The Study Of Screening Enzyme Inhibitors By High Performance Capillary Electrophoresis

Among the listed drug, the proportion of drugs whose target is enzyme has beenup to fifth. And at present,the main disease which has a threat to human life andhealth has a significant relationship with the enzyme disordered, such as cancer,diabetes and so on. So the research of inhibitors on enzyme is important for R&D fornew drug research. Today the commonly methods for screening enzyme inhibitors arespectrophometric, microtiter plate and so on. But spectrophometric can not avoid theinfluence of complex matrix, and the cost of microtiter plate is so high. While theHPCE has a series of advantages, for example, efficient, fast and less reagentconsumption. This paper aims to research the enzyme inhibitors by CE, which isintended to develop into a simple, reliable high-throughput screening platform forR&D for innovative drug research.On chapter one, a new on-capillary method was established, a micro-immobilizedcapillary enzyme reactor whose ionic-bonding-agents was HDB for screeningtyrosinase inhibitors was created. In the experiment with L-tyrosine as a substrate,the stability of micro-immobilized capillary enzyme reactor was studied and only10times, reaction time was optimized and2min, the Michaelis-Menten constant oftyrosinase and the inhibition constant of kojic acid as a positive sample wasdetermined, the result was0.5334mM and22.54μM respectively, at the same time,the type of inhibiton of kojic acid was confirmed as mix-inhibition, so the model wasfeasible.18metal complexes and20crude extracts of traditional Chinese herbs wereused to screening the tyrosinase inhibitors while Rhizoma Anemarrhenae and RadixAngelicae Pubescentis have an inhibitive effect on tyrosinase, the IRE%was29.51%�'�47.34%respectively.On chapter two, a pre-capillary method that DPP-IV inhibitors were screened byHPCE was established for the first time. With Gly-Pro-PNA as the substrate, theseparation conditions of substrate and product, the reaction conditions of substrate andDPP-IV were optimized, the optimum conditions were established: the separationcondition: run buffer-10mM NaH2PO4+10mM SDS pH7.5, tempreture37℃, runvoltage30Kv, the length of caplliary was40cm; the reaction condition:85μL10mM NaH2PO4pH7.5+10μLsubstrate solution+5μL enzyme solution，the reaction time-40min, and detection wavelength-214nm and405nm. The Michaelis-Menten constantof DPP-IV and the inhibition constant of sitagliptin as a positive sample wasdetermined as0.3708mM and1.2169mM respectively. The type of inhibiton ofsitagliptin was confirmed as mix-inhibiton, so the model was feasible. The DPP-IVinhibitors activity of50compounds and16crude extracts of nature products wereassayed, while the IRE%of TN-LGQ-94#crude extract was63.29%and the IRE%of4compounds which had the inhibitive effect on DPP-IV was56.98%、65.32%、48.61%、60.58%respectively.