Phytochemical and Antimicrobial Investigation of Ochna Thomasiana Engl

1 PHYTOCHEMICAL AND ANTIMICROBIAL INVESTIGATION OF OCHNA THOMASIANA ENGL. & GILG MBITHI JUSTUS MUEMA (B.ED, Sc) I56/10372/2007 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in the School of Pure and Applied Sciences, Kenyatta University JUNE, 2015 2 DECLARATION DECLARATION BY CANDIDATE This thesis is my original work and has not been presented for a degree in any other university or any other award. Mbithi Justus Muema Reg No.I56/10372/2007 Signature Date Department of Chemistry DECLARATION BY SUPERVISORS This thesis has been submitted in partial fulfillment of Master of Science degree of Kenyatta University with our approval as supervisors. Prof. Alex K. Machocho Department of Chemistry Kenyatta University Signature Date Prof. Nicholas K. Gikonyo Department of Pharmacy and Complementary/Alternative Medicine Kenyatta University Signature Date 3 DEDICATION To my parents, my wife Merceline and children, Ndunge, Mwongeli and Wanza. 4 ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my supervisors, Prof. Alex K. Machocho and Prof. Nicholas K. Gikonyo. Special thanks to Prof. Paul K. Tarus for initiating this project. I wish to thank Stephen Musyoka and Julius Mwenda who carried out the NMR analysis, Elias Maina the retired Chief Technician and Dennis Osoro the acting Chief Technician for assisting with chemicals and glass ware, my classmates for encouraging me to continue with the course especially Ronald Okwemba, Mbarak Mohamed, Stanley Tumbo, Philip Mayeku and the entire staff of Chemistry Department of Kenyatta University. I would like to extend my profound gratitude and appreciation to my wife Merceline, no words in this world can express my gratitude, my daughters Ndunge, Mwongeli and Wanza, for the loneliness they suffered when they missed my company and care. The understanding was a special source of inspiration to me. My heartfelt gratitude goes to my parents, sisters, brothers, Cousin David Mwania, Uncle David Mutiso, in-law James Muya and friends for their encouragement and support both financially and otherwise. My deepest appreciation goes to Higher Education Loans Board of Kenya and Ministry of Education of Science and Technology for the financial support they gave me. I am also grateful to my employer, TSC for granting study leave to advance my education. Last but not least, I am most indebted to God for giving me good health and strength during the entire period of this work. 5 TABLE OF CONTENTS Page DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv TABLE OF CONTENTS v LIST OF TABLES viii LIST OF PLATES ix LIST OF FIGURES x LIST OF SCHEMES xi LIST OF ABBREVIATIONS AND ACRONYMS xii ABSTRACT xiv CHAPTER ONE INTRODUCTION 1 1.1 Background 1 1.2 Plants as a source of antimicrobials 2 1.3 Phytochemicals 3 1.4 Herbal medicine 3 1.5 Statement of the problem 5 1.6 Hypotheses 6 1.7 Objectives 6 1.7.1 General objective 6 1.7.2 Specific objectives 6 1.8 Justification and Significance 7 CHAPTER TWO LITERATURE REVIEW 8 2.1 Antimicrobial agents 8 2.1.1 Antibacterial agents 8 2.1.2 Antifungal agents 12 2.2 Antiviral agents 13 2.3 Flavonoids 16 2.4 Distribution of the Ochnaceae family 17 2.5 Economic importance of the Ochnaceae family 17 2.6 Medicinal uses of plants of Ochnaceae family 18 2.7 Medicinal uses of Ochna species 19 2.8 Phytochemical and Biological Activities of the Genus Ochna 22 2.9 Kenyan Ochna species 35 CHAPTER THREE METHODOLOGY 39 3.1 Plant collection and processing 39 3.2 Reagents 39 3.3 Cleaning procedures 39 3.4 Sequential extraction of root and stem barks 40 3.5 Thin layer chromatography 42 3.6 Melting point 43 3.7 Infrared (IR) spectroscopy 43 6 3.8 Ultraviolet (UV) spectroscopy 43 3.9 Nuclear magnetic resonance (NMR) spectroscopy 44 3.10 Mass Spectroscopy (MS) 45 3.11 Antibacterial tests 45 3.11.1 Bacterial strains tests 45 3.11.2 Preparation of media 46 3.11.3 Screening procedure 46 3.11.4 Minimum inhibitory concentrations (MIC) and MBC 47 3.11.5 Disc diffusion and MIC ratings of the extracts 48 3.12 Isolation of compounds from Ochna thomasiana 49 3.13 Physical and spectroscopic data of isolated compounds 54 3.13.1 Compound 18 54 3.13.2 Compound 20 54 3.13.3 Compound 17 55 3.13.4 Compound 23 55 3.13.4 Compound 74 56 3.13.5 Compound 75 56 CHAPTER FOUR RESULTS AND DISCUSSION 58 4.1 Plant material yield of extracts 58 4.2 Antibacterial disc diffusion screening test for the O. thomasiana extracts 59 4.2.1 The DCM extracts 59 4.2.2 The ethyl acetate extracts 59 4.2.3 The methanol extracts 59 4.2.4 MIC and MBC of O. thomasiana methanol extracts 60 4.3 Structure elucidation 61 4.3.1 Compound 18 61 4.3.2 Compound 20 66 4.3.3 Compound 17 69 4.3.4 Compound 23 and 74 71 4.3.5 Compound 75 75 4.4 Antibacterial activity test for the isolated compounds 77 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 78 5.1 Conclusions from the study 78 5.2 Recommendations from the study 79 5.3 Suggestions for further research 79 REFERENCES 80 APPENDICES 92 1 Appendix 1a: H NMR (600 MHz CD3OD) of compound 18 92 13 Appendix 1b: C NMR (150 MHz CD3OD) of compound 18 93 13 Appendix 1c: C DEPT (CH) NMR (150 MHz CD3OD) of compound 18 94 13 Appendix 1d: C NMR APT (150 MHz CD3OD) of compound 18 95 1 1 Appendix 1e: H- H COSY (600 MHz CD3OD) of compound 18 96 13 Appendix 1f: C NMR HSQC (150 MHz CD3OD) of compound 18 97 13 Appendix 1g: C NMR HMBC (150 MHz CD3OD) of compound 18 98 Appendix 1h: IR of compound 18 99 Appendix 1i: UV of compound 18 100 7 1 Appendix 2a: H NMR (400 MHz CD3OD) of compound 20 101 13 Appendix 2b: C NMR (100 MHz CD3OD) of compound 20 102 Appendix 2c: IR of compound 20 103 1 Appendix 3a: H NMR (400 MHz CD3OD) of compound 17 104 13 Appendix 3b: C NMR (100 MHz CD3OD) of compound 17 105 Appendix 3c: IR of compound 17 106 13 Appendix 4a: C NMR (100 MHz CDCl3) of compound 23 and 74 107 13 Appendix 4b: C NMR (100 MHz CDCl3) of compound 23 and 74 108 Appendix 4c: IR of compound 23 and 74 109 Appendix 4d: MS of compound 23 and 74 110 1 Appendix 5a: H NMR (600 MHz CDCl3) of compound 75 111 1 Appendix 5b: H NMR (600 MHz CDCl3) of compound 75 112 1 Appendix 5c: Expanded part H NMR (600 MHz CDCl3) of compound 75 113 13 Appendix 5d: C NMR (150 MHz CDCl3) of compound 75 114 13 Appendix 5e: Part of C DEPT-135 (150 MHz CDCl3) of compound 75 115 13 Appendix 5f: Section of C NMR (150 MHz CDCl3) of compound 75 116 13 Appendix 5g: C NMR DEPT (150 MHz CDCl3) of compound 75 117 Appendix 5h: IR of compound 75 118 Appendix 5i: UV of compound 75 119 8 LIST OF TABLES Page Table 2.1: Medicinal uses of Ochna species 20 Table 4.1: Plant material yield of O. thomasiana extracts 58 Table 4.2: The inhibition zones (in mm) of crude extracts of O. thomasiana 60 Table 4.3: The MIC and MBC of crude extracts of O. thomasiana 60 1 Table 4.4: H NMR (600 MHz, CD3OD) and COSY for 18 63 13 Table 4.5: C NMR (150 MHz, CD3OD), DEPT, HSQC and HMBC 18 65 1 13 Table 4.6: H and C NMR (100 MHz, CD3OD) data for 20 68 1 13 Table 4.7: H and C NMR (100 MHz, CD3OD) data for 17 71 13 Table 4.8: C NMR DEPT (100 MHz, CDCl3) for 23 and 74 74 13 Table 4.9: C NMR (150 MHz, CDCl3) for 75 76 Table 4.10: Inhibition zones (in mm) of antibacterial activity of the compounds 77 9 LIST OF PLATES Page Plate 2.1: Map of distribution of Ochna species 18 Plate 2.2: Twig, leaves and fruits Ochna serrulata 20 Plate 2.3: Photograph of aerial part of Ochna thomasiana 38 Plate 2.4: Photograph of flowers and fruit of Ochna thomasiana 38 Plate 3.1: Photograph of Petri dish showing disk diffusion method 48 Plate 3.2: Test of bacterium 48 10 LIST OF FIGURES Page Figure 4.1: HMBC correlations in partial structure 18 62 Figure 4.2: HMBC correlations in the partial structure 18 64 Figure 4.3: Compound 18 66 Figure 4.4: Compound 20 69 Figure 4.5: Compound 17 70 Figure 4.6: Compound 23 73 Figure 4.7: Compound 74 73 Figure 4.8: Compound 75 76 11 LIST OF SCHEMES Page Scheme 3.1: Sequential extractions of O. thomasiana stem bark. 41 Scheme 3.2: Sequential extractions of O. thomasiana root bark. 42 Scheme 3.3: Chromatographic separation of O. thomasiana root bark extract. 52 Scheme 3.4: Chromatographic separation of O. thomasiana root bark extract. 53 12 ABBREVIATIONS AND ACRONYMS ¹³C NMR Carbon 13 Nuclear Magnetic Resonance ¹H NMR Proton Nuclear Magnetic Resonance 2D NMR Two Dimensional Nuclear Magnetic Resonance AA Antimicrobial Activity Ac Acetyl AIDS Acquired Immune Deficiency Syndrome APT Attached Proton Test ASFMT Arubuko Sokoke Forest Management Team ATCC American Type Culture Collection CC Column Chromatography CD3OD Deuterated Methanol CDCl3 Deuterated Chloroform COSY Correlation Spectroscopy d Doublet DCM Dichloromethane DEPT Distortionless Enhancement by Polarization Transfer DMSO Dimethyl Sulphoxide DR Democratic Republic DST Diagnostic Sensitivity Test Et Ethyl EtOAc Ethyl Acetate HETCOR Heteronuclear Correlation HIV Human Immunodeficiency Virus HMBC Heteronuclear Multiple Bond Coherence HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantam Coherence ICU Intensive Care Unit IR Infrared Jr Junior KEMRI Kenya Medical Research Institute KNPHL Kenya National Public Health Laboratories LD Lethal Dose 13 m Multiplet mb Millibar MBC Minimum Bactericidal Concentration MDRS Multi-Drug Resistance Strain MRSA Methicillin Resistant Staphylococcus Aureus MeOH Methanol MP Metalloporphyrin MIC Minimum Inhibitory Concentration MS Mass Spectroscopy NA Nutrient Agar NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Enhancement Spectroscopy PDA Potatoes Dextrose Agar ppm Parts Per Million PTLC Preparative Thin Layer Chromatography s Singlet Sw Swahili Rf Retention factor t Triplet TLC Thin Layer Chromatography TMS Tetramethylsilane µg Microgram UV Ultraviolet VLC Vacuum Liquid Chromatography WHO World Health Organization δ Chemical Shift 14 ABSTRACT Infectious diseases are the leading cause of death world-wide despite the vigorous campaigns that have been made to combat them.

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