Preparation, Identification And Biological Evaluation Of Oxidation Products Of (-)-epigallocatechin-3-o-gallate

Researches about the chemical and biological activities of tea polyphenol and tea catechins have been done systematically and deeply. But studies on tea catechins oxypolymers have been just carried out. Very few studies on the transformation of tea catechins to tea catechins oxypolymers and relations between the structure and functions of tea catechins oxypolymers have been found reported. Oxidation was regarded to have negative effect on the biological activities of tea catechins in the past, but some recent researches illustrated that tea catechins oxypolymers may have more significant activities than tea catechins. In order to further research the oxidation products of polyphenol, in this paper, (-)-epigallocatechin-3-O-gallate (EGCG) which is the most powerful catechin was oxidized chemically in vitro and we studied its optimum oxidation condition in vitro, dynamic model and discussed the seperation, purification, structure identification as well as biological activities of EGCG oxidation products. The main results were shown as follows:1 Determination of the optimum oxidation conditon of EGCG in vitroThrough the single-factor experiments and the analysis of response surface method, the influences of reaction temperature, time, pH value, the mass ratio of oxidant to EGCG on inhibiting·OH of EGCG oxidation mixture have been discussed. According to the inhibiting·OH activities, the optimum oxidation condition of EGCG in vitro was determined as follows: temperature 25℃, reaction time 15min, pH 7.0, the mass ratio of oxidant to EGCG 1.5: 1.·OH inhibiting activity could reach the highest (53.40%) under this condition, which higher than that of EGCG (41.50%). Those indicated that EGCG oxidation mixture could possess strong activity when EGCG was oxidized properly.2 Research of the reaction dynamic of EGCG oxidation in vitroHPLC/MS analysis showed that it existed three EGCG oligomeric products (EOP) in EGCG oxidation mixture and EOP belonged to EGCG dimers. The research of reaction dynamic indicated that both the oxidation of EGCG and the formation of EOP had accordance with the second order reaction in the fomer phase (0-10min). The reaction rate of them increased with increase of temperature and the proportion of oxidant; The reaction rate was much higher when pH value was in alkaline.For the purpose of the formation of EOP and keep its stability, the condition of temperature 25℃, reaction time 15min, pH 7.5, the mass ratio of oxidant to EGCG 1.5 : 1 was the optimum formation condition of EOP and this condition had consistence with the optimum oxidation condition of EGCG in vitro. It was indicated that EOP was the important component on inhibiting .OH.3 Fractionation of EGCG oxidation products and their biological activitiesEGCG oxidation products were extracted to obtain two fractions: Fra.A which was extracted at neutral pH contained product 1, 2 and EGCG; Fra.B which was extracted at acid pH contained product 1 and 3. Product 1-3 were all EGCG dimers.The antioxidant effects of Fra.A and Fra.B in vitro were examined. The study showed that Fra.A and Fra.B could effectively scavenge superoxide anion, hydroxyl radical, hydrogen peroxide and inhibit DNA oxidative damage. Fra.A exhibited a stronger antioxidant activity than EGCG while Fra.B elicited less. The study of antimicrobial activities suggested that Fra.A and Fra.B had strong inhibition effects on the bacteria but no distinct inhibition on yeast and mold. With a comparision with EGCG, the antimicrobial activities of Fra.A and Fra.B were less than those of EGCG.4 Preparation and structure identification of EGCG oxidation prodcutsA purification and preparation method of EGCG oxidation products has been established: Fra.B was applied to a Sephadex LH-20 column (40cm×1.5cm i.d.). The column was eluted with 70% methanol at the flow rate of 1mL/min and the main chromatography peaks were collected to give product 3 and the faint yellow powder was subsequently obtained by concentrating and freeze-drying. Fra.C was applied to a D₁₀₁ macroporous absorption resin column (20cm×1.0cm i.d.), eluted with distilled water and 80% ethanol at the natural flow rate and 80% ethanol elution was collected and extracted with ethyl acetate to give product 1 and the orange powder was then obtained by concentrating and freeze-drying.Product 1 and 3 were deduced to dehydrotheasinensin A (m/z 929) and theacitrin C (m/z 911), respectively by using the methods of UV-VIS, HPLC-ESI-MS, NMR. Those two dimer products were all formed through the polymerization of B ring of EGCG.5 Antioxidant effects of EGCG oxidation productsThe antioxidant effects of dehydrotheasinensin A and theacitrin C in vitro were futher studied. The results showed that dehydrotheasinensin A and theacitrin C exhibited strong antioxidant activities in four testing systems. The scavenging rate on superoxide anion of dehydrotheasinensin A, theacitrin C was 1.48 times and 1.32 times respectively stronger than that of EGCG; The scavenging rate on hydroxyl radical of dehydrotheasinensin A, theacitrin C was 1.41 times and 1.27 times respectively stronger than that of EGCG; The scavenging rate on hydrogen peroxide of dehydrotheasinensin A, theacitrin C was 2.04 times and 1.50 times respectively stronger than that of EGCG; The inhibiting rate on DNA oxidative damage of dehydrotheasinensin A, theacitrin C was 2.86 times and 2.37 times respectively stronger than that of EGCG. The antioxidant activities of those two products were much stronger than those of Fra.B.