University of Cape Coast Phytochemistry, Anti
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UNIVERSITY OF CAPE COAST PHYTOCHEMISTRY, ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITIES OF THE ROOT BARK OF ANTHOSTEMA AUBRYANUM (BAILL) PATRICK MALCOLM FYNN 2016 Digitized by UCC, Library © Patrick Malcolm Fynn University of Cape Coast Digitized by UCC, Library 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: Patrick Malcolm Fynn 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. Yaw Opoku-Boahen Co-Supervisor’s Signature: ........................................... Date:......................... Name: Dr. (Mrs) Genevieve Adukpo ii Digitized by UCC, Library ABSTRACT The work presented in this thesis involves the scientific investigation into the traditional uses of the root bark of Anthostema aubryanum (Baill., family, Euphorbiaceae) as an anti-inflammatory and antioxidant agent. It also describes the isolation and characterization of two compounds from the alkaloid extract of the root bark of Anthostema aubryanum Baill. The anti-inflammatory activity was investigated using the acute carrageenan – induced foot pad edema model in six weeks old rats. The extracts were given orally to the rats at 30, 100 and 300 mg/kg body weight, 1 hour after induction of oedema with carrageenan using diclofenac as the reference drug. All extracts of the root bark were demonstrated to display a time-and dose-dependent anti-inflammatory effects in rats with methanolic extract showing the highest activity (ED50 = 5.29± 0.02 BDW) compared to the standard drug, diclofenac (ED50 = 1.99± 0.01). The antioxidant properties of the extracts were investigated using three assays; total antioxidant capacity, total phenolic content and DPPH scavenging activity. The antioxidant activity of the methanolic crude extract (IC50=8.84±0.02 µg/ml) was equivalent to the standard vitamin E (IC50=8.61±0.01 µg/ml) with total phenolic content of 74.53±0.004. Comprehensive chromatographic and spectroscopic analyses of the alkaloid extract led to the isolation and characterization of two major anti-inflammatory and antioxidant agent as 5-methoxycanthin-6-one and canthin-6-one with the former showing the highest pharmacological activity (ED50=60.84±0.01, IC50=27.62±0.010 and ED50=96.64±0.01, IC50=33.60±0.01 respectively). This is the first report of the isolation of these compounds from the family Euphorbiaceae. iii Digitized by UCC, Library ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisors, Professor Yaw Opoku-Boahen and Dr (Mrs) Genevieve Adukpo, both of the Department of Chemistry, for their professional guidance, advice, encouragement and the goodwill with which they guided this work. I am really very grateful. I am again grateful to my good friend Dr Francis Armah for providing us with the plant sample and assisting in the pharmacological activities. I also express my appreciation to the laboratory technicians of the Departments of Chemistry, University of Cape Coast, Biomedical and Forensic Sciences, University of Cape Coast and Pharmacognosy, Kwame Nkrumah University of Science and Technology, Kumasi, for their excellent technical assistance. I am forever grateful. I would like to thank Professors Solomon Habtemariam of the Department of Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, United Kingdom and Baldwyn Torto, Chemical and Behavioral Ecology Department, International Centre for Insect Physiology and Ecology, Kenya for generously running and providing us with the NMR and MS spectra of the isolated compounds. I would like to thank Rev. Sr. Elizabeth Amoako-Arhen, the Principal of OLA College of Education, Cape Coast for her unflinching support throughout the programme. The sponsorship from the Ghana Education Trust Fund (GETFUND) is gratefully acknowledged. Finally, I wish to thank my family and friends for their support, especially, my friend, Justice Owuraku Addo. iv Digitized by UCC, Library DEDICATION To my lovely wife, Naomi Arthur Fynn (Mrs) and children, Nhyiraba, Nyameyie, Judalyn and Jedida v Digitized by UCC, Library TABLE OF CONTENTS Page DECLARATION ii ABSTRACT i ii ACKNOWLEDGEMENTS iv DEDICATION v TABLE OF CONTENTS vi LIST OF TABLES xiii LIST OF FIGURES x i v LIST OF ABBREVIATIONS xviii CHAPTER ONE: INTRODUCTION Background to the Study 1 The Plant Anthostema aubryanum (Baill) 3 Botanical Description of Plant Species 4 Ethnomedicinal Uses 5 Statement of the Problem 6 Justification of the Study 8 Main Objectives of the Study 11 Specific Objectives of the Study 11 CHAPTER TWO: LITERATURE REVIEW Introduction 12 The Family Euphorbiaceae 12 Ethnomedicinal Uses of Euphorbiaceae 14 Phytochemistry of Euphorbiaceae 16 Diterpenes 17 vi Digitized by UCC, Library Triterpenes 22 Alkaloids 23 Flavonoids and other phenolic compounds 25 Tannins 28 Coumarins 30 Cyanogenic Glycosides 31 Fatty Alcohols 33 Other Classes of Compounds 34 Alkaloids 35 Properties of Alkaloids 3 6 Structure and Classification of Alkaloids 37 Biosynthetic Classification 37 Chemical Classification 38 Pharmacological Classification 39 Taxonomic Classification 39 Types of Alkaloids 40 True Alkaloids 40 Protoalkaloids 42 Pseudoalkaloids 4 2 Nomenclature of Alkaloids 43 Pharmacological Uses of Alkaloids 44 Distribution of Alkaloids 44 The Family Euphorbiaceae 46 The Family Apocynaceae 47 The Family Asteraceae 4 8 vii Digitized by UCC, Library The Family Loganiaceae 49 The Papaveraceae Family 50 The Family Rutaceae 51 The Family Solanaceae 5 3 The Family Erythroxylaceae 54 The Family Boraginaceae 55 The Family Fabaceae 56 The Family Menispermaceae 57 The Family Berberidaceae 59 The Family Ranunculaceae 60 The Family Liliaceae 61 The Family Rubiaceae 62 The Family Amaryllidaceae 64 The Family Elaeagnaceae 65 The Family Zygophyllaceae 65 Mushroom 66 Moss 67 Fungi and Bacteria 68 Animals 69 Tests for Alkaloids 73 Extraction and Isolation of Alkaloids 76 Acidic Water Extraction 76 Aqueous-Alcohol Extraction 77 Organic Solvent Extraction 77 Beta-carboline Alkaloids 78 viii Digitized by UCC, Library Nomenclature of Beta-carboline Alkaloids 78 Distribution of Beta-carboline Alkaloids 78 Biosynthesis of Beta-carboline Alkaloids 82 Synthesis of Beta-carboline Alkaloids 82 Pharmacological Uses of Beta-carboline Alkaloids 86 Inflammation 94 Inflammatory Pathway 98 Experimental Models of Inflammation 99 Models of Acute Inflammation 99 Carrageenan-induced Paw Edema 100 Oxidative Stress 101 Antioxidants 103 Determination of Antioxidant Properties 104 Total Antioxidant Capacity 105 DPPH radical scavenging activity 106 Total Antioxidant Activity by the Phosphomolybdenum Method 107 Total Phenolic Activity by Folin-ciocalteau Method 107 CHAPTER THREE: MATERIALS AND METHODS Chemicals 109 General Experimental Procedures 109 Collection and Authentication of Plant Sample 110 Processing of Plant Material 110 Phytochemicals Screening of Crude Plant Extract 110 Extraction of Plant Material 116 Anti-Inflammatory Assay of Extracts 117 ix Digitized by UCC, Library Experimental Animals 117 Carrageenan-Induced Edema in Rats 117 Anti-inflammatory Assay of Crude Methanolic Extract 118 Anti-inflammatory Assay of Crude Alkaloid Extract 119 Antioxidant Assay of Extracts 119 Total Phenolic Content Assay 119 Total Antioxidant Capacity Assay 119 In Vitro Qualitative DPPH Test 120 Quantitative Antioxidant Assays of Extracts 120 Statistical Analysis of Data 121 Fractionation of Alkaloid Extract 122 Chromatographic Materials 122 Detection for Analytical thin Layer Chromatography 122 Column Chromatography 123 Preparative-Layer Chromatography 123 Development of Thin Layer Chromatogram 124 Isolation of Compounds from the Crude Alkaloid Extract 125 Column chromatographic separation 0f the crude alkaloid extract 125 Isolation of Compound M1 128 Isolation of Compound M2 and M3 128 Isolation of Compounds M4 and M5 129 Anti-inflammatory Activity of Isolated Compounds 130 In Vitro DPPH Radical Scavenging Activity of Isolated Compounds 130 CHAPTER FOUR: RESULTS AND DISCUSION Introduction 131 x Digitized by UCC, Library Characterization and Identification of Isolated Compounds 133 Identification of M1 as 5-Methoxy-Canthin-6-one (1) 133 Identification of M5 as Canthin-6-one (2) 138 Bioassays 142 Anti-inflammatory Activity of Root Bark Extract 142 Anti-inflammatory Activity of Crude Alkaloid Extract 147 Anti-inflammatory Activity of the Isolated Compounds 147 Antioxidant Activity of Extracts 151 Antioxidant Activity of Crude Extracts and Isolated compounds 151 Quantitative Antioxidant Assay of Extracts 152 Total Phenolic Content 152 Total Antioxidant Capacity 153 DPPH Radical Scavenging Activity of Extracts of A. Aubryanum 156 Antioxidant Activity of Isolated Compounds 157 Quantitative DPPH Radical Scavenging Test 157 CHAPTER FIVE: SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Introduction 161 Summary 161 Conclusions 163 Recommendations 165 Suggestions For Further Research 167 REFERENCES 1 68 xi Digitized by UCC, Library 1 APPENDIX A: H-NMR of M1 in MeOD at 500 MHz 197 1 APPENDIX B: Integrated H-NMR of M1 in MeOD at 500 MHz 198 13 APPENDIX C: