There is a growing demand for natural and potent α-glucosidase inhibitors due to the rising prevalence of diabetes. In this study, newly identified α-glucosidase inhibitory peptides were identified from the tryptic hydrolysate of hemp seed proteins based on peptidomics and in silico analysis. A total of 424 peptides, primarily derived from four cupin-type-1 domain-containing proteins, were identified, and 13 ultimately were selected for validation based on their higher PeptideRanker scores, solubility, non-toxicity, and favorable ADMET properties. Molecular docking revealed that these 13 peptides primarily interacted with α-glucosidase via hydrogen bonding and hydrophobic interactions. Among them, three novel peptides—NPVSLPGR (−8.7 kcal/mol), LSAERGFLY (−8.5 kcal/mol), and PDDVLANAF (−8.4 kcal/mol)—demonstrated potent α-glucosidase inhibitory activity due to their lower binding energies than acarbose (−8.1 kcal/mol), the first approved α-glucosidase inhibitor for type 2 diabetes treatment. The molecular mechanism analysis revealed that the peptides NPVSLPGR and LSAERGFLY inhibited α-glucosidase by simultaneously blocking substrate entry through occupying the entrance of the active site gorge and preventing catalysis by binding to active sites. In contrast, the peptide PDDVLANAF primarily exerted inhibitory effects by occupying the entrance of the active site gorge. Molecular dynamics simulation validated the stability of the complexes and provided additional insights into the molecular mechanism determined through docking. These findings contribute essential knowledge for the advancement of natural α-glucosidase inhibitors and offer a promising approach to effectively manage diabetes.

This study aimed to identify novel angiotensin-converting enzyme (ACE)-inhibitory peptides from royal jelly proteins (RJPs) by integrating in silico digestion, virtual screening, and in vitro evaluation. Three major royal jelly proteins (MRJP1-3) were subjected to in silico digestion using 16 enzymatic systems, yielding 1411 unique peptides. Virtual screening based on predicted bioactivity, toxicity, water solubility, and ADMET profiles resulted in the selection of 27 candidate peptides. Molecular docking revealed strong binding affinities for these peptides compared with the positive control captopril, among which PYPDWSFAK and RPYPDWSF exhibited potent ACE-inhibitory activity, with IC₅₀ values of 110 ± 1.02 μmol/L and 204 ± 0.61 μmol/L, respectively. Kinetic analysis indicated that PYPDWSFAK acts as a mixed-type ACE inhibitor. Docking visualization demonstrated that PYPDWSFAK forms multiple hydrogen bonds with key residues in the ACE active pocket and directly coordinates with the catalytic Zn²⁺ ion. Cellular assays showed that PYPDWSFAK was non-cytotoxic, suppressed Ang II–induced endothelial cell migration, restored NO and ET-1 balance, and enhanced SOD and GSH-Px activities. Overall, this study enriches the repertoire of ACE-inhibitory peptides derived from royal jelly proteins. Furthermore, PYPDWSFAK is identified as a promising ACE-inhibitory peptide with potential for incorporation into natural antihypertensive ingredients or functional foods.