Antioxidant peptides from shrimp by-products: preparation, identification and protective function on H₂O₂-induced HepG2 cell

The objective of this study is to identify the antioxidant peptides from shrimp by-products and clarify the underlying protective mechanism involved in HepG2 cells with oxidative stress induced by H₂O₂. Protein from shrimp by-products was hydrolyzed by three enzymes (neutral protase, alcalase, and Protamex) and the hydrolysates were separated by using Sephadex G-15 gel filtration, among which the A3 (fraction of alcalase-hydrolysate) displayed a significant 1,1-diphenyl-2-picrylhydrazyl radical and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) cation radical scavenging ability. A total of 3 480 peptides were identified through nano-high performance liquid chromatography-tandem mass spectrometry, with the prediction of five discovered antioxidant peptides (DYPLVPPYF, HFVPVYEGF, GFPPFTGGPFR, EGYPFNPLL, and RVSDGPWLGR). Notably, HFVPVYEGF and EGYPFNPLL emerged as the potent antioxidant peptide, displaying lower half maximal inhibitory concentration. Furthermore, HFVPVYEGF and EGYPFNPLL obviously relieved oxidative stress in HepG2 cells, which strengthened the activity of total-antioxidant capocity, catalase, glutathione peroxidase, and superoxide dismutase, with diminishing the intensity of malondialdehyde and intracellular reactive oxygen species. Molecular docking results revel that HFVPVYEGF and EGYPFNPLL can bind to Kelch-like ECH-associated protein 1 with hydrophobic interactions. The results provided theoretical basis for the production and application of the by-product of shrimp. And a further study should be carried out to examine the bioavailability and in vivo activity of HFVPVYEGF and EGYPFNPLL which identified from shrimp by-products.