Crab Pentapeptide and Its Anti-inflammatory Activity on Macrophage Cells

Marine fishes have long been known due to their nutritional and pharmaceutical importance. In this study, marine crab (C. natator) body muscle (CBM) was utilized for the separation, purification, and identification of anti-inflammatory peptide using enzymatic hydrolysis. The amino acid composition of CBM depicted the presence of inflammation suppressing amino acids such as serine, aspartic acid, and glutamic acid. Furthermore, the CBM protein hydrolysates prepared using three enzymes (alcalase, trypsin, and papain) at different time intervals were analyzed for anti-inflammatory activity using albumin denaturation and membrane protection assays which exhibited 12th h papain with the highest denaturation inhibition (78.55 +/- 1.36%) and membrane protection (69.02 +/- 2.05%) activity. Moreover, separation of this active hydrolysate was performed by ultrafiltration using 10, 3 kDa cut-off membranes and gel chromatographic purification using Sephadex G-25 beads. The active peak (II) determined using anti-inflammatory assays was subjected to sequencing which revealed the peptide sequence of ESPVL (544 Da). Further, the functional aspects of the peptide were studied which showed increased stability and solubility of the peptide at varied pH range. Moreover, the effect of CBM derived peptide was studied on lipopolysaccharide-stimulated RAW264.7 cells. The purified peptide was found to be non-cytotoxic and suppressed cytokine expression which was estimated using enzyme immunoassay and western blot analysis.