The Study On The Protective Effect Of Scutellrein Against Brain Ischemia And Synthesis Metabolic Stability Scutellrein Derivatives

This work was supported by National Nature Science Foundation Of China "Study based on in vivo metabolism of scutellarin aglycone structural modification, structure-activity relationship and bioavailability"(No.81001382).This dissertation was divided into four major chapters.In Chapter1, literature reviewA detail review was given about the latest research progress of Erigeron breviscapus (vant) Hand Mass including resource chemistry, pharmacological effects and the problems and solutions of scutellarin clinical use were also summarized, which provided a new approach to improve its solubility and bioavailability for the further development.In Chapter2, the vivo study of scutellarein was performed, and the results were summarized in two section.Section1:Identification of scutellarein metabolites in rat using ultraperformance liquid chromatography/quadrupole-time-of-fligh mass spectrometryScutellarin, extracted from the dried plant of Erigeron breviscapus, has protective effects against neuronal injury. Scutellarein, which is the main metabolite of scutellarin in vivo, has better protective effects. However, there were few studies on the metabolic profile of scutellarein in vivo. In this study, a method based on ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was applied to identify the scutellarein and its metabolites in rat plasma, urine and feces after oral administration of scutellarein. Using MSE and mass defect filter techniques, scutellarein (M1), Scutellarein-5-O-β-D-glucuronide (M2) Scutellarein-6-O-β-D-glucuronide (M3), scutellarin (M4), diglucuronide conjugate of scutellarein (M5), glucuronide conjugate of apigenin (M6),4’-O-sulfate scutellarein (M7), methylated conjugate of scutellarein (M8), methylated and glucuronide conjugate of scutellarein (M9), and acetylated conjugate of scutellarein (M10) in rat urine were detected; M1, M2, M4, and M10in rat plasma were detected; and only the M10was found in feces. Our studies indicated that scutellarein can be metabolited as the forms of glucronidation, loss of hydroxyl group, glucronidation, sulfation, methylation and acetylation in vivo after oral administration.Section2:Phamacokinetics and tissues distribution of scutellarein in normal and ischemia model ratsTo develop a UPLC-MS method for determining scutellarein in rat plasma and tissues and to study the pharmacokinetics and distribution character of scutellarein in normal rats and ischemia model rats. The solution of scutellarein was administered to rats (34mg·kg-1) by oral gavage and caudal vein injection, respectively. In plasma, scutellarein (1.05-1.05×104ng·mL-1) and the metabolite scutellarin (2.38-2.38×103ng·mL-1) presented a fine linear relationship (r2=0.999). The bioavailability of scutellarein was2.57%in the normal rats and3.22%in the ischemia model rats, indicating that ischemia model promoted the scutellarein use. In normal rats, the concentration of scutellarein is the most highest in brain tissue than other tissues, and the overall trend was Cbrai>Ckideny>Cspleen>Cheart>Clung>Cliver.In Chapter3, neuroprotective effects and underlying mechanisms of scutellarin and scutellarein were investigated and the results were summarized in four sections.Section1:Investigation on the interactions of scutellarin and scutellarein with bovine serum albumin using spectroscopic and molecular docking techniques.Scutellarin, extracted from the dried plant of Erigeron breviscapus, has been reported to protect the neural injury induced by ischemia. Scutellarein, which is the main metabolite of Scu in vivo, has better neuroprotective than Scu. In this paper, the binding abilities of Scu and Scue with bovine serum albumin (BSA) were investigated using equilibrium dialysis, high performance liquid chromatography (HPLC), fluorescence spectroscopy, competitive site marker and molecular docking. The results showed that the average protein binding ratios of Scu and Scue with BSA were (79.85±1.83)%and (85.49±1.21)%respectively. Under simulated physiological conditions, the fluorescence data indicated that the binding procedure of Scu and Scue with BSA mainly occurred through a static mechanism. The thermodynamic parameters indicated that the interaction of Scu-BSA and Scue-BSA were driven mainly by van der Waals forces and hydrogen bonds and it was easier for Scue to bind BSA than Scu, indicating that the glycosylation decreased the binding affinity. Site competitive marker experiments showed that the binding site of Scu and Scue mainly located within sub-domain IIA of BSA. Furthermore, molecular docking indicated that one BSA can bind three Scue, while one BSA can only carry two Scu. All of these results clearly indicated the interactions of Scu and Scue with BSA, which will lay the foundation for further research to determine the pharmacology and pharmacodynamics of Scu and Scue for treating ischemic cerebrovascular disease.Section2:Studies on the protective effects of scutellarein against neuronal injury by ischemia through the analysis of endogenous amino acids using HILIC-MS/MS and evaluation of Ca2+concentration together with Ca2+-ATPase activityScutellarin, extracted from the dried plant of Erigeron breviscapus, has been reported to protect the neural injury against excitotoxicity induced by ischemia. However, there are few studies on the protective effect of scutellarein, which is the main metabolite of scutellarin in vivo. Thus, the aim of this study was to investigate the protective effects of scutellarin and scutellarein against neuronal injury through the analysis of endogenous amino acids using HILIC-MS/MS, and evaluation of Ca2+concentration together with Ca2+-ATPase activity. With the administration of scutellarein and scutellarin respectively, the changes of amino acids contents, intracellular Ca2+concentration and Ca2+-ATPase activity were then used to compare their neuroprotective effects on cerebral ischemia/reperfusion in rats by bilateral common carotid artery occlusion model (BCCAO). The results showed that scutellarein had better protective effect on cerebral ischemia/reperfusion than scutellarin, which suggested that scutellarein could be a promising potent agent for the therapy of ischemic cerebrovascular disease.Section3:Neuroprotective effects of scutellarin and scutellarein on repeatedly cerebral ischemia reperfusion in rats.Scutellarin had protective effects against neuronal injury, however, there are few studies on the protective effect of scutellarein, which is the main metabolite of scutellarin in vivo. This study investigated whether the neural injury by ischemia/reperfusion would be influenced by different doses of scutellarin and scutellarein. Male Wistar rats were orally administered with scutellarin and scutellarein at the doses of0.09,0.17,0.35,0.70,1.40mmol-kg-1, respectively, then after six consecutive days, they were subjected to global ischemia by occlusion of the bilateral common carotid arteries (BCCAO). After reperfusion for about21h, neurological and histological examinations were performed. The present results showed that scutellarein attenuated neuronal cell damage, reduced cerebral water content, regulated the expression of glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), y-aminobutyric acid (GABA) and taurine (Tau), and improved the Ca2+-ATPase and Na+,K+-ATPase activity. Meanwhile, significant difference was found among various doses of scutellarin and scutellarein. Our studies indicated that scutellarin and scutellarein could improve neuronal injury, and scutellarein had better protective effect than scutellarin in rat cerebral ischemia.Section4:Metabolomics study on the neuroprotective effects of scutellarin and scutellarein against ischemia stroke using UPLC-Q-TOF-MS coupled with pattern recognition approach.Scutellarin, extracted from the dried plant of Erigeron breviscapus, has been clinically used to treat acute cerebral infarction and paralysis in China since1984. Scutellarein, which is the main metabolite of scutellarin in vivo, has a better neuroprotective activity than scutellarin. In this paper, metabolic feature profiling and metabolite network of scutellarin and scutellarein against neural injury were investigated comprehensively through the analysis of the urine/plasma/hippocampus tissue samples by ultra-high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). Total23endogenous metabolites (9in urine,8in hippocampus tissue and6in plasma) contributing to separation of model group and control group, up-regulated or down-regulated (P<0.05or0.01) were tentatively identified, and11metabolic pathways in urine,14metabolic pathways in hippocampus tissue and3metabolic pathways in plasma were found through the pathway analysis. These endogenous metabolites were mainly involved in sphingolipid metabolism, lysine biosynthesis together with alanine, aspartate and glutamate metabolism. The metabolic deviations could be regulated closer to control-like level after scutellarin and scutellarein intervention. Moreover, scutellarein had better protective effect against ischemia injury than scutellarin according to the relative distance in PLS-DA score plots and the administration of scutellarin and scutellarein could provide protective effects through regulating the perturbed pathways.In the four Chapter, structural modificationScutellarein was taken as a lead compound and the synthesis routes on the structural modification of the eighth hydrogen atom on the A ring was designed. And the sulfonation and hexacyclic scutellarein derivatives were synthesized. The vitro antioxidant activity of scutellarein derivatives were evaluated by DPPH radical-scavenging activity assays and testing their reducing capacity on Fe3+.