Capture of anti-coagulant active ingredients from Moutan Cortex by platelet immobilized chromatography and evaluation of anticoagulant activity in rats

Moutan Cortex (MC) is a well-known Chinese medicine for promoting blood circulation and relieving blood stasis. The intent of this study was to evaluate the anticoagulant activity of MC and capture the bioactive compounds by platelet immobilized chromatography. Sprague Dawley (SD) rats were randomly divided into the control group, aspirin group and MC group (1.25, 2.5, 5 g/kg/d). Coagulation system and platelet activity were investigated to evaluate the anti-coagulation effect of MC. The effective components of MC were captured by platelet immobilized chromatography. High performance liquid chromatography-diode array detection (HPLC-DAD) and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis were used to identify the binding ingredients. Meanwhile, the efficacy of active ingredients was assessed through inhibiting platelet adhesion and regulating the expression of platelet related proteins. Principal findings showed that 2.5 g/kg/d MC significantly prolonged thrombin time (TT) and 5 g/kg/d MC significantly prolonged TT and prothrombin time (PT). MC exhibited an inhibitory potency on adenosine diphosphate-induced platelet aggregation. Four active compounds were found by platelet immobilized chromatography including oxypaeoniflorin, tetragalloylglucose, pentagalloyl glucose and benzoylpaeoniflorin; these active ingredients significantly up-regulated the expression of hsp-70 and coronin-1B, reduced the ratio of adhesion platelets. These results suggest that MC markedly promoted blood circulation and relieved blood stasis by inhibiting platelet activation, as an anti-coagulant, elucidating its potential capacity to treat cardiovascular diseases.