2.6. Cytotoxic activity assay

In the present study, two cancer cell lines were tested: HCT116 (colorectal carcinoma) and MCF-7 (breast cancer) (American Type Culture Collection, USA). Fetal calf serum (FCS: 10 %) and L-glutamine (2 mM) were used to make complete growth medium to conserve the cell lines (RPMI: Roswell Park Memorial Institute, France). Cell lines were incubated at 37 °C under atmosphere conditions of CO₂ (5%). All tests were performed when the cells reached 80% confluence, and a trypan blue exclusion assay was used to test the viability of the cell lines (Louis and Siegel, 2011).

The cytotoxicity of the isolated compounds against HCT116 (colorectal carcinoma) and MCF-7 (breast cancer) cells was evaluated spectrophotometrically through an MTT assay. The assay was performed in 96-well microtiter plates (Bekir et al., 2013). The concentrations of HCT-116 and MCF-7 cells were fixed at 10 × 10³ and 12 × 10³ cells/well, respectively. The adherent cells were incubated overnight (37 °C) in a 5% enriched CO₂ atmosphere. For the cells in the exponential growth phase, further incubation (37 °C) was maintained for 72 h, and the concentration of the tested compounds was 100 μM (DMSO does not exceed 1%). After removing the medium, incubated cells (37 °C) were mixed with 50 μL of MTT solution (3 mg/mL in phosphate-buffered saline, PBS) for 20–40 min. The dissolution of the cell mitochondria and the precipitation of violet formazan crystals proceeded by adding 80 μL of 100% DMSO. The measurement of the obtained reaction was taken spectrophotometrically at 540 nm. As a positive control, Doxorubicin, a conventional anticancer drug, was used. Cell viability was measured using formula II:

where Abs _control is the absorbance measured of the total cell activity as described without any inhibition; Abs _{blank control} is the absorbance of MTT substrate; and Abs _sample is the absorbance of each inhibitor compound sample.