3.5. Measurement of LPS-Induced Nitric Oxide (NO) Production and Cell Viability

The nitric oxide (NO) concentration in the medium was measured as an indicator of NO production according to the Griess reaction [26]. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at 2 × 10⁵ cells/mL and stimulated with 1 μg/mL of LPS in the presence or absence of sample. After incubation at 37 °C for 24 h, 50 μL of cell-free supernatant was mixed with 50 μL of Griess Reagent I and 50 μL of Griess Reagent II to determine nitrite production. Absorbance was measured at 546 nm against a calibration curve with sodium nitrite standard. Indomethacin was selected as a positive control. As stock solutions, all samples were dissolved in DMSO. The cell viability was examined using the MTT assay following the previously published protocols [27] to make sure the tested oleanolic triterpenes exhibited no cytotoxicity against RAW 264.7 macrophage cells at their effective concentrations. All experiments were run in triplicate (see Supplementary Materials).