Evaluation of metabolic activity

After one day of incubation with the extracts, cell metabolic activity was evaluated using resazurin assay (Fig. 1, day 3)¹⁷. Cells were washed by phosphate buffer saline (PBS) and resazurin solution (final concentration 25 µg · ml^-1) in MEM + 10% FBS without phenol red was added. After 1 h of incubation, fluorescence at 560/590 nm (excitation/emission) was measured. Cytotoxicity of the extracts was depicted as a percentage of metabolic activity of the control, i.e., as relative metabolic activity (RMA). First, blank measurement (no cells) was subtracted from all values. The formula used was RMA = (F_sample)/(F_control), where F_sample stands for the average fluorescence of resorufin produced by the affected cells (i.e. cells incubated with extracts) and F_control stands for the average fluorescence of resorufin produced by of the unaffected cells (i.e. control cells). Extracts causing the decrease below 70% of the activity of the control were considered cytotoxic, as described in the standard ISO 10993-5.