Study Of The Different Metabolites Of Different Tea Types And Their Water Extracts And Differential Metabolites Neuronal Cell Protective Effects And Mechanism

Fujian is a famous tea-producing province in China,mainly producing green tea,black tea,oolong tea and white tea.Tea is rich in specialized metabolites such as catechins,proanthocyanidins,flavonol glycosides,theanine(Thea),and caffeine.These specialized metabolites vary among different types of tea made from the same fresh leaves.It is meaningful for directional design of tea processing technology and the development of tea products beneficial to human health to reveal the different metabolites of different tea types,explore the influence of processing on the formation of tea quality and functional active substances.Alzheimer's disease(AD)is a serious neurodegenerative disease which has a painful course and a high cost of care,and brings misfortune to the entire family.As the average life expectancy of humans keeps increasing,the number of patients diagnosed with Alzheimer's disease also increases.Tea is important natural product with the potential neuronal cell protect activity and prevent and treat AD,but the effect still unclear.Clarifying the neuronal cell protect effect and mechanism of tea extracts and characteristic metabolites is benefit for further use of tea resources and provides a theoretical basis for the coordinated prevention and treatment of AD by multi-component natural products of tea.In order to clarify the regulation mechanism of the influence of different tea processes on the tea functional quality formation and the role of tea on the prevention and treatment of AD.Metabolomics approach was used for metabolite profiling of these tea types to understand their metabolic differences.Set PC-12 cells as study object,this study compare the neuronal cell protect effect and mechanisms of different tea types.The main findings were as follows:1 Study on Differential Metabolites among Different Tea Types Based on Targeted Metabolomics ApproachThe contents of main metabolites of green tea,oolong tea,black tea and white tea made from the same leaves were studied based on targeted metabolomics approach.The results showed that,compared with fresh leaves,the content of total catechins in four tea types was reduced which showed the highest retain in green tea,followed by oolong tea and black tea.For white tea,the ECG and EGCG contents decrease 5.19%and 25.35%respectively comprared with fresh leaves which closed to oolong tea,but others reduced 66.37%?74.04%which closed to black tea.The white tea showed different transform model compared with other tea types.Thea,glutamic acid,and aspartic acid were higher in green tea.Glutamine,phenylalanine and tryptophan were accumulated in oolong tea.Contents of most amino acids in black tea were relatively low compared with other tea types apart from leucine.Most of amino acids content in white tea was higher than other tea types except leucine and glutamine.Rutin and caffeine also present higher in white tea.2 Study on Differential Metabolitess among Different Tea Types Based on Non-targeted MetabolomicsThe contents of main metabolites of green tea,oolong tea,black tea and white tea made from the same leaves were studied based on non-targeted metabolomics approach.A total of 381 differential compounds were found,of which 38 were identified,including 8 flavanols,1 amino acid,6 proanthocyanidins,12 flavonol glycosides,2 hydrolysable tannins,5 phenolic acids and 4 theaflavins.Principal component analysis showed that green tea was closer to oolong tea and separate with black and white tea which formed a triangles.In the first principal component,from negative axis to positive is green tea,oolong tea,white tea and black tea in order.In the second principal component,from negative axis to positive is white tea,green tea,oolong tea,and black tea in order.In the first principal component,the catechins and theaflavins located in the negative and positive axis.In the second principal component,the flavone or flavonol glycosides and theaflavins located in the negative and positive axis.That means the flavone glycosides maybe the representative compounds in white tea.The green tea which went through fixation process have more catechins,methyl catechins,proanthocy anidins,thean,hydrolysable tannins and chlorogenic acid.That compounds showed lower in oolong tea but accumulated proanthocyanidins,coumaroylquinic acid,gallic acid and theaflavin.In black tea,coumaroyluinic acid,gallic acid and theaflavin highly accumulated because of formation process.In white tea,higher accumulated flavone or flavonol glycosides which followed by accumulation of ECG,EGCG,methyl catechins,theaflavin and gallic acid.3 Protective Effect of Different Tea Types Water Extracts on PC-12 Neuronal CellsSet up the amyloid-beta(A?)and oxidative damage injury models of PC-12 and interfered by water extracts of green tea,oolong tea,black tea and white tea.The safe dosages of water extracts of green,oolong,black and white tea for neuronal cells were 100 ?g/mL.Higher dosage(150?g/mL)of tea water extracts decreased neuronal cell viability,especially green tea,oolong tea and white tea,while black tea showed the lowest cytotoxic effect.Instead,black tea could enhance cell viability when used in the low concentration.In A? injury model,the cell viability of neuronal cells was significantly enhanced to over 95%after treatment with differences tea water extracts.In oxidative injury model(t-bhp and H2O2),different tea water extracts failed to protect the neuronal cells.On the contrary,whether low concentration of oxidizing agents was present,tea water extracts aggravated the cell injury.4 Fluorescence kinetics and isomerization effects of different tea water extracts against A? tanglesEstablished and applied of Thioflavin T(ThT)dynamic fluorescence analysis and Transmission Electron Microscopy(TEM)to investigate different tea water extract and Ap effect directly.The fluorescence continuously increased in the whole 48 h and reached over 3 × 103 RFU with A? alone,and the total fluorescence value was 1.0 × 106 RFU.ThT fluorescence analysis showed that all tea water extracts significantly inhibited A? fluorescence to below 300 RFU,with total fluorescence being 5.5 × 103 RFU TEM analysis showed that a largearea of thick fiber entanglement appeared after 48 h of culture with A? alone whereas the entanglement area,density,and thickness of Ap fibers were reduced after adding different tea extracts.A? morphology in green and white tea treatments amorphous scattered points were present however clear fiber tangles were still visible in the A? morphology treated with oolong tea and black tea.5 Protection effects and mechanisms of characteristic constituents of different tea types on PC-12 neuronal cellsThe different tea metabolites were used to clarify the function and principle of neuronal cell protection of tea extract.No cytotoxic effects on PC-12 neuronal cells were observed when the cell were treated with Epicatechin(EC)?theaflavin(TFa)?kaempferol-3-O-glucoside(K-Glu)?kaempferol-3-O-rutinoside(K-Rut)?quercetin-3-O-glucoside(Q-Glu)?quercetin-3-O-rutinoside(Q-Glu)?GABA and thea under 100 ?M.However,cell viability significantly decreased when treated with EGC(100?M),ECG(50 ?M and 100 ?M),EGCG(100 ?M)and TFb(100?M).In A? injury model,cell viability significantly enhanced after EC(50?M and 100 ?M)?EGC(50 ?M)?EGCG(10 ?M and 50 ?M)?TFa(50?M and 100 ?M)?TFb(10 ?M and 50 ?M)treatment.K-Glu,Q-Gluand Q-Rut also increased the cell viability but not significantly.GABA and theanine showed no neuronal protection effects.In oxidative injury model(t-bhp),only ECG could protect PC-12 cells when used in 50 ?M and 100?M.EC?EGC?EGCG?TFa?TFb?K-Glu?K-Rut?Q-Glu?Q-Rut?GABA and theanine all failed to protect PC-12 cells when applied in 100?M.On the contrary,EGC and EGCG aggravated cell injuries when low ROS existed.ThT fluorescence showed that the fluorescence value continuously increased in the whole 48 h which sharply increased in the first 10 h and reached over 6 × 10~3 RFUwith the total value reaching 1.7 × 10~6 RFU.EC?EGC?ECG?EGCG?TFa?TFb?K-Glu?K-Rut?Q-Gluand Q-Rut significantly inhibited A? fluorescence and continuously decreased in whole 48h but not GABA and theanine.The function of ECG?EGCG?TFa ? TFb were better which fluorescence decrease under 1.3 × 10~5 followed by ECG,Q-Rut,EC,K-Glu,K-Rut and Q-Glu.The TEM analysis showed that a largearea of thick fiber entanglement appeared after 48 h of culture with A? alone,and the entanglement area,density and thickness of Ap fibers decreased after adding catechins and theaflavins.The morphologies of A? under EC,EGC and TFa treatments were similar,and cavities of various sizes occurred within the tangles.The morphologies of A? under ECG?EGCG and TFb treatments were similar,and A? fibers changed markedly which became concentrated and smaller.The densities and areas of A? entanglement were slightly reduced after the addition of flavonol glycosides,especially Q-Glu.In contrary,the densities and areas of A? tangles under theanine and GABA treatments did not change compared with the control.Computer simulation had shown that EGC,ECG,EGCG,and TFa interact with important site Lys16 and Lys28 of A? monomer and A? pentamer.