Antioxidant activity and protective effects of Alcalase-hydrolyzed soybean hydrolysate in human intestinal epithelial Caco-2 cells

Soybeans are known as a promising source of bioactive peptides. However, knowledge on the antioxidant behaviors of soybean protein hydrolysate (SPH) in the human intestinal epithelium is limited. In this study, SPH was prepared with Alcalase and subsequently ultrafiltered into four peptide fractions as SPH-I (< 3 kDa), SPH-II (3-5 kDa), SPH-III (5-10 kDa) and SPH-IV (> 10 kDa). The antioxidant properties of SPH and membrane fractions were investigated using different chemical assays and their protective effects against oxidative stress were evaluated using H2O2 -stressed human intestinal Caco-2 cells. Results showed that SPH-I exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 2.56 mg/mL) and reducing capacity while SPH-III had the best metal ion-chelating activity (IC50 = 0.29 mg/mL). Both SPH and the peptide fractions dose-dependently suppressed intracellular reactive oxygen species (ROS) accumulation induced by H2O2 in Caco-2 cells, but the strongest inhibitory effect was observed for SPH-I. Amino acid (AA) results revealed that SPH-I was rich in hydrophobic and antioxidant AM, which could contribute to its stronger antioxidant properties. Additionally, SPH-I protected Caco-2 cells from H2O2-induced oxidative stress via inhibiting lipid per-oxidation and stimulating antioxidant enzyme activities. These results suggest that SPH-I and constitutive peptides can be beneficial ingredients with antioxidant properties and protective effects against ROS-mediated intestinal injury.