A Novel Integrated Strategy for Discovering Absorbable Anticoagulant Bioactive Peptides: A Case Study on Leech Protein Hydrolysates.

Medicinal plants and animal-derived proteins represent valuable natural sources of bioactive components with pharmaceutical potential. Whilst some medicinal plants and animal-derived proteins also offer rich sources of anticoagulant bioactive peptides, their development faces multiple challenges: anticoagulant evaluation relies on single-parameter assays with limited reliability, native proteins demonstrate suboptimal activity without enzymatic treatment, and few researchers investigate bioavailable peptides. Our study establishes an innovative framework using the leech as a case study to overcome these barriers. A novel anticoagulant evaluation model was first established with the Critic-G1 weighting method. And we optimized the enzymatically hydrolyzed extracts with high activity using Box-Behnken response surface methodology. Subsequently, the everted gut sac model was implemented to simulate intestinal absorption and screen for absorbable peptide fractions. Furthermore, peptidomics was employed to identify the bioactive peptides. Lastly, we identified the bioactivity using anticoagulation assays. Results indicated that the optimal hydrolysis conditions were achieved with trypsin at 50.48 °C, an enzyme-to-substrate ratio of 6.78%, 7.51 h, and pH of 8.06. The peptide DLRWM was identified through integrated peptidomics and molecular docking approaches, with subsequent activity validation demonstrating its potent anticoagulant effects. This study has successfully identified a novel anticoagulant peptide (DLRWM) with confirmed intestinal absorption properties and provides a template for unlocking the pharmaceutical potential of medicinal animal proteins.