| Large-leaf yellow tea(LYT)belong to the yellow tea in China with a long history.In the Ming Dynasty,there was a systematic record of its origin,manufacturing process,flavor and health benefits.The mature fresh tea leaves with stems are harvested mechanically in the summer or autumn,and then processed through six steps to get the final LYT product.The main processes are fixation,rolling,first drying,yellowing,second drying and roasting.The special sensory features of LYT mainly shaped in the unique yellowing and roasting peocesses,which is atttibute to the changes of chemical composition,and eventually lead to differences in health benefits.Previous studies have shown that LYT had a better hypoglycemic efficacy,and mice had a better tolerance to high dose of LYT infusion compared with other teas,the hypoglycemic mechanism related to suppress the early elevation of thioredoxin interaction protein in mouse liver.However,it is still unclear the formation mechanism of LYT sensory characteristics,the transformation of sensory components,and the potential hypoglycemic substances formed in the processing.Therefore,it is necessary to clarify the hypoglycemic compounds,and provide evidences to elucidating the excellent hypoglycemic effect of LYT.Based on the above-mentioned reasons,this study was systematicly investigated the changes of chemical composition during seven processing(including fresh leaves)of LYT by HPLC,LC-Q-TOF-MS,LC-QQQ-MS,metabolomics and other methods.We have explored the transformation mechanism of the marker compounds,and carried out the synthesis and quantification of parts of them.The roasting process,which caused the greatest changes in chemical composition during the process of LYT,produed the effects on the glucose and lipid metabolism in non-alcoholic fatty liver disease(NAFLD)mice induced by high-fat diet were also studied.α-glucosidase,a hypoglycemic target protein,was used to investigate the hypoglycemic effect and its mechanism in vitro and in vivo of the marker compounds produced in roasting process.The main results listed as below:(1)The chemical composition of tea leaves were not significantly changed among fixation,rolling,first drying,yellowing and second drying,while roasting was the most obvious process on chemical composition of tea leaves.Compared with second drying tea leaves,the main epicatechins were changed significantly,with more than 50%reduction,epimerized catechins,gallic acid and pyrogallic acid were multiplied.Chlorophyll,total free amino acids and water-soluble polysaccharides,flavonoids and other components were also decreased by thermal action.Meanwhile,Strecker aldehyde of L-theanine conjugated with catechins’ Aring produced twenty-two N-ethyl-2-pyrrolidone substituted flavan-3-ols during the high-temperature roasting process,which was proved by a model thermal reaction with epigallocatechin gallate(EGCG)and L-theanine.Further synthesis and quantitative analysis showed that 8-C N-ethyl-2-pyrrolidone-gallocatechin gallate was about 1.05 mg/g in LYT,while the total content of N-ethyl-2-pyrrolidone substituted galloylated catechins is about 7.5 mg/g in LYT.Therefore,roasting was the critical process of LYT processing,in which the chemical composition of LYT has undergone dramatically changes.(2)According to the intense changes of chemical composition before and after roasting of LYT,we evaluated the effects of roasting process on the glucose and lipid metabolism of NAFLD mice induced by high-fat diet(HFD).Both the second drying and roasting tea infusion could significantly inhibited the increase of body and liver weight;decreased fat deposition in perirenal,mesenteric and epididymal tissue.Serum levels of triglyceride(TG),total cholesterol(TC),lowdensity lipoprotein cholesterol(LDL-C)and fasting blood glucose(FBG)were reduced,while they were also improved glucose tolerance,hepatic steatosis and damage caused by long-term HFD intake.In addition,the expression of fatty acids synthesis and β-oxidation genes were reversed.After roasting,LYT had a more obvious advantage in regulating glucose and lipids metabolism.The biochemical parameters of LYT group tended to be the low-fat diet group,which was showed in body weight,liver weight,weight gain and abdominal fat(perirenal,mesenteric and epididymal adipose tissue).The normalization of serum levels of TC,LDL-C and FBG,glucose tolerance,liver glucose and lipids metabolism related genes(CPT1,PPARα,PEPCK and G6Pase)were significantly different from before roasting.These results suggested that the roasting process has improved the ability of LYT in regulating the homeostasis of glucose and lipids metabolism.(3)In order to reveal the potential hypoglycemic compounds and their possible mechanisms caused by roasting process in LYT,α-glucosidase was selected the hypoglycemic target protein,and the potential hypoglycemic marker compounds were evaluated in vitro and in vivo.The results showed that the infusion of roasted LYT had significant inhibition on α-glucosidase better than unroasted LYT.As the two main α-glucosidase inhibitors,gallocatechin gallate(GCG)exhibited a higher inhibitory effect on α-glucosidase than its corresponding epimer(EGCG),with had about 3-fold lower in IC50 value.Modeling of molecular docking suggested that GCG preferably bound to the target α-glucosidase,which was confirmed by in vitro protein-polyphenol binding,GCG had about 4-times higher than EGCG in binding rate.Comparative studies in vivo using oral starch tolerance test in mice verified that GCG exhibited lower postprandial blood glucose compared to EGCG.These findings suggested that high-temperature roasting process of LYT enhanced the levels of epimerized catechins with strong α-glucosidase inhibition effect,which had a contribution to improving the ability of LYT in regulating postprandial blood glucose. |
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