2.6. Determination of Nitric Oxide (NO) Production

RAW 264.7 cells were plated at a density of 2 × 10⁵ cells/well in a 96-well plate. The cells were incubated with 100 ng/mL lipopolysaccharide (LPS) in the presence of ethanol extracts of PL or PF (100, 200, and 500 µg/mL) for 18 h. After incubation, culture supernatants (100 µL) were mixed with an equal volume of Griess reagent and allowed to react for 15 min. Then, the absorbance was measured at 540 nm using a microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA). The nitrite concentration in the sample was calculated using a standard curve prepared with NaNO₂.