A multidimensional strategy for exploring active peptides combining chemical analysis and bioassays for medicinal leeches.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches were first recorded in Shennong Bencao Jing, and they have been traditionally used to treat blood stasis-related disorders through decoction and oral administration more than 2000 years in China. This practice involves heating and gastrointestinal transformation. However, whether it converts native constituents into pharmacologically active substances remains unresolved, representing a critical gap in understanding bioactive substance formation in traditional medicine.

AIM OF THE STUDY: To elucidate the component transformation of Hirudo nipponia (HN) and Whitmania pigra (WP) during decoction and enzymolysis, and identify their effective anticoagulant and antiplatelet peptides.

MATERIALS AND

METHODS

A multidimensional strategy integrating decoction, enzymolysis, chemical profiling, and bioactivity evaluation was employed. Component transformation was characterized by gel permeation chromatography, protein/peptide determination, gel electrophoresis, and nano LC-MS. Anticoagulant activity was assessed using in vivo thrombosis models and coagulation assays. Bioactive peptides were prioritized by molecular prediction and validated by in vitro bioassays.

RESULTS

HN and WP significantly reduced mice tail thrombosis incidence invivo (10.5% and 13.8%). Decoction and enzymatic hydrolysis induced proteins-to-peptides transformation was essential for pronounced bioactivity. In <3 kDa fractions with strongest antiplatelet effects, 23905 (HN) and 18783 (WP) peptides were identified, and among them, 70 candidates targeting thrombin and P2Y12 receptors were prioritized. Six peptides exhibited significant antiplatelet activity at 31.3 μM. Notably, the combined activity of peptides could be attributed to clinical efficacy of leeches.

CONCLUSION

Leech antiplatelet activity may be primarily mediated by decoction and digestion-generated peptides rather than native constituents, providing a mechanistic basis for the efficacy of non-hematophagous WP. This strategy offers a framework for identifying bioactive substances formed during traditional practice of animal-derived medicines.