Discovery of a potent and digestively stable DPP4 inhibitory peptide from rice bran protein via an integrated peptidomics and in silico/in vitro strategy.

Rice bran protein (RBP) is rich in hydrophobic amino acids and serves as an ideal precursor for DPP4 inhibitory peptides. In this study, an integrated strategy combining peptidomics, virtual screening, molecular simulation, and in vitro validation was established to identify DPP4-inhibitory peptides from RBP hydrolysates. LC-MS/MS analysis of the active <3 kDa hydrolysate fraction identified 1029 peptides. After bioactivity prediction, toxicity evaluation, molecular docking, and database comparison, five novel candidate peptides were synthesized and evaluated using a Caco-2 cell-based DPP4 activity assay. Among them, FGAP (FP4) showed the strongest inhibitory activity, with an IC50 value of 68.13 ± 6.22 μM. Molecular docking and 100 ns molecular dynamics simulations further revealed that FP4 formed a stable complex with DPP4 by interacting with key catalytic pocket residues, particularly Ser630 and His740. MM-PBSA analysis showed that the FP4-DPP4 complex had the most favorable total binding free energy (-27.50 kcal/mol), supporting its stronger inhibitory potency compared with PL4 and SF5. Simulated gastrointestinal digestion showed that FP4 exhibits significant digestive stability and retains substantial inhibitory activity. These findings highlight rice bran protein as a promising source of DPP4-inhibitory peptides and demonstrate the efficacy of an integrated strategy for discovering food-derived peptides with antidiabetic potential.