MOLECULAR PHYLOGENY, LEAF MICROMORPHOLOGY AND ANTIMICROBIAL ACTIVITY OF PHYTOCONSTITUENTS OF KENYAN Plectranthus SPECIES IN THE COLEUS CLADE FREDRICK MUTIE MUSILA Bsc. Biology (UoN), Msc. Botany (UoN) Reg. No. I80/92761/2013 A THESIS SUBMITTED IN FULFILMENT OF DOCTOR OF PHILOSOPHY DEGREE IN PLANT TAXONOMY AND ECONOMIC BOTANY OF THE UNIVERSITY OF NAIROBI December, 2017 DECLARATION This is my original work and has not been presented for a degree in any other University. Signed : _______________________________ Date: ________________________ Mr. Fredrick Mutie Musila, BSc Msc. School of Biological Sciences, College of Biological and Physical Sciences, University of Nairobi Supervisors This thesis has been submitted with our approval as university supervisors Signature: _______________________________ Date: _______________________ Prof. Dossaji Saifuddin F., BSc, MSc, PhD. School of Biological Sciences, College of Biological and Physical Sciences, University of Nairobi Signature: _______________________________ Date: _______________________ Dr. Catherine Lukhoba W. B.Ed, MSc, PhD. School of Biological Sciences, College of Biological and Physical Sciences, University of Nairobi Signature: _______________________________ Date: ________________________ Dr. Joseph Mwanzia Nguta, BVM, MSc, Ph.D. (UON). Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, College of Agriculture and Veterinary Sciences, University of Nairobi i DEDICATION This thesis is dedicated to my family and friends who provided me with moral and financial support throughout my studies. ii ACKNOWLEDGEMENTS I am very grateful to the following individuals and organizations who contributed towards this thesis. First, I express my sincere gratitude to my supervisors Prof. S. F. Dossaji, Dr. C. Lukhoba and Dr. J. M. Nguta for their patience, guidance, suggestions, encouragement and excellent advice through the course of this study. I would like to thank Mr. Patrick Mutiso (University of Nairobi) and Mr. Mwandime Nyage (National Museums of Kenya) for their help in collection and identification of the studied Plectranthus species. Secondly, I would like to express my gratitude to Mr. Joseph Geda for facilitating DNA sequencing at Inqaba Biotec, South Africa. I am grateful to Mr. Victor Shikuku of CSI international for facilitating structure elucidation of pure compounds through NMR and Mass spectroscopy at Stellenbosch University, South Africa. I would like to express my appreciation to Prof. Abby Yenesew and Mr. Yoseph Atlaw (Chemistry Department, University of Nairobi) for their expert interpretation of NMR spectra, structure illustrations and identification of pure compounds. Lastly my acknowledgement goes to the University of Nairobi for provision of laboratory space for doing my entire research and DAAD (Germany Academic Exchange Service) for funding this research (Reference Number: A/13/94804). iii TABLE OF CONTENTS DECLARATION ..................................................................................................................... i DEDICATION ....................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................... iii TABLE OF CONTENTS ....................................................................................................... iv LIST OF TABLES .............................................................................................................. viii LIST OF FIGURES ............................................................................................................... ix LIST OF APPENDICES ....................................................................................................... xii LIST OF ABBREVIATIONS AND ACRONYMS .............................................................. xiii ABSTRACT .........................................................................................................................xiv CHAPTER ONE: INTRODUCTION ......................................................................................1 1.1. Background information ...................................................................................................1 1.2. Economic importance of Plectranthus...............................................................................2 1.3. A taxonomic review of Plectranthus .................................................................................4 1.4. Taxonomic difficulties in Plectranthus genus....................................................................5 1.5. Problem Statement ............................................................................................................8 1.6. Justification ......................................................................................................................8 1.7. Hypotheses .......................................................................................................................9 1.8. Objectives of the study .................................................................................................... 10 1.8.1. General objective...................................................................................................... 10 1.8.2. Specific objectives .................................................................................................... 10 2. CHAPTER TWO: LITERATURE REVIEW ..................................................................... 11 2.1. MOLECULAR PHYLOGENY OF Plectranthus SPECIES ............................................ 11 2.1.1. Introduction .............................................................................................................. 11 2.1.2. Existing molecular phylogeny of Plectranthus and related genera............................. 12 iv 2.1.3. Universal molecular markers in plants ...................................................................... 13 2.1.4. MatK and Rbcl molecular markers ............................................................................ 14 2.1.5. Importance of molecular characters in phylogenetic studies ...................................... 15 2.1.6. Steps in molecular phylogeny ................................................................................... 16 2.2. LEAF MORPHOLOGY/MICROMORPHOLOGY OF Plectranthus SPECIES .............. 17 2.2.1. Morphological characters and phylogeny .................................................................. 17 2.2.2. Diagnostic morphological/micromorphological characters in plants .......................... 18 2.2.3. Existing micromorphological studies of Plectranthus and related genera .................. 19 2.3.4. Description of the ten Plectranthus species ............................................................... 21 2.3. ANTIMICROBIAL ACTIVITY AND PHYTOCONSTITUENTS OF Plectranthus SPECIES ............................................................................................................................... 21 2.3.1. Introduction .............................................................................................................. 21 2.3.2. Ethnopharmacological uses of Plectranthus species.................................................. 22 2.3.3. Phytochemical composition and bioactivity of Plectranthus species ......................... 23 CHAPTER THREE: MATERIALS AND METHODS .......................................................... 27 3.1. Collection of Plectranthus species samples ..................................................................... 27 3.2. Deoxyribonucleic acid (DNA) extraction ........................................................................ 29 3.3. Polymerase Chain Reaction (PCR) of two genes in Plectranthus..................................... 30 3.4. DNA Sequencing of amplified genes in Plectranthus ...................................................... 31 3.5. Phylogenetic analysis techniques .................................................................................... 31 3.5.1. Assembling and aligning datasets ............................................................................. 31 3.5.2. Building phylogenetic trees ...................................................................................... 31 3.6. Stomatal distribution and density in Plectranthus leaves ................................................. 32 3.7. Leaf anatomy .................................................................................................................. 33 3.8. Scoring of charaters. ....................................................................................................... 34 v 3.9. Cluster analysis based on leaf characters ......................................................................... 34 3.10. Evaluation of antimicrobial activity of Plectranthus species .......................................... 34 3.10.1. Extration and preparation of crude extracts of Plectranthus species ........................ 34 3.10.2. Determination of antimicrobial activity of the crude extracts. ................................. 35 3.10.3. Preparation of Plectranthus test extracts ................................................................. 36 3.10.4. Disc diffusion technique ......................................................................................... 36 3.10.5. Broth dilution technique ........................................................................................