Anti-inflammatory Activity of Extracts via NO Assay

The anti-inflammatory activity of the extracts (400 to 6.25 μg/mL) was evaluated based on nitric oxide (NO) production in a RAW 264.7 murine macrophage cell line (ECACC 91062702) due to lipopolysaccharide (LPS, 1 mg/mL in DMEM; Sigma-Aldrich, Saint Louis, MO, USA) stimulation. For that purpose, the Griess Reagent System kit (Promega, Madison, WI, USA) was used as described in previously published protocols [32 (link)]. Dexamethasone (50 mM) (Sigma-Aldrich, Saint Louis, MO, USA) and samples without the addition of LPS were used as positive and negative controls, respectively. The NO generated was monitored at 540 nm (ELX800 Biotek microplate reader; Bio-Tek Instruments Inc., Winooski, VT, USA). The extract concentrations that trigger 50% of NO production inhibition were expressed as EC₅₀ values (μg/mL).