Synthesis And Bioactivity Of Hesperetin-7-O-glucoside By Green Enzymatic Transformation Method

Hesperidin monoglycoside(HMG),as the precursor of high-power sweetener glucosyl hesperetin dihydrochalcone(GHDC),has anti-inflammatory,free radical scavenging,anti-cancer and other pharmacological and biochemical properties as well as calming,increasing yield,preserving freshness,inhibiting melanin,etc.However,the poor solubility of hesperidin restricts the high value conversion of HMG by enzymatic method.In our previous study,we found that the solubility and biological activity of hesperidin were greatly improved after coordination modification.This work aims to propose and practice a new enzymatic modification method based on hesperidin complex.Using the pulsed electric field method to extract hesperidin from tangerine peel,constructing the green cross-linked immobilization platform,separating HMG through coordination-enzymatic hydrolysis-decoordination method,preliminarily verifying the technical feasibility of the preparation of the sweetener,were beneficial exploration for efficient biosynthesis of HMG.The main findings were presented as follows:(1)With the extraction rate of hesperidin as the index,the effects of four single factors of liquid to solid ratio,electric field intensity,pulse times and extraction time on the extraction of hesperidin by the method of pulsed electric field were investigated.On this basis,the regression model was established by response surface test as:Y=-446.81+15.23A+250.36B+6.48C+0.66AB-0.016AC-0.095BC-0.13A²-36.20B²-0.07C².This model had high accuracy and could be used to optimize extraction conditions of the pulsed electric field.(2)Using FeCl₃ and FeSO₄ as raw materials,Fe₃O₄ was synthesized by precipitation method,and the immobilized material was prepared by combining with graphene oxide(GO).The structural changes and magnetic properties of the material before and after preparation were studied by characterization methods such as electron microscopy,infrared spectroscopy,XRD,thermal analysis,and magnetic detection.The results showed that the crystallinity of Fe₃O₄@GO was lower than that of GO,and thermal stability was better.The hysteresis curve of Fe₃O₄@GO measured by a magnetometer was S-shaped,without remanence and coercivity,indicating that Fe₃O₄@GO had good superparamagnetism and could be used as a good carrier for immobilized enzymes in the next experiment.(3)The biological enzymatic hydrolysis path of HMG synthesis was explored,using the green cross-linking method to synthesize magnetic immobilized rhamnosidase and studying the immobilization rate of rhamnosidase on Fe₃O₄@GO under different conditions and the changes in enzymatic properties.The separation and purification of HMG was achieved by decomplexing,and the product was confirmed to be HMG by means of high-performance liquid chromatography(HPLC)and mass spectrometry.(4)The antioxidant activities of Hesperidin and HMG were evaluated by ABTS free radical scavenging ability and oxygen free radical absorption capacity(ORAC),and the inhibition ability of?-amylase was studied.The CCK-8 method was used to study the proliferation effects of the two on human breast cancer cells MCF-7,liver cancer cells Hep G2and normal human kidney cells 293A.The results showed that both hesperidin and HMG had a certain free radical scavenging ability,and HMG performed better anti-oxidation and anti-proliferation properties and less cytotoxicity.In addition,the ring-opening hydrogenation was carried out in a reaction kettle,and the feasibility of GHDC synthesis from HMG was preliminarily analyzed through liquid phase,which provided a reference for the development of sweeteners in the future...