Rapid Screening of Active Components with Thrombin Inhibitory Activity from Peach Kernel Based on Affinity Ultrafiltration coupled with UPLC-Q-TOF-MS.

INTRODUCTION: Peach kernel (PK), one of the medicinal and edible plants, has been widely used in the treatment of clinical thrombotic diseases and exhibited great therapeutic effects. However, the effective substances and targets are still obscure. METHODS: A method consisting of affinity ultrafiltration (AUF) coupled with ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectroscopy (UPLC-Q-TOF-MS) was established to rapidly screen active components that inhibit thrombin, a key mediator in coagulation cascade. The binding energy and sites were analyzed by molecular docking to evaluate the binding capacity of thrombin-ligand complexes, and the thrombin inhibitory activity of screened ligands was further validated via in vitro and in vivo tests. RESULTS: two potential thrombin ligands (L-arginine and cytidine) were screened by AUF-HPLC and identified by UPLC-Q-TOF-MS. The ligands with anti-thrombin structure exhibited great affinity with thrombin. The anticoagulant bioactivity of ligands was validated by a significant reduction in thrombosis in zebrafish tails and the potential mechanism could be related to direct inhibition of thrombin. CONCLUSION: The systematic screening of anti-thrombin active components in PK based on AUF-UPLC-Q-TOF-MS is a feasible and effective method, providing valuable information for the future development of direct thrombin inhibitors.

Thrombin-targeted screening of anticoagulant active components from Polygonum amplexicaule D. Don var. sinense Forb by affinity ultrafiltration coupled with UPLC-Q-TOF-MS.

INTRODUCTION: Polygonum 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. OBJECTIVES: This 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). METHODS: Different 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. RESULTS: Eleven potential thrombin ligands from PAF were screened by the AUF-UPLC-Q-TOF-MS method, and two compounds (butyl gallate and ß-sitosterol) with significant anticoagulant activity were discovered via in vitro and in vivo activity testing. CONCLUSION: A 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.

Screening of anti-thrombin active components from Ligusticum chuanxiong by affinity-ultrafiltration coupled with HPLC-Q-Orbitrap-MSn.

INTRODUCTION: Ligusticum chuanxiong ('chuanxiong') is a traditional Chinese medicine for promoting blood circulation and removing blood stasis, which is often used to treat thrombotic diseases. However, its potential anticoagulant active ingredients have been unexplored. OBJECTIVES: The study aims to establish an affinity ultrafiltration mass spectrometry (AUF-MS) method for rapid screening of anti-thrombin active components of chuanxiong and to verify it in vitro. METHOD: In this study, the chemical constituents of different parts of chuanxiong were determined. A method for rapid screening of anticoagulant active ingredients by AUF-MS was established using thrombin as an affinity receptor target. Subsequently, the anticoagulant effect of such ligands was verified by in vitro anticoagulation experiments such as chromogenic substrate method and in vitro coagulation assay. Then the possible interaction mechanism between these ligands and thrombin was further studied by molecular docking. RESULTS: Twenty-one components were detected from different parts of chuanxiong. And three potential anti-thrombin active components were screened: ferulic acid, chlorogenic acid, isochlorogenic acid A by AUF coupled with high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (HPLC-Q-Orbitrap-MSn ). The in vitro activity experiments and molecular docking revealed that these potential ligands exhibited strong binding ability and inhibitory activities on thrombin. CONCLUSION: The present study revealed that chuanxiong is a traditional Chinese medicine with excellent anticoagulation effects. Meanwhile, the integrated strategy based on AUF-MS, in vitro experiments and molecular docking also provided a powerful tool for further exploration of active ingredients responsible for the anticoagulant activity in chuanxiong.

Screening of anticoagulant active components in Xuefu Zhuyu decoction via affinity-ultrafiltration coupled with mass spectrometry.

This study aimed to establish an effective affinity-ultrafiltration coupled with mass spectrometry (AUF-MS) method to rapidly screen the active components of antithrombin in Xuefu Zhuyu decoction (XFZYD). The HPLC fingerprint of different batches of XFZYD was established to verify the stability of the compound ingredients. AUF-MS was established for the rapid screening of the active ingredients in XFZYD. Eight active ingredients were screened using AUF-MS: oxypaeoniflorin, chlorogenic acid, amygdalin, isoliquiritin, neohesperidin, hesperidin, glycyrrhizic acid and chikusetsu saponin IVa. Simultaneously, the anticoagulant effect of the active ingredients was verified by performing in vivo and in vitro experiments. The results of this study indicate that AUF-MS can be used for high-throughput screening of active components of traditional Chinese medicine compounds, thus providing a basis for the discovery of thrombin inhibitors and promoting the research progress of antithrombotic drugs. The compounds screened from XFZYD showed an inhibitory effect against thrombin and a certain antithrombotic effect.