Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and prostaglandin-E2 (PGE2 ) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.