ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL PROFILES OF WARBURGIA UGANDENSIS SPRAGUE (CANELLACEAE) EXTRACTS FROM DIFFERENT POPULATIONS ACROSS THE KENYAN RIFT VALLEY Abuto John Otieno (B.Sc.) I56/23055/2011 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University NOVEMBER, 2016 ii DECLARATION iii DEDICATION This research work is dedicated to my beloved wife and friend Dorca Auma Murono and our dear sons Benard Abuto Otieno and Benjamin Jesse Otieno. iv ACKNOWLEDGEMENTS I am grateful to the almighty God for His continued love and faithfulness in enabling me go through the entire research work with strength, confidence and determination. Surely, I can do all things though Jesus Christ who strengthens me. I am greatly indebted to my supervisors Prof. Alex Machocho, Dr. David Mburu and Dr. Alice Muchugi for their wise counsel, tireless effort, great support, intelligent criticism and advice in the whole process of planning and implementing this research work. Without your wonderful guidance, this work would not have been realized. My appreciation goes to Kenyatta University and the Department of Biochemistry and Biotechnology for providing me with the opportunity to take my M.Sc studies. Many thanks go to Dr. Christine Bii and the entire team at CMR-KEMRI, Nairobi Kenya for their support in bioassay activities. Much gratitude goes to Dr. Geoffrey Karau and Mrs. Jacqueline Kang’iri of KEBS for assisting in GC-MS analyses. I wish to acknowledge Dr. Alice Muchugi for funding this research through Warburgia ugandensis Phytochemical Research Project (IFS) Grant number (F/3829-2). I also wish to thank the National Commission for Science, Technology and Innovation (NACOSTI), Kenya for funding my M.Sc research work through grant number NCST/ST&I/RCD/4th Call M.Sc./157, 2012/2013. I appreciate my beloved wife Dorca Auma Murono for her love, moral and financial support and our sons Benard Abuto Otieno and Benjamin Jesse Otieno for being patient with me throughout the entire course. I’m also grateful to my Dad and Mum for their encouragement and support. v TABLE OF CONTENTS DECLARATION..................................................................................................................... ii DEDICATION........................................................................................................................ iii ACKNOWLEDGEMENTS .................................................................................................. iv TABLE OF CONTENTS ........................................................................................................v LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES .................................................................................................................x ABBREVIATIONS AND ACRONYMS ............................................................................ xiii ABSTRACT .......................................................................................................................... xiv CHAPTER ONE ......................................................................................................................1 INTRODUCTION....................................................................................................................1 1.1 Background information ..................................................................................................1 1.2 Statement of the problem and justification ......................................................................3 1.3 Research questions ...........................................................................................................4 1.4 Objectives of the study .....................................................................................................5 1.4.1 General objectives .....................................................................................................5 1.4.2 Specific objectives .....................................................................................................5 CHAPTER TWO .....................................................................................................................6 LITERATURE REVIEW .......................................................................................................6 2.1 The Canellaceae plant family ...........................................................................................6 2.2 Distribution and morphological description of Warburgia ugandensis ...........................7 2.3 Propagation of Warburgia ugandensis .............................................................................8 2.4 Medicinal uses of Warburgia ugandensis ........................................................................9 2.5 Non medicinal uses of Warburgia ugandensis ................................................................9 2.6 Pharmacological properties of tree species in the genus Warburgia ...............................9 2.7 Effects of geographic and genetic variation on yield and composition of plant secondary metabolites .......................................................................................16 2.8 Gas Chromatography Mass Spectrometry (GC-MS) .....................................................23 CHAPTER THREE ...............................................................................................................25 vi MATERIALS AND METHODS ..........................................................................................25 3.1 Sample collection ...........................................................................................................25 3.2 Sample preparation .........................................................................................................27 3.3 Solvent extraction ...........................................................................................................28 3.4 Antimicrobial activity ....................................................................................................28 3.4.1 Test strains and standard drugs ................................................................................28 3.4.2 Preparation of extracts for antimicrobial activity ....................................................29 3.4.3 Media and plates preparations .................................................................................29 3.4.4 Preparation of the McFarland standard for bacterial suspension turbidity ..............30 3.4.5 Preparation of inocula suspensions .........................................................................30 3.4.6 Calculation of concentrations of the extracts ..........................................................31 3.4.7 Bioassay screening of pant extracts .........................................................................32 3.4.8 Minimum inhibitory concentration (MIC) analysis ................................................32 3.4.9 Preparation of samples for GC-MS analysis ...........................................................34 3.5 Gas Chromatography Mass Spectrometry (GC-MS) analysis ...........................................34 3.6 Data management and statistical analysis ..........................................................................35 CHAPTER FOUR ..................................................................................................................37 RESULTS ...............................................................................................................................37 4.1 Preliminary bioassay screening of extracts of W. ugandensis .......................................37 4.1.1 Bioassays for dichloromethane extracts of W. ugandensis .....................................37 4.1.2 Bioassays for methanol extracts of W. ugandensis .................................................39 4.2 Minimum inhibitory concentration of DCM extracts of W. ugandensis ........................41 4.3 Minimum inhibitory concentration of MeOH extracts of W. ugandensis ......................43 4.4 Interactions plant sites, samples, parts and solvent types on antibacterial and antifungal activities .............................................................................45 4.5 Gas Chromatography Mass Spectrometry analyses of extracts of W. ugandensis .........60 CHAPTER FIVE .................................................................................................................108 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ..................................108 5.1 DISCUSSION .............................................................................................................108 5.1.1 Antimicrobial activity of extracts of W. ugandensis .............................................108 5.1.1.1 The effects of plant parts, solvents and extract types on vii antimicrobial activity of W. ugandensis .......................................................109 5.1.2 Phytochemical profiles of extracts of W. ugandensis ............................................116 5.1.2.1 The effects of plant parts, solvents and extract types on phytochemical profiles of W. ugandensis .....................................................117 5.2 CONCLUSIONS ............................................................................................................125 5.3 RECOMMENDATIONS