Study And Application On The Conversion And Hypoglycemic Activity Of Ethyl-rutinoside In Tartary Buckwheat

In recent years,the number of people with diabetes and prediabetes has increased rapidly worldwide.As a chronic basic metabolic disease,diabetes poses an increasingly serious threat to people’s health,and the effective treatment methods are still limit.Therefore,the demand for blood sugar intervention through dietary intake is in urgent.Tartary buckwheat is a dietary resource that shows an excellent hypoglycemic activity.The exploration of bioactive hypoglycemic components in tartary buckwheat,has experienced different stages including flavonoids,rutin,quercetin,D-chiral inositol,etc.Recent research shows that Ethyl-rutinoside（ER）,generated from rutin during the process of tartary buckwheat products,may be the active ingredient that plays a key role in regulating blood sugar.However,the systematic research on the process of ER formation,regulating mechanism,and the effectiveness of hypoglycemic activity in final products is limit.In terms of these problems,this study based on UPLC-MS/MS detection method,filtered tartary buckwheat materials with high ER potential.Additionally,the study evaluated the hypoglycemic activity of ER and speculated its potential mechanism.In order to improve ER yield during processing,a superfine grinding combined with enzymatic technique was established based on the optimized enzyme catalysis condition.This technique was finally applied to prepare high ER tartary buckwheat steam bread,and the hypoglycemic effect of this product was verified.Therefore,the main research conclusions were summarized as follows:（1）Tartary buckwheat materials with the highest ER potential was filtered.The UPLC-MS/MS method was applied to detect ER conversion of different tartary buckwheat tissues（flowers,leaves,stems,roots,hulls,bran,outer flour,and inner flour）during enzymatic catalysis.Among these tissues,tartary buckwheat bran had the highest ER conversion potential,and the ER content after enzyme catalysis was the most abundant compared to other tissues,reaching to 20.59 mg/g,which is 66.67 times higher than the one without catalysis.Bran belongs to the seed part of tartary buckwheat.In order to harvest the bran with the highest ER generation potential,the ER conversion capability of tartary buckwheat seeds at different maturity stages was also investigated.Compared with other maturity stages,tartary buckwheat seeds with 21 days after flowering,could produce the highest content of ER（18.82 mg/g）after enzyme catalysis.（2）This study verified that ER is an important hypoglycemic profile in tartary buckwheat.First,the Hep G2 insulin resistance model was constructed.After ER intervention,the glucose consumption level was up to 1.34 times than that of model group,and the glucose consumption level in ER group was significantly higher than that of flavonoids such as rutin and quercetin（p<0.05）.Second,for gluconeogenesis,two glycolytic rate-limiting enzyme（G-6-P and PEPCK）acivities with ER treatment were 37.79%and 75.69%compared to the model group,and the expression of FOXO1 gene was down-regulated by 47.39%of model group;in terms of glycogen synthesis,the glycogen synthesis in ER treatment group was increased by 57.12%than insulin-resistant cells,while ER significantly downregulated GSK-3βgene expression（p<0.05）.The study shows that ER can improve hepatic glucose metabolism by inhibiting gluconeogenesis and promoting glycogen synthesis.（3）Study of ER transformation conditions and patterns during enzyme catalysis.First,tartary buckwheat endogenous glucosyltransferase（TBEG）,which is responsible for ER genetation,was purified by ammonium sulfate precipitation,gel chromatography and anion chromatography.In the simulate system for TBEG catalysis,the effects of p H value,reaction temperature,ethanol concentration,substrate addition ratio,metal ions,different types of chemical reagents on ER production were systematically evaluated.The results showed that TBEG had the highest catalytic activity in the conditions of p H=5,temperature 40°C,and 20%ethanol concentration,which was suitable for the ER generation.Additionally,TBEG could maintain a high catalytic efficiency for ER production when the addition ratio of substrate/enzyme（w/w）was less than 6.The presence of metal ions(Zn²⁺,Al³⁺and Cu²⁺)and chemical reagents（Tween 20,SDS and DMSO）is not not suitable for ER generation.Secondly,the recognition pattern of TBEG to flavonoid glycosides shows that this enzyme is suitable for catalyzing 3-O-rutin（3-O-Rut Fla）flavonoid glycoside type to produce ER,especially for rutin and kaempferol-rutinoside,which exhibited high catalytic efficiency constants（Vmax/Km）.A total of 13 3-O-Rut Fla type flavonoid glycosides were identified in tartary buckwheat materials by high-resolution mass spectrometry.With the addition of exogenous TBEG,the raw material,which does not contain TBEG activity,was successfully catalyzed to generate ER.（4）A superfine grinding combined with enzymatic technique was established to improve the ER yield.The abovementioned tartary buckwheat bran with high ER conversion potential was applied as the material in this study,and the tartary buckwheat bran micronized powder with different particle sizes were prepared by superfine grinding.Combined with the optimized enzymatic reaction conditions（p H=5,temperature 40°C,and ethanol concentration of 20%）in the previous simulation system,tartary buckwheat bran micronized powders with different particle sizes were catalyzed.The result showed that tartary buckwheat bran micronized powder with 42μm average particle size had an efficient conversion capability,with the ER yield increased from 30%（unground tartary buckwheat bran powder）to 82%,and DPPH,ABTS,FRAP andα-glucosidase inhibitory activities of 42μm group also increased.According to these results,the tartary buckwheat bran micronized powder with an average particle size of 42μm was selected,and the tartary buckwheat bran micronized powder product with high ER content was prepared by enzymatic conversion and drying process.The ER content of this product was increased from 0.31 mg/g（untreated bran）to 28.34 mg/g.（5）ER-enhanced tartary buckwheat steam bread with hypoglycemic activity was prepared.The tartary buckwheat micronized bran material,which prepared by the abovementioned superfine grinding combined with enzymatic technique,was used as ingredients to develop tartary buckwheat micronized bran steam bread（TMBB）,and ER content in TMBB was 7.80mg/g.In vivo hypoglycemic experiments,the TMBB intervention improved indexes of body weight,water intake,food intake and urine volume of Type 2 diabetes mellitus mice.Compared to other tartary buckwheat steam bread that does not have ER,feeding TMBB leads to the best hypoglycemic effect on diabetes mellitus mice.After four weeks of feeding,the blood glucose value decreased by 48.52%compared with the model group,and the GSP and AUC value decreased by 40.69%and 36.77%,respectively.Insulin resistance-related indicators of diabetes mellitus mice were also significantly improved by feeding TMBB（p<0.05）.In terms of lipid metabolism complications in diabetes mellitus mice,TMBB diet can significantly down-regulate serum triglyceride,cholesterol and low-density lipoprotein cholesterol levels in diabetes mellitus mice（50.72%,37.11%and 58.37%of the model group,respectively）,and the serum high-density lipoprotein cholesterol content was significantly up-regulated（2.32 times higher than that of the model group）.According to the results of liver and islet tissue sections,4 week TMBB supplementation can effectively repair Type 2 diabetes mellitus-induced liver and pancreas damage in mice.The animal experiment verified the hypoglycemic effect of high ER content tartary buckwheat products.Conclusively,this paper has systematically studied ER,clarified that this compound is an important hypoglycemic profile in tartary buckwheat,revealed the ER transformation pattern,and increased ER content in the processing scene of tartary buckwheat,finally developed the steam bread with hypoglycemic activity.This study has provided a theoretical basis for the processing of tartary buckwheat products with high hypoglycemic activity.