Fast screening of tyrosinase inhibitors from traditional Chinese medicinal plants by ligand fishing in combination with in situ fluorescent assay.

A simple and rapid method for screening of tyrosinase (TYR) inhibitors present in traditional Chinese medicines (TCMs) was developed by combining ligand fishing and the fluorescent enzymatic assay based on dopamine-functionalized carbon quantum dots (CQDs-Dopa). Ligands of the enzyme present in the TCM extractions were firstly adsorbed on the enzyme-modified magnetic beads, and then the beads were magnetically separated and subjected directly to the CQDs-Dopa-based fluorescent assay. Finally, compounds were desorbed from the "active" beads and identified with ultra-performance liquid chromatography-triple quadrupole mass spectrometry. A known natural TYR inhibitor quercetin was selected to assess the feasibility and quantification performance of this method, and good linearity in the range of 0.01-0.16 mM (R2 = 0.992) with a low detection limit of 0.004 mM was obtained. This method was then applied to screen TYR inhibitors present in Scutellaria baicalensis and Sophora flavescens. Six TYR inhibitors including baicalin (1), baicalein (2), wogonin (3), oroxylin A (4), kurarinone (5), and sophoraflavanone G (6) were found, among which 1-4 were firstly discovered in this work. This is the first report on the in situ assessment of the target compounds obtained by ligand fishing in the form of a mixture, which exhibited the combined advantages of specific extraction ability of ligand fishing and the high sensitivity of CQDs-based fluorescent assay, showing great potential for fast screening of enzyme inhibitors from TCMs.

Screening of lipase inhibitors from Scutellaria baicalensis extract using lipase immobilized on magnetic nanoparticles and study on the inhibitory mechanism.

Scutellaria baicalensis is a traditional Chinese medicinal plant possessing a wide variety of biological activities. In this work, lipase immobilized on magnetic nanoparticles (LMNPs) was used as solid phase extract absorbent for screening of lipase inhibitors from this plant. Three flavonoids were found to bind to LMNPs and were identified as baicalin, wogonin, and oroxylin A by liquid chromatography-mass spectrometry (HPLC-MS). Their IC50 values were determined to be 229.22 ± 12.67, 153.71 ± 9.21, and 56.07 ± 4.90 µM, respectively. Fluorescence spectroscopy and molecular docking were used to probe the interactions between these flavonoids and lipase. All the flavonoids quenched the fluorescence of lipase statically by forming new complexes, implying their affinities with the enzyme. The thermodynamic analysis suggested that van der Waals force and hydrogen bond were the main forces between wogonin and lipase, while hydrophobic force was the main force for the other two flavonoids. The results from a molecular docking study further revealed that all of them could insert into the pocket of lipase binding to a couple of amino acid residues.