Phytochemical and Biological Activity Studies on the Twigs of Baikiaea Plurijuga and the Tubers of Ipomoea Bolusiana University of Botswana Faculty of Science By Yaone Moabi ID: 200304668 Supervisor: Dr G. Bojase August, 2016 A DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY AT THE UNIVERTISY OF BOTSWANA Certification The undersigned certify that they have read and hereby recommend, for the acceptance by the Department of Chemistry, University of Botswana, a dissertation entitled “Phytochemical Studies on the Twigs of Baikiaea Plurijuga and the Tubers of Ipomoea Bolusiana” as part of the work recommended in fulfilment of the requirements for a Master’s of Science Degree in Chemistry of the University of Botswana. --------------------------- Dr. G. Bojase (Supervisor) Department of chemistry, university of Botswana -------------------------- Internal Examiner -------------------------- External Examiner i Copyright and Declaration The work contained in this thesis was completed by the author at the University of Botswana between June, 2014 and August 2016. I hereby declare that the work is original except where due reference has been made. The work in this thesis has never been nor will ever be submitted for the award of any degree at any other University. -------------------------- ----------/----------/2016 Yaone Moabi Date ID: 200304668 Copyright © University of Botswana 2016 ii Acknowledgements I wish to sincerely and humbly thank my supervisor Dr G.Bojase-Moleta for advice, support and encouragement she offered throughout my research work. Much appreciation also goes to my supervisory committee members; Prof R.T. Majinda, Prof I.B. Masesane and Dr A. Munyaneza for providing guidance and encouragement through their valuable suggestions. I am grateful to Dr I. Morobe and the Biological Sciences technical stuff for their assistance during the antimicrobial tests. I wish to also thank Prof R. T. Majinda and Dr Tsopitlo for the plant material. Special thanks goes to Prof R. T. Majinda’s research group; Miss E. Muriithi, and Dr M. Abubakar who were always there for me, Mr K. Bedane, Mr K. Chaka, Mr P. Mokgweetsi and Mr G. Gumessa for their support. I also acknowledge all the Chemistry Department personnel and fellow graduate students for input in one way or the other throughout my journey. Above all, I thank the Almighty God and my family for everything thus far. iii List of Abbreviations DEPT: Distortionless Enhancement by Polarization Transfer HMQC: Heteronuclear Multiple Quantum Correlation HMBC: Heteronuclear Multiple Bond Correlation COSY: Correlation spectroscopy NOESY: Nuclear Overhauser Enhancement Spectroscopy HR: High Resolution LR: Low Resolution DPPH: 2,2-Diphenyl-1-picrylhydrazyl MIQ: Minimum Inhibitory Quantity TPC: Total Phenolic Content IC50: 50 % Inhibitory Concentration NADPH: Nicotinamide adenine dinucleotide phosphate (reduced form) NADP+: Nicotinamide adenine dinucleotide phosphate (oxidized form) ESI: Electrospray Ionization TLC: Thin Layer Chromatography MTT: Methylthiazolyldiphenyl-Tetrazoliumbromide iv Abstract Phytochemical studies on the twigs of Baikiaea plurijuga afforded four compounds, (+) – catechin, taxifolin, eriodictyol and sucrose. The dissertation describes the methods of their isolation and discusses their characterization. All the isolated compounds are being reported for the first time from this plant species. The isolated compounds and extracts from this plant were screened for their antioxidant activity using DPPH radical. Eriodictyol showed the highest radical scavenging activity among the tested flavonoid derivatives with an IC50 value of 0.047 ± 0.007 μM. The IC50 value was better than that of the standard ascorbic acid (0.090 ± 0.01 μM). The total phenolic content of the extracts was determined using the Folin-Ciocalteau method and the ethyl acetate extract exhibited the highest amongst the twigs of Baikiaea plurijuga extracts (350 mg GAE/g). Antimicrobial studies on both the extracts and pure isolates was done using the agar-overlay method and (+) – catechin exhibited the best inhibitory activity against gram-positive bacteria S. aureus at an MIQ of 5 µg. Eriodictyol on the other hand showed the best inhibition against gram- negative bacteria E. coli and P. aeruginosa at MIQs of 50 µg. Taxifolin exhibited the best activity against the test fungus C. albicans at an MIQ of 5 µg. Phytochemical studies on the tubers of Ipomoea bolusiana afforded seven compounds, (E)- octadec-11-en-1-yl (E)-3-(4-hydroxyphenyl) acrylate, (E)-icos-13-en-1-yl (E)-3-(3,4- dihydroxyphenyl) acrylate, methyl (E)-octadec-11-enoate, sucrose, maltose, C4C and IBMF2, all of which are reported for the first time from this plant species. Trans esterification of C4C afforded caffeic acid methyl ester. The extracts and isolated compounds from these tubers were screened for antimicrobial as well as antioxidant activities. Amongst the cinnamic acid derivatives isolated, (E)-icos-13-en-1-yl (E)-3-(3,4-dihydroxyphenyl) acrylate showed better radical scavenging activity with an IC50 value of 0.023 ± 0.006 μM as compared to (E)-octadec-11-en-1-yl (E)-3-(4- hydroxyphenyl) acrylate which showed an IC50 value of 0.47 ± 0.06 μM. Antimicrobial studies of the isolated compounds as well as the extracts revealed that C4C exhibited the best activity against gram-negative bacteria P. aeruginosa with an MIQ of 10 μg, while (E)-icos-13-en-1-yl (E)-3-(3,4- dihydroxyphenyl) acrylate exhibited weak activity against gram-positive bacteria S. aureus with an MIQ of 50 μg. Extracts of the tubers of this plants exhibited moderate activity against all the test microorganism at an average MIQ of 50 µg. The total phenolic content of the extracts revealed that the aqueous methanol (80 %) extract showed the highest among the extracts at 150 mg GAE/g. v Table of Contents Certification ..................................................................................................................................... i Copyright and Declaration .............................................................................................................. ii Acknowledgements ........................................................................................................................ iii List of Abbreviations ..................................................................................................................... iv Abstract ........................................................................................................................................... v List of Tables ............................................................................................................................... viii List of Schemes ............................................................................................................................... x List of Figures ................................................................................................................................ xi Chapter One .................................................................................................................................... 1 1.1 Introduction ........................................................................................................................... 1 1.2 Plants Selected for The Study ............................................................................................ 4 1.3 Previously Isolated Metabolites of B. Plurijuga and I. bolusiana ................................... 10 1.4 Aim of The Study ................................................................................................................ 22 1.5 Objectives ............................................................................................................................ 22 References ................................................................................................................................. 23 Chapter Two.................................................................................................................................. 33 Materials and Methods .............................................................................................................. 33 2 General Experimental Procedures ...................................................................................... 33 2.1 Physical and Spectroscopic Measurements ..................................................................... 33 2.2 Procedure for Acquisition of UV Spectra Using Shift Reagents ..................................... 34 2.3 Chromatographic and Separation Methods ..................................................................... 35 2.4 Extraction and Isolation Procedure for I. Bolusiana ....................................................... 36 2.5 Trans esterification of Compound 73 .............................................................................. 38 2.6 Extraction and Isolation Procedure for B. Plurijuga ....................................................... 39 2.6 Physical and Spectroscopic Data for The Isolated Compounds .......................................... 42 References ................................................................................................................................. 45 Chapter Three................................................................................................................................ 46 Results and Discussion .............................................................................................................