Activity Testing, Toxicity Assay and Characterization of Chemical Constituents of Medicinal Plants Used to Treat Tuberculosis in Ethiopian Traditional Medicine
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Date
2016-04
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Addis Ababa University
Abstract
Background: Tuberculosis (TB) is the leading killer disease worldwide. In 1993, WHO
declared TB as a ‘global emergency,’ which requires emergency action and launched
several programs to curb the disease, including the search for newer remedies and/or anti-
TB agents to complement currently used agents. Hence, herbal remedies have become the
readily available alternatives in the search for new antimycobacterial compounds.
Objective: To investigate antimycobacterial activity and toxicity of selected Ethiopian
medicinal plants (Otostegia integrifolia, Vernonia amygdalina, Persea americana,
Pterolobium stellatum and Carissa edulis) as well as to isolate the main active principles
through a bioassay guided process.
Methods: Antimycobacterial activity test was conducted using the broth microdilution
and microtitre resazurin assay methods in 96 well microtitre plates and MIC was
determined by colony counting and resazurin color change observation for all test
materials. Cytotoxicity test was performed based on the CellTiter 96® AQueous One
Solution Cell Proliferation Assay on HepG2 cells. Genotoxic effects of extracts were
evaluated using SCGE method on HepG2 cells.
Results: Chloroform and 80% methanol extracts of P. stellatum and O. integrifolia as
well as 80% methanol and acetone extracts of P. americana had significant
antimycobacterial activity (p < 0.001) against M. tuberculosis H37Rv, while chloroform
extract of V. amygdalina and C. edulis didn’t show significant activity compared to
negative controls. The MIC of positive control was 0.125 μg/ml against the standard
strain. However, MDR-TB clinical isolates were isoniazid resistant. Fractionation and
activity testing of the chloroform extract of P. stellatum revealed that ethyl acetate
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fraction to be the most active fraction against M. tuberculosis H37Rv with MIC of 0.195
μg/ml. The MICs of compound 1, 2 and 3 were 1.25, 2.5 and 0.625 μg/ml, respectively.
In the cytotoxicity test, V. amygdalina chloroform extract showed the highest IC50 value
(3.202±0.3375), which suggests its safety. O. integrifolia and P. stellatum chloroform
extracts were the most toxic in dose dependent manner as one can see the steepness of the
dose-response curve. DNA damage in the form of comet tail has been observed for 1 and
0.5 mg/ml P. stellatum chloroform and 80% methanol extracts on HepG2 cells,
respectively. The rest of test extracts seemed to be without genotoxic effect up to a
concentration of 0.5 mg/ml. Cytotoxicity test was not in the objectives of my study.
Conclusion: P. stellatum, O. integrifolia and P. americana have potential to be
developed into new anti-TB drugs or standardized herbal medicines. P. stellatum
chloroform extract was the most active extract and hence, three compounds were isolated
from ethyl acetate fraction and they were active against M. tuberculosis H37Rv. The
results have also validated indigenous medical knowledge from the local people
regarding the use of these species to treat TB. The IC50 value of P. stellatum chloroform
extract was relatively higher compared to other extracts, suggesting its safety. In addition,
its selectivity index was 13.5, which demonstrated > 10 selectivity index, considered as
being of interest to the pharmaceutical companies. The genotoxicity assay findings
revealed that the chloroform and 80% methanol extracts of P. stellatum caused DNA
damage at 1 mg/ml and 0.5 mg/ml concentrations. Thus, necessary precautions should be
taken during utilization of this plant.
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Keywords
Otostegia integrifolia, Vernonia amygdalina