Cns Active Principles from Selected Chinese Medicinal Plants
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THE FACULTY OF MEDICINE IN THE UNIVERSITY OF LONDON CNS ACTIVE PRINCIPLES FROM SELECTED CHINESE MEDICINAL PLANTS Thesis presented by MIN ZHU (BSc., MSc.) for the degree of Doctor of Philosophy Department of Pharmacognosy The School of Pharmacy University of London 1994 ProQuest Number: 10105154 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 10105154 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 ABSTRACT In order to identify potential central nervous system (CNS) active principles from plants, 10 Chinese herbs have been selected from literature reports, namely Schejflera hodinieri, Schejflera delavayi, Celastrus angulatus, Celastrus orbiculatus, Clerodendrum mandarinorum, Clerodendrum bungei, Periploca callophylla, Periploca forrestii, Alangium plantanifolium and Uncaria rhynchophylla. These plants were extracted by 70% ethanol and biologically screened by receptor ligand binding assays which included a 1-adrenoceptor, a2-adrenoceptor, p-adrenoceptor, 5HT1, 5HT1A, 5HT1C, 5HT2, opiate, benzodiazepine, Ca^-ion channel(DHP), K^-ion channel, dopamine 1, dopamine 2, adenosine 1, muscarinic, histamine 1, Na'^/K^ ATPase, GABA^ and GABAg receptors. The results of extract screening showed that all these plants were able to inhibit the specific binding of radioligands to at least one receptor at concentrations of 1 mg/ml. Four of the species were then selected according to their distinct biological activities for further investigation in order to isolate their active principles . A total of forty-two compounds have been obtained by combination of various chromatographic techniques and their structures were determined by spectroscopic methods. The complete interpretation of all the spectra has been achieved and twelve compounds were found to be of novel structure. From Schejflera bodinieri leaves and roots, fourteen triterpenoids and oligosaccarides have been obtained and eleven of them are novel. The extract screening showed that the plant was able to bind to a l and a2 adrenoceptors, 5HT1,5HT2, opiate, adenosine 1, Ca^-ion channel (DHP), K^-ion channel, dopamine 1, dopamine 2, GABA^ and GABAq receptors. In further screening, two compounds isolated from the plant bound to the 5HT2 receptor, one compound bound to the Ca^-ion channel receptor, one compound bound to the dopamine 2 receptor and three compounds bound to the muscarinic receptor (IC5 0 0.9-8.0 pM). Five of the compounds had interactions with agonists or antagonists of p-adrenoceptor, Ca^^-ion channel, 5HT1C, 5HT1A, 5HT2, histamine 1, K^-ion channel or adenosine 1 receptors decreasing their IC50 values and some of these compounds were able to influence the binding sites of the receptors. Fifteen compounds have been obtained from Clerodendrum mandarinorum root bark and one of them is novel. In ligand binding assays, the plant extract was able to bind to 5HT1, 5HT2, opiate, dopamine 2, adenosine 1, K^-ion channel, GABA^ and GABAg receptors. Three isolated compounds were able to bind to adenosine 1, muscarinic and K^-ion channel receptors (IC 5 0 3-7.5pM). Six compounds had the ability to lower the IC50 values of agonists or antagonists of P-adrenoceptor, 5HT1 A, 5HT2, histamine 1, adenosine 1, Ca^-ion channel and K"^-ion channel receptors and change the binding sites of some of the receptors. The extract of Alangium plantanifolium root bark was able to bind 5HT1, 5HT1A, 5HT2, histamine 1, adenosine 1 , dopamine 1 , opiate, Ca^^-ion channel, GABA^ and GABAg receptors. Five compounds have been obtained, two of them bound to the muscarinic receptor (IC50 6.7-S.5 pM) and two compounds have the ability to decrease the IC50 value of agonists or antagonists of adenosine 1, 5HT1A and 5HT1C receptors and alter the characteristics of some of the receptors. The extract of Uncaria rhynchophylla bound to 5HT1A, 5HT2 and opiate receptors and eight compounds were obtained. Three compounds were able to bind to the K^-ion channel, 5HT1 A, 5HT2, opiate, a l and p-adrenoceptors (IC50 0.14-6.7 pM) and three compounds decreased the IC50 values of agonists or antagonists of 5HT1C, opiate, histamine 1 and K^-ion channel receptors and influenced some of the binding site. The present investigation has resulted in the isolation of different chemical types of CNS active principles including triterpenoids and their glucosides, oligosaccarides, flavonoids, phenols and alkaloids. The majority of clinical medicines in use are nitrogen-containing compounds, therefore it is possible that new non-nitrogen containing drugs may be developed from a knowledge of the structures identified in the present investigation. Plant medicines contain a mixture of chemical substances and their clinical efficacy cannot always be correlated with a single chemical component. The present results show that individual compounds may react with a single receptor or with a number of different receptors or affect other compound binding to receptors. The application of receptor ligand binding assays to plant extracts and their individual principles will further our understanding of the action of plant medicines. ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Professor J. David Phillipson, Head of the pharmacognosy, for his conscientious supervision on phytochemistry research, and his constant encouragement, kindness and friendship. I am deeply grateful to Doctor Pam M. Greengrass (Discovery Biology Department, Pfizer Limited) for her invaluable guidance on the radioligand receptor binding work and her interest and kindness. Appreciation is also expressed to Professor Norman G. Bowery (Head of Pharmacology, The School of Pharmacy) for capable supervision on the pharmacological study, especially on the G ABA receptors research. These three supervisors gave me great help and consideration during the course of this study, without their encouragement, this work would not be possible. I acknowledge the colleagues in Discovery Biology Department, Pfizer Limited for their friendly cooperation, in particular I would like to mention Mr. Mike Russell for his patient guidance in the practical ligand binding work, and Dr. Mike William, Head of the Department, for his carefulness and guidance in the whole Ph.D course. I would also like to express my sincere thanks to Doctor David V. Bowen (Head of Spectroscopy Department, Pfizer) for his kindly running partial NMR spectra and MS spectra in his leisure time. My thanks also go to Professor Zhengyu Liu (Head of Department of Botany, Institute of Medicinal Plant Cultivation, Chong Qing, China) and his colleagues for their help in collection and identification the plant material. I thank Mrs. J. Hawkes for running the NMR experiments ( University of London Intercollegiate Research Service at King’s College) and Dr. K. Welham and his colleagues for running the mass spectra at the Mass Spectrometry Unit at The School of Pharmacy, University of London. I am grateful to my friends and colleagues in the Department of Pharmacognosy, The School of Pharmacy with whom I have collaborated in various aspects of this work, particularly Doctor Shiling Yang, Doctor Ya Cai, Mrs. Hong-Wen Yu, Mrs. Janice Hallsworth, Miss Maria Camacho, Miss Caroline Lang’at and Dr. Pablo Solis. Also, I would like to thank Mrs. M. Pickett and Mr. G. Ronngren for their willing technical assistance and Mrs A. Cavanagh for graphical work in the preparation on posters for conferences. I would like to express my appreciation and thanks to Professor J. David Phillipson, Doctor Pam M. Greengrass and Doctor Ya Cai for their attentively consideration, constructive suggestions and valuable discussions on the manuscript, from which I learned a great deal. Undoubtedly, I am eternally indebted to my family members, for their encouragement, comprehension, cooperation and patience during these three years. Finally, I gratefully acknowledge Pfizer limited for providing a research scholarship and modem facilities for the radioligand receptor binding assays. CONTENTS Page Abstract 2 Acknowledgement 5 List of Abbreviations 11 List of Figures 14 List of Tables 16 CHAPTER 1 INTRODUCTION 18 1.1 Central nervous system (CNS) disorders 19 1.1.1 Pain and analgesics 20 1.1.2 Depression and antidepressants 23 1.1.3 Anxiety, insomnia and related medicines 28 1.1.4 Schizophrenia and antipsychotic drugs 33 1.2 Receptors related to the CNS 37 1.3 Receptor ligand binding assays 47 1.4 Other pharmacological approaches for the CNS drug development 51 1.5 Plants as medical resource for the treatment of CNS disorders 52 1.5.1 Plants used for CNS disorders 52 1.5.2 Traditional Chinese medicine used for the CNS disorders 54 1.5.2.1 History of traditional Chinese medicine 54 1.5.2.2 Traditional Chinese medicine used for CNS disorders 56 1.6 The purpose and strategy of the present study 60 CHAPTER 2 MATERIALS AND METHODS 61 2.1 Plant materials 62 2.2 Preparation of plant extracts 62 2.3 Chromatographic techniques used for isolation 62 2.4 Spectroscopy for structure identification 64 2.4.1 Ultraviolet spectroscopy 64 2.4.2 Mass spectrometry (MS) 64 2.4.3 Nuclear magnetic resonance spectroscopy (NMR) 64 2.5 Hydrolysis of glycosides 65 2 . 6 The protocol of ligand binding assays for extract screening 65 2.7 The protocol of ligand binding assays for fraction screening 6 8 2 . 8 The protocol of ligand binding assays for compounds screening 6 8 2.9 The protocol of ligand binding assays for testing the interaction of the isolated compounds with agonists or antagonists of various receptors 6 8 2 .