| Tea(Camellia sinensis)is a popular beverage worldwide and one of the most important functional foods.Oolong tea is a kind of partially-fermented tea of excellent quality made through the processes of picking,withering,rocking,stir-frying,rolling,and drying.During the fermentation process of oolong tea,a chemical reaction centered on the enzymatic oxidation of tea polyphenols occurred,and the chemical components in the fresh leaves changed greatly,resulting in the production of catechin oxidation polymerization product such as theasinensins(TSs).Theasinensin A(TSA)and theasinensin B(TSB)are the two main monomers.It has been reported that theasinensins have a variety of biological activities,such as antioxidation,antiinflammatory,anti-cancer,anti-microbial,anti-obesity and hypoglycemic and so on.However,the content of theasinensins in oolong tea is extremely low,accounting for only 0.65%of the dry weight of oolong tea.Therefore,preparing a sufficient amount of high-purity samples is very important to research its biological activity.In this paper,epigallocatechin gallate(EGCG)and epigallocatechin(EGC)were used as raw materials to establish a method for the preparation of high-purity TSA and TSB monomers in large quantities.The antioxidant abilities of TSA and TSB were evaluated by in vitro chemical antioxidant experiments and the protective effect of TSA on oxidative damage of RAW264.7 cells was investigated.In addition,TSA and TSB were used as samples to study their inhibitory effect on α-amylase and α-glucosidase,and their inhibitory mechanism was explored by enzyme kinetics,fluorescence spectroscopy,circular dichroism(CD),and molecular interaction.The contents and results of research are as follows:1、Synthesis,purification,structural identification and antioxidant activity of TSA and TSBUsing EGCG as raw material,with the treatment of cuprous chloride for 24 hours at room temperature,and then excess ascorbic acid(Vc)was added and the mixture was heated at 85℃ for 15 minutes to obtain crude TSA.In a similar manner,TSB was prepared as mentioned above with EGCG and EGC as raw materials.The mixture was loaded onto a Diaion HP20 column(3.0×25.0 cm),and the column was washed with distilled water to remove some impurities such as Vc that cannot be adsorbed by the resin.Then the column was eluted with different concentrations of MeOH(from 20%to 60%),the desired fractions were combined,concentrated and loaded onto an AKTA purifier system equipped with YMC-Pack ODS-A column and UV detector.The optimized mobile phase was ultrapure water/acetic acid/acetonitrile(125/10/11,v/v/v).All the fractions were checked by HPLC,and the target fractions were combined,concentrated by rotary evaporation and freeze-dried.The structures of resulting TSA and TSB were characterized by HPLC,MALDI-TOF-MS and 1H nuclear magnetic resonance(NMR)spectrometry and the purity of TSA and TSB were more than 97%.The results of chemical antioxidant experiments showed that TSA and TSB had potent ability with scavenging 2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS)radicals,scavenging 1,1-dipheny1-2-picrylhydrazyl(DPPH)radicals,the ferric reducing antioxidant power(FRAP)and reducing power.Especially,they exhibited stronger capability than Vc.When the concentration of TSA and TSB was 25 μg/mL,the scavenging ability on ABTS radical were both close to 100%.When the concentration of TSA and TSB was 50 μg/mL,the scavenging ability on DPPH radical were 70.76%and 72.57%,respectively.In the cell experiment,TSA showed the strong protection against oxidative damage induced by H2O2 in RAW264.7 cells and at the same time,it could significantly reduce intracellular ROS levels(p<0.05).2、The inhibitory of TSA and TSB on α-amylaseIn this section,TSA and TSB were used as samples to study their inhibitory effect on α-amylase,and their inhibitory mechanism was explored by enzyme kinetics,fluorescence spectroscopy,circular dichroism(CD),and molecular interaction.The results showed that the inhibitory effect of TSA on α-amylase was significantly better than that of TSB,and it was probably that the galloyl group in the structure enhanced the inhibitory effect.The results of enzyme kinetics showed that the type of inhibition of α-amylase by TSA was competitive and reversible inhibition.The interaction of TSA and α-amylase changed the internal structure of α-amylase and exposed the chromophore to a more polar microenvironment.The secondary structure of the protein was changed and the protein peptide chain was caused certain curl,which disrupted the stability of the protein.TS A entered into the active center of α-amylase,and interacted to α-amylase through hydrogen bond and hydrophobic forces,which destroyed the protein structure and eventually lead to decrease in α-amylase activity.3、The inhibitory of TSA and TSB on α-glucosidaseIn this part,TSA and TSB were used as samples to study their inhibitory effect on αglucosidase,and their inhibitory mechanism was explored by enzyme kinetics,fluorescence spectroscopy,CD,and molecular interaction.The results showed that both TSA and TSB have good inhibitory effect on α-glucosidase and TSA had better inhibitory effect than TSB,and the IC50 was lower than that of acarbose.The type of inhibition of α-glucosidase by TSA and TSB was non-competitive reversible inhibition.The results of fluorescence spectrum and CD showed that the combination of TSA or TSB with α-glucosidase changed the microenvironment of α-glucosidase and the secondary structure of the protein which improving the hydrophobicity of αglucosidase.The structure stability of α-glucosidase was enhanced,thereby preventing the substrate from binding to the enzyme,and reducing the activity of α-glucosidase.The molecular interaction results showed that TSA and TSB were bound to αglucosidase through hydrogen bond and hydrophobic force.Compared with TSB,TSA was closer to the active sites of-glucosidase that TSA had better inhibitory effect on α-glucosidase. |
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