A fast and accurate method for the identification of peroxidase inhibitors from Radix Salvia Miltiorrhizae by on-flow biochemical assay coupled with LC/Q-TOF-MS: comparison with ultrafiltration-based affinity selection.

Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates. Graphical abstract Compared with a classical method of ultrafiltration LC-MS, the newly developed method of on-flow bioassay shows advantages in saving time, avoiding false positives and improving the accuracy.