High-throughput Screening Of α-amylase Inhibitors From Tea And Their Hypoglycemic Effect

Diabetes mellitus(DM)is a chronic metabolic disease characterized by hyperglycemia and insulin resistance,and has attracted wide attention because of its high morbidity,high mortality and many complications.Dietary starch is one of the main sources of human energy and blood sugar,when human body ingests starch,α-amylase can randomly cut off the α-1,4-glucosidic bond in starch,and the rapid degradation of starch into glucose can lead to a sharp increase in blood glucose level,which is called postprandial hyperglycemia and recognized as an important index of type 2 DM.At present,oral hypoglycemic drugs is the main method to treat and prevent diabetes,but long-term use produce a series of side effects.Therefore,screening safe and effective α-amylase inhibitors from natural resource,and regulating blood sugar by inhibiting a-amylase activity to delay the digestion and absorption of glucose in starchy foods,it is one of the effective ways to reduce the risk of diabetes mellitus and its complications.Tea(Camellis sinensis var.sinensis)originated in China,is one of the three non-alcoholic beverages in the world.According to the production technology and fermentation degree,Chinese tea is divided into six categories:green,white,yellow,oolong,black,and dark tea.Tea contain a variety of bioactive components such as tea polyphenols,theanine,and caffeine,which endow tea with neuroprotective,anticancer,hypoglycemic and other health effects.Among them,the hypoglycemic effect of tea is gaining increasing attentions,a large number of epidemiological investigations and research experiments have shown that tea and bioactive components of tea play a positive role in the prevention and treatment of diabetes mellitus and its complications.In this study,we select different teas as experimental objects,analyze the inhibitory effect of tea onα-amylase and screen out potential α-amylase inhibitors from tea,finally clarify their inhibitory mechanism and verify the hypoglycemic effect of tea.The main results are as follows:(1)Study on the chemical compounds analysis and the inhibitory effect ofα-amylase activity of different tea.The chemical compounds of six types of tea were analyzed by metabonomics method based on ultrahigh performance liquid chromatography-quadrupole orbitrap mass spectrometer(UHPLC-Q-Exactive/MS),and a total of 166 compounds were identified,including 12 alkaloids,14 catechins,17 amino acids,16 dimer catechins,16 phenolic acids,30 flavonoid glycosides,6 organic acids,7 aroma glycosides,8 N-ethyl-2-pyrrolidinone-substituted flavan-3-ols(EPSF),15 lipids and 25 other compounds,among them,145 compounds had significant differences between six types of tea(P<0.05).Heat-map cluster analysis showed that most catechins,dimer catechins,phenolic acids,organic acids,alkaloids and flavonoid glycosides were relatively high in not-and sight-fermented green,white,and yellow tea,the contents were relatively low in half-and fully-fermented oolong,black,and dark tea.The contents of theaflavins in four black teas ranged from 2.01 to 5.73 mg/g,which were significantly higher than in other teas.By measuring the inhibition rate of six types of tea on α-amylase activity,it was found that the inhibitory effect against a-amylase were positively correlated with the fermentation(polyphenol oxidase-mediated oxidation)degree of teas.Black tea showed the strongest inhibitory effect on α-amylase,which is speculated that the contents of theaflavins in black tea are significantly higher.(2)Study on high throughput screening of α-amylase inhibitors from tea.Potential α-amylase inhibitors from tea were screened with high throughput by"protein-metabolite" interaction-based affinity selection-mass spectrometry.A total of 150 compounds were identified,including 11 catechins,6 organic acids,9 dimer catechins,15 amino acids,10 alkaloids,8 EPSFs,12 phenolic acids,31 flavonoid glycosides,7 aroma glycosides,17 lipids and 24 other compounds.Among them,11 compounds were screened to specifically bind to α-amylase,respectively were 4 theaflavins(theaflavin,theaflavin-3-gallate,theaflavin-3’-gallate,and theaflavin-3,3’-digallate),2 catechins(epigallocatechin and epigallocatechin gallate),3 lipids(LysoPC(18:3),LysoPC(16:0),and MG(18:3)),and 2 other compounds(dihydroactinidiolide and theasinensin A).By comparing the inhibitory effects of theaflavins and other main compounds(such as epigallocatechin gallate,gallic acid,theanine,caffeine,quercetin,and so on)in tea on a-amylase activity,it is determined that theaflavins are the key compounds that can inhibit the activity of α-amylase in tea,which is inferred to be related to the unique benzotrianone skeleton structure of theaflavins,and it is also the reason why black tea has the strongest inhibitory effect on α-amylase activity among the six types of tea.(3)Study on the mechanism of theaflavins in inhibiting α-amylase activity and hypoglycemic effect.The inhibition kinetics indicated that the inhibition type of theaflavins on a-amylase belonged to non-competitive inhibition,theaflavins inhibit its activity by acting on the non-active site of α-amylase.Fluorescence spectroscopy indicated that theaflavins had static quenching effect on α-amylase,hydrogen bond and van der waals force were main interaction forces.Circular dichroism indicated that theaflavins destroyed the secondary structure of a-amylase,affected the hydrophobicity of a-amylase and thus inhibit its activity.The results of molecular docking showed that the inhibitory effect of theaflavins on α-amylase activity were mainly formed hydrogen bonds and π-πinteractions with the residues of α-amylase,and the formation of hydrogen bonds mainly depended on the benzotrianone skeleton structure,π-π interactions were mainly formed by gallic groups in theaflavin-3’-gallate and theaflavin-3,3’-digallate.After 28 days of gavage to db/db mice with water solution of theaflavins and black tea,the long-term change of blood glucose,the content of blood glucose in serum on the 29th day and oral starch tolerance test showed that theaflavins and black tea extracts have hypoglycemic effect,among them,theaflavin,theaflavin-3,3’-digallate,and Yinghong 9 extract can reduce blood sugar of db/db mice by inhibiting the activity of a-amylase.At the same time,it was found that theaflavin,theaflavin-3,3’-digallate and Yinghong 9 extract had better hypoglycemic effect in db/db mice than acarbose,theaflavin-3-gallate and Fuliang black tea.