Molecular phylogeny, leaf micromorphology and antimicrobial activity of phytoconstituents of

171 Pages·2017·6.46 MB·English

by  Fredrick Mutie Musila

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).

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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
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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
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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 ......................................................................................... 37
3.10.6. Bioassay guided fractionation of P.barbatus leaf extracts ....................................... 37
3.10.6.1. Partition Liquid Chromatography (PLC) of P. barbatus leaf extracts ................... 38
3.10.6.2. Liquid column chromatography (LCC) of the DCM partition of P.barbatus ........ 38
3.10.6.3. Antimicrobial activity of isolated compounds ...................................................... 40
3.11. Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) ............................. 40
3.12. Compilation of data and data analysis ........................................................................... 41
CHAPTER FOUR: RESULTS .............................................................................................. 42
4.1. Plectranthus species investigated .................................................................................... 42
4.2. Molecular phylogeny of ten Plectranthus species ............................................................ 47
4.2.1. Genomic DNA ......................................................................................................... 47
4.2.2. Sequencing and multiple alignment of MatK and Rbcl genes .................................... 48
4.2.3. Phylogenetic trees based on MatK and Rbcl gene sequences ..................................... 49
4.2.4. Comparison of the Plectranthus species genes with genbank sequences ................... 53
4.3. Leaf micromorphology of Plectranthus species ............................................................... 57
4.3.1. Stomatal distribution in Plectranthus species ............................................................ 57
4.3.2. Leaf micromorphology of Plectranthus species ........................................................ 58
4.3.3. Dendrogram based on the leaf micromorphology data .............................................. 64
4.3.4. Dendrogram based on morphological characters described in the FTEA ................... 66
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4.3.5. Comparison of leaf micromorphology dendrogram with MatK and Rbcl phylogenetic
trees ................................................................................................................................... 68
4.4. Antimicrobial activity of Plectranthus species ................................................................ 70
4.4.1. Disc diffusion results ................................................................................................ 70
4.4.2. Analysis of antimicrobial activities of Plectranthus species ...................................... 75
4.4.3. Minimum inhibitory concentration (MIC) values of total extracts from studied
Plectranthus species ........................................................................................................... 79
4.5. Antimicrobial activity of fractionated extracts of P.barbatus ....................................... 81
4.5.1. Disc diffusion results ................................................................................................ 81
4.5.2. Analysis of antimicrobial activities of P. barbatus fractionated crude extracts .......... 86
4.5.3. Minimum Inhibitory Concentrations of P. barbatus fractionated crude extracts ........ 90
4.6. Thin Layer Chromatography (TLC) profiles of P. barbatus isolated compounds ......... 91
4.7. Determination of the presence of isolated compounds A and B in other Plectranthus
species ................................................................................................................................... 91
4.8. Antimicrobial activity of the isolated compounds A and B .............................................. 93
4.9. Interpretation of NMR Spectra for identification of Compounds A and B........................ 95
4.10. Comparison of Compound A and B ............................................................................ 102
CHAPTER FIVE: DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .......... 105
5.1. Discussion .................................................................................................................... 105
5.2. Conclusions .................................................................................................................. 123
5.3. Recommendations......................................................................................................... 126
REFERENCES .................................................................................................................... 128
APPENDICES ..................................................................................................................... 140
PUBLICATIONS ................................................................................................................ 152

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LIST OF TABLES

Table 2.1: Description of the ten Plectranthus species ............................................................... 19
Table 3.1: Primers used in DNA amplification .......................................................................... 30
Table 3.2: Yields and percentage recovery from DCM: MeOH (1:1) leaf extracts of Plectranthus
species ...................................................................................................................................... 35
Table 4.1: Stomata distribution in the ten Plectranthus species .................................................. 57
Table 4.2: Leaf anatomical characters in the ten Plectranthus species........................................ 58
Table 4.3: Leaf micromorphology character states matrix of the ten Plectranthus species ......... 64
Table 4.4: Gross morphology character state matrix of the ten Plectranthus species .................. 66
Table 4.5: Comparison of growth inhibitions of test microbes among Plectranthus species at 200,
100 and 50 mg/ml using ANOVA. ............................................................................................ 76
Table 4.6: Comparison between growth inhibitions of test microbes by Plectranthus species at
200, 100 and 50 mg/ml and the positive controls ....................................................................... 78
Table 4.7: Minimum inhibitory concentration of Plectranthus species ....................................... 80
Table 4.8: Comparison of growth inhibitions of test microbes among P.barbatus fractionated
crude extracts at 200, 100 and 50 mg/ml.................................................................................... 87
Table 4.9: Comparison between growth inhibitions of test microbes by P.barbatus fractionated
crude extracts at 200, 100 and 50 mg/ml and positive controls .................................................. 89
Table 4.10: Minimum inhibitory concentrations of P.barbatus fractionated crude extracts ........ 90
Table 4.11: Determination of MICs of Compound A & B against the test mirobes .................... 94
Table 4.12: Compound A: Interpretation of 1D NMR (13C NMR, 1H NMR) and 2D NMR
(COSY, HSQC and HMBC) spectra .......................................................................................... 95
Table 4.13: Compound B: Interpretation of 1D NMR (13 CNMR, 1H NMR) and 2D NMR
(COSY, HSQC and HMBC) spectra. ......................................................................................... 99

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LIST OF FIGURES
Figure 2.1: Steps in molecular phylogenetic analysis ................................................................. 16
Figure 3.1: Specific localities where Plectranthus species were collected within the regions of
the Flora of Tropical East Africa ............................................................................................... 28
Figure 4.1: Pictures of ten Plectranthus species showing aerial parts ......................................... 46
Figure 4.2: Gel electrophoresis of genomic DNA from the ten Plectranthus species .................. 47
Figure 4.3: Multiple alignment of MatK sequences .................................................................... 48
Figure 4.4: Multiple alignment of Rbcl sequences ..................................................................... 49
Figure 4.5: A phylogenetic tree for the ten Plectranthus species based on MatK gene sequences
................................................................................................................................................. 50
Figure 4.6: A phylogenetic tree for the ten Plectranthus species based on Rbcl gene sequences. 51
Figure 4.7: Comparison of MatK (a) and Rbcl (b) phylogenetic trees of the ten Plectranthus
species ...................................................................................................................................... 53
Figure 4.8: Comparison of current project MatK sequences with those obtained from genbank
Plectranthus sequences ............................................................................................................. 55
Figure 4.9: Comparison of current project Rbcl sequences with those from genbank Plectranthus
sequences .................................................................................................................................. 56
Figure 4.10: Adaxial surface of P.caninus leaf .......................................................................... 59
Figure 4.11: Abaxial surface of P.pseudomarrubioides leaf ....................................................... 59
Figure 4.12: Transverse section of P.lanuginosus leaf ............................................................... 60
Figure 4.13: Transverse section of P.ornatus leaf ...................................................................... 61
Figure 4.14: Transverse section of P.barbatus leaf .................................................................... 62
Figure 4.15: Transverse section of P.edulis leaf ......................................................................... 62
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