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

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 (IC₅₀) 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.