Research On Enzymatical Synthesis Of Tea Catechin Derivatives With PUFA(C22-EGCG) And Their Antioxidant Activity

(-)-Epigallocatechin gallate(EGCG)which contains several phenolic hydroxyl groups exerts important antioxidant activity.EGCG shows great potential in prevention and treatment of various diseases such as cardiovascular disease prevention.Studies showed that protective effects of EGCG to diseases is related to its potent antioxidant activity.Nevertherless,the applications in areas such as food,cosmetics and pharmaceuticals are always limited due to its several phenolic hydroxyl groups.In order to increase solubility and stability of catechins in lipid,new groups are introduced to active sites of catechins for modification,which can improve bioavailabilities and certain activities,etc.There are many researches on the structure modification of EGCG.However,it is rarely reported that the biological activity of polyunsaturated fatty acids is designed directionally for liposoluble catechins derivatives.In this study,EGCG and DHA were used in targeted molecular structure modification to generate new derivative C22-EGCG under the catalysis of lipase,and synthesis system of different enzymes was also studied.The influence of different mole ratio of substrates,different reaction solvent and reaction time on production rate of C22-EGCG derivative were explored.The structure elucidation of derivative was carried out by HPLC and UPLC-MS.Antioxidant ability of C22-EGCG derivative was studied.In the single-factor experiment of C22-EGCG synthesis catalyzed by lipase,C22-EGCG conversion catalyzed by Novozyme435 was better than it by Lipase A.Therefore,Novozyme435 was selected as the optimal catalyst.When mole ratio of EGCG/DHA was 1:4,conversion rate of C22-EGCG catalyzed by Novozyme435 was 83.36% which was significantly different from 1:3,so DHA/EGCG=1:4 was selected as the best substrate ratio.When solvent in the system was acetonitrile,C22-EGCG conversion catalyzed by Novozyme435 had a maximum 79.75% which was significant difference from methanol and water,so acetonitrile was considered as best solvent.When reaction time was 12 h,conversion rate of C22-EGCG catalyzed by Novozyme435 was up to 94.05% which was significant difference from 4h,but there was no significant difference with 8h,so 8h was considered as best reaction time.In summary,the optimal reaction conditions of enzymatic synthesis system: with Novozyme435 as catalyst,1:4 as molar ratio of DHA and EGCG,acetonitrile as reaction solvent,8 h as reaction time,C22-EGCG has the highest conversion rate.The results of C22-EGCG structure elucidation showed that retention time of EGCG was 15.108 min at ? = 280 nm and DHA was 34.615 min at ? = 195 nm.The derivative is a single peak separated from DHA which retention time is 36.968 min at ? = 195 nm.The spectrum of EGCG can be observed in the range of 260-300 nm and EGCG has the highest absorption at 274 nm.DHA and derivative have the same characteristic spectrum which can be observed in the range of 190-210 nm,and DHA and derivative have the highest absorption at 196 nm.Combining MS scan,daughter mode and MRM mode of UPLC-MS,three subsegments of EGCG were obtained which were m/z= 169,m/z= 305,m/z= 457.Five subsegments of DHA were respectively m/z=59,m/z= 135,m/z= 152,m/z= 283,m/z= 327.The sample structure elucidation showed the high frequency subsegments m/z=293,m/z=355,m/z=381,m/z=518,m/z=653,m/z=727 were found which were formed by the combination of EGCG and DHA.The analytical results verify the main structure of C22-EGCG and provide the basis for its accurate molecular configuration analysis.In the antioxidant experiment,ethanol was used as blank control and VC was positive control in DPPH system.Results showed that C22-EGCG had the same ability with VC and EGCG to eliminate DPPH free radicals and DHA also had ability to a certain extent.The ability to clear DPPH free radicals is: C22-EGCG>EGCG>VC>DHA.DPPH free radical clearance rate of C22-EGCG was 87.84% which was significantly higher than that of EGCG and VC.Water was used as blank control and VC as positive control in ABTS system.Results showed that C22-EGCG had the same and strong ability to remove ABTS free radicals with VC and EGCG.DHA had no ability to remove ABTS free radicals.Ability to clear ABTS is: C22-EGCG>EGCG>VC and ABTS free radical clearance rate of C22-EGCG was 92.63% which was significantly higher than that of EGCG and VC.In this study,antioxidant activity of derivative C22-EGCG was enhanced with DHA introduced.