Anti-inflammatory activity assay

The murine macrophage cell line RAW 264.7 was purchased from Shanghai Institute of Cell Biology (Shanghai, China). RAW 264.7 cells were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS. Cells were incubated in a 5% CO₂ atmosphere at 37 °C and were subcultured every day.

Cell viability was determined using the MTT method according to a previous study [9]. Cells were seeded and treated with the indicated concentrations of MPE with LPS for 22 h and then were incubated with a solution of 5 mg/mL MTT for the next 2 h. DMSO (1% final concentration) was added to each well, and formazan was dissolved by gentle shaking. The plates were evaluated in a microplate reader at 540 nm.

To determine NO levels, RAW 264.7 cells were seeded at a density of 2 × 10⁵ cells/well in a 96 well plate and grown for 2 h to adhere. Then, they were treated with various concentrations of MPE for 1 h and incubated for 24 h in fresh DMEM with or without LPS (1 µg/mL). The Griess reagent was used to determine nitrite levels. Briefly, an equal volume of culture supernatant (100 µL) was mixed with the Griess reagent, and absorbance was measured at 540 nm using a microplate reader.

The secretion of TNF-α, IL-6, and PGE2 was measured by ELISA [15]. Briefly, RAW 264.7 cells (1 × 10⁴ cells/well) were seeded in 24-well plates, stimulated with LPS (1 µg/mL), and incubated with different concentrations of MPE for 24 h. Then, TNF-α, IL-6, and PGE2 levels in the culture medium were determined with an ELISA kit according to the manufacturer’s instructions. All experiments were performed in triplicate.