This study aimed to discover novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from defatted walnut meal and elucidate their molecular mechanisms. Enzymatic hydrolysis coupled with ultrafiltration yielded fractions of different molecular weights, of which the <1 kDa fraction demonstrated the strongest DPP-IV inhibitory activity. Based on LC-MS/MS analysis and multi-stage bioinformatics screening, 18 high-potential candidate peptides were obtained. Molecular docking and dynamics simulations revealed that core peptides (LPQY, FPAG, WDPNN, LPQNFA, LPSYQPTP) could stably bind to the DPP-IV active site via hydrogen bonds and hydrophobic interactions, with FPAG and LPSYQPTP exhibiting the most favorable binding. Inhibition kinetics identified FPAG (IC₅₀ = 105.6 μM) and LPSYQPTP (IC₅₀ = 132.4 μM) as mixed-type inhibitors with dominant competitive characteristics, functioning through active site competition and enzyme conformation alteration. Importantly, both peptides retained significant DPP-IV inhibitory activity after simulated gastrointestinal digestion. In STC-1 enteroendocrine cells, they effectively inhibited cellular DPP-IV and enhanced active GLP-1 secretion. Structure-activity relationship analysis highlighted the crucial role of X hy P-type peptides containing hydrophobic amino acids. Network pharmacology further predicted that these peptides might exert hypoglycemic effects through multi-target and multi-pathway synergistic mechanisms. This study provides a new strategy for the high-value utilization of walnut meal and offers a theoretical foundation for developing food-derived DPP-IV inhibitory peptides. • Integrated in silico and experimental screening identified novel DPP-IV inhibitory peptides from defatted walnut meal. • FPAG and LPSYQPTP were identified as mixed-type DPP-IV inhibitors, exhibiting notable gastrointestinal stability along with potent cellular activity, effectively inhibiting DPP-IV and enhancing GLP-1 secretion in STC-1 cells. • Network pharmacology revealed their multi-target potential for synergistic regulation of glucose metabolism.
Yan et al. (Mon,) studied this question.