Synthesis,Purification And Application Of Hesperetin-7-O-glucoside By Enzymatic Method

Hespertin-7-O-glucoside(HMG)is a product of hesperidin after the removal of a molecule of rhamnose.It is also a precursor of a new sweetener-neohesperidin dihydrochalcone,and its physiological activity and solubility are greatly improved compared with hesperidin.In this paper,the enzymatic properties of hesperidinase were studied,and the preparation process of HMG was optimized,HMG was isolated and purified and the antioxidant activity of HMG was determined.The main findings are as follows:(1)The enzyme activity and enzymatic properties of hesperidinase were determined by HPLC method.The results showed that the total enzyme activities of hesperidinase,?-L-rhamnosidase and ?-D-glucosidase were 12.28,3.5 and 0.413 U/g,respectively.The hesperidinase had good stability at 20-70? and pH 3.0-7.0,the optimum temperature and pH was 60? and 4.0,respectively.Furthermore,at the optimum reaction temperature and pH,the kinetic constant K_m of hesperidinase was 296.69?g/mL,and the maximum reaction rate V_m was 0.8723?g/(mL·min).The precision and accuracy of the established HPLC method are good,and the method can be used for the quantitative detection of HMG.(2)Taking the conversion rate of hesperidin and the amount of HMG production as indexes,the effects of enzyme dosage,cosolvent addition of dimethylformamide(DMF),glucose concentration and hesperidin concentration on the preparation of HMG were investigated.On this basis,taking the yield of HMG as an index,the regression model of HMG is established by response surface test.The regression model is Y=45.72-1.18A+0.35B+1.48C-0.76AB+0.08AC-0.29BC-3.74A~2-1.92B~2-3.55C~2.This model has a high accuracy and can be used to predict the yield of HMG in a certain range of conditions.(3)HMG from enzymatic hydrolysate was separated by macroporous adsorption resin and semi-prepared liquid chromatography.The HPD300 resin was selected for the preliminary purification of the hydrolysate.After purification,the purity of the three main flavonoids in the hydrolysate was increased from 80.37% to 97.83%.The purity of monomer HMG was increased to 98.21% by further separation and purification in semi-preparative liquid phase.The product was confirmed to be HMG by UV-Visible spectrum(UV-Vis),infrared spectrum(FTIR),X-ray diffraction(XRD)and mass spectrometry(TOF-MS).(4)The antioxidant activity of the enzymatic product and hesperidin were evaluated by using DPPH free radical,hydroxyl radical,superoxide anion radical,iron reduction force and metal ion ionization force test.The results showed that hesperidin,HMG and hesperetin all had certain antioxidant effects,but their free radical scavenging ability was not as good as that of Vc,and the scavenging effect of each antioxidant was concentration-dependent within a certain concentration range.