Study On Enzymatic Acetylation Of Epigallocatechin Gallate And The Antioxidant Properties Of Its Derivatives

(-)-Epigallocatechin-3-O-gallate (EGCG) is an active compound with the biologicalactivities of anti-oxidation, anti-mutation, radiation resistance, preventing cancer andcardiovascular diseases, modulating the immune system, anti-aging, etc.. However, with itslow solubility in lipophilic systems, the application of EGCG in food industry is limited. Inthis study, modification of EGCG by enzymatic acylation was carried out in order to improveits hydrophobic property. The acylated EGCG products were separated and purified, themolecular structure of the productes were determined and the acylation positions for EGCGwere obtained. And then the in vitro antioxidant properties and solubility for the acylatedEGCG products were further evaluated.Firstly, the enzymatic acylation process of EGCG was established. Different C chains ofvinyl ester had great effect on the conversion of EGCG. The conversion yield reduced withthe increasing of C chains. The highest conversion yield was obtained with the reactionbetween EGCG and vinyl acetate. By response surface methodology, the optimal reactioncondition was obtained under40°C, with the enzyme concentration of2.1%, the reactiontime was12h and EGCG/vinyl acetate mole ratio was1.1, respectively. The conversionyield of acylated EGCG products reached87.37%under the optimal conditions. The acylatedproducts were determined by liquid chromatography tandem mass spectrometry and infraredspectrometry. The presence of mono-, di-and tri-acetylated derivatives in acetylated EGCGwas confirmed by LC-MS-MS. The acylation reaction of EGCG catalyzed by lipase wascontrolled by the kinetic theory. There was no substrate inhibition with the concentrationrange of EGCG <15mmol/L and vinyl acetate <10mmol/L. The reaction was in accordancewith the Michaelis-Menton equation, which followed the ping-pong mechanism.High speed counter current chromatography (HSCCC) and preparative high performanceliquid chromatography was used to separate and purify the acylated EGCG products. Theconditions for HSCCC were: rotation rate of700r/min under20?at a flow rate of5mL/min.And two-phase solvent system contained n-hexane, ethyl acetate, methanol and water withthe ratio of1.5:5:1.5:5was selected.The structures of acylated EGCG products wereidentified as5?-O-acetyl-EGCG,3?,5?-2-O-acetyl-EGCG and5?,3?,5?-3-O-acetyl-EGCGby TOF MS/MS,1H NMR and13C NMR. The light transmittance experiments and soluble properties of acylated EGCG showedthat the acetylation products had excellent lipid soluble property and high transparency inedible oil. The solubility in soybean oil was425mg/kg under30?. There was little effect oncolor and brightness of soybean oil with the addition of200mg/kg of acylated EGCG asshowed by chromatic aberration experiments. Furthermore, the antioxidant effects ofacetylated EGCG, EGCG, butylated hydroxytoluene (BHT)?tert-butyl hydroquinone (TBHQ)on edible oils were evaluated and compared by POV values and Rancimat apparatus. Theresults indicated that the solubility of acetylated EGCG in edible oils was significantlyimproved by modification. And the antioxidant capabilities of acetylated EGCG in edible oilswere superior to that of EGCG and BHT, but slightly inferior to that of TBHQ. The residueof acylated EGCG products was under the level of the European pharmacopoeia. Thephysicochemical property of the oil was not changed after adding the acetylated EGCGproducts.The in vitro antioxidant activity of acetylated EGCG products was evaluated bymeasuring its scavenging effect on superoixide anion (O2·-), hydroxyl radical (·OH) and1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical. The acetylated EGCG was found to havegood scavenging effects on O2·-,·OH and DPPH·and the50%inhibitory concentrations(IC50) were0.52mg/mL,0.43mg/mL and11.5mg/L, respectively. Anti lipid peroxidationexperiments showed that with acetylated EGCG concentration of320mg/L, the inhibitionrate of malonyldialdehyde (MDA) formation in rat liver mitochondrial induced by H2O2was61.11%and that of hemolysis for rats red blood cells induced by H2O2was93.54%. Itindicated that acetylated EGCG had strong in vitro antioxidant activity. The antioxidantactivity was dependent on the concentration of acetylated EGCG products.