Antibacterial Efficacy and Safety of Selected Kenyan Medicinal Plants
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1 ANTIBACTERIAL EFFICACY AND SAFETY OF SELECTED KENYAN MEDICINAL PLANTS ALFRED OGAO ABIBA HD (Kenya Polytechnic) CE (University of Westminster UK), MSc. Brunel University of West London, UK. (Reg. No. I84/7602/98) A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of a Doctor of Philosophy Degree (PhD) in the Department of Biochemistry and Biotechnology in the School of Pure and Applied Sciences of Kenyatta University JUNE 2013 2 DECLARATION This is my original work and has not been presented for a degree award in any other University or for any other award. Alfred Ogao Abiba I84/7602/98 Signature Date We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Prof. Eliud N.M. Njagi Department of Biochemistry & Biotechnology Kenyatta University Sign Date Prof. Paul O. Okemo Department of Plant and Microbial Sciences Kenyatta University. Sign Date Prof. Isaiah O. Ndiege Department Chemistry Kenyatta University Sign Date 3 DEDICATION I dedicate this work to my late father Michael Abiba Omodo, my Mother Margaret Dwera Abiba, my late wife Josephine Ngute Ogao who continually loved, encouraged and prayed for me to complete this research work and also to my late brother Patrick Lubondi Abiba, for sacrificing all they had to facilitate my education. 4 ACKNOWLEDGEMENT Blessings, honour, glory, and power be to God through His son Christ Jesus my Lord for the grace and strength He provided to me to complete this work. I profoundly thank my supervisors; Prof. E.N.M. Njagi, Prof. Paul O. Okemo and Prof. Isaiah O. Ndiege for their guidance in designing experiments and performance, thesis editing, without which this research work would not be possible. I highly appreciate Kenyatta University through successive Chairmen of the Department of Biochemistry and Biotechnology for their support and making available laboratory space, equipments and chemicals to me during my research. I also would like to thank Kenyatta University again through successive Deans, School of Pure and Applied Sciences (SPAS) for availing research grants to me which greatly enabled me to purchase chemicals and other materials without which it would have been difficult to complete this work. I thank Dr. Oundo of Kenya Medical Research Institute (KEMRI-Nairobi), Centre for Microbiological Research (CMR) for allowing me to perform some of my bacterial work at their laboratories. I appreciate Simon Mathenge a taxonomist from University of Nairobi, Botany Department who identified my plant samples assisted by Stephen Gichobi of Kenyatta University herbarium, in Department of Plant and Microbial Sciences. I acknowledge the assistance from the following technical staff that helped me in my laboratory analysis, James Adino and the late Solomon Buleti of the Department of Biochemistry, Laurence Alaro of the Department of Plant and Microbial Sciences at Kenyatta University who helped me with statistical analysis of my research data. I also thank all the staff of the Department of Biochemistry and Biotechnology, Kenyatta University, especially the most immediate Chairman, Dr. J.J. Ngeranwa, for his encouragement and support. My heartfelt gratitude goes to all my relatives and friends who supported and encouraged me in one way or the other throughout my study. Finally, I wish to express my deepest appreciation to my family, first to my beloved late wife Josephine Ngute, my loving wife Patricia Nzyoka, then to my dear children Zelda Dwera, Duren Abiva Victo Itati and Pendo Dweko. Their love, prayers and encouragement kept me going on even when it became difficult. To them all I say THANK YOU AND GOD BLESS YOU. 5 TABLE OF CONTENTS TITLE PAGE……………………………………………………………………….i DECLARATION……………………………………………………………………ii DEDICATION………………………………………………………………………iii ACKNOWLEDGEMENT…………………………………………………………..iv TABLE OF CONTENTS…………………………………………………………...v LIST OF TABLES…………………………………………………………………..xiv LIST OF FIGURES…………………………………………………………………xvi LIST OF PLATES…………………………………………………………………..xviii ABBREVIATION AND ACRONYMS…………………………………………….xix ABSTRACT…………………………………………………………………………xxiii CHAPTER ONE…………………………………………………...1 1 INTRODUCTION…………………………………………………1 1.1 Background Information……………………………………………1 1.2 Statement of the Problem…………………………………………...9 1.3 Research Questions…………………………………………………9 1.4 Research Hypotheses……………………………………………….10 1.5 Study Objectives……………………………………………………10 1.5.1 General Objective…………………………………………………...10 1.5.2 Specific Objectives………………………………………………….10 1.6 Justification…………………………………………………………11 1.7 Significance of the Study and the Anticipated Outcome………….11 CHAPTER TWO…………………………………………………..13 2 LITERATURE REVIEW…………………………………………13 6 2.1 WHO Policy on Medicinal Plant Research…………………………13 2.2 Kenya National Policy on Medicinal Plants………………………..15 2.3 Kenyan Medicinal Plant Research………………………………….16 2.4 The History of Global Uses of Medicinal Plants…………………..17 2.5 Current Global Uses of Medicinal Plants…………………………..24 2.6 The Use of Medicinal Plants in Africa……………………………...26 2.7 Antimicrobial Activities of Medicinal Plant Extract ………………30 2.8 Active Compounds Identified in Medicinal Plants………………...31 2.9 Resistance of Bacteria against Conventional Antibiotics………….34 2.10 Mode of Action of Medicinal Plant-Derived Medicines……………36 CHAPTER THREE………………………………………………..37 3 MATERIALS AND METHODS………………………………….37 3.1.0 Sampling Sites………………………………………………………37 3.1.1 Mwingi District……………………………………………………..37 3.1.2 Busia County……………………………………………………….38 3.2 Plants of this Study………………………………………………...41 3.2.1 Ethnobotanical Survey……………………………………………..41 3.2.1.1 Dichrostachys cinerea………………………………………………42 3.2.1.2 Combretum molle…………………………………………………...43 3.2.1.3 Euclea divinorum…………………………………………………...44 3.2.1.4 Ficus sur……………………………………………………………..44 3.2.1.5 Schkuhria piñata…………………………………………………….45 3.2.1.6 Carisa edulis………………………………………………………...45 3.2.1.7 Zanthoxylum usambarense............................................................46 3.2.1.8 Securidaca longipedunculata……………………………………….46 7 3.2.1.9 Crinum linn………………………………………………………….48 3.3.1 Plant Sample Collection…………………………………………….48 3.3.2 Processing and Extraction of Plant Extracts ………………………48 3.3.2.1 Percolation Extraction ………………………………………………49 3.4 Bioassays……………………………………………………………50 3.4.1 Bacterial Culture Used……………………………………………...50 3.4.1.1 Escherichia coli……………………………………………………...50 3.4.1.2 Staphylococcus aureus………………………………………………50 3.4.1.3 Pseudomonas aeruginosa…………………………………………..51 3.4.1.4 Salmonella typhi…………………………………………………….52 3.4.1.5 Shigella dysenteriae…………………………………………………52 3.4.1.6 Klebsiella pneumonia……………………………………………….52 3.5 Agar Paper Disc Diffusion Screening of Medicinal Plant Extracts ………………………………………………………53 3.5.2 Preparation of Media……………………………………………….53 3.5.3 Preparation of the Inoculum………………………………………..54 3.5.3 Preparation of McFarland Standard……………………………….54 3.5.4 Inoculation of Bacteria……………………………………………...54 3.5.5 Preparation of Paper Discs…………………………………………55 3.5.6 Impregnation of Extracts on to Paper Discs ………………………55 3.5.7 Concentration of Extract on the Paper Disc……………………….55 3.5.8 Placement of Extract Impregnated on Paper Discs on Petri Dish………………………………………………………………….55 3.5.9 Measuring of Zones of Inhibitions………………………………….56 3.6 Determination of Minimum Inhibitory Concentrations (MICs)…...56 8 3.7 Determination of Minimum Bactericidal Concentrations (MBCs)...58 3.8 The Time-Kill Kinetics………………………………………………59 3.8.1 Test Cultures………………………………………………………...60 3.8.2 Time Kill Kinetics Bioassay Method ………………………………60 3.9 In Vivo Single Dose Toxicity Test…………………………………61 3.9.1 Materials and Methodology………………………………………...61 3.9.1.1 Experimental Animals………………………………………………61 3.9.1.2 Determination of Body Weight……………………………………..62 3.9.1.3 Absolute Organ Weight……………………………………………..62 3.9.2. Biochemical Assays…………………………………………………62 3.9.2.1 Preparation of Sera Samples………………………………………..62 3.9.3. Laboratory Determination of Enzyme Activities and Analytes……63 3.9.3.1 Determination of the Activity of Aspartate Aminotransferase (AST)………………………………………………………………..63 3.9.3.2 Determination of the Activity of Alanine Aminotransferase (ALT)………………………………………………………………...64 3.9.3.3 Determination of the Activity of Alkaline Phosphatase (ALP)……64 3.9.3.4 Determination of the Blood Levels of Urea (BUN)………………..65 3.9.3.5 Determination of the Activity of Creatine Kinase (CK)……………65 3.9.4.0 Determination of Hematological Parameters ……………………….66 3.9.5.0 Determination of the Mineral Content of the Plant Extracts ……….67 3.9.5.1 Preparation of Plant Extracts ……………………………………….67 3.9.5.2 Elemental Analysis by Total Reflection X-Ray Fluorescence System (TXRF System)……………………………………………..67 3.9.6.0 Histopathology……………………………………………………...68 3.10.0 Phytochemical Screening……………………………………………68 3.10.1 Testing for the Presence of Alkaloids ………………………………69 9 3.10.2 Testing for Tannins…………………………………………………70 3.10.3 Testing for the Presence of Anthraquinones……………………….70 3.10.4 Testing for the Presence of Triterpenes and Steroids ………………70 3.10.5 Testing for Saponins………………………………………………..71 3.10.6 Testing for the Presence of Flavonoids…………………………….71 3.10.7 Coumarines…………………………………………………………71 3.10.8 Reducing Sugar……………………………………………………..72 3.11.0 Data Analysis ………………………………………………………72 CHAPTER FOUR…………………………………………………73 4 RESULTS………………………………………………………….73 4.2.1 Yields in Milligrams for each Plant Extract……………………….73 4.2.2 Agar Disc Diffusion Bioassays Results……………………………74 4.2.1 Antibacterial Activity of D. cinerea Extract by Agar Disc Diffusion…………………………………………………………….74 4.2.3 Antibacterial