Purification and Characterization of Antioxidant Peptides from Alcalase-Hydrolyzed Soybean ( Glycine max L.) Hydrolysate and Their Cytoprotective Effects in Human Intestinal Caco-2 Cells.

This study aimed to purify and identify antioxidant peptides from the low-molecular-weight fraction (SPH-I, MW < 3 kDa) of Alcalase-hydrolyzed soybean ( Glycine max L.) hydrolysate and further evaluate the cytoprotective effects of synthesized peptides against oxidative stress in human intestinal Caco-2 cells. After purification by gel filtration chromatography and reversed-phase HPLC, four major peptides were sequenced by nano-LC-ESI-MS/MS as VVFVDRL (847 Da, SPH-IA), VIYVVDLR (976 Da, SPH-IB), IYVVDLR (877 Da, SPH-IC), and IYVFVR (795 Da, SPH-ID). The antioxidant peptides were synthesized and displayed desirable DPPH radical-scavenging activity (from 16.5 ± 0.5 to 20.3 ± 1.0 μM Trolox equivalent (TE)/μM), ABTS^•+ radical-scavenging activity (from 3.42 ± 0.2 to 4.24 ± 0.4 mM TE/μM), ORAC (from 143 ± 2.1 to 171 ± 4.8 μM TE/μM), and FRAP (from 54.7 ± 1.2 to 79.0 ± 0.6 mM Fe²⁺/μM). Moreover, the synthesized peptides protected Caco-2 cells against H₂O₂-induced oxidative damage via significantly downregulating intracellular ROS generation and lipid peroxidation ( p < 0.05). Additionally, SPH-IC and SPH-ID statistically upregulated total reduced glutathione synthesis, enhanced activities of catalase and glutathione reductase, and suppressed ROS-mediated inflammatory responses via inhibiting interleukin-8 secretion ( p < 0.05).