UPLC-Q-TOF-MS.

ABSTRACT

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.