3.6. Anti-Inflammatory Assay

Anti-inflammatory activity was measured by nitric oxide (NO) and prostaglandin E₂ (PGE₂) productions. RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/well. Adhered cells were then incubated for 24 h with or without 1 μg/mL of lipopolysaccharide (LPS; Sigma-Aldrich) in the absence or presence of samples. The supernatants detected were NO and PGE₂. For the product of NO, the supernatants were mixed with an equal volume of Griess reagent (Sigma-Aldrich) and incubated for 15 min at room temperature. The nitrite concentration was measured at 540 nm using an ELISA microplate reader. PGE₂ production in supernatants was measured using an ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.