4.5. Cell Proliferation Inhibition by SSE and HPLC-Purified Fractions

To evaluate the potential effect on the inhibition of cell proliferation, SSE and HPLC-purified fractions were tested at different concentrations (2 µg/mL, 4 µg/mL, 8 µg/mL, 16 µg/mL, 32 µg/mL, 64 µg/mL and 128 µg/mL) by triplicate in two independent experiments in normal human dermal fibroblasts (HDFa, ATCC^® PCS-201-012), human epithelial cells from colorectal adenocarcinoma (CaCo2, ATCC^® HTB-37) and human epithelial cells from breast adenocarcinoma (MCF7, ATCC^® HTM-22). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 5% fetal calf serum in flat-bottomed 96-well microtiter trays at 5 × 10⁴ cells per well at 37 °C in a humidified incubator with 5% CO₂. After 24 h incubation, culture media was removed and fresh media containing SSE or HPLC-purified fractions was added to each well and incubated for 48 h at 37 °C in a humidified incubator with 5% CO₂. To estimate proliferation of viable cells, 20 µL of phenazine methosulfate 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide mix-based (MTS) CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) were added to each well and after 45 min incubation at 37 °C, absorbance at 490 nm was recorded using a 96-well plate reader. Percentage of viable treated cells was calculated in relation to untreated controls (viability percentage = treated cells Optical Density/untreated cells Optical Density × 100%) [46].