Activity Testing Toxicity Assay And Characterization Of Chemical Constituents Of Medicinal Plants Used To Treat Tuberculosis In Ethiopian Traditional Medicine

Background: Tuberculosis (TB) is the leading killer disease worldwide. In 1993, WHOrndeclared TB as a ‘global emergency,’ which requires emergency action and launchedrnseveral programs to curb the disease, including the search for newer remedies and/or anti-rnTB agents to complement currently used agents. Hence, herbal remedies have become thernreadily available alternatives in the search for new antimycobacterial compounds.rnObjective: To investigate antimycobacterial activity and toxicity of selected Ethiopianrnmedicinal plants (Otostegia integrifolia, Vernonia amygdalina, Persea americana,rnPterolobium stellatum and Carissa edulis) as well as to isolate the main active principlesrnthrough a bioassay guided process.rnMethods: Antimycobacterial activity test was conducted using the broth microdilutionrnand microtitre resazurin assay methods in 96 well microtitre plates and MIC wasrndetermined by colony counting and resazurin color change observation for all testrnmaterials. Cytotoxicity test was performed based on the CellTiter 96® AQueous OnernSolution Cell Proliferation Assay on HepG2 cells. Genotoxic effects of extracts werernevaluated using SCGE method on HepG2 cells.rnResults: Chloroform and 80% methanol extracts of P. stellatum and O. integrifolia asrnwell as 80% methanol and acetone extracts of P. americana had significantrnantimycobacterial activity (p < 0.001) against M. tuberculosis H37Rv, while chloroformrnextract of V. amygdalina and C. edulis didn’t show significant activity compared tornnegative controls. The MIC of positive control was 0.125 μg/ml against the standardrnstrain. However, MDR-TB clinical isolates were isoniazid resistant. Fractionation andrnactivity testing of the chloroform extract of P. stellatum revealed that ethyl acetaterniirnfraction to be the most active fraction against M. tuberculosis H37Rv with MIC of 0.195rnμg/ml. The MICs of compound 1, 2 and 3 were 1.25, 2.5 and 0.625 μg/ml, respectively.rnIn the cytotoxicity test, V. amygdalina chloroform extract showed the highest IC50 valuern(3.202±0.3375), which suggests its safety. O. integrifolia and P. stellatum chloroformrnextracts were the most toxic in dose dependent manner as one can see the steepness of therndose-response curve. DNA damage in the form of comet tail has been observed for 1 andrn0.5 mg/ml P. stellatum chloroform and 80% methanol extracts on HepG2 cells,rnrespectively. The rest of test extracts seemed to be without genotoxic effect up to arnconcentration of 0.5 mg/ml. Cytotoxicity test was not in the objectives of my study.rnConclusion: P. stellatum, O. integrifolia and P. americana have potential to berndeveloped into new anti-TB drugs or standardized herbal medicines. P. stellatumrnchloroform extract was the most active extract and hence, three compounds were isolatedrnfrom ethyl acetate fraction and they were active against M. tuberculosis H37Rv. Thernresults have also validated indigenous medical knowledge from the local peoplernregarding the use of these species to treat TB. The IC50 value of P. stellatum chloroformrnextract was relatively higher compared to other extracts, suggesting its safety. In addition,rnits selectivity index was 13.5, which demonstrated > 10 selectivity index, considered asrnbeing of interest to the pharmaceutical companies. The genotoxicity assay findingsrnrevealed that the chloroform and 80% methanol extracts of P. stellatum caused DNArndamage at 1 mg/ml and 0.5 mg/ml concentrations. Thus, necessary precautions should berntaken during utilization of this plant.

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