Evaluation of cytotoxic activity

MTT assay [16] was performed to evaluate cytotoxicity of aqueous, methanol, and acetone extracts at concentration ranging from 12.5 μg/ml - 200 μg/ml at 24, 48 and 72 h against HCT 116 cells as well as acetone extract at 24, 48 and 72 h against CCD-18co. The cells were counted to achieve a concentration of 5.0 x 10⁴ cells/ml. A total volume of 200 μl of cell suspension was seeded in each well of the 96-microtiter plate. The seeded cells were incubated for 24 h before they were treated with each of the extract. Menadione was used as a positive control whereas the untreated cells comprised the HCT 116 cells in the media was used as negative control. A total volume of 200 μl of sample treatment was added in each well and incubated for 24, 48 and 72 h prior to addition of 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at 5 mg/ml to each of the well and left for 4 h incubation. About 200 μl of DMSO solution was added to each well to dissolve the formazan crystals at room temperature and was then incubated for 15 min. The plate was shaken on an automatic mixer for 5 min and the absorbance was read by using an ELISA plate reader at a wavelength of 570 nm. The percentage of cell viability was calculated using the formula given below:

The percentage of cell viability against the concentration of test compounds was plotted. The half maximal inhibition concentration (IC₅₀) which is the concentration of sample that inhibits cell growth was obtained from the plotted graph.