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© University of Cape Coast https://erl.ucc.edu.gh/jspui UNIVERSITY OF CAPE COAST PHARMACOLOGICAL EVALUATION OF EXTRACT AND ISOLATED COMPOUNDS FROM THE ROOT BARK OF ZIZIPHUS ABYSSINICA HOCHST EX. A RICH (RHAMNACEAE) ISAAC TABIRI HENNEH 2019 Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui ©2019 Isaac Tabiri Henneh University of Cape Coast Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui UNIVERSITY OF CAPE COAST PHARMACOLOGICAL EVALUATION OF EXTRACT AND ISOLATED COMPOUNDS FROM THE ROOT BARK OF ZIZIPHUS ABYSSINICA HOCHST EX. A RICH (RHAMNACEAE) BY ISAAC TABIRI HENNEH Thesis submitted to the Department of Biomedical Sciences of the School of Allied Health Sciences, College of Health and Allied Sciences, University of Cape Coast, in partial fulfilment of the requirements for the award of Doctor of Philosophy degree in Drug Discovery and Development SEPTEMBER, 2019 Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui DECLARATION Candidate’s Declaration I hereby declare that this thesis is the result of my own original research and that no part of it has been presented for another degree in this university or elsewhere. Candidate’s Signature:.................................................... Date:........................... Name: Isaac Tabiri Henneh Supervisors’ Declaration We hereby declare that the preparation and presentation of the thesis were supervised in accordance with the guidelines on supervision of thesis laid down by the University of Cape Coast. Principal Supervisor’s Signature:.................................... Date:......................... Name: Prof. Martins Ekor Co-Supervisor’s Signature: ........................................... Date:......................... Name: Dr. Francis Ackah Armah ii Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui ABSTRACT The diversity offered by natural products has timelessly positioned it as a good source for novel therapeutics for the management of various medical conditions, including pain and CNS disorders. This study evaluated hydro- ethanolic root bark extract (ZAE) and isolated compounds from Ziziphus abyssinica for analgesic, anti-inflammatory, anxiolytic and antidepressant properties. Established in vitro and in vivo experimental models were adopted in assessing ZAE and the isolated compounds for these pharmacological properties. Elucidation of structure of the isolated compounds was carried out using infra-red spectroscopy, mass spectrometry, nuclear magnetic resonance and X-ray crystallography. Two pentacyclic triterpenes, β-amyrin and polpunonic acid, were for the first time isolated from the plant. This is also the first time the crystal structure of polpunonic acid is reported. ZAE was found to be relatively safe in acute and sub-chronic toxicity studies in rats. ZAE exhibited analgesic effect that is possibly mediated via opioidergic, ATP- sensitive potassium channels and nitric oxide - cyclic guanosine monophosphate pathways. ZAE also exhibited anti-inflammatory activity via membrane stabilisattion as well as inhibition of protein denaturation, neutrophil degranulation and activity of inflammatory mediators (TNF-α, IL- 1β, prostaglandin E2 and bradykinin). The isolated compounds, β-amyrin and polpunonic acid, also exhibited analgesic, anti-inflammatory (anti-arthritic), anxiolytic and antidepressant properties in murine models. It is concluded that ZAE, β-amyrin and polpunonic acid possess analgesic, anti-inflammatory, anxiolytic and antidepressant properties. iii Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui KEYWORDS β-Amyrin Biological activity Drug discovery Herbal medicine Phytochemistry Polpunonic acid iv Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui ACKNOWLEDGMENTS Doctoral studies is a long expedition which is not possible without the help of God and numerous other people. To this, I say a big thanks to God Almighty for making this dream a reality. I would also like to thank my supervisors Prof. Martins Ekor and Dr. Francis Ackah Armah for their guidance. I acknowledge the support of the Provost, Prof. J. N. Boampong for his encouragement and fatherly role. Also, I appreciate the support given me by the Head of Department, Dr. Desmond Omane Acheampong. My next appreciation goes to Dr. Elvis Ofori Ameyaw, Dr. Robert Peter Biney, Mr. Ernest Obese, Dr. Christian Adokoh, Mr. Dubois Asante and all the lecturers and staff of the Department of Biomedical Sciences and the Department of Pharmacology, University of Cape Coast. I am also very grateful to Prof. Martin Safo and his team at Virginia Commonwealth University, USA, who assisted me with X-ray crystallography analysis of my compounds. I also acknowledge the support of Dr Ben Harley. I would also like to express my gratitude to Emmanuel Adakurugu, Daniels Konja, Eric Otumi, Joseph Acquah-Mills, John Alake, John Afortude, Wisdom Ahliadzi and my colleague students who assisted me in one way or the other in carrying out this work. I cannot end without aknowledging the support of my father, Nana Kwabena Hinneh and my siblings: Mr. T.K. Donkor, Mr. Anthony Asamoah, Lawyer Barima A. Hinneh, Flt. Lt. Dr. Kyere Hinneh, Georgina Ohenewaa and Cecilia Nketia. To my wife, Benedicta Obeng, and my kids: Maame Tabuaa, Obaapa and TK, I say thank you for your love. v Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui DEDICATION This thesis is dedicated to the loving memory of my dear mum: Maame Akua Tabuaa. vi Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui TABLE OF CONTENTS Content Page DECLARATION II ABSTRACT III KEYWORDS IV ACKNOWLEDGMENTS V DEDICATION VI TABLE OF CONTENTS VII LIST OF TABLES XI LIST OF FIGURES XII LIST OF ACRONYMS XXII CHAPTER ONE 1 INTRODUCTION 1 Background to the Study 1 Statement of the Problem and Justification 3 Hypothesis 6 General Objective 6 Significance of the Study 7 Limitation of the Study 8 Definition of terms 9 The Organisation of the Study 10 CHAPTER TWO 12 LITERATURE REVIEW 12 Introduction 12 vii Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui Inflammation 12 Acute and Chronic Inflammation 12 Mediators of Inflammation 14 Rheumatoid Arthritis 17 Models of Inflammation 18 Inflammatory Pain 24 Inflammation and Depression 27 Anxiety 31 Toxicity of Herbal Medicine 34 The Family Rhamnaceae 36 Genus Ziziphus 36 Ziziphus abyssinica 41 CHAPTER THREE 50 MATERIALS AND METHODS 50 Collection of Plant Material 50 Preparation of Plant Extract 50 Phytochemical Tests 50 Biological Activity of the Extract 52 Animals 52 Drugs and Chemicals 52 Toxicity Studies 53 Anti-nociceptive Effect of the Extract 55 Anti-inflammatory Property of the Extract 61 Antidepressant Effect of the Extract 66 Anxiolytic Effect of the Extract 67 Extraction and Isolation of Compounds 68 Biological Activity of the Isolated Compounds 71 viii Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui Analgesic effect of the isolated compounds 75 Antidepressant effect of the isolated compounds 75 Anxiolytic Effect of the isolated compounds 75 Statistical analysis 76 CHAPTER FOUR 77 RESULTS 77 Phytochemical screening 77 Toxicity Studies on the Extract 77 Anti-nociceptive Effect of the Extract 90 Mechanisms of Antinociceptive Action of the Extract 95 Assessment of Involvement of nociceptive Pathways Using the Formalin Test 95 TNF-alpha and Interleukin 1β Induced Hyperalgesia 98 Bradykinin and Prostaglandin E2-induced Hyperalgesia 100 Anti-inflammatory Effect of Extract 102 Antidepressant Effect of the Extract 115 Structural Elucidation of Isolated Compounds 119 Anti-arthritic Effect of the Isolated Compounds 127 Analgesic Effect of the Isolated Compounds 139 Antidepressant Effect of Isolated Compounds 141 Anxiolytic Effect of the Isolated Compounds 144 CHAPTER FIVE 146 DISCUSSION, SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 146 Discussion 146 Summary 169 Conclusions 171 ix Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui Recommendations 172 REFERENCES 173 APPENDICES 199 Appendix A: Spectra Data of Β-Amyrin 199 Appendix B: Spectra Data of Polpunonic Acid 208 Appendix C: Crystal Structure of Polpunonic Acid 218 Appendix D: Results from activity-guided isolation of compounds from the plant. 220 x Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui LIST OF TABLES Table Page 1 List of local names for Ziziphus abyssinica in different parts of the world 42 2 Taxonomy of Ziziphus abyssinica 42 3 Results from phytochemical screening 77 4 The effect of varying doses of ZAE in an acute toxicity study 78 5 13C NMR assignments for compounds 1 and 2 122 6 Crystal data and structure refinement for Compound 2. 123 7 For compound 2 Bond lengths 124 8 Bond Angles for compound 2 125 9 Hydrogen-bond geometry (Å, o) for compound 2 126 xi Digitized by Sam Jonah Library © University of Cape Coast https://erl.ucc.edu.gh/jspui LIST OF FIGURES Figure Page 1 Inflammatory mediators coordinated release initiated by different stimuli. 26 2 Cyclopeptide alkaloids isolated from various species of Ziziphus 37 3 Triterpenoids that have been isolated from various species of Ziziphus 39 4 Flavonoids isolated from various species of Ziziphus 39 5 Ziziphus abyssinica tree, leaves and fruits 43 6 The map above shows countries with the documented