Rapid screening of the antithrombotic active components of AgrimoniaPilosaLedeb with thrombin binding affinity ultrafiltration mass spectroscopy

Traditional Chinese Medicine (TCM) holds significant potential as a natural source of medication, providing effective alternatives for preventing and treating thrombotic diseases. Although research on the anticoagulant activity of Agrimonia Pilosa Ledeb (Rosaceae, A. pilosa) has been paid attention to, the monomer component and its mechanism of action have not been studied deeply. Therefore, this study aimed to screen and identify the antithrombotic active components of A. pilosa by affinity ultrafiltration (AUF) combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The affinity between compounds and thrombin was studied through molecular docking, and the activity of thrombin inhibitors was evaluated from multiple dimensions by the zebrafish thrombus model, chromogenic substrate method, and coagulation factor test. Eleven ligands with potential antithrombotic activity were screened from A. pilosa. Among them, compounds hyperoside, quercitrin, afzelin, baicalin, quercetin, kaempferol, and 3-O-caffeoylquinic acid were shown to have antithrombotic activity. This study is the first to report that kaempferol-3-rhamno-glucoside, tiliroside, and taxifolin exhibit thrombin-inhibitory effects through both in vivo and in vitro experiments. The results of this study indicate that AUF-HPLC and UPLC-Q-TOF-MS can be used for high-throughput screening of active components from TCM, thus laying a groundwork for the discovery of thrombin inhibitors and promoting the research progress of antithrombotic drugs.