2.8. Cytotoxicity Assay in Caco-2 Cells

The cells were maintained as monolayers in 175 cm² culture flasks containing an RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, at 37 °C in humidified air with 5% CO₂. Caco-2 cells were seeded at a density of 2 × 10⁴ cells/well in transparent 96-well plates and allowed to grow as a confluent and non-differentiated monolayer that can mimic the human intestinal epithelium. This cell model shares some characteristics with crypt enterocytes, and thus it has been considered to be an accepted intestinal model widely implemented to assess the effect of chemical, food compounds, and nano/microparticles on the intestinal function [17]. The medium was changed every 2 days. On the day of the experiment, cells were washed twice with pre-warmed PBS at ~37 °C. Water and ethanol extracts of peony were solubilized in H₂O and EtOH, respectively. All extracts were prepared with a final concentration of 16.7 mg/mL. Cell-based assays were performed using a maximum concentration of solvent—50% and 5% for H₂O and ethanol, respectively.

Cytotoxicity was assessed by the MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide) method [17]. Prepared cells monolayers were incubated with various concentrations of peony extracts (100 µL) for 4, 24, and 48 h and washed with PBS. Their viability was determined by adding a 100 µL 10-fold diluted MTS reagent (according to the manufacturer’s guidelines), incubating for 2.5 h at 37 °C 5% CO₂, measuring the absorbance at 490 nm in an Epoch Microplate Spectrophotometer (Bio-Tek, Instruments, Winooski, VT, USA). Data were expressed in a cellular viability percentage relative to control (%). The experiments were performed in triplicate.