3.2.2. Cytotoxicity Measurement

The anticancer activity against HCT-116, HepG2, A549, and MCF-7 human cancer cell lines was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in viable cells [49,50,51,52]. Cells were dispensed in a 96-well sterile microplate (5 × 10⁴ cells/well) and incubated at 37 °C with a series of different concentrations, in DMSO, of each tested compound or doxorubicin (positive control) for 48 h in a serum-free medium prior to the MTT assay. After incubation, media were carefully removed, and 40 µL of MTT (2.5 mg/mL) were added to each well and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 570 nm using a Spectra Max Paradigm Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. All experiments were conducted in triplicate and repeated on three different days. All the values were represented as mean ± SD. IC₅₀s were determined by probit analysis by SPSS Incprobit analysis (IBM Corp., Armonk, NY, USA).