3.7. Anti-Inflammatory Assay

RAW264.7 macrophages were plated into 96-well plates. Then, the macrophages were incubated with 1 μg/mL LPS and the sample. Nitric oxide synthase inhibitor (NOSI) was used as positive control, and the culture medium without sample was used as control group. After the cells were cultured overnight, the culture medium was used to detect the production of NO. The optical density was measured to detect nitrite (NO₂⁻) at 570 nm wavelength by Griess method. To remove the effect of the sample on the cell toxicity, MTS was added to the remaining medium to detect the cell viability [43]. Anti-inflammatory activity was calculated according to the following Equation (5).

where A₁ is optical density at 570 nm in non-drug treatment group, and A₀ is optical density at 570 nm in control group.