Establishment And Application Of The Chromatography Models Of GQDs-P2Y12 Receptor

Bioactive components screened from traditional Chinese medicine(TCMs)provide a great source for new drugs development.TCMs for dissipating blood stasis drug have special advantages for treating thrombotic diseases.The aggregation of platelet by activating P2Y12 receptor is an important process of thrombotic diseases,Therefore,P2Y12R has been the most ideal target of anti-thrombotic drugs.This paper aimed to establish a P2Y12R chromatography system for the quick screening,separation and identification of active components which could react with platelet P2Y12 receptor from dissipating blood stasis TCMs.This paper mainly falls into two parts:1.The GQDs was first prepared by Photo-Sono with H2O2 in the advanced oxidation process(AOP);using EDC/NHS as coupling agent to bind BSA to GQDs/silica.The stationary phase was characterized by scanning electron microscopy(SEM)and surface analyzing technology.Then the chromatographic behavior was investigated.The results showed that this novel method could bind BSA to GQDs/SiO2 and maintain the activity of the protein.Furthermore,the interactions between ligands and proteins were analyzed by the direct-injection technique.The binding constants of the interaction between BSA and gallic acid,protocatechualdehyde,ferulic acid,vanillic acid,protocatechuic acid and caffeic acid were determined.The results showed that there was one kind of binding sites for the six drugs and BSA.The study of this part proved that BSA chromatography model can be used to study the interaticon between drug molecules and protein,which provided a methodology for the high throughput analysis of online protein and drug interactions.This system would be applied in the quick screening of active components in the complex system.2.Using platelet P2Y12 receptor as a target protein,we attempted to immobilized P2Y12 receptor on the surface of functional GQDs/SiO2 uniformly and covalently with the mode of moderate chemical coupled reaction,using EDCI/NHS as coupling agent.The stationary phase would be characterized with the bond quantity of receptor,bioactivity and retention mechanism.This P2Y12 receptor chromatography models were applied to screen DBZ,IDHP and active ingredients from Salvia miltiorrhiza and Dan Hong injection.With the online-coupling technique of HPLC-Q-TOF-MS/MS,this system was applied in the quick screening,separation and identification of active components and the results,supplemented with platelet aggregation tests.The results shown that elinogrel,ticagrelor,ADP and ATP can be separated from the P2Y12 receptor chromatography models.And we found 3 active components from Salvia miltiorrhiza(ethanol extracted),including dihydrotanshinone I,Tanshinone ?B,etc;10 active components from Salvia miltiorrhiza(water extracted),including danshensu,salvianolic acid H,etc;7 active components from Dan Hong injection,including protocatechualdehyde,chlorogenic acid,etc.Besides,we found that DBZ and IDHP had interaction with P2Y12 receptor.These results suggest that P2Y12 receptor chromatography models based on GQDs/SiO2 can be applied to screening of components which have anti-platelet activity and applied in the quick screening of active components in the complex system.