ANTIBACTERIAL, ANTIFUNGAL AND PHYTOCHEMICAL SCREENING OF THE PLANT SPECIES Lannea schweinfurthii (ENGL.) ENGL. KIHAGI REGINA WAMUYU (B.Ed, Sc) I56/CE/10703/2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (CHEMISTRY) IN THE SCHOOL OF PURE AND APPLIED SCIENCES, KENYATTA UNIVERSITY NOVEMBER 2016 ii DECLARATION This thesis is my original work and has not been presented for any other degree in any other universities or for any other award. Signature…………………………………………….. Date…………………………… Kihagi Regina Wamuyu - I56/CE/10703/2006 Department of Chemistry SUPERVISORS We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Signature…………………………………………….. Date…………………………… Prof. Alex K. Machocho Department of Chemistry Signature…………………………………………….. Date…………………………… Dr. Alphonse W. Wafula Department of Chemistry iii DEDICATION To my parents, siblings, nieces and nephew for your continued love and support iv ACKNOWLEDGEMENTS I am grateful to God for giving me strength to carry on with this study, without which I would not have come this far. My sincere gratitude goes to my able supervisors, Prof. Alex Machocho and Dr. Alphonse Wanyonyi for their immeasurable advice, remarkable commitment to supervise my work, limitless encouragement and patience throughout this research and writing of thesis. Many thanks to Dr. Omari Amuka of Maseno University for his assistance in the collection of plant samples, Mr. Lucas Karimi of the Department of Pharmacy and Complementary/Alternative Medicine for the authentication of the plant materials, Dr. Margaret Ng’ang’a and Dr. Evelyne Mahiri, both of Kenyatta University, for offering the much needed moral support to carry on with the research and Prof. Martin Onani of the University of Western Cape for his assistance in running NMR of isolated compounds in South Africa. I acknowledge the support of the entire Departments of Chemistry and Microbiology Laboratory staff at Kenyatta University for the help they accorded me during my research. The technical staff of Kenya Bureau of Standards as well, for running GC-MS of crude extracts. My appreciation also goes to all my research assistants for the important role they played in the research. I highly appreciate Tabitha, Ombuna, Keru and all my course mates at Kenyatta University for their timely contributions throughout the study. Special thanks to my parents, siblings, relatives and friends for their tolerance, emotional and material support they offered throughout the entire process. May the Lord bless you all. v TABLE OF CONTENTS DECLARATION .................................................................................................................ii DEDICATION .................................................................................................................. iii ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF FIGURES ............................................................................................................ ix LIST OF TABLES .............................................................................................................. x LIST OF SCHEMES .......................................................................................................... xi ABBREVIATIONS AND ACRONYMS ..........................................................................xii ABSTRACT .................................................................................................................... xiii CHAPTER ONE ................................................................................................................ 1 INTRODUCTION ............................................................................................................... 1 1.1 Background .................................................................................................................... 1 1.2 Phytomedicine ............................................................................................................... 3 1.3 Antibiotics ..................................................................................................................... 6 1.3.1 Bacteriostatic agents ................................................................................................... 7 1.3.2 Bactericidal agents ...................................................................................................... 7 1.4 Active compounds in plant extracts .............................................................................. 8 1.5 Statement of the problem ............................................................................................... 8 1.6 Hypotheses .................................................................................................................. 10 1.7 Objectives .................................................................................................................... 10 1.7.1 General objective ...................................................................................................... 10 1.7.2 Specific objectives .................................................................................................... 10 1.8 Justification and significance of the study ................................................................... 11 CHAPTER TWO ............................................................................................................. 12 LITERATURE REVIEW .................................................................................................. 12 2.1 The family Anacardiaceae ........................................................................................... 12 2.2.1 The Genus Lannea .................................................................................................... 13 2.2.2 Chemical systematics and bioactivity of Genus Lannea .......................................... 13 vi 2.3.1 Lannea schweinfurthii (Engl.) Engl. ........................................................................ 18 2.3.2 Medicinal uses of Lannea schweinfurthii ................................................................. 19 2.3.3 Other uses of Lannea schweinfurthii ........................................................................ 20 2.4 Bioassay ....................................................................................................................... 21 2.4.1 Role of bioassay in isolation of pure active compounds from plants ....................... 21 2.4.2 Antimicrobial assays ................................................................................................ 22 2.5 Flavonoids ................................................................................................................... 23 2.5.1 Biosynthesis of flavonoids ....................................................................................... 24 2.5.2 Structural elucidation of flavonoids ......................................................................... 27 2.5.2.1 Nuclear Magnetic Resonance (NMR) ................................................................... 28 2.5.2.1.1 2D-NMR ............................................................................................................. 28 2.5.2.2 Mass spectroscopy (MS) ....................................................................................... 30 2.5.2.3 Ultra-Violet (UV) spectroscopy ............................................................................ 31 2.6 Terpenoids ................................................................................................................... 31 2.6.1 Biosynthesis of lupeol .............................................................................................. 31 2.6.2 Biosynthesis of β-sitosterol ...................................................................................... 32 CHAPTER THREE ......................................................................................................... 34 MATERIALS AND METHODS ...................................................................................... 34 3.1 General procedures ...................................................................................................... 34 3.1.1 Laboratory equipment and instruments used ............................................................ 34 3.1.2 Chromatographic materials and solvents .................................................................. 34 3.1.3 Spray reagents .......................................................................................................... 35 3.1.4 Detection of compounds ........................................................................................... 36 3.1.5 Nuclear magnetic resonance (NMR) spectroscopy .................................................. 36 3.2 Plant collection and identification ............................................................................... 37 3.3 Extraction procedure of Lannea schweinfurthii stem bark .......................................... 37 3.4 Bioassay of the plant extracts ...................................................................................... 39 3.4.1 Selected test strains ................................................................................................... 39 3.4.2 Antifungal screening tests ........................................................................................ 40 vii 3.4.2.1 Preparation