The cytotoxic activity of extracts was evaluated in 96-well flat-bottomed microtiter plates (Corning) by using the standard MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) colorimetric assay with a slight modification. For this purpose, HeLa human cervical carcinoma cells and U251 human glioblastoma cells were cultured in T25 flasks in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 1% of penicillin/streptomycin, and 1% L-glutamine and kept at 37°C in a 5% CO2 incubator [39]. The cells were seeded in 96-well plates (1 × 104 U251 cells/well and 2 × 104 HeLa cells/well) in 100 μL of the medium and incubated in a 5% CO2 incubator at 37°C for 24 h. Following attachment and cell confluence, the cells were treated with different concentrations of the plant extracts (50, 100, 200, and 400 μg/mL) for 48 h. Crude extracts were diluted by dissolving them in 0.1% DMSO (dimethyl sulfoxide, Merck, ≥99.9%) and the EMEM culture medium. The assay was performed in triplicate. Following 48 h of incubation, the supernatant was removed and 150 μL of the medium was combined with 50 μL of MTT for each well. Following 4 h of incubation, a purple formazan crystal was produced. Then, 100 μL of 0.1% DMSO was added and the plates were incubated for a further 15 min at room temperature to dissolve the formazan crystals. The absorbance of samples was measured with a microplate reader (iMarkTM, Bio-Rad) at a wavelength of 595 nm. A commercially available cis-platin drug was used as positive control. The percentage of cytotoxic activity was calculated by the following formula:

where Ab1 is the absorbance of cells with all components except plant extracts and Ab2 is the absorbance of cells with all components, including plant extracts.

A dose-response curve was plotted for each extract to calculate the 50% inhibition of cell growth (IC50μg/mL of the extract). The cytotoxic capacities of extracts (IC50) were calculated using a regression equation.

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