3.11. Inhibition of Nitric Oxide (NO) Production in Lipopolysaccharide (LPS)-Stimulated RAW 264.7 Cells

Inhibition of NO production was assessed as previously reported [31]. Briefly, RAW 264.7 cells were seeded in 24 well plates, at a density of 2 × 10⁵ cells/well and incubated overnight in complete medium. The following day, medium was replaced with fresh DMEM and then cells were treated for 24 h with LPS (1 µg/mL) and different concentrations of extracts (ranging from 0.1 to 10 µg/mL). DMSO was used as a vehicle control. After 24 h, 100 µL of cell culture supernatant were incubated with 100 µL of Griess reagent (Sigma-Aldrich) at room temperature for 10 min in a 96-well plate and then, to quantify nitrites, stable products of NO, the absorbance was measured at 550 nm using a microplate reader (Synergy H1 microplate reader, BioTek, Winooski, USA). The absence of treatment-induced cytotoxic effects on RAW 264.7 cells was verified monitoring cell viability by MTT assay, as previously described [32]. LPS-stimulated RAW 264.7 cells were treated with increasing doses (0.1 to 10 μg/mL) of the extracts (as indicated) or DMSO (Ctrl). Data were expressed as percentage of nitrite production versus control (DMSO-treated cells).