Purification and identification of novel antioxidant peptides from watermelon seed protein hydrolysates and their cytoprotective effects on H2O2-induced oxidative stress

Abstract

The aim of this study was to purify and identify antioxidant peptides from watermelon seed protein hydrolysates (WSPHs-I: Mw < 1 kDa) and further evaluate their cytoprotective effects against H2O2-induced oxidative stress in HepG2 cells. After purification by Sephadex G-15 and semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), five peptides, RDPEER (P1), KELEEK (P2), DAAGRLQE (P3), LDDDGRL (P4), and GFAGDDAPRA (P5) were sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. These peptides showed desirable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity (IC50: 0.216 ± 0.01–0.435 ± 0.03), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity (IC50: 0.54 ± 0.02–1.23 ± 0.03), and oxygen radical absorbance capacity (ORAC) (82.36 ± 1.2–130.67 ± 2.2 μM TE/mg). Among them, peptide P1 exhibited the strongest antioxidant capacity. Moreover, the results suggested that peptide P1 may protect HepG2 cells from H2O2-induced oxidative damage by significantly inhibiting reactive oxygen species (ROS), [Ca2+]i, malondialdehyde (MDA) levels and increasing antioxidative enzyme activities.