Abstract: In this study, neutral protease enzymatic hydrolysates of wheat germ albumin were separated step by step using ultrafiltration and chromatography techniques. Oxygen radical absorbance capacity (ORAC) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid (ABTS) cation radical scavenging capacity were used as antioxidant activity evaluation indicators for selecting hydrolysate components with high antioxidant activity. The selected component was served as the initial material to further screen small peptides with high antioxidant activity by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with PeptideRanker database activity prediction technology. High glucose-induced vascular smooth muscle cells (VSMCs) and H2O2-induced HepG2 and VSMCs were served as oxidative stress injury models to explore the protective effect of obtained small peptides. Results: Component V with small molecular weight obtained by ultrafiltration and chromatography had the highest antioxidant activity. Twenty small peptides were identified with LC/MS-MS analysis from this fraction. PeptideRanker prediction and antioxidant experiment validation showed that the peptide sequence LNYPPY (765.3 Da) had the highest antioxidant activity (ORAC was 3.01 μmol TE/μmol), and ABTS cation radical clearance rate was 95.58%). This peptide had a good protective effect on the H2O2-induced oxidative damage in HepG2 cells and vascular smooth muscle cells (VSMCs), but had no significant effect on the abnormal proliferation and the increase of reactive oxygen species (ROS) levels in VSMCs induced by high glucose. In conclusion, the peptide LNYPPY has good antioxidant properties in vitro and has protective effect against cellular oxidative damage induced by H2O2, which provides an important theoretical reference for the development and utilization of wheat germ albumin.

Key words: wheat germ albumin; isolation; antioxidant peptide; cellular oxidative damage