Anti-inflammatory activity of peptide from chicken feather meal in macrophage RAW 264.7

Abstract

Inflammation is a defense mechanism in the body induced by foreign chemicals or pathogens. Thus, inflammation is one of the most critical factors implicated in vasodilation, carcinogenesis and other degenerative disorders. However, the study of the anti-inflammatory mechanism of peptides has been limited. So studies on anti-inflammatory peptides from natural sources are increasing in interest. The objective of this study, to investigate the optimal condition of enzymatic hydrolysis to prepare an anti-inflammatory peptide from chicken feather meal by using 3 types of microbial protease (Alcalase®, Flavourzyme®, and Neutrase®) in various concentrations (1% 2.5% and 5%). Peptide hydrolysate with 1% Flavourzyme® produced the best results for inhibition of NO production (IC50 5.48±1.0 µg/ml) when compared with other proteases. These peptide fractions were fractionated by ultrafiltration with 10, 5, 3 and 0.65 kDa membranes and further analyzed for NO scavenging activity. Among the fractions, MW ≤ 0.65 kDa exhibited a high level of antioxidant activity toward the NO radical scavenging assay. The ≤ 0.65 kDa fractions were selected for further fractionation by gel filtration chromatography. The F2 fraction was the strongest in inhibiting nitric oxide production. After that, F2 fraction was selected to study the NO production and cytotoxicity test by MTT assay in LPS-stimulated macrophage RAW 264.7. The result shows that the peptide also inhibited NO production and wasn’t toxic to macrophage RAW 264.7. The peptide fraction was further purified by HPLC and identification by mass spectrometry with amino acid sequence characterized into 4 peptides. In addition, the peptides are suppressed in the expression mRNA of iNOS, COX-2, IL-6 and TNF-α which are responsible for pro-inflammatory cytokine production. The results indicate that bioactive peptide from chicken feather meal can be developed to medical, pharmaceutical and cosmetic products.