Title page for ETD etd-02242010-193059

Document Type Master's Dissertation
Author Kaikabo, Adamu Ahmad
Email kavkafa@yahoo.co.uk
URN etd-02242010-193059
Document Title Isolation and characterization of antibacterial compounds from a Garcinia livingstonei (Clusiaceae) leaf extract
Degree MSc (Veterinary Science)
Department Paraclinical Sciences
Advisor Name Title
Dr B B Samuel Co-Supervisor
Prof J N Eloff Supervisor
  • South Africa
  • infectious microorganisms
  • antibiotics
  • plant species
Date 2009-11-27
Availability unrestricted

Although pharmaceutical industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by infectious microorganisms has increased. For a long period of time, plants have been a valuable source of natural products for maintaining human and animal health. The use of plant compounds for pharmaceutical purposes has gradually increased worldwide. This is because there are many bioactive constituents in plants which hinder the growth or kill microbes. Plants could be considered a potential gold mine for therapeutic compounds for the development of new drugs.

In this study, sixteen South African plant species were selected based on their antibacterial activity after a wide screening of leaf extracts of tree species undertaken in the Phytomedicine Programme, University of Pretoria. Literature search excluded eleven plants because of the work already performed on their antibacterial activities, while Pavetta schumaniana was found toxic and thus not included in the screening. The remaining four plants namely; Buxis natalensis, Macaranga capensis, Dracaena mannii and Garcinia livingstonei were screened for antibacterial activity by determining the minimum inhibitory concentrations (MIC) against 4 nosocomial bacterial pathogens Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, and also by using bioautography. The extracts of Macaranga capensis, Garcinia livingstonei, Diospyros rotundifolia and Dichrostachys cinerea had good antibacterial activity with MIC values of 0.03, 0.04, 0.06 and 0.08 mg/ml against different pathogens. The average MIC values of the plant extracts against all the tested pathogens ranged from 0.23-1.77 mg/ml. S. aureus was the most susceptible bacterial pathogen with average MIC of 0.36 . The extract of Diospyros rotundifolia was the most active with an average MIC against all the organisms of 0.23 mg/ml. The extracts of Buxus natalensis, Dracaena mannii, and Pittosporum viridiflorum, Acacia sieberiana, Erythrina lattissima, Cassine papillosa and Pavetta schumanniana had lower antibacterial activity. G. livingstonei was selected for further work on the basis of its good activity.

The bulk acetone extract of Garcinia livingstonei (20g) was subjected to solvent-solvent fractionation which yielded seven fractions. Only the chloroform and ethyl acetate fractions showed good bioactivity in the microdilution assay and bioautography. Column chromatography was used to isolate two bioactive biflavonoids from the ethyl acetate fraction. The structures of the two compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy, and were identified as amentoflavone (1) and 4′ monomethoxyamentoflavone (2). These two compounds have been previously isolated from plants that belong to the Clusiaceae. The two compounds were isolated in sufficient quantity with a percentage yield of 0.45% for amentoflavone and 0.55% for 4′ monomethoxyamentoflavone from 20 g crude acetone extract. The antibacterial activity was determined against four nosocomial bacterial pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa). The MIC values ranged from 8-100 μg/ml. Except for Staphylococcus aureus which showed resistance to amentoflavone at >100 μg/ml. All the other tested organisms were sensitive to both compounds.

It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. Based on this, the antioxidant activities of the two isolated compounds were tested using the Trolox assay. The two flavones had good antioxidant activity. Amentoflavone had a Trolox equivalent antioxidant capacity (TEAC) of 0.9. The second compound 4′ monomethoxyamentoflavone had a TEAC value of 2.2 which is more than double the antioxidant activity of Trolox, a vitamin E analogue.

To assess the safety of the two compounds on cell systems, cytotoxicity was determined using a tetrazolium based colorimetric assay (MTT assay) using Vero monkey kidney cells. The compounds indicated little to low toxicity against the cell line with cytotoxic concentration (CC50) of 386 μg/ml and >600 μg/ml for compound 1 and 2 respectively. Berberine (used as the control toxic substance) had a CC50 of 170 μg/ml.

The Ames genotoxicity assay is used to assess the mutagenic potential of drugs, extracts and phytocompounds. The compounds isolated in this study were assayed for genotoxicity using the Salmonella typhimurium TA98 strain. Amentoflavone was genotoxic at the concentration of 100 μg/plate, but 4′ monomethoxyamentoflavone was inactive at the highest concentration of 400 μg/plate tested.

The results of the antibacterial, antioxidant and cytotoxicity testing were encouraging and indicated the potential usefulness of Garcinia livingstonei in traditional medicine and drug discovery. However, the genotoxicity assay revealed potential mutagenic effects of amentoflavone, a compound isolated from the plant. Therefore, it is suggested that application of Garcinia livingstonei extracts in the treatment of human and animal ailments be done with caution to avoid mutagenic effects on the treated subjects.

A relatively small change in the structure of the two compounds by replacing an hydroxyl group with a methoxy group had a major effect in increasing antibacterial and antioxidant activity and in decreasing cellular and genotoxicity. This illustrates the potential value of modifying a molecule before its possible therapeutic use.

Copyright 2009, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Kaikabo, AA 2009, Isolation and characterization of antibacterial compounds from a Garcinia livingstonei (Clusiaceae) leaf extract, MSc (Veterinary Science) dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02242010-193059/ >

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