antibacterial, antioxidant and phytochemical investigation of albizia anthelmintica leaves, roots and

72 Pages·2017·2.45 MB·English

ANTIBACTERIAL, ANTIOXIDANT AND PHYTOCHEMICAL INVESTIGATION
OF ALBIZIA ANTHELMINTICA LEAVES, ROOTS AND STEM BARK

A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILMENT

OF THE REQUIREMENT FOR THE DEGREE OF

MASTER OF SCIENCE IN INDUSTRIAL BIOCHEMISTRY

OF
THE UNIVERSITY OF NAMIBIA

BY
Teopolina N NAWINDA
201148757
October 2016

Supervisor: Dr P. Kapewangolo
DECLARATIONS
I, NAWINDA TEOPOLINA N, declare hereby that this study is a true reflection of my own
research, and that this work, or part thereof has not been submitted for a degree in any other
institution of higher education.
No part of this thesis/dissertation may be reproduced, stored in any retrieval system, or
transmitted in any form, or by means (e.g. electronic, mechanical, photocopying, recording or
otherwise) without the prior permission of the author, or The University of Namibia in that
behalf.
I, NAWINDA TEOPOLINA N, grant The University of Namibia the right to reproduce this
thesis in whole or in part, in any manner or format, which The University of Namibia may deem
fit, for any person or institution requiring it for study and research; providing that The University
of Namibia shall waive this right if the whole thesis has been or is being published in a manner
satisfactory to the University.

………………………………. Date……………………………..

ii
Summary
Albizia anthelmintica belongs to the family Fabaceae. The plant is traditionally used to treat
symptoms of microbial infections in both humans and animals. The present study was designed
to evaluate the phytochemical content, antibacterial and antioxidant activities of A. anthelmintica
leaves, roots and stem bark ethanol extracts. Antibacterial activity was carried out by means of
the agar disc diffusion method. The anti-oxidative activities of the extracts were determined by
means of ferric reducing antioxidant power (Fe 3+- Fe 2+), phosphomolybdenum reduction and
2,2-diphenyl-1-picrylhydrazyl (DPPH) assays and phytochemical screening was used to
determine the major classes of compounds present in the specific plant parts. Phytochemical
screening revealed the presence of alkaloids, saponins and diterpenes in roots and bark extracts,
flavonoids in leaves extract, tannins in roots extracts, and phenolic compounds were detected in
all three extracts. The root extract exhibited the highest total phenolic and flavonoid content of
1741.08 ± 0.05 mg GAE/100 g and 366.80 0.02 ± mg QEE/100 g, respectively. The extracts
demonstrated dose dependent varying degrees of anti-oxidative efficacy in the
phosphomolybdenum reduction, iron (III) reduction and 2,2-diphenyl-1-picrylhydrazyl radical
scavenging assays. For the DPPH assay, IC values of 0.018 ± 0.01, 0.019 ± 0.01 and 0.024 ±
50
0.01 mg/mL were obtained for the root, leaf and bark extracts, respectively. All extracts were
active against Staphylococcus aureus, Shigella sonnei and Serratia marcescens but did not show
activity against Enterococcus faecalis and alcaligenes faecalis. The results from the study show
that Albizia anthelmintica extracts possess compounds with antibacterial as well as anti-oxidative
properties, making this plant species a good source for potent antibacterial and anti-oxidative
agents. The findings of this study will add value to the traditional uses of A. anthelmintica, and
conservation is necessary due to the significant medicinal properties demonstrated by the plant.
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Table of Contents
List of Tables .................................................................................................................. viii
List of Figures ................................................................................................................... ix
List of Abbreviations ......................................................................................................... x
ACKNOWLEDGEMENT ................................................................................................ xi
DEDICATION ................................................................................................................. xii
CHAPTER 1 ...................................................................................................................... 1
INTRODUCTION ............................................................................................................. 1
1.1 Background .................................................................................................................. 1
1.2 Statement of the problem ............................................................................................. 3
1.3 Objectives of the study................................................................................................. 3
1.4 Significance of the study .............................................................................................. 4
CHAPTER 2 ...................................................................................................................... 5
LITERATURE REVIEW .................................................................................................. 5
2.1. A review of Albizia species....................................................................................... 5
2.1.1 Albizia procera.......................................................................................................... 5
2.1.2 Albizia antunesiana ................................................................................................... 6
2.1.3 Albizia julibrissin ...................................................................................................... 6
2.1.4 Albizia lebbeck .......................................................................................................... 6
2.1.5 Albizia amara ............................................................................................................ 7
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2.2 Description and geographical locations of Albizia anthelmintica ............................... 9
2.3 Traditional uses and studies done on Albizia anthelmintica ...................................... 10
2.4 Natural phytochemicals as a source of medicine ....................................................... 11
2.5 Natural Products as antioxidants ................................................................................ 11
2.6 Natural Products as antibacterial agents .................................................................... 13
2.7 Test bacteria ............................................................................................................... 14
2.7.1 Staphylococcus aureus ............................................................................................ 14
2.7.2 Serratia marcescens ................................................................................................ 15
2.7.3 Shigella sonnei ........................................................................................................ 15
2.7.4 Alcaligenes faecalis ................................................................................................ 16
2.7.5 Enterococcus faecalis ............................................................................................. 16
2.8 A review of the methods employed ........................................................................... 16
2.8.1 Antioxidant assays .................................................................................................. 17
2.8.2 Antibacterial activity screening assays ................................................................... 18
CHAPTER 3 ................................................................................................................... 20
METHODS ..................................................................................................................... 20
3.2 Research design ......................................................................................................... 20
3.3 Plant material ............................................................................................................. 21
3.4 Preparation of crude extracts ..................................................................................... 21
3.5 Phytochemical screening ........................................................................................... 21
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3.5.1 Qualitative Phytochemical screening ...................................................................... 21
3.5.2 Quantitative phytochemical screening .................................................................... 23
3.6 Determination of antioxidant activity ........................................................................ 24
3.6.1 DPPH free radical scavenging assay....................................................................... 24
3.6.2 Ferric reducing antioxidant power assay ................................................................ 25
3.6.3 Determination of total antioxidant capacity ............................................................ 25
3.7 Antibacterial activity assay ........................................................................................ 26
3.7.1 Antibacterial activity screen test ............................................................................. 26
3.7.2 Test microorganisms ............................................................................................... 26
3.7.3 Disc agar diffusion method ..................................................................................... 27
3.7.4 Minimum inhibitory concentration determination .................................................. 27
3.7.5 Statistical analysis ................................................................................................... 28
CHAPTER 4 .................................................................................................................... 29
RESULTS ........................................................................................................................ 29
4.2 Plant extraction yield ................................................................................................. 29
4.3 Qualitative phytochemical data.................................................................................. 29
4.3 Quantitative phytochemical data................................................................................ 30
4.3.1 Total Phenolic Content ........................................................................................... 30
4.3.2 Total Flavonoid Content ......................................................................................... 31
4.4 Antioxidant activity ................................................................................................... 31
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4.4.1 DPPH radical scavenging activity .......................................................................... 31
4.4.2 Ferric reducing antioxidant power .......................................................................... 33
4.4.3 Total Antioxidant Capacity ..................................................................................... 34
4.5 ANTIBACTERIAL ACTIVITY ................................................................................ 35
4.5.1 Antibacterial preliminary screening ........................................................................ 35
4.5.2 Antibacterial susceptibility data .............................................................................. 35
4.5.3 Minimum inhibitory concentration ......................................................................... 38
CHAPTER 5 .................................................................................................................... 39
DISCUSSION .................................................................................................................. 39
CHAPTER 6 .................................................................................................................... 42
CONCLUSION ................................................................................................................ 42
CHAPTER 7 .................................................................................................................... 43
RECOMMENDATIONS ................................................................................................. 43
REFERENCES ................................................................................................................ 44
APPENDIX 1 ................................................................................................................... 51
APPENDIX 2 ................................................................................................................... 55
APPENDIX 3 ................................................................................................................... 57

vii
List of Tables
Table 1: Various compounds isolated from some Albizia species .................................... 8
Table 2: Crude extracts yielded……………………………………………………...….29
Table 3: Phytochemical constituents of A. anthelmintica roots, stem bark and leaf
ethanolic extracts. ............................................................................................................. 30
Table 4: Total phenolic and flavonoid contents in leaves, roots and stem bark ethanol
extracts of Albizia anthelmintica. ..................................................................................... 31
Table 5: IC values of leaf, root and stem bark ethanolic extract of Albizia anthelmintica
50
.......................................................................................................................................... 32
Table 6: Total antioxidant capacity of leaves, roots and stem bark ethanol extracts of A.
anthelmintica. ................................................................................................................... 34
Table 7: Antibacterial activity screen test results of leaf, root and stem bark ethanolic
extracts of A. anthelmintica.............................................................................................. 35
Table 8: Albizia anthelmintica ethanolic leaf extract bacterial growth inhibition zones at
varying extract concentrations ......................................................................................... 36
Table 9: Root extract bacterial growth inhibition zones at varying extract concentrations
.......................................................................................................................................... 37
Table 10: Bark extract bacterial growth inhibition zones at varying extract
concentrations .................................................................................................................. 37
Table 11: MIC values for leaf, root and stem bark ethanolic extracts of Albizia
anthelmintica. ................................................................................................................... 38
viii
List of Figures
Figure 1: Albizia anthelmintica (local flora). Picture was taken during sampling. .......... 10
Figure 2: A general reaction mechanism of DPPH with an antioxidant. ......................... 17
Figure 3: A summary of the study methods. .................................................................... 20
Figure 4: DPPH free radical scavenging activity of leaves, roots and stem bark ethanolic
extracts of A. anthelmintica.............................................................................................. 32
Figure 5: Reducing power of ethanolic extracts of A. anthelmintica leaves, roots and
stem bark. ......................................................................................................................... 33
Figure 6: Bacterial growth inhibition by A. anthelmintica leaf ethanolic extract at
varying concentrations against S. marcescens, S. sonnei and S. aureus. ......................... 55
Figure 7: Bacterial growth inhibition by A. anthelmintica root ethanolic extract at
varying concentrations against S. marcescens, S. sonnei and S. aureus. ......................... 55
Figure 8: Bacterial growth inhibition by A. anthelmintica stem bark ethanolic extract at
varying concentrations against S.marcescens, S. sonnei, and S. aureus. ......................... 56
Figure 9: Albizia anthelmintica stem ............................................................................... 57
Figure 10: Albizia anthelmintica fresh leaves .................................................................. 57
Figure 11: Albizia anthelmintica dry stem bark ............................................................... 58
Figure 12: Albizia anthelmintica crushed stem bark ........................................................ 58
Figure 13: Albizia anthelmintica dry roots....................................................................... 59
Figure 14: Albizia anthelmintica crushed roots ............................................................... 59
Figure 15: Albizia anthelmintica crushed leaves ............................................................. 60
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List of Abbreviations
AAE Ascorbic Acid Equivalents
DPPH 2,2 – Diphenyl – 1 – Picrylhydrazyl
ET Electron Transfer
FRAP Ferric Reducing Antioxidant Power
GAE Gallic Acid Equivalents
HAT Hydrogen Atom Transfer
MIC Minimum Inhibitory Concentration
MRSA Methicillin Resistant Staphylococcus aureus
PBP2 Penicillin Binding Protein 2
QEE Quercetin Equivalents
SPA Synthetic Phenolic Antioxidant
TAC Total Antioxidant Capacity
TFC Total Flavonoid Content
TPC Total Phenolic Content
UTI Urinary Tract Infection
WIND National Herbarium of Namibia
x