| Objective:Fuzhuan tea has benefits on regulating blood lipid metabolism and improving obesity.More and more studies have found that Fuzhuan tea also has potential hypoglycemic activity.As a traditional tea,Fuzhuan tea has a large audience and potential medicinal value.However,the current research on the active compounds of Fuzhuan tea often only focused on the main components,such as polyphenols and polysaccharides,which was not sufficient.Moreover,the mechanism of hypoglycemic activity of Fuzhuan tea was not clear enough.Therefore,this study intended to comprehensively investigate the hypoglycemic active components and mechanism of the hypoglycemic activity in Fuzhuan tea,following the holistic view of traditional Chinese medicine.There are the objectives:To explore the hypoglycemic effect of extract/fractions from Fuzhuan Brick-Tea in streptozotocin-induced diabetic mice;To screen potential hypoglycemic active ingredients by the spectrum–effect relationship based on α-glucosidase inhibition;To explore the effect of drink-treatment on the composition of gut microbiota,and discover the potential target gut microbes and influenced physiological processes of Fuzhuan tea’s hypoglycemic activity;To discover the potential target metabolites,potential target genes and mechanisms of the hypoglycemic activity in Fuzhuan tea.Method:Experiment 1 Study on hypoglycemic activity and active fractions of Fuzhuan tea water extract1.The water extract of Fuzhuan tea was fractionated by D101 macroporous resins to obtain fraction A,fraction B,and fraction C by eluted with water,70% Et OH/H2 O and95% Et OH/H2 O,respectively.2.The experimental diabetic mice were established by high-fat diet feeding and STZ intraperitoneal injections.After diabetic model established,the mice were intragastrically administered with extract or fractions for 4 weeks.Food intake,water intake,body weight,oral glucose tolerance,serum glucose level,TC,LDL-C,HDL-C,SOD,and MDA were measured to determine hypoglycemic active fractions.3.By UPLC-QTOF-MS/MS,the main compounds in each fraction were qualitatively analyzed to the chemical information of Fuzhuan tea.Experiment 2 Study on potential hypoglycemic metabolites in Fuzhuan tea based on UPLC-QTOF-MS/MS combining global metabolomic and spectrum-effect relationship analyses1.Based on UPLC-QTOF-MS/MS,global metabolomic analysis was used to screen the differential variables between Fuzhuan tea and its unfermented raw materials.2.The α-glucosidase inhibition rate of Fuzhuan tea and unfermented raw materials was measured,and the compounds with high correlation with α-glucosidase inhibition were screened using spectrum-effect relationship analysis.3.Combined with the results of metabolomics and spectrum-effect relationship analysis,the potential active components of hypoglycemic were screened.Experiment 3 Study on the hypoglycemic activity mechanism of Fuzhuan tea based on 16 S r DNA sequencing analysis of gut microbiota1.Simulating the real drinking method,Fuzhuan tea drinking was used as treatment to STZ induced diabetic mice.2.By 16 S r DNA amplicon high-throughput sequencing technology,the gut microbiota ecology of the normal group,model group and treatment group were sequenced.3.Through a series of analyses of gut microbiota diversity,the gut microbiota composition of three groups were compared and analyzed to explore the potential target gut microbes for the hypoglycemic activity of Fuzhuan tea.Experiment 4 Study on the hypoglycemic activity mechanism of Fuzhuan tea based on serum global metabolomics and network pharmacology analyses1.The serum target biomarkers of STZ induced diabetic mice were screened by global metabolomic analysis.2.Based on the chemical information of Fuzhuan Tea,network pharmacology analysis was applied to predict the targets and pathways of hypoglycemic activity in Fuzhuan tea.3.The relevant action targets of screened target metabolites were predicted,and the interaction analysis with the network pharmacology results was carried out to uncover the potential action target genes and pathways.Result:Experiment 1 Study of hypoglycemic activity and active fractions of Fuzhuan tea water extract1.The results of animal experiments showed that the water extract and its three fractions of Fuzhuan tea could improve the symptoms of diabetic mice,reduce blood sugar levels,improve glucose tolerance,and regulate blood lipid metabolism to different extents.Among them,fraction C was confirmed as the active fraction which showed the most prominent hypoglycemic effect.2.There were 7,13 and 20 compounds identified and characterized in three fractions by using UPLC-QTOF-MS/MS technology,respectively.The main compounds in fraction A and fraction B were catechins and flavonoid glycosides.Fraction C contains a lot of lipid compounds,including fatty acids,Lyso-PCs and a class of new theophylline-fatty acid derivatives.3.Due to the considerable hypoglycemic activity of fraction C,these lipid compounds in fraction C might be the hypoglycemic active compounds of Fuzhuan tea.Experiment 2 Study on potential hypoglycemic metabolites in Fuzhuan tea based on UPLC-QTOF-MS/MS combining global metabolomic and spectrum-effect relationship analyses1.Based on UPLC-QTOF-MS/MS,PCA and PLS-DA had been used to screen 730 variables with VIP > 2.0 from Fuzhuan tea and unfermented raw materials.2.By spectrum-effect relationship analysis,the top 200 and bottom 200 peaks were screened by correlation coefficients,which were considered as potential variables closely related to hypoglycemic activity.3.Combined the results of metabolomics and spectrum-effect relationship analysis,10 potential hypoglycemic active components were screened and characterized,including long-chain fatty acids,phospholipids,triterpenes and theophylline-fatty acid derivatives.Experiment 3 Study on the hypoglycemic activity mechanism of Fuzhuan tea based on 16 S r DNA sequencing analysis of gut microbiota1.The results of Alpha diversity analysis,Chao1 index and Observed species index showed that tea-drinking intervention effectively improved the diversity and uniformity of species distribution in the gut microbiota of mice.The results of PCA,PCo A and UPGMA in beta diversity analysis showed that the composition of intestinal flora in model mice had changed.And tea-drinking intervention could improve this trend.2.Through a series of analyses of gut microbiota diversity,the results showed that the ratio of Firmicutes to Bacteroidetes decreased significantly in the model mice,which was reversed after tea-drinking intervention.In addition,gut microbiota diversity was significantly improved after tea-drinking intervention,which showed an improvement of Euryarchaeota,Cyanobacteria,Enterococcaceae and Erysipelatoclostridium.Experiment 4 Study on the hypoglycemic activity mechanism of Fuzhuan tea based on serum global metabolomics and network pharmacology analyses1.By PCA and PLS-DA models in global metabolomics analysis,31 differential metabolites with VIP > 1.5 were screened.After relative quantitative analysis,metabolite which showed a significant difference between the model group and the normal group and caused a correction trend after administration in treatment group,was considered as activity target metabolites.Finally,11 target metabolites were selected,including Lyso-PCs,fatty acids,acylcarnitines,sphingomyelins,sphingosines,and sterols.2.According to the chemical information from experiment 1,919 potential corresponding targets were retrieved,and 243 targets were retained after deduplicating.Searching with "type II diabetes mellitus" as the "Keywords",selecting targets with "correlation > 20",990 targets were obtained.Through PPI network analysis,24 key targets were screened,which included TNF,AKT1,EGFR,VEGFA,PPARG,PTGS2,HIF1 A,MAPK1,MTOR,IL2,MAPK14,PPARA,STAT1,MMP9,KDR,NR3C1,AR,MET,CYP19A1,APP,PIK3 CA,PTPN1,PTPN11 and AKR1B1.3.Based on the selected target metabolites,relevant targets were searched.There were 14 common target genes with type II diabetes targets,and PPI analysis was performed on these 14 target genes.Metabolite-disease core target PPI analysis and component-disease core target PPI analysis were integrated,and 6 common target genes were screened,which included AKT1,VEGFA,PTGS2,MAPK14,PPARA and KDR.Both target metabolites and target genes point to physiological activities such as the response to vascular endothelial growth factor,the metabolism of fatty acids,and the regulation of inflammatory response.Combined with the results of experiment 3,it was speculated that the hypoglycemic activity of Fuzhuan tea involved two important mechanisms of improving fatty acid metabolism and inflammatory response.Conclusion:1.Through the pharmacological experiment of STZ induced diabetic mice,the fraction C was determined to be the hypoglycemic active fraction.The main compounds in fraction C were identified and characterized,which included phospholipids,fatty acids and theophylline-fatty acid derivatives.2.Through a comprehensive analysis strategy combining global metabolomic analysis and spectrum-effect relationship analysis,10 key active and positively correlated compounds were screened from Fuzhuan tea.These compounds were the key potential hypoglycemic active ingredients,including octadecanoic acids,phospholipids and theophylline-fatty acid derivatives.3.Through a series of analyses of gut microbiota diversity in many groups,the results showed that the gut microbiota of diabetic mice could be increased after treatment with Fuzhuan tea.And the dysregulated bacteria species were corrected.Then,the related physiological processes such as fatty acid metabolism,inflammatory response and intestinal barrier function were improved,which finally contributed to regulate glucose metabolism and blood glucose level in diabetes mellitus.4.Combined with serum global metabolomic and network pharmacology analysis,it was speculated that Fuzhuan tea could regulate expression of AKT1,VEGFA,PTGS2,MAPK14,PPARA and KDR by affecting metabolites such as phosphatidylcholines,fatty acids,acylcarnitines,sphingomyelins,dihydrosphingosines,and sterols.These results were pointed to physiological activities related to vascular endothelial growth factor response,fatty acid metabolism and inflammatory response.Combined with the analysis of gut microbiota,it was speculated that the hypoglycemic activity of Fuzhuan tea involved two important mechanisms,including fatty acid metabolism and inflammatory response. |
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