Anticoagulant activity of crude extract of Holotrichia diomphalia larvae.

ETHNOPHARMACOLOGICAL RELEVANCE: Holotrichia diomphalia larvae are one classical folk medicinal material in East Asia which has clinically been used to promote blood circulation and dispel blood stasis for several hundred years.

AIM OF THE STUDY: The anticoagulant activity of crude extract of H. diomphalia larvae (CEHDL) in vitro and in vivo was evaluated to explore its mechanism as antithrombotic medicine.

MATERIALS AND METHODS: The effects of CEHDL on plasma recalcification time, platelet aggregation, bleeding time, hydrolysis of fibrinogen and fibrin were measured with normal human plasma, plasma-rich platelet, transected mouse tails and bovine fibrinogen; the anti-thrombosis activities of CEHDL in vitro and in vivo were analyzed with clots lysis assay and carrageenan-induced mouse tail thrombosis model.

RESULTS: CEHDL was found to contain large numbers of proteins and could inhibit blood coagulation and platelet aggregation in a dose-dependent manner. Furthermore, CEHDL preferentially cleaved α- and β-chains followed by γ-chains of fibrinogen. Besides, CEHDL could directly degrade fibrin rather than activate plasminogen. It has been noted that fibrinogenolytic activity of CEHDL could be unaffected by metal ions such as Mg(2+), Ca(2+), Zn(2+), Fe(2+), Fe(3+), Cu(2+) and buffers with pH 3-10. Moreover, protease inhibitors like TPSI, aprotinin, leupetin, PMSF, DTT and EDTA only slightly or not inhibited fibrinogenolytic activity of CEHDL. However, CEHDL could be completely inactivated at 75°C and 100°C. In addition, CEHDL exhibited anti-thrombosis activities in both blood clot lysis assay and carrageenan-induced thrombosis model.

CONCLUSION: CEHDL possessed potent anticoagulant activity and several fibrin(ogen)olytic agents from H. diomphalia larvae were responsible for its antithrombotic effect as medicine.