The cytotoxicity of the plant sample extracts was evaluated in cell lines using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay35. HepG2 (human hepatoma), MCF-7 (human breast adenocarcinoma), and CACO2 (human colon adenocarcinoma) cells were maintained in RPMI medium (Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum. MCF-7 cells were cultured at 37 °C and 5% (v/v) CO2 in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum, 1% (w/v) l-glutamine, 1% sodium pyruvate, and 0.4% (w/v) antibiotics (50 U/mL penicillin, 50 mg/mL streptomycin). Cells were obtained from the American Type Culture Collection (Rockville, MD, USA; HPACC, Salisbury, UK) and sub-cultured twice per week. All chemicals and reagents were purchased from Sigma Aldrich (Darmstadt, Germany). To normalize cell viability values, each plate included a triplicate of cells treated with the compound carrier dimethyl sulfoxide to define 100% viability as well as a triplicate of cells incubated with a cytotoxic mixture [200 ng/mL tumor necrosis factor, 200 ng/mL CD95L (Fas ligand), 200 ng/mL tumor necrosis factor–related apoptosis-inducing ligand, 25 g/mL cycloheximide, 1% (w/v) sodium azide] to define maximal cell death and thus 0% viability. All other viability values were normalized according to the averages of these triplicates and analyzed using Graph Pad Prism 5 software (La Jolla, CA, USA). 5-Flurouracil was used as a positive control.

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