Phytochemical Investigation of the Stem Bark of Millettia Oblata Ssp
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v\ PHYTOCHEMICAL INVESTIGATION OF THE STEM BARK OF MILLETTIA OBLATA SSP. TEITENSIS FOR ANTIPLASMODIAL AND LARVICIDAL PRINCIPLES BARASA LEONARD A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE AWARD OF MASTERS OF SCIENCE CHEMISTRY DEGREE UNIVERSITY OF NAIROBI 2011 University of NAIROBI Library 0478799 0 DECLARATION This thesis is my original work and has not been presented for a degree in any other university. The research is carried out in the Department of Chemistry of the University of Nairobi. BARASA LEONARD This thesis has been submitted for examination with my approval as a University supervisor. DEPARTMENT OF CHEMISTRY DEPARTMENT OF CHEMISTRY UNIVERSITY OF NAIROBI UNIVERSITY OF NAIROBI DEDICATION This thesis is dedicated to my late beloved mum; Esther Nyongesa. Thank you for your great love and for giving me direction in life. My love for you is immeasurable. I in ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisors Dr. Solomon Derese and Prof. Abiy Yenesew for their prompt guidance, support and inspiration throughout my MSc work. I am deeply indebted to Prof. Martin G. Peter and Dr. Matthias Heydenreich for organizing and analyzing the samples on high resolution NMR and MS. Mr. Akala, H., Waters, N. C and Ms. Liyala, P. o f Kenya Medical Research Institute and United States Army Medical Research Unit - Kenya; are greatly acknowledged for their collaboration in carrying out the antiplasmodial tests described in this work. I wish to sincerely acknowledge the financial support o f International Foundation for Science (IFS); grant No. F/4581 for funding this project. I would like to sincerely thank Dr. Albert Ndakala for day to day updates of the latest developments from the Department of Chemistry and the School of Postgraduate. Thank you very much. I owe much gratitude to Dr. Jacques Kabaru and the technicians of the Zoology Department, University of Nairobi, for the supply and hatching of the mosquito larvae of the Aedes aegypti used in the larvicidal tests. Sincere acknowledgements go to Mr. Patrick C. Mutiso of the University of Nairobi Herbarium, School of Biological sciences, for identification o f the plants investigated in this study. The academic and technical staff of the Department of Chemistry, my MSc colleagues and PhD students are sincerely IV thanked for their endless support and encouragement. Lastly, I wish to thank the almighty God for giving me life, strength and protection during the entire study period v LIST OF ABBREVIATIONS m/z Mass to charge ratio [M]+ Molecular ion CHS Chalcone synthase CHI Chalcone isomerase PKR Polyketide reductase EIMS Electron ionization mass spectroscopy IC50 Concentration o f 50% LC 50 Concentration of 50% lethality inhibition COSY Correlated spectroscopy DEPT Distortionless enhanced polarization transfer HMBC Heteronuclear multiple HMQC Heteronuclear multiple quantum bond correlation ( 2J c h , coherence ( 1J c h ) * 3J c h ) Nuclear magnetic Maximum wavelength of NMR ^-max resonance absorption UV Ultra violet 5 Chemical shift MS Mass spectroscopy J Coupling constant Hz Hertz MHz Mega hertz d Doublet S Singlet t Triplet M Multiplet (multiplicity) dd Doublet of a doublet TLC Thin layer chromatography AP Aerial parts FL Flowers HW Heart wood L Leaves RB Root bark RW Root wood SB Stem bark SD Seeds SDP Seedpods WD Wood DCM Dichloromethqne Mp Melting point TABLE OF CONTENTS DEDICATION.....................................................................................................................................iii ACKNOWLEDGEMENTS..............................................................................................................iv LIST OF ABBREVIATIONS.......................................................................................................... vi TABLE OF CONTENTS.................................................................................................................vii ABSTRACT...........................................................................................................................................x CHAPTER O NE................................................................................................................................... 1 1.0 INTRODUCTION.1..................................................................................................................1 1.1 GENERAL............................................................................................................................. 1 1.2 Malaria....................................................................................................................................3 1.3 Biodegradable larvicides.....................................................................................................5 1.4 Problem statement................................................................................................................ 6 1.5 Justification of the Research..............................................................................................7 1.6.0 Objectives........................................................................................................................ 9 CHAPTER TW O................................................................................................................................10 2.0 LITERATURE REVIEW .....................................................................................................10 2.1 Botanical information....................................................................................................... 10 2.2 Phytochemistry of the genus M illettia........................................................................18 CHAPTER THREE...........................................................................................................................28 3.0 METHODOLOGY............................ 28 3.1 General..................................................................................................................................28 3.2 Extraction and Isolation of Compounds...................................................................... 28 3.3 Physical and Spectroscopic Data for the Isolated Compounds............................... 30 3.4 Biological Activity A ssays..............................................................................................33 CHAPTER FOUR.............................................................................................................................. 36 RESULTS AND DISCUSSION................................................................................................36 4.0 PRELIMINARY TEST RESULTS................................................................................... 36 4.1 COMPOUNDS FROM THE STEM BARK OF MILLETTIA................................ 36 OBLATA SSP. TEITENSIS......................................................................................................36 4.2 Biological Activities of the Isolated Compounds...................................................... 51 CHAPTER FIV E............................................................................................................................... 54 vii CONCLUSIONS AND RECOMMENDATIONS...............................................................54 5.1 CONCLUSIONS................................................................................................................ 54 5.2 RECOMMENDATIONS................................................................................................. 55 REFERENCES...................................................................................................................................56 APPENDICES....................................................................................................................................62 LIST OF FIGURES Figure 2.1: Millettia oblata ssp. teitensis (Catalogue of life, 2009)..................................... 12 Figure 4.1: EIMS fragmentation pattern o f compound 2 ..................................... „.................40 Figure 4.2: HMBC Correlation in Compound 2 .........................................................................42 Figure 4.3: HMBC Correlation in Compound 4 .........................................................................44 LIST OF TABLES Table 2.1: Distribution of Millettia oblata Dunn subspecies................................................. 12 Table 2.2: Anti-plasmodial activities of flavonoids from Kenyan Millettia species......... 13 Table 2.3: The larvicidal activity, LC50, o f the rotenoids after 24 hours on 2nd instar......17 larvae o f Aedes aegypti......................................................................................................................17 Table 2.4: Traditional uses of some Kenyan Millettia species...............................................18 Table 2.5: A summary of isoflavones of Millettia reported..................................................... 21 Table 4.1: 'H (CD2C12, 600 MHz) and 13C (CDC13, 50 MHz) - NMR Chemical Shift....38 Values for Compound 1 ....................................................................................................................38 Table 4.2: 'H (CD2C12, 600 MHz) and 13C (CD2C12, 150 MHz) -NMR Chemical Shift..41 Values for Compound 2 ....................................................................................................................41 Table 4.3: 'H (CD2C12, 600 MHz) -NMR Chemical Shift Values for Compound 3 .........43 Table 4.4: 'H (CD2C12, 600 MHz)-NMR