*3.8. Anti-Inflammatory Effectiveness*

RAW 264.7 cells were loaded on a 96-well plate at a concentration of 1 × 105 cells/well and incubated for 1 d in a 37 ◦C incubator. Lipopolysaccharide (LPS) was added at a concentration of 500 ng/mL to induce NO production, and the samples were allowed to react for 24 h. After the reaction, the upper solution layer was reacted with Griess reagent, and the absorbance was measured at 540 nm to estimate the NO production. The cells were treated with 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent and incubated for an additional hour. The cytotoxicity was determined by removing all the upper fluid and treating it with 100 μL DMSO to completely dissolve the cells. Subsequently, the absorbance was measured at 550 nm. In addition, the significance of the test substance was determined by the two-sample test to meet the biological statistical criterion of (α): 1%. It treated nordihydroguaiaretic acid (NDGA) as a benign control group [28,29].
