Preliminary Study On The Chemical Composition In Vivo And Vitro Of Bushen Huoxue Prescription And The Mechanism Of Treating DR

Diabetic retinopathy(DR)is one of the most serious and common complications of diabetes.It can cause the abnormality of capillary vessel of the tissues and organs in the body.A lot of modern pharmacological studies shown that the main ingredients of Bushen Huoxue prescription contains Rehmannia glutinosa Libosch.,Salvia miltiorrhiza Bge.Panax ginseng C.A.Mey.and Pueraria thomsonii Benth.,which is mainly used in the treatment of DR.Previously,the pharmacological material basis and mechanism of Bushen Huoxue prescription have been studied by our research group.few studies on the chemical composition of Bushen Huoxue prescription in vitro and vivo and the mechanism of treating DR was described systematically.In this paper,an ultra-high performance liquid chromatography couple with hybrid quadrupole-orbitrap mass spectromertry method was developed and used to identify the chemical constituents from Bushen Huoxue prescription.we research the pharmacological material of the compounds as the fallowing aspects:1.Study on the chemical composition of Bushen Huoxue prescription in vitro and vivo1)studies on the chemical composition of Bushen Huoxue prescription in vitro and vivo.An ultra-high performance liquid chromatography couple with hybrid quadrupole-orbitrap mass spectromertry method was developed and used to rapidly recognize and identify the chemical constituents from Bushen Huoxue prescription.The constituents from Bushen Huoxue prescription were rapidly recognized and identified by HRMS in the negative ion mode using both full scan and automatic triggering of the function of the two stage mass spectrometry scanning.A total of 86 compounds were identified by refering to mass spectrometric data and comparing with reference substances.Bushen Huoxue prescription mainly contains flavonoids,salvianolic,ginsenosides and a small quantity of iridoids.2)The preliminary study of plasma pharmacochemistry in rats.An UPLC-Q-Exactive method was employed inthe analysis of the constituents absorbed into blood after the oral administration of Bushen Huoxue prescription,After comparing the mass information of blank plasma and Bushen Huoxue prescription extract,a total of 28 prototype compounds of Bushen Huoxue prescription were observed in the plasma.2.Study on the mechanism of Bushen Huoxue prescription in protect DR1)Study on the mechanism of action based on cell molecular level.The animal model of DR was induced by a high-fat diet and intrperitoneal injection of low-dose streptozotocin(STZ).Expression of vascular endothelial growth factor(VEGF)and hypoxia-inducible factor(HIF-1?)in retina were shown by immunohistochemical staining and toluidine blue-eosin staining,and were quantitated by image analysis system.Compared with the normal control group,The content of VEGF and HIF-1?were increase obviously(P<0.01).Compared with the model group,The content of VEGF and HIF-1? were decrease obviously(P<0.05).The results showed that Bushen Huoxue prescription has protective effects on DR.Combined with previous studies,the mechanism of action was discussed.2)To observe the metabolic profile changes by Bushen Huoxue prescription in treatment of DR rats.On the basis of previous experiment,An ultra-high performance liquid chromatography couple with hybrid quadrupole-orbitrap mass spectromertry method was developed for the study on metabolic variations of urine from all groups.The principal component discriminant analysis(PCA-DA)and partial least-squares discriminant analysis(PLS-DA)were used for multivariate analysis.In urine,The 9 endogenous metabolites were identified as potential biomarkers,namely Phenylacetylglycine,3-Methyldioxyindole,Hippuric acid,p-Cresol sulfate,p-Cresol glucuronide,Phenyllactic acid,Cholic acid,5-L-Glutamyl-taurine.The results revealed that Bushen Huoxue prescription administration could provide satisfactory effect on DR through partially regulating the perturbed metabolic pathways including intestinal bacteria metabolism,lipid metabolism and tryptophan metabolism in diabetic rats.which could help us to elucidate the mechanism of Bushen Huoxue prescription in protect DR.The researches above mentioned lay a foundation for pharmacological material basis of Bushen Huoxue prescription,which also provide an experimental basis forfurther study of pharmacological mechanism in the prevention and treatment of DR.It provided a new methodological cue for studying the holistic efficacy of the other TCM formulae.