BIOPROSPECTING FOR EFFECTIVE ANTIBIOTICS FROM SELECTED KENYAN MEDICINAL PLANTS AGAINST FOUR CLINICAL Salmonella ISOLATES 2017 PETER OGOTI MOSE DOCTOR OF PHILISOPHY PhD (Biochemistry) JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY 2017 MOSE P.O. Bioprospecting for Effective Antibiotics from Selected Kenyan Medicinal Plants against four Clinical Salmonella Isolates Peter Ogoti Mose A thesis submitted in fulfillment for the Degree of Doctor of Philosophy in Biochemistry in the Jomo Kenyatta University of Agriculture and Technology 2017 DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Signature………………………………………………Date…………………………. Mose Peter Ogoti This thesis has been submitted for examination with our approval as University Supervisors: Signature………………………………………………Date…………………………. Prof. Esther Magiri, JKUAT, Kenya Signature……………………………………………...Date…………………………. Prof. Gabriel Magoma, JKUAT, Kenya Signature………………………………………………Date…………………………. Prof. Daniel Kariuki, JKUAT, Kenya Signature………………………………………………Date…………………………. Dr. Christine Bii, KEMRI, Kenya ii DEDICATION This thesis is dedicated to my beloved wife and children for their support and prayers. iii ACKNOWLEGEMENT I am grateful to the almighty God for giving me life and strength to overcome challenges. I appreciate the cordial assistance rendered by my supervisors, Prof. Esther Magiri, Prof. Gabriel Magoma, Prof. Daniel Kariuki, all from the department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology and Dr. Christine Bii, Centre of Microbiology Research-Kenya Medical Research Institute for their keen guidance, supervision and persistent support throughout the course of research and writing up of thesis. I would also like to thank the Deutscher Akademischer Austauschdienst for their financial support, Centre of Microbiology Research-Kenya Medical Research Institute for providing clinical Salmonella isolates and microbiology laboratory. I also thank Department of Chemistry and Biochemistry, Jomo Kenyatta University of Agriculture and Technology for providing Laboratory facilities. I am grateful to Ms Hyline Mochabo of Department of Medical microbiology, Jomo Kenyatta University of Agriculture and Technology and Everyn, Centre of Microbiology Research-Kenya Medical Research Institute for providing technical assistance throughout the research program. I would also like to thank Mr. John Kamau, Department of Botany, Jomo Kenyatta University of Agriculture and Technology, for identification of plant specimens. I appreciate the cooperation of my fellow postgraduate students: Mr. Macharia Mumbi and Mr. Osano. I am greatly indebted to my Wife, Edna Kwamboka for social and psychological support she accorded me throughout the entire period of the research work. Finally, but not leastly, I am grateful to my brothers and sisters in the Lord who have unceasingly been praying for me as I struggled with the research work. iv TABLE OF CONTENTS DECLARATION ........................................................................................................ ii DEDICATION ........................................................................................................... iii ACKNOWLEGEMENT ........................................................................................... iv TABLE OF CONTENTS ........................................................................................... v LIST OF TABLES .................................................................................................... xi LIST OF FIGURES ................................................................................................. xii LIST OF PLATES ................................................................................................... xiv LIST OF APPENDICES ......................................................................................... xvi ABBREVIATIONS AND ACRONYMS .............................................................. xvii ABSTRACT ............................................................................................................. xxi CHAPTER ONE ........................................................................................................ 1 INTRODUCTION ...................................................................................................... 1 1.1 General introduction ............................................................................................. 1 1.1.1 Typhoid fever ................................................................................................. 1 1.1.2 Prevention and Control strategies of typhoid fever ........................................ 3 1.1.3 Challenges associated with typhoid fever management ................................. 6 1.1.4 The role of Dehydrofolate reductase enzyme (DHFR) in S. typhi ................. 7 1.1.5 Ethnopharmacological uses of selected medicinal plants .............................. 8 v 1.2 Statement of the problem ..................................................................................... 9 1.3 Justification of the study ....................................................................................... 9 1.4 Null hypothesis ................................................................................................... 10 1.5 Objectives ........................................................................................................... 10 1.5.1 Broad objective ............................................................................................. 10 1.5.2 Specific objectives ........................................................................................ 11 CHAPTER TWO ..................................................................................................... 12 LITERATURE REVIEW ........................................................................................ 12 2.1 Clinical Salmonella typhi and its impacts .......................................................... 12 2.2 Plants with antibacterial activity ........................................................................ 13 2.3 Secondary metabolites from plants with antibacterial activity .......................... 14 2.3.1 Flavonoids .................................................................................................... 15 2.3.2 Alkaloids ...................................................................................................... 17 2.3.3 Terpenes ....................................................................................................... 18 2.3.4 Phenolics and Polyphenols ........................................................................... 18 2.3.5 Coumarins .................................................................................................... 22 2.4 Medicinal properties and uses of the five selected plant species ....................... 23 2.4.1 Tithonia diversifolia ..................................................................................... 23 2.4.2 Warburgia ugandensis ................................................................................. 24 2.4.3 Croton megalocarpus ................................................................................... 24 vi 2.4.4 Carissa edulis ............................................................................................... 25 2.4.5 Launaea cornuta ........................................................................................... 25 2.5 Evaluation of antimicrobial plants ..................................................................... 26 2.5.1 Extraction of bioactive compounds from plants ........................................... 27 2.5.2 Column chromatograpy ................................................................................ 28 2.6 Dihydrofolate Reductase .................................................................................... 29 2.6.1 Function and clinical significance of DHFR ................................................ 30 2.6.2 Mechanism of action of DHFR .................................................................... 32 2.6.3 Therapeutic applications of DHFR ............................................................... 33 2.7 Natural products in drug development ............................................................... 33 CHAPTER THREE ................................................................................................. 35 MATERIALS AND METHODS ............................................................................ 35 3.1 Experimental study site ...................................................................................... 35 3.2 Experimental design ........................................................................................... 35 3.3 Clinical Salmonella isolates ............................................................................... 36 3.4 Plant Materials .................................................................................................... 36 3.5 Preparation of plant materials for extraction ...................................................... 37 3.5.1 Extraction of phytochemicals from plant materials ..................................... 37 3.5.2 Aqueous extraction ....................................................................................... 38 3.5.3 Determination of phytochemical constituents .............................................. 39 vii 3.6 Controls .............................................................................................................