Human embryonic kidney (HEK293), breast cancer (MCF-7) and human lung cancer (A549) cells were donated by the Department of Biotechnology, Durban University of Technology. Cells were grown at 37 °C in a humidified incubator under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Foetal Bovine Serum (FBS) and antibiotics (Penicillin; 10,000 U mL−1 and Streptomycin sulphate; 10,000 U mL−1 [Penicillin/Streptomycin]). Antibiotics change the phenotype and morphology of cells; therefore, the use of the antibiotics should be in very low concentrations, thus for this study, 1% Penicillin/Streptomycin was used. The cells were grown until 80% confluence was reached with media replaced as necessary. After confluence was reached, cells were trypsinized and sub-cultured. The 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the extracts. The MTT assay was conducted according to Dwarka et al. [40 (link)] with minor modifications. Briefly, cells (50 μL of 1 × 102 cells mL−1), as well as 50 μL of DMEM, were seeded into a 96-well flat bottom plate and incubated (37 °C for 24 h) in a humidified incubator under 5% CO2. Cells were then treated with 50 μL of extracts at varying concentrations (7.8–1000 μg mL−1) prepared in 5% DMSO and incubated for 24 h. Camptothecin was used as a positive control. MTT reagent (20 μL, 5 mg mL−1) was added to the cells and incubated at 37 °C for 4 h. One hundred microliters of DMSO was then added to each well to solubilise the formazan salt formed, and absorbance was read at 570 nm on a microplate spectrophotometer (Multiscan Go, Thermo Scientific, Waltham, MA, USA) for both treated and untreated cells. The percentage viability was calculated using the following formula:
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