Isolation and characterization of bioactive compound from Combretum erythrophyllum leaf extracts
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Date
2012-08
Authors
Ledwaba, I.P
Journal Title
Journal ISSN
Volume Title
Publisher
Vaal University of Technology
Abstract
Combretum erythrophyllum has been used for medicinal purposes and several
studies have been carried out to investigate the bioactive compounds present in the
leaves of this plant. The World Health Organization reported that 80% of the people
living in the developing countries almost exclusively use traditional medicine.
Combretum species are used in many human medicines for the treatment of
microbial infections and several anti-inflammatory conditions. Members of the
Combretaceae family are widely traded in the traditional medicine market in
Southern Africa. The family is also used for medicinal purposes in the rest of Africa
and Asia for close to 90 medicinal indications. Many of these indications are related
to infective agents. Traditional healers have long used plants to prevent or cure
infectious conditions.
Plants are rich in a wide variety of secondary metabolites, such as tannins,
terpenoids, alkaloids and flavonoids, which have been found in vitro to have
antimicrobial properties. This review highlights the current status of traditional
medicines, its contribution to modern medicine, recent trends in the evaluation of
anti-microbial activity with a special emphasis upon some tribal medicine, in vitro and
in vivo experimental design for screening and therapeutic efficacy in safety and
human clinical trials for commercial outlet. Many of these commercially available
compounds are crude preparations administered without performing human clinical
trials.
The leaves of Combretum erythrophyllum were extracted with acetone to obtain the
crude extract. Liquid-liquid fractionation was performed on the crude using different
solvents of different polarity. The crude and obtained fractions were investigated for
antimicrobial activity. The crude and fractions were tested against certain bacterial
and fungal microorganisms. The assay methods used included the microtitre dilution
method for determining the minimum inhibitory concentration and bioautographic
methods used to detect the inhibition of bacterial and fungal growth by active
compounds separated from the crude and fractions. The antioxidant activity was
performed using TLC-DPPH, DPPH, ABTS and hydroxyl radical scavenging.
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The toxicity of crude extract and fractions was determined using MTT assay.
Isolation of compounds was performed using column chromatography. Structural
elucidation was done using NMR and MS spectrometry.
In microtitre dilution assay acetone fraction inhibited the growth of S. aureus, E.
faecalis, P. aeruginosa with the lowest MIC value of 0.02, 0.32, 0.16 μg/ml and ethyl
acetate fraction inhibited the growth of E. coli with the lowest MIC value of 0.16
μg/ml. All fractions were active against C. neoformans with the MIC value of 0.02
μg/ml. Dichloromethane was the least active against C. albicans with the MIC value
of 0.16 μg/ml while the rest had the MIC value of 0.08 μg/ml. Dichloromethane was
found to be active against A. fumigatus with the lowest MIC value of 0.16 μg/ml.
Bioautography showed the presence of various inhibitory chemical compounds. Ethyl
acetate and hexane fraction had a very good separation and showed various zones
of inhibition on exposure to E. faecalis, E. coli, S. aureus and P. aeruginosa with the
Rf values ranging from 0-0.98. Crude and fractions showed slight zones of inhibition
against C. neoformans, C. albicans and A. fumigatus with the Rf values ranging from
0-0.76. TLC-DPPH assay displayed that ethyl acetate and water fraction had the
highest antioxidant activity in CEF. Ethyl acetate fraction had a strong antioxidant
activity in DPPH assay with the EC50 of 0.04272 μg/ml, water fraction showed a good
antioxidant activity with the EC50 of 0.01825 μg/ml in ABTS assay and in the hydroxyl
radical scavenging the crude extract scavenged 77.62 ± 1.41% at the highest
concentration of 0.250 mg/ml and 47.21 ± 3.20% at the lowest concentration of
0.003 mg/ml. The toxicity level of the crude extract and fractions were found to be
between 34 and 223 mg/ml which were all below doxorubicin (LC50 = 7.1855 μg/ml)
which was used as the positive control.
Column chromatography was used in a bio-guided assay fractionation and led to
isolation of one compound. The antimicrobial activity was determined against
pathogenic bacteria. The isolated compound had a good activity against Pa and Sa
with the lowest MIC value of 0.32 μg/ml. Nuclear magnetic resonance spectroscopy
(NMR) and mass spectroscopy were used for the identification of isolated
compounds. One compound was isolated and identified as Friedelin. The results
obtained confirm that the leaves of Combretum erythrophyllum have a good
antimicrobial activity and strong antioxidant activity.