*2.11. Anti-Inflammatory Activity*

For inflammatory experiments, murine macrophage cell line RAW 264.7 was used. Cells were seeded in a 96-well plate at density of ~5 × 104 cells/well. Nitrite accumulation, indicator of nitric oxide (NO) synthesis, was measured in the culture medium of treated and control cells by the Griess reaction. After 24 h of incubation, the medium was removed, the cells were washed with 200 μL PBS and treated with PEP (1 mg/mL) and 10 μg/mL of LPS from *Escherichia coli* O55:B5 for 24 h. Control cells were

incubated in serum-free medium with LPS (Control+) and without LPS (Control−) for 24 h. Finally, the media were collected and used for NO quantification. Briefly, 100 μL of collected cell supernatants were plated in 96-well plate and an equal amount of Griess reagent constituted by 1% (*w*/*v*) sulfanilamide and 0.1% (*w*/*v*) N-1-(naphthyl) ethylenediamine-diHCl in 2.5% (*v*/*v*) H3PO4, was added. The plate was incubated for 5 min and the absorbance measured at 550 nm in a microplate reader Synergy HT. The amount of NO was calculated using a sodium nitrite standard curve (0–10 μg/mL). Data were expressed as percentage of NO production calculated relative to Control+. Data represent the mean and standard deviation of three independent experiments (*n* = 3). All experiments were carried out between passage 10 to passage 30 to ensure cell uniformity and reproducibility.
