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SCREENING for ANTIBACTERIAL and ANTIFUNGAL COMPOUNDS in Bersama Abyssinica FRESEN

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SCREENING for ANTIBACTERIAL and ANTIFUNGAL COMPOUNDS in Bersama Abyssinica FRESEN SCREENING FOR ANTIBACTERIAL AND ANTIFUNGAL COMPOUNDS IN Bersama abyssinica FRESEN ONG’ERA TABITHA NYANCHOKA (B.ED, SC.) (I56/CE/11221/2007) A Research 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 Science, Kenyatta University MAY 2016 ii Declarations I declare that this thesis is my original work and has not been previously presented for a degree in Kenyatta University or in any other University. Ong’era Tabitha Nyanchoka Reg. No. I56/CE/11221/07 Sign: _________________________Date _____________________________ Department of Chemistry We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Prof. Alex K. Machocho Department of Chemistry Kenyatta University Sign: _______________________ Date_____________________________ Prof. Nicholas K. Gikonyo Department of Pharmacy, Complementary and Alternative Medicine Kenyatta University Sign: ____________________________Date__________________________ iii Dedication To my daughter Flavian Bonareri, sons Adrian Nyaenya and Fabian Ong’era and my beloved husband Evans Nyaenya. iv Acknowledgements I would like to display my sincere gratitude to the following persons for their support, assistance and encouragement throughout the course of my research and compilation of this thesis. My supervisors: Prof. Alex Machocho of the Department of Chemistry and Prof. Nicholas Gikonyo of the Department of Pharmacy, Complementary and Alternative medicine for their guidance, motivation, support and time invested in me. Also, Dr. Omari Amuka of Maseno University for assisting me in the plant collection exercise. Dr. Margret Ng’ang’a of the Department of Chemistry for her encouragement and support throughout my research in the laboratory. I would also like to thank the staff in the Chemistry laboratory, Kenyatta University for their assistance in the laboratory and the staff in the microbiology laboratory more especially Daniel Ng’ang’a for providing expertise knowledge on in vitro antibacterial and antifungal assays. To my classmates and research partners; Regina Kihagi, Moses Oswago, Stephen Kamau, Ombuna Naftal, Shylock Onduso and Nyaenya Evans for their valuable suggestions. In addition, my sincere gratitude goes to Keru Kamitha, University of Kwazulu Natal for assisting me in running the NMR of the isolated compounds and to the technical staff of Kenya Bureau of Standards for running GC-MS of the crude extracts. Special thanks go to my husband and children, brothers, sisters and parents for their patience, support, encouragement, prayers and motivation when it was most needed and also to Vicres foundation for partially sponsoring my research work. Last but not least, God without whom nothing is possible. v TABLE OF CONTENTS Declarations ............................................................................................................................... ii Dedication ................................................................................................................................. iii Acknowledgements …....................................................................................................................iv Table of contents…………………………………………………………………………………..v Abbreviations and Acronyms………………………………………………………………......xiii Abstract ..................................................................................................................................xvi CHAPTER ONE .......................................................................................................................1 INTRODUCTION .....................................................................................................................1 1.1 Background ...........................................................................................................................1 1.2 Antibiotic resistance …………………………………………………………………………..2 1.3 Phytomedicine and Phytopharmaceutical agents ....................................................................5 1.4 Bacterial infections ................................................................................................................8 1.4.1 Staphylococcus aureus ................................................................................................9 1.4.2 Escherichia coli ...........................................................................................................9 1.4.3 Pseudomonas aeruginosa .......................................................................................... 10 1.5 Antibacterial drugs .............................................................................................................. 11 1.5.1 Tetracyclines ............................................................................................................. 11 1.5.2 Penicillins ................................................................................................................. 12 1.5.3 Sulphonamides .......................................................................................................... 12 1.5.4 Flouroquinolones ....................................................................................................... 13 1.5.5 Quinolones ................................................................................................................ 14 1.6 Fungal infections ................................................................................................................. 14 1.7 Antifungal drugs.................................................................................................................. 16 1.7.1 Flucytosine ................................................................................................................ 16 1.7.2 Amphotericin ............................................................................................................ 17 1.8 Statement of the problem ..................................................................................................... 18 1.9 Hypotheses…………………………………………………………………………………..19 1.10 Objectives of the study ...................................................................................................... 19 1.10.1 General objective ..................................................................................................... 19 vi 1.10.2 Specific objectives ................................................................................................... 19 1.11 Justification and significance of the study .......................................................................... 20 CHAPTER TWO .................................................................................................................... 21 LITERATURE REVIEW ....................................................................................................... 21 2.1 The family Melianthaceae ................................................................................................... 21 2.1.1 Bersama yangambiensis Toussaint ............................................................................ 21 2.1.2 Bersama engleriana Gurke ........................................................................................ 22 2.1.3 Bersama swinnyi Phil…………………………………………………………………23 2.1.4 Bersama abyssinica Fresen ........................................................................................ 24 2.2 Biosynthesic pathway of anthraquinone ............................................................................... 27 2.3 Biosynthesis of lupeol ......................................................................................................... 29 2.4 Biosynthetic pathway of steroids ......................................................................................... 30 2.5 Biosynthetic pathway of oleanolic acid……………………………………………………... 31 CHAPTER THREE …………………………………………………………………………….32 MATERIALS AND METHODS ............................................................................................ 32 3.1 General procedures .............................................................................................................. 32 3.2 Chromatographic techniques ............................................................................................... 33 3.2.1 Spray reagents ........................................................................................................... 33 3.2.3 Detection of terpenoids ............................................................................................. 33 3.2.4 Column chromatography ........................................................................................... 33 3.3 Plant material ...................................................................................................................... 32 3.4 Bioactivity screening of the plant extracts............................................................................ 34 3.4.1 Anti-bacterial activity test .......................................................................................... 34 3.4.1.1 Preparation of nutrient agar media and growing of bacteria cultures …………35 3.4.1.2 Introduction of the crude plant extract into inoculated Petri dishes.………..…36 3.5 Antifungal activity test ........................................................................................................ 36 3.5.1 Preparation of the drugs ............................................................................................. 37 3.6 Extraction
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