Identification of active ingredients mediating anti-platelet aggregation effects of BuyangHuanwu decoction using a platelet binding assay, solid phase extraction, and HPLC-MS/MS

BuyangHuanwu decoction (BHD) is widely used as a traditional herbal medicine because of its antithrombotic effect, which is attributed to the inhibition of platelet aggregation; however, its active compounds remain unknown. In this study, we developed a method involving platelet binding, solid-phase extraction, and HPLC-MS/MS for screening BHD compounds with potential anti-platelet aggregation properties. Five compounds showing platelet binding affinity were identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-β-d-glucoside, galloylpaeoniflorin, and formononetin-7-O-β-d-glucoside. The results of anti-platelet aggregation experiments in vitro confirmed that these compounds inhibited adenosine diphosphate-induced platelet aggregation. Our results suggest that a platelet binding assay combined with solid-phase extraction and HPLC-MS/MS is an effective method for screening anti-platelet aggregation agents in traditional Chinese medicines. •An efficient method was developed for screening potential anti-platelet-aggregation compounds in BHD.•SPE technology was used for enrichment of the ingredient before analysis.•Five compounds showed platelet-binding activity and were identified using HPLC/MS and 1HNMR.•Five of binding components were confirmed to have pharmacological activity.