figure 1.3 Vincristine Vinblastine Strychnine, derived from the seeds of Strychnos ntcx vomica (Loganiaceae) is an indole alkaloid and has CNS stimulatory activity. It is a highly toxic member of this class of alkaloids.
23 Figure 1.4 Strychnine Physostigmine from Physostigma venenosum (Leguminosae) is used therapeutically in the form of eye drops as a miotic, and is toxic in large amounts.
cC
CH, CH,
Figure 1.5 Physostigmine Isoquinoline alkaloids are the largest single group of plant alkaloids. They are derived from phenylalanine and/or tyrosine to produce an array of complex structural types. Morphine, from Papaver somniferum (Papaveraceae) is most notable in this group of alkaloids. It is the major base in opium poppy latex and produces the most potent state of analgesia. Also derived from P.somniferum is papaverine which is used clinically as a muscle relaxant and an antitussive.
CM.
‘OCH,
OCH,
Figure 1.6 Morphine Papaverine
24 Tetrandrine, found in Stephania tetrandra (Menispermaceae) is employed as an analgesic, an anti-inflammatory and an antipyretic;
OCM, CKfi. .OCM,
CM,
CM;
•CM,
OCM,
Figure 1.7 Tetrandrine Monoterpene alkaloids are formed biosynthetically from the iridoids loganin and secologanin by condensation with ammonia (Harbome et al, 1993). Sesquiterpene alkaloids derived biosynthetically from a C15 famesol precursor may be divided into three groups, according to the plant source fi'om which they were derived e.g. the tetracyclic alkaloids, such as dendrobine which characterise orchid plants of the large genus Dendrobium,
CM, H
CM,.
CH,
Figure 1.8 Dendrobine the Celastraceae alkaloids which includes cassinine from Cassine metabelica (Celastraceae) which has a weak tranquilising effect in animals, and the alkaloids such as those in the water lily family e.g. deoxynupharidine.
.CM,
■CM, 0 "^M y — o . ...
5". ... CM, Figure 1.9 Deoxynupharidine 'CM,
Cassinine
25 Dendrobine from Dendrobium nobile (Orchidaceae) has weak analgesic and antipyretic therapeutic activities and thiobinupharidine located in Nuphar luteum (Nymphaceae) which has weak anti-bacterial activity.
Figure 1.10 Thiobinupharidine The pyrrolizidine or Senecio alkaloids are one of the most poisonous groups of alkaloids, causing death and liver damage to humans and animals. The pyrrolizidine alkaloids are distinguished by the presence of a fused two 5-membered ring system which has a bridge head nitrogen shared between the two rings. They occur largely in the Compositae and it is thought that all 1500 known species in the Compositae contain varying amounts of alkaloids. Examples are jacobine found in Senecio jacobbaea, senecionine from Senecio vulgaris and symphytine from Symphytum officinale.
.CM,
CM,
Figure 1.11 Jacobine Senecionine Steroidal alkaloids are triterpenoid in origin and are derived biosynthetically from six isoprene units (Harbome et al, 1993). Steroidal alkaloids are found in four unrelated families: the Apocynaceae, Buxaceae, Liliaceae and Solanaceae, each containing different sets of alkaloids within their tissues. Medicinally, important steroidal alkaloids are present in the genus Veratrum, in particular protoverine from Veratrum alba (Liliaceae) which is
26 hypertensive. The alkaloids conessine from Holarrhena sp., funtumine from Funtumia sp. and kurchessine from the Holcarhena antidysentria are all members of the Apocynaceae, and irehine derived from Narcissus sp. is from the Amaryllidaceae. These alkaloids all provide starting materials for the partial synthesis of useful steroids e.g.aldosterone.
Om CH,
Figure 1.12 Protoverine Conessine Funtamine
CH,
CM, ÇH,
Figure 1.13 Kurchessine frehine Tropane alkaloids occur widely in the Solanaceae family and have been widely utilised for their medicinal, hallucinogenic or poisonous properties (Evans, 1979). Examples are cocaine derived from Erythroxylum coca which has CNS stimulatory activity and is used as an anaesthetic and a mydriatic, and hyoscyamine from Hyoscyamus niger which has a number of effects including CNS depression, antispasmolysis and anti-emesis.
27 OCM,
Figure 1.14 Cocaine Hyoscyamine Other classes of alkaloids with differing degrees of clinical activity are the betalains, the peptide, quinoline and quinolizidine alkaloids.
1.7.2.PHENOLICS
Phenolic compounds, or polyphenols, are plant substances possessing an aromatic ring containing one or more hydroxyl groups. The majority of phenols are water soluble and occur naturally combined with a sugar in glycosidic form (Harbome et al, 1993). Phenohcs are classified according to stmctural complexity and biosynthetic origin. The simplest class, the phenols, includes phenolic acids and phenolic ketones. The phenylpropanoids, a much larger group, are based on a C 5 -C3 -C5 nucleus and are distinguished into the coumarins, chromones and chromanes, benzofurans and dimeric lignans. The xanthones, stilbenoids and quinones are also phenolic compounds. These plant metabolites contribute to the colour, taste and flavour of many foods and drinks and some are used therapeutically for their anti inflammatory and antihepatotoxic properties.
Tannins are a class of naturally occurring phenolic compound which are able to react with protein forming water-insoluble copolymers. There are two main types of tannin : condensed (proanthocyanidins) and hydrolysable. Condensed tannins, or flavolans, are formed by the condensation of catechin units to form dimers and higher oligomers. Hydrolysable tannins fall into two groups; gallotannins in which a glucose is surrounded by five or more galloyl ester groups, and ellagitannins where units of hexahydroxydiphenic acid are present (Harbome et al, 1993). Condensed tannins have been used medicinally to aid the healing of wounds and bums, producing an impervious layer under which healing takes place, when applied to the skin. Some hydrolysable tannins have been documented for their antiviral and antitumour
28 properties and some examples are agrimonin, found in Agrimonia pilosa (Rosaceae), an ellagitannin, rugosin D from Rosa rugosa, (Rosaceae), an ellagitannin, and corilagin from Terminalia chehula (Combretaceae), which is a gallotannin.
MO OH MO OH
OH HO HO R. OH
HO.
) C H , :0
OH OH
Rugosin D
1.7.3.FLAVONOIDS
Isoflavonoids differ from other classes of flavonoids by way of the structural configuration of their 3 membered ring system e.g. flavonoids have the structure:
II
compared to isoflavonoids which have the following:
29 Isoflavonoids occur in plants in the fi'ee form state rather than in the glycosidic combination (Harbome et al, 1993) and are found in the families Leguminosae, Compositae, Iridaceae, Myristaceae and Rosaceae. Approximately six hundred isoflavonoids have been described (Dewick et al, 1988) and they are divided into subclasses according to the oxidation level of the central pyran ring. Formononetin occurs widely in the Leguminosae family e.g. in
Trifolium and Baptisia spp. and has been documented for its antifungal activity. Genistein occurs in the wood of Prunus spp. (Rosaceae) and also has antifungal activity.
OCH,
Figure 1.16 Formononetin Genistein
Flavones and flavonols occur widely in plants, existing as co-pigments to anthocyanins in petals and in the leaves of higher plants (Harbome et al, 1993). They occur most frequently in glycosidic combinations. Three flavonol aglycones commonly occur and they are kaempferol, which is located in Aesculus hippocastanum (Hippocastanaceae), quercetin, found in the families of Compositae, Solanaceae, Rhamnaceae and Passiflorae and myricetin which occurs in the heart wood of Soymidia fehrifuga, and is a member of the Meliaceae family.
.OH S ^ o c H , .OH HO. r “OH
HO. OH “OH
OH “OH
OH
Figure 1.17 Kaempferol Quercetin Myricetin Flavones have a slightly different structure to flavonols as they lack a 3-hydroxyl substitution which affects their U. V. absorption, chromatographic mobility and colour
30 reactions. Simple flavones can be distinguished from flavonoids e.g. acacetin isolated from Tilia japonica (Liliaceae), is a flavone and isorhamnetin, isolated from the flowers o f Arnica
^ / 7.(Compositae) is a flavonol.
.OCH,
ocH,
Figure 1.18 Acacetin Isorhanmetin Two commonly occuring flavones are apigenin, found in Amorpha fruticosa (Leguminosae) and luteolin, present in Antirrhinum majus (Scrophulariaceae). Both of these flavones have antibacterial and anti-inflammatory activities.
,OCH,
MO.
OH o
Figure 1.19 Apigenin Luteolin Minor flavonoids have a more limited natural distribution than the widespread anthocyanins, flavones and flavonols (Harbome et al, 1993). Examples include naringenin, a flavonone from Artemisia and Dahlia spp. (Compositae), Catechin, a flavan-3-ol which is widespread in woody plants e.g. Salix caprae (Saliaceae) and hesperetin, a flavonone fi'om Citrus spp. (Rutaceae).
■|<0 HO
HO
OH OM
Figure 1.20 Naringenin Catechin Hesperetin
31 1.7.4. TERPENOIDS The terpenoids comprise the largest group of plant natural products and more than 20,000 structures have been classified from plant sources (Harboune et al, 1993). Derived biogenetically from a C 5 precursor, isoprene, they are also known as isoprenoids.
Monoterpenoids are derived from the condensation of two C 5 precursors dimethylallyl pyrophosphate and isopentyl phosphate to produce geranyl pyrophosphate which is the starting point for sesquiterpenoids which have 15 carbon units. Diterpenoids with a C 20 based structure are synthesized from famesyl pyrophosphate. Two molecules of the famesyl pyrophosphate condense together to form the triterpenoids which have a C 30 basic structure.
The steroid saponins fall into this category. Two molecules of geranylgeranyl pyrophosphate
(C20) condense together to produce a C 40 intermediate, phytoene, which is the immediate precursor of the yellow carotenoid pigments. Examples of monoterpenoids are camphor, where the positive form occurs in the camphor tree, Cinnamomum camphora (Lauraceae), and the negative form occurs in Matricaria parthenium (Compositae). Camphor affects the CNS and its effects include those of a mild analgesic and a rubefacient.
CH, CH,
>
Figure 1.21 Camphor Menthol is also a monoterpenoid and occurs in Mentha piperita and other Mentha spp.
(Labiatae). It has analgesic and antiseptic activity (Harbome et al, 1993).
Figure 1.22 Menthol Sesquiterpenoids can be classified according to their biosynthetic origin into over 200 different skeletal types (Nakanishi et al, 1974) and several thousand have been found in
Nature (Harbome et al, 1993). Examples of some sequiterpenoids are abscisic acid found in Gossypium hirsutum (Malvaceae), famesene in the coating of the peel of apples and pears,
Malus and Pyrus spp. respectively (Rosaceae) and gossypol, which occurs in the seeds of Gossypium sp. (Malvaceae).
CH, CH, CH, CMC C m OM CMC
O,
OM MO CM, CM,
■CM, CM, CM, CM, CM,
Figure 1.23 Abscisic acid Famesene Gossypol Diterpenoids can be divided into two groups: the resin acids and the giberellins. An example of a resin acid is abietic acid, present as the primary constituent in many conifer resins. Gibberellic acid is one of the most frequently occuring giberellins and is found in the fungus Gibberella fujikuroi.
Figure 1.24 Giberellic acid
Triterpenoid saponins consist of water soluble sugars attatched to a lipophilic steroid (C 2 7 ) or triterpenoid (C 3 0 ) moiety. This type of compound produces a fi'oth in aqueous solution due to the ability to lower surface tension which is possible due to its hydrophobic and hydrophilic asymmetry (Harbome et al, 1993). These compounds also cause haemolysis of blood erythrocytes. Frequently occuring triterpenoid saponins are aescin from the
33 horsechestnut Aesculus hippocastanum (Hippocastanaceae), which has strong haemolytic activity and anti-inflammatory activity, glycyrrhizin from the roots of Glycyrrhiza glabra (Leguminosae) which has antiulcerogenic activity and a number of ginsenosides isolated from
Panax ginseng (Araliaceae) which have a range of pharmacological activities.
o CH,
Figure 1.25 Aescin Glycyrrhizin Steroid saponins are similar in terms of biogenesis, structure and biological activity to the triterpenoid saponins as they have the same number and location of sugar components. Largely, they are used as detergents, fish poisons and as foaming agents in fire extinguishers. Digitogenin and digitonin, which are both located in the seeds of Digitalis sp., were the first natural source of steroid saponins. Diosgenin is obtained from acid hydrolysis of many different saponins from e.g. Dioscorea spp. (Dioscoreaceae) and Trigonella sp.
(Leguminosae) and is used for the partial synthesis of hormones with a steroid structure e.g. progesterone.
Figure 1.26 Digitogenin Digitonin Diosgenin
34 Further classes of terpenoid compounds are cardenolides and bufadienolides which are used therapeutically for controlling congestive heart failiure, phytosterols which include a number of insect moulting hormones e.g.ecdysone, and nortriterpenoids which include limonoids and quassinoids. Quassinoids are known for their antitumour activities but are very toxic and limonoids are also of interest as they are able to counteract the bitter taste they impart to citrus drinks (Harbome et al, 1993).
35 1.8 NOCICEPTION IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM
Pain, whether it is sharp, stinging, aching or burning, is still one of the most difficult and widespread medical problems to understand and treat accordingly. The clinical assessment of pain and the disorder stimulating it requires an understanding of the drugs used to treat it and the effects the drug may have on the patient being treated. Opiate drugs mediate their effects via opiate receptors and have been the major class of drug for the management of pain for many years. The opiate receptors were first characterised in the early 1970’s.
However, the use of opioid drugs to treat pain have a number of alarming side effects e.g. addiction, increased tolerance, respiratory depression, constipation and sedation which limit their use. Some non-opioid centrally acting analgesic drugs have also been found and others which act peripherally cause effects which are not mediated via the Central Nervous
System(CNS). Many of the non-opioid drugs do not produce the same side effects as opioid drugs which makes them very important clinically.
More than 20 endogenous neuropeptides have been implicated in the mediation of pain within the mammalian central nervous system (Conn, 1991) including Bradykinin(BK),
Calcitonin Gene Related Peptide(CGRP), and Substance P. Bradyldnin is a nine amino acid neuropeptide which acts on sensory fibres and neurones. It has been implicated in the pathophysiological processes that accompany tissue damage, especially inflammation and hyperalgesia. CGRP consists of thirty seven amino acids and is deemed to be a mediator of migraine and other vascular headaches. As well as being a neuropeptide it is thought to be a neuromodulator of other peptides and hormones in the mammalian CNS. The bradykinin BK n and the CGRP receptor binding assays were used (Personal communication S.Fogarty) in which plant extracts selected for their use in traditional medicine were screened to identify
36 new leads to analgesic drugs. Bradykinin and CGRP were chosen in particular as, in the case of bradyldnin, a number of peptide antagonists are available for use in vitro but the need for antagonists which are selective and stable in vivo are desired, and in the case of CGRP, which is thought to be a mediator of migraine and other vascular headaches, there is no single drug which will prevent the development of migraines after the initial onset. Thus, new non-peptide lead molecules to CGRP antagonists are required.
There are two prototypes for analgesic drugs: (a) drugs which are used to relieve severe or deep pain, e.g. morphine and (b)drugs which are used to relieve mild or moderate pain e.g. headache and backache, for which the prototype drug is aspirin.
1.9. THE FIRST OPIOID AGONISTS AND ANTAGONISTS
The most widely used analgesic and the first to be therapeutically used was the alkaloid morphine derived from the seeds of the opium poppy, Papaver somniferum, family
Papaveraceae (Bradley, 1989). Morphine decreases the sensation of pain but in therapeutic doses the drug also produces sedation and drowsiness, and large doses of morphine cause
CNS excitement and convulsions. At normal therapeutic doses the alkaloid also causes depression of respiration due to a direct action on the respiratory centre in the pons and medulla and further side effects include nausea and vomiting. Morphine acts peripherally on the gastro intestinal tract, increasing the resting tone of the stomach and the small and large intestine, causing decreased motility which leads to constipation. Large doses of morphine cause hypotension and bradycardia in the cardiovascular system, due to its effect on the medulla (Bradley, 1989). Morphine is generally administered intramuscularly or intravenously as when it is taken orally only a small amount reaches target organs due to the first pass effect as the drug is metabolised in the liver.
There are a number of drugs related to morphine which are used clinically for the management of pain, in particular, pethidine (meperidine) which has one tenth the potency of morphine but produces the same degree of analgesia and sedation, however it has a shorter
37 duration of action and produces a dry mouth and blurred vision (Bradley, 1989). Methadone is similar in terms of potency to morphine and its pharmacological properties have a longer duration of action with less associated sedation, however it is bound to proteins in various tissues and accumulates with repeated administration. When the administration of this drug is discontinued, methadone is released from these binding sites, resulting in a milder abstinence syndrome than with morphine and other opioid analgesics. Naloxone and nalorphine are opioid antagonists, the latter being the first antagonist to morphine to be discovered.
Nalorphine has some agonist properties while naloxone is a pure antagonist.
1.10. NON OPIOID ANALGESIC DRUGS
Aspirin is the prototype non narcotic drug, and it does not produce dependence or addiction, it also has antipyretic and anti-inflammatory properties (Bradley, 1989). It is a non steroidal anti-inflammatory drug (NSAID) with relatively weak analgesic action which is inefrective for the relief of severe pain at any dosage, and therefore is used to treat mild pain e.g. headaches and toothache. Aspirin, or acetylsalicylic acid, is a synthetic derivative of salicin derived from salicylic acid from the bark of willow, Salix alba, family Saliaceae. Aspirin has a number of side effects, the major one being an irritant action on the gastric mucosa which results in bleeding. Paracetamol is an analgesic with antipyretic but no antiinflammatory action (Bradley, 1989). Very large doses of paracetamol cause necrosis of the liver. Other drugs in this class include ibupofren, naproxen and fenoprofen, all derivatives of propionic acid which have similar properties to aspirin and are used for the treatment of rheumatoid arthritis and related conditions.
1.11. MEDIATION OF PAIN IN THE MAMMALIAN CNS
Pain is elicited by noxious stimuli in ‘normal’ individuals, where it acts as a warning of impending damage to tissues, or it may indicate the damage has already occured, e.g. in trauma, and it is the outstanding symptons in many diseases. Free nerve endings have been identified as pain receptors in pain sensitive tissues, e.g. skin and muscle. Most nociceptive receptors at the terminals of C fibres are polymodal receptors, i.e. they respond to different
38 modalities of noxious stimuli (Bradley, 1989). The cell bodies of the spinal nociceptive afferent fibres are in the dorsal ganglia and fibres enter the spinal cord through the dorsal roots and terminate in the grey matter of the spinal cord, the substantia gelatinosa of the dorsal horn (Figurel.27). The terminals of the primary afferent pain fibres in the dorsal horn of the spinal cord release substance P as their neurotransmitter. The second order neurones in the spinal cord give rise to ascending fibres, most of which cross the cord to form the contralateral ascending spinothalamic tract which projects to various sites in the brain (Bradley, 1989). This pathway is polysynaptic and relays in the reticular formation of the brain stem before passing to the ventral and medial thalamus, and then to the sensory cortex. This pathway connects with the limbic system and the hypothalamus, where emotional responses and reactions of the autonomic nervous system (ANS) can be evoked.
Sensory Cortex
Limbic System Thalamus Periaqueductal Grey Brain Stem
5-HT Spinal Cord Pain Afferents
Substantia Nigra
Figure 1.27. Simplified diagrammatic representation of the main neuronal pathways involved in the transmission of pain (Bradley, 1989).
There is also a descending pathway which is involved in pain sensation. This is a polysynaptic pathway which arises fi’om the periaqueductal grey matter of the midbrain, a small area surrounding the central canal (Bradley, 1989). The pathway passes through the dorsal raphe nucleus in the dorsolateral funiculus in the spinal cord to terminate in the dorsal horn, the descending pathway is mainly inhibitory. Electrical stimulation of either the periaqueductal grey or the dorsal raphe nucleus in experimental animals produces analgesia. The neurotransmitter released by the terminals of the descending pathway is 5-hydroxytryptamine
(5-HT). Opiate receptors and opiate peptides, especially enkephalins, are concentrated in the
39 dorsal horn of the spinal cord, the periaqueductal grey matter, and in the thalamus the
enkephalins may function as neurotransmitters.
Nociceptors encode the occurrence, intensity, duration and location of noxious stimuli and signal pain sensation (Sherrington, 1906). Nociceptors can be classified according to their response to different modalities of intense stimulation, the conduction velocity of their peripheral axons and differences in the characteristics of their response to stimuli. Cutaneous nociceptive afferents may be myelinated (A fibres) or unmyelinated (C fibres). Acute pain, such as that elicited by bradykinin, is transmitted via A and C fibres. Most of the myelinated afferents (71%) can be excited by intra-arterial injections of algesic substances such as bradykinin (Kitamura et al, 1977) and approximately 50% of unmyelinated afferents are activated by bradykinin (Foch et al, 1976). Bradykinin causes acute pain when injected into the base of human blisters and two BK II receptor antagonists produced significant antagonism of bradykinin-induced pain responses, concluding that the kinin receptor mediating pain on the human blister base is of the BK II type (Whalley et al, (1987).
1.12 INFLAMMATION AND NOCICEPTION
Inflammmation is often associated with pain and hyperalgesia and some of the agents
produced during inflammation can activate nociceptive sensory neurones either directly or
indirectly. Sensory neurones can influence the inflammatory process through the release of
neuropeptides (notably Substance P and CGRP) which act on blood vessels and
inflammatory cells. Inflammation is also associated with vasodilation and increased blood
flow as well as extravasation of the blood vessels which allow the entry of cells, e.g.
monocytes and platelets, and humoural mediators e.g. bradykinin. A number of substances
are released during inflammation which either cause pain or sensitise pain fibres.
Eicosanoids are substances derived from the metabolism of arachidonic acid, most notable
are the prostaglandins which are released from tissues during inflammation. Prostanoids
generally sensitise nociceptors to bradykinin and other stimuli which are present in the
inflammatory exudate, and the sensitisation of nociceptors by prostanoids may have a role in
the state of peripheral hyperalgesia. Leukotrienes are lipoxygenase products of arachidonic
40 acid metabolism, and when produced during tissue inflammation, they may act indirectly on sensory neurones by stimulating other cells to release neuroactive agents. In inflamed or damaged tissue leukotriene D4 produces rapid alterations in gene expression in cells such as macrophages and basophils, which stimulate an increase in the synthesis and release of eicosanoids (Crooke et al, 1989). Leukotriene D4 has also been shown to release Substance
P (Bloomquist et al, 1987) and may also have a role in hyperalgesia. Cytokines are interleukins, e.g. IL-1, IL3, tumour necrosis factor (TNF) and interferons, which are released by phagocytic and antigen presenting cells of the immune system. They are inflammatory
agents and may interact with peripheral nerves that involve mediators produced by other cell types. Interleukin IL-1 may activate eicosanoid metabolism in cells, e.g. fibroblasts, and this brings about the release of prostaglandins which in turn are thought to activate cellular cyclic
AMP dependent secondary mechanisms. Levels of Nerve Growth Factor (NGF) are elevated
in inflamed tissue (Weskamp, et al, 1987) and may influence the properties of sensory nerves by regulating the expression of various genes. NGF stimulates the synthesis of Substance P
and CGRP and elevated levels of NGF may lead to increased synthesis and the release of
peptides in vivo. Tissue NGF levels may also be regulated by peptides released fi'om sensory
neurones. Hyperalgesia occurs when inflammation is present and it is characterised by a
decrease in pain threshold, as well as an increase in pain to suprathreshold stimuli. The state
of analgesia may be primary, referring to changes that occur within the site of injury, or
secondary which refers to changes that occur in the area surrounding the site of injury
(Hardy et al, 1950).
Thus, many different compounds mediate pain in the mammalian CNS either directly or via
inter-related mechanisms and different types of pain are provoked through injury and disease.
41 1.13. DISCOVERY OF BRADYKININ AND ITS PHARMACOLOGICAL EFFECTS.
Bradykinin is a nonapeptide with the structure Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, the sequence first being published in 1960 by Boissonas. It is a neuropeptide and belongs to the family of kinins which were grouped together because of their similar pharmacological properties, notably their contractile effect on isolated smooth muscle and the production of hypotension. Bradykinin and its precursor Lys-Bradykinin (Kallidin) are generated by proteases (kallikrein enzymes) fi'om their inactive precursor Kininogen (Miller
et al, 1988).
In the course of some experiments on the physiological action of the venom of Bothrops jaracara, blood samples taken from a dog after the injection of minute doses of the venom had a stimulating effect upon the isolated gut of the guinea pig ileum. It was not thought that the effect was due to the venom as the gut had been made refractory to it prior to the addition of the snake venom. Following several additions of the serum to the bath
containing the guinea pig ileum there was no observed desensitisation. Addition of the venom to the defibrinated blood of a dog resulted in the release of a potent stimulating agent very similar to that which was detected in the blood after injection in vivo. The active
principle was dialysable through cellophane paper and was rapidly destroyed by the venom
itself and by trypsin, and was not antagonized by histamine, choline or acetylcholine.
This as yet unknown substance had certain analogies with the principle described by
Feldberg et al (1938), which was then called ‘slowly reacting substance’(SRS). SRS was
deemed to be released from rabbit spleen by snake venoms and trypsin (Trethewie, 1941).
Although there was no indication that the unidentified substance was identical with the
SRS, the contraction produced by it was of a slow type, commencing after a short latent
interval. Silva et al, (1949) therefore named it ‘Bradykinin’ which indicates a substance
42 which produces a slow movement of the gut, from the Greek nomenclature ‘kinin’ meaning movement and ‘brady’ meaning slow. Bradykinin was found to stimulate all the smooth muscle preparations assayed by Silva, notably the intestine of the rat, rabbit and guinea pig ileum.
1.14. KININOGENS. KALLIKREINS AND KININS
In the blood, the sequence of events leading to the production of bradykinin is initiated by the activation of Hageman factor, which also initiates coagulation and fibrinolysis (Figure 1.28). Plasma kallikrein circulates in the blood as an inactive zymogen from which active kallikrein is released by Hageman factor. Kallikrein appears to be located predominantly on the outer surface of epithelial cell membranes at sites thought to be involved in transcellular electrolyte transport (Miller et al, 1988). Negatively charged surfaces (collagen, urate) pH changes ------Temperature changes"
Hageman Factor ^ Activated Hageman Factor
Prekallikrein \Jy v Kallikrein
(plasma) HMW Kininogen N/ ^Bradykinin
LMW Kininogen .Kallidin
Kallikrein
(tissue)
Figure 1.28. Kinin Formation
High molecular weight kininogens may be cleaved by plasma kallikrein to produce bradykinin or by tissue kallikrein to produce kallidin. The kininogenase family includes
43 enzymes such as plasma and tissue kallikreins, trypsin, plasmin, snake venom
(B. jararaca) and proteases. Kininogenases are multifunctional proteins involved in cascade reactions during clotting and inflammation, and as inhibitors protecting cells from damage by cysteine proteases (Muller-Esterl, 1989). The primary function of tissue kallikrein is thought to be to form kinins with the additional function of processing enzymes for protein precursor molecules, enzymes and hormones (Lazure et al, 1983; Seidah et al,
1986; Drinkwater et al, 1988).
Low molecular weight kininogen is a substrate for kallidin only (Miller et al, 1988). Both high and low molecular weight kallikreins are synthesised in the liver and are derived from the alternative splicing of a single gene product (Kitamura et al, 1983). There may be conversion of kallidin to bradykinin as plasma aminopeptidase may remove the N-terminal
‘Lys’ of Kallidin (Burch et al, 1990). Kinins have a short half life of approximately 15 seconds and are degraded largely by the enzyme kininase II (angiotensin converting enzyme) and to a lesser extent by kininase I enzyme (carboxypeptidase N) which removes the C-terminal Arginine (Erdos et al, 1979). Ward et al (1991) found that in addition to angiotensin I converting enzyme (ACE) and carboxypeptidase N bringing about the degradation of bradykinin in plasma, aminopeptidase P enzyme was also active and hydrolysed the N-terminal Arg-Pro bond. The algesic effect of bradykinin is thought to be due to the stimulation of chemoreceptors on free nerve endings which are located in the paravascular connective tissue spaces around capillaries and venules (Lim, 1986;Armstrong, 1970). To show that bradykinin elicits algesia and that bradykinin receptors were present on sensory fibres, by inhibiting the production of bradykinin due to wounding or trauma, pain should be alleviated at these positions. Steranka et al (1988) have shown that bradykinin receptors were localised to sensory neurones and that a variety of bradykinin receptor antagonists selectively inhibited algesia in a number of
44 models of tissue damage. Steranka (1988) found that tritiated bradykinin receptors were concentrated in a narrow band in the dorsal horn of the spinal cord, the substantia gelatinosa, the dorsal root ganglion and the dorsal root, proving that bradykinin receptors are localised to nociceptive pathways (sensory nerve terminals) and small cells in the dorsal root ganglion.
1.15.THE MECHANISMS OF BRADYKININ SENSORY NEURONE EXCITATION AND SENSITISATION
A number of techniques have been used to try and elucidate exactly how bradykinin elicits pain and inflammation. Examples are the direct recording of C-fibre nociceptors in vitro and bradykinin elicited nociceptive reflexes (Lang et al, 1990; Juan et al, 1980; Griesbacher et al,
1987) and recording bradykinin mediated excitation of nociceptors in vivo in skin, skeletal muscle, joints and visceral organs (Foch et al, 1976). The activation of sensory fibres by bradykinin also causes the release of other neuropeptides e.g. Substance P and Neurokinin A (Maggi et al, 1990). The release of these peptides mediates a local proinflammatory effect which contributes to nociceptor sensitisation and hyperalgesia. These peptides stimulate vascular endothelial cells to release a smooth muscle relaxant, nitric oxide, which increases tissue blood flow and induces an inflammatory flare due to dilation of arterioles. Substance P also induces the contraction of the endothelium which facilitates the leakage of plasma proteins which accounts for neurogenic oedema. Other blood borne factors, e.g.SHT and ATP from platelets may interact with nociceptors and contribute to sensitisation (Rueff et al, 1992). Bradykinin is also thought to participate in inflammatory reactions where the activation of the kallikrein-kinin system by different pathological conditions is characterised by inflammation. These conditions include thermal and chemical injury, allergic reactions, rheumatoid, psoriatic and gouty arthritis, asthma, pancreatitis and inflammatory disease of the colon (Colman et al, 1979).
1.16.BRADYKININ RECEPTORS
Newly formed bradykinin acts locally, close to its site of production on neural and non
45 neural tissues producing a wide range of effects including smooth muscle contraction, glandular secretion, cell stimulation and sensitization and activation of sensory and sympathetic neurones (Dray, 1993). In addition to these well known effects, there is increasing evidence for the involvement of bradykinin in other pain conditions such as cardiac pain, inflammation and rheumatoid diseases (Bathon et al, 1991; Farmer et al, 1992; Meller et al, 1992). The effects elicited by bradykinin are hypotension, bronchoconstriction, gut and uterine contraction, epithelial secretion in airways, gut and exocrine glands, vascular permeability, connective tisue proliferation, cytokine release and eicosanoid formation
The presence of bradykinin, and its effects are stimulated by pathophysiological processes such as tissue injury (wounding), low pH, inflammation and anoxia (Figure 1.29), due to the action of tissue and plasma kallikreins at the site of injury in blood and tissue (Dray et al,
1993). ______Injuiy Anoxia Inflammation Low pH
Kallikreins and Kininogens
Bradykinin Inactive Prostanoid Production Metabolite Endothelium and Epithelium [DesArg] BK Immune Cells Smooth Muscle Mast Cells Sympathetic Nociceptive Neurones Neurones Figure 1.29. Physiological stimuli required for the production of bradykinin and mediation of effects via BK II receptors. 131 is the same as BK I, and for this thesis the nomenclature of BK I and BK II is used, as opposed to B1 and BII.
The role of bradykinin BK I receptors is not relevant to this thesis but it suffices to say they have a putative role in the state of hyperalgesia. Bradykinin BKl receptors have largely been studied in rabbit vascular smooth muscle (Marceau et al, 1991; 1993) although their existence has also been demonstrated in human fibroblasts (Goldstein et al, 1984) and P338-D1 murine
46 macrophage cells (Burch et al 1989). The expression of BKl receptors has been observed to increase over a number of hours whether in vitro or in vivo and may be enhanced by agents producing inflammation (Marceau et al, 1983). This may be an adaptive mechanism which occurs peripherally and centrally following the prolonged activation of nociceptors during inflammation and injury (Dray et al, 1993), with respect to algesia and inflammation, BKl receptors may have a role only in the state of prolonged hyperalgesia. Bradykinin acts acutely on a variety of tissues via the interaction with BK II receptors, stimulating pain directly or indirectly (Dray et al, 1993). Specific effects mediated by the bradykinin BKII receptor include contraction of the guinea pig intestine, contraction and relaxation of rat duodenum, contraction of the rat uterus and relaxation of arterial smooth muscle from a number of species including humans (Dray et al, 1993 ). Bradykinin effects on sensory and efferent autonomic nerve fibres include membrane depolarisation and modulation of neurotransmitter release. Bradykinin B receptors have been subdivided into different classes depending on their affinities for the analog [Thi^"^,D-Phe^]BK (Seguin et al, 1993). It was suggested that the bradykinin BK 11(a) subtype is coupled to a G-protein and the BK U (b) and (c) subtypes are either not coupled to G proteins or may be coupled to a different type of G protein, i.e.Go, not Gi, or the (b) and (c) subtypes are less sensitive to guanine nucleotides. The possibility of a BK III receptor arose when the bradykinin - induced bronchoconstriction and contraction of guinea-pig ileum was only partially inhibited by the standard second generation of BK II antagonists (Farmer et al, 1989b). The existence of BK III receptors have been reported in the intestinal epithelia, fibroblasts, primary brain cultures (Lewis et al, 1985;Braas et al, I988;Roberts et al, 1989) and three on a neuroblastoma cell line (Snider et al, 1984).
Bradykinin has long been thought to be a mediator of inflammatory pain based on its increased presence in injured tissue (Garcia, 1978; Armstrong, 1970; Clark, 1979). Injection of kinins (bradykinin, Lys-BK, kallidin) into the skin of animals or humans produces pain due to
47 the direct stimulation of sensory C fibre terminals and Substance P is also released which adds to the neurogenic inflammation. Axon-reflex mediated flare is caused by local vasodilation and oedema is created by the increase in vascular permeability and extravasation of proteins and fluid. Kinin stimulated release of cytokines fl*om monocytes attract leukocytes to the site of injection. Kinins release cytokines e.g.IL-1 and TNF (Tiffany et al, 1989) and many secondary generation mediators including prostaglandins and leukotrienes which are formed through the activation of phospholipase A2 (PA2). Bradykinin and kallidin have similar pharmacological potency in causing vasodilation and increasing permeability which results in local oedema (Bhoola et al, 1960). Bradykinin produces pain by stimulating C and A fibres in the peripheral nerve trunk which encodes the occurrence and intensity, duration and location of noxious stimuli (Kahn, 1986;Szolcsany,1987;Franz et al 1985). In humans, bradykinin causes a burning, stinging pain when applied to a blister base as well as after intradermal, intra-arterial or intra peritoneal injection (Clark, 1979). The algesic effect of bradykinin is potentiated by thromboxanes, prostaglandins and 5-hydroxytryptamine (5-HT). Kinins generated in injured or inflamed tissue activate sensory receptors that relay nociceptive information through C and A fibre afferent fibres to the substantia gelatinosa of the spinal cord. Writhing movements are produced in mice when bradykinin is injected intraperitoneally (Emele et al, 1963).
Bradykinin also exerts a vasodilatory effect which brings about a reduction in blood pressure.
Because of this, it has been implicated in the pathogenesis of several shock syndromes, particuarly septic and endotoxic shock (Colman et al, 1979).
1.17. CHARACTERISATION OF BRADYKININ RECEPTORS
A series of bradykinin agonists and antagonists have been used to classify the bradykinin receptors according to their relative potencies. The initial experiments carried out by Regoli and Barabe (1980) placed bradykinin receptors into BKl and BKII classes with the BKII receptor having a higher affinity for kallidin or bradykinin than for the kininase II metabolites
48 Des-Argl^ kallidin or Des-Arg^ kallidin. BKl receptors were more sensitive to the Des-Arg metabolites. It was not until 1985 that selective antagonists to the BK II receptor were available to use as tools for classifying these receptors (Vavrek et al, 1985). A number of bradykinin agonists and antagonists were used to determine the nomenclature of a receptor. For bradykinin there exists a series of compounds which have been used to classify bradykinin receptors into the I, II or III type. Bradykinin BKl receptor agonists have a higher affinity for the compounds [DesArgHypThiD-Phe]BK and [DesArg^JBk, and which
BK n receptors have a much lower afiBnity, and a higher affinity for [D-Arg^,Hyp^,Thi^,D- Tic7,0ic8]BK (Hoe-140) and [D-Phe^JBK. BK n receptors have been subdivided into different classes on the basis of the properties of the analogue [Thi^“^,D-Phe^]BK which has antagonist activity on the muscular receptor(Braas et al, 1988). Differences in agonist and antagonist profiles on different tissues fi-om the same organism may be due to species differences or the characteristics of G-protein coupling in different cell types. Saturable binding of monoiodo-Tyr-Kallidin to bovine and rat uterus membranes was first reported by Odya (1980). Following this, tritiated binding sites were demonstrated on membranes fi’om guinea pig intestine (Innis et al, 1981; Manning et al, 1982) and in 1983 Manning and Snyder published data revealing bradykinin attatchment sites were localised in the spinal cord and sensory ganglia by autoradiographic techniques. Further binding studies have identified bradykinin receptors on sensory fibres and neurones in the dorsal root, substantia gelatinosa and trigeminal ganglia (Steranka et al, 1988). A BK n receptor was cloned fi’om rat uterus by McEachem et al in 1991. The sequence predicted a protein of 366 amino acids, with a molecular mass of 41,696 Da. The receptor belonged to the superfamily of G protein coupled receptors containing seven transmembrane domains which span the membrane. The receptor was expression cloned using Xenopus oocytes and both bradykinin and kallidin activated the oocyte-expressed receptor equipotently. The human BK II receptor gene, as reported by Powell et al (1993) codes for a 364 amino acid protein with a molecular mass of 41,442 Da, which is highly homologous
49 to the rat BK II receptor oDNA (81% homology). Innis et al (1981) reported that tritiated bradykinin binds to membranes from a variety of mammalian tissues in a saturable fashion with a Kd of 5nm. The highest levels of binding were detected in guinea pig ileum, colon, duodenum and uterus.
Eggerickx et al (1992), cloned the gene which encodes a putative G-protein coupled receptor from human genomic DNA which was found to be homologous to a recently described rat BK II receptor (McEachem et al, 1991). The receptor was expressed in Xenopus oocytes and stably transfected to Chinese Hamster Ovary cells (CHO).
Displacement by BK antagonists and agonists allowed the characterisation of the receptor as a BK n subtype. The characterisation using bradykinin analogues did not match strictly the pharmacological profiles described for the rat or guinea pig BKII receptor subtypes or the putative BK II subtypes, as acknowledged by Farmer in 1989. This difference may be attributed either to species variability or differences in the coupling efficiency of the receptor to the transduction cascade in different cell types.
I.18.THE RATIONALE FOR NEW BRADYKININ RECEPTOR ANTAGONISTS
Bradykinin receptor antagonists which are effective at specific sites involved in the mediation of inflammatory, nociceptive, and/or vasodilatory processes would require bradykinin antagonists selective to BK II receptors or to a BK II receptor subtype. A bradykinin antagonist that would selectively inhibit either the PGI 2 or EDRF-mediated endothelium dependent vasodilation elicited by bradykinin would present a major advance with respect to both tissue selectivity and specificity of action. Resistance to the actions of Kininase I and II enzymes has largely been overcome by substituting non natural amino acid residues into the chemical stmcture of bradykinin. The first specific competitive sequence related antagonists of bradykinin were described by Vavrek and Stewart in 1986. Proline at position 7 in the bradykinin peptide was replaced by
D-phenylalanine and this conferred significant antagonistic effects on the molecule. Other
50 positions were subsequently modified in this compound which increased potency, tissue selectivity and metabolic stability (Stewart and Vavrek, 1987(a),(b). A competitive inhibitor of the BK II receptor should have a high receptor affinity with no intrinsic agonist activity.
Some of the bradykinin BK II antagonists e.g.[D-Phe^)]BK, have weak agonist and antagonist activity rather than pure antagonistic activity. Regulatory influences that modify bradykinin action may exert their action at three different levels;(l) at the prereceptor position, influencing the bioavailability of kinin receptor agonists ( 2 ) at the receptor level, by events which directly modify cellular kinin receptor in terms of biosynthesis, turnover, degradation, number and affinity or (3) at the post receptor level by signal transduction events that interact in a feedback mechanism or influence receptor characteristics. The biological effects observed with an antagonist are generally interpreted as the result of its ability to block receptor activation produced by an endogenous agonist. [D-Arg- (Hyp^Thi^D-Tic,Oic^)]BK (Hoe-140) is a potent and long acting bradykinin BK II antagonist in in vitro studies (Hock et al, 1991), differing from the known bradykinin antagonists by replacement of two residues with the unnatural amino acids 'Tic' and 'Oic'. It is a highly potent antagonist with an antagonist potency value (PA 2 ) 2-3 times greater than the antagonist D-Arg[Hyp^ ,Thi^"^,D-Phe^]BK, representing a new class of bradykinin antagonists. However, Hoe-140 may be less efficacious in vivo than expected in light of its potency at BK II receptors in vitro, as noticed when Hoe-140 activity was tested in models of persistent inflammatory hyperalgesia (Griesbacher et al, 1989). New bradykinin BK II antagonists are needed which have the potency of Hoe-140, are non-peptide and which have a high level of in vivo efficacy. If unusual amino acids are substituted into positions 2,3,5,7, or 8 and the N-terminal extended with 1 or 2 amino acids, classical bradykinin antagonists are produced, however these peptide compounds are subject to rapid enzymatic degradation and their short duration of action limits their influences as potential therapeutic agents (Griesbacher et al, 1989).
The bradykinin molecule is cleaved at a number of different sites. Aminopeptidase(APP)
51 brings about degradation by cleavage of the arginine-proline bond, neutral endopeptidase(NEP) cleaves between the amino acids proline and glycine, angiotensin converting enzyme I (ACE) cleaves the bond between serine and proline and carboxypeptidase N (CPN) cleaves the bond between proline and phenylalanine. The antagonists [D-Phe^]BK are not cleaved by ACE, and [Pro^] analogues should add greater stability. CPN is the major enzyme limiting lifetime of antagonists with a C- terminal
Arginine. Thus, a number of enzymes cleave the bradykinin molecule. The first bradykinin
BK n antagonists were partial agonists rather than antagonists in various biological systems, and promoted the release of histamine and prostaglandins, or they were not selective for BK
II receptors, having some affinity for BKl receptors, and a low affinity for BKII receptors.
1.19.SIGNAL TRANSDUCTION MECHANISMS OF BRADYKININ VIA BK n RECEPTOR SUBTYPES
The huge variety of post receptor events reported to be activated in various cells in response to kinin receptor occupancy may be explained on the basis of the same receptor being coupled to different signal transduction pathways in each cell type. It may also be possible that different receptor subtypes are each coupled to a specific signal transduction pathway.
The secondary messenger systems of bradykinin and its mediated effects fall into two classes, one leading to biologically active lipids, the other to activation of calcium sensitive systems.
In nearly all tissues, bradykinin induces the release of arachidonic acid and metabolites which result in the production of a variety of products. Depending on the tissue upon which bradykinin interacts, prostaglandins, leukotrienes, hydroxyeicosatetraenoic (HETES) or platelet activating factor (PAF) may be released. Inhibition of the release of these metabolites often blocks the effects of bradykinin. The release of arachidonic acid after the interaction of bradykinin with BKII receptors may be mediated by several different pathways, although
52 phospholipase A2 appears to be the most important (Burch et al, 1987). PA2 releases arachidonate directly from a phospholipid with a lysophospholipid as the other product. In some cells, activation of bradykinin receptors leads to the activation of a phosphatidylcholine and subsequent conversion to phosphorylcholine and diacylglycerol containing a saturated fatty acid and arachidonate (Clark et al, 1986). The mechanisms by which diacylglycerol is liberated to produce arachidonate is uncertain but it may involve a diacylglycerol or monoacyglycerol lipase (Majerius et al, 1986), or, after phosphorylation, a phosphatidic acid specific phospholipase A2. In nearly all tissues, bradykinin activates PI-PLC to release phosphate inositol and diacylglycerol and inositol phosphates are involved in the release of calcium from the endoplasmic reticulum (Berridge, 1987). Bradykinin has been reported to stimulate a variety of intracellular events including the accumulation of cyclic AMP and the activation of phospholipase C (Portilla et al, 1988) which lead to enhanced levels of inositol phosphates, cytosolic calcium, diacylglycerol and the activation of PA2 (Dixon et al, 1989).
1.20.SEVEN TRANSMEMBRANE RECEPTORS
There are approximately 150 seven - transmembrane segment receptors characterised, each has its own attendant ligand and comprises a highly selective combination which is responsible for signalling in virtually every organ in the body. This family comprises adrenoreceptors, neurokinins and dopamine receptors (Frielle et al, 1988). Bradykinin BK II receptor mediated inositol lipid hydrolysis and eicosanoid release are dependent on the coupling of receptors to G-proteins (Burch et al, 1987; Francel et al, 1989; Higashida et al,
1986; Murayama et al, 1987). Bradykinin BK II receptor activation often leads to the enhanced cellular accumulation of cyclic AMP (Stoner et al, 1973;Brunton et al, 1976)
53 which is often due to the stimulation of the biosynthesis of eicosanoids which subsequently bind to their respective receptor and activate adenylate cyclase (Brunton et al, 1976). In some tissues the bradykinin receptor may be directly coupled to adenylate cyclase by G proteins (Liebmann et al, 1991). In rat myométrial membranes two tritiated bradykinin binding sites with Kd values of 19pM and InM were identified. At IpM concentration bradykinin stimulated high affinity GTPases.
This effect was abolished by the treatment of membranes with pertussis toxin. Myométrial membranes contained two pertussis toxin substrates of 40 and 41 KDa which correspond immunologically to a - subunits, of type Gi G-proteins. In uterine smooth muscle, G- proteins of the Gi family couple high afiBnity bradykinin BKII receptors to their effector enzymes. Burch et al, (1987) showed that rat myometrium possess two binding sites and the high afiBnity binding site represented a receptor coupled to pertussis-sensitive G-proteins of the Gi family. At concentrations above IpM, bradykinin stimulated the high afiBnity GTPase.
The stimulatory effect of bradykinin was not observed if the bradykinin antagonist [D-
Arg(Hyp^Thi^"^,D-Phe^)]BK, which did not affect GTPase activity in the absence of bradykinin, was present in the incubation medium at a concentration of lOuM. The treatment of membranes with pertussis toxin did not affect basal GTPase activity and abolished the stimulatory effect of bradykinin on the high afiBnity GTPase activity. This indicates that the high afiBnity bradykinin binding site represents a receptor capable of activating pertussis toxin-sensitive G-proteins. Data indicates that in rat myométrial membranes the G-proteins which mediate bradykinin effects are members of the Gi family (G 12, Gig). They functionally couple a high afiBnity bradykinin receptor to effector systems which generate signals e.g. adenylate cyclase or phospholipase.
54 1.2 1 .THE STRUCTURE OF G-PROTEIN COUPLED RECEPTORS
Common features to members of the family are the presence of seven hydrophobic protein sequences which span the cell membrane (Dohlman et al, 1987) and interact with one or more GTP-binding proteins to promote high affinity binding with a ligand and transduce intracellular signals (Figure 1.30.) (Gillman, 1987).
intracellular IM |
membrane
extracellular
Figure 1.30. A model of the structural domains of G-protein coupled receptors (Lee et al, 1993). The transmembrane domains are depicted as cylinders perpendicular to the plane of the plasma membrane. Transmembrane domains 1-7 (TM1-TM7) are proposed to traverse the membrane in an alpha-helical fashion, connected by alternating extracellular (el-e3) and intracellular (il-i3) loops. The amino- (NH2) and carboxyl-(COOH) terminal regions of G- protein-coupled receptors are situated at the extracellular and intracellular sides of the plasma membrane, respectively. The second messenger system initiated due to the interaction of bradykinin with the BK II receptor may be a function of the receptor coupled G-protein in each cell type which may indicate the occurrence of BK2 receptor subtypes (Bhoola et al, 1992). The linkage of each bradykinin receptor and subtypes to a specific G-protein coupled secondary messenger
system could explain the differences in cellular actions on the same cell by bradykiniiL G-protein coupled receptors mediate the actions of a diverse range of extracellular signals in many biological systems. The first mammalian G-protein coupled receptors to be characterised were rhodopsin (Hargrave, 1982) and the beta adrenoreceptor (Nathans
1987;Tota et al, 1990). G-protein coupled receptors feature a seven transmembrane domain which spans the intracellular and extracellular domains of a membrane. The seven transmembrane domains are formed by alpha helices of hydrophobic amino acid sequences
55 (Kyte et al, 1982). Members of this receptor superfamily mediate cellular responses to diverse ligands e.g.light, hormones, alkaloids and peptides. Most of the G-protein linked receptors consist of single polypeptide chains forming seven putative transmembrane helices through the plasma membrane.
1.22.SIGNALLING BY SEVEN TRANSMEMBRANE RECEPTORS
G-protein coupled receptors exist in two states, either complexed with a G-protein, where they have a high affinity for agonists, or uncomplexed where they have a low agonist affinity. Addition of Guanosinediphosphate, (GDP), Guanosinetriphosphate(GTP) or their analogues causes G-protein dissociation and a reduction in agonist affinity. When seven transmembrane receptors bind to agonists, they catalyse the exchange of GTP for GDP which is bound by the alpha subunit of an associated heterotrimeric G protein(Clapham et al, 1993; Hepler et al, 1992; Neer et al, 1988). When GTP binds to the a sub unit it activates the G-protein which promotes the dissociation into a GTP and a P-y complex. Free forms of a GTP and p-y directly regulate the activity of effector enzymes. The intrinsic GTPase activity of the G- protein a-subunit hydrolyses the GTP, and a-GDP reassociates with P-y. Protein(s) stimulated by individual seven transmembrane receptors determine the type(s) of signal transduction pathways triggered by hormone binding (Spiegel et al, 1992). The P-y subunits of G- proteins also regulate the functions of effector proteins. Insights into the structure- function relationship of G-protein coupled receptors has been made possible with the use of techniques such as in vitro mutagenesis (Savarese et al, 1992). This technique is used to identify the amino acids critical for ligand binding, to determine the domains on the receptor responsible for interacting with G-proteins and to analyse the molecular basis of receptor desensitisation.
1.23.REGULATION OF BRADYKININ ACTIVITY
As soon as bradykinin is produced and is mediating algesic and inflammatory effects, other factors are coming into play which counteract the effects of bradykinin and limit its actions in vivo, mainly the desensitisation of the BK II receptor and the rapid degradation of bradykinin
56 by the proteolytic enzymes ACE and kininase I. Cyclic GMP dependent kininase which phosphorylates the bradykinin receptor brings about desensitisation of the receptor. The response of sensory neurones to bradykinin is desensitised by the production of nitric oxide, which activates guanyl-cyclase to increase cellular cyclic GMP (Burgess, 1989).
Desensitisation possibly involves membrane glycoproteins and down regulation of receptor proteins following aggregation and internalisation of receptor proteins (Dray et al, 1993).
Whether receptor desensitisation occurs during tissue injury is not clear as endogenous bradykinin may be metabolised too quickly to bring about desensitisation and local levels may not be high enough to induce sensitisation (Dray et al, 1993). Desensitisation may involve several mechanisms including the dislocation of receptors from secondary messengers or translocation and internalisation of membrane receptor protein (Roscher et al,
1984).
57 1.24.CALCITONIN GENE RELATED PEPTIDE AND MEDIATION OF NOCICEPTION IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM
The hormone Calcitonin is synthesised in the C cells of the thyroid. During studies of calcitonin gene expression in rat medullary carcinoma of thyroid cell lines, it was noted that cells could be switched from the production of mRNA for calcitonin to a second related mRNA species that was 50-250 nucleotide bases longer but did not encode calcitonin (Dockray, 1988). The two mRNA species were shown to be produced from a single gene by alternative splicing of the initial transcript. The sequence of the hypothalamic type cDNA indicated it encoded a peptide different to that of a preprocalcitonin in the C-terminal region which had the features of a regulatory peptide precursor. Rosenfeld et al (1983) assumed that this precursor molecule was processed by steps known to apply to other peptides, and predicted that a 37 residue C-terminally amidated peptide would be liberated on cleavage. This was Calcitonin Gene Related Peptide (CGRP), isolated from rat and had the amino acid sequence :
Ala-Cy s-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu- S er-Arg- S er-Gly- Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Glu-Ala-Phe-NH2 A second rat gene closely related to CGRP was later found by Amara et al(1985) so that the two CGRP peptides were known as alpha(a) and beta(P) and differed in the substitution of
Glutamine (alpha) for Lysine(beta) in the third amino acid adjacent to the N-terminal end of the molecule. The calcitonin encoding region was absent in the P CGRP gene. Humans also have two genes which encode CGRP, differing by three residues to each other. There are some suggestions that rat and human a-CGRP may differ slightly in biological activity
(Marshall et al, 1986), but structure-activity relationships would have to be ascertained to prove differences in function between the two types of peptide. The calcitonin gene was found to encode two different mRNA’s with identical 5’ primer sequences but different 3’ primer sequences(Rosenfeld et al, 1983). The mRNA’s encode either the 17,500 MW precursor protein which is proteolysed to yield calcitonin (CT) and two other peptides, or a 16,000MW protein which is the predicted translation product of CGRP mRNA(Rosenfeld et al, 1983). From the nucleotide sequence of cloned CGRP
58 mRNA, it was predicted that the encoded protein was proteolytically processed, yielding three peptides including the 37 amino acid CGRP. The synthesis of mRNA for CGRP by alternative splicing in the formation of mRNA from calcitonin genes has been shown in medullary thyroid carcinoma cell lines(Amara et al 1982). CGRP genes have also been reported in the human ventral spinal cord and pituitary gland(Tschopp et al, 1985). The structure of a hCGRP, as predicted by nucleotide sequencing has been confirmed by Fast
Atom Bombardment Mass Spectroscopy (FAB-MS) (Morris et al, 1984).
1.25.CGRP IMMUNOREACTIVITY
CGRP immunoreactivity and binding sites have been mapped throughout the central and peripheral nervous systems(Yamamoto et al, 1989). CGRP immunoreactivity in the spinal cord is concentrated in the areas which receive sensory input into the dorsal horn, and to a lesser extent, areas associated with autonomic inputs. There is evidence that some motor neurones contain CGRP in the ventral horn. In the brain, CGRP is present in the nuclei of sensory and motor cranial nerves and cell bodies in the hypothalamus, preoptic area, ventromedial thalamus, medial amygdala, hippocampus, superior colliculus, lateral lemniscus and dendate gyrus(Goodman et al, 1986). In the peripheral system, CGRP is located in the sensory and motor nerves. In the autonomic system it is present pre-ganglionically in sympathetic and parasympathetic nerve fibres and postganglionically in nonadrenergic and non cholinergic neurones(Poyner 1992).
CGRP has been found in the gastro intestinal tract, tongue, oesophagus, pancreas, salivary
glands, lungs and the kidney (Yamamoto et al, 1989; Mulderry et al, 1985,1988; Kummer et
al, 1991). CGRP receptors are widely distributed in the nervous system(Tschopp et al, 1985; Henke et al, 1987; Wimalawansa et al, 1987) and in the cardiovascular system(Wimalawansa et al, 1988; Sigrist et al, 1986; Chatteijee et al, 1991). The highest density of CGRP receptors was found in the cerebellum and the substantia nigra (Tschopp et al, 1985; Wimalawansa et al, 1987).
59 1.26.PHYSIOLOGICAL EFFECTS MEDIATED BY CGRP
The wide distribution of CGRP suggests its involvement in a range of processes, and the distribution of the a and P forms of CGRP differ. In the enteric nervous system, a CGRP is located in sensory nerves and the intrinsic submucosa whilst myenteric neurones express P CGRP(Mulderry et al, 1988). The independent regulation of the two forms of CGRP suggest different actions, possibly mediated by different receptors. CGRP often coexists with other substances e.g. at the neuromuscular junction with acetyl choline in motor neurones (Mora et al, 1989) and in the monkey bulbospinal tract with 5-hydroxytiyptamine and preprotachykinin mRNA (Arvidsson et al, 1990). A study of the rat colon by Ekbald et al (1988) reported four populations of CGRP containing nerves. Some sensory fibres contained CGRP and Substance P, other perivascular fibres contained CGRP and vasoactive intestinal peptide, some submucosal nerves contained CGRP and somatostatin and some myenteric and submucosal ganglions contained CGRP only (Kummer et al, 1991). Goodman et al (1986) noted the colocalisation of CGRP and substance P in the stellate ganglion, and co-localisation with substance P in the CNS and periphery was common.
1.27.CENTRAL NERVOUS SYSTEM ACTIONS OF CGRP
When CGRP was injected into the rat CNS the molecule had actions which indicated it activated neuronal pathways. CGRP increases noradrenergic sympathetic outflow(Fischer et al, 1983) leading to an increased heart rate and blood pressure. Further pharmacological
effects mediated by CGRP are a decrease in appetite (Tannebaum et al, 1985) and gastric acid secretion (Lenz et al, 1985), and a decrease in intestinal activity (Fargeas et al, 1985). CGRP decreases growth hormone release via a central action (Tannebaum et al, 1985) and it increases rectal temperature which suggests a role in thermoregulation(Dennis et al, 1990). CGRP has also been found to decrease motor activity and causes catalepsy in rats, and there are indications that CGRP interacts with dopaminergic neurones of the basal ganglia, this is consistent with the presence of CGRP receptors in the substantia nigra (Jolicoeur et al,
60 1989). Sana et al (1986) have shown that CGRP is released from capsaicin-sensitive neurons in a calcium dependent manner in a number of peripheral organs and central terminals. High capsaicin concentrations and nicotine evoke CGRP release via other mechanisms. CGRP released due to the irritation of peripheral branches may evoke a number of cardiovascular actions and influence motility in the gastrointestinal and urogenital tracts.
1.28.THE RATIONALE FOR THE USE OF A CGRP ANTAGONIST IN MIGRAINE
CGRP may be an important mediator in migraine and other vascular headaches. It is localised in the trigeminal nerve which innervates the cranial vasculature and is involved in the perception of pain in migraine. It has been shown clinically that plasma levels of CGRP are increased during a migraine attack and in response to trigeminal nerve stimulation. CGRP released from the trigeminal nerve terminals is thought to produce a vasodilatation of cranial blood vessels and together with Substance P evokes extravasation of plasma proteins from cranial blood vessels. Inflammation and vasodilatation lead to a further activation of trigeminal nerve terminals which sets up reflex activity and additional peptide release. A non peptide antagonist may alleviate the pain of migraine by attenuating the vasodilatation and inflammation induced by CGRP and by reducing the efficacy of Substance P. Recent research into the mechanisms underlying migraine has gained an insight into the processes which may explain the prolonged periods of dizziness occurring in some migraine sufferers. Some treatments are thought to work by suppressing the antidromic release of
neuropeptides from sensory neurones into the durai circulation e.g. Substance P and CGRP (Moskowitz et al, 1990). In one animal experimental model, the release of these peptides is elicited by repetitive stimulation of the trigeminal ganghon, which results in inflammation of durai blood vessels with extravasation of plasma proteins(Moskowitz et al, 1990; Buzzi et al, 1990). It is suggested that neuropeptide release is involved in the migraine associated
dizziness. CGRP-positive neurons have also been identified in the trigeminal sensory nucleus
and the closely associated pontine nuclei of the vestibular efferent system (Kruger et al, 1988).
61 The cochlea, taste buds and olfactory epithelium are richly innervated by CGRP-containing afferent fibres (Silverman 1989). In common with the vestibular system, the cochlea receives efferent fibres rich in CGRP (Takeda et al, 1986).
Phonophobia and a heightened sensitivity to taste and smell are commonly reported by migraine patients. Drugs are required that block the release and uptake of neuropeptides into the vestibular system, stopping the dizziness and sensitivity to motion. This type of drug may possibly also block the release of neuropeptides into durai blood vessel walls, thus preventing headaches (Moskowitz 1990).
Lance et al (1977) hypothesise that in migraine dilatation and pain are causally related, with dilatation developing as a consequence of perivascular pain fibre stimulation (Humphrey et al, 1991; Moskowitz, 1984). Sensory fibres projecting to meningeal arteries, veins and sinuses fi’om the ipsilateral trigeminal ganglion (Moskowitz, 1984,1990) contain potent vasodilating neuropeptides e g CGRP, NK A and Substance P. Due to depolarisation these neuropeptides are released into the vessel wall by calcium dependent mechanisms(Moskowitz et al, 1983) therefore vessels dilate and flow may increase. In support of this, plasma levels of released CGRP increase within the internal jugular vein or sagittal sinus during trigeminal stimulation in experimental animals (Buzzi et al, 1991) and humans (Goadsby et al, 1990). Vasodilatation may not be the cause of migraine pain but develops as a consequence of sensory neurogenic activation. The cause of activation is unknown although algogenic substances from the brain, blood vessel wall or vessel lumen are implicated (Moskowitz et al, 1991).
1.29.CO-LOCALISATION OF CGRP WITH OTHER NEUROPEPTIDES The spinal (as well as medullary) dorsal horn receives various sensory inputs, including nociceptive information from the peripheral tissues. Nociceptive information is conveyed by unmyelinated and finely myelinated primary afferent neurons, most of these terminate in the superficial laminae of dorsal horn. Immunohistochemical studies have shown that nociceptive primary afferents contain many different peptides, e.g. Substance P (Hokfelt et al,
62 1975)Somatostatin (Hokfelt et al, 1976) and CGRP (Amara et al, 1982). Moore et al (1989) has shown that all somatic motor cranial nerve nuclei contain CGRP and galanin-like immunoreactivity and both peptides were also found to be present in nerve fibres which innervate striated musculature. CGRP and galanin-like immunoreactivity appeared to be present in spinal motor neurons, galanin was not. Moore’s observations suggest that galanin and CGRP participate in the process of synaptic transmission at the neuroefifector junction of
cranial motor neurons. CGRP has been reported to potentiate the action of Substance P when the peptides are co administered into the CNS (Hokfelt et al, 1986). The extensive co-distribution suggests that in vivo, CGRP is more likely to modify the response of target tissues to other substances. In some areas there are high numbers of receptors and low CGRP immunoreactivity e.g. the molecular layer of the cerebellum. This pattern of distribution and activity does not entirely fit with the localised actions associated with classical neurotransmitters such as bradykinin, and is more suggestive of a neuromodulator.
1.30.CGRP AND CALCITONIN RECEPTORS
Receptors may be classified into different classes depending on how their effects are
mediated, e.g. receptors may be ligand-gated ion channels, or intrinsic intracellular enzymatic
domains or the receptor may be coupled to a guanine nucleotide binding regulatory
protein(G protein) which in turn activates a separate effector such as an ion channel or
enzyme. Generally, CGRP and calcitonin (CT) interact with separate and different receptors.
Calcitonin receptors have distinct distributions (Fischer et al, 1981;Tschopp et al, 1985) and
structure activity relationships(Findlay et al, 1985;Nicholson et al, 1986). Salmon calcitonin
acts on ‘true’ calcitonin receptors at concentrations in the nanomolar range or below, but at
the CGRP receptor it is only active at micromolar concentrations(Poyner et al, 1992). The
opposite pattern exists with CGRP (Zaidi et al, 1990).
63 Based on the effects of CGRP antagonists, it has been suggested that there are two classes of CGRP receptors. CGRPj receptors are more sensitive to the analogue CGRP^"^^ and
CGRP2 is more sensitive to [Cys(ACM)^’^]CGRP. CGRPj receptors are typified by those in the guinea-pig heart and CGRP 2 receptors in the rat vas deferens (Dennis et al, 1990). This classification relies on the comparisons between rat and guinea pig tissues, however
species differences between CGRP receptors do exist, and this classification of receptor may need to be revised. There is a lack of suitable CGRP analogues to characterise the CGRP receptor and any further subtypes fully. Human and rat a and P variants are available but as they only differ by 5 amino acids out of 37, differences between agonists are very small. Amylin, which has 46%
sequence identity with CGRP is a potent CGRP analogue although tests using this compound have shown variation in experimental results (Leighton et al, 1990 ; Poyner et al, 1992).
1.31.CGRP RECEPTOR AGONISTS AND ANTAGONISTS
Three distinct binding sites for Calcitonin (CT)/CGRP peptides were reported in rat
brain(Sexton et al, 1988). C% exhibited high selectivity for Calcitonin, C 2 was selective for
CGRP and C3 displayed affinity for both CT and CGRP.
1.31.1. CGRP 8-37
As an antagonist, this peptide was first described by Chiba et al (1989) who showed that the compound produced a parallel rightwards shift in the dose response curve to human alpha CGRP in a cyclic AMP production assay in rat liver membranes. CGRP had no agonist
activity at concentrations upto 10 micromolar, however it had weakly agonist effects on the calcitonin receptor of LLC-PK cells (Yamaguchi et al, 1988). Chiba et al (1989) have reported a demonstration of the activity of a related fi^agment, CGRP 12-37 and this study
indicated an antagonist which could discriminate between different CGRP responses. Other
experiments have been carried out using this peptide, and these have shown that CGRP^"^^ does not appear to bind to all receptors with equal affinity and may act non-competitively under some conditions.
64 1.31.2. [TvrlCGRP ^8-37
The most detailed description of this compound was for its actions on the oppossum internal anal sphincter (Chakder et al, 1990). Data suggests that human and rat CGRP interact at separate receptors in this tissue but they have different affinities for the analogue [Tyr]CGRP^^"^^ which is a potentially useful antagonist. However, to use it as a reliable tool to probe receptor heterogeneity, further experiments need to be carried out.
1.31.3. Human Calcitonin Hirata et al, (1988) reported that human calcitonin could displace iodinated CGRP from cultured rat vascular endothelial cells, indicating it can interact with the CGRP binding site.
There were no agonist effects reported using concentrations of the peptide of upto 5 micromolar. To conclude that this peptide is an antagonist in this system would require further experiments to be carried out. For antagonist activity using the CGRP molecule as a base, Dennis et al (1989) found that the N-terminal portion of the CGRP molecule encompassing the disulphide bridge between positions 2 and 4 was not critical for affinity to the receptor. Data to support this was shown by the relatively high affinity of the C-terminal fi*agment hCGRP^^"^^, whereas a variety of N-terminal fragments (e.g. cyclo^’^-CGRP^"^, cyclo^"^hCGRP^“^, cyclo^»^hCGRP^'^ or cyclo^’^-CGRP-hCGRP^"^) displayed no affinity for iodinated hCGRP and binding sites. The relatively limited importance of the disulphide bridge for the affinity of the CGRP molecule to its’ receptor is revealed further by the relative potencies and affinities of the linear CGRP analogue [Cys(ACM)^’^]hCGRP and cyclo^’^-[Asp^,Lys^]hCGRP. In the rat CNS a high affinity CGRP binding site with a Kd of 0.25nmol was characterised with a Bmax of 8 finol/mg protein and a low affinity site was also located with a Kd of 80nmol and a Bmax of 120finol/mg protein (Wimalawansa et al, 1987; Nakamura et al,
1986). In cardiovascular tissues a single class of CGRP receptors was reported with a Kd of 3nmol and a Bmax of 120finol/mg protein(Wimalawansa et al, 1988;Chatteijee et al, 1991). Stangl et al(1991) characterised and photoaffinity labelled a CGRP receptor from human cerebellum. This was a single class of binding sites with a Kd of 50pM for intact iodinated human CGRP^"^^ and 160pM for the antagonist iodinated human CGRP^"^^. 65 1.32.CGRP RECEPTORS EXPRESSED IN THE SK-N-MC CELL LEVE Van Halen et al (1990) found that CGRP receptors were linked to cyclic AMP production in SK-N-MC human neuroblastoma cells. The human SK-N-MC cell line was originally prepared from a tumour diagnosed as a neuroblastoma. Scatchard analysis indicated the presence of a homogeneous population of binding sites with a Kd of 52pM and a Bmax of 15frnol/mg protein. It was found that CGRP regulated cyclic AMP production in human neuroblastoma cells via interaction with highly selective CGRP 2 receptors.
1.33.CLEAVAGE OF CGRP
CGRP is cleaved by an endopeptidase which is also known to hydrolyse substance P. This enzyme was isolated from human cerebrospinal fluid and converted rat CGRP into the two products rCGRP^"^^ and rCGRP^^"^^ (LeGreves et al, 1989). Amino acid analysis indicated that the cleavage occurred at the bond between the amino acids Leu^^ and Ser^^ for both Substance P and CGRP. Substance P and CGRP are assumed to adopt an a-helical conformation in the region of the respective cleavage sites, thus it is possible that the endopeptidase requires this kind of conformation to be active and that cleavage is directed to the amino acid sequence which is exposed. Le Greves(1989) did not locate any biological activities of the generated CGRP fragments.
6 6 1.34. SUBSTANCE P AND THE NEUROKININ 1 (NK1> RECEPTOR.
The Neurokinin 1 receptor (NKl) receptor ligand substance P (SP) is an eleven amino acid peptide which belongs to the tachykinin group of neuropeptides, which have a common five amino acid carboxy terminal sequence. SP has the structure Arg-Pro-Lys-Pro-Gln-Gln-Phe-
Phe-Gly-Leu-Met-NH2 with the receptor binding area at the carboxy terminal end.
The NK 1 receptor is a seven-domain transmembrane protein and structurally the receptor has seven hydrophobic segments of 20-25 uncharged amino acid residues. The cytoplasmic
carboxy termini of the receptor has a pattern similar to the G-protein coupled receptors, with
significant sequence similarity, and serine and threonine resisues present. This similarity to
other G-protein-receptor systems allows the tachykinin receptor to participate in intracelllar
second messenger systems (Nakanishi, 1991),
Substance P has a wide distribution in the body. The organs in which it has been found
include the CNS, peripheral nervous system, sympathetic ganglia, cardiovascular system,
gastrointestinal tract, skin and salivary glands (Pemow, 1983).
The U373 MG cell line has been previously studied by Lee et al, 1989 and has been
demonstrated to express the NK 1 receptor.
67 1.35. AIMS The search for analgesic compounds from higher plants required that a number of aims should be set to facilitate the discovery of natural product novel compounds from plants. The aims of this project were to identify and isolate bio-active compounds which could either be used as extracted from the natural source or which would have a potential structure on which ‘lead molecules’ could be based to produce antagonists to the Calcitonin Gene Related Peptide CGRP) and the bradykinin BK II receptors. The following aims are set in an effort to isolate natural product neuropeptide antagonists to the bradykinin BK II and CGRP receptors : 1.A tissue or cell source which expresses the appropriate receptor will be identified and radioligand binding assay methods will be fully developed which involve expression of the receptor in in vitro conditions. 2. The scientific and medicinal literature of China, the West Indies, West Africa and South America will be searched to select plants which have been, or are being used as analgesics in traditional medicine. 3.The methanol plant extracts which produce a positive result in the bradykinin BK II assay will be investigated for their potency and selectivity to the BK II receptor. 4.The methanol plant extracts which produces the most potent and specific activity will be selected in an effort to elucidate the compound(s) responsible for bioactivity in the bradykinin BK II assay.
5.The methanol plant extracts which are not selective to the BK II receptor but which have potent activity in the BK II and Neurokinin 1 (NKl) receptor - binding assays will be screened in a model of acute pain to determine their in vivo effects. ô.Methanol extracts of the same plants which are to be screened in the bradykinin BK II assay will also be screened against the CGRP receptor to identify selective and specific activity to the CGRP receptor.
6 8 CHAPTER TWO MATERIALS AND METHODS 2.1. CHAPTER TWOiEXPERIMENTAL METHODS AND MATERIALS
Radioligand receptor binding techniques have been used extensively to identify and characterise a whole range of receptors and enzymes involved in hundreds of different therapeutic areas. (Sweetman et al, 1993). Many drug discovery programmes utilise this type of assay to identify lead compounds which interact with receptors that have roles in neuronal, gastrointestinal, immunological and cardiovascular therapeutic areas. A number of advantages exist in utilising radioligand binding assays; binding studies require small amounts of test compounds, (microgrammes to milligrammes), while whole animal studies may routinely require gramme quantities. Small amounts of tissue are required as compared to the cost and maintenance of live animals for in vivo screening. The structure activity data generated in binding assays are a reflection of the ligand/receptor interaction without the added complications which result from secondary effects in animals, once the drug has interacted with the receptor, e.g. bioavailability, side effects and pharmacodynamic interactions. Differences in the supply and labour costs for a radioligand receptor binding assay and experiments requiring animals may be negligible, although increased speed, efficiency and the potential for screening thousands of compounds in a short period of time are
advantageous when using in vitro binding assays. The use of radioligand receptor binding assays has largely been based on the preparation and use of membranes from tissues dissected from an animal. Such a method is that of the solubilisation and characterisation of a bradykinin BKII receptor
from rat myométrial membranes (Snell et al, 1988). Uteri were removed from female rats to produce a membrane preparation which was used to screen compounds and plant extracts. More recent has been the molecular cloning, functional expression and pharmacological characterisation of a human bradykinin BKII receptor gene (Eggerickx et al, 1992). This method involved cloning a gene which encoded a
69 putative G-protein coupled receptor from human genome DNA which was expressed in stably transfected Chinese hamster ovary (CHO) cell lines. A number of BK I and
BK II analogues were tested against the BK II receptor expressed in CHO cells to verify that its expression fitted that of the pharmacological profile of the BK II receptor. This method was advantageous to use in terms of selectivity as only the BK II receptor was expressed in the CHO cell line, whereas in membrane preparations from rat uteri, BK II, and to a lesser extent, BK I receptors may also be expressed. Receptor radioligand binding assays were used for the human bradykinin BKII receptor and the human Calcitonin Gene Related Peptide (CGRP) (Methods 2.3 and 2.9). Radioligand binding assays accurately determine the specific binding of a radioligand to the targeted receptor through the delineation of its total and non specific binding components. Total binding, involving the receptor and radioligand in a biological system, is defined as the amount of radioligand remaining following the rapid separation of unbound radioligand from radioligand bound to the receptor. The nonspecific binding (NSB)component is defined as the amount of radioligand remaining fbllovying separation of the reaction mixture consisting of the receptor, radioligand and excess unlabelled ligand. Very high concentrations of the drug used to determine NSB may also inhibit NSB, thus it is more useful to use a drug which is chemically dissimilar from the radioligand, due to the possibility of the drug inhibiting 'specific' but not non - receptor binding sites. The radioligand remaining represents that which is bound to components other than the receptor. The specific radioligand bound is determined by subtracting the nonspecific bound from total radioactivity bound and is generally expressed as a percentage of total binding. The greater the percentage value, the more reproduceable and reliable the assay is, as a tool for drug discovery. Assays with a specific binding component of greater than 80% are excellent and are often suited to high throughput (high volume) screening, assays with values of specific binding in the area of 50 to 60% are still workable, but associated with this is an
70 experimental variation. A number of problems are associated with radioligand binding assays e.g. many
radioligands have the potential to interact non selectively with other membrane proteins in a receptor preparation or they may bind to the glass fibre filters. This may be decreased by ( 1) pretreating filters, plates and test tubes with a solution of polyethylenimine (PEI) which decreases non specific binding (NSB) to the filter, (2) limiting the amount of receptor preparation per reaction volume since NSB often increases as the concentration of receptor preparation increases. The kinetic properties which describe the interaction between transmitters and/or drugs are similar to those used for enzyme studies; binding has to be competitive, specific, reversible and saturable. The equilibrium condition for a particular ligand/receptor interaction is one of the first parameters to establish when developing a radioligand binding assay. Equilibrium is defined as the time it takes 'specific' binding to reach a maximum. All experiments should be determined under steady state conditions so that results are valid and standardised. Saturation analysis determines whether the specific binding component of an assay is saturable, which reflects the finite number of receptors available in any tissue or cell preparation. Specific bound values are plotted against concentration of radioligand to produce a Scatchard plot for the linear analysis of binding data. This gives a value for Bmax, which is defined as the maximum amount of drug (expressed in picomoles/milligramme of protein) which can bind specifically to the receptor in a
membrane preparation. If one drug binds to one receptor, it indicates the concentration of receptor in the tissue. The Kd, defined as the dissociation constant for a radiolabelled drug is also determined by saturation analysis, and is measured in moles per htre, and is the concentration of drug at equilibrium which occupies 50% of the receptors.
Pharmacological specificity of the radioligand for the receptor is verified by carrying out a displacement curve of the compound or extract in the assay. The radioligand
71 concentration and all other experimental conditions are kept constant, while the concentration of the inhibiting compound or extract is varied. The data generated determines the concentration of drug or test compound required to inhibit 50% of the
specific binding in the assay (IC 5 0 ). IC50 values may vary slightly, the most common reasons being the concentration of radioligand used and the apparent Kd of the receptor. To correct for these differences, IC 50 values are transformed to inhibition constants, (Ki) using the Cheng Prussof equation (1973) :
Ki = IC5o/l+([L]/Kd) where Ki = inhibition constant for a drug: the concentration of competing ligand in a competition assay which would occupy 50% of the receptor if no radioligand were present. [L] = concentration of free ligand used in the assay. Kd = Dissociation constant of the radioligand for the receptor.
When carrying out the full characterisation of a receptor, it is also necessary to
establish a rank order of potency of various agonists and antagonists known to interact with a specific receptor. This determines the selectivity of radioligands for the receptor being targeted for testing compounds against, and such a method produces a profile of the receptor in relation to agonists and antagonists which are active in the
assay. For displacement curves see Figures 3.4,3.5 and 3.6, pages 106-108 and Table 3.2pl09.
72 2.2. THE INITIAL DEVELOPMENT OF THE BRADYKININ RADIOLIGAND BINDING ASSAY USING A RAT UTERUS MEMBRANE PREPARATION.
The bradykmin radioligand binding assay that was initially developed by Snell et al (1988) was used to test the affinity of methanol plant extracts for the bradykinin BK II receptor, and was measured by the displacement of tritiated bradykinin jfrom the BK II receptor, the bradykinin BK II receptor being expressed in the uterus of female rats.
Briefly, the method for the preparation of rat uteri membranes was as follows ;
The uteri were dissected out from female Sprague - Dawley rats. The rats weighed between 150 - 200 grammes, and the dissected uteri were placed in buffer G. The uteri were homogenised using a
‘Polytron’ homogeniser on setting number 6, for 15 seconds. The homogenate was centrifuged at
2000rpm for 10 minutes, after which the supernatant was discarded and the pellet was resuspended in buffer G. The homogenisation and centrifugation procedure was repeated three times and the final pellet was resuspended in buffer H. The volume of buffer H added to the pellet was calculated by multiplying the weight of the uterus tissue by 70mls to give a tissue concentration of 1 mg/ml.
Thus, the membrane preparation was stored at - 70°C and prior to its use in the raioligand binding assay, was thawed out and re - homogenised.
For testing the plant extracts, lOuL of extract was added to the 96 well plate, 12 hours prior to addition of the other reagents, and dried in vacuo. The membrane preparation was the final reagent to be added to the 96 well plate when carrying out the assay. The reaction was terminated onto
Whatman GF/C filters using a ‘Skatron’ harvester with buffer G at 4°C. The filter was placed in a filter bag and lOOuL of liquid scintillant was added. The bag was heat - sealed and the radioactivity counted using a ‘Rackbeta’ counter.
This was the first method developed utihsing the bradykinin BK II receptor which was expressed in the female rat uterus.
73 2.3.CLONING OF THE BRADYKININ BK II RECEPTOR IN CHINESE HAMSTER OVARY CELLS.
Chinese Hamster Ovary cells expressing the Bradykinin BK II receptor were prepared by Dr. Gilbert
Vassart at The University of Brussels, Belgium, according to the method of Eggerickx et al, (1992).
Briefly, the gene encoding a putative G - protein coupled receptor, termed HGIO, was cloned from human genomic DNA by low astringency PGR and was found to be homologous to the recently described rat BK II receptor (McEachem, et al, 1991). The receptor was expressed in a stably transfected CHO cell line, according to Eggerickx, et al (1992). The sail - Bam HI PCR fragment of clone HGIO was cloned in the PSVL vector (Pharmacia). The resulting construct was co - transfected with PSV2Neo in CHO - KI cells as described by Velu et al (1989), with the exception that no carrier DNA was added. Two days later, selection for transfectants was initiated by the addition of400ug/ml of Geneticin (G418, Gibco) and resistant clones were isolated at day 10. CHO
- KI cells were cultured in HAMS F12 medium supplemented with ImM sodium pyruvate, lOOiu/ml penicillin, lOOug/ml streptomycin, 2.5ug/ml amphotericin B andl% fetal calf serum.
Membranes were prepared fi'om transfected CHO - KI cells, after clonal selection.
Cells were rinsed once with calcium and magnesium free phosphate buffered saline. After low speed centrifugation, the cell pellet was resuspended in buffer A (15mM Tris at pH 7.5, 2mM MgCl 2 ,
0.3mM EDTA, ImM EGTA, ImM PMSF, luM leupeptin) and mixed in a glass homogenizer. The crude membrane fraction was collected by centrifugation at 40,000g for 30 minutes, washed once and resuspended in buffer B (75mM Tris - HCL at pH 7.5, 12.5mM MgCl 2 , 0.3mM EDTA, ImM
EGTA, 250mM sucrose) and flash frozen in liquid nitrogen. BK II receptors expressed in CHO cells were thus received for use in the BK II radiohgand binding assays.
74 2.4. EXPRESSION OF THE BRADYKININ BKII RECEPTOR IN CHINESE HAMSTER OVARY CELLS.
A vial containing 2mls of suspended CHO cells e?q)ressing the human BK II receptor were thawed at room temperature (20®C) for approximately 20 minutes. Cells were maintained in buffer A (see section 2.25 ) throughout the studies. Following thawing of CHO cells, they were placed in a 20 ml sterile universal (Sterilin) and 18mls of buffer A were added. The cells were spun for 5 minutes at 10,000g in a refrigerated bench centrifuge, model lEC 4R. The supernatant was removed and the pellet was resuspended in lOmls of buffer A. The cells were placed in a Nunclon sterile flask
(Intermed) and approximately lOmls of buffer A were added. Cells were maintained in an incubator, at 20^C with a level of C0% not less than 5.0%.
Using 5mls of PBS - EDTA to lift cells from the flask, they were decanted into a 50ml universal
(Sterilin) and filled upto a volume of 5Omis with buffer A. Cells were spun at 10,000g for 5 minutes, the supernatant was removed and the pellet resuspended in lOmls of buffer A. Cells were passaged into a Nunclon flask, size 25ml, where they remained in the incubator until confluent. They were then passaged into a 260ml flask and when confluent, furftier passaged into a 600ml flask. When the cells reached confluency at passage number four, they were at an appropriate level of growth to be used in the radioligand binding assay.
When the cells appeared fully confluent they were removed with PBS - EDTA, and were spun in the centrifuge for 5minutes. The pellet was resuspended in 5 Omis of Buffer A and 250uL of cells were dispensed into 96 well polyethylene terepthalate (PET) plates prior to their use. This gave approximately 18 hours for the cells to adhere to the plate. Cells remained sterile until used in the assay.
For all radiohgand receptor assays (see section 2.17) buffer B was used, this was non sterile.
75 2.5. ASSAY CONDITIONS FOR THE BRADYKININ BKII SCREEN.
The time period of incubation for an assay has to be sufficient to reach a steady state of equiUbrium, for the bradykinin and CGRP assays, this was 90 minutes. Both assays were carried out at room temperature, (20°C) under non - sterile conditions.
2.5.1.RADIOCHEMICALS
The advantage of using a tritiated ligand is that it can be left chemically unaltered for a period of months to years, depending on the half life (tritium has a half life of 12.3 years, compared to iodine which is sixty days), however, iodinated radiohgands have a higher specific activity which makes them very useful if the receptor density is low. Ligands with higher affinities are preferable as lower concentrations can be used in an assay, resulting in lower levels of NSB. Ligands with high affinities also have high specific activity. Radiohgands labelled with iodine -125 have affinities in the picomolar range, whereas tritiated ligands have affinities in the nanomolar range.
It is generally accepted that the specific activity measured for a radiotracer is accurate to plus or minus 10%. A 10% error in the measured specific activity results in a corresponding 10% error or over estimate of the affinity, compared to the value obtained with 'correct' specific activity, therefore differences in radioligand specific activity are not likely to contribute significantly to the variability of measured ligand affinity in receptor assays with well designed methods.
The radioligand may be an agonist or an antagonist. Agonist radiohgands may only label a portion of the total receptor population (the high affinity state for G - protein coupled receptor) whereas antagonist hgands generally label all available receptor. However, agonist radiohgands may reflect more accurately receptor alterations of biological significance, as it is agonists which are biologically active in producing an effect.
2.5.2.STOCK RADIOLIGAND OF BRADYKININ
^[H]BK was supplied by Dupont NEN. The purity of commercially available ^[H]BK was 99% pure
(as determined by HPLC on a Zorbax ODS column using acetonitrile and trifluoroacetic acid.
Sigma). 5 OUCi of tritiated Bradykinin contained 37MBq/ml (1.0mCi/ml)of radioactivity. ^[H]BK
76 was stored at 4®C, and the concentration used in the bradykinin assay was 0.5nM.
2.5.3.NON-RADIOACTIVE LIGAND
[D-Arg, Hyp^jThi^’D-Tic^-Oic^JBk, (Hoe - 140). 0.2mgs was purchased from Peninsula
Laboratories, and was diluted with ethanol to give a stock solution of lOOuM. 60uL of stock solution were diluted with 5mls of buffer B to give a final concentration in the assay of 75nM.
2.5.4.DETERMINATION OF NON- SPECIFIC BINDING
The non specific binding of the radioligand was estimated in the presence of an excess of non radioactive drug, and for the bradykinin BK II and CGRP assays, this was one hundred fold excess.
The value generated was subtracted from the total amount of radioactivity bound in the presence of any drug to yield the amount specifically bound. The level of specific binding was expressed as a percentage of total radioactivity bound.
2.6. SEPARATION OF BOUND FROM FREE LIGAND
For all receptor - Ugand reactions the separation of bound ligand from free Ugand is a crucial component of receptor binding assays. Significant dissociation of the receptor - hgand complex must be prevented when performing the separation as this is the parameter measured. Reducing the temperature of the assay to slow the rate of dissociation and perform the separation as rapidly as possible, is crucial to terminating the reaction. Vacuum filtration through glass fibre filters is the most commonly used method. Membrane fragments containing the radioligand - receptor complex are retained by the filter and the free radioligand passes through it. Membranes and the filter were washed with excess buffer which preferentially decreases NSB. In binding assays using peptides, a number of protease inhibitors were added to the buffer to prevent degradation of the peptide. The combination of inhibitors vary for each peptide. Adherent cells in PTE plates were washed
(removing buffer A) and the reaction terminated using Titertek Microplate Washer, Mode M96V,
Flow, ICN, on programme No. 1 using phosphate buffered saline as the wash buffer.
77 2.7. MEASUREMENT OF RADIOACTIVITY FOR BRADYKININ
200uL of Optiphase 'Supermix', purchased from Wallac, was added to the microtitre plates following termination of the binding reaction. Scintillant was contained within wells using plate sealers purchased from ICN {in vitro diagnostic use) and were counted for 30 seconds per well on a microbeta liquid scintillation counter. Counting efficiency was measured using prepared standards.
2.8. DATA ANALYSIS AND CAPTURE
Data for the analysis of results from bradykinin BK II binding sites was collected from the 'SCINT' database which incorporates a 'Lotus 123' spreadsheet containing the data from the binding assays.
Data is converted from ‘bound’ and ‘non-specific bound’ DPM’s into values of percentage inhibition of binding.
2.9. THE PREPARATION AND USE OF CALCITONIN GENE RELATED PEPTIDE (CGRP) MEMBRANES.
The methods for preparing Calcitonin Gene Related Peptide membranes were derived from Heuillet et al,(1993) and Cascieri et al, (1992) and were prepared by J. Coote (Glaxo Research and
Development). Briefly, for the preparation of membranes which express a human CGRP receptor,
SK-N-MC cells from a human neuroblastoma cell line were used to make the preparation. Isolated
SK-N-MC cells were grown in 500mls Dulbecco's Modified Eagle Medium (DMEM) which contained 56mls of fetal calf serum and 5.6mls of 200mM glutamine (HyQ cell reagents) and this was filtered once prior to use. Isolated SK-N-MC cells were detatched from the surface they adhered to using ethylenediaminetetraacetic acid, (EDTA, BDH Laboratories) after reaching confluence using
(HBSS) and were spun for seven minutes at 1200g. All subsequent steps were performed at 4°C using pre-cooled reagents and apparatus. The supernatant was removed and the pellet resuspended in lOmls of buffer C. The cells were homogenized in a Waring blender three times at lOOOg for ten minutes. The suspension was spun at 500g for 20 minutes. The pellet was discarded and the supernatant was spun at 48,000g for 30 minutes. Elimination of the unbroken cells and nuclei is crucial in avoiding aggregation either straight away or after freeze - thawing. The pellet was resuspended in buffer D and was vortexed for 5 seconds subsequently being forced through a 0.8mm
78 needle using a syringe, followed by the same process through a 0 .6mm bore.
The resulting membrane preparation was prepared in wash buffer at a concentration of lOOug/ml final concentration/microtitre well and was stored at -70 °C. Thus, CGRP receptors expressed in SK-
N-MC membranes were received for use in the CGRP radiohgand binding assay.
For the radiohgand binding assays buffers E and F were used (see section 2.17).
2.10. ASSAY CONDITIONS FOR THE CGRP ASSAY
I-l^^CGRP was supphed by Amersham. 50uCi of iodinated CGRP contained 74TBq of activity, and was stored at 4°C. The purity of commercially available iodinated CGRP is greater than 90% with less than 5% free iodinated iodine (as determined by reverse phase HPLC, Sigma). The final concentration used in the assay was 15pM.
2.10.1 NON - RADIOACTIVE LIGAND
Human CGRP (Sigma) was used to displace radioactive CGRP from the receptor. 0.5g of CGRP was added to 1.32mls of buffer F to give a stock solution of lOOuM. 50uL aliquots were stored at -
70° C to give a working stock solution of 6nM in the assay, 50uL were diluted 1:100 followed by a
1:166.7 dilution in buffer F. All binding assays were carried out in polypropylene plates from
Titertek. The assay incubation period was 90 minutes and plates were harvested onto GF/B single thickness filter mats (Wallac) which had been soaked for 4-5 hours prior to use in 0.5% w/v polyethylenimine, (PEI, Sigma), in wash buffer G using a TomTec harvester on programme number
8 . Filter mats were dried for 2 minutes at medium heat in a microwave oven.
2.10.2.MEASUREMENT OF RADIOACTIVITY AND DATA ANALYSIS
Filters were heat sealed in filter bags (LKB Wallac). One end was cut off and lOmls of hquid beta plate scintiUant (LKB, Wallac) were added and the bag was resealed. Filters were counted on a
WaUac beta plate counter on programme 2 0 . A 3 and 1/4" disc was inserted to capture data and results were processed using CGRP macro.
79 2.11. DETERMINATION OF THE PROTEIN CONTENT FOR BKII AND CGRP
The method for determining the amount of protein in the BKU receptor preparation and the CGRP membrane preparation was based on that of Bradford (1976).
20mls of Bradford reagent, (Biorad Protein Assay Dye Reagent Concentrate) was added to 80mis of distilled water. This was mixed and filtered through tissue. A standard protein curve was produced using Bovine Serum Albumen, (Sigma, Fraction V, 96-99% pure by gel electrophoresis) at a concentration of 1 mg/ml. Into glass bijoux 1 - lOOuL of BSA were pipetted, and 4mls of Bradford
Reagent were added. 200uL from each bijoux were pipetted in quadruplicate into a 96 well Nunclon plate and absorbance was monitored at 595nm.
To determine the protein concentration in the sample, and in the assay buffer used for binding studies, 2,5,10,25,50 and lOOuL of assay buffer and the same volume of cells were placed in bijoux and 4mls of bradford reagent were added. 200ul were pipetted into 96 well plates and absorbance was measured at 595nm.
2.12. TIME COURSE STUDIES FOR BRADYKININ AND CALCITONIN GENE RELATED PEPTIDE.
BRADYKININ BKII.
To determine the optimal incubation period for radioligand binding assays, the percentage of specifically bound counts (Dpm's) were counted for a period of 0 - 110 minutes. For the BK II assay,
150uL of buffer B was added to each well in quadruphcate and 50uL of 0.5nm ^[H]BK were added once every ten minutes. For NSB, lOOuL of buffer B were added to each well and 50uL of 75nm
Hoe-140. 50uL ^[H]BK was added every ten minutes from 1-180 minutes. The reaction was terminated on the Titertek Microplate Washer by washing with phosphate buffered saline solution.
2 12.1CALCIT0NIN GENE RELATED PEPTIDE.
Specific binding was monitored for a period of 0-180 minutes. To determine bound specific counts, lOOul of membrane was added to 5Oui of buffer F and 5Oui of 1.5pM I-^^^CGRP was added once every 10 minutes. To determine NSB, lOOul of receptor was added to 50ul of non radioactive CGRP,
80 at a concentration of 15pM, and 5Oui of I-^^^CGRP was added every ten minutes. The reaction was terminated using the Tomtek harvester with buffer E, programme number eight.
2.13. SATURATION ANALYSIS OF THE BKII AND THE CGRP RECEPTOR
Determination of the Kd and Bmax for the BKU and CGRP receptors:
Bmax is defined as the maximum amount of drug (usually expressed in picomoles/mg protein) which can bind specifically to the receptor in a membrane preparation.
Kd is defined as the dissociation constant for a radiohabelled drug which is determined by saturation analysis. Units are in moles per htre, and it is the concentration of drug which at equihbrium occupies
50% of the receptors.
2 13 1 SCATCHARD ANALYSIS
When determining the number of classes of receptor site, data may fit into one of a number of possible configurations. The transformations of the saturable components of ^[H]BK and I-125_
CGRP is possible using the method of Scatchard (1949) where generated plots are one of the following:
(a)Non-linear least squares optimisation technique : A simple hyperbola plot is generated signifying one receptor site per class of receptor.
Data generated from binding studies will represent one or more classes of a distinct receptor type.
The equation for this is :
B = Bmax.S/Kd + S
B = specifically bound hgand (picomoles/nanomoles)
Bmax = maximum hgand binding capacity(picomoles/nanomoles)
S = hgand concentration (picomoles/nanomoles)
Kd = equihbrium dissociation constant (picomoles/nanomoles)
Type (b) Simple hyperbola plus a non - saturable component.
B = Bmax.S/Kd + S + D.S
D = linear component (picomoles/mg protein)
81 Type (c) Two simple hyperbola with differing constants.
B = Bmaxl.S/Kdl + S + Bmax2.S/Kd2 +S
Type (d) Two simple hyperbola plus a non-saturable component.
B = Bmaxl.S/Kdl+S + Bmax2.S/Kd2 + S + D.S.
For many hgand - receptor systems studied, binding data are inconsistent with the predictions of the
Law of Mass Action for the simplest model of a reversible, bimolecular interaction between a ligand and a single set of identical, independent receptors (Kermode, 1989). The Scatchard plot, which has a predicted straight line relationship may be altered so that a curvilinear plot is produced instead. To conclude that a plot signifies greater than one receptor class, one must reahse a number of experimental artefacts could produce the alteration to the straight line normally expected, these should be accounted for before a conclusion is reached.
Theoretical analyses have indicated that experimental artefacts may explain the curvilinear nature of a Scatchard plot which does not include the concept of receptor heterogeneity (Taylor, 1975) and the artificial explanations outnumber the real models of heterogeneity (Kermode, 1989).
The term 'functional heterogeneity' may encompass differences in behaviour between individual receptors as well as any actual molecular differences between receptors or it may allude to different subclasses of a single receptor e.g.BKI and BK U.
Principal artefacts include; (a) imprecise estimate of non specific binding, which usually produces an upward curvature to the Scatchard plot and is a fi’cquent occurrence (b) impure labelled hgand which produces an upward or downward curvature of the plot, which is a firequent problem, (c) irreversible binding and intemaUsation of the receptor will produce a downward curvature, a problem specific to whole cell assays, (d) ligand degradation produces a downward curvature, and (e) non equilibrium bninding conditions produce a downward curvature.
As well as representing binding data as a Scatchard plot, a second method is that of applying linear regression methods to raw (untransformed) data. The total ligand concentration is regarded as essentially an error-free independant variable and curvilinear regression can be applied to the
82 relationship between this and the bound radiohgand concentration (an independent variable).
Scatchard plots were analysed using the RSI data package.
2.14. ASSOCIATION AND DISSOCIATION STUDIES.
To test that the ligand-receptor interaction was reversible, binding was measured over a period of 110 minutes, which was 20 minutes longer than the incubation period for the bradykinin assay. Bound and NSB was determined upto 110 minutes.
Non specific binding was measured in the prescence of a 100 fold excess of non radioactive hgand to determine Wiether displacement of radiohgand from the receptor occurs.
The method for measuring the association and dissociation of hgand to and from the receptor was the same as for time course studies, with radioactive hgand added every ten minutes to a 96 well plate, and non specific binding measured in the presence of excess non radoactive hgand. The reaction was terminated using the Titertek plate washer for the BKU assay.
2.15. CHARACTERISATION OF THE BRADYKININ BKII RECEPTOR.
A number of agonist and antagonist compounds are available which interact with the bradykinin
BKU receptor. The classification of an individual receptor is determined by its interaction with these compounds which produce a profile with an accepted order and potency values. The value determined is the Ki, the inhibition constant for a drug which is the concentration of competing hgand in a competition assay which would occupy 50% of the receptor if no radiohgand were present.
2.16. THE NEUROKININ 1 (NKl) RECEPTOR RADIOLIGAND BINDING ASSAY
Plant extracts which were active in the bradykinin BKU assay were subsequently screened against the Neurokinin 1(NK1) receptor. These assays were done by Ms. T. Shaw-Hamilton. This facihtates a means of testing for selective neuropeptide interactions of each plant extract. The methods used to develop the NK 1 receptor radiohgand binding assay was based on those used by Jung et al (1991).
Briefly, the Neurokinin 1 receptor hgand. Substance P, is an eleven amino acid peptide which is one of the tachykinin group of neuropeptides, along with bradykinin and Calcitonin Gene Related peptide.
83 Substance P has a wide distribution in the body and has been located in the Central Nervous System, the Peripheral Nervous System, the gastrointestinal tract, the cardiovascular system, the skin, kidneys,
sahvary glands and pancreas (Pemow, 1983).
The U373MG cell has been found to express NK 1 receptors (Lee et al, 1989). The cell line grows
readily in tissue culture and is an adherent cell line. Cells were grown at 37®C at a level of 5% CO 2
in tissue culture flasks in culture media. Cells were grown to confluence after 14 days and were then
passaged in a 1:4 spht using phosphate buffered saline with 0.02% w/v EDTA to harvest the cells.
Cells were diluted to give a density of 500,000 cells/ml and were dispensed into 96 well plates in a
volume of 200ul/well, equivalent to 100,000 cells/well.
Plates were incubated overnight to ensure they adhered to the well of the plate to give a confluent
monolayer. Prior to use in the assay the media was discarded and the wells washed twice with 200ul
of PBS on a Titertek plate washer.
84 2.17.COMPOSITION OF SOLUTIONS
BUFFER A
500 mis HAMS F12 Nutrient Mixture ( HyQ Cell Culture Reagents)
5 mis 200mM L - glutamine (HyQ Cell Culture Reagents)
5 Omis bovine fetal calf serum (Sigma)
5mls lOOmM Sodium pyruvate (HyQ Cell Culture Reagents)
Geneticin, 0418 sulphate. 5 Omis of phosphate buffered saline were added to 5g of powdered 0418.
This was filtered twice through O.SuM sterile acrodiscs, purchased from Oelman Sciences.
4mls of the filtered solution were added per 500 mis of Hams F12 nutrient mixture.
The preparation of buffer was carried out under sterile conditions at room temperature.
BUFFER B
500 mis Dulbecco's Modified Eagle Medium (DMEM) (Hyclone Ltd) lOOmg Sodium azide (Sigma)
500mg Bovine serum albumen (Sigma)
70mg Bacitracin (Sigma)
77.1 mg Dithiothroeitol (DTT) (Sigma)
5Oui of a 0.0 IM stock solution of Captopril (Sigma)
All reagents were non sterile.
BUFFER C
50mM Hepes at pH 7.4 (Sigma)
47ug/ml Leupeptin (Sigma)
25ug/ml Bacitracin (Sigma)
ImM EDTA (BDH Laboratories)
ImM PMSF (Sigma)
3uM Pepstatin A (Sigma)
PMSF and Pepstatin A were added prior to homogenisation in ethanol. 85 BUFFER D
50mM Hepes at pH 7.4 (Sigma)
47ug/mL Leupeptin (Sigma)
25ug/mL Bacitracin (Sigma)
ImM EDTA (BDH Laboratories)
BUFFER E
6.057g/L Tris HCL (Sigma)
1.017g/L MgCL2.6H20 (Fisons Analytical Reagents)
0.372 g/L EDTA disodium (BDH Laboratories) pH 7.4
BUFFER F
As for buffer E plus:
O.lg/L Bacitracin (Sigma)
Ig/L Bovine Serum albumen (Sigma)
Prepare buffer F fresh on the day required.
BUFFER G
6.28g/L TES buffer (Sigma)
0.6g/L 1,10 Phenanthroline (Sigma) dissolved using a minimum quantity of 5M HCL pH to 6.8 using IM HCL
BUFFER H
6.28g/L TES buffer (Sigma)
ImM Dithiothroeitol (Sigma) luM Captopril (Sigma)
0.14mg/L Bacitracin (Sigma)
Ig/L BSA (Sigma)
86 2.18.ASSAY PROTOCOLS FOR THE BRADYKININ BKII AND CGRP RADIOLIGAND BINDING ASSAYS.
96 WELL ASSAY FORMAT FOR THE BRADYKININ BK II ASSAY.
To test methanol plant extracts: lOOuL assay buffer
50uL 0.5nm ^[H]BK
50uL plant extract or control compound
Control Value Determinations:
Bound Non Specific Bound (NSB)
15 OuL assay buffer B lOOuL assay buffer B
50uL O.Snm ^[H]BK 5OuL Non radioactive Hoe -140
50uL 0.5nm 3[H]BK Assay incubation period : 90 minutes.
2.19. ASSAY PROTOCOL FOR THE I-125 ç g r p BINDING ASSAY.
To test methanol plant extracts: lOOuL membrane ata concentration of lOOmgs/ml.
50uLofl.5pM 1-125 CGRP
5OuL plant extract or control drug.
To determine control values: Bound
5 OuL assay bufifer F
50uL 1.5pM 1-125 CGRP lOOuL membrane at a concentration of lOOmgs/ml
Non Specific Binding
50uL1.5pM 1-125 CGRP lOOuL membrane at a concentration of lOOmgs/ml
5OuL 15pM non-radioactive CGRP
87 2.20.ASSAY PROTOCOL FOR THE BRADYKININ BK H ASSAY USING RAT UTERUS MEMBRANES
To test methanol plant extracts :
50uL 0.5nM ^[H]BK
5 OuL rat membrane preparation lOOuL buffer H lOuL of plant extract, added to the 96 well plate 12 hours prior to the assay, and dried in vacuo.
To determine control values: Bound______Non-Specific Bound
50uL 0.5nM ^[H]BK 50uL 0.5nM ^[H]BK
5 OuL rat membrane preparation 5 OuL rat membrane preparation lOOuL buffer H 5 OuL Buffer H
5OuL 300nM Hoe-140
2.21.THIN LAYER CHROMATOGRAPHY FOR THE DETECTION OF TERPENOID COMPOUNDS. WAGNER ET AL. fl983)
A: Ig Vanillin dissolved in 100ml of 50% phosphoric acid.
B : 2 parts 24% phosphoric acid and 8 parts 2% ethanolic vanillic acid. After spraying with either A or B, the plate is heated for 10 minutes at 100°C and evaluated in vis.
88 2.22.PREPARATION OF PLANT EXTRACTS FOR SCREENING IN IN VITRO BINDING ASSAYS
In the search for leads to novel analgesic compounds, plants traditionally used for the treatment of pain were selected in the present investigation and subjected to screening in the in vitro bradykinin and calcitonin gene related peptide binding assays. The selection of Chinese plants was made from Chang and Butt (1986), the middle and south American plants were from Morton (1981), the West Indian plants were from Ayensu (1981), African plants were selected from Ohver-Bever (1986) and the Australian plants were from Law(1990). Other higher plants which were ethnomedically selected were derived from the 'Napralert' database (Loub, 1985). In as many instances as possible, the plant part used traditionally in the original reference cited was used. However, if for example, the stem was used traditionally but was unavailable, but seeds or leaves were available, an extract was made and screened. For a complete list of all plants, and plant parts screened, see Appendix I, pages 198 to 209. Initial e?q)eriments for selection of solvent for extraction of plant material utihsed chloroform, ethyl acetate, water and methanol. When these extracts were screened by the BK II binding assay there was no significant difference in the results, and as methanol was most compatible with the BK II assay it was selected as the solvent of choice for all further work. Ig of dried plant material was macerated in lOmls of methanol overnight at room temperature. Filtered extracts were evaporated to dryness in vacuo and were redissolved m 1ml of methanol. lOpL were used for in vitro screening. Plant extracts producing greater than 50% inhibition in the binding assays were treated with Polyclar AT (insoluble polyvinylpyrrohdone, PVP, BDH laboratories) to remove tannins (Figure 2.1). To ensure the extract had as high a concentration of tannins removed as possible, 5mls of extract (using the ratio of w/v as above) were passed through a 5ml Bectcn
Dickinson syringe which was packed to a depth of 1cm with cotton wool and upto 2cm with PVP. The extract was collected under vacuum (lOOmmHg) and lOuL of this extract was used for screening.
89 2.23.EXTRACTION AND ISOLATION OF BIOACTIVE ENTITIES. The extraction of compounds from plants with activity in the BKII assay (defined as greater than 50% inhibition of binding, or greater ) were carried out according to the following procedure:
An Analytical CIS Bond Elut Sohd Phase Extraction (SPE) cartridge was used in conjunction with a
Baker SPIO Vacuum Manifold System. Bond Elut columns were "primed" (wetted) with lOmls of methanol and re-equilibrated with 10% aqueous methanol. The column was washed with 10% aqueous methanol, samples of ImL in 10% methanol were applied to the column and then increasing concentrations (20-80% ) of acetonitrile in water, followed by 100% methanol were apphed to the column. Fractions of lOmls were collected and 50pL aliquots were assayed in the radioligand binding assay. Following identification of the active fractions, further purification was achieved using high performance liquid chromatograpy (HPLC) using either CIS bonded sUica columns or columns containing cross-linked styrene-divinyl benzene co-polymers. HPLC columns with a diameter of 4.6mm x 150mm in length (typical flow l-2mL/min) were used for analytical chromatography and 21mm X 250mm columns (typical flow 20mL/min) were used for preparative chromatography. The gradient systems used for the HPLC of the extract of Symplocos leptophylla were : Svstem 1: 0-5mins------20% MeOH 6-20mins -—20%-S0% MeOH
21-SOmins —S0% MeOH
Svstem II: 0-5 mins------70% MeOH
6-20 mins 70-85% MeOH
21-30 mins —85% MeOH
90 Svstem III 0-10 mins 70% MeOH 1 l-20mins — 70-80% MeOH
21-80 mins —80% MeOH High Pressure Liquid Chromatography of samples was performed using a Waters 601 pump, a Waters 600E Gradient Module, a Waters 484 Tunable Absorbance Detector for preparative work and a Waters 990+ Photodiode array Detector for analytical apphcations.
2.24.PLANT SAMPLE PREPARATION FOR EXTRACTION AND ISOLATION. For scaling up the amount of active plant material an "open" glass column packed with Whatman P40 sorbent was employed using gravity feed. P40 is similar to the type of C18 packing used. 300g of the plant sample were macerated overnight at room temperature in 3000mls of methanol. The extract was filtered and evaporated to approximately lOOmls. To this, 5g of P40 packing material was stirred to form a slurry. Following preparation of the P40 column with IL of methanol and IL of 10% acetonitrile, the sample was applied to the top of the column and eluted with a stepwise gradient as previously detailed.
2.25.SAMPLE PROCESSING ON LH20 COLUMNS Sephadex LH20 was prepared by hydroxypropylation of Sephadex 025 (see Discussion, Chapter 4.4,pl83).
2.26. AUTOMATED MULTIPLE DEVELOPMENT - HIGH PRESSURE LIQUID THIN
LAYER CHROMATOGRAPHY Purification of plant extracts was fiirther carried out using the solvent system described by Menziani et al, (1990), the details of which are described below. Multiple development was carried out in the AMD apparatus manufactured by CAMAG. Merck 5641 Silica Gel HPTLC precoated plates (10 x 20 cm) were twice prewashed with methanol. luL spots of concentrated fractions were apphed to the HPTLC plate at 8 mm fi’om one edge and the plate developed (see below). Zones were detected by scanning the plates at 210 and 280nm using a Camag Scanner U in a reflective mode and CATS software. Preparative runs were carried out in a similar manner except that the sample was apphed as a 160mm band using an Automated TLC Sampler II (CAMAG).
91 The experimental AMD parameters used in this study were as follows: The total number of steps involved in this study was 15. For drying the plate in between steps 1-4 the period of time was six minutes, and for steps 5 to 15 this was decreased to 4 minutes. The period of drying time for the final step was 10 minutes. Nitrogen bubbled through the water provided the conditioning atmosphere. The solvents, and the steps for which they were used, were made up according to the instructions for the CAMAG AMD system (1986) and Menziani et al (1990). For the first step, solvent one was used which contained methanol, dichloromethane, water and formic acid in the ratio of 70.5 : 25.0 : 4.5 : 1.0, for step numbers 2-5 solvent two was used which contained the same chemicals as in step one but in the ratio of 23.5 : 74 : 1.5 : 1.0, for step numbers 6-10 the ratio of chemicals was 9.4 : 90.0 : 0.6 : 1.0 and for the final steps 11-15, 100% dichloromethane was used. This sequence thus provided the chemical environment for separating the bioactive fractions obtained fi'om Symplocos leptophylla on HPTLC plates using the AMD system. 2.27. PREPARATION OF PLANT EXTRACTS FOR IN VIVO STUDIES INTO MALE MICE. 40g of dried plant material were prepared by maceration overnight in 200mls of methanol (Fisons). The extract was filtered and evaporated to dryness and 20 mis of acetone (BDH Laboratories) was added, together with 4mls of Cremophor el (a derivative of castor oil and ethylene oxide. Sigma).
Cremophor el is the vehicle in which plant extracts were dissolved in order that they could be used for intraperitoneal injections. It was also used with acetic acid (the control compounds) against which the effects of the plant extracts were compared. The acetone was blown off under nitrogen gas at room temperature and 16 mis of 0.9% saline were added to the extract prior to inter-peritoneal injection of mice.
92 2.28.PREPARATION OF MICE FOR THE IN VIVO SCREENING OF PLANT EXTRACTS
IN THE ACETIC ACID WRITHING TEST.
Male mice, supplied from Charles Rivers, of approximately 35-45g, were weighed prior to injection. The volume of extract injected was calculated as a ratio of body weight at a concentration of Img/kg. Mice were observed singly in glass bell jars for one hour prior to injection and for different time periods following injections. Groups consisting of five mice were taken randomly from a cage and were marked using waterproof pen. Methanol extracts of Panax ginseng, family Arahaceae, and Ipomea pes-caprae, family Leguminosae, were prepared for injection (see 2.27).
Mice were injected with the appropriate volume of extract, and were observed for five minutes. A 0.6% solution of acetic acid was injected and the number of writhes were recorded for 15, 30 and 45 minutes. For control animals, Chromophor el was injected at time zero and a 0.6% solution (w/v) of acetic acid was injected five minutes later. Mice were observed for 15, 30 and 45 minutes. The volume of extract injected was calculated as a ratio to body weight.
93 FIGURE 2.1
PLANT SAMPLE PREPARATION FOR SCREENING IN THE IN VITRO RADIOLIGAND BINDING ASSAYS.
Ig of dried plant material in lOmls of methanol i Macerate overnight
\i/ Filter
Redissolve in 1ml of methanol
Assay (BKII, CGRP,NK1)
Nl/ Treat with PVP if the extract produces 50% inhibition or greater in the radioligand binding assay
\ y Reassay in radioligand binding assay
94 CHAPTER THREE RESULTS CHAPTER THREE : RESULTS
3.1 THE RESULTS OF SCREENING METHANOL PLANT EXTRACTS IN THE IN VITRO BRADYKINEV BK H RAT UTERUS MEMBRANE PREPARATION.
The initial development studies of the bradykinin BK II radioligand binding assay which utilised the membrane preparation derived from female rat uteri, produced results which were inconsistent for two reasons: ( 1) the parameters which needed to be satisfied in order to screen the methanol plant extracts, e.g. the expression of a single class of BK II receptors as determined by Scatchard analysis, could not be determined from the Scatchard plot (see Appendix V p217), and (2) there was experimental variation greater than 10% between the values for the % inhibition of plant extracts both for identical plant extracts screened in the assay and for the replicate screening of non - identical extracts. An example of some of the inconsistent values produced from the repeated screening of a Ig/lOml methanol plant extract can be seen in Table 3.1. Table 3.1 Repeated screening of methanol plant extracts in the bradvkinin BK II assav.
Plant Family % Inhibition in MeOH Salvia miltiorrhiza Menispermaceae 9,64,45,47 Stephania tetrandra Menispermaceae 14,21,90,62 Corydalis decumbens Papaveraceae 0,24,45,30,
The % inhibition values are a mean value of duphcate assay points. Due to the inconsistencies in the % inhibition values produced when screening methanol plant extracts in the bradykinin BK II assay, further sources of tissue and cell lines were investigated which expressed the bradykinin BK II receptor. For the discussion of the inconsistencies observed using the rat uterus membrane preparation to screen plant extracts (Chapter Four, Section 4.6., pl90-193).
95 3.2 RESULTS FOR THF DEVELOPMENT STUDIES OF THE IN VITRO BRADYKININ BK n RECEPTOR EXPRESSED IN CHINESE HAMSTER OVARY CELLS. The development studies for the in vitro bradykinin BK II receptor expressed in Chinese Hamster Ovary (CHO) cells and the CGRP receptor (expressed in SK - N - MC cells) radioligand binding assays produced a series of parameters which were applied to the screens for testing the methanol plant extracts. The determination of the protein concentration of the Chinese Hamster Ovary cells which expressed the bradykinin BK II receptor gave a value of 0.68mg/ml, (Appendix 11(a), p210) which was comparable to that of Eggerickx et al, (1993).
3.3 TIME COURSE STUDIES CHO cells were incubated with a fixed concentration of ^H[BK] at 20®C for periods up to 110 minutes to assess the time when maximal binding occured. Equilibrium appeared to have been achieved by 70 minutes subsequent to which the amount bound remained constant (Figure 1, p 98). As a consequence, an incubation time of ninety minutes was used routinely for the screening assay. The data presented in figure 1 are mean values for quadruplicate determinations. 3.4 DISSOCIATION STUDIES To confirm that the interaction of tritiated bradykinin with the bradykinin BK II receptor was reversible, the dissociation of tritiated bradykinin from the BK II receptor was initiated by adding excess non-radioactive bradykinin to the bradykinin BK II assay at the point of equilibrium (90 minutes) (Figure 2, pi GO) and continuing the reaction for a fiirther 90 minutes. Addition of excess non - radioactive ligand caused the endogenous ligand to dissociate from the bradykinin BK II receptor, thus concluding that the binding of tritiated bradykinin to the BK II receptor was reversible. The data presented in Figure 2 are mean values for duplicate determinations.
96 Figure 3.1 Time course study for the bradvkinin BK II receptor ligand binding assav.
Chinese Hamster Ovary cells which expressed the bradykinin BK II receptor were incubated with 0.5nM ^H[BK] at 20°C for periods upto 110 minutes to assess the time at which maximal binding occured. Equilibrium was determined at 90 minutes.
97 Figure 3.1 A time couse study to determine the incubation period for the bradykinin BK II assay. 3500
3000
2500
cu Û 2000 TJ C O3 u 1500 - I u O. C/D
1000
Time (minutes) Non-specific binding was subtracted from bound to give specific bound.
98 Figure 3.2 Dissociation studies for the bradvkinin BK II radioligand binding assav.
The dissociation of ^[H]BK from the BK II receptor was initiated by adding excess non-radioactive bradykinin (250 nM) to the assay at the point of equilibriation, indicated by the arrow, and continuing the reaction for a further 90 minutes. The dissociation of tritiated bradykinin from the BK II receptor indicates that the binding was reversible.
99 Figure 3.2 The association and dissociation of 3H[BK] binding to the bradykinin BK II receptor.
S 1500
1
20 40 60 80 _100 Time (minutes) The value for non-specific binding (NSB) was subtracted from the value for bound to give specific bound. Excess non-radioactive bradykinin was added to the reaction at 90 minutes to initiate the dissociaUon of [H]BK from the bradykinin BK n receptor. 100 3.5 SCATCHARD ANALYSIS OF THE BRADYKININ BK H RECEPTOR The Scatchard plot for the BK II receptor was analysed using the RS1 computer programme. A single straight line fit was produced which pertained to a single class of receptors. The equation to which this plot corresponds to is :
B = Bmax./Kd + S
The value for the Bmax was 0.0125pmoles/mg protein and the Kd was 0.5 InM (Figure 3.3, pl03).
101 3 Figure 3.3 Scatchard analysis of the binding of [H]BK to the bradykinin BK II _
receptor.
The Scatchard analysis indicated that only the bradykinin BK II receptor binding site was expressed in the CHO cells.
1 0 2 Figure 3.3 Scatchard analysis of the binding of ^[H]BK to the bradykinin BK II receptor expressed in Chinese Hamster Ovary Cells.
0 . 16-
O.K
0 . 12-
S P Ç 0.10 I c 8 i
0 . 06-
0.06
0 . 02-
^urve 0.2E-10 0 .6 E -1 0 l.O E -1 0 Specific Bound
103 3.6 DISPLACEMENT CURVES FOR THE BRADYKININ BK n ASSAY The displacement curve of the agonist bradykinin in the bradykinin BK II assay (Figure
3.4, pl05) gave an IC 50 value of 0 .12nM and a Ki value of 10.28. The displacement curves for a number of bradykinin analogues were constructed by serially diluting the analogue 1: 2 in the bradykinin BK II assay, whilst maintaining the tritiated bradykinin at a constant concentration. The standard bradykinin BK II antagonist Hoe-140 [DesArg(Hyp^Thi^D-TicOic^)] (figure 3.4, p i06), inhibited binding of tritiated bradykinin to the BK II receptor and was the most potent antagonist in this assay, with an IC 50 value of 2.07nM, compared to all other antagonists which had IC 50 values in the micromolar range. The bradykinin analogues with agonist and antagonist activity only partially inhibited binding of tritiated bradykinin to the BK II receptor. These were [DesArgl^]Hoe (Figure 3.5, p i07), DesArg^Leu^]BK (Figure3.5, p i07), [DesArgHypThi^"^,D-Phe^]BK (Figure 3.6, p i08) and [DesArg^JBK (Figure 3.6). These analogues produced a profile which is expected for that of the bradykinin BK II receptor expressed in CHO cells (Table 3.2, p i09) (Eggerickx et al, 1993). Two fijrther analogues were serially diluted in the bradykinin BK II assay and they were bradykinin BK I analogues. [DesArg^]BK and [DesArgHyp^D-PheThi^"^]BK had no intrinsic antagonist activity at the bradykinin BK II receptor. [DesArg^]BK produced a maximum inhibition value of 16% at O.OluM and [DesArgHyp^D-
PheThi^ ^]b K inhibited the binding of tritiated bradykinin to the BK II receptor with a maximum value of 28% at l.OuM. The data for the displacement curves are mean values for duplicate determinations.
104 Figures 3.4 -3.6 Displacement curves of a series of bradvkinin analogues in the
bradvkinin BK II receptor radioligand binding assav.
25nM bradykinin and 25nM Hoe-140 were serially diluted 1:2 in the bradykinin BK II assay to determine their IC 50 values. Bradykinin produced the most potent IC 50 value.
10 Q a 7 [DesArg ]Hoe, [DesArg Leu ]BK, [D-Phe ]BK, [DesArgHyp,D-Phe]BK,
5-8 9 [DesArgHypThi ,D-Phe]BK and [DesArg ]BK produced a series of I C 5 0 values, the profile of which fitted that expected for the bradykinin BK II receptor.
105 Figure 3.4 Displacement curves for (l)bradykinin (2)Hoe-140 in the bradykinin BK II assay.
100
X3 I
W
(l)bradykinin (nM) and (2)Hoe-140 (nM) Figure 3.5 Displacement curves for (l)(DesArglOJHoe, (2)[DesArg9Leu8JBK and (3)[D- Phe7]BK in the bradykinin BK II assay.
80
70 I
60
50 KB mm 30
iiiis 20 msm
10 (2). (3)
(1 ) 0 4 0 2 3 4 5 6 7 8 9 10 (1)IDesArg10IHo«, 0.2 0.3 0.4 0.5 0.6 0.7 0.8 (1)IDesArgHyp,D4>t»lBK, (uM), (2)IDesArgHypThl5^,0-Phe]BK, (uM) (3){DesArg9]BK (uM) TABLE 3.2. THE IC^n AND Kl VALUES OF BRADYKININ ANALOGUES IN THE BRADYKININ BKH ASSAY (METHOD 2.23 FIGURES 4-6). BRADYKININ ANALOGUES IC^O oKi Ki Bradykinin 0.12nM 6.09x10" 1 0 .2 Hoe-140 2.07nM 1.05x10'^ 8.97 [D-Phe^JBK 8.7uM 4.40x10"^ 5.35 [DesArg l^]Hoe-140 II.39uM 5.70x10^ 5.20 [DesArg^JBK 2.98uM 1.51x10-^ 5.80 [DesArgHypThi^"^»DPhe]BK 0.68uM 3.45x10"^ 6.46 [DesArg^Leu^]BK 1.18uM 5.90x10'^ 6 .2 0 [DesArgHyp,D-Phe]BK 0.42uM 2.13x10’^ 6.60 The pharmacological profile of the human BK II receptor (as expressed in CHO cells) matches that of Eggericx et al (1992). The profile does not match strictly that of other profiles, as determined on other cell lines and which express the BK II receptor (Steranka et al ,1988). This may be due to species differences or the fact that the BK II receptor is linked to different G-proteins. 109 3.7. RESULTS FROM THE CGRP RADIOLIGAND BINDING ASSAY. TIME COURSE STUDIES FOR THE CGRP ASSAY. SK-N-MC membranes were incubated with a fixed concentration of ^^^I-CGRP at 20®C for periods of upto 180 minutes to assess the time at which maximum binding occurred. Equilibrium appeared to be achieved between the time period of 70 - 90 minutes subsequent to which the amount bound decreased (Figure 3.7, pi 12). As a consequence, an incubation time of 90 minutes was used routinely for the screening assay. The determination of the protein content of the SK-N-MC membranes which expressed the CGRP receptor gave a value of 0.87 mg/ml (Appendix II, page 211). 3.8 SCATCHARD ANALYSIS OF THE CGRP RECEPTOR The Scatchard plot for the CGRP receptor was analysed using the RSI programme. A single straight line was produced which indicated a single class of receptor, defined by the equation : B = Bmax.S/Kd + S The value for the Bmax was 4.47 finoles/mg protein and the Kd was 8.9pM (Figure 3.8, pi 14). 3.8 DISPLACEMENT CURVE FOR THE CGRP RECEPTOR A displacement curve for non - radioactive CGRP was constructed by serially diluting CGRP 1 : 2 in the assay whilst maintaining a fixed concentration of iodinated CGRP (Figure 3.9, pi 16). The IC 50 of CGRP was 0.67pM and this was the only CGRP analogue available with which to displace iodinated CGRP from the CGRP receptor. The data for the CGRP displacement curve is a value for duplicate determinations. 110 Figure 3.7 A time course study of the CGRP receptor radioligand binding assay. SK-N-MC membranes which expressed the CGRP receptor were incubated with -125 1,5nM I CGRP for a period of upto 180 minutes. Maximum binding occured at 90 minutes and this time period was used routinely for the binding assay. I ll Figure 3.7 A time course study to determine the incubation period for the CGRP binding assay. 1200 1000 ii ii 800 p 600 CO 400 m■ m I 200 20 40 60 80 100 120 140 160 180 Tim* (minutes) Excess non-radioactive CGRP was added to the reaction at 90 minutes to initiate the dissociation of I CGRP from the CGRP receptor. Non-specific binding was subtracted from bound to give specific bound. -125 Figure 3.8 Scatchard analysis of I CGRP binding to the CGRP receptor which was expressed in SK-N-MC membranes. The Scatchard analysis of the SK-N-MC membranes indicated a single class of CGRP receptors were present, as defined with a straight fit analysis. The CGRP receptor was expressed in a human neuroblastoma SK-N-MC cell line. 113 Figure 3.8 Scatchard analysis of the binding of I-125CGRP to the CGRP receptor expressed in SK-N-MC membranes. CGRP_SKAT 0.420" e 0.400" 0.380" 0.360" 0.340" 0.320" 0.300" S P 0.280" e c i 0.260" f i 0.240" c 0 . 220" B / 0. 200^ F 0.180" 0.160" 0.140" 0 ,120-' 0 , 100" 0.080T 0.060 \------1------1— O.Oe+00 l.Oe-12 2 . 0e-1 2 3.0e-12 4.0e-12 ^oecific Douncl Figure 3.9. A displacement curve of non-radioactive CGRP in the CGRP radioligand binding assav. Non-radioactive CGRP was serially diluted 1:2 in the assay to determine an IC 50 value, and this was 0.67nM. Non-radioactive CGRP was the only analogue available with which to perform a displacement curve. 115 Figure 3.9 A displacement curve of non - radioactive CGRP in the CGRP binding assay. OXI ’"5c CGRP (nM) non-specific binding (NSB) was subtracted from the value for bound to give specific 3.9 RESULTS OF SCREENING 300 ETHNOMEDICALLY SELECTED AND 335 NON SELECTED PLANTS IN THE BRADYKININ BK H AND CGRP RADIOLIGAND BINDING ASSAYS. Six hundred and thirty five methanol plant extracts were screened in both the bradykinin BK II and CGRP radioligand binding assays (see Appendix I, p i98-209) . Three hundred plants were ethnomedically selected from the medicinal and scientific literature of China, south America, Africa and the West Indies. ‘Napralert’ was also accessed for a further source of higher plants which were selected for their use as analgesics. Three hundred and thirty five plants, which were not selected on the basis of their ethnopharmacological use, but which were also screened in radioligand binding assays, were termed ‘non-selected’ A positive result in the assay was defined as 50% inhibition of tritiated bradykinin binding to the BK II receptor or iodinated CGRP binding to the CGRP receptor (Table 3.3, pi 19-120).The methanol extracts of 300 ethnomedically selected plants and 335 ‘non-selected’ plants were screened in the bradykinin BK II receptor binding assay. A one gramme in ten millilitres of methanol extract was made for each one of 635 plants screened in the bradykinin BK II assay. Seventy nine extracts produced greater than 50% inhibition of tritiated bradykinin binding to the BK H receptor, which was deemed to be a positive result. From the 79 plant extracts which produced greater than 50% inhibition of binding of tritiated bradykinin to the BK II receptor, forty eight of the plants were ethnomedically selected and 31 were non-selected. 117 TABLE 3.3 The positive results of screening 635 methanol plant extracts in the bradykinin BK II assay. A Ig in lOmls of methanol extract was made for each one of the 635 plants screened in the bradykinin BK H assay and SOuL were used in the assay. The inhibition of tritiated bradykinin binding to the BK II receptor, by a methanol plant extract, at a yalue of 50% or greater was deemed to be a positiye result. Seyenty nine methanol plant extracts produced a positiye result. The yalue for % inhibition is a mean of duplicate assay points. 118 TABLE 3.3 Positive results of screening 635 methanol plant extracts in the bradykinin BKII binding assay. PLANT SPECIES , % INHIBITION OF BINDING Acorus calamus 100 A^eratum conyzoides 98 A^apanthus praecox 100 Allium albopilosum 82 Asiarum heteotropoides 54 Anemmarhena asphodeloides 88 Allopectus tetragous 100 A^eratum houstonianum 83 Acacia auriculiformis 100 Aconitum napellus 55 Ale tris farnosa 100 Arisaema consanguineum 88 Artemisia carna 90 AesculusX hippocastanum 95 Aesculus marylandicus 99 Angelica dauhrica 61 Allium christophii 64 Berheris thunhergii 54 Barringtonia edulis 100 Bos-wellia carterii 85 Bletilla striata 68 Betula pendula 72 Carapa guineensis 100 Citrus aurantium 63 Corvdalis decumbens 71 Croton tiglium 97 Chelidonium majus 59 Cymboviatus law’sonianus 68 Cocos nucifera 53 Cypella herbetii 52 Caryopteris x canodensis 75 Calpocalyx x brevibracteus 78 Commiphora myrhh 100 Dioscorea bulbifera 60 Evodia rutaecarpa 90 Erinus alpinus 64 Ercilla volubilis 97 Ervthrina corallodendron 57 119 Table 3.3 continued PLANT SPECIES % INHIBITION Cff BINDING Fri til lari a persica 50 Guaiacum officinale 57 Gomphrena ffiobosa 89 Gunnera mannicata 70 Heliconia wagneriana 76 Hardenbergia comptoniana 76 Hyacinthus 'white pearl ’ 54 Haworthia chlorantha 54 Holarrhena floribunda 77 Ipomea pes-caprae 83 Juniperus communis 100 Kalanchoe kewensis 64 Ledebouriella divaricata 100 Maesobotrya barteri var sparsifolia 53 M aesa sp. 100 Nandina dornestica 66 Panax ginseng 63 Potentilla erecta 75 Parthenium hysterophorus 68 Piptadeniastrum qfricanum 53 Punica granatum 58 Paeonia moutan 100 Pittosporum arborescens 100 Recaisea fargesii 51 Rheum palmatum 58 Ruschia stenophylla 62 Ricinus communis 95 Rosmarinus officinalis 73 Schleffera arboricolor 96 Scutellaria baicalensis 79 Solidago virgaurea 100 Sophora flavescens 100 Stephania tetrandra 52 Symplocos leptophylla 100 Salvia miltiorrhiza 83 Sinomenium acutum 94 Stephania dinklagei 64 Sphaerulacea ambigua 57 Schinus molle 73 Thymus vulgaris 64 Teucrium Jruticans 51 120 All extracts producing a positive result were treated with PVP at three concentrations, to determine whether their potency could be atributed to phenolic components in the bradykinin binding assay. The methanol extract of Sinomenium acutum produced 94% inhibition of binding in the initial screening in the bradykinin BK II assay (Table 3.3). In the subsequent screening (Table 3.4), this extract did not produce a positive result following treatment with PVP. The methanol extracts of Ageratum conyzoides, Anemarrhena asphodeloides and Solidago virgaurea inhibited the binding of tritiated bradykinin to the BK II receptor prior to treatment with PVP. The addition of PVP reduced the inhibition of binding indicating the effect may have been due to the non specific binding effect of tannins in the methanol extract. The methanol extracts of Citrus aurantium, Erythrina corallodendron, Ipomea pes- caprae, Juniperus communis, Pittosporum arborescens, Rosmarinus officinalis and Schleffera arboricolor all produced an inhibition of tritiated bradykinin binding to the BK n receptor following treatment of the extract with PVP, and also when the extract was diluted to 1:5 (Table 3.5, pl27-128). Thus the activity of these plants did not appear to be due to the non-specific binding effect of tannins. The methanol extracts of Barringtonia edulis, Maesa sp., Panax ginseng and Symplocos leptophylla all produced a potent inhibition of the tritiated bradykinin binding to the BK II receptor with and without PVP at all three concentrations of plant extract. The activities of these three plant extracts appeared to be specific and potent in the bradykinin BK II binding assay. The methanol extracts of Acacia auriculiformis and Croton tiglium produced an inhibition of tritiated bradykinin binding to the BK II receptor following treatment with PVP, at a concentration of Ig: lOmls of methanol {Croton tiglium) and 1:4 {Acacia auriculiformis). However, adequate amounts of both of these plants were unavailable for subsequent investigations. The methanol extracts of 556 plants inhibited the binding of tritiated bradykinin to the BK II receptor at values of less than 50% and these were deemed to be negative results. Appendix I (pages 198-209) contains a list of all the methanol plant extracts 121 screened in the BK II and CGRP radioligand binding assays. The methanol extracts of the most potent plants (Table 3.3) were serially diluted 1:2 and screened in the bradykinin assay to determine which plant extract produced the most potent and selective inhibition of the BK II receptor. The methanol extracts of Schleffera arboricolor, Citrus aurantium, Juniperus communis, Panax ginseng and Rosmarinus officinalis produced 100% inhibition of tritiated bradykinin binding to the BK II receptor at the lowest dilution factor, which within three serial dilutions of 1:2 fell to below 50% inhibition of binding (Table 3.4). The methanol extracts o ï Ipomea pes-caprae, Anemarrhena asphodeloides, Barringtonia edulis, Maesa sp,, Pittosporum arborescens and Erythrina corallodendron produced responses in the bradykinin assay which suggested their effects were more specific to the BK II receptor as the 1:2 serial dilution of the extract appeared to have a concentration dependent effect (Table 3.5). The methanol extract of Symplocos leptophylla produced the most potent and concentration -dependent inhibition of tritiated bradykinin binding to the BK II receptor. The bioactivity of the extract reached a level of inhibition lower than 50% at a dilution factor of 1:512. Therefore, the methanol extract of the stem and bark of S. leptophylla was chosen to further investigate the nature of the compound(s) producing a potent inhibition of tritiated bradykinin binding to the BK II receptor. The methanol extracts of all the plants with initial activity greater than 50% inhibition of binding in the bradykinin BK II assay were screened in eighteen other cell, enzyme and receptor assays to determine their selectivity and specificity to the BK II receptor. These screens were enzyme and other radioligand binding assays routinely ran at Glaxo Group Research. These were used in order to determine the selectivity and specificity of the plant extracts which produced a positive result in the BK II assay. 122 TABLE 3.4 The influence of PVP on the activity of plant extracts in the bradykinin BK n assay. The 79 plant extracts which produced greater than 50% inhibition of binding of tritiated bradykinin binding to the BK H receptor (Table 3.3) were treated with PVP at three concentrations of extract, to determine whether their potency could be attributed to phenolic components, in the bradykinin binding assay. A Ig of dried plant material in lOmls of methanol extract was diluted 1:5 and 1:25 to produce three points in the bradykinin assay. The extracts producing the most potent inhibition which appeared not to be due to phenols were to be selected for further investigations into their bioactivity. 123 TABLE 3.4 The influence of PVP on the activity of plant extracts in the bradykinin BKII assay with PVP. PLANT SPECIES %INHD3mOM OF BINDING ' -PVP MeOH 1:5 1: 25 MeOH 1:5 1:25 Acorus calamus 100 10 8 31 26 19 Ageratum conyzoides 100 100 78 4 20 10 Agapanthus praecox 63 37 21 25 21 27 Allium albopilosum 82 79 34 24 22 3 Asiarum heteotropoides 54 58 48 30 8 7 Anemmarhena asphodeloides 88 42 51 44 26 14 Allopectus tetragous 100 49 51 14 7 5 Ageratum houstonianum 83 100 40 40 33 27 Acacia auriculiformis 100 100 40 60 59 25 Aconitum napellus 55 59 19 32 32 27 A le tris farnosa 100 60 41 38 15 13 Arisaema consanguineurn 97 16 10 34 31 10 Artemisia carna 76 27 20 43 25 15 Aesculus X hippocastanum 91 76 15 100 68 2 Aesculus marylandicus 97 78 72 100 84 35 Angelica dauhrica 43 31 40 43 45 9 Allium christophii 62 41 26 28 13 0 Berheris thunbergii 51 43 37 22 26 0 Barringtonia edulis 100 100 100 81 80 72 Boswellia carterii 50 32 27 19 1 6 Bletilla striata 20 16 8 2 0 0 Betula pendula 50 28 10 31 0 0 Carapa guineensis 44 21 19 22 11 5 Citrus aurantium 63 60 67 50 4 0 Corydalis decumbens 42 39 29 31 24 17 Croton tiglium 82 60 39 60 32 10 Chelidonium majus 61 24 10 30 6 1 Cymboviatus lawsonianus 22 44 17 29 29 1 Cocos nucifera 53 52 58 42 36 18 Cypella herbetii 61 42 51 42 40 25 Caryopteris x canodensis 36 32 0 0 0 0 Calpocalyx x brevibracteus 69 54 40 28 12 0 Commiphora myrhh 100 86 74 45 34 17 Dioscorea bulbifera 51 47 21 57 55 49 Evodia rutaecarpa 94 52 34 47 21 3 124 Tabic 3.4 continued. PLANISPECIES , % INHIBITION OF BINDING . - PVP M e O H 1:5 1:25 M e O H : 1:5 1:25 Ernius alpinus 60 60 35 39 33 0 Ercilla volubilis 24 6 2 11 8 0 Ervthrina corallodendron 83 58 16 56 18 14 Fritillaria persica 56 11 0 12 0 0 Guaiacum officinale 77 22 3 36 7 6 Gomphrena globosa 93 19 6 17 12 5 Gunnera mannicata 62 40 29 2 1 0 Heliconia wagneriana 28 33 0 0 0 0 Hardenbergia comptoniana 73 30 10 13 8 0 Hyacinthus 'white pearl' 50 20 9 0 0 0 Haworthia chlorantha 48 36 8 19 0 0 Holarrhena floribunda 76 54 12 22 8 0 Ipomea pes-caprae 66 55 34 83 80 16 Juniperus communis 100 79 64 73 32 8 Kalanchoe kewensis 58 32 28 18 5 2 Ledebouriella divaricata 100 100 85 6 7 3 Maesobotrya barteri var 51 37 12 25 0 0 sparssifolia M aesa sp. 100 100 100 941 72 87 Nandina domestica 63 28 12 8 0 1 Panax gingseng 100 85 22 100 91 61 Potentilla erecta 95 43 27 49 33 16 Parthenium hysterophorus 57 50 42 41 17 10 Piptadeniasstrum african 48 48 16 18 15 14 Punica granatum 54 42 16 14 8 1 Paeonia moutan 95 43 27 49 33 16 Pittosporum arborescen 100 100 78 100 96 36 Recaisea fargesii 49 35 10 8 0 0 Rheum palmatum 50 33 16 0 0 0 Ruschia stenophylla 48 22 20 32 12 6 Ricinus communis 89 54 30 49 39 10 Rosmarinus officinalis 76 47 40 64 18 4 Schefflera arboricolor 98 68 57 100 15 0 Scutellaria baicalensis 68 55 33 50 13 2 Solidago virgaurea 100 34 14 40 55 21 Sophora flavescens 100 90 52 37 24 0 Stephania tetrandra 23 12 0 23 3 0 Symplocos leptophylla 100 100 100 100 87 82 Salvia miltiorrhiza 43 37 20 27 15 13 Sinomenium acutum 48 45 32 48 39 24 Stephania dinklagei 52 21 34 44 31 19 Sphaerulacea ambigua 48 20 13 19 12 4 Schinus molle 66 17 10 15 4 3 Thymus vulgaris 64 10 9 9 3 1 Teucrium fruticans 49 20 3 15 16 0 125 TABLE 3.5 Serial dilutions of the most potent methanol plant extracts in the bradykinin BK II assay. Thirteen methanol plant extracts produced an inhibition of tritiated bradykinin binding to the BK n receptor, after treatment with PVP. Serial dilutions of the methanol extracts were carried out to determine which extracts produced a dose-dependent inhibiiton of binding. The methanol extracts of Schefflera arboricolor. Citrus aurantium, Juniperus communis, Panax ginseng, Ricinus officinalis, Ipomea pes- caprae, Anemmarrhena asphodeloides, Barringtonia edulis, Maesa sp. Pittosporum arborescens, Erythrina corallodendron and Symplocos leptophylla produced the most potent and concentration - dependent inhibition of tritiated bradykinin binding to the BK II receptor when serially diluted 1:2 in the bradykinin assay. 126 TABLE 3.5 (a-d) Serial dilutions of the plant extracts with positive activity in the bradykinin BKII assay. (a) METHANOL % INHIBITION OF BINDING BXTRAGT PLANT SPECIES jDilutioam assay Ipomea pes- caprae Anemarrhena asphodeloides 1:2 100 100 1:4 100 95 1:8 100 99 1:16 100 100 1:32 100 77 1:64 94 65 1:128 67 66 1:256 56 60 1:512 25 37 1:1024 14 18 (b) METHANOL % INHIBITION OF BINDING EXTRACT PLANT SPECIES Diliitioti in ass^ BeaHngtonia Maesa Sp Symplocos Pittosporum eduiis leptophylla arborescens 1:4 72 94 87 100 1:8 80 72 82 96 1:16 81 87 100 100 1:32 89 85 89 36 1:64 73 91 85 41 1:128 78 79 95 31 1:256 47 23 93 20 1:512 41 15 70 6 1:1024 38 14 41 0 1:2048 14 2 6 0 (c) % INHIBITION OF BINDING PLANT SPECIES DUution in assay Erythrina Scutellaria Schleffera O irus coralhdendrm baicalensis arboricolor aurantium 1:20 81 88 100 96 1:40 63 36 100 9 1:80 51 32 89 0 1:160 50 28 27 0 1:320 36 43 1 0 1:640 17 32 3 0 1:1280 21 31 0 0 1:2560 18 37 0 0 1:5120 18 39 0 0 1:10240 0 2 0 0 127 Table 3.5 continued (d) METHANOL % INHIBITION OF BINDING EXTRACT PLANT s m c m Dilation in assay Jtiniperus communis Panax ginseng Rosmarinus officinalis 1 20 91 100 91 1 40 45 100 58 1 80 12 65 27 1 160 15 23 12 1 320 5 15 6 1 640 0 8 17 1 1280 0 7 7 1 2560 0 1 6 1 5120 3 0 13 1 10240 0 0 5 128 Eighteen plant extracts had specific activty in the bradykinin screen only, including the methanol extract of Symplocos leptophylla (Table 3.6). The plant extract producing the most potent and selective activity in the bradykinin BK II assay was Symplocos leptophylla and so was selected for further investigations, in an effort to isolate the bioactive entities. The stem and bark of Symplocos leptophylla, family Symplococaceae, was used to make a methanol extract with a concentration of Ig; lOmls of methanol which was applied to a Cl 8 bond elut colunm. The column was serially eluted with increasing concentrations of acetonitrile and 100% methanol and the most bioactive fractions, which were 20%, 30% and 40% acetonitrile, were combined, evaporated to dryness and used for the investigation into bioactive entities (Table 3.7). These investigations resulted in the isolation of eight fractions from Symplocos leptophylla (Figure 3.18, p i65) which appeared well resolved by HPLC analysis, each containing principal peaks. Displacement curves of fractions 1-8, which were isolated from the stem and bark of Symplocos leptophylla, were carried out in the bradykinin BK 11 assay (Figures 3.10 - 3.17, p i62-163) prior to NMR analysis. There was variation between the dry weights of fractions 1 to 8 , with fractions 1 and 8 producing the most potent and specific displacement curves. Fraction numbers 2 - 7 (Figures 3.11-3.16, p i62-163) caused an apparent inhibition of tritiated bradykinin binding to the BK n receptor, but the displacement curves were very steep. This may indicate that these compounds were lysing the cells at the highest concentration and were having little effect at the lowest concentrations, thus their interaction with the BK n receptor may be non - specific. 129 TABLE 3.6 Plant extracts with selective activity to the bradykinin BK II receptor. The methanol extracts of all the plants with initial activity greater than 50% inhibition of binding in the bradykinin BK II assay (Table 3.3, 3.4) were screened in eighteen other cell, enzyme and receptor assays to determine their selectivity and specificity to the BK II receptor. Eighteen plant extracts appeared to show selective activity to the bradykinin BK II assay. 130 TABLE 3.6 Plant extracts with selective activity to the bradykinin BK II receptor. PLANT SPECIES FAMILY Acorus calamus Araceae Af^eratum houstonianum Compositae Allopectus tetragous Gesneriaceae Angelica dauhrica Umbelliferae Arisaemia consanguineum Araceae Asiarum heterotropoides Aristolochiaceae Barringtonia edulis Barringtonaceae Boswellia carterii Burseraceae Commiphora myrrh Burseraceae Discorea bulbifera Discoreaceae Gomphrena globosa Amarranthaceae Guaiacum officinale Zygophyllaceae Holarrhena floribunda Apocynaceae Paeonia moutan Ranunculaceae Pittosporum arborescens Pittosporaceae Ricinus communis Euphorbiaceae Scutellaria baicalensis Labiatae Symplocos leptophylla Symplococaceae 131 TABLE 3.7 Isolation of the bioactive fractions from Symplocos leptophylla in the bradykinin BK II assay. The stem and bark of Symplocos leptophylla, family Symplococaceae, produced the most potent inhibition of tritiated bradykinin binding to the BK II receptor (Table 3.3, 3.4, 3.5) and was selected to further investigate its bioactive entities in the bradykinin assay. Extraction of the stem and bark into methanol, and eluting with increasing concentrations of acetonitrile, produced a series of fractions which indicated the majority of the bioactivity of Symplocos leptophylla was present in 20%, 30% and 40% acetonitrile. These fractions were combined, evaporated to dryness, resuspended in methanol and used for the investigation of bioactive compounds. 132 TABLE 3.7 Isolation of the bioactive fractions from the stem and hark of Sympolocos leptophylla in the bradykinin BKII assay. Bympiocos iep^phyüa % INHIBm ONO F BINDING DILUTION IN ASSAY Sanple 20% 30% 40% 50% 60% 70% 80% MeOH 1 10 100 91 76 100 31 0 10 17 80 1 20 100 100 100 100 0 0 0 0 80 1 40 100 75 75 91 0 0 0 0 21 1 80 55 49 37 33 10 0 0 0 19 1 160 40 42 35 28 10 0 0 0 14 1 320 28 37 15 26 6 0 0 0 18 1 640 39 36 24 20 8 0 0 0 2 1 1280 11 30 25 20 13 0 0 0 0 1 2560 10 57 13 16 14 0 0 0 0 1 5120 0 22 0 0 2 0 0 0 0 Control values (DPM’s): Bound 1003 NSB: 119 % Sp.Bound = 88 133 TABLE 3.8 The results of screening the methanol extracts of Panax ginseng and Ipomea pes-caprae in the Neurokinin 1 (NKl ) receptor ligand binding assay. The methanol extracts of the plants producing greater than 50% inhibition of binding in the bradykinin BK II assay (Table 3.3) were screened in the Neurokinin 1 receptor radioligand binding assay. The Ig : lOmls of methanol extracts of Panax ginseng and Ipomea pes-caprae inhibited the binding of Substance P to the NKl receptor, and thus appeared to have non - selective activity in the bradykinin BK II assay. 134 TABLE 3.8 The results of screening the methanol extracts of Panax ginseng and Ipomea pes-caprae in the Neurokinin 1 assay. OPBÎMDÏNG -PW + PVP ' : + P W 1 20 100 100 100 100 1 40 100 78 100 100 1 80 82 21 100 26 1 160 42 9 73 15 1 320 46 6 61 10 1 640 61 5 50 16 1 1280 67 5 35 11 1 2560 57 10 31 6.5 1 5120 39 0 26 0 1 10240 30 0 18 0 Control values (DPM’s):Bound:1397 NSB; 95 % Sp Bound : 93 135 TABLE 3.9 The results of screening the methanol extracts of Panax ginseng and Ipomea pes-caprae in the in vivo acetic acid writhing test. The concentrated extracts of Panax ginseng, family Araliaceae, and Ipomea pes- caprae, family Convovulaceae, were screened in the acetic acid writhing test I. pes-caprae significantly decreased the number of writhes as compared to the control values, for all test periods. The extract of P.ginseng did not appear to have any effect on the number of writhes as induced with acetic acid. 136 TABLE 3.9(a-i) The results of Panax ginseng and Ipomea pes-caprae in the in vivo acetic acid writhing test. a)Group 1 controls - 15 minutes. 350 300 300 300 b) Group 1 controls - 30 minutes. c) Group 1 controls - 45 minutes ^ K' ‘ i 1 ^ 1 35 350 52 2 40 400 38 3 35 350 41 4 30 300 28 d) Group 2. Panax ginseng - 15 minutes 1 35 350 45 2 35 350 20 3 30 300 33 4 35 350 27 e) Group 2. Panax ginseng - 30 minutes 1 35 350 38 2 30 300 53 3 30 300 59 4 30 300 50 137 Table 3.9 continued f) Group 2. Panax ginseng - 45 minutes Wetgbl/||,, Voliacelic NtmW rof (ul> y m ê m 1 35 350 20 2 30 300 29 3 35 350 24 4 35 350 43 g) Group 3. Ipomea pes-caprae - 15 minutes Mouse miBBlJer . aeadfeattMclW::! w w . 1 35 350 22 2 30 300 12 3 40 400 12 4 30 300 28 h) Group 3. Ipomea pes-caprae - 30 minutes „>WS«rQf> J*; 1 30 300 17 2 35 350 20 3 30 300 23 4 35 350 17 i) Group 3. Ipomea pes-caprae - 45 minutes wndMS 1 35 350 25 2 25 250 19 3 35 350 27 4 30 300 31 138 Fraction numbers 1 and 8 (Figures 3.10 and 3.17, p i62-163) produced displacement curves which appeared to have a concentration dependent efifect indicating the effect may be a more specific inhibition of tritiated bradykinin binding to the BK II receptor than observed by fractions 2-7. The IC50 of fraction 1 was between 1.66 and 3.33ugs/ml, and for fraction 8 the value was very much lower (Table 3.19, pl59). The mass spectral data (Table 3.10) for the Time Of Flight (TOF) analysis of fractions 1 - 8 are in Appendix IV, pages 215-216. The TOF analysis of fractions 1 - 8 indicated that these fractions were a mixture of impure compounds. The TOF data for each fraction (where applicable) was not reinforced by the FAB data, indicating the presence of a number of impure compounds. TABLE 3.10 RESULTS OF THE MASS SPECTRAL ANALYSIS AND THE RETENTION TIMES OF FRACTIONS 1-8 FRACTION NUMBER______MASS SPECTRA \M \^ at m/z TOF______FAB______Retention Time (minutes) 1. 782,1260,1502 506,1237,1375 2. 0 - 3. 0 2.1140,1233,1299, 2386 1095,1259,1301 9. 0 - 10. 0 3. 1134,1254,1379,2534 21. 0 - 22. 0 4.1251,1397,2555 20. 5 - 22. 0 5. 1246,1398,2526,3920 26. 5 - 28. 0 6. 1244,1275,2525,3855 33. 0 - 34. 0 7. 1200,1229,2423,3636 41. 0 - 43. 0 8. 1220,1242,1259,2480,3720 57. 0 - 58. 5 The eight fractions which were inhibiting the binding of tritiated bradykinin to the BK n receptor were analysed using NMR spectroscopy (figures 3.19 - 3.26, p i67-74), with fractions 1 and 8 producing the clearest and strongest signals. 139 From the NMR spectra of fraction 8 , which can also be applied to those of fractions 2-7, the following chemical signals are present : Methyl groups, CH 3 at 50.7 - 51.4 CH2 groups at Ô 1.5 - 52.2 O = C-CH3 groups at 62.5-63.0 CH - O groups at 63.5 - 64.5 C = CH groups at 64.8 - 64.9 As the retention times of fractions 1 -8 differed but the NMR spectra were very similar, (Table 3.10) it would appear that they are mixtures of related compounds. 140 3.11 THE IN VITRO AND IN VIVO RESULTS OF THE METHANOL EXTRACTS OF PANAX GINSENG AND IPOMEA PES-CAPRAE. The 635 methanol plant extracts were screened against the BK II receptor with a positive result being defined as greater than 50% inhibition of binding. This yielded 79 extracts which were positive in the BK II assay. In an effort to determine their selectivity to the BK II receptor, these 79 plant extracts were screened against the NKl receptor expressed in U373 MG cells. A second extract was made of these 79 plants (Table 3.3) and was screened in the NKl assay. A positive result in the NK 1 assay was that of lOuL of a Ig in lOmls of dried plant material in methanol, producing 50% inhibition of binding of ligand to receptor. Out of the 79 plant extracts tested, 2 gave a positive result in the NK 1 assay. They were Ipomea pes-caprae and Panax ginseng. As these two plant extracts were active against the NK 1 receptor, in addition to the BK II receptor, they were not producing a selective interaction with the BK II receptor, and thus their bioactivities did not fit the criteria for the selection of a plant extract which would be followed through to isolation in order to yield a non-peptide BK II receptor antagonist. However, because the extracts were active against a receptor other than bradykinin, they were deemed to be non-selective to the BK II receptor, but it was possible that they could have some broad based analgesic effects or activities due to different components in the methanol extract. The inhibition of binding of Substance P to the NKl receptor, by the methanol extracts of Ipomeapes caprae and Panax ginseng in the NKl assay, upto a dilution factor of 1:40, did not appear to be due to the non specific binding effect of tannins. 3.11.1. The In vivo results of Panax ginseng and Ipomea pes-caprae in the acetic acid writhing test. The concentrated extracts of Panax ginseng, family Araliaceae, and Ipomea pes- 141 caprae, family Convovulaceae, were screened in the in vivo acetic acid writhing test (see Table 3.9). The mean number of writhes for the control groups were 28.0, 39.0 and 44.0 (for 15, 30 and 45 mins.) (n=4 for each group) writhes. The mean value for the extract of Panax ginseng, for 15 minutes, was 31 (n=4) writhes. For 30 minutes the number of writhes was 50 (n=4), and for 45 minutes the number was 29 (n=4). The mean value for the number of writhes, as produced by the extract of Ipomea pes- caprae, for 15 minutes, was 18.5 (n=4). For 30 minutes the number was 19.2 (n=4), and for 45 minutes the number of writhes was 25.5. Statistical analysis of the results of Panax ginseng and Ipomea pes-caprae suggested Ipomea pes-caprae has trends which are different to that of Panax ginseng in that the number of writhes in the latter two periods of observation (30 and 45 mins.) were decreased when mice were injected with extracts of Ipomea pes-caprae. Kruskal-Wallis 1 Way ANOVA compared the mean values for the number of writhes in 15 minutes for the control, versus the values for Ipomea pes-caprae and Panax ginseng. At 15 and 45 minutes there was no measurable statistical difference between the groups (p > 0.05). At 30 minutes p < 0.05 indicating a significant difference between Ipomea pes-caprae (decrease in the number of writhes) and Panax ginseng ( no effect on the number of writhes). For time at 45 minutes, the number of writhes produced by the extracts of Ipomea pes-caprae and Panax ginseng were both less than the control values, but as the sample size is low (n=4), this difference cannot be statistically measured. The Mann-Whitney U-Wilcoxon Rank Sum W Test was also used, to test the significance between two experimental groups i.e. P.ginseng vs I. pes-caprae, P.ginseng vs Control group and I. pes-caprae vs Control group. There was no measurable difference between the number of writhes in the acetic acid writhing test ÏOV P.ginseng and I. pes-caprae at times 15 and 45 minutes (p > 0.05). For 30 minutes there was a significant difference between the two plants with the extract of Lpes- 142 caprae decreasing the number of writhes (p < 0.05). For the extract of Lpes-caprae versus the control values, there was no significant difference in the number of writhes at time 15 minutes (p > 0.05). For 30 and 45 minutes there was a significant difiference in the number of writhes (p < 0.05) when the extract of Lpes-caprae was injected into mice. For the extract of P.ginseng and the control values there was no significant difference in the number of writhes at 15, 30 or 45 minutes (p > 0.05). These results suggest that the extract of I.pes-caprae has different effects on the number of writhes, as compared to P.ginseng. This effect is more pronounced at the time periods of 30 and 45 minutes. However, the low sample numbers (n=4) do not give a large degree of statistical confidence and the experiment requires replication using larger numbers of mice ( at least n= 8 ) to clarify the potential analgesic effect of the extract o ïI.pes-caprae. Thus, P.ginseng did not appear to have any effect on the number of writhes induced by acetic acid. / .pes-caprae decreased the number of writhes as compared to control values for all test periods (Table 3.9), which with the bioactivity in the bradykinin and Neurokinin 1 assay, suggests that this plant may have some broad - based analgesic activity. 143 3.12 THE RESULTS OF SCREENING 300 ETHNOMEDICALLY SELECTED AND 335 NON SELECTED PLANT EXTRACTS IN THE CGRP ASSAY. A duplicate methanol extract was made for each of the 635 plants which were to be screened in the CGRP assay. The methanol extracts of thirteen plants produced greater than 50% inhibition of binding in the CGRP assay prior to treatment with PVP (Table 3.11, p i46), and these were all ethnomedically selected from the literature of China, West Africa, the West Indies, South America and by accessing ‘Napralert’(Farnsworth, 1994). The remaining 622 methanol plant extracts did not produce values of 50% inhibition or greater, and were deemed to be negative results (Appendix I p i98-209). The methanol extracts of Coptis chinensis and Stephania dinklagei maintained bioactivity greater than 50% inhibition of binding prior to treatment with PVP (Table 3.12, pl49). The levels of inhibition fell to below 50% after treatment with PVP, suggesting the activity was due to the non-specific binding effects of tannins. The methanol extract of Physostigma venenosum maintained an inhibition of iodinated CGRP binding to the CGRP receptor, at a Ig: lOmls methanol concentration following treatment with PVP. This bioactivity decreased with and without PVP at the 1: 5 and 1:25 concentrations of methanol extract. The methanol extract of Typhonium giganteum inhibited the binding of iodinated CGRP to the CGRP receptor prior to treatment with PVP at the three concentrations of methanol extract screened. A value of 69% inhibition was produced by the extract at a lg:10mls concentration, following treatment with PVP. This value fell to 34% at the 1:5 dilution, and to 35% at the 1:25 dilution. Serial dilutions of the methanol extracts of Physostigma venenosum and Typhonium giganteum indicated that these were the most potent extracts (Table 3.13), but neither of these extracts were selective to the CGRP receptor only (Table 3.14). The extract of Typhonium giganteum had positive activities in antiviral and anti inflammatory assays, and the extract of Physostigma venenosum had positive activities in anticancer and anti-inflammatory assays, therefore the methanol extracts were 144 thought to be unlikely to yield a selective antagonist to the CGRP receptor. It was a possibility that the activity of the methanol extract of Physostigma venenosum was due to the presence of the alkaloid physostigmine, thus this alkaloid was screened in the CGRP assay. A serial dilution of this alkaloid in the CGRP assay, with an initial concentration of 2.5mgs/ml, indicated that the activity oiPhysostigma venenosum was not due to the presence of this secondary metabolite as the alkaloid did not inhibit the binding of iodinated CGRP to the CGRP receptor at any concentration tested (Table 3.14, p i51). The methanol extracts of plants with greater than 50% inhibition of binding were screened in eighteen other cell, receptor and enzyme assays. Seven methanol extracts appeared to show selective activity to the CGRP receptor only after testing in 18 other screens which included antiviral, anticancer and anti-inflammatory therapeutic targets (Table 3.15, pl53). 145 TABLE 3.11 The positive results of screening 635 methanol plant extracts in the Calcitonin Gene Related Peptide (CGRP) binding assay. A Ig in lOmls of methanol extract was made for each of the 635 plants which were to be screened in the CGRP binding assay. The methanol extracts of thirteen plants produced inhibition of iodinated CGRP binding to the CGRP receptor. 146 TABLE 3.11 The positive results of screening 635 methanol plant extracts in the Calcitonin Gene - Related Peptide (CGRP) assay. PLANT SPECIES FAMILY % INHIBITION OF BINDING Ananas canosus Bromeliaceae 84 Buxus sempervirens Buxaceae 64 Chionathus virginiana Oleaceae 56 Clematis chinensis Ranunculaceae 94 Coptis chinensis Ranunculaceac 100 Ecballium elateuriurn Cucurbitaceae 79 Galanthus nivalis Amaryllidaceae 100 Ligusticum sinense Umbelliferae 87 Physostigma venenosum Leguminosae 72 Stephania dinklagei Menispermaceae 100 Typhonium giganteum Araceae 100 Urtica pilulifera Urticaceae 65 Vernonia cinerea Compositae 83 147 TABLE 3.12 The results of treating the methanol extracts producing a positive result in the CGRP assay with PVP. The percentage inhibition values produced by the methanol extracts of Coptis chinensis and Stephcmia dinklagei decreased after treatment with PVP. The methanol extract of Physostigma venenosum maintained an inhibition of iodinated CGRP binding to the CGRP receptor at a Ig : lOmls methanol concentration following treatment with PVP. The methanol extract of Typhonium giganteum inhibited the binding of iodinated CGRP to the CGRP receptor prior to treatment with PVP at the three concentrations of methanol extract screened. 148 TABLE 3.12 The results of treating the methanol extracts producing a positive result in the Calcitonin Gene Related Peptide (CGRP) assay with PVP. % inhibtion of binding % inhibition of binding PLANT SPECIES -PVP + PVP ^ < ■ l:î5 Ananas canosus 100 28 7 35 0 0 Buxus sempervirens 62 35 7 23 9 10 Clematis chinensis 93 31 32 16 9 9 Chionathus virginiana 56 40 56 9 15 11 Coptis chinensis 100 92 36 28 20 0 Ecballium elaterium 79 38 21 20 16 8 Galanthus nivalis 100 34 32 14 11 0 Ligusticum sinense 53 9 33 35 21 7 Physostigma venenosum 71 26 77 55 25 8 Stephania dinklagei 100 100 93 31 24 0 Typhonium giganteum 100 100 0 69 34 35 Urtica pilulifera 65 30 36 31 0 0 Vernonia cinerarea 83 37 0 18 8 0 The value for % inhibtion of binding is a mean of duplicate assays. 149 TABLE 3.13 Serial dilutions of the methanol extracts of Physostigma venenosum and Typhonium giganteum in the CGRP binding assay, after treatment with PVP. Serial dilutions of the concentrated extracts of Physostigma venenosum and Typhonium giganteum were carried out in the CGRP assay. The extract was treated with PVP and serially diluted 1 : 2. TABLE 3.14 A serial dilution of Physostigmine in the CGRP binding assay. In an attempt to determine whether or not the activity in the CGRP assay was due to physostigmine a 2.5mg/ml solution of the alkaloid physostigmine was made, and serially diluted 1 : 2 in methanol in the CGRP binding assay. The alkaloid did not inhibit the binding of iodinated CGRP to the CGRP receptor at any concentration tested. 150 TABLE 3.13 Serial dilutions of the methanol extracts of Physostigma venenosum and Typhonium giganteum in the CGRP assay, after treatment with PVP. DE.DT10NINTHE % INHIBITION OF BINDING PLANT SPECIES Physostigma venenosum Typhohium giganteum 1 4 72 100 1 8 86 100 1 16 97 100 1 32 55 71 1 64 69 73 1 128 53 65 1 256 78 58 1 512 54 43 1 1024 48 30 1 2048 28 15 Control values (DPM’s) : Bound : 2330 NSB ; 857 % Sp Bound = 63 TABLE 3.14 Serial dilution of Physostigmine in the CGRP binding assay. CONCENTRATION OF ALKALOID % INHIBITION m BINDING mgs/ml 2.5 25 1.25 12 0.62 0 0.31 8 0.15 0 0.078 0 0.039 0 0.019 0 0.009 0 0.0048 0 Control values (DPM’s) : B:1870 NSB :630 %Sp.Bound = 66 151 TABLE 3.15 Plants with selective activity to the CGRP receptor. The methanol extracts of the plants with greater than 50% inhibition of iodinated CGRP binding to the CGRP receptor (Table 3.12) were screened in 18 other cell, receptor and enzyme screens to determine their selectivity to the CGRP receptor. Seven methanol plant extracts were selective to the CGRP receptor only. 152 TABLE 3.15 Plants with selective activity to the CGRP receptor. PLANT SPECIES FAMILY Clematis chinensis Ranunculaceae Chionathus vir^iniana Oleaceae Galanthus nivalis Amaryllidaceae Lif^usticum sinense Umbelliferae Stephania dinklagei Menispermaceae Urtica pilulifera Urticaceae Vernonia cinerea Compositae 153 3.13.THE EXTRACTION OF BIOACTIVE ENTITIES FROM SYMPLOCOS LEPTOPHYLLA. The methanol extract of the stem and bark of Symplocos leptophylla produced the most potent and selective inhibition of binding in the bradykinin BKII low throughput radioligand binding assay. Analytical and preparative reverse phase chromatography was used to isolate the compound(s) which inhibited the binding of tritiated bradykinin to the bradykinin BK II receptor. 3.14. THE PROCEDURE FOLLOWED FOR THE EXTRACTION OF BIOACTIVE ENTITIES FROM SYMPLOCOS LEPTOPHYLLA. The initial chromatography on Symplocos leptophylla used a PRPl analytical column, with a flow rate of 2mls/minute and a sample load of 25 ul of a Ig : lOmls methanol extract (Appendix Ill(a), p212), using gradient system I. Eighty 1ml fractions were collected and evaporated to dryness overnight in a’Savant’ bench centrifuge. The fractions were resuspended in lOOul of methanol and lOul were used for assays. The bioactive fractions were numbers 55 upto 59 and had retention times between 27.5 and 29.5 minutes. 500ul of a concentrated extract of S.leptophylla (lg:5mls methanol, evaporated and resuspended in 500ul methanol) was loaded onto a preparative CIS PRPl column and eluted with a flow rate of 20mls/minute. The gradient system No. 1 was used and eighty fractions with a volume of 20ml were collected. One millilitre was removed from each, evaporated to dryness and resuspended in 50ul of methanol, lOul were assayed. The active fractions eluted between 32 and 37 minutes. 300g of dried and ground S.leptophylla stem and bark was macerated overnight in 3 154 litres o f methanol, (see method 2.24). The extract was filtered and evaporated to dryness under reduced pressure to 50mls. A 1:2 serial dilution o f the extract was carried out to verify the bioactivity: Table 3.16 The bioactivity o f Symplocos leptophylla in the BK II assay prior to PVP tr e a tm e n t. 1 :4 8 9 1:8 9 5 1 :1 6 9 5 1 :3 2 9 5 1 :6 4 9 0 1 :1 2 8 9 0 1 : 2 5 6 9 0 1 : 5 1 2 9 0 1 : 1 0 2 4 9 4 1 : 2 0 4 8 7 3 Control values (DPM ’s) B:1504 NSB : 304 Specific Bound : 80% lOOul o f the concentrated extract w as eluted on a 1ml PVP column using methanol. Ten millilitre fractions were collected, evaporated to dryness and resuspended in lOOul o f methanol. lOul were diluted 1:10 and and a sequential dilution w as carried out on the extract. 155 Table 3 .17. The bioactivitv o ïSymplocos leptophylla in the BK II assay following treatment with PVP. ...... A a s f g r - — ...... 1 :4 0 9 7 1 :8 0 9 8 1 : 1 6 0 1 0 0 1 : 3 2 0 1 0 0 1 :6 4 0 1 0 0 1 : 1 2 8 0 1 0 0 1 : 2 5 6 0 7 7 1 : 5 1 2 0 4 5 1 : 1 0 2 4 0 2 5 1 : 2 0 4 8 0 1 6 The bioactivity in the bradykinin BK II assay remained potent and did not appear to be due to the non specific binding effects o f tannins. The remaining extract was evaporated to dryness in vacuo, after which it was resuspended in l.Sm ls of methanol and applied to a Sephadex LH20 column. A flow rate o f O.Sml/minute was operated and I ml fractions were collected for 160 minutes. Each fraction was assayed for its ability to inhibit tritiated bradykinin binding to the BK II receptor. The active fractions had retention tim es of 6.5-14.5 minutes and 22-23 minutes. These fractions were evaporated to dryness and lul o f each fraction w as applied to an HPTLC plate, placed in a chamber containing methanol and left overnight. The fractions were viewed under U.V. light on the CAM AG AM D system (see method 2 . 2 6 ) . 3.14.1. Results o f the separation o f Symplocos leptophylla using the AM D system. lul of fraction numbers 4 - 1 1 , which most strongly absorbed ultra-violet light, were re-applied to the base o f a second HPTLC plate. They were placed in enclosed chambers containing 2mls o f methanol and left for 18 hours. Viewing in U .V . light indicated plant-derived material w as present at the top o f the plate. The end o f the 156 plate w as separated by cutting, and the silica gel scraped off and centrifuged in methanol. The supernatant was removed, evaporated to dryness and resuspended in lOOul o f methanol. The eight fractions were assayed at three concentrations to determine whether there was a dose-dependent relationship. T a b le 3.18. Determination of a dose-dependent relationship of Symplocos leptophylla bioactivitv in the BK II assav. 1 5 u L 3 0 u L 4 5 u L 1 4 5 4 6 5 2 2 1 9 3 4 4 8 3 1 0 0 1 0 0 1 0 0 4 3 1 4 0 9 4 5 3 2 3 9 1 0 0 6 9 5 1 0 0 1 0 0 7 3 2 2 1 6 8 2 2 4 9 9 2 Control values (DPM ’s)B : 2396 NSB : 263 Specific Bound : 89% The remaining methanol extract of Symplocos leptophylla was evaporated to 2mls in vacuo and applied to an LH20 column and processed as described previously. The bioactivity in the bradykinin assay corresponded to fractions eluting at 55-75 minutes. These were combined, evaporated to dryness and resuspended in 2.5m ls of methanol. ISOuls o f extract were applied to a HPTLC plate and run in the CAM AG system (see method 2.26). A wide band o f material strongly absorbing U.V. light was scraped off into 4mls o f methanol. This was centrifuged, the supernatant removed, evaporated to dryness and resuspended in lOOul o f methanol. Ten microlitres of 157 fraction 1 were loaded onto a CIS PRPl column. A 0.1% solution of formic acid was added to SOOmls of mobile phase and 1ml fractions were collected over 60 minutes. Gradient system No.l was applied to the extract. The fractions eluted were evaporated to dryness and resuspended in 5Oui of methanol and lOul were assayed. The bioactive fractions eluted at between 24-26.5 minutes. Gradient System No.2 was used to try and further separate the peaks. Bioactive fractions eluted at 3-6, 8-9 and 9-11 minutes, (220nm U.V.absorption). An analytical PRPl column was used with Gradient No.3 to increase the separation of the peaks (see Chromatogram 2 in Appendix IH(c), p214). Bioactive fractions eluted at 2.5-10 minutes. To separate these peaks further, an isocratic concentration of 70% methanol together with 0.1% formic acid was used. A sample load of 20ul was applied to a PRPl analytical column and bioactive fractions eluted at 5.0-13.5 minutes. The remaining 19mls of each fraction were evaporated to dryness, resuspended in 1ml of methanol and applied to a PRPl analytical column with an isocratic concentration of 70% methanol containing 0.1% formic acid, and the above procedure followed. Eight peaks which were quite well resolved (Figure 3.18, p i65) were weighed and resuspended in 1ml of methanol. These were submitted to mass spectral and NMR analysis after which sequential dilutions were carried out on fractions 1- 8 in the bradykinin BK II assay (Figures 3.19-3.26, p i67-174). Fractions 1-8 were spotted onto TLC plates and sprayed with Dragendorffs reagent and Vanillin-Phosphoric acid (VPA). The fractions developed a blue-green colour with VP A indicating the presence of terpenoids, but were negative for alkaloid compounds as indicated with Dragendorffs reagent. 158 3.15. THE DISPLACEMENT CURVES OF FRACTIONS 1 - 8 FROM SYMPLOCOS LEPTOPHYLLA IN THE BRADYKININ BK II ASSAY. The displacement curves o f the eight fractions which inhibited tritiated bradykinin binding to the BK II receptor (Figures 3 . 1 0 -3 .1 7 , p i62-163) were separated and partially purified from Symplocos leptophylla. All fractions were colourless and absorbed in the U.V. spectrum at 210nm. Fraction numbers 1 and 8 (Figures 3.10 and 3.17) produced displacement curves which suggested that the binding o f these extracts in the bradykinin BK II assay was more selective than those o f fractions 2 to 7 (Figures 3.11 to 3.16). Fraction numbers 2 -7 (Figures 3.11 -3.16) produced steep displacement curves with high percentage inhibition values at the maximum concentration o f extract, falling within tw o dilutions to values 10% inhibition o f binding. This result may indicate that fractions 2 - 7 were producing a detergent type o f effect causing lysis o f CHO cells at higher concentrations. Fractions 1 and 8 may be causing inhibition o f tritiated bradykinin binding to the BK II receptor in a more specific manner, which can only be verified by com plete characteristion o f these fractions followed by mechanism o f action studies. TABLE 3.19 VALUES FOR FRACTION NUMBERS 1-8 FROM Svmplocos leptophylla IN THE BRADYKININ BK B ASSAY. ...... , • MBHteB) ' < ****# 1 2.0-3.0 2.36 2 9.0-10.0 6.67 3 21.0-22.0 6.08 4 22.5-23.0 3.64 5 26.5-28.0 3.53 6 33.0-34.0 8.93 7 41.0-43.0 7.71 8 57.0-58.5 77.1 Fraction numbers one and eight produced displacement curves which suggested that 159 the impure mixtures of compounds were inhibiting the binding of tritiated bradykinin to the bradykinin BK H receptor in a more specific manner than the mixtures of compounds in fractions two to seven. The IC 50 value for fraction number one was 32.6 times more potent than that of fi*action eight (see Table 3.19). 160 Figures 3.10-3.17 Displacement curves of the partially pure fractions 1-8. isolated from Symplocos leptophylla. Eight fractions were isolated from Symplocos leptophylla which produced an inhibition of binding of tritiated bradykinin binding to the bradykinin BK H receptor. Semi-purification of the eight fractions causing the activity gave similar NMR spectra and had differing retention times. It was concluded that the fractions were mixtures of related compounds. 161 ggj.lO -3.17. Displacmcnt curves of the eight partially separated fractions Nr ^yfttplocos leptophylla in the bradykinin BK II binding assay. Figure 3.13 TTTTT 100 80 80 60 60 o 40 40 a JZc 20 0 S . lllL 1 10 Fraction 1.(ugs/ml) Fraction 4. (ugs/ml) 03.11 Figure 3.14 100 100 60 c .9 .1 £5 40 c 0.1 10 Fraction 2. (ugs/ml) Fraction 5. (ugs/ml) W 3.I2 Figure 3.15 100 80 80 60 c 40 I 20 1 10 0.1 1 10 Fraction 3. (ugs/ml) Fraction 6. (ugs/ml) 162 Figures 3.16 and 3.17. Displacement curves of the eight partially purified fractions from Symplocos leptophylla in the bradykinin BK II assay. Figure 3.16 80 60 40 0 1 10 100 Fraction 7 (ugs/ml) Figure 3.17 80 60 40 asz c 20 0 1 10 100 Fraction 8. (ugs/ml) 163 Figure 3 .18 The HPLC chromatogram of eight bioactive fractions isolated from Svmplocos leptophylla using preparative and analytical C l 8 reverse phase chromatography. Eight fractions with principal peaks having different retention times were isolated from a methanol extract of the stem and bark of the plant Symplocos leptophylla. 164 Symplocos leptopHylla using preparali-ve and analytical chromatography. I . I . I i . I . t . 1 . I . I . I . I . I Rt =41.0 mins | R t = 33.0mms 6 1 Rt = 57.0 mins 1 Rt = 2.0mins Rt = 21.0 mins. 2 Rt = 9.0mins 0\Ul Rt = 20.5 mins Rt = 26.5mins > i I I 3 I ! i I 1 I I : i I ; I 1 ...... A, V__ • I 1 • I • I • I • I • t - r * 1 •* Figures 3.19-3.26 NMR spectra of fractions 1 -8 separated from Symplocos leptophylla. The eight partially purified fractions which were inhibiting the binding of tritiated bradykinin to the bradykinin BK II receptor were analysed using NMR spectroscopy. 166 NPD19940494 B1950/52/1 Corrcnl ÜJIA n.ir,imcii"' NAME NP019940A9 Figure 3.19 SPECTRA 1 EXPNÜ pnocNO FRACTION NUMBER 1 F2 - Acquisition Param D a te 94 0 5 0 - t i n e 9 2 PU LPnoc 4 g J( SOLVENT HeO' AO 3 2 I I 2 I U ' n c 2041 n u c l e u s 11 HLl 1 01 0 JOOPCPf PI e ( OE 7C f SfOI 500 njppoc SWH 10204 TO 65531 NS 64 OS r ^2 - Proces' i.ng o*'“i)‘tet «1 3 c7G ‘; V 500 1300227 MOM CM 5E9 r LO •1 I'O CO ? 1 PC 1 4-3 10 NHP OIO I Ojrafaetî''-- CX 34 v..' F |P 8 500 P2P -0 tCO PP mCm 0 25C0C NZCM 125 r !.;5l iNPDl9940J95 81960/52/2 Figure 3.20 ^ ^ ^MR SPECTRA 2 C u r r e n t Data Parameter* FRACTION NUMBER 2 same NP0199A0490 EXPNO 1 POOCMO 1 f ? • ACQuisition P » r « i e t G a te 940504 T iar 9 S3 “ UCPPOC <933 •iCH.VcNf HeOM AO 3 ^112839 • 9E 1024 NUCLEUS IH 1^1 I 1 01 0 lOOOPCO ■ 8 r 1 oe 70 0 » S fQ l 500 1330000 A S S>#M 10204 08 ^ ro 65536 NS 64 o s r r ; - ProccJSing oa'‘ 1 0 n m o c 191 oa'ûieiî''* .« 34 SO •. f IP S *00 01 r j o -0 200 Cl PPwCM : (5 0 0 0 01 MiCN 125 03250 N. >4 % ><»4yyi r : s / 52 ! Figure 3.21 Ig ^MR SPECTRA 3 . . . r - ç n i n.iirt f’.i'-.iTipif'-'. FRACTION NUMBER 3 sam £ NPO 119^0 «Vl; f IPNO ; nnoCyO 1 • *rnuisil 131 P .ir,i$rt,' O o ie qyo"i04 I l i e 1: k . '•f ' C ■•' . ■•'■CI 1 • ; i r r r :r a'-i !•• ••• :n '*:■ :: « ■ S «■; ■ P r ; l i e . '. , i q v r 5cr yQw ss» , >.n •1 •r ! 1 4C ir> MMn 1 i3i D.i-.neve--. . » > n - r,o - '-.00 n- r^o -0 c’cr ï'j OOMTM r uMc: :r ',ZC" izr- ' 'L"-: ••; y y y f' 7 5 6 ’) 6 n fi 0 1 ", -10 1 1 n i; •> a r t s I r 0 0 NPD19940497 81960/52/4 Figure 3.22 NMR SPECTRA 4 .-rr»ni O.ua Poramete'-s FRACTION NUMBER 4 N*ne NPOI99AOA9/ EtPNO 1 nnoc^iC 1 • * c n u i s i 1tion Par^nei O.UC 94050A I imc 10 48 "in.pno{j :g JO MH.VENI M ePH a O ?. < ! ltî9 2 9 0{, 1024 wUCl Eu S IM ^ 1 l 01 0 iCOOPPO -'1 3 r OE 70 P SfQ I 50P l ’ OOPCP 1 0 2 :4 r e '0 6053b S'j 128 rj . Orcic".'' ,nq oj'àocir 3 2 7 6 “ • r OOP 13PCP90 - 0- 6 “ LO -1 r : : ! =c 1 40 10 NMR o n i n r t 'd K C t f s 3 4 or c ,0 9 OOP fÿC -C 2 0 f oom Cm C c5 0 C f mZCm 125 03 2 5 0 I NPnji,'94349(.' ei9ün/52/5 Figure 3.23 H NMR SPECTRA 5 Current Outa Oaraneter;; name NPOlS9A049fl FRACTION NUMBERS EXPNO 1 PnoCNO I E2 - Acquis il 100 PAnj,,, SAO^OA PüLPnoü I ltgjf or so lv en t Me Oh ? «ÎI12PJ9 4006 ►AÆcEUS IM I 0 lOPOOC.' H f 70 r 50P l3J0P?r 1C2P4 rp e'-s'îc 12“ - Prjif ,ng Od^aoci! 3J7ÔI: 50.' no.'vo^ ÛM •l PCf ? I I 4C 10 NHP 0131 0 4 ''< i e te ''., ?4 s r-cr •Cf c-or ooh;h 0 jr.ûor 125 P J20C NP0Ï994049S 0 1 9 6 0 / 5 2 / 6 Figure 3.24 NMR SPECTRA 6 0.H.1 P.iri>«ctrrs FRACTION NUMBER 6 NAME NPQigsa0499 fXPNO I raocN O 1 F2 - AcoolSilioo Pir»ret* D a te 940504 TIac II 12 p u u p n cb IÇ20 SOLVENT Me OH All 3 2 II2 B 2 9 i no 1024 NUCLEUS IH HLl 1 0 til C 1000000 3 "1 0 0 u Of 70 0 L s f c i 500 1330000 M ;;mh 1 0 2 0 4 .OP H to 6553Ü Nb 126 n:. T ■ urg oaraT.ete'' • 1 327ES 50.' 1.300099 4 wOa CM SS" 0 l O - 1 .0 0 H cn 0 1 PC 1 40 10 nmo 0131 o . - a n e i e r ; LX 3 4 .9 0 c CJP fl 500 01 X2P -0 (0 0 01 PPMCM 0 25000 01 MZCM 125,02250 H, NPD19940500 B1950/52/7 Figure 3.25 NMR FRACTION 7 ob FRACTION NUMBER 7 U uccnl Ooca P.T'ameters EXPNOname NP019940500 I PnoCNO 1 F? • AC nul a,tio n Paramcte O o ie 94 0 5 0 4 I lr.e 1 1 .2 4 PULPPOO 1930 SOLVENT MeOH AO 2 2 1 1 2 8 3 9 s no 1024 NUCLEUS IH HLl 1 0 01 0 1000000 S "I 3 0 V OE 70 0 u S^OI 530 1330000 M ..S 4 H 11 0 2 0 4 .0 9 H “ • TO 65036 NS 129 OS 0 ^2 - PPicesiir.g O A - a n e iî- SI 22769 s r 500 1200090 y A0« GH SS3 0 l P - i . c r - M. 30 0 1 PC 1.4 0 10 NHfl o io c p m 'a c e t c ) 2 4 .0 0 c 8 lOO 01 -0 iOO 0( 0 ^SOOO 01 125 02250 h; T— 0 P Figure 3.26 ^ g NMR FRACTION 8 Current 0«ta Parameter;: NAME NP019940SOI FRACTION NUMBER 8 f.XPMO 1 PflOCNO i F2 - Acqutx it ion Paramctr Date 14010.1 rtme Il 14 PtJLPflOO ig.TO SOLVENT Melwi AQ 3 i!ll28J1 s AC 2048 NUCLEUS IH Kl I Ü 01 0 1000000 4 PI 8 0 u OE 70 0 u SfOI 500 I.1.10000 y SWH 10204.0(1 M ro 65511, NS 64 or. ^2 - Processing naratete" S I 22781 V 500 1100105 H- wow CM 5 5 9 C lB I (?0 H C9 0 I PC I 40 10 NMfl olot pa'am ete'". CX 24 10 ..• f|P 8 500 01 F2P -0 200 0. PPNCM 0 25000 ni M2CN 125 0.1250 M n 7 POn 8 0 7.5 7 0 ti 5 b 0 5 5 5 0 4 5 4 0 1 5 l'o 2^ 0 15 10 0 5 0 0 CHAPTER FOUR DISCUSSION CHAPTER FOUR. 4.1. A DISCUSSION OF THE METHODS AVAH.ABLE FOR SELECTING PLANTS FOR SCREENING FOR ANALGESIC BIOACTIVITY In obtaining plants for biological screening of active principles, the question arises as whether it is more beneficial to screen plants collected on a random basis, or to select plants which have been used ethnopharmacologically for the treatment of a particular disease, or to collect plants which are noted for the production of a particular class of metabolite, e.g. the production of alkaloids by members of the Solanaceae, or to collect plants fi’om distinct geographical areas known for their rich sources of plant species e.g. the Amazonian rain forests. Possible sources for gleaning plant samples include a phytopharmacological approach where medicinal folklore is evaluated for new leads to biologically active compounds (Farnsworth, 1966). Herbal literature and medical botany books contain a wealth of information concerning the physiological appearance of plant species, from which plants may be selected for a particular therapeutic use. Phytochemical tests used to indicate different classes of compounds e.g. terpenoids or alkaloids, present information on the type of secondary metabolites likely to be present in a plant species and these methods may help to increase the likelihood of obtaining new leads to drugs. Some of the most important drugs of the past fifty years or so have been isolated fi"om plant sources, and often fi"om plants which for one purpose or another have been employed in primitive or ancient societies (Schultes, 1986). These drugs include the curare alkaloids, cortisone, reserpine, vincaleucoblastine and the veratrum alkaloids (Schultes, 1976). It has been estimated (Farnsworth et al, 1985) that as much as 74% of the biologically active plant derived compounds presently in use worldwide have been discovered through follow up research to verify the authenticity of information concerning the folk and/or ethnomedical uses of the plants (Farnsworth et al, 1985). In addition to searching medicinal and scientific literature for plants used traditionally as analgesics, a computerised database, Napralert, was also searched. This is a database which provides textual-numeric collections of records regarding the chemistry and pharmacology of natural products and their appropriate taxonomic data. (Loub et al, 1985). It covers the chemistry and biological 175 activities of extracts and/or secondary constituents from identified plants, marine organisms, microbes, and to a lesser extent, animals. Napralert records information which is capable of predicting or rank ordering organisms as to their probability of having specific biological properties if properly investigated. It is a valuable source of information when searching for plants which are used in traditional medicine for a particular pharmacological effect. Ethnopharmacological data on the use of plants was utilised, and plants were screened primarily against a single receptor involved in the mediation of acute pain. The extract was tested further against a number of other receptors involved in the mediation of pain to identify other analgesic effects, and in doing so, hopefully reinforce the data for its traditional analgesic use. Plant extracts exhibiting activity in the bradykinin assay only, were assayed in eighteen other enzyme, receptor and whole cell based assays for other non-related therapeutic effects, e.g. anticancer, antifungal, and antiviral activities, as well as the other analgesic screens of CGRP and Neurokinin 1, to gain a measure of the selectivity and specificity for the bradykinin BK U receptor. Plant extracts with potent, selective bioactivity against a single in vitro receptor (bradykinin BK II) were to be followed up by extracting the bioactive compound(s) responsible for the apparent inhibition of the radiolabelled ligand binding to the receptor. 4.2.DISCUSSION OF RESULTS FROM SCREENING NON-SELECTED AND ETHNOMEDICALLY SELECTED PLANTS FOR NOVEL LEADS TO ANALGESICS Six hundred and thirty five methanol plant extracts were screened in total in the bradykinin BK II and the CGRP radioligand binding assays (Appendix 1, p i98-209). Three hundred of these plants were ethnomedically selected for their use as analgesics in traditional medicine throughout the world, and the remaining 335 were plants which were non-selected. Plants which were classified as non selected were taken from areas which afforded maximum biodiversity within the constraints of cost and availability. A truly random approach is one which blindly collects everything encountered, without giving considerations even to the possibility of duplication. This is not practical and so the approach used in most 176 drug discovery programmes falls between this approach and selecting plants for screening based on an ethnomedical approach (Soejarto, 1993). Plant extracts were screened in the bradykinin and CGRP neuropeptide ligand binding assays to identify novel leads to analgesic compounds. In total, 79 out of 635 plant extracts produced 50% inhibition of binding or greater in the bradykinin BK II assay (Table 3.3), which was deemed to be a positive result. Twenty four of these plants maintained activity after treatment with PVP (Table 3.4). Out of the 79 plant extracts which were active prior to PVP treatment, 48 were ethnomedically selected and 31 were non-selected. After PVP treatment, 2 2 plants which were ethnomedically selected and 2 plants which were non- selected, maintained activity greater than 50% inhibition of binding in the bradykinin BK II binding assay. Serial dilutions were carried out on the most potent methanol plant extracts with positive activity in the bradykinin BK II assay following treatment with PVP (Table 3.5). The plant extracts were Schleffera arboricolor, Citrus aurantium, Juniperus communis, Panax ginseng, Rosmarinus officinalis, Ipomea pes-caprae, Anemmarhena asphodeloides, Barringtonia edulis, Mae sa sp., Pittosporum arbore scens, Erythrina corallodendron and Symplocos leptophylla. The methanol plant extracts which produced greater than 50% inhibition of binding after PVP treatment were screened in the Neurokinin 1 assay which is a further in vitro model of acute pain. Two methanol plant extracts produced potent dose responses in the NKl assay as well as in the bradykinin assay, (Table 3.8) and these were tested in an /w vivo model of acute pain, which was the acetic acid writhing test. Ipomea pes-caprae, family Convovulaceae, produced a decrease in the number of writhes as compared to a control value, and Panax ginseng, family Araliaceae, had no effect on the number of writhes (Results, Chapter 3.0 Table 3.9 and 3.11). These experiments require further detailed replication to ascertain whether the in vivo effects are real when compared to control values. The 22 ethnomedically selected plants with a positive result in the bradykinin BK II assay only were submitted to eighteen enzyme, whole cell and receptor based assays, to gain a measure of selectivity in the bradykinin assay. Eighteen plants maintained activity in the 177 bradykinin screen only, and this interaction was deemed to be selective to the BK2 receptor (Table 3.6) thus, the traditional use for many of these plants which are used for pain relief, was reinforced with this data. The results of three hundred and thirty five non-selected plants screened in the bradykinin BK II assay were contrasted with the plants which were ethnomedically selected, as a means of comparing two different methods for selection for screening plants, and the subsequent likelihood of finding novel chemical templates to analgesic compounds. Out of 335 non- selected plants tested in the bradykinin BK II assay, 22 had activity greater than 50% inhibition, this fell to 2 after treatment with PVP. Therefore there is a ten fold difference between the number of ethnomedically selected plants with activity ( 6 %) and the number of non selected plants active (0.6%) after treatment with PVP. Statistical analysis of the mean values for the non-selected and ethnomedically selected plants indicated that collecting plants which have been ethnomedically selected was significantly more likely to yield plants which are new leads to analgesic compounds. = 8 .6 8 (1 degree of freedom), when p= 0.01 is 6.63. Therefore, as p > 0.01, the null hypothesis which states that there is no advantage gained by selecting plants which have been used ethnomedically for the treatment of pain, is rejected. Thirteen plants had activity greater than 50% inhibition of binding in the CGRP assay prior to treatment with PVP (Table 3.11), and these were all ethnomedically selected plants. After PVP treatment, two plants, Physostigma venenosum and Typhonium giganteum, retained activity which was greater than 50% inhibition of binding (Table 3.12). Serial dilutions of the methanol extracts of these plants were carried out following treatment with PVP (Table 3.13). Of these plants, seven had activity in the CGRP assay only, after screening the methanol extracts which had been treated with PVP in 18 other cell, enzyme and receptor assays(Table 3.15). Symplocos leptophylla, family Symplococaceae, produced the most potent and selective inhibition of binding of the bradykinin BK II receptor (Table 3.5) and was selected to elucidate the compound(s) responsible for the bioactivity. The active fractions were initially identified using C l 8 bond elut columns (Table 3.7) and displacement 178 studies were carried out on the final eight bioactive fi-actions which were eluted using reverse phase analytical columns (Figures 3.10-3.17, pl62-163). A large number of plants which were ethnomedically selected and screened in the bradykinin BK II assay either did not produce potent, selective effects in this screen, or the methanol extract produced an inhibition of binding greater than 50% prior to the addition of PVP. The value of percentage inhibition after treatment with PVP subsequently decreased, suggesting the initial positive result was due to tannins binding non-specifically to proteins. Examples of such plants were Stephania tetrcmdra, family Menispermaceae, (Table 3.4) which is used in Traditional Chinese Medicine (TCM) as a sedative, a tranquiliser and an analgesic (Anonymous, 1975). Sinomenium acutum^ also fi*om the Menispermaceae family contains the alkaloid sinomenine (Peigen, 1980)and is used as an analgesic and sedative in TCM. The methanol extract of this plant produced a negative result in the bradykinin assay, and would suggest mediation of its analgesic effects was not via the BK II receptor. The use of PVP to remove phenolic components in methanol extracts is questionable, as it is possible that non-phenolic compounds may also be removed, or phenolic components which are novel and thus require characterisation. However, when trying to identify and isolate antagonists to the bradykinin BK II receptor, the removal of as high a percentage of phenolic compounds as possible was required in an effort to identify a compound with specific and selective activity to the bradykinin BK II receptor. A number of reasons explain why a plant extract which initially produced a positive result in a binding assay may subsequently produce a negative result. Tannins exert an inhibitory effect on many enzymes and PVP is used to adsorb polyphenols and tannins from plant extracts (Loomis et al, 1966). The procedure relies on the formation of hydrogen bonds between phenolic groups present in the molecule and the amide groups of the PVP, and many of the methanol plant extracts in Table 3.3 subsequently became negative due to the adsorption of polyphenolic compounds by PVP, thus the initial positive effect was due to non specific binding effects of tannins. The methanol extracts of Aesculus hippocastanum and Aesculus marylcmdicus, family Hippocastanaceae, both inhibited the binding of tritiated 179 bradykinin to the BK II receptor (see Table 3.3 and 3.4). This species of plants are known to contain high concentrations of the secondary metabolites saponins which lyse cells at high concentrations. Thus, the activity of these two methanol extracts in the bradykinin assay could be attributed to saponins, as the CHO cells, when viewed in the 96 well plate used for the assay, were seen to lyse as the plant extracts were added. Parallel testing of methanol at the same concentration used for extracting the dried plant samples did not produce lysis of the CHO cells. Thus, this type of inhibition of binding is not specific to the bradykinin BK ü receptor and the extracts of these plants would not be suitable for further investigation as a potential lead molecules for analgesic drugs. Two methanol plant extracts. Acacia auriculiformis and Croton tiglium, produced inhibition of tritiated bradykinin binding to the BK II receptor (Table 3.3 and 3.4) but there was inadequate amounts of plant sample available to follow up investigations into their bioactivity, thus displacement curves in the bradykinin BK II assay were not carried out. Investigation into plant materials can be associated with a number of problems which may prevent the conclusive characterisation of bioactive compounds. As mentioned in the introduction, (Section 1.16), there may be variation in the amounts of a particular metabolite in a plant depending on the time of year it is collected, or in the geographical region in which it was collected fi*om. The initial investigations in to Acacia auriculiformis were on samples collected in Australia. A subsequent collection fi*om Singapore (when the amount of dried material available was low) did not yield the same bioactivites (unpublished observations), thus, as a second sample of material fi’om Australia was unavailable, investigations into this plant were halted. Levels of particular metabolites may vary within a plant in different organs, so if, for example the activity of a metabolite was identified from the leaf, and only seeds were available for fijrther analysis, the same degree of potency, and occurrence would possibly not be found. Physostigma venenosum, family Leguminosae, is used to treat individuals with senile dementia and headaches in the Northwest Amazon (Schultes 1993). The methanol extract produced greater than 50% inhibition of binding in the CGRP assay and produced a dose- 180 dependent response (Table 3.13). Percentage inhibition of binding was slightly decreased after treatment with PVP (Table 3.14). The indolecholinergic alkaloid physostigmine is commercially available and a full dose response in the CGRP assay indicated the activity of the methanol extract of Physostigma venenosum was not due to the prescence of this alkaloid (Table 3.15). Typhonium giganteum, family Araceae, is used in a decoction with Pinellia temata, Arisaema consanguineum and Buthus martensi for the treatment of headaches (Anonymous, 1975). Many TCM remedies involve a number of plants used together in a single recipe. The combination of extracts may synergise to produce a particular pharmacological effect which is not apparent if the plants are used individually for the same effect. The extract of Typhonium giganteum maintained activity in the CGRP assay and had a dose dependent response after treatment with PVP (Table 3.13). The observed activities of Physostigma venenosum and Typhonium giganteum in the CGRP in vitro binding assay reinforce the data on their use in traditional medicine for the relief of pain. This preliminary data suggests that ethnomedical selection of plants significantly improved the outcome of obtaining a novel ligand to new analgesic compounds. 4.3.NON PEPTIDE BRADYKININ ANTAGONISTS The methanol extract of Symplocos leptophylla produced the most potent and selective bioactivity in the bradykinin assay (Table 3.4) and was, therefore, chosen to isolate the biochemical compound(s) in the methanol extract which inhibited the binding of tritiated bradykinin to the BK II receptor. The development of non-peptide bradykinin receptor antagonists which retain the pharmacological profile of active peptide compounds with a greater chance of retaining their activity after oral administration are required. A number of compounds of plant origin have been screened for bradykinin antagonistic activity including the flavonoids apiin, khellin, quercetin and escuhn (Calixto et al, 1990). Khellin produced a competitive type of inhibition, apiin and hesperetin affected responses to the angiotensin II enzyme and eledosin. The biflavonoids amentoflavone, extracted from Ginko biloba and cupressuflavone extracted from Cupressus tarubosa antagonised bradykinin action in the guinea pig ileum 181 (Ramaswamy, 1970). The flavonoids 0-(B-hydroxyethyl) rutin and vitexin were also reported to have anti-bradykinin BK II activity (Barbor, 1979). Jatrophine, a diterpene extracted from Jatropha elliptica exhibited some antagonism to bradykinin-induced contractions of the rat uterus and guinea pig ileum, in a non-competitive manner (Calixto et al, 1987). The saponin aescin, from Hippocastanum sp. exhibited functional antagonistic activity against the bradykinin induced increase in vascular permeability (Vogel, 1971). As the majority of these effects are non-competitive and non-selective, they may be exhibiting antagonistic properties due to the interaction with one of the many intracellular transduction mechanisms of bradykinin effects, rather than a direct interaction with the bradykinin receptor. Blockage of protein kinase C activation, inhibition of calcium mobilisation, eicosanoid function or phospholipase A interaction would all prevent the cellular mediated effects of bradykinin but would not involve inhibition of the receptor. To confirm the mode of action of these plant-derived compounds, detailed studies need to be pursued to investigate the secondary messenger transduction system through which inhibition of binding is being mediated e.g. the flavonoid quercetin has been shown to modulate protein kinase C activation in rat brain and pig thyroid (Picq et al, 1989). A plant extract with anti-bradykinin activity under investigation by Calixto et al (1985) is that ofMandevilla velutina, from the Apocynaceae family. Folk medicine in Brazil prescribes the use of infusions or alcoholic extracts of the rhizomes of the Mandevilla plant for the treatment of venomous snakes, including that of Borthops jaracara. Calixto (1986) tested the plant extracts for anti-bradykinin activity in the rat uterus. Research has resulted in the isolation of several crystallised compounds which selectively antagonise bradykinin induced contractions in the rat uterus (Calixto, 1985). These are the first reports of a selective bradykinin antagonist of plant origin. The crude methanol extract selectively and competitively inhibited bradykinin induced contraction of rat uterus and blocked bradykinin action at the BK II receptor(Calixto et al, 1986). Oral administration of the extract inhibited carrageenan induced paw edema and pleurisy (1987). The compounds inhibiting bradykinin 182 activity were reported to be terpene glycosides(Calixto et al, 1988). The isolation and subsequent characterisation of compound(s)from Symplocos leptophylla producing positive activity in the bradykinin in vitro binding assay would permit greater insight into the structural configuration of compounds with anti-bradykinin activity. 4.4.THE PARTIAL PURIFICATION OF SYMPLOCOS LEPTOPHYLLA The HPLC techniques used for the elucidation of bioactive compounds fi’om Symplocos leptophylla used different HPLC columns which were essentially the same but which were used for different sized samples. The analytical C l 8 Solid Phase Bond Elut cartridges contained octadecasilyl bonded silica gel. This sorbent is a high capacity 40|i particle size which affords highly selective isolations. Compounds with non-polar functional groups e.g. alkyl chains and aromatic rings bind strongly to the sorbent from aqueous solution and can be eluted selectively by stepwise gradient elution using increasing concentrations of solvents such as acetonitrile or methanol in water. Following the identification of the active fractions, further purification was achieved using either C18 bonded silica columns or columns containing cross-linked styrene-divinyl benzene co-polymers. This chromatography is essentially the same as solid phase extraction except that a smaller particle size is used, typically 5|i diameter which leads to more rapid equdibriation times which in turn allow higher flow rates to be used. The Whatman P40 sorbent which was used for scaling up the amount of active plant material was similar to the Cl 8 packing (40p diameter) and allows the processing of larger samples. Sephadex LH20, used in the penultimate stage of the semi-purification of S. leptophylla, is a bead-formed dextran gel in which chains are cross linked to give a three dimensional polysaccharide network. The introduction of hydroxypropyl groups, which attach ether linkages to glucose or to units of the dextran chain, increase the ratio of carbon to hydroxyl 183 groups. This produces a gel which then has both hydrophilic and lipophilic properties. The technique of AMD was used to develop silica gel HPTLC plates using a solvent gradient. The TLC plate was first developed for a short distance using a polar solvent system, and was then developed over successively greater distances using less polar solvent systems. This process reduces band broadening, leading to tightly focussed zones and enables compounds of widely different polarities to be separated in the same chromatographic run. Thin layer Chromatography of the eight colourless fi'actions, which all absorbed in the U.V. region at 210nm, gave a positive result with Vanillin-Phosphoric acid (VPA) spray reagent (see Methods 2.21). Spraying spots of the fi-actions with Dragendorffs reagent tested negative for the presence of alkaloids. A positive result with VPA was observed with the development of blue-green coloured spots. This indicated the presence of terpenoid compounds, and together with the mass spectral and NMR data suggested the compounds with activity in the bradykinin BK II assay were triterpenoid in nature. Thus, a number of similar chromatographic techniques were used in an effort to isolate the fi-actions causing bioactivity in the bradykinin BK II assay. The very low weight of material isolated fi-om Symplocos leptophylla precluded further spectroscopic investigation and it was not possible to characterise and identify the active compound(s). A comprehensive search carried out using Napralert (Farnsworth, 1994) did not reveal any references to the anti-bradykinin activity of Symplocos leptophylla. Thus these results show new information on the bioactivity of the stem and bark of this plant. 184 Table 4.1. DOCUMENTED PHYTOCHEMTSTRY OF THE SYMPLOCOS SPECIES. Species Class of Compound Identity Reference Symplocos celastrinea Isoquinoline alkaloid Caaverine Tschesche et al 1964 S.confusa Flavonoid Confusoside Tanaka et al, 1982 S.glauca Iridoid glycoside Verbenalin lida et al, 1990 Iridoid glycoside 6-dihydro- Verbenalin SJancifolia Flavonoid Phlorizin Mancini et al, 1979 S, microcalyx Flavonoid Confusoside Tanaka et al, 1982 Flavonoid Trilobatin S.racemosa Triterpene Leucopel- Heller et al, 1985 argonidin-3 -glycoside Triterpene 12,18-tarax Ali et al, 1990- astadien 3,28,diol Triterpene 12-olean- ene-3,24-diol Triterpene 24-hydroxy- Tanaka et al, 1988 12-oleanene- 3-one Triterpene 28-hydroxy- Ali et al, 1983 20x-urs,12,18 19)-dien-3-b-yl- acetate S.spicata Terpenoid Alpha-spino Frotan et al, 1983 -sterol Flavonoid 3)28-bis-|3- D -glucopyr- Higuchi et al, 1982 anosyl Flavonoid 3jO-P-D- Higuchi et al, 1982 glucopyran - oside S spicata Flavonoid 3-0-[P-D]- Tiwari et al, 1976 galactopyr- P-D-galacto- SMnctoria Carbohydrate D-fructose Carbohydrate Galacturonic acid Carbohydrate D-glucose Carbohydrate Glucuronic acid Carbohydrate Mannose S. uniflora Flavonoid Symplocoside Tschesche et al, 1980 Symplocos spp. Alkaloid Isoboldine Chan et al, 1966 185 One of the first steps in the structural determination of an unknown substance is the establishment of its empirical formula and molecular weight. For the eight individual fractions isolated from Symplocos leptophylla (Tables 3.18 and 3.19), sufficient purified material was not available to reach a full conclusion on the identity of the compound(s) producing the inhibition of binding in the bradykinin BK II assay. However, speculative ideas about the nature of the bioactive compounds in S.leptophylla may be put forward from observations on the mass spectral, NMR data and the retention times of the individual fractions. The evidence collected in this study suggests that the bradykinin BK II antagonistic activity is not due to any of the previous reported metabolites isolated from Symplocos (Table 4.1) The NMR spectra of fractions 1 to 8 are very similar and the chemical shifts of the molecular groups in spectra 1 also apply to those in fractions 2 to 8 . Fraction 8 appeared to be the cleanest fraction and produced the strongest signals. From the NMR spectra of fraction 8 the following chemical signals were detected : methyl groups, CH 3 at Ô0.7-Ô1.4, CH2 groups at 51.5 to 62.2, 0 =C-CH3 groups at Ô2.5-Ô3.0 and CH-0 groups at Ô3.5-Ô4.5, C=CH groups at Ô4.8-54.9 and aromatic proton signals at 56.7-56.8. From the integrals on the NMR spectra of fraction 8 the ratio of CH-0 groups to CH 3 groups was 1 : 3 indicating the presence of one sugar group to three methyl groups. The presence of large numbers of methyl groups suggests compounds which are triterpenoid in character. The results of the TOF, FAB mass spectral analysis, and retention times of fractions 1 to 8 are in Table 3.10, pl39 (see Figures 3.19 -3.26 for the ^H NMR spectra, and Appendices IV, p215-216 for mass spectral results.) The HPLC chromatograms of fractions 1-8 which were eluted from Symplocos leptophylla, using C18 analytical columns, are in Appendix II, for the mass spectral data of fractions 1 -8 see Appendices IV. The TOF mass spectrum of fraction 1 produced three [M]'*’ ions at m/z 782, 1260 and 1502 (see Table 3.10). These values were not substaniated by the FAB mass spectrum which gave prominent ions at m/z 506, 1237 and 1375. The conclusion from these results is that fraction 1 is a mixture of several samples. The presence of [M]"*" ions at m/z 186 1140, 1254, 1379 and 2534 produced in the TOF of fraction 2 were not substantiated by the ions produced in FAB at m/z 1095, 1259 and 1301. Thus, fraction 2 is also a mixture of several compounds. The TOF data produced from fractions 3 - 8 also indicated that these fractions were also a mixture of compounds. The FAB spectra of fractions 3 - 7 did not yield sufficient data to produce conclusive m/z ions. To summarise, the data obtained from the mass spectral analysis of fractions 1 to 8 indicates that all fractions are a mixture of samples. The NMR traces are very similar but the retention times of Fl to F 8 are all different, suggesting that fractions 1 to 8 are mixtures of related compounds. If the molecular weights of these compounds are in the region of 1200, (as suggested by the mass spectral data) there are three possible common groups of plant metabolites to which they could belong to, namely saponins, polysaccharides, or polypeptides. If the compounds were polypeptides, one would expect to observe functional groups with aromatic character in the region of 56.5- 57.0, and these are present in the NMR spectrum of fractions one to eight. The presence of polypeptide or amino acids could be confirmed by the application of ninhydrin spray to the fractions on a TLC plate. Larger polysaccharides with molecular weights in the region of 1 0 0 0 -1 2 0 0 do not contain aromatic protons and hence it is unlikely that these compounds are polysaccharides. The observation of frothing while preparing methanol extracts of Symplocos leptophylla for HPLC suggested the presence of saponins. These compounds consist of sugars linked to a triterpene or steroid aglycone. The presence of many methyl groups in the region of 50.9- Ô1.3 (see NMR traces 1-8, p i67-174) suggest the compounds may be triterpenoid saponins. The presence of triterpenoid saponins were noted in S.spicata (Higuchi, 1982; Ali, 1990), and the molecular weights were 898 and 452 respectively (Ali, 1990). Further observation on the possible identity of the compound(s) causing inhibition of binding in the bradykinin BK II assay is the nature of the displacement curves of fractions 1 to 8 (Figures 3.10 - 3.17, p i62-163) in the bradykinin BK II assay. Fractions 2-7 produced very steep displacement curves when the extract was serially diluted in the bradykinin assay. 187 This may suggest that fractions 2 - 7 are producing a detergent type effect which causes lysis of the CHO cells at the highest concentration and little or no cell lysis at the lowest concentration. Fraction numbers 1 and 8 have produced slightly different displacement curves in that when serially diluted in the bradykinin assay the values for percentage inhibition appear to have a dilution - dependent effect. Fractions 1 and 8 may be interacting in a more specific manner, rather than causing lysis of the CHO cells. From the data on the semi-purification of the methanol extract of Symplocos leptophylla it is possible that the compounds which have caused inhibition of tritiated bradykinin binding to the BK II receptor are triterpenoid saponins in nature. 4.5 NON SELECTIVE ANALGESIC EFFECTS OF PLANTS USED IN TRADITIONAL MEDICINE: A DISCUSSION OF IN VIVO RESULTS. The methanol extracts of Ipomea pes-caprae^ family Convovulaceae, and Panax ginseng, family Araliaceae, inhibited binding of tritiated bradykinin to the BK II receptor and Substance P to the Neurokinin 1 receptor. The interactions with the BK II receptor were thus non selective, but were interesting in that both plants were selected from ethnomedical literature for their use in pain relief and both were effective in two in vitro models of pain, therefore it is possible that they may have some broad based analgesic activity. Ipomea pes-caprae has been documented for its use in the treatment of neuralgia and arthritis and is used externally for rheumatism in the form of a decoction and internally as a colic in Afiican traditional medicine (Christensen et al, 1983). This plant is known to contain a volatile oil, resin and a phytosterol. Different parts of Ipomea pes-caprae were used in traditional medicine to treat rheumatoid arthritis and diverse skin lesions (Perry and Metzger, 1980). A clinical study carried out in Bangkok where patients with varying degrees of skin involvement after exposure to toxic jellyfish showed that a 1% ointment based upon the IP A extract of Ipomea pes-caprae was clinically effective in the relief of pain or inflammation. The extract was obtained through 188 petroleum-ether extracts of the water distillate from steam distillation of dry ground leaves (Sunthonpalin and Wasuwat, 1985). Bohlin et al (1993) showed that the IPA extract of Ipomea pes-caprae was able to neutralize the proteolytic and haemolytic activities of several jellyfish toxins. The IPA extract showed a reversible, concentration-dependant inhibition of contractions in guinea-pig ileum induced by histamine, acetylcholine, bradykinin and barium chloride. Isoprenoids, diastereomeric actinidols and beta-damascenone were isolated from Ipomea pes- caprae and were found to possess anti-inflammatory effects. Panax ginseng is documented for its use as an analgesic in CTM (Chang and Butt 1986). Ginsenosides derived from this plant have a wide array of pharmacological activities and because of the manner of use, are known as ‘Adaptogens’. Adaptogens are general well being drugs and are used to stimulate a state of unspecific resistance towards stress factors of different kinds, maintaining the body stamina at a regular level without detrimental side effects. Saponins termed ginsenosides have been extracted from the root, stem, leaves and flowers of P.ginseng. Ginseng is also prescribed as a tonic, stimulant and aphrodisiac and is incorporated into tonics for the treatment of amnesia, headaches, convulsions and dysentry. The acetic acid writhing test is a simple test to carry out and is sensitive to all known clinically useful analgesics (Wood, 1984). Analgesic activity of a compound or extract is deemed positive if either the latency to the first writhe is prolonged or the frequency of writhing is reduced. The extract of Lpes-caprae reduced the number of writhes as compared to control values using acetic acid (Table 3.9, p i36). P.ginseng did not significantly affect the number of writhes, as compared to the control values. Many investigators have described the non-specificty of this test as drugs not commonly considered to be analgesics have been effective (Emele et al, 1961; Hendershot et al, 1969) and the 189 writhing test does not distinguish between narcotic and non-narcotic analgesic drugs. However, it is possible to determine whether or not broad based analgesic effects are produced, as observed with the positive effect of Lpes-caprae and the negative effect of P.ginseng. To further substantiate the possibility that I.pes-caprae does have some degree of analgesic action, more specific tests on concentrated extracts need to be investigated using in vivo tests that are designed to screen non-narcotic drugs to reinforce the data obtained from the acetic acid writhing test, such as the rat tail flick technique, which is a more applicable method for measuring acute pain (Duboisson et al, 1977) and the formalin test (Amodei, 1980) which are more specific models for identifying analgesic activity. 4.6. CONSIDERATIONS OF THE DIFFICULTIES ENCOUNTERED IN BIOASSAY GUIDED FRACTIONATION OF PLANT EXTRACTS. A number of problems may occur when using bioassay guided fractionation of plant extracts to identify lead molecules to new drugs. The successful isolation of compounds which show bioactivity in a receptor binding assay relies on the reproducibility and consistency of the developed assay. The initial bradykinin BK II radioligand binding assay utilised a membrane preparation. The source of membranes was from female rat uteri which expressed the BK II receptor (Method 2 .2 ), however a number of problems arose when routinely using the membranes for screening methanol plant extracts. There was poor replication for the percentage inhibition of binding results of identical methanol plant extracts and so the results could not be relied upon to have any real value (see Results, Table 3.1, p95). The activity of the uteri membrane preparation which expressed the bradykinin BK II receptor was decreased after thawing following 190 storage at -20°C. A decrease in activity of approximately 10% is expected when using membranes which are frozen, however the frozen rat uterus membranes were only 50% active as compared to freshly prepared rat membranes (unpublished observations). The consistency of uteri membranes was generally ‘lumpy’ which would account for the irreproduceability of results. The Scatchard plot, which indicates the number of binding sites present in a cell hne or membrane preparation, was not conclusive for the bradykinin receptor expressed in rat uteri (see Appendix V, p217) in that the plot did not produce data which pertained to one or more binding sites after repeated experiments. For these reasons the bradykinin BK II assay was redeveloped using the recombinant Chinese Hamster Ovary cell line which expressed the bradykinin BK II receptor. This low throughput screen was then used to test methanol plant extracts and to isolate the bioactivity of Symplocos leptophylla. Variation occurs from assay to assay but there has to be limits within the parameters being measured, to ascertain that a bioactive compound is being isolated and not an artefact in the assay, or an artefact from the HPLC column which is being used to effect seperations. Retention times may shift slightly as a result of slight changes in experimental conditions and the assay has to be able to reflect this change in time. If changes in the retention time of the bioactive components do occur, but are not detected, the assay is not a reliable indicator of the bioactivity. Reasons such as loss of the receptor, death of the cells or contamination of the cells by mycoplasma, bacteria or fungal sources could cause misinterpretation of results. The value which signifies a positive result often varies from assay to assay. A radioligand binding assay which produces many positive results if the value of a positive result is 50% is made more selective by increasing the value of a positive result to 60% or higher. In this way it is hoped that compounds or extracts are identified which are more selective to the receptor, cell or enzyme in which the compound is being screened. Conversely, an assay 191 which identifies very few bioactive compounds may have the value which is deemed to be a positive result decreased, to increase the likelihood of obtaining new leads to drug molecules e.g. from 50% inhibition of binding to 40% or less. Experimental variation occurs from assay to assay e.g. the initial value produced by a methanol plant extract, if deemed to be a positive result with a value just over 50% inhibition of binding, due to experimental variation, may produce a value slightly below 50% inhibition of binding in a replicate assay. Thus this extract would not be viewed as having positive activity in the binding assay. Methanol extracts which consistently produce over 50% inhibition of binding after replicate assays are deemed to be positive results. Experimental variation within 10% of an initial inhibition value is to be expected, however values which differ by more than 10% indicate the radioligand binding assay is not producing valid data. Thus, a methanol extract may produce similar or identical values for inhibition of binding with repeated assays, but a level exists where variation from assay to assay is not acceptable. The choice of solvent used for extracting dried plant samples into may also influence the diversity of plant metabolites in the extract. Polyphenolic compounds are insoluble in chloroform, therefore the use of this solvent would possibly reduce the number of ‘false’ positives identified in the assay, however, chloroform is incompatible with the 96 well assay plates used, causing the plate to dissolve. This problem could be overcome by evaporating the chloroform extract to dryness and resuspending it in methanol. The use of PVP to adsorb polyphenolic compounds from methanol plant extracts is a matter of preference when screening plant extracts in high or low throughput assays. The use of this compound may remove polyphenolic compounds which are producing effects which are selective to the receptor to which new compounds are being sought. However, the advantage of removing the majority of compounds which are producing non - specific effects, thus reducing the likelihood of pursuing the isolation of a non - specific compound into isolation, is thought to outweigh the disadvantages. The receptor tissue available for developing a receptor ligand binding assay will also affect the outcome of results, for example, a receptor which is expressed in in vivo tissues and is 192 used to produce a membrane preparation to express the receptor in in vitro may behave differently to receptors which are genetically engineered and expressed in recombinant cell lines. To ensure the receptor is expressed adequately in in vivo or in vitro, control compounds are used to ensure the assay has a high level of replication and consistency, as shown by carrying out a serial dilution curve with a standard antagonist compound known to selectively inhibit the binding of ligand to receptor, as well as using the endogenous ligand which binds to the receptor in vivo. Plant extracts screened in receptor radio-ligand binding assays possibly contain several hundred different compounds and identification of a single compound can be very difficult. In common gradient-elution techniques using reverse phase HPLC with standard columns of 5- lOum, typical available peak capacities are about 50-100 (Dondi et al, 1986). As uncontrolled peak overlapping always occurs and as part of the chromatographic space may not be completely available for sample separation, complete resolution can be difficult to achieve. The chromatograms of plant materials often have complex appearances with no quantitative measure of the extent of separation or of individual peak purity. Therefore, when using bioassay guided fractionation techniques, an error margin has to be set and maintained to ensure that only the bioactive compound of interest is isolated in as pure a form as possible, and this can only be acieved when the assay used to identify the active 'peaks' or fractions is consistent, reliable and reproduceable. 4.7. FINAL CONCLUSIONS This research work has identified different aspects of searching for novel leads to new drugs with analgesic action. With regard to the number of plants which were selected for testing because of their documented use as analgesics, many of them did have some degree of activity in in vitro binding assays which substantiated the claims made for these plants. Many radioligand assays are potentially able to screen thousands of samples per year because of their efficiency, reliability and the short time periods for performing the assay through to the analysis of results. This type of assay allows the collection of detailed data on the selectivity and potency of a plant in many screens with differing therapeutic targets, thus they are quick 193 and reliable methods for screening large numbers of samples. However, one must also realise that this type of screening may overlook less potent or less selective biologically active plant extracts which are interesting for reasons of novelty or occurrence, but which do not fit the criteria for which the screen was developed. One step on from identifying in vitro activity is to continue with an in vivo model of the disorder being investigated. In vivo tests aim to confirm the original activity of a plant extract and the range of in vivo methods available also makes the identification of a bioactive extract or compound quick, easily accessible and relatively inexpensive for gathering large amounts of information. With any type of research, difficulties may be encountered which may delay reaching a conclusion. With research involving plants there is also concern for the dissapearance of many species due to widespread deforestation in tropical, and to a lesser extent, temperate areas, before the first steps can be taken to identify a particular plant and its pharmacological effects. Research into the elucidation of lead molecules to new drugs from natural sources such as plants requires that strict standards should be adherred to (see Introduction 1.14, 1.16). Once considered a renewable resource, tropical moist rain forests are now a non renewable source through an increase in human pressure (Gomez et al, 1972) and it is possibly only a matter of time before temperate species of plants and trees become extinct. With the dissappearance of tropical moist forests, human cultures that have developed within them, and the traditions and knowledge concerning medicinally useful plants also dissappears before there is a chance to learn fi*om them. The areas to suffer the greatest losses due to deforestation are the tropics where medicinal plants are most important to daily health. The tropics contain the largest number of species per unit area and tropical plants display huge variability in their genetic make up e.g. the biochemical compounds they produce. The importance of ethnobotanical inquiry as a cost-effective means of locating new and useful tropical plant compounds cannot be over emphasised as as mentioned previously, 74% of the 121 pure chemical substances extracted fi’om higher plants used in medicines throughout the world had the same or a related use as the plants fi*om which they were 194 derived. High throughput screening (HTS) of plant extracts is an extremely useful method for screening large numbers of samples quickly and effectively. High and low throughput screens can be used in conjunction with ethnobotanical literature as a means of identifying particuarly usefiil plants, where it is possible to produce lists of hundreds of plants (as opposed to thousands for HTS) to substantiate a particular plant use for screening in this type of assay. With the vast disappearance of thousands of plant species it is imperative that as many of the plant species as possible which are available for collection, are screened quickly in assays. By doing this, it is not only possible to identify novel leads to new drug compounds for many different therapeutic disorders but also by doing this, the documentaion on many more plant species is increased before they become extinct. 4.8. FUTURE WORK The methanol plant extracts of Symplocos leptophylla and Ipomea pes-caprae have produced some degree of bioactivity in the bradykinin BK II assay and many questions are raised due to their pharmacological activités, which would be interesting to follow up, in order to produce some conclusive answers. To achieve a complete characterisation of the compounds from Symplocos leptophylla which are producing inhibition of binding in the bradykinin assay, a weight of dried plant material not less than two kilogrammes would be required. As the solvent system for isolating these compounds was elucidated, the identification of the compound(s) producing inhibition of tritiated bradykinin binding to the BK II receptor should be straightforward in the future. Following the isolation of the compounds responsible for producing the inhibition of tritiated bradykinin binding to the BK II receptor, the molecular weights of the compounds and their 1 13 structures (determined from mass spectral, H NMR and NMR spectroscopy) would be screened against all other known compounds to verify whether the metabolites were already documented or were novel structures. The in vitro and in vivo bioactivity of Ipomea pes- caprae is also intriguing as to what type 195 of compounds are causing this apparent activity, and how the in vivo effects are mediated. Replication of the experiments performed here along with bioassay guided fractionation and parallel testing of the active fractions in in vivo assays should result in the isolation of the bioactive metabolites in this plant extract. When further in vitro assays are developed to target particular secondary messenger systems involved in the mediation of pain by bradykinin e.g. arachidonic acid, phospholipase A2, eicosanoids or epidermal derived releasing factors (EDRF's), the structure-activity relationship of these compounds and how they are producing an inhibition of bradykinin binding to the BK II receptor may be identified. The plant extracts with selective activity in the CGRP assay require the identification of a solvent system for the isolation of bioactive compounds. If the activity of these extracts remains potent and reproduceable in the CGRP assay, complete characterisation should be completed and in vivo tests identified to investigate fiirther the effects of known or novel t ligands present in the plant extract. 196 APPENDIXES APPENDIX I A Key for Identifying the Part of the Plant Used for Screening in the BK n. CGRP and NKl Assays. BL Bulb WF Whole Plant and Flower BK Bark WP Whole Plant CM Micropropogation TW Twig FF Flower and Fruit FL Flower FR Fruit HB Herb LF Leaf LS Leaf and Stem LT Leaf, Flower and Stem ND Needles RC Root and Cortex RH Rhizome RS Root and Stem RT Root RW Root and Wood SB Stem and Bark SG Style and Stigma ss Stem, Seed and Leaf ST Stem SD Seed SF Seed and Flower SH Seed Husk TB Tuber TL Twig and Leaf 197 APPENDIX I The ethnomedicallv selected and non-selected plant species which were screened in the bradvkiilin BK II and the CGRP radioligand receptor binding assays. Species Genus Family Geosraphic Plant R/T Location part used. Abelia x grandi/lora Caprifoliaceae China LF R A belia triflora Caprifotiaceae China LS R Abutilon indica M alvaceae China WP T Acacia auricuUformis Leguminosae Australia SH T Acacia farnesiana Leguminosae Middle America SD T Acacia longifotia Leguminosae AustraUa LS T Acanthopanax senticosus AraUaceae China RT T Achillea millefolium Compositae Europe SD T Achyranthes bidentata Amaranthaceae Arabia RT T Aconitum carmichaeli Ranunculaceae China RT T Aconitum napellus Ranunculaceae China SD T Acorus calamus Araceae India RT T Actinidia chinensis Actinidiaceae China LS R Actinidia ploygama Actinidiaceae China ST R Adenodolichos paniculatum Papilionaceae A frica WP R Aesculus glabra Hippocastanaceae India LF R Aesculus glaucescens Hippocastanaceae India LF R Aesculus hippocastanum Hippocastanaceae India LF R Aesculus hybrida Hippocastanaceae India LF R Aesculus mdica Hippocastanaceae India LF R Aesculus marylandica Hippocastanaceae India FL R Aesculus neglecta Hippocastanaceae India FL R Aesculus parviflora Hippocastanaceae India LF R Aesculus pavia Hippocastanaceae India LF R Aesculus sylvatica Hippocastanaceae India LF R Aesculus turbinata Hippocastanaceae India LF R Afrormosia laxiflora FabaceaelVest A frica SD T Agapanthus praecox LiUaceae A frica SD R Ageratum conyzoides Compositae Middle America WP T Ageratum houstonianum Compositae AustraUa WP T Agrimoniadorata Rosaceae Europe WP R Agrimonia pilosa Rosaceae China HB T Akebia quinata Lardizabalaceae A sia ST T Alchomea latifotia Euphorbiaceae West Africa SD T Aletris farinosa LiUaceae North America SD T Allium albopilosum LiUaceae Europe SD R AUium christophii LiUaceae Europe BL R Allium giganteum LiUaceae Europe BL R AUium sativum LiUaceae West indies SD T AUium sepa LiUaceae West Africa SD T AUoplectus tetragous Gesneriaceae Am erica LF T A loe ferox LiUaceae Africa FR T A loe vera LiUaceae Africa WP R A loysia triphyUa Verbenaceae Middle America SD T Alpinia officinarum Zingiberaceae China RT T Alstonia boonei Apocynaceae West Africa LF T Ambrosia peruviana Compositae Middle America SD R Althaea officinalis M alvaceae Europe RT R Key : RiRandomly Collected T:Targeted 198 Species Genus Familv Geoeranhic Location Plant R/T part used Anacardium occidentale Anacardiaceae North Africa BKT Ananas contosus Bromeliaceae Am erica SFT Anaphalis margaritacea Compositae North America SDT Anchomanes difformic Araceae A frica TB T An^ographis paniculata Acanthaceae India LFT Anemarrhena asphodeloides LiUaceae China RH T Anemone blanda Ranunculaceae China BL R Angelica dahurica Umbelltferae Europe RT T Angelica sinensis UmbeUiferae Europe RT T Angelonia angustifolia Scrophulariaceae Middle America SD T Annona muracata Anonaceae Africa LFT Antignon leptopus Polygalaceae Am erica SD R Aphanes arvensis Rosaceae Europe WP R Apium graveolens UmbeUiferae Middle America SD T Aquilaria sinensis Thymelaceaceae China SD T Arbutus unendo Ericaceae South Europe WP T Arctostaphylos uva-ursi Ericaceae North America LF R Areca catechu Palmae AustraUa FR T Arisaema consangmneum Araceae China TB T Aristolochia bottae Aristolochiaceae Am erica SD T Aristolochia debilis Aristolochiaceae America RT T Aristolochia elegans Aristolochiaceae Am erica LTR Aristolochia odoratissima Aristolochiaceae Middle America SDT Arnica chamissonis Compositae Europe WP R Arnica montana Compositae Europe SD T Artemisia absinthum Compositae Europe SD R Artemisia annua Compositae Europe WP T Artemisia argyii Compositae China LF R Artemisia cana Compositae Europe TW R Artemisia capilliaria Compositae China HBR Artemisia vulgaris Compositae Middle America HB T Asclepias curassavica Asclepiadaceae Middle America HBT Asiarum heterotropioides Aristolochiaceae Asia SD T Asparagus sprengeri LiUaceae Africa SD R Asparagus tenuifoUus LiUaceae Africa WPR Aperula odorata Rubiaceae Europe HB R Asperula orientalis Rubiaceae Europe LT R Asperula sp. Rubiaceae Europe SD R Asphodeline libumica Asphodelaceae Mediterranean FRR Asphodelus aestivus LiUaceae Mediterranean SD R Aspidistra lurida LiUaceae A sia WPR Aspilia ttfricana Compositae A sia LS R Asplénium nidus Aspleniaceae Europe LF R Aster acris Compositae America LS R A ster m atsumoto Compositae Am erica SD R Astralagus membranaceus Leguminosae Europe RTT Asystasia gangetica Acanthaceae Africa WP R Ateleia herbert -smithii Leguminosae Am erica WP R Athamanta turbith UmbeUiferae Mediterranean WP R Athyrium feli-femina Athyricaceae Europe LF R Atlantia monophylla Rutaceae Thailand FRR Atractylodes macrocephala Compositae China RH T Atractylodes chinensis Compositae China WP T Atriplex canescens Chenopodiaceae Europe WP R Atriplex hortensis Chenopodiaceae Europe SD R Atriplex hymenelytra Chenopodiaceae Europe LF R Atropa belladonna Solanaceae Europe SD R Atropa komarovii Solanaceae Japan LFR Aucuba japonica Cornaceae Japan SB R Key : RiRandomly Collected T:Targeted 199 Species Genus Familv Geoeranhic Plant R /T Location part used Autococalyx jasminijlora Rubiaceae Africa SB R Avenu sativa Graminae Europe WP T Avicennia alba Verbenaceae Thailand BK R Azadirachta indica M eliaceae West Africa BK T Barringtonia edulis Barringtonaceae Africa LF R Berberis thunbergii Berberidaceae Africa FR R Betula pendula Betulaceae North Am erica LF R Bixa Orellana Bixaceae Middle America RT R Blechum gibbur Acanthaceae Europe LST B letilla striata Orchidaceae Asia RHR Blighia sapida Sapindaceae Africa RB T Bocconia cordata Papaveraceae East Asia ST R Boltonia incisa Compositae East Asia LS R Bombax aquaticum Bombaceae Asia SH T Boswellia carteri Burseraceae A sia WPR Bouerria ovata Boraginaceae Sierra Leone SD T Brassica nigra Cruciferae Europe SDT Brunfelsia uniformis Solanaceae Am erica LF T Brunnera macrophyla Boraginaceae E ast Mediterranean LS T Bryonia cretica Cucurbitaceae North Africa LT T BryophyUum pinnatum Crassulaceae Sierra Leone FL T Buddleia crispa Loganiaceae East Asia LT R Bupleurum longeradiatum UmbeUiferae North A frica SD T Buxus sempervirens Buxaceae West Africa RT T Byrsonima coccineus Malpighiaceae America TL R Calendula officinalis Compositae West indies FL T Calendula suffruticosa Compositae West Indies LF R Calpocalyx brevibracteatus Mimosaceae West Africa LF R Carapa guianensis MeUaceae Africa ST R Carica papya Capricaceae West Indies WP T Carfuncellus pinnatus Compositae No record SD T Carissa lanceolata Apocynaceae Australia SD T Carpentaria californica Hydrangaceae Am erica SS R Carthamus tinctorius Compositae Meditteranean FL T Caryopteris clandonensis Verbenaceae East Asia LT R Cassia corymbosa Leguminosae Europe LT R Cassia fruticosa Leguminosae West Indies SD R Cassia occidentalis Leguminosae West Indies ST R Cassia senna Leguminosae West Indies LF R Catharanthus roseus Apocynaceae China WPT Castonospermum australe Leguminosae A ustralia SD R Casuarina equisetifolia Casuarinaceae A ustralia ND T Cecropia obtusifotia Moraceae Middle America SD T Cedrela odorata M eliaceae West Indies SD T Cedronella canariensis Labiatae Canary Islands HB T Centella asiatica UmbeUiferae South Africa HBT Ceratonia siliqua Leguminosae Arabia FR R Chelidonimu mqjus Papaveraceae China WP T Chenopodium album Chenopodiaceae Europe SD R Chenopodium ambrosioides Chenopodiaceae Middle America LF T Chinuqthila umbellata Ericaceae Europe HB R Chionathus virginiana Oleaceae Africa SD T Chondrus crispus Gigartinaceae UK SD R Chusquea culeou Gramineae Am erica WP R Chrysanthemum leucanthemum Compositae Europe SD R Chrysanthemum morifolium Compositae Europe FL R Cimicifuga heracleifolia Ranunculaceae China RHT Cimicifuga simplex Ranunculaceae China SD T Cinnamontum camphora Lauraceae West Indies SD T Key : RiRandomly Collected TiTargeted 200 Svecies Genus Familv Geoeraohic Plant WT Location £ o r t used Cinnamomum zeyUmicum Lauraceae West Indies BK T CUruUus colocynthis Cucurbitaceae South A frica FR R Citrus auratUium Rutaceae Middle America FL T Citrus limonum Rutaceae Middle America FRT Citrus medica Rutaceae China SD T Citrus reticulata Rutaceae South East Asia FR T Citrus sinensis Rutaceae Middle America LF T Clematis chinensis Ranunculaceae China RZT Cleome hassleriana Capparidaceae Africa WP R Clerodendron trichotomum Verbenaceae Japan HB R Clerodendron umbellatum Verbenaceae Japan LFR Clethra barbinermis Clethracaea Am erica SF R Cleyera theaeoides Theaceae Japan ST R Clianthus formosus Leguminosae Australia SD R Clidemia conglomerata Melastomataceae America LF R Clidemia hirsuta Melastomataceae America LF R Clinopodium vulgare Labiatae Europe SD R Clitoria tematea Leguminosae UK SDT Clivia miniata Amaryllidaceae South Africa WPR Cocos nucifera Palmae East Malaysia LS R Coffea ligustrifolia Rubiaceae Africa TL T Collinsonia canadensis Labiatae America RTR Colutea media Leguminosae Africa RT R Commiphora molmol Burseraceae A frica SD T Commiphora myrrha Burseraceae Africa SD T Commiphora sp. Burseraceae Africa SDR Conopharyngia longifoUa Apocynaceae Sierra Leone LFR Conyza canadensis Compositae Middle America WP T Coptis chinensis Ranunculaceae Asia RT T Coreopsis maritima Compositae Asia LF R Coreopsis pubescens Compositae Am erica LF R Coreopsis verticillata Compositae Am erica LF R Coriandrum sativum UmbeUiferae Middle America SD T Corokia cotoneaster Cornaceae New Zealand LF R Coronilla galuca Leguminosae Europe LFR Corydalis decumbens Papaveraceae China SDT CorydaUs yanhuso Papaveraceae China SD T Cotoneaster horizontalis Rosaceae China LS R Crescentia cujete Bignoniaceae MidMe America LF T Crinum xpowelUi Amaryllidaceae South Africa WPT Croton humilis Euphorbiaceae Sierra Leone SDT Croton leonensis Euphorbiaceae Sierra Leone TL R Croton tiglium Euphorbiaceae China SDT Crucianella stylosa Rubiaceae Europe WP R Cucurbita maxima Cucurbitaceae Middle America SD T Cucurbita moschata Cucurbitaceae M id^ America SD T Cucurbita pepo Cucurbitaceae Middle America WP T Curcuma longa Zingiberaceae India RTT Cymbopogon citratus Graminae India WP T Cymbopogon sp. Graminae India SD R Cymboviotus lawsonianus Graminae UK LF T Cynometra ananta Leguminosae Africa TL T Cypella herbertii Iridaceae South America LT R Cyperus odoratus Cyperaceae Australia ST R Cyperus rotundas Cyperaceae Australia TB T Cystopteris butbifera Aspidiaceae Europe WP R Cytisus aJbus Leguminosae Africa HB T Cytisus maderensis Leguminosae Africa SDR Daboecia praegerae Ericaceae Europe LS R Key : R;Randomly Collected T;Targeted 201 Species Genus Familv Geoeraohic Plant R/T Location part used Dalbergia rufa Leguminosae B razil ST R Datura meteloides Solanaceae Middle America SD T Datura stramoniunm Solanaceae Middle America SD T Daucus carota UmbeUiferae Africa SDR Davidia mvolucrata Davidiaceae China STR Delonix regia Leguminosae Africa FRR Dianthus barbatus Leguminosae China SD T Diascia Jlanaganii Scrophulariaceae South Africa WP R Dichroa grandi/lora Hydrangeaceae China HB R DicUptera elliotti Acanthaceae Sierra Leone WP R Dictamnus dasycarpus Rutaceae Europe RC R Dierama pulcherrimum Iridaceae East Africa SD R Digitalis purpurea Scrophulariaceae UK WPR Dimorphotheca aurantiaca Compositae Africa LF R Dioscera bulbifera Dioscoreaceae Asia TB T Dipteryx odorata Leguminosae West Indies SD T Doronicum hirsutum Compositae Europe LF R Duboisia myroporoides Solanaceae Australia WP T EcbalUum elaterium Cucurbitaceae Meditteranean SD T Echinacea angustifolia Compositae America RTT Echinaceae purpurea Compositae Am erica SD T Echinocereus salmdyckianus Cactaceae America WP R Echinopsis hoUygate Cactaceae South America WP R Echinopsis spachianus Cactaceae South America WP R Eclipta alba Compositae China SD T EcUpta prostarata Compositae America HB R Edgeworthia chrysantha Thymelaceae China HB R Ehretia thyrisifolia Boraginaceae Japan LS R Elaeagnus angustifolia Elaeagnaceae North Am erica HBR Elaeagnus commutata Elaeagnaceae North America ST R Elaeis guineensis Palmae West Africa RT R Elaeocarpus coorooylooloo Elaeocarpaceae India LF R Eleaodendron laneanum Celastraceae Africa LS R Elettaria cardamomum Zingiberaceae India WP R Eleusine coracana Gramineae Africa WP R Eleusine indica Grammeae Middle America WP T Elodia canadensis Hydrocharitaceae North America LS R Enantia chlorantha Annonaceae West Africa LF T Epimedium sagittaum Berberidaceae China WP T ErciUus volubilis Phytolaccaceae Chile SD R Erinus alpinus Scrophulariaceae North Africa RT R Eremophila maculata Myroporaceae Australia SDT Erymomastax polysperma Acanthaceae Ghana LS R Eryngium agavifoUum UmbeUiferae South A frica SDR Eryngium campestre UmbeUiferae South Africa WP R Eryngium foetidum UmbeUiferae Middle America WP T Eryngium giganteum UmbeUiferae South Africa SD T Erysimum linifolium Cruciferae Europe SDR Erysimum scoparia Cruciferae Europe LSR Erythrina corallodendron Leguminosae Middle America SDT Erythrina vogelii Leguminosae America LF R EscaUonia macrantha Grossulariaceae Chile HB R Eschscholzia californica Papaveraceae Am erica SD T Eschscholzia macrantha Papaveraceae America HB T Eucalyptus globulus Myrtaceae Middle America LF T Eucalyptus sp. Myrtaceae Middle America LF T Eucommia ulmoides Eucommiaceae China BK T Key : RiRandomly Collected TiTargeted 202 Svecies Genus Familv Geoeravhic Plant R /T Location part used Euonymous atropurpurea Celastraceae Am erica BK R Euonymus europaeus Celastraceae West AustraUa SF R Eupatorium cannabinum Compositae Asia SD T Eupatorium odorata Compositae West Indies RTT Euphobia amyloides rubra Euphorbiaceae West Africa WPR Euphorbia hirta Euphorbiaceae West Africa HB T Evodia rutaecarpa Rutaceae China FR T Fallopia convolvulus Polygonaceae UK HBR Faucaria tigrina Aizoaceae Africa WP R Ficus carica Moraceae Middle America LF T Forsythia suspensa Oleace Europe FR T Fragaria vesca Rosaceae Europe LF R Fraxinus excelsior Oleaceae Europe RS T Freesia 'smgle mixed* ridaceae South A frica BLR FritiUaria imperialalis LiUaceae China SD R Fritillaria meleagris LiUaceae Europe BLR FritiUaria persica LiUaceae Europe BL R Galanthus nivaUs Amaryllidaceae Britain WP T Gardenia jasminoides Rubiaceae A frica WPT Gasteria 'spotted beauty' LiUaceae A frica WP R Gelsemium sempervirens Loganiaceae Am erica HBT Gentiana lutea Gentianaceae China LF T Gentiana macrophyila Gentianaceae China LFT Gentiana manshura Gentianaceae China SD T Gibbaeum anguUpes Aizoceae A frica WP R Glaucium flavum Papaveraceae Europe SD T Glehnia littoralis UmbeUiferae Asia RT T Glycyrrhiza glabra Leguminosae Meditteranean RTT Glycyrrhiza uralensis Leguminosae China RTT Gomphrena globosa Amaranthaceae Middle America SD T Guaiacum officUude Zygophyllaceae Middle America SD T Guibourtia copalUfera LegunUnosae Africa RTR Gunnera manicata Gunneraceae ChiU LSR Hamamelis virginiana Hamamelidaceae Am erica LF T Hardenbergia comptoniana Leguminosae AustraUa LSR Hannoa klaineana Simaroubaceae Sierra Leone FR R Harpagophytum procumbens PedaUaceae A frica RTT Harungana madagascariensis Guttiferae A frica RT T Haworthia chloracantha LiUaceae A frica WPR Haworthia rigola var expans LiUaceae A frica WP R Hedysarum coronarium LegunUnosae Europe WPR Helenium autumnale Compositae Am erica HBR HeUconia wagneriana Heliconiaceae Am erica LF R HeUotropium arborescens Boraginaceae Europe ST R HeUotropium digynum Boraginaceae Europe SD R HeUotropium peruviana Boraginaceae Europe WP R Helleborus corsicus LiUaceae Europe WP R Heritiera utilis SterctiUaceae Africa Africa BK R Hippocratea wehvitschii Celastraceae Africa ST R Hippuris vulgaris Hippuridaceae UKWP R Holarrhena floribunda Apocynaceae West Africa RT T HolboeUia latifoUa Lardizabalaceae China LFR Hordeum jubatum Gramineae China SD T Hordeum vulgare Gramineae China FR T H osta sieboldiana LiUaceae China WP R Key : RiRandomly Collected TiTargeted 203 Species Genus Familv GeoeroDhic Plant R/T Location part used Hosta ventricosa LiUaceae China LT R Houttuynia cordata Sauraceae China HB R Hovenia dulcis Rhamnaceae Asia SDR Hoya bella Asclepiadaceae China WP R Hoya fraterna Asclepiadaceae China LS R Hoya longifoUa Asclepiadaceae China WPR Huemia hislopii Asclepiadaceae Africa CM R HunuUus lupulus Cannabinaceae Britain LS R Hyacinthus 'white pearV LiUaceae Europe BL R Hyoscyamus niger Solanaceae America SD T Hypericum perforatum Gutiferae Europe SD T Hyptis suaveolens Labiatae West Indies TL T Iberis umbellatum Cruciferae Meditteranean WF R Ilex paraguensis AquifoUaceae Brazil LFR Ipomea pes - caprae Convolvulaceae A frica LS T Indigofera suffruticosa Leguminosae Middle America TW R Iris reticulata 'cantab' Iridaceae Europe BL R Irlbachia caerulescens Gentianaceae South Am erica LF R Irvingia gabonensis Irvingaceae Africa BK R Isatis tinctoria Cruciferae Europe LFR Isertia coccinea Rubiaceae America FR R Isertia spiciformis Rubiaceae Am erica LF R Isolana campanulata Annonaceae Africa BK T Isotoma axillaris Campanulaceae South America LT R Itea ilicifolia Iteaceae China HB R Jacaranda filicifoUa Bignoniaceae America LF R Jacaranda mimosifoUa Bignoniaceae America WP R Jacobmia carnea Acanthaceae South America HB R Jasminum laurifoUum f. niti Oleaceae China HB R Jatropha curcas Euphorbiaceae West Indies LF T Jatropha gossypifoUa Euphorbiaceae West IntUes LF T Juniperus Communis Pinaceae Europe FR T Kalanchoe daigremontiana Crassulaceae Madagaskar LFR Kalanchoe kewinsii Crassulaceae Madagaskar WP R Kalanchoe schumacheri Crassulaceae Madagaskar WP R Kleinhovia hospita StercuUaceae Africa TL R Knautia drymeia Dipsaceae Meditteranean HB R Lactuca sativa Compositae MidMe America SD T Lagarus ovatus Gramineae UK SD R Lantana camara Verbenaceae America SD T Laurus nobilis Lauraceae Middle America WP T Lavandula angustifolia Labiatae Britain HB T Lavandula offidnaUs Labiatae Britain FL R Lavatera species Malvaceae Mediterannean SDT Ledebouriella dharicata UmbeUiferae China R T T LedebourieUa seseloides UmbeUiferae China RT T Leonotis leonurus Labiatae West Indies SD R Leonatis nepetaefolia Labiatae West Indies WP T Leonurus cardiaca Labiatae Europe HB R Leucaena leucocephala Leguminosae Middle America WP T Ligusticum sinense UmbeUiferae Europe SD T Ligusticum chuangxiong UmbeUiferae Europe RH R Linaria vulgaris Scrophulariaceae America LF R Lindera benzoin Lauraceae America LS T Liriodendron tuUpifera MagnoUaceae America ST R Liriope muscari LiUaceae China WP T Lisianthius grandifloris Gentianaceae America LFR Lithocarpus densiflorus Fagaceae America LFR Key : RiRandomly Collected TiTargeted 204 Svecies Genus Familv Geouravhic Plant R/1 Location part used Lithodora zahnii Boraginaceae Mediterranean LS R LUhops karasmontana bella Aizoceae A frica WP R Lithops lesUei Aizoaceae A frica WP R Lithops saUcola Aizoaceae Africa WPR Lithops terricolor Aizoaceae Africa WPR Littonia modesta LiUaceae A frica WPR Lmstonia chinensis Palmae A frica SD R Loasa vulcanica Loasaceae Am erica SD R Lobelia chinensis Campanulaceae Am erica HB T Lobelia erinus Campanulaceae Africa LS T Lobelia inflata Campanulaceae Africa HBT Lobelia radicans Campanulaceae China HB T Lobivia schieleana Cactaceae South Andes WPR Lonicera japonica Caprifoliaceae Japan FLT Lophatherum gracile Gramineae Australia LF T Lycium chinense Solanaceae China FR T Lycopodium clavatum Lycopodiaceae Europe HB T LycopoMum serratum Lycopodiaceae Europe SDT Maesa ramentacea Myrsinaceae Africa LF T Maesa species Myrsinaceae Africa TF T Maesobotrya barteri var sparsifolia Euphorbiaceae A frica RT R Mandevilla xamabilis Apocynaceae Am erica LF R MandevilUa laxa Apocynaceae America LST Mandevilla splendens Apocynaceae Am erica ST T Mandevilla suaveolens Apocynaceae Am erica LF T Mammilaria erythrsperma Cactaceae America WP R Mammilaria picta Cactaceae Am erica WPR Mandragora o/fidanarum Solanaceae Europe WP T Manihot esculenta Euphorbiaceae America LS T Manihot isoloba Euphorbiaceae Am erica SD R Manihot palmata Euphorbiaceae A m erica WP R Manmophyton africana Euphorbiaceae Africa LS R Manotes expansa Connaraceae Africa RT R Margyricarpus pinnatus Rosaceae Andes SF R Marsdenia reichenbachii Asclepiadaceae Madagascar BK T Melaleuca quinquenervia Myrtaceae Middle America LF T Melasphaerulea graminea Iridaceae Africa WPR Melia azedarach Meliaceae Australia TWT Melissa officinalis Labiatae Middle America SD T Melilotus officinalis Leguminosae Africa SDR M entha h^^tocafyx Labiatae Middle America LF R Mentha piperita Labiatae Middle America SD T Mentha pulegium Labiatae Middle America LFR Mesembryanthenuun crinifolium Aizoaceae A frica HB R Metasequoia glyptostroboides Taxodiaceae China HB R Michelia figo MagnoUaceae China LT R Micromera thymifoUa Labiatae Meditteranean HBR Mikania scandens Conytositae A frica LF T Mimosa pudica Leguminosae Middle America SD T Momordica charantia Cucurbitaceae Middle America FR T Monanthes polyphylla Crassulaceae Canary Islands WP R Morinda officinalis Rubiaceae West Indies RT T Moringa oleifera Moringaceae West In ^ es LFT Moms alba Moraceae West Africa RT T Mucuna pruriens Fabaceae West Africa SD T Musa paradisiaca Musaceae West Africa HB T Musari plumosum LiUaceae West Asia BL R Myrianthus cerratus Moraceae Africa TWR Myristicia fragrans Myristicaceae West Africa LFT Key ; R;Randomly Collected TiTargeted Species Genus Familv OeosraDhic Plant R/T Location part used Myristicia fragrans Myristicaceae West Africa LFR Myrrhis odorata UmbeUiferae Europe WP T Myrtus domestica Myrtaceae Africa RT R Nandina nmdinaceae Berberidaceae Jfgfan SD R Nauclea diderichii Rubiaceae West Africa BK T Nauclea latifolia Rubiaceae West Africa TL R Nectaroscordiun siculum LiUaceae Europe WPR Neurolaena lobata Con^ositae West Indies LF T Newhouldia laevis Bignoniaceae Africa STT Nicotiana tabacum Solanaceae West Indies SDT Nigella damascena Ranunculaceae Meditteranean SD R Notopterygium incisum UmbeUiferae China SDR Ocimum basilicum Labiatae West Indies SD T Oldenlandia diffusa Ritifiaceae A frica HB R Olearia albida Compositae AustraUa LF R Olearia phlogopappa Compositae AustraUa STR Orobanche hederae Orobanchaceae Europe SD T Osmanthus yunnanensis Oleaceae A sia LF R Osteospermum vallantii Compositae A frica LF R Paeonia lactiflora Ranunculaceae Africa RW R Paeonia moutan Ranunculaceae China RWT Paeonia suffruticosia Ranunculaceae China RB T Panax ginseng AraUaceae China RTT Panax notoginseng AraUaceae China RTT Papaver dubium Papaveraceae A frica HBR Parthenium hysterophorus Compositae West Indies STT Passiflora caerulea Passijloraceae Africa WPR Passiflora foetida Passifloracea Middle America SF T Passiflora incamata Passifloraceae Am erica WPT Pausinystalia yohimbe Rubiaceae Africa BK T Peganum harmala Zygophyllaceae Meditteranean LS T Peltophorum ferrugineum Leguminosae Sierra Leone SB R Persea americana Lauraceae West Indies WP T Petivera aUiaceae Phytolaceae Middle America WPT Phaseolus vulgaris PapiUonaceae West Africa SD T Phellodendron amurense Rutaceae A sia BKT Phoenix rectinata Palmae A frica SD R Phyllanthus discoideus Euphorbiaceae West Africa TL T Physostigma venenosum Leguminosae Africa SDT Picrasma excelsa Simaroubaceae America LF T Pimenta dioica Myrtaceae Middle America LF T Pimpinella anisum UmbeUiferae Africa FR T PinelUa temata Araceae China TB T Pinus tabuUformis Pinaceae Am erica FC T Piper longum Piperaceae India WPR Piper nigrum Piperaceae India WP T Piper methysticum Piperaceae Fiji RT T Piper sarmentosum Piperaceae InMa WP R Piptadeniastrum qfricanum LegunUnosae Africa BKR Piscidia erythrina Leguminosae West Indies SD T Pistacia lentiscus Anacardiaceae MeiUtteranean LF T Pittosporum arborescens Pittosporaceae Africa TL T Plantago m(ÿor Plantaginaceae Europe WP R Pogostemon cablin Labiatae Africa WP T Polygala senega Polygalaceae Am erica RT R Polyganatum odoratum LiUaceae China LF T Polygonum hydropiper Polygonaceae Europe SDR Poncirus trifoUata Rutaceae China LF T Populus species Saliaceae America BK R Key ;R:Randomly Collected TiTargeted 206 Species Genus Familv Geosraohic Plant R /T Location part used Populus tremula Salicaceae America LF T Parla cocos Polyporaceae China SD T Portulacaria afra variegata Portulaceae Africa WP R Portulaca oleraceae Portulaceae West Indies WP T Potentilla erecta Rosaceae Europe RT R Prunella vulgaris Labiatae Africa SD T Prunus mume Rosaceae Japan FR T Prunus spinosa Rosaceae Europe FR T Psoralea coryUfoUa Leguminosae Africa FR R Ptychopetalum olacoides Oleaceae America LFT Pueraria lobata Leguminosae Asia RT R Pulsatilla chinensis Ranunculaceae China RT T Punica granatum Puniaceae Europe BK R Pyrola rotundifolia Pyrolaceae Europe HB T Quillqja saponaria Rosaceae Chile LSR Rebutia xanthocarpa salmonea Cactaceae BoUvia WP R Recaisrea fargessi Not Located Not Located LF R Rehmarmia glutinosa Scrophulariaceae China RH T Rheum officinale Polygonaceae China LSR Rheum palmatum Polygonaceae China LF T Rhigio carya racemifera Menispermaceae Africa ST T Rhodedendron UUeum Ericaceae Europe HB T Rhodotypos scandens Rosaceae China LS R Rhus toxicodendron Anacardiaceae America SD T Richea scorparia Epacridaceae Australia LS R Ricin communis Euphorbiaceae Africa LF T Rinorea microdon Voilaceae America TL R Rosa species Rosaceae Europe FLR Rosmarinus officinalis Labiatae MidtUe America HB T Rotula aquatica Boraginaceae BrazU TL R Ruhia cordifolia Rubiaceae Africa RTT Rubus idaeus Rosaceae Britain LF R Rungia grandis Acanthaceae A frica TL R Rushia stenophylla Aizoaceae Africa WP R Sabal texenensis Palmae Am erica WP R Saccharum officianarum Gramineae West Indies LS T Saggittaria sagittifolia Alismataceae Europe SDT Salacia erecta Celastraceae Sierra Leone ST R Salix discolor Saliaceae Europe BKR Salvia cacalifoUa Labiatae Europe LT R Salvia dorisiana Labiatae Europe LS R Salvia guarantica Labiatae China LS R Salvia haematodes Labiatae China SDT Salvia interrupta Labiatae China LT R Salvia miltiorrhiza Labiatae China RT T Samobis valerandi Primulaceae Europe WP R Sanguinaria canadensis Papaveraceae Am erica RT T Sanguisorba officinalis Rosaceae Am erica RT R SantoUna chaemaecyparis Compositae Meditteranean SD T Saponaria officinalis Caryophyllaceae America LF R Scabiosa atropurpurea Dipsacaceae Europe SD T Schefflera arboricola AraUaceae China SDT Schinus molle Anacardiaceae UKLFR Schisandra chinensis Schisandraceae America FL T Schizonepta tenuifolia Labiatae Asia LS T Scrophularia ningpoensis Scrophulariaceae China SDT Scoparia dulcis Scrophulariaceae Middle America RT T Scopolia camiolica Solanaceae Mediterranean WP T Scorzonera hispanica Compositae Mediterranean SD R Key ;R:Randomly Collected TiTargeted 207 Svecies Genus Familv Geo2raohic Plant R/T Location £O rt used Scutellaria baicalensis Labiateae China TW T Securidaca longepeduncidata Polygalaceae A frica ST T Securinega suffruticosa Euphorbiceae China FR T Selenicereus grandijloris Cactaceae Am erica WP R Senecio rowleyanus Compositae China WPR Semiaquilega adoxoides Ranunculaceae Asia SD T Sequoiadendron giganteum Taxodiaceae Am erica LS R Siegesbeckia pubescens Compositae China RT T Sinomenium acutum Menispermaceae China SD T Smilax species LiUaceae South America RT R Solanum nigra Solanaceae Africa HB T Solanum torvum Solanaceae A frica TL R Solidago graminifolia Compositae Am erica LF R Solidago virgaurea Compositae Am erica SD T SoUya heterophyUa Pittosporaceae Australia LF R Sonchus oleraceus Compositae Middle America LF T Sophora flavescens Leguminosae China RT T Sorbus aucuparia Rosaceae Europe FR R Sorbus intermedia Rosaceae Europe LS R Sphaerulacea ambigua Not Located UK RT R Spiropetalum solanderi Connaraceae Africa LF R Stachys olympia Labiatae Asia LT R Stachys officinalis Labiatae A sia HBR Stellaria media Caryophyllaceae Europe WPR Stephania dinklagei Menispermaceae China ST T Stephania tetrandra Menispermaceae China RT T Stembergia lutea Amaryllidaceae Europe BL R Stillingia sylvatica Euphorbiaceae Am erica RT R Stratiotes aloides Hydrocharitaceae Britain WPR Strychnos nux - vomica Loganiaceae Asia SDT Swertia chirata Gentianaceae Africa HB T Symplocos leptophylla Symplocaceae Am erica BK T Symphytum officinale Boraginaceae America HB T Syzygium cumini Myrtaceae Middle America SD T Tamarindus indica Leguminosae Middle America FR T Tamus communis Dioscoraceae Africa SF T Tanacetum vulgare Compositae A frica HB T Taraxacum officinale Compositae Middle America HB T Tecoma stans Bignoniaceae Middle America LF T Tecomanthe speciosa Bignoniaceae Australia LS R Terminalia catappa Combretaceae India TL T Tetrapleura tetraptera Leguminosae West Africa SD T Teucrium fruticans Labiatae Meditteranean LT R Thevetia neriifoUa Apo KEY 1 R= Randomly Collected Plant T=Ethnomedically Selected Plant (Targeted for use in traditional medicine as having analgesic properties) 209 Appendix n(a) ^ standard curve using BSA to determine the concentration of protein in CHO cells which express the bradykinin BK II receptor. 1.2 O o E c 1 to O) If) 0.8 TO cs 0.6 CO -2 0 10 100 BSA (microlitres) Linear Regression Simple weighting Correlation Coefficient (r) = 0.7435 Variable Value Std. Err. Intercept 0.2606 0.1996 Slope 0.0101 0.0032 210 n(b) A standard curve using BSA to determine the concentration of protein in SK-N-MC membranes which express the CGRP receptor. 2 0.8 c m O) m (0 0.6 Ü0) c CO •e 0.2 10 100 BSA (microlitres) Linear Regression Simple weighting Correlation Coefficient (r) = 0.9882 Variable Value Std. Err. Intercept 0.2550 0.0165 Slope 0.0049 0.0003 211 10 20 25 The initial HPLC trace of lOuL of a methanol extract of Symplocos leptophylla on a CIS Reverse Phase Analytical Column (see Methods 2,27-2.30). 10 2 0 30 T i ri i e 212 [(b)HPLC chromatogram of the methanol extract Symplocos leptophylU lüally separated (for details see Methods 2.27-2.30). I 0 1G xl AU Cl . 0 2 5 I O ' ) — • ' AU •u. a< o a . '/I - h t . 10 T I n, 213 ni(c)HPLC chromatogram of fractions 1 to 8 of Symplocos leptophylla partially 2*30)™**** ***'“8 analytical reverse phase chromatography (see Methods 2.27- f - 2 2 0 n r . i •n ,1' »1< '.' I £ - H Cill tTi£ Ü- a*+-•• •n tit o-t iL. iL AU ti. c c Vi Û. =: —« 3 Vi £ —t tji #:! til iTi t;i iTi C' ' - t i_ i-t-t t_t ü_ SI U . ü_ -I tfi (JLt J - c Hi i—i 20 U nm L t—t I O £ ’T U It, n, •- £ ^ • '7, u litt i t 1:1C $ *?l ÛL • 7 C •It Vi +-• I — .TI I II r_ 0 7 5 • «i. •_• Vl til S V l C Ü . I t | •f* I .1 . .TI .1 . 10 15 :: - t.t-t ct- i«: T i ftia 0 2 0 214 pendix IV Ktss Spectral Data from the Time of Flight Analysis of Fractions 1- . I& * . o ( I I e s t I KOO idOO %K0 yjdO K « t 2500 X<0 3XC « 4 (ru:> NC F IL M ; SAMPLE ALSfA&l/ MADE UP, SU. LOAKO NC FILM; SA/fLE ALF£A£«Y MA[( UP; GUL .OA[(D wAS/tO WASHED ,Sv* o/ II ch«nn#l«l «< 11 CM#AA«ts) 2W) 1400) !S r 120» I 1(0» lOCCO- axM 1500 ZOOO KOO XCO J5C0 215 Ipendix IV |#ss Spectral Data from the Time of Flight Analysis of Fractions 1-8, <5o* of I? «h*rv%*Ul ill» 3 i : 1 cf.kM .4i A IfM ÏI940/S2/Î I1960/SZ/7 NC FILM, SAnH.£ ALfKAOr f«t< UP| BJU K r i u i l SWI?LC ALREAOr HADE UP, 8UL LOAOCO «ASHCO MO »0 tAJW lX AUCADT MAOC UP I PUL LOAOCO PC r i U i lAMPLC UP I t u t LOADED 216 A p p e n d ix V The Scatchard analysis of the bradykinin BKII receptor from the rat uterus preparation. <>neiat Ljh.:- OZWH-i::. : now :- 0-06- 0}0i-r T ' ♦ k ...... ♦ • • — » » * • • • iiz'éO-poK:.: '7O T Q .fôçO; ♦. . * . . 7^^ V ... .4...... - • • • I ' 217 REFERENCES REFERENCES Akitt, J.W. (1973) N.M.R. and Chemistry. Chapman and Hall, London. Ali,M., Bhutani,K.K., Srivastava,T.N.(1990)Triterpenoids from Symplocos racemosa bark. Phytochemistry vol 29 p3601-4 Amara, S.G., Jonas,V., Rosenfeid,M.G., Ong,E.S., Evans,R.M. (1982) Alternative RNA processing in Calcitonin Gene Expression generates mRNA’s encoding different polypeptide products. Nature vol 280 p240-44 Amara, S.G., Arriza,J.L, Left,S.E., Swanson,L.W., Evans R.M., Rosenfeld, M.G.(1985)Expression in brain of a mRNA encoding a novel neuropeptide homologous to CGRP. Science vol 229 p1094-97 Anonymous (1982) Markets for selected medicinal plants and their derivatives. Geneva International Trade Centre 206pp Anonymous (1975) American Herbal Pharmacology Delegation. Herbal Pharmacology in the People’s Republic of China. A Trip Report. Washington, DC. National Academy of Sciences Armstrong, D, Jepson, J.B, Keele,C.A, Stewart, J.W. (1957) Bradykininrpain producing substance in human inflammatory exudates and plasma. J.Physiology vol 135 p350-70 Armstrong,D. (1970) Pain. In:Handbook of Exp.Pharmacol. vol 25(suppl) E.G. Erdos (ed) Springer-Verlag, New York pp434-81 Arvidsson,U., Schalling,M., Cullheim,S., Ulfhake,B., Terenius,L., Hokfelt,T. (1990) Evidence for the presence of CGRP in the monkey serotonergic bulbospinal pathway Eur.J.Neurosci. (suppl. 1) p30 Aschoff, J. (ed) (1965) In:Circadian clocks. Proceedings of the Feldaying Summer School. N.Holland Publishing Co. Amsterdam Ayensu, E.S. (1981) Medicinal plants of the West Indies. Reference Publications Baerheim-Svendsen, A. (1984) Biogene Arzneistoffe-Heute noch oder heute wieder. In:Biogene Arzneistoffe. Czygan,F.C.(eds) pp27-42 Braunschweig:Vieweg. Koumare, M. (1983) Traditional medicine and psychiatry in Africa. In : Traditional Medicine and Health Care Coverage. Bannennan, R.H., Burton, J., Wen-Chieh, C.(eds). WHO, Geneva, pl-326 Banwell, C.N.(1966) Fundamentals of Molecular Spectroscopy. McGraw-Hill Publ. London Bathon, J.M. and Proud, D.(1991) Bradykinin antagonists. Ann.Rev.Pharmacol. and Toxicol, vol 31 pl29-62 Berridge, M.J.(1987) Inositol trq)hosphate and diacylglycerol : Two interacting second messengers. Ann.Rev.Biochem. vol 56 p 159-93 Bhoola, K.D. and Schachter, M. (1960) Some properties of kallidin, bradykinin and wasp venom kinin In:Polypeptides which affect smooth muscles and blood vessels. M.Schachter. (ed) Pergamon Press, Oxford pp232-46 Bhoola,K.D. (1961)Properties of kallikrein,kallidin and related substances Ph.D Thesis, London University. Bhoola, K.D., Figueroa,C.D., Worthy; K.(1992) Bioregulation of kinins:kallikreins,kininogens and kminases. Pharmacol.Rev. vol 44 pl-80 Bloomquist, E.I. and Kream, R.M. (1987) Leukotriene D4 acts in part to contract guinea pig ileum smooth muscle by releasing substance P. J. Pharmacol. Exp. Ther. vol 240 p523-8 218 Bohlin, L, (1993)Research on ethnopharmacologically active natural products at the Department of Hiarmacognosy. Uppsala University. J.Ethnopharmacology. vol 38 p225-31 Boissonnas, R.B., Guttmann, S., Jaquenoud, D.A., Konzett, H., Stunner, E. (1960) Synthesis and biological activity of peptides related to bradykinin. Experimentia vol 16 p326 Braas, K.M., Manning, D.C., Perry, D.C., Snyder, S.H. (1988) Bradykinin analoguesidifferential agonist and antagonist activities suggesting multiple receptors. Br.J.Pharmacol. vol 94 p3-5 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantitieso^rotein utilizing die principle of protein dye binding. Anal.Biochm. vol 72 p248-54 Bradley, P.B. (1989) Introduction to Neuropharmacology. Butterworth and Co. Publishers Ltd. Brinkworth R.I., Lloyd, E.J., Andrews, P.R. (1988) Brain Chemistry and Central Nervous System Drugs. Natural Products Reports p363-385 Brock, N., Barding, H., Wilk, W.(1962)Technische Problème und Erfahrungen bei der Herstellung und Darreichung von Altromin R-Standarddiaten fur Versuchstiere, Arzneimittel-Forsch 12 507 Brodie, D C. and Smith,W.E. (1985) Implications of new technology for pharmacy education and practice. Am.J.Hosp.Pharm. vol 42 p81-95 Bruhn, J.G. and Holmstedt, B. (1982)Natural products as Medicinal agents. E.Reinhard and J.L.Beal (eds) pp405-30 Hippokrates and Stuttgart. Brunton, L.L., Wikhind, R.A., VanAesdale, P.M., Gilman, A.G. Binding of ^[Hjprostaglandin to El putative receptors linked to adenylate cyclase of cutured cell clones. (1976) J.Biol.Chem. vol 251 p3037^ Burch, R.M.,and Axelrod, J. (1987) Dissociation of bradykinin induced prostagalandin formation from inositol phosphate in swiss 3T3 fibroblasts. Evidence for a G-protein regulation of phospholipase A2. J.Proc.Natl.Acad.Sci.USA vol 84 p374-78 Burch, R.M., Connor, J.R., Axelrod, J.(1988) Interleukin-1 amplifies receptor mediated activation of PA2 in 3T3 fibroblasts. Proc.Natl.Acad.Sci.USA vol 85 p6306-9 Burch, R.M. (1989a) Bradykinin stimulates tumour necrosis factor and interleukin-1 release from macrophages. FEBS Letts, vol 247 pl89-92 Burch, R.M., Connor, J.R.,Tiffany, W. (1989)The kallikrein-kininogen-kinin system in chronic inflammation. Agents Actions vol 27 p258-60 Burch, R.M., Farmer, S.G., Steranka, L.R. (1990) Bra(fykinin Receptor Antagonists. Med.Res.Rev. vol 10 p237-69 Burgess, G.M., Mullaney, L, Mcneill, M., Dunn, P., Rang, H.P. (1989)Second messengers involved in the mechanism of action of bradykinin in sensory neurones in culture. J.Neuroscience. vol 9 p3314-25 Burgess, G.M. (1989) Activation of guanylate cyclase by bradykinin in rat sensory neurones is mediated by Calcium Influx ; Possible role of the increase in cyclic AMP. J.Neurochem. vol 53 pl212-18 Buzzi, M.G. and Moskowitz, M.(1990) The anticholinergic drug. Sumatriptan (GR43175) selectivley blocks plasma extravasation from blood vessels in dura mater. Br.J. Pharmacol, vol 99 p202-6 Buzzi, M.G., Carter, B., Moskowitz, M., Shimizu, T., Heath, H.H. (1991) Dihydroergotamine and sumatriptan attenuate the increase in plasma CGRP levels witiiin rat superior sagittal sinus during electrical trigeminal stimulation. Neuropharmacology vol 30 pi 193-200 Calixto, J.B., Nicolau, M.,Yunes, R.A. (1985) The selective antagonism of bradykinin action on rat isolated uterus by crude Mandevilla velutina extract. Br, J, Pharmacol, vol 85 p729-31 219 Calixto, J.B., Nicolau, M.,Trebien, H., Henrique, M.G.O, Weg, V.B., Cordiero, R.S,B.,Yunes, R.A. (1986) Antiedematogenic actions of a hytkoalcoholic crude water-alcohol extract of Mandevilla velutina. Braz. J. Med. Biol. Res. vol 19 p575 Calixto, J.B.,Yunes, R.A. (1986) Effect of a crude Mandevilla velutina extract on contractions induced by bradykinin and [DesArg^BK in isolated vessels of the rabbit. Br.J.Pharmacol, vol 88 p937 Calixto, J.B, Nicolau, M, Pizzolatti, M.G, Yunes, R.A. (1987)Kinin antagonist activity of compounds from Mandevilla velutina in the rat isolated uterus. Br. J. Pharmacol.vol 91 pl99-204 (Zalixto, J.B., Sant’Ana, A.E.G. (1987) Hiarmacological analysis of the inhibitory effect of jatrophine, a diterpene isolated isolated from Jatropha elliptica, on smooth and cardiac muscles. Phytotherapy Res. vol 1 pi22-26 Calixto, J.B., Pizzolatta, M.G.,Yunes, R.A. (1988)The competitive antagonistic effect of conqxmnds from Mandevilla velutina on kinin induced contraction of rat uterus and guinea pig ileum in vitro. Br.J.Pharmacol. vol 94 pi 133-42 Calixto, J.B. (1990) Non-peptide bradykinin antagonists. Inflamm.Dis.Ther.vol 5 p97-129 Cascieri, M.A., Liang, T. (1983) Characterisation of the Substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides. J.Biol.Chem. vol 258 p5158-64 Cascieri, M.A., Ber, E., Fong, T.M., Sadowski, S., Bansal, A.,Swain, C., Seward, E., Frances, B., Burns, D., Strader, C.D. (1992) Characterisation of the binding of a potent, selective radioiodinated antagonist to the human neurokinin-1 receptor. Mol.Pharmacol. vol 42 p458-63 Chan,W. W.C. (1966) Organic reactions in aqueous solution at room ten^)erature. Part IV. In vitro oxidation of bisbenzylisoquinoline alkaloid to an aporphine alkaloid. J.Chem.Soc.,C. p753 Chance, M.R.A. ( 1956)Environmental factors influencing gonadotrophin assay in the rat. Nature vol 177 p228 Chang, H. and Butt, P. (1986 and 1987) Pharmacology and Applications of Chinese Materia Medica. Vols I and H. World Scientific Publishing Co. Pte.Ltd Chatterjee,T.K., Moy, J.A., Fisher, J.A.( 1991 )Characterisation and regulation of high affinity CGRP receptors in cultured neonatal rat cardiac myocytes. Endocrinology vol 128 p2731-38 Cheng, Y.C., Prusoff, W.H. (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (ICgQ) of an enzymatic reaction. Biochem.Pharm. vol 22 p3099-3108 Chernov, H.I.,Wilson, D.E., Fowler, F., Plummer, A.J.(1967) Non-Specificity of the mouse writhing test. Arch.Int.Pharmacodyn. vol 167 pl71-78 Chiba, M., Hisada, S., Mshibe, S.,Thieme, H.(1980)^^C NMR analysis of symplocosinand (+) - epipinoresinol glucoside. Phytochemistry vol 19 p335-6 Chiba,T.,Yamaguchi, A.,Yamatant, T., Nakamura, A., Morishita, T., Inui, T., Fukase, M., Foda,T. Fujita,T.(1989)CGRP receptor antagonist human CGRP-°"^^ Am. J. Physiol, vol 256 p331-5 Christensen, B.V., Reese, J.A. (1938) A Study of the Leaves of Ipomea pes Caprae. Am.Pharm.Assoc, vol 27 pl95-99 Clark,W.G. (1979) Kinins and the peripheral CNS. Handbook Exp.Pharmacol. (suppl) E.G. Erdos (ed) Springer-Verlag New York vol 25 pp311-56 220 Clark, M.A., Bomalaski, J.S., Conway, T.M., Wartell, J., Crooke, S.T. (1986)Differential effect of aspirin and dexomethasone on phospholipase A2 and C activities and arachidonic acid release firom endothelial cells in response to bradykinin and leukotriene D4. Prostaglandins vol 32 p703 Code,C.F. (1937)The histamine-like activity of white blood cells J.Physiol. vol 90 p485-500 Cohnan, R.W., Wong, P.Y. (1979) In : Bradykinin, kallidin and kaUikrein vol XXV, Supplement.Handbook of Exptl.Pharmacology. E.G.Erdos (ed) Springer-Verlag, New York pp569- 607 Conn, P. ( 1991 )Neuropeptide Technology : Gene expression and neuropeptide receptors. Methods in Neurosciences, vol 5 Academic Press Cordell, G.A. (1990) Pharmacognosy-A High Tech Pharmaceutical Science. Pharmacia JTPA vol 30 pl69-81 Crooke, S.T., Mattem, M., Sarau, H.M.(1989)The signal transduction system of the leukotriene D4 receptor. TIPS vol 10 p 103-7 Croom, E.M. (1983) Documenting and Evaluating Herbal Remedies. Econ.Bot.vol 37 pi3-27 Culvenor, C.C., Clarke, M.,Edgar, J.A., Frahn, J.L., Jago, M.V., Peterson, J.E. Smith, L.W.(1980) Structure and toxicity of the alkaloids of russian comfrey {Symphytum uplandicum Nyman), a medicinal herb and item of human diet. Experientia vol 36 p377-9 Dennis,T., Fournier, A.,Pierre, S. ,Quirion, R. (1989) Structure and activity profile of CGRP in peripheral and brain tissues. Evidence for receptor multiplicity. J. Pharmacol.Exp. Ther. vol 251 p718-25 Dennis,T., Fournier, A., Cadieux, A., Pomerlau, F., Jolicoeur, F.B., St.Pierre, S., Quirion, R.(1990) hCGRP8-37: a CGRP antagonist revealing CGRP receptor heterogeneity in brain and periphery. J.Pharmacol. Exp.Ther. vol 254 p 123-8 De Silva, L.B.,and De Silva, U.L.L.(1979)The chemical constituents of Symplocos racemosa Roxb. J.Natn.Coun.Sri Lanka vol 7 pl-3 Didcott, E.F. (1978)The encyclopaedia of alternative medicine and self help. Hulke,M. (ed). London, Rider and Co. pp 48-101 Dixon, B.S., Breckton, R., Fortune, J., Sutherland, E., Simon, F.R., Anderson, R.J. (1989) Bradykinin activates protein kinase C in cultured cortical collecting tubular cells. Am.J.Physiol. vol 257 p808-17 Dockray, G.J. (1988) Calcitonin Gene Related Peptide. ISI Atlas of Science : Pharmacology p40-45. Dohlman, H.C., Caron, M.G., Lefkowitz, R.J. (1987) A 6mily of receptors coupled guanine nucleotide regulatory proteins. Biochem. vol 26 p2657-64 Dondi, F., Kahie, Y.D., Lodi, G., Remelli, M., Reschiglian, P., Bighi, C. (1986) Evaluation of the number of conqwnents in multi-component liquid chromatograms of plant extracts. Analytica Chimica Acta vol 191 p261-73 Dray, A. and Perkins, M.N. (1988) Bradykinin activates peripheral capsaicin sensitive fibres via a secondary messenger system. Agents Actions vol 25 p214-15 Dray,A. and Perkins, M. (1993) Bradykinin and inflammatory pain. TINS vol 16 p99-104 221 Dray, A. and Bevan, S. (1993) Inflammation and hyperalgsia : highlighting the team effort. TIPS vol 14 p287-90 Drinkwater, C.C., Evans, B.A., Richards, R.I.(1988) Kallikreins, kinins and growth factor Hosynthesis TIBS vol 13 p 169-72 Edbrooke, M.R., Parker, D., Mcvey, J.H., Riley, J.H., Sorenson, G.D., Pettergill, O.S., Craig, R.K. (1985) Expression of the human calcitonin/CGRP gene in lung and thyroid carcinoma. E.M.B.O.I. vol 4 p715-24 Eggerickx, D., Raspe, E., Bertrand, D.,Vassart, G., Parmentier, M.(1992) Molecular cloning, functional expression and pharmacological characterisation of a human bradykinin B2 receptor gene vol 187 pl306-13 Ekblad, E., Hakanson, R., Sundler, F. (1988) Projections of peptide-containing neurons in rat colon. Neuroscience vol 27 p655-74 Elisabetsky, E. and Castilhos, Z.C. (1990) Plants used as analgesics by Amazonian Caboclos as a basis for selecting plants for investigation. Int.J.Crude.Drug.Res. vol 28 p309-320 Emele, J.F., Shanaman, J.,Warren, M R. (1961)The analgesic activity of phenelzine and other compounds. J.Pharmacol. vol 134 p206 Emele, J.F. and Shanaman, J.(1963) Bradykinin writhing: a method for measuring analgesia. Proc.Soc.Exp.Biol.Med. vol 114 p680-82 Fargeas, M.J., Fioramonti, J., Bueno, L. (1985) CGRPibrain and spinal action on intestinal motility. Peptides vol 6 pi 167-11 Farmer, S.G., Burch, R.M., Meeker, S.A.,Wilkins, D.E. (1989b) Evidence for a pulmonary B3 bradykinin receptor. Mol. pharmacol. vol 36 pi-8 Farmer, S.G., and Burch, R.M. (1992) Biochemical and molecular pharmacology of kinin receptors. Ann. Rev. Pharmacol. Toxicol. vol 32 p511-36 Farnsworth, N.R., Henry, L.K., Svoboda, G.H., Blonster, K.N., Yates, M.J., Euler, K.L. (1966)Plant Evaluation I. vol 29 plOl-22 Farnsworth, N.R. (1966)Biological and Phytochemical Screening of Plants. J.Pharm.Sci. vol 55 p225-76 Farnsworth, N.R. and Morris, R.W.(1976) Higher plants.:The sleeping giant of drug development. Am.J.Pharmac. vol 148 p46-52 Famswordi, N.R., and Soerjarto, D.D. (1985)Potential consequence of plant extinction in the United States on the current and future availability of prescription drugs. Econ. Bot vol 39 p231-40 Farnsworth, N. (1986) Screening plants for new medicines. Nat.Acad. Sci.Symposium, Washington DC 22-29 Sept. Farnsworth, N.R. (1988) In: Akerele,0. ,Heywood, V.and Synge,H.(eds)Conservation of medicinal Plants, Cambridge University Press, Cambridge, pp25-31 Farnsworth, N.R. (1994) Ethnopharmacology and Drug Development. Ciba Foundation Synçosium vol 185 42-51 discussion 51-59 222 Feldberg,W., Holden, H.F., Kellaway, C.H. (1938)The formation of lysocidiin and of a muscle- stimulating substance by snake venoms. J.Physiol. vol 94 p232-48 Feldberg,W. and Kellaway, C.H. (1938) Liberation of histamine and formation of lysocithin-like substances by cobra venom. J.Physiol.vol 94 pi87 Ferreira, S.H. (1972) Prostaglandins, aspirin-like drugs and analgesia. Nature vol 240 p2(X)-3 Findlay, D.M., Michelangeli, V.P., Martin, T.J., Orlowski, R.C., Seyler, J.K. (1985) Conformational requirements for activity of salmon calcitonin. Endocrinology vol 117 p801-5 Fischer, J.A., Tobler, P.H., Kaufman, M., Henke, H, Cooper, P.E., Sagar, S.M., Martin, J.B.(1981) Calcitonin : regional distribution of the hormone and its binding sites in the human brain and pituitary Proc. Natl. Acad. Sci. vol 78 p7801-5. Fischer, J.A., Kikkawa, D.O., Rivier, J.E., Amara, S.G., Evans, R.M., Rosenfeld, M.G., Vale, W.W., Brown, M.R. (1983)Stimulation of noradrenergic synçathetic outflow by CGRP. Nature vol 305 p534-6 Foch, S., Mense, S. (1976) Excitatory effects of 5-hydroxytryptamine, histamine and potassium ions on muscular group IV afferent units : A comparison with bradykinin. Brain Res. vol 105 p459-69 Foote, W.R., Achini, R., Romer, D. (1988) FS 205-397: A new antipyretic analgesic with a paracetamol-like activity but lack of acute hepatotoxicity in mice. Life Sciences vol 43 p905-12 Foster, G.M. (1983) An Introduction to Ethnomedicine. In: Traditional Medicine and Health Care Coverage, WHO, Geneva, Switzerland pp 17-24 Francel, P.C., Keefer, J.F., Dawson, G. (1989)Bradykinin analogs antagonise bradykinin-i nduced second messenger production in a sensory neuron cell line. Am.Soc.for Pharm. and Exp.Ther. vol 35 p34-8 Franz, M., Mense, S. (1987) Muscle receptors with group IV afferent responding to application of bradykinin. Brain Res. vol 92 p369-83 Frielle,T., Kobilka, M.G., Lefkowitz, R.S., Caron, M.G. (1988) Human B1 and B2 adrenergic receptors structurally and functionally related receptors derived from distinct genes. TINS vol 11 p321-24 Frotan, M.H., Acharya, S.B., Frotan, R., Pathak, N.K.R., Biswas, M.(1983)Pharmacological investigations on alpha spinosterol isolated from Symplocos spicata. Ind.J.pharmacol. vol 15 pl97- 201 Garbor, M. (1979) Anti-inflammatory substances of plant origin. In : Handbook of Experimental Pharmacology. J.R.Vane, S.H. Ferreira (eds) vol 50 pp 698-739 Springer-Verlag, Berlin Garcia, L.J. (1978) The potent algesic effects of exogenously administered bradykinin. Handbook Exp.Pharmacol. E.G.Erdos (ed) Springer -Verlag New York vol 50 pp 464-522 Geppetti, P.,Tramontana, M.,Santicioloi, E., Del-Branco, E.,Giuliana, S., Maggi, C.A.(1990)Bradykinin induced release of CGRP from capsaicin-sensitive nerves in guinea pig atria : mechanism of action and calcium requirements. Neuroscience vol 38 p687-92 Gibson, S.J., Polak, J.M., Bloom, S.R. (1984) Calcitonin Gene-Related Peptide immunoreactivity in the spinal cord of man and of eight other species. J.Neurosci. vol 4 p3101-ll 223 Gillman, A.G. (1987) G-proteins ; Transduction of receptor generated signals. Ann.Rev.Biochem. vol 56 p615-49 Goadsby, P.I., Edvinsson, L., Ekman, R. (1990)Vasoactive peptide release in the extracerebral circulation of man during migraine headaches. Ann. Neurol, vol 28 p i83-7 Goldstein, R.H.,Wall, M., Graeff, E.G., Corrado, A.P., Pela, I.R., Cape, K.R. (1984) Activation of protein formation and cell division by bradykinin and Des-Arg^BK. J.Biol.Chem. vol 259 p9263- 68 Gomez-Ponpa, A.,Vasquez-Yanes, C., Guerara, S. (1972)The tropical forest : A non-renewable source. Science vol 177 p762-65 Goodman, B.C. and Iversen, L.L. (1986) CGRPiNovel neuropeptide (1986) Life Sci.vol 38 p2169- 78 Griesbecker,T. and Lembeck, F. (1987) Effects of the bradykinin B4310 on smooth muscles and blood pressure in the rat, and its enzymatic degredation. Br.J.Pharmacol. vol 92 p333-40 Griesbecker, T., Lembeck, F. (1992) Analysis of the antagonistic actions of Hoe-140 and other novel bradykinin analogues on die guinea-pig ileum. Eur.J.Pharm. vol 211 p393-98 Hamann,0. (1991)The Joint lUCN-WWF plants conservation programme and its interest in medicinal plants. In: The Conservation of Medicinal Plants. Akerele, O., Hey wood, V., Synge, H.(eds) pp 13-21 Cambridge University Press Harbome, J.B. and Baxter, H., (eds) (1993) Phytochemistry dictionary : A handbook of bioactive compounds from plants. Taylor and Francis Publishing Hardy, J.D., Wolff, H.G., Goodell, H.(1950) Experimental evidence on the nature of cutaneous hyperalgesia. J.Clin.Invest, vol 29 pi 15-40 Hargrave, P.A. (1982) Rhodopsin chemistry, structure and topography. Prog.Retinal Res. vol 1 pl- 52 Haslam, E. (1966) Chemistry of vegetable tannins. Academic Press Inc. London. Haslam, E. and Lilley, T.H. (1988) Natural astringency in foodstuffs : A molecular interpretation vol. 27 pl-40 Haslam, E., Lilley, T.H., Cai, Y., Martin, R., Magnolato, D. (1989) Traditional herbal mediciæs - The role of polyphenols. Planta Med,vol 55 p i-8 Hay ley, J.E., Dickenson, A.H., Schachter, M. (1989) Electrophysiology evidence for a role of bradykinin in chemical nociception in the rat. Neurosci.Lett pl98-202 Hendershot, L.C. Forsaith, J. (1959)Antagonism of the frequency of pheiylquinone-induced writhing in the mouse by weak analgesics and non-analgesics. J.Pharmacol. vol 125 p327 Heftmann, E. (1977)Functions of steroids in plants. Prog. Phytochem. vol 4 p257-76 Pergamon Press Heinz, J. and Seewan, S. (1980) Selective reduction by some vasodilators and the prostagalndin antagonist SC-19220 of a response to the algesic effect of bradykinin. Eur. J. Pharmacol, vol 65 p267-78 224 Heller,w ., Forkmann, G., Britsch, L., Griesbach, H. (1985) Enzymatic reduction of (+ ) - dihydroflavonols to flavan -3,-4 - cis - diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis. Planta vol 165 p284-87 Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, P., Beckman, E., Downing, K.H. (1990)Model for the structure of bacteriorhodopsin based on high resolution electron cryo- microscopy. J.Mol.Biol. vol 213 p899 Henke, H., Sigrist, S., Lang, W., Schneider, J., Fischer, J.A. (1987) Comparison of binding sites for CGRP I and II in Man. Brain Res. vol 410 p404-8 Hepler, J.R. and Gilman, A.G. (1992) G-Proteins. TIBS vol 17 p383-87 Heuillet, E., Menager, J., Fardin, V., Flamand, O., Bock, M., Garret, C.A., Crespo, A.M., Fallourd, A.M., Doble, A. (1993) Characterisation of a human NKl tachykinin receptor in the astrocytoma cell line U373MG. J.Neurochem. vol 60 p868-76 Higashada, H., Streaty, R.A., Klee, W,N., Nirenberg, M. (1986) Bradykinin activated transmembrane signals are coupled via NO or Ni to production of inositol 1,4,5-triphosphate, a second messenger in NG108-15 neuroblastoma glioma hybrid cells. Proc.Natl.Acad.Sci. vol 83 p942-46 Higuchi, R., Kawasaki, T., Biswas, M., Pandey, V.B., Dasgupta, B. (1982)Triterpenoid saponins from the stem bark of Symplocos spicata. Phytochemistry vol 21 p907-10 Hirata, Y.,Takagi, S., Fukuda,Y.,Yoshimi, H., Fujita, T. (1988) CGRP receptors in cultured vascular smoodi muscle and endodielial cells. Biochem.Biophys.Res.Commun vol 151 pi 113-21 Hock, F.J.,Wirth, K., Albus, U.,Linz, W., Gerhaas, H.J., Weimer, G., Henke, S., Breipohl, G., Konlig, W., Knolle, J., Scholkens, B.A. (1991) Hoe-140, a new potent and long acting bradykinin antagonist:in vitro studies. Br.J.Pharmacol. vol 102 p769-73 Hokfelt,T.,Kelleth, J.O., Nilsson, G., Pemow,B. (1975) Immunohistochemical evidence for the prescence of somatostatin, a powerful inhibitory pepyide, in some primary sensory neurons. Neurosci.vol 1 pl31-6 Hokfelt,T., Everitt, B., Holets, V.R., Meister, B., Melander,T., Schalling, M., Staines, W., Lundberg, J.M. (1986) In:Fast and slow chemical signalling in the nervous system. Iversen, I.I, Maudnent, J, Goodman, E.C (eds) Oxford University Press. Oxford Hollister, L.E. (1978) Clinical Pharmacology. In:Melmon,L.K. and Morelli,H.F.(eds)2nd ed. Macmillan Publ.Co. Ltd. New York, USA pp874-912 Holmstedt, B. and Bruhn, J.G. (1983) Ethnopharmacology-A challenge. J.of Ethnopharmacol.vol 8 p251-56 Hostettmann, K., Hostettmann, M. (1982) In:Flavonoids:Advances in Research. J.B.Harborne and T.J.Mabry (eds) Chapman and Hall, London Humphrey, P.P.A., Apperley, E., Fenuik, W., Perren, M.J. (1991) ImSaxena P.R, Wallis,D.I, Wouters,W, Veran,P(eds) Kluwer Academic Publishers pp417-31 Huskisson, E.C. (1974) Measurement of pain. Lancet vol 2 pi 127-31 Hussain, R.A., Lin, Y.M., Poveda, L.J., Bordas, E., Chung, B.S., Pezzuto, J.M., Soejarto, D.D., Kinghom. (1990) Plant derived sweetening agents : saccharide and polyol constituents of some sweet-tasting plants. J.Ethnopharmacol. vol 28 pl03-15 225 lidaj., Hayashi, M., Murata, T., Ono, M., Inoue, K., Fujita, T. (1990) Identification of a new minor iridoid in Symplocos glauca by thermospray liquid chromatography-mass spectrometry. J.Chromatog. vol 515 p503-8 Innis,R.B., Manning, D.C., Stewart, J.M., Snyder, S.H. (1981)^[H]BK Bradykinin receptor binding in mammalian tissue membranes. Proc. Nad. Acad.Sci. USA vol 78 p2630-34 Irwin, H.S. (1978)In;Extinction is forever, 3rd edition. Prance,G.T. and Elias,T.S.(eds). Bronx, New York Botanical Gardens. Jaffe, J.H. and Martin, W.R. (1980) Opioid analgesics and antagonists. In : The pharmacological basis of therapeutics. A.G.Gilman, L.S. Goodman and A.Gilman (eds) pp 494 - 534, MacMillan, New York Jolicoeur, F.B., Michaud, J.N., Menard, D., Fournier, A., St.Pierre,S. (1989) Neurobehavioural effects of CGRP. Soc. Neurosci. Abstr. vol 15 p288 Juan, H. and Seewann, S. (1980)Selective reduction by some vasodilators and the prostaglandin antagonist SC-19220 of a response to the algesic effect of bradykinin. Eur.J.Pharmacol. vol 65 p267-78 Kahn, A.A., Raja, S.N., Campbell, J.N., Hartke, T.V., Meyer, R.A. (1986) Bradykinin sensitisation of nociceptors. Neurosci. vol 12 p218 Kahn, A.A., Raja, S.N., Campbell, J.N., Hartke, T.V., Meyer, R.A. Bradykinin sesitises nociceptors to heat stimuli. Neurosci. Abstracts vol 388 p9-23 Kapoor, S.L. and Mitra, R. (1979)Herbal drugs in the Indian pharmaceutical Industry. National Botanical Research Institute, Lucknow, India. Keele, C.A. and Armstrong, D. (1964) Substances producing pain and itch. Arnold Publ. London Kermode, J.C. (1989) The curvilinear scatchard plot: experimental artifact or receptor heterogeneity?. Biochem.Pharm. vol 38 p2053-2060 Kitamura, N., Takagaki,Y., Farato, S., Tanaka, T., Nawa, H., Nakanishi, S. (1983) A single gene for bovine high molecular weight and low molecular weight kininogens. Nature vol 305 p545-49 Kremer, E., Atkinson, J.H., and Ignelzi, R.J. ( 1981 )Measurement of pain : Patient preference does not confound pain measurement. Pain vol 10 p241-48 Kruger, L., Mantyh, P., Sterini, C., Brecha, N., Mantyh, C. (1988) CGRP in the rat CNS : Patterns of immunoreactivity and receptor binding Sites. Brain Res. vol 463 p9223-44 Kummer, W. and Heym, C. (1991) Different types of CGRP immunoreactive neurons in the guinea pig stellate ganglion as revealed by triple-labelling immunofluorescence. Neurosci. Letts, vol 128 pl87-90 Kurup, P.N.V. (1983)Ayurveda. In : Traditional medicine and psychiatry in Africa. P.Copo (eds) Kyte, J., Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J.Mol.Biol. vol 157 pl05 Lance, J.W.(1977) The cephalgias with special reference to vascular and muscle-contraction headaches. In : Goldenstein, E.S., Aphel, S.H (eds) Scientific Approaches to Clinical Neurology : Phila PA:Lea and Febiger ppl958-77 226 Lang, E., Novak, A., Reeh, P.W., Handwerker, H.O. (1990)Chemosensitivity of fine afferents from rat skin in vitro. J.Nenrophys. vol 63 p887-901 Lanly, J.P. (1982) Tropical Forest Resources. Forestry Paper No.30 Rome, FAO Law,T. (1990) Bush Medicine. A Pharmacopoeia of Natural Medicines. Angus and Robertson, London Lazure, C., Seidah, N.G., Pelaprat, D., Chtetien, M. (1984) Proteases and post translational processing of prohormones : a review. Can. J. Biochem. Cell. Biol, vol 61 p501-15 Lee, N.H, and Kerlavage (1993) Molecular biology of G-protein -coupled receptors. TIBS vol 6 P488-497 Lee, Y., Kawai, Y., Shiosaka, S., Takami, K., Hiyama, H., Hillyard, C.J., Girgis, S.L, Maclnytre, L, Emson, P.C., Tohyama, M. (1985) Coexistence of CGRP and substance P-like peptide in single cells of the trigeminal ganglion of the rat. Brain Res. vol 330 pl94-6 Lee, C M., Kum, W., Cockram, W.S., Teoh, R., Young, J.D. (1989) Functional Substance P receptors on a human astrocytoma cell line (U373MG). Brain Res. vol 488 p328-31 Le Greves, P., Nyberg, F., Terenius,T., Hokfelt, T. (1985) CGRP is a potent inhibitor of Substance P degredation. Eur.J.Pharmac. vol 115 p309-ll Le Greves, P., Nyberg, F., Hokfelt, T.,Terenius, T. (1989) CGRP is metabolised by an endopeptidase hydrolysing Substance P. Reg.Pept.vol 25 p277-86 Lenz, H.J., Mortrud, M.T., Rivier, I.E., Brown, M.R. (1985) CNS actions of CGRP on gastric acid secretion in the rat. Gastroenterology vol 88 p539-44 Leung, A. Y. (1980) In : Encyclopaedia of common natural ingredients used in food,drugs and cosmetics. J. Willey (ed) New York Lewis,T. (1927) Blood vessels in human skin and their responses, pp 322 Shaw and Son Ltd, London Lewis,T.(1933)Clinical observations and experiments relating to burning pain in the extremities, and to so-called ’erythromelalgia’ in particullar. Clinical science vol 1 pl75-211 Lewis,R.,Childens,S.R.,Phillips,M.I.(1985) t^^^ll-Tyr-BK binding in primary rat brain cultures. Brain Res. vol 346 p263-72 Liebmann, C., Offermanns, S., Spicher, K., Hinsch, K., Schnittler, M., Morgat, J.L., Reissmann, S., Schultz, G., Rosenthal, W. (1990) A high affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin sensitive G-proteins of the Gi family. Biochem. Biophys.Res.Commun, vol 167 p910-17 Liebmann, C., Reissmann, S., Roberecht, P., Arnold, H. (1991) Bradykinin action in the rat duodenum ; Receptor binding and influence on the cyclic AMP system. Biomed. Biochem. Acta, vol 46 p469-78 Liebmann, C., Schnittler, M., Stewart, J.M., Reibmann, S. (1991) Antagonist binding reveals two heterogeneous B2 bradykinin receptors in rat myométrial membranes. Eur. J. Pharmacol, vol p363- 65 Leighton, B. and Foot, E.A. (1990)The effects of amylin on carbohydrate metabolism in skeletal muscle in vitro and in vivo. Biochem. J. vol 269 pl9-23 Lim, R.K.S., Miller, D.G., Guzman, F., Rogers, R.W.,Wang, S.K., Chas, P.Y. Shih,T.Y. (1967) Pain and analgesia evaluated by the intraperitoneal bradykinin evoked pain method in man. Clin. Pharmacol. Ther. vol 8 p521-42 227 Lindsay, R.M., Lockett, C., Sternberg, J.,Winter, J. (1989)Neuropeptide expression in cultures of adult sensory neuronesimodulation of Substance P and CGRP levels by NGF. Neurosci. vol 33 p53- 65 Logothetis, D.E., Kurachi, Y., Galper, J., Neer, E.J., Clapham, D.E. (1987) The beta gamma subunits of GTP binding proteins activate the muscarinic potassium channel in heart. Nature vol 325 p321-26 Loub, W.B., Farnsworth, N.R., Soejarto, D.D., Quinn, M.L. (1985) NAPRALERT : Conçuter Handling of Natural Product Research Data. J.Chem. Inf. Conq>ut. Sci. vol 25 p99-103 Maggi, C.A., Patacchini, R., Rovero, P., Giachetti, A. (1990)Immunoblackade by a specific tachykinin antiserum of the non-cholinergic contractile responses in the guinea-pig isolated bronchus. J. Auton. Pharmacol, vol 10 pl73-9 Majerius, P.W., Connolly, T.M., Deckym, H., Ross,T.S., Bross, T.E., Ishii, H., Bansal, V.S., WUson, D.B. (1986) The metabolism of phosphoinositide-derived messenger molecules. Science vol 234 pl519 Mancici, S.D., Edwards, J.M. (1979) Cytotoxic principles from the sap of Kalnda Latifolia . J.NatProd. vol 42 p483-82 Manning, D.C., Snyder, S.H., Kachur, J.F., Miller, R.J., Field, M. (1982)Bradykinin receptor mediated chloride secretion in intestinal function. Nature vol 299 p256-59 Manning, D C., and Snyder, S.H. (1983) ^[H]BK receptor localisation in spinal cord and sensory ganglia - evidence for a role in primary afferent function. Soc. Neurosci. Abstracts vol 9 p590 Manning, D C., Dehaas, C.J., Ferkany, J.W., Borosky, S.A., Connor, J.R., Vavrek, R.J., Stewart, J.M., Snyder, S.H. (1988) Bradykinin as a pain mediator;receptors are localised to sensory neurones and antagonists have analgesic action. Proc. Natl. Acad. Sci. USA vol 85 p3245-49 Marceau, F., Lussier, A., Regoli, D., Giroid, J.P. (1983) Pharmacology of kinins:Their relevance to tissue injury and inflammation. Gen. Pharmacol, vol 14 p204-229 Marceau, F. and Regoli, D.(1991) Kinin receptors of the B1 type and their antagonists. In:Bradykinin antagonists ; basic and clinical research. R.M.Burch (ed) Marcel Dekker New York pp33^9 Marshall, I., AlKazwini, S.J., Roberts, P.M., Shepperson, N.B., Adams, M., Craig, R.K. (1986)Human and rat alpha-CGRP but not calcitonin cause mesenteric vasodilation in rats. Eur.J. Pharmacol.vol 123 p207-16 Martin, D., Lilley, T.H., Bailey, N.A., Falshaw, C.P., Haslam,E, Magnolata, D., Begley, M.J. (1986)Polyphenol-caffeine complexation, J.Chem.Soc.Chem. Commun. pl05-6 Mason, R.T., Peterfi-eund, R.A., Sawchenko, P.E., Corrigan, A.Z., Rivier, J.E., Vale, W.W. (1984) Release of die predicted CGRP from cultured rat trigeminal ganglion cells. Nature vol 308 p635-38 Mattison, N.,Trimble, A.G., Lasagna, L. (1988) New drug developments in the U.S.A. 1963 through 1984. Clin.Pharmacol. Therapeutics, vol 43 p295-301 McEachem, A.E., Shelton, E.R., Bhakta, S., Obemolte, R., Bach, C., Zippan, P., Fujisaki, J., Aldrich, R.W., Jamagin, K. (1991) Expression cloning of a rat B2 bradykinin receptor. Proc.Natl.Acad.Sci. USA vol 88 p7724-28 McGehee, D.S., Goy, M.F., Oxford, G.S. (1992) Involvement of the nitric oxide-cyclic AMP pathway in the desensitisation of bradykinin responses of cultured rat sensory neurones. Neuron vol 9 p315-24 McLean, E.K. (1970) Derivatives of 1,8-dihydroxyanthraquinone. Pharmacol. Rev. vol 22 p429 228 Meller, S.T., Gebhart, G.F.(1992)Bradykinm Antagonists. Neuroscience vol48p501-24 Menziani, E.,Tosi, B., Bonora, A., Reschiglian, P., Lodi, G. (1990) Automated mutiple development high performance thin layer chromatographic analysis of natural phenolic confounds. J.Chromatography vol 511 p396-401 Miller, D M. and Margolius, H.S. (1988)KaUil^ein-kininogen-kinin system. 2nd Edition Endocrinology. DeGroot, L.J., Cahill, G.E. Jr. (eds) Grune and Stratton, USA. Mimeault, M., Fournier, A., Dumont., Y., St.Pierre, S., Quirion, R. (1991) Comparative affinities and antagonistic potencies of various human CGRP fragments on CGRP receptors in brain and periphery. J.Pharmacol.Exp.Ther. vol 258 pl084-89 Moore, R.Y. (1989) Cranial motor neurons contain either galanin or CGRP-like immunoreactivity. J.Comp.Neurol. vol 282 p512-22 Mora, M., Marchi, M., Polak, J.M., Gibson, S.J., Comelio, F. (1989) CGRP immunoreactivity at die human neuromuscular junction. Brain Res. vol 492 p404-7 Morley, J.E. (1987)Neuropeptide regulation of appetite and weight Endocr. Rev. vol 8 p256-87 Morris, H.R., Punico, M., Etienne, T.,Tippins, J., Girgis, S.L, Macintyre, I. (1984) Isolation and characterisation of human CGRP. Nature vol 308 p746-48 Morton, J.F. (1981) Atlas of Medicinal Plants of Middle America. Charles C.Thomas, Publishing. Moskowitz, M.A., Brody, M., Liu-Chen, L.Y. (1983) In vitro Release of immunoreactive Substance P from putative afferent nerve endings in bovine pig arachnoid. Neusosci. vol 9 p809-14 Moskowitz, M.A. (1984) Neurobiology of vascular head pain. Ann.Neurol, vol 4 p 157-68 Moskowitz, M.A. (1990) Basic mechanisms of headache. Neurol. Clin.vol 8 p801-15 Moskowitz, M.A. (1991) The visceral organ brain:Inq>lications for the pathophysiology of vascular head pain. Neurology vol 41 pl82-6 Moskowitz, M.A. (1992)Interpreting Vessel Diameter Changes in Vascular Headaches. Cephalagia vol 12 p5-7 Mulderry, P.K., Ghatei, M.A., Rodrigo, J., Allen, J.M., Rosenfeld, M.G., Polak, J.M., Bloom, S.R. (1985) CGRP in cardiovascular tissues of the rat. Neurosci. vol 14 p947-54 Mulderry, P.K., Ghatei, M.A., Spokes, R.A., Jones, P.M., Pierson, A.M., Hamid, Q.A., Kanse, S., Amara, S.G., Burrin, J.M., Legon, S., Polak, J.M., Bloom, S.R.(1988) Differential expressions of a - CGRP and p - CGRP by primary sensory neurons and enteric autonomic neurons of die rat. Neurosci.vol 25 pi95-205 MuUer-Esterl,W. (1990) Kininogens,Kinins, and Kinships. .J.Car.Pharm. vol 15 (suppl. 6) S1-S6 Murrayama, T., Ui, M. (1987) Phosphatidic acid may stimulate membrane receptor mediating adenylate cyclase inhibition and phosphatidic breakdown in 3T3 fibroblasts. J.Biol.Chem. vol 262 p5522-29 Myers, N. (1984) The Primary Source. W.W.Norton and Co.Publ. New York Myers, N. (1987) Tropical deforestation and a mega-extinction spasm. In: Conservation Biology.M.E.Soule (ed) Massachusettes : Sin auer Associates Inc. Nakamura, H., Fukuda, Y., Koida, M., Fujii, N., Otaka, A., Funakoshi, S., Yajima, H., Mitsuyasu, N., Orlowaski, R. (1986) Jpn.J.Pharmacol. vol 42 p 175-80 Nakanishi, K., Goto, T., Ito, S., Natori, S., Nozoe, S.(eds) Natural Products Chemistry. Vol. 1 New York Academic Press Nakanishi, S. (1991) Mammalian tachykinin receptors. Ann. Rev.Neurosci. vol 14 pl23-126 229 Nathans, J. (1987) Molecular Biology of Visual Pigments. Ann. Rev.Neurosci. vol 10 pl63 Neer, E.J., Clapham, D.E. (1988)Roles of G-protein subunits in transmembrane signalling. Nature vol 333 pl29-34 Nicholson, G.C., Moseley, J.M., Sexton, P.M., Mendelsohn, F.A.O., Martin, T.J. (1986) Abundant calcitonin receptors in isolated rat osteoclasts ; biochemical and autoradiographic characterisation. J.Clin.Invest vol 265 p355-61 Odya, G.E., Goodfriend,T.L., Pena, C. (1980) Bradykinin receptor-like binding studies with iodinated analogues. Biochem. Pharmacol, vol 29 pl75-85 Oliver-Bever, L. (1986)Medicinal plants in tropical West Africa. Cambridge University Press Pasternak, G. (1984) Analgesics : Neurochemical, behavioural and clinical perspectives. Kuhar, M. (ed) Raven Press new York, USA. Pecile, A., Guidobono, P., Netti, C., Sibila, V., Biella, G., Braga, P.C. (1987) CGRP : Antinociceptive activity in rats, conq)arison with calcitonin. Reg.Pept. vol 18 pl89-99 Peigen, X. (1980) Traditional experience of Chinese herb medicineilts application in drug research and new drug searching. Planta Med. vol 351-94 Penso, G. (1982)Index Plantarum Medicilinalium Totius Mundi Eorumque Synonymoru. Organizzazione Editoriale Medico Farmaceutica, Milan ppl-1026 Pemow, B. (1983) Substance P. Neuropharmacol. Rev. vol 35 p85-41 Phillipson, J.D. and Anderson, L.A. (1984) Plants as Sources of New Medicines. Pharm.J. vol 232 p41-# Portilla, D.,Morrissey, J., Morrison, A.R.(1988) Brdaykinin activates membrane-associated phospholipase C in Madin-darby canine kidney cells. J.Clin.Invest, vol 81 pl896-1902 Powell, S.J., Slynn, G., Thomas, C., Hopkins, B., Briggs, I., Graham, A. (1993)Humanbradykinin B2 receptor : nucleotide sequence analysis and assignment to chromosome 14. Genomics vol p435- 38 Poyner, D R. (1992) CGRP : Multiple actions, multiple receptors. Pharm. Therap. vol56 p23-51 Poyner, D.R., Andrew, D.P., Brown, D., Bose, C., Hanley, M.R. (1992) Pharmacological characterisation of a receptor for Calcitonin Gene-Related Peptide on rat, L6 Myocytes. Br.J.Pharm.Prac. vol 105 p441-47 Prance, G.T. (1977) Floristic Inventories of the Tropics : where do we stand?. AnmMissouri Bot. Guard, vol 64 p659-84 Prance, G.T. (1978) Extinction is forever. New York Botanic Garden, Bronx Prescott-Allen, R., Prescott-Allen, P.(1982) What’s wildlife worth?. London Int. Institute for Environment and Development. Principe, P. (1991) Valuing the biodiversity of medicinal plants. In : Conservation of Medicinal Plants. Akerele, O., Hey wood, V., Synge, H. (eds) pp79-124 Cambridge University Press, Cambridge Ramabadra, K., Bansinath, M., Tumdorf, H., Puig, M.M. (1989) Tail immersion test for the evaluation of a nociceptive reaction in mice. Methodological considerations J.Pharmacol. Methods, vol 21 p21-31 Ramaswamy, A S. (1970) Pharmacological action of the biflavonoids from the gymnosperms widi special reference to Ginko biloba, L. J. Ind. Med.Assoc, vol 55 pl63-5 Regoli, D., Barabe,J. (1980)Pharmacology of bradykinin and related kinins Pharmacol.Rev.vol 32 pl-46 230 Regoli, D. (1984) Neurohormonal regulation of precapillary vessels : The kaUikrein-kinin system. J.Cardiovascular Pharmacol, vol 6 p5401-412 Regoli, D., Rhaleb, N.E., Dion, S., Drapeau, G. (1990) New selective bradykinin receptor antagonists and bradykinin B2 receptor characterisation. TIPS vol 11 pl56-61 Reid, G., Bick, M. (1942) Pharmacologically active substances in serum. Aus.J.Exper.Biol. and M.Sc. vol 20 p33 Regoli, D., Barabe, J,(1980) Pharmacology of bradykinin and related kinins. Pharmacol. Rev.vol 32 pi Roberts, R.A., Gullick, W.J. (1989) Bradykinin receptor number and sensitivity to ligand stimulation of mitogenesis by expression of mutant vas oncogene. J.Cell Sci. vol 94 p527-35 Robbins, S.L., Cotran, R.S. (1979) Pathologic basis of disease. 2nd ed. W.B.Saunders co. Philadelphia. Romer ,D. (1980) Pharmacological evaluation of mild analgesics. J.Clin.pharmac. vol 10 p247-251 Roscher, A, A., Manganiello,V., Jeselma, C.L., Moss, J. (1984) Autoregulation of bradykinin receptors and bradykinin -induced prostacyclin formation in human fibroblasts. J.Cli. Invest, vol 74 p552-58 Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., Evans, R.M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature, vol 304 p 129-35 Rueff, A, Dray,A .(1992) 5-HTP induced sensitisation and activation of peripheral fibres in the neonatal rat are mediated via different 5-HTP receptors. Neurosci. vol 50 p899-905 Rustogi, R.N. (1980) The search for new medicinal plants from Indian flora. In : Advance notes on Syn^sia and discussions, 67th Session of Indian Science Congress.ppl6-17 Saria, A., Gamse, R., Peterman, J.A., Fischer, E., Norheim, T., Lundberg, J.M. (1986) Simultaneous release of several tachykinins and CGRP from rat spinal cord slices. Neurosci. Letts, vol 63 p310-14 Savarese, T.M., Fraser, G.M. (1992) G - Protein coupled receptors. J.Biochem. vol 283 pl-19 Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann.New York.Acad. Sci. vol p660-72 Schultes, R.E. (1972) In : Plants in the development of modem medicine. Swain,T(eds) Cambridge. Harvard University Press, pl03-24 Schultes, R.E., Swain,T. (1976) In : The recent chemistry of natural products, including tobacco. New York Proceedings of Phillip Morris Science Symposium. Schultes, R.E. (1986)Ethnopharmacological conservation ; a key to progress in medicine. Opera Botanica vol 92 p217-24 Schultes, R.E., Raffauf, R.F. (1990) The Healing Forest-Medicinal and toxic plants of the Northwest Amazonia. Dioscorides Press. Schultes, R.E. (1993) Plants in treating senile dementia in the Northwest Amazon. J.Edmopharmacol. vol 38 pl29-35 Seguin, L. and Widdowson, P.S. (1993 )Effects of nucleotides on ^[H]BK binding in guinea pig : further evidence for multiple B2 receptor subtypes J.Neurochem. vol 60 p752-57 Seidah, N.G., Cromlish, J.A., Hamelin, J.,Thibault, G., Chretien, M. (1986) Homologous ICRM- serine proteases from pititary, heart atrium and ventricle : a common pro-enzyme maturation enzyme. Biosci. vol 6 p835-44 231 Seidah, N.G., Faquin, J., Hamelin, J., Benjannet, S., Chretien, M. (1988) Structural and immunological homology of murine and porcine pituitary and plasma ICRM-serine proteases 1 to plasma kaUikrein : marked selectivity fiar pairs of basic residues suggests a widespread role in pro hormone and pro-enzyme processing. Biosci. vol 70 p33-46 Seifert, H., Sawchenko, P., Chestnut, J., Rivier, J., Vale, W.W., Pandol, S.J. (1985)Receptor for calcitonin gene-related peptide : binding to exocrine pancreas mediates biological actions. Am. J. Physiol, vol 249 p 147-51 Sexton, P.M., McKenzie, J.S., Mendelsohn, F.A.O. (1988)Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding site in rat brain. Neurochem. Int.vol 12 p323-35 Sherrington, C.S. (1906) The integrative action of the nervous system. New York Scribner pp226- 230 Sibley, D.R., Lefkowitz, R.J. (1985) Molecular mechanisms of receptor desensitisation using the p- adrenergic receptor coi^led adenylate cyclase system as a model. Nature vol 317 pl24-29 Sigrist, S., FrancoCereceda, A., Muff, R., Henke, H., Lundberg, J.M., Fischer, J.A. (1986) Specific receptor and cardiovascular effects of CGRP. Endocrinology vol 119 p381-80 Silva, R.E., Beraldo, W.T., Rosenfeld, G. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. J. Physiol, vol 156 pl56 Silverman, J., Kruger, L. (1987) CGRP immunoreactive innervation of tiie rat head with emphasis on specialised sensory structures. J.Comp. Neurol, vol 280 p303-30 125 Skofitsch, G., Jacobwitz, D.M. (1985)Autoradiographic distribution of I -CGRP binding sites in the rat CNS Peptides vol 6 p975-86 Smith,}.J., McCaan, J.D., Welsh, M.J. (1990)Bradykinin stimulates airway epithelial Cl ‘ secretion via two second messenger pathways. Am. J.Physiol. vol 258 p369-77 Snell, P.H., Snell,C.R. (1988) Solubilisation of brarfykinin binding sites from rat uterine membranes. Biochem. Soc. Transac. vol 17 p569-70 Snider, R.M., Richelson, E. (1984) Bradykinin mediated cyclic GMP formation in a nerve ceU population(murine neuroblastoma clone NlE-115) J.Neurochem. vol 43 pl749-54 Soejarto, D.,D.(1993) Logistics and Politics in Plant Drug Discovery : The Other End of the Spectrum In : Human Medicinal Agents From Plants. Kinghom, D.A.,Balandrin, M.F.(eds) ACS Syn^sium Series No. 534. American Chemical Society. Washington D.C. pp96-lll Spiegel, A.M., Shenker, A., Weinstein, L.S. (1992)Receptor-effector coupling by G proteins : Implications for normal and abnormal signal transduction. End.Rev. vol 13 p536^5 Stangl, D., Bom, W., Fischer, J.A. (1991)Characterisation and photoaffinity labelling of a CGRP receptor solubilised fi:om human cerebellum. Biochemistry vol 30 p8605-ll Steranka, L.R., Manning, D C., Dehaas, C.J., Ferkaity, J.W., Borosky, S.A., Connor, J.R., Vavrek, R.J., Stewart, J.M., Snyder, S.H. (1988)Bradykinm as a pain mediator : receptors are localised to sensory neurons and antagonists have analgesic actions. Proc. Natl.Acad.Sci. USA vol 85 p3245^9 Steranka, L.R., Burch, R.M., Vavrek, R.J., Stewart, J.M., Enna, S .J. (1988)Multçle bradykinin receptors : results of studies using a novel class of receptor antagonists. Adv.Exp.Med.Biol. vol 236 pi 11-27 Steranka, L.R., Farmer, S.G., Burch, R.M. (1989) Antagonists of BK2 receptors. FASEB J.vol 3 p2019-25 232 Stewart, J.M., Vavrek, R.J. (1986) Bradykinin competitive inhibitors fi)r classic kinin systems. In: Kinins IV. Greenbaum, L.M and Margolius,H(eds) Plenum Press, New York Stewart, J.M., Vavrek, R.J. (1987a)Bradykinin con^titive antagonists : design and activities In: Enzymes and enzyme inhibitors. R.L. Schwen and Barth, A. (eds) Pergamon Press, New York pp73-80 Stewart, J.M., Vavrek, R.J.(1987b) Design of bradykinin antagonists. In : Proceedings of the 10th American Peptide Symposium. Marshall,G.R (eds) ESCOM Science Publishers, Nedierlands. Stewart, J.M., Vavrek, R.J. (1990) Kinin antagonists : design and activities. J.Cardiovascular Pharmacol, vol 15 (suppl) s69-74 Stoner, J, Maganiello, V.C, Vaughan, M. (1973) Effects of bradykinin and indomethacin on cyclic GMP and cyclic AMP in lung slices. Proc.Natl.Acad. Sci. vol 70 p3830-33 Sunthonopalin, P., Kritalugsana, S., Chantarakul, N., Thianprasit, M. (1985) J.Med.Assoc.Thailand vol 68 p505-ll Sweetman, P.M., Caldwell, L., Lancaster, J., Bauer, C., McMillan, B., Kimiier, W.J., Price, C.H. (1993 )The role of receptor binding in drug discovery. J.Nat.Prod. vol 56 p441-455 Szolcsaityi, J. (1987) Selective responsiveness of poly modal nociceptors of the rabbit ear to capsaicin, bradykinin and U.V. irradiation. J.Physiol. vol 388 p9 - 23 Taiwo, Y.O. and Levine, J.D. (1992) Serotonin is a directly acting hyperalgesic agent in the rat. Neurosci. vol 48 p485-90 Tanaka,T.,Yamasaki, K., Kohda, H., Mahato, S.B. (1980) Dihydrochalcone-glucosides as sweet principles of Symplocos spp. Planta Med. suppl. p81-3 Tanaka,T., Kawamura, K., Kohda, H., Yamasaki, K., Tanaka, O. (1982) Glycosides of the leaves of Symplocos 5p/?.(Symplococaceae) ChenLPharm.BuU. vol 30 p2421-23 Takeda, M., Kitjin, N., Girgis, S., Hillyard, C.J., MacIntyre, I., Emson, P.C., Shiosakei, S., Tohyama, M. (1986)The presence of a CGRP receptor in the olivocochlear bundle in rat. Exp. Brain Res. vol 61 p575-78 Tamm, R. (1978) Helv.Chim.Acta. vol 61 pl814 Tannebaum, G.S., Goltzman, D. (1985) CGRP mimics calcitonin actions in brain on growth hormone release and feeding. Endocrinology vol 116 p2685-87 Taylor, B. (1978) J.Chem.Res. vol 301 Taylor, J.E., DePeudis, F.V., Moreau, J.P.(1989) Bradykinin antagonists : therapeutic perspectives. Drug Dev.Res. vol 16 pl-11 Tiffany, C.W., Burch, R.M. (1989) Bradykinin stimulates tumor necrosis fector and interleukin-1 release from macrophages. FEBS letts vol 247 pi 89-92 Tiwari, R.D., Tripathi, H.L. (1976) A new flavonol glycoside from die leaves of Symplocos spicata. Phytochemistry vol 15 p833 Tiwari, R.D. and Tripathi, H.L. (1976)The structure of an arabinogalactan from the leaves of Symplocos spicata. Planta Med. vol 29 p376-79 Togo J., Burch, R.M., Dettaas, C.J., Cannov, J.R., Steranka, L.R. (1989) D-phe^ subsdtuted peptide bradykinin analogues are not substrates for kininase n. vol 10 pl09-l 12 Tota, M.R., Strader, C.D. (1990) Molecular biology of visual pigments. J.Biol.Chem vol 265 pl6891 Trease, G.E., Evans,W.C.(1978) Pharmacognosy. Balliere Tindall, London 233 Trethewie, E.R. (1941)The liberation of pharmacologically active substances from tissues by chloroform. Aus.J.Exp.Biol. and Mol. Sci. vol 19 pl75 Tschesche, R., Welzel, P., Moll, R., Legler, G. (1964) Uber 2 Alkaloide aus der rinde von Symplocos celastrinea mart. Tetrahedron vol 20 p1435-47 Tschesche, R., Braun, T.M., Sassen, W.V. (1980) Symplocoside, a flavanol glycoside from Symploœs uniflora. Phytochemistry vol 19 p i825-29 Tschopp, Henke, H., Petermann, J.B., Tobler, H., Janzer, R., Hockfelt, J.M., Lundberg, C., Cuello, C., Fischer, J.A. (1985) CGRP and its binding sites in the human CNS and Pituitary Proc. Natl. Acad.Aci. vol 85 p248-52 Tyler, V.E., Brady, L.R., Robbers, J.E. (1976) Pharmacognosy 7th ed. Lea and Febiger, Philadelphia USA. Tyler,V.E.(1986)Plant drugs in the twenty first century. Econ.Bot. vol 40 p279-88 Vane, J.R. (1969) The release and fate of vasoactive hormones in the circulation. B.J.Pharmacol. vol 35 P209-42 Van Halen, F., Piechot, G., Jurgens, H. (1990) CGRP receptors are linked to cyclic adenosine monophosphate production in SK-N-MC neuroblastoma cells. Neurosci.Letts, vol 119 pl95-8 Vavrek, R.J., Stewart, J.M. (1985) Competitive antagonists of bradykinin. Peptides vol 6 pl61 Velu, T.J., Beguinot, L., Vass, W.C., Zhang, K., Pastan, I., Lowy, D.R. (1989)Retroviruses expressing different levels of the normal epidermal growth factor receptor : Biological properties and new Bioassay. J.Cell.Biochem. vol 39 pl53-166 Vitousek, P.M., Ehrlich, P.R., Ehrlich, A.H., Matson, P.A. (1986) Human appropriation of the products of photosynthesis. Bioscience vol 36 p368-73 Vogel, G.(1971)The effect of drugs of plant origin on capillary permeability and the lymphatic system. In : Pharmacognosy and Phytochemistry. H.Wagner, L.Horhammet (eds). Springer-Verlag, Berlin, pp 370-86 Wagner, H., Bladt, S., Zgainski, E.M. (1983) Plant Drug Analysis : A Thin Layer Chromatography Atlas. Springer-Verlag Publ. Wallenstein, S.L. (1984) Analgesics : Neurochemical, behavioural and clinical perspectives. M.Kuhar and G.Pasternak (eds) Raven Press, New York Ward, P.E., Chow, A., Drapeau, G. (1991) Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P. Biochem.Pharmacol. vol 42 p721-27 Weskamp, G., Otten, U. (1987) An enzyme-linked immunoassay for nerve growth factor(NGF) : a tool for studying regulatory mechanisms involved in NGF production in brain and in peripheral tissues. J. of Neurochem. vol 48 pl779-86 Whalley, E.T., Clegg, S., Stewart, J.M., Vavrek, R.J.(1987) The Effect of Kinin Agonists and Antagonists on the Pain Response of the Human Blister Base. Archives of Pharmacology vol 336 p652-55 Williams, D.H., Fleming, I. (1980) Spectroscopic Methods in Organic Chemistry. MsGraw-Hill Publ, London. Wilson, E.G. (1981) Threats to Biodiversity. Sci.Am. vol 261 p 60 - 108 Wimalawansa, S.J., Emson, P.C., MacIntyre, I. (1987) Regional distribution of CGRP and its specific binding sites in rats with particular reference to the nervous system. Neuroendocrinology vol 46pl31-6 Wimalawansa, S.J., MacIntyre, I. (1988CGRP and its specific binding sites in the cardiovascular system of the rat. Int. J. Cardiol, vol p29-37 234 Wimalawansa, S.J., Emson, P.C., MacIntyre, I. (1989) Both alpha and beta CGRP are present in plasma, cerebrospinal fluid and spinal cord in man. J. Mol. Endocrinology vol 3 p247-52 Wise, R.A. (1987) The role of reward pathways in the development of drug dependence. Pharmac. Ther. vol 35 p227-263 Wood, P.L. (1984) Animal models in analgesic testing. In : Analgesics : Neurochemical, biobehavioural and clinical perspectives, M. Kuhar, G. Pasternak (eds) Raven Press, New York ppl75 Yamaguchi, A., Chiba, T., Okimura,T., Yamatini, T., Morishito, Y., Nakamura, A., Kadowaki, S., Fukase, M., Fujita, T. (1988) Receptors for CGRP on the rat liver plasma membrane. Biochem. Biophys.Res.Commun, vol 152 p383-91 Yamamoto, A.I., Tohyama, M. (1989) CGRP in the nervous tissue. Prog.Neurobiol. vol 33 p335-86 Zaidi, M., Brain, S.D., Tippins, J R., Marzo, V., Moonga, B.S., Chambers, T.J., Morris, H.R., MacIntyre, 1. (1990) Structure-activity relationship of human CGRP. Biochem. Journal vol 269 p775-80 Zucker, M.B. (1944)A study of the substances in blood serum and platelets which stimulate smooth muscle. Am. J. Physiol, vol 142 pl2 235