Abstract: This study isolated and identified novel antioxidant peptides from an enzymatic hydrolysate of rice bran protein and elucidated their potential mechanisms of action. Based on the scavenging capacity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, bioactivity-guided fractionation was employed to obtain the antioxidant peptides through sequential ultrafiltration, gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). The peptide sequences were identified using liquid chromatography-mass spectrometry (LC-MS). Computer-aided screening combined with molecular docking technology was utilized to reveal the antioxidant mechanisms of the identified peptides. The results showed that 183 peptide segments were identified from fraction R-2 with high antioxidant activity, obtained after RP-HPLC separation. Finally, five antioxidant peptides were selected: WCY, YFC, HWC, AHWC, and QGYY. Among these, WCY, YFC, HWC, and AHWC exhibited potent in vitro antioxidant activity. Molecular docking results indicated that the binding energies of these four peptides to Kelch-like ECH-associated protein-1 (Keap1) were all below −7 kcal/mol, with hydrogen bonding and hydrophobic interactions as the primary intermolecular forces. This suggests their antioxidant mechanism might be through inhibiting the Keap1/nuclear factor E2-related factor 2 (Nrf2) signaling pathway. In conclusion, the antioxidant peptides derived from rice bran protein possess potential as natural antioxidants. This study provides a theoretical foundation for the development and application of bioactive peptides from rice bran protein in the food and medicinal industries.

Key words: rice bran protein; antioxidant peptides; molecular docking; structure-activity relationship; functional activity