3.8. Cytotoxicity Assay

The toxicity of the extracts on cell viability was evaluated on MRC-5 cells, a fibroblast derived from normal lung tissue (ECACC, reference 05090501) using the MTT test, based on the reduction of MTT in formazan crystals by metabolically active cells [41]. MRC-5 cells is a line of normal cells (non-cancerous) which have the capacity to multiply under in vivo culture conditions. This cell line is commonly used as a model for the evaluation of toxicity. Cells were plated at 10000 cells per well in complete medium in 96-well tissue culture plates and grown for 48 hours at 37 °C. Then medium was discarded and replaced by fresh medium containing extracts previously dissolved in dimethyl sulfoxide. Three different controls were added: medium alone, cells in medium and extract in medium. After 24 h incubation, medium was replaced by 100 µl of 0.5 mg MTT solution in medium per well and plates were incubated for 4 hours at 37 °C. Then formazan crystals were dissolved by 100 µl of dimethyl sulfoxide. Absorbance was measured at 540 nm using a 96-well plate reader. Percentages of survival and half maximal inhibitory concentration (IC₅₀) were calculated using MS Office Excel. The water extract was able to convert MTT in formazan. That is why a confirmation of its toxicity was carried out by microscopic observation.