Discovery of garlic-derived peptides as natural DPP4 inhibitors: An integrated computational, network pharmacology, and in situ evaluation approach.

Type 2 diabetes mellitus (T2DM), marked by impaired glucose homeostasis and hyperglycemia, remains a major global health burden. Although dipeptidyl peptidase-IV (DPP4) inhibitors improve glycemic control, safety concerns about synthetic agents drive the search for safer food-derived alternatives. Garlic (Allium sativum L.), a widely consumed functional food, is underexplored as a source of antidiabetic peptides. Here, simulated gastrointestinal digestion, virtual screening, molecular docking, and experimental validation were integrated to identify garlic-derived DPP4 inhibitory peptides. Five peptides-WPHY, IASW, WPQY, PPSPSY, and VAPGW-were synthesized and evaluated, with WPHY showing the strongest activity (IC₅₀ = 81.57 μM). Extended 150 ns molecular dynamics simulations elucidated the atomistic basis for this potency, revealing that WPHY achieves exceptional stability through the formation of a persistent N-terminal salt bridge and a stable C-terminal backbone interaction. Network pharmacology suggested additional targets (ACE, PTGS2) and involvement in glucose metabolism and inflammation. In situ assays using early-confluent Caco-2 cells showed preserved bioactivity, particularly for WPHY and IASW. Furthermore, in vitro digestion assays demonstrated that WPHY exhibits high gastric stability and retains bioactivity under intestinal conditions. These results highlight garlic peptides as promising natural DPP4 inhibitors, supporting their potential application in functional foods for glycemic regulation.