4.7. Biological Properties

According to the MTT cytotoxicity test method of ISO 10993-5, the number of L929 cells per well was adjusted to 1 × 10⁴, and 100 μL was dispensed onto the wells and cultured for 24 h. After incubation, 100 μL of the natural extract diluted to various concentrations was applied to the cells for 24 h. As a control, RPMI 1640 without the natural extract was used. After application, the extract was discarded and washed with 100 μL of DPBS (Gibco BRL, Life Technologies, NY, USA). Subsequently, DPBS was removed and 50 µL was added per well and cultured with 1 mg/mL of MTT (Sigma, UK) for 2 h. In order to dissolve the as-formed MTT (3-(4–dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) formazan, 100 mL of isopropanol (Sigma, UK) was added to 100 µL/well and reacted for 20 min. Thereafter, the absorbance was measured at 570 nm on a spectrophotometer and analyzed. The result was normalized to 100% of the MTT reduction rate of the control group and expressed as a percentage. For the microscopic observation, the images of the L929 cells exposed to sample extracts were observed using an EVOS FL microscope (Advanced Microscopy Group USA Ltd, Mill Creek, WA, USA) at 20× magnification.