Screening thrombin inhibitors from Yangxinshi tablets by online capillary electrophoresis‐based immobilized enzyme microreactor and molecular docking

Abstract Introduction Yangxinshi tablet (YXST) is a effective traditional Chinese medicine in treating cardiovascular diseases such as heart failure and myocardial infarction. Objectives This study aims to develop a method for screening thrombin inhibitors from YXST using an online immobilized enzyme microreactor (IMER) based on capillary electrophoresis (CE). Materials and methods Thrombin (THR) was immobilized on the capillary's inner wall using polydopamine (PDA). The chromogenic substrate S‐2238 was employed to assess thrombin (THR) activity and kinetic parameters. The stability and repeatability of the constructed thrombin‐immobilized enzyme microreactor (THR‐IMER) were evaluated over 40 runs, maintaining 85% of initial activity. The Michaelis–Menten constant ( K m ) for THR was determined to be 11.98 mM. The half‐maximal inhibitory concentration (IC 50 ) and inhibition constant ( K i ) for argatroban on THR were calculated. Ten compounds in YXST were screened for THR inhibitory potency using the THR‐IMER. Results Salvianolic acid B and caffeic acid were identified as potential THR inhibitors in YXST, with inhibition rates at 200 μg/mL of 55.06 ± 6.70% and 31.88 ± 4.79%, respectively, aligning with microplate reader assay results. Molecular docking analysis confirmed their interactions with key THR residues, verifying their inhibitory activity. Conclusion The CE‐based THR‐IMER method was successfully developed for screening thrombin inhibitors from YXST, offering a reliable approach for identifying potential therapeutic compounds.