Identification of anti-inflammatory active peptide from black soybean treated by high hydrostatic pressure after germination

Abstract

This study was conducted to isolate and identify the anti-inflammatory active peptide in black soybean treated by high hydrostatic pressure after germination. The anti-inflammatory activity was examined using RAW 264.7 macrophages. Black soybean was germinated for 4 days and subjected to 150 MPa for 24 h, followed by preparation of protein extracts. The extracts were sequentially filtered using membranes with a molecular weight cutoff of 30, 10, and 3 kDa. A strong inhibitory effect was observed with the 3–10 kDa fraction. The active peptide from this fraction was purified by gel permeation column chromatography and first and second semi-preparative HPLC. After isolation and purification of the active peptide, secretion of inflammatory markers (nitric oxide, TNF-α, IL-1β, and IL-6) by LPS-stimulated RAW 264.7 macrophages was measured. The purified anti-inflammatory peptide was analyzed by UV, LC–MS, 1H and 13C NMR, COSY, HMBC, and HSQC and was identified as a tripeptide.