Journal of ethnopharmacology · 2026
Bioactivity-guided identification of antithrombotic peptides from an ethanol-soluble fraction of Hirudo nipponia with anticoagulant and antiplatelet activities.
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Zhong Wanling, Shen Huijuan, Ma Yunnan, Fan Xiaoyu, Ye Jinhong, Liu Zhuyuan, Du Shouying
Аннотация
ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal leeches have long been administered orally in traditional Chinese medicine for blood stasis-related disorders. The thermal instability and poor oral bioavailability of hirudin suggest that other heat- and digestion-resistant constituents may contribute to the pharmacological activity of orally administered leech preparations.
AIM OF THE STUDY: To characterize the antithrombotic activity of an ethanol-soluble fraction derived from processed Hirudo nipponia decoction pieces (Hn70) and identify peptide candidates with anticoagulant and antiplatelet activities.
MATERIALS AND METHODS: Hn70 was prepared by aqueous extraction followed by 70% ethanol fractionation. Its anticoagulant and antiplatelet activities, in vivo antithrombotic effects, and retention of anticoagulant activity under thermal and simulated gastrointestinal conditions were evaluated. Antithrombin activity was quantified using recombinant hirudin as an activity reference. The major active fraction was isolated by bioactivity-guided gel filtration, followed by coagulation factor and platelet aggregation assays, nLC-MS/MS-based de novo sequencing, bioinformatics screening, molecular docking, and functional validation of selected peptides.
RESULTS: Hn70 showed concentration-dependent anticoagulant and antiplatelet activities and significantly reduced carrageenan-induced thrombosis in vivo. Six batches showed a specific antithrombin activity of 1.156 ± 0.020 ATU·mg-1 dry Hn70 (CV, 1.70%). Hn70 retained substantial anticoagulant activity after thermal and simulated gastrointestinal treatment. Hn70-D was identified as a major active fraction that reduced multiple coagulation-factor activities and inhibited platelet aggregation. Twenty-eight peptides were identified, and seven selected synthetic peptides exhibited both activities in single-concentration assays.
CONCLUSION: Hn70 is an antithrombotic fraction of Hirudo nipponia that retains substantial anticoagulant activity after thermal treatment and simulated gastrointestinal digestion. Bioactivity-guided analysis identified Hn70-D and candidate peptides for further investigation of its antithrombotic pharmacology.
AIM OF THE STUDY: To characterize the antithrombotic activity of an ethanol-soluble fraction derived from processed Hirudo nipponia decoction pieces (Hn70) and identify peptide candidates with anticoagulant and antiplatelet activities.
MATERIALS AND METHODS: Hn70 was prepared by aqueous extraction followed by 70% ethanol fractionation. Its anticoagulant and antiplatelet activities, in vivo antithrombotic effects, and retention of anticoagulant activity under thermal and simulated gastrointestinal conditions were evaluated. Antithrombin activity was quantified using recombinant hirudin as an activity reference. The major active fraction was isolated by bioactivity-guided gel filtration, followed by coagulation factor and platelet aggregation assays, nLC-MS/MS-based de novo sequencing, bioinformatics screening, molecular docking, and functional validation of selected peptides.
RESULTS: Hn70 showed concentration-dependent anticoagulant and antiplatelet activities and significantly reduced carrageenan-induced thrombosis in vivo. Six batches showed a specific antithrombin activity of 1.156 ± 0.020 ATU·mg-1 dry Hn70 (CV, 1.70%). Hn70 retained substantial anticoagulant activity after thermal and simulated gastrointestinal treatment. Hn70-D was identified as a major active fraction that reduced multiple coagulation-factor activities and inhibited platelet aggregation. Twenty-eight peptides were identified, and seven selected synthetic peptides exhibited both activities in single-concentration assays.
CONCLUSION: Hn70 is an antithrombotic fraction of Hirudo nipponia that retains substantial anticoagulant activity after thermal treatment and simulated gastrointestinal digestion. Bioactivity-guided analysis identified Hn70-D and candidate peptides for further investigation of its antithrombotic pharmacology.