SCREENING, ISOLATION AND PURIFICATION OF BIOACTIVE COMPOUNDS WITH ...

134 Pages·2011·2.81 MB·English

SCREENING, ISOLATION AND PURIFICATION OF BIOACTIVE COMPOUNDS WITH
ANTIBACTERIAL ACTIVITY AGAINST MYCOBACTERIUM SMEGMATIS

by

MMUSHI TSHEPO JOSEPH

RESEARCH DISSERTATION
Submitted in fulfilment of the requirements for the degree of

MASTER OF SCIENCE
in
MICROBIOLOGY
in the
FACULTY OF SCIENCE & AGRICULTURE
(School of Molecular & Life Science)
at the
UNIVERSITY OF LIMPOPO

Supervisor: Prof. R.L. Howard
Co-Supervisors: Dr. P. Masoko and Prof L.J. Mampuru

2011
DECLARATION
I declare that the dissertation hereby submitted to the University of Limpopo for the
degree of Master of Science in Microbiology has not been previously submitted by me
for a degree at this or any other University; that it is my own work in design and in
execution, and that all materials contained therein has been duly acknowledged.

--------------------------------------- ---------------------------------------
Initials & Surname (Title) Date

Student Number: ---------------------------------------
ii
DEDICATION
I dedicate this work to my family; my mother Sophy, my sisters Isabella and Tshidi,
and my brothers Neo and Rorisang for having faith in me and for their support and
encouragement throughout the project.
ACKNOWLEDGEMENTS
I would like to sincerely thank the following:
 The Almighty God, for giving me courage, guidance and strength to carry this
project to the end.
 The University of Limpopo for giving me a chance to do a Masters degree and
offering materials to carry the project through.
 Department of Water Affairs (DWA) and University of Limpopo Research
Development and Administration office for their financial support.
 My supervisor, Prof. R.L. Howard, for helping me with the scientific education,
inspiration and words of encouragement.
 My co-supervisors for their support, Dr. P. Masoko and Prof. L.J. Mampuru for
giving me the incredible insight into medicinal plants and also for the outstanding
assistance.
 Dr. M.P. Mokgotho; thanks for your help during the collection of plant materials at
Lowveld Botanical Garden (Nelspruit).
 The Lowveld Botanical Garden for allowing us to collect the plant materials for
the project.
 Dr. L. Mdee for the remarkable project assistance with isolation and identification
of bioactive compounds.
 Mr. F.H. Makhubela for generating the NMR sprectra for the two samples.
 My friends and colleagues from the discipline of microbiology.
 My family, for the encouragement and support throughout my studies.

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TABLE OF CONTENTS
Page
Declaration………………………………………………………………………............. ii
Dedication…………………………………………………………………………........... iii
Acknowledgement………………………………………………………....................... iv
Table of Content…………………………………………………………………............ v-ix
List of Abbreviations……………………………………………………………............ x
List of Figures……………………………………………………………………............ xi-xv
List of Tables……………………………………………………………………….......... xvi
Abstract……………………………………………………………………………........... xvii
Publications from this thesis…………………………………………………............. xviii

Chapter 1
LITERATURE REVIEW
1.1 Introduction………………………………………………………………..… 1-3
1.2 Literature review.................................................................................... 3
1.2.1 Plants as a source of new drug development……………...................... 3-5
1.2.2 Bioactive compounds in medicinal plants…………………………......…. 5-6
1.2.3 Nitrogen containing compounds…………………………………...……… 6
1.2.3.1 Alkaloids…………………………………………………………….............. 6-7
1.2.3.2 Terpenoids………………………………………………………………....... 7-8
1.2.3.3 Phenolic compounds……………………………………………................ 8
1.2.3.3.1 Flavonoids……………………………………………………………........... 8-9
1.2.3.3.2 Tannins………………………………………………………………............ 9
1.3 Identification of bioactive compounds…………...................................... 10
1.3.1 Collection of plant materials…………………………………………..…… 10-12
1.3.2 Preparation of plant extracts………………………………………………. 12-13
1.3.2.1 Solvent extraction…………………………………………………………... 13
1.3.2.2 Hot extraction……………………………………………………………….. 13
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1.3.2.3 Soxhlet extraction………………………………………………………… ... 14
1.3.3 Separation and purification of bioactive compounds…………………… 14
1.3.3.1 Thin layer chromatography……………………………………………… ... 14-15
1.3.3.2 Column chromatography…………………………………………………... 15
1.3.3.3 High pressure liquid chromatography…………………………………..... 16
1.4 Bioassays for antimicrobial activity……………………………………….. 16
1.4.1 Agar diffusion assays............................................................................. 16-17
1.4.2 Disc diffusion.......................................................................................... 17
1.4.3 Well diffusion.......................................................................................... 17 -18
1.4.4 Dilution assays (minimum inhibitory concentration)............................... 18
1.4.4.1 Agar-dilution method............................................................................ 18
1.4.4.2 Serial dilution method........................................................................... 18 -19
1.4.5 Bioautography........................................................................................ 19
1.5 Identification of chemical structures....................................................... 19-20
1.6 The classification, distribution, description and uses of the fifteen 20
plants used in this study.......................................................................
1.6.1 Albizia gummifera.................................................................................. 20-21
1.6.2 Annona senegalensis............................................................................. 21
1.6.3 Antidesma venosum.............................................................................. 22
1.6.4 Apodytes dimidiata subsp. dimidiata..................................................... 22-23
1.6.5 Barringtonia racemosa........................................................................... 23-24
1.6.6 Kigelia africana...................................................................................... 24
1.6.7 Kirkia acuminata…………………………………………………………….. 25
1.6.8 Macaranga capensis.............................................................................. 25-26
1.6.9 Maytenus species.................................................................................. 26-27
1.6.10 Millettia stuhlmannii............................................................................... 27
1.6.11 Sclerocarya birrea.................................................................................. 28
1.6.12 Vangueria infausta subsp. infausta....................................................... 28-29
1.6.13 Warburgia salutaris................................................................................ 29-30
1.6.14 Xanthocersis zambesiaca...................................................................... 30
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1.7 Microbiology and pathogenicity of Mycobacterium................................ 31
1.7.1 Treatment of Mycobacterium tuberculosis............................................ 31-32
1.7.2 Medicinal plants as a potential source of anti-Mycobacterium
tuberculosis............................................................................................ 32-33
1.7.3 Mycobacteium smegmatis and Rhodococcus erythropolis.................... 34-35
1.8 Rationale and hypothesis for the study.................................................. 35-36
1.9. Aim and objectives................................................................................. 37
1.9.1 Aim......................................................................................................... 37
1.9.2 Objectives.............................................................................................. 37

CHAPTER TWO
MATERIALS AND METHODS
2.1 Collection of medicinal plants................................................................ 38
2.2 Preparation of crude extracts................................................................. 38-39
2.3 Test organisms...................................................................................... 39
2.4 Screening of plants................................................................................ 39
2.4.1 Phytochemical screening....................................................................... 39
2.4.2 Antioxidant assay................................................................................... 40
2.4.3 Minimal inhibitory concentration............................................................. 40
2.4.4 Bio-autographic assays.......................................................................... 40-41
2.5 Isolation of bioactive compounds........................................................... 41
2.5.1 Preparation of crude extracts................................................................. 41
2.5.2 Column chromatography........................................................................ 41
2.5.3 Analysis and bioassays of fractions....................................................... 42
2.5.4 Purification of pooled and un-separated compounds............................. 42-43
2.5.4.1 Preparative TLC..................................................................................... 43
2.5.5. Characterization of pure compounds by nuclear magnetic resonance.. 43

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CHAPTER THREE
RESULTS
3.1 Mass of plants extracts.......................................................................... 44
3.2 Phytochemical analysis of plant extracts………………………………… 45
3.2 Qualitative (DPPH) assay on TLC (Antioxidant activity)…………...…… 50
3.4 Minimum inhibitory concentrations (MIC) and total activities of the
extracts.................................................................................................. 55
3.5 Qualitative antibacterial activity assay by bioautography……………..... 58
3.6 Isolation of bioactive compounds from acetone fraction of A. dimidiata 68-69
3.7 Nuclear magnetic resonance of purified compounds…………………… 80

CHAPTER FOUR

DISCUSSION
Discussion................................................................................................................. 93-96
Conclusion and future work....................................................................................... 96- 97

CHAPTER FIVE
REFERENCES
References................................................................................................................ 98-112

CHAPTER SIX

APPENDICES
6.1 Appendix A: Solutions…………………………………………………… 113
6.1.1 Preparation of vanillin sulphuric acid……………………………………... 113
6.1.2 Preparation of DPPH solution……………………………………………... 113
6.1.3 Preparation of p-iodonitrotetrazolium chloride (INT) solution………..... 113-114
6.2 Appendix B: Media preparation………………………………………… 114
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6.2.1 Preparation of 1000 ml Middlebrook7H9 broth...................................... 114
6.2.2 Preparation of 1000 ml Luria Bertani (LB) broth……………………….... 114-115
6.1 Appendix C: Mobile phases in 100 ml……………………….………… 115
6.1.1 EMW ………………………………………………………………………… 115
6.1.2 BEA……………………………………………………….………………….. 115
6.1.3 CEF………………………………………………………………………....... 115
6.2 Appendix D: TLC plates and other equipment…………………….. 115-116

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LIST OF ABBREVIATIONS
ATCC American Type Culture Collection
BEA Butanol, ethanol and ammonia
13C Carbon-13
CEF Chloroform, ethyl-acetate and formic acid
DCM Dichloromethane
DPPH 2,2-diphenyl-1-picrylhydrazyl
EMW Ethyl acetate, methanol and water
ETAC Ethyl acetate
1H Hydrogen-1
HEX Hexane
INT ρ-Iodonitro-tetrazolium violet salt
NMR Nuclear magnetic resonance
MEOH Methanol
MIC Minimum inhibitory concentration
MCC Microbial culture collection
TB Tuberculosis
TLC Thin layer chromatography

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