Screening Target Enzyme Inhibitors Of Traditional Chinese Medicine By AS/UF-LC-MS

Enzyme inhibitors were used to treat human diseases caused by the abnormal expression of enzymes in medicine. Traditional Chinese medicine(TCM) resources are rich in our country. It is great significance to the discovery of original new drug lead compounds and creation of new drug with independent intellectual property by screening low toxicity, high efficiency enzyme inhibitors from the TCM.The traditional isolation and screening methods of TCM active ingredients are complex, time-consuming and poor selectivity. It is seriously restrict the fast discovery of TCM active constituents. AS/UF-LC-MS has many advantages, such as high sensitivity, rapid analysis, simultaneous activity selection and structure identification, which is mainly used for screening of the active ingredients in the combinatorial chemistry library, however, there are few applications in the selection of active constituents of TCM. The research has developed the method of screening target enzyme inhibitors from TCM by AS/UF-LC-MS. It provided the new ideas and methods for the rapid selection and identification of target enzyme inhibitors in the complex system of TCM.1. Screening acetylcholinesterase inhibitors in TCM by AS/UF-LC-MS.The experiment adopted the electrophorus electrics acetylcholinesterase as target enzyme, AChI for the substrate, DTNB as the chromogenic agent, SDS as the reaction termination agent. the acetylcholinesterase(AChE) inhibitory activity of the extracts of 49 kinds of TCM were investigated by microplate reader based on the Ellman’s method, the inhibitory rates of the 80% ethanol extracts of Cortex Phellodendri, Rhizoma Corydalis, ethyl acetate extract of Salvia miltiorrhiza and 80% ethanol extract of Coptis chinensis were the highest, and their half inhibitory concentration values were measured.The research established the AChE AS/UF-LC-MS system by optimizing the incubation conditions and chromatographic conditions. Five compounds were screened from the 80% ethanol extract of Coptis chinensis by the system.The five compounds in Coptis chinensis were separated and prepared by half the preparation of liquid phase, and identified by MS and 1H NMR. They were columbamine, jateorhizine, coptisine, palmatine and berberine. Half inhibitory concentrations were determined by microplate reader, IC50 values were 25.09, 37.86, 20.64, 13.90 and 48.51 mg L-1, respectively.2. Screening tyrosinase inhibitors in TCMThe experiment adopted tyrosinase of mushroom as the target enzyme, L-DOPA as the substrate, the tyrosinase inhibitory activities of extracts of 21 kinds of TCM were investigated by microplate reader, the inhibitory rates of the 80% ethanol extract of Moutan Cortex, Rhizoma Corydalis, ethyl acetate extract of Glycyrrhiza, Sopjora flavesens and Cortex Mori were the highest, and their half inhibitory concentration values are 746.20, 62.18, 63.82 and 210.17 mg L-1, respectively.The radical scavenging capacities of TCM extracts had an antagonism effect on tyrosinase activity, the radical scavenging capacities of four TCM extracts were investigated in the study, and the IC50 values are 45.55, 298.87, 1096.63 and 992.27 mg L-1, respectively.