| Large-leaf yellow tea is produced in Huoshan County,Lu 'an City,Anhui Province.It is a kind of yellow tea roasted at high temperature.The process of "roasting under over-fired " is the most special and important process of large-leaf yellow tea,and this process not only have given the large-leaf yellow tea unique aroma and taste,but also enhanced the hypoglycemic activity.Studies have shown that large-leaf yellow tea could significantly reduce blood sugar levels in mice,and the inhibitory against ?-glucosidase on tea samples after roasting was significantly improved than before,but the key compounds against ?-glucosidase have not been reported.Metabonomics analysis of the processing samples of large-leaf yellow tea showed that: in the roasting of large-leaf yellow tea generated a lot of new compounds polymerized by catechins and theaine,which might play an important role in hypoglycemic activity.Therefore,this paper further to study the key compound and it's formative mechanism against ?-glucosidase in the large-leaf yellow tea.In this study,solvent extraction,separation by liquid chromatography,analysis technology were used to study the large-leaf yellow tea and five fractions.The inhibitory rate of carbohydrate hydrolase activity was used to further trace the key hypoglycemic active compounds of large-leaf yellow tea,then spectroscopic analysis was used to analyze the structure of the key compounds and in vitro simulation experiment was used to further explore the pathway and mechanism of its formation.The main content and results of this study are as followed:(1)The analysis results of large-leaf yellow tea extractive by LC-Q-TOF-MS showed that the main micromolecule compounds(50?1000 m/z)in the tea were catechins such as EGCG(Epigallocatechin gallate),GCG(Gallocatechin gallate),GC(gallocatechin),EC(Epicatechin)and ECG(Epicatechin gallate),phenolic acids(such as gallic acid and quinic acid)and flavonoid glycosides(such as quercetin 3-glucoside and quercetin 3-O-locinoside).At the same time,there are some polymers formatted during the roasting,such as N-ethyl-2-pyrrolidone substituted flavane-3-ols(EPSFs).(2)The experimental results of inhibitory against ?-glucosidase and ?-amylase in vitro showed that the ethyl acetate fraction of large-leaf yellow tea had best inhibition ability after being extracted with different polarity organic solvents.Then the fluorescence quenching detection confirmed that the ethyl acetate fraction can cause the fluorescence quenching to?-glycosidase,and the protein(?-glycosidase)interactions and separation experiment also showed that the compounds of ethyl acetate fraction can inhibit ?-glycosidase by binding or collision,the compounds such as EGCG,GCG and GC can bind with ?-glycosidase.(3)In order to quickly screen the key enzyme-inhibiting compounds in the ethyl acetate layer,LC-Q-TOF-MS/MS was used to identify the main 31 compounds,the liquid phase preparation was used to isolate the 31 components,and the ?-glucosidase activity inhibition was detected respectively.The results showed that in addition to the eight catechins and alkaloids that have been reported,several N-ethyl-2-pyrrolidone substituted flavane-3-ols and quercetin-3-O-glucoside showed high inhibitory with low concentration in large-leaf yellow tea than other compounds.(4)To study the formative mechanism of EPSFs,different ratio of EGCG and L-theanine was roasted at 150? for different time,respectively.It was found that the mixture ratio of1:20 of EGCG and L-theanine and the roasting time of 120 min caused to the highest productivity of EPS-EGCG/GCG.Under these conditions,the ?-glucosidase activity inhibition rate of samples(the ratio of EGCG and L-theanine is 20:1)before and after reaction and EPS-EGCG was detected.The inhibitory of EPS-EGCG is higher than EGCG,so we speculate that the EPSFs have a big impact on the promotion of inhibitory against ?-glucosidase of large-leaf yellow tea after roasting. |
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