Abstract

Objective Since screening compound active ingredients is more complex than screening single ingredients, we developed a one-step high-throughput screening method using Affinity Ultrafiltration-Mass Spectrometry (AUF-UPLC-Q-TOF-MS) to rapidly identify the active anti-thrombotic components in Xuefu Zhuyu Decoction. Method In this study, 50 g of Xuefu Zhuyu Decoction was subjected to reflux extraction with 90% ethanol, followed by concentration and sequential extraction using ethyl acetate, n-butanol and water. The n-butanol fraction was selected as the key research target. Using thrombin as the target, the fraction was incubated with active/inactivated enzyme at 37 °C for 40 min, and the complexes were separated via a 10 kDa ultrafiltration tube. Potential ligands were preliminarily screened by UPLC-Q-TOF-MS combined with blank control experiments. Subsequent in vitro and in vivo validation was performed using molecular docking, chromogenic substrate assay, three coagulation indicators (APTT/PT/TT) and a zebrafish thrombosis model. By omitting the tedious steps of repeated column chromatography and activity tracking, this method exhibits advantages such as minimal sample consumption, high screening efficiency, and reduced experimental cost and time. Conclusion AUF-UPLC-Q-TOF-MS identified seven components with binding rates exceeding 10% from 11 differential peaks, among which Rhoifolin, Ginsenoside Ro, and Licoricesaponin G2 were confirmed for the first time to exhibit significant thrombin inhibitory efficacy. Compared to the traditional “separation-purification-verification” approach, this method reduces sample consumption by 90% and cuts both time and cost by 80%. It provides a replicable template for rapid, eco-friendly, and cost-effective screening of active components in traditional Chinese medicine formulations, while also establishing a solid experimental founda