Thrombin Immobilized on Magnetic Core‐Shell Mesoporous Silica Nanoparticles for Screening of the Enzyme Inhibitors From <i>Notopterygium Incisum</i>

Thrombin was immobilized on magnetic core-shell mesoporous silica nanoparticles (Fe₃O₄@nSiO₂@mSiO₂) for the first time and used to screen enzyme inhibitors from extracts of Notopterygium incisum. The immobilized thrombin was characterized by Fourier-transform infrared spectroscopy, transmission electronic microscopy, thermal gravimetric analysis, and vibrating sample magnetometry. The results demonstrated that the immobilized thrombin possessed good thermal stability and high-temperature resistance for feasible storage and reuse. Two ligands, notopterol (1) and isoimperatorin (2) were fished out from the extract of N. incisum using the immobilized thrombin. They inhibited thrombin with IC₅₀ values of 59.35 ± 1.420 and 107.8 ± 1.660 µM, respectively. This is the first report of thrombin-inhibitory activity associated with notopterol and isoimperatorin. Meanwhile, the inhibition mechanism of both compounds was investigated by enzyme kinetic analysis and molecular docking. The method used enables rapid screening for thrombin inhibitors from medicinal plants, providing an efficient tool for the discovery of anti-thrombotic active compounds.