Investigation of Antithrombotic Activity and In Vivo Effective Forms of Kaempferitrin Using FeCl3-Induced Rat Arterial Thrombosis and UHPLC-Q-Exactive Orbitrap MS

Kaempferitrin (KAE) is a natural flavonol dirhamnopyranoside with various pharmacological activities, isolated from the antithrombotic fraction of <i>Celastrus orbiculatus</i> Thunb. This study aimed to investigate the antithrombotic activity and "effective forms" of KAE. The results showed that KAE significantly prolonged rabbit plasma recalcification time in vitro. In the FeCl₃-induced rat arterial thrombosis model, KAE demonstrated antithrombotic effects by inhibiting coagulation, platelet aggregation, and fibrinolysis, with a lesser risk of bleeding compared to aspirin. KAE was orally administered to rats, and a total of 192 metabolites were characterized. These included 25 phase I metabolites, 8 hydroxylated and methylated metabolites, 57 sulfated metabolites, 74 glucuronidated metabolites, 26 sulfated and glucuronidated metabolites, and 2 glycosylated metabolites. Twenty-eight compounds were considered the in vivo "effective forms" of KAE for their antithrombotic activity. Network pharmacology, molecular docking, and molecular dynamics simulations collectively predict that these "effective forms" may exert antithrombotic effects by suppressing the SRC/PI3K/AKT pathway. This study provides a foundation for a better understanding of the in vivo "effective forms" and mechanisms underlying KAE's antithrombotic activity, which is essential for understanding of "hexue" traditional efficacy of <i>C. orbiculatus</i>.