Enzymatic Synthesis,Artificial Simulated Digestion,Anti-inflammatory And Antioxidant Effects Of Theaflavin-3,3'-o-digallate

Black tea is produced by withering,rolling,fermentation and drying of tea(Camellia sinensis(L.)O.Ktze.)leaves or buds,originated in Fujian China,and it is of diverse physiological activities and delicious.Black tea contains many kinds of active substances,such as polyphenols,polysaccharides and theanine,related to its physiological activities,and theaflavins is the most important substances among them.The content of theaflavins in Black tea is extremely low,which account for only 0.5%-2%of the dry weight of Black tea;it is difficult to better study the bioactivity of theaflavins because of the lack of sufficient samples.Therefore,we selected theaflavin-3,3'-O-digallate(TFDG)as a model to develop an enzymatic synthesis method by which could prepare a large quantity of TFDG.Theaflavins,which have prominent biological functions such as anti-oxidation,anti-inflammatory and tumor suppression,have been extensively studied by scholars domestic and abroad.However,the protective effect of TFDG on the hydroxide peroxide induced RAW 264.7 cell oxidative damage has not been reported.Because of its molecular structure with large molecular weight and high hydroxyl content,TFDG were generally considered that it cannot be absorbed directly by the intestinal tract,while it is rarely reported that it can be used in the intestinal tract with its inherent molecular structure.In this study,we synthesized TFDG by pear polyphenol oxidase;The stability of TFDG in artificial simulated digestive fluids was studied,furthermore,the anti-oxidation effects of TFDG on chemical system and H2O2 induced RAW 264.7 cell were studied.The main contents of the research and results are presented as follows:1.Enzymatic synthesis,isolation,purification and structural identification of TFDGPolyphenol oxidase extracted from pear,was applied to synthesis EGCG and ECG to produce TFDG.To optimized the synthesis condition,factor of enzymatic reaction including pH,reaction temperature and molar ratio of substrate were determined by single-factor assays,and they were 4.0-6.5,35? and 3-9,respectively.Based on this,a three-level,three-variables Box-Behnken design(BBD)was applied to test the optimal levels of synthesis variables for synthesis of TFDG,and the optimal levels of synthesis variables were determined as follow:ratio of EGCG and ECG was 3.1,pH4.1,reaction temperature was 37?,respectively.The yield of TFDG under optimal synthesis condition was 85.14%±0.4%,which was pretty close to the yield predicted by the Design expert software.TFDG was synthesis under the optimal condition,reaction mixture was loaded on the AB-8 column,the column was washed by water to remove impurities which cannot attach to AB-8,then 90%ethanol was applied to elute the column to afford crude TFDG,after that,Crude TFDG was purified by HSCCC with the solution system containing water-ethyl acetate n-hexane-methanol?finally the purity of TFDG monomer could reach 97%.The structural identification of TFDG monomer was performed by1H NMR and MALDI-TOF-MS.2.Simulated digestion of TFDG in vitroIn this charter,artificial simulated digestive fluids was utilized to investigate the stability of TFDG in oral cavity,stomach and small intestinal.Furthermore,LPS-induced Caco-2 cell line inflammation model was used to determine the anti-inflammatory effect of TFDG.The results showed that TFDG was stable under simulated saliva and gastric fluids,while a slight hydrolysis could happen in simulate pancreatic fluid,but the degradation products were not detected.In Caco-2 monolayer cell model,TFDG could neither be transformed from the apical to the basal nor hydrolyzed by the digestive enzymes.Caco-2 cell inflammation was induced by LPS,compared with the positive control group,the level of TNF-? and IL-8 were significantly inhibited by TFDG.3.Anti-oxidation effects of TFDGIn this part,chemical systems(including superoxide anion radical scavenging,reducing power,scavenging of ABTS free radicals and anti-lipid peroxidation ability)and H2O2 induced RAW 264.7 cell injury model were introduced to systematically investigate the anti-oxidation capacity of TFDG.Results showed that TFDG had potent superoxide anion radical,ABTS free radicals,reducing power and anti-lipid peroxidation ability.In H2O2 induced RAW 264.7 cell injury model,TFDG could significantly reduce the content of malondialdehyde(MDA)caused by lipid peroxidation and increase the activity of GSH-PX,CAT and SOD.It is suggested that TFDG can effectively reduce the oxidative damage of RAW264.7 cells caused by hydrogen peroxide.