IntroductionPolygonum amplexicaule D. Don var. sinense Forb (PAF), a medicinal plant, has the effect of promoting blood circulation and removing blood stasis. However, the active compounds and targets of its anticoagulant effect are still unclear.ObjectivesThis study aims to establish an effective reversely thrombin‐targeted screening method for anticoagulant active components in PAF by affinity ultrafiltration (AUF) coupled with ultrahigh‐performance liquid chromatography–quadrupole time‐of‐flight mass spectroscopy (UPLC‐Q‐TOF‐MS).MethodsDifferent polar parts of PAF were screened for potential thrombin ligands by AUF‐HPLC and identified by UPLC‐Q‐TOF‐MS. After studying the affinity between ligands and thrombin by molecular docking, the antithrombotic activity of ligands was detected in vivo by zebrafish thrombus model, and in vitro by chromogenic substrate method. The mechanism of such ligands on thrombin was further studied by coagulation factor assay.ResultsEleven potential thrombin ligands from PAF were screened by the AUF‐UPLC‐Q‐TOF‐MS method, and two compounds (butyl gallate and β‐sitosterol) with significant anticoagulant activity were discovered via in vitro and in vivo activity testing.ConclusionA method system based on AUF‐UPLC‐Q‐TOF‐MS, molecular docking and in vivo and in vitro experiments also provided a powerful tool for further exploration of anticoagulant active components in PAF.

中文翻译：

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凝血酶靶向筛选蓼茎抗凝活性成分亲和超滤结合 UPLC-Q-TOF-MS 法测定中华草

介绍抱茎蓼D. 唐 var.中华香茅（PAF），一种药用植物，具有活血化瘀的功效。但其抗凝活性成分及其作用靶点尚不清楚。 目的 本研究旨在通过亲和超滤（AUF）结合超高效液相色谱-四极杆时间建立有效的反向凝血酶靶向筛选PAF中抗凝活性成分的方法飞行质谱（UPLC-Q-TOF-MS）。方法通过AUF-HPLC筛选PAF的不同极性部分，寻找潜在的凝血酶配体，并通过UPLC-Q-TOF-MS进行鉴定。通过分子对接研究配体与凝血酶的亲和力，通过斑马鱼血栓模型体内检测配体的抗血栓活性，并通过显色底物法检测体外配体的抗血栓活性。结果通过AUF-UPLC-Q-TOF-MS方法从PAF中筛选出11种潜在的凝血酶配体，其中两种化合物（没食子酸丁酯和β-谷甾醇）具有显着的抗凝作用结论基于AUF-UPLC-Q-TOF-MS、分子对接以及体内外实验的方法体系也为进一步探索PAF抗凝活性成分提供了有力的工具。 。