4.6. Anti-Inflammatory Bioassays

Cell culture: RAW264.7 cells were cultured in DMEM complete medium supplemented with 10% neonatal bovine serum. Cells were maintained at 37 °C under a humidified atmosphere of 5% CO₂ in an incubator.

Cell viability assay: Cell viability was evaluated by MTT reduction assay. Briefly, RAW 264.7 cells were seeded on 96-well microtiter plates at 1.0 × 10⁵ cells/well for 24 h and treated with each compound (0, 6.25, 12.5, 25, 50, and 100 µM). After being treated with tested samples for 24 h, the medium was removed and the cells were incubated with MTT (1.0 mg/mL, 10 µL for 3 h at 37 °C. The formazan crystals in the cells were dissolved in DMSO. The levels of MTT formazan were measured as absorbance at 490 nm. The cell survival rate was calculated [20,21,22,23].

Measurement of NO release and NO inhibition rate: Accumulation of nitrite, an indicator of NO synthase activity, in culture medium was measured using the Griess reaction. Cells (2 × 10⁵ cells/well) were cultured on 24-well microtiter plates for 12 h, then treated with each compound (20, 50 µM) for 1 h. LPS (1 µg/mL) was added to the medium and cultured for 24 h. Fifty microliter culture medium supernatants were mixed with 50 µL Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthyl ethylene diamide dihydrochloride and 2% phosphoric acid) at 37 °C. After 10 min, the absorbance was measured at 540 nm. Inhibition rate of NO was calculated [20,21,22,23].

Statistical Analysis: All the results were expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). Dunnett’s multiple comparison test was employed to perform the one-way analysis of variance to identify the significance of differences among groups. p < 0.05 was considered to be statistically significant.