Browsing by Author "Kahaliw, Wubayehu"

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    Activity Testing, Toxicity Assay and Characterization of Chemical Constituents of Medicinal Plants Used to Treat Tuberculosis in Ethiopian Traditional Medicine
    (Addis Ababa University, 2016-04) Kahaliw, Wubayehu; Engidawork, Ephrem (PhD); Abebe, Markos (PhD)
    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 ii 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.