Discovery of novel DPP-IV inhibitory peptides from bullfrog (Lithobates catesbeianus) muscle: Molecular interactions, stability, and in vivo hypoglycemic effects

Dipeptidyl peptidase-IV (DPP-IV) is a key therapeutic target for type 2 diabetes, and food-derived inhibitory peptides are promising alternatives to synthetic drugs. In this study, bullfrog muscle protein hydrolysates were prepared using different proteases, among which the compound protease hydrolysate showed the strongest DPP-IV inhibitory activity. Ultrafiltration revealed that the <3 kDa fraction exhibited the highest potency. Three peptides (VPVG, LPVQ, and VPLG) were identified by LC–MS/MS combined with in silico screening. Among them, synthesized VPVG showed the strongest inhibitory activity (IC 50 = 128.91 μM). Enzyme kinetics indicated a mixed-type inhibition mechanism, while molecular docking and molecular dynamics simulations demonstrated stable interactions with the DPP-IV active site. The peptides showed good stability under heat treatment, varying pH conditions, and simulated gastrointestinal digestion. They exhibited no cytotoxicity in Caco-2 cells, inhibited cellular DPP-IV activity, and significantly reduced blood glucose levels in a hyperglycemic zebrafish model.