Study On Enzymatic Acylation Of Flavonoid Glycosides

Flavonoid glycosides are the main components of many natural drugs.Due to the polyhydroxyl nature,however,it usually suffers from low oral bioavailability,short half-life in human plasma and various side effects in the clinical application.Fortunately,the modifications of this kind of natural drugs by regioselective acylation have been proved to be one of the most successful approaches to improve their physicochemical properties,pharmacokinetics,and bioavailabilities.Hence,enzymatic preparations of gastridin unsaturated ester derivatives was firstly explored.Besides,the catalytic performances exhibited by lipases in various reaction systems were chatacterized and the effects of some variables on the enzymatic acylation were examined.Furthermore,the acyl donor recognition by lipases of different sources in the acylation of gastrodin was explored using various acyl donors of differing structures.In this paper,magnetic nanoparticles with large specific surface area were used as the carriers for lipase immobilization,which was modified by dopamine to make it have more active groups such as amino and mercapto groups.On this basis,Aspergillus Niger lipase was stabilized on the surface of magnetic nanoparticles by schiff base reaction.According to the optimization experiments,the optimal carrier/enzyme(mass ratio),immobilized temperature,p H and immobilized time was 5/1,40 °C,8.5 and 10 h,respectively.Under this condition,the immobilized enzyme enzyme activity and protein load could reach up to 1305 U/g and 155 mg/g,respectively.The crotonylation of gsatrodin was selected as a model reaction to elucidate the influences of the organic solvent,substrate molar ratio,enzyme dosage and temperature on the ANL-mediated reaction.Under the optimal conditions(THF,12,70 U,50 °C),the enzymatic reaction rate,1,6'-regioselectivity,and maximal substrate conversion were as high as 12.34 m M/h,100%,and 98.24%,respectively.Moreover,the lowest apparent Km value of 41.76 m M seemed to indicate that TLL exerted an extremely higher affinity for the substrate in THF than in the other organic solvents(69.82-74.46Mm)tested,which suggested that THF was the most appropriate solvent for the givenreaction.Based on the above studies,the identification of enzymes and substrates in the acylation process of flavonoid glycosides was explored by using 12 differing structures of acyl donors and 11 flavonoid glycosides.The identification of acyl donors showed that gastrodin could be effectively converted into corresponding diester derivatives,especially,decanoylation was the fastest reaction(14.10 m M/h)and butazole was the slowest reaction(6.74 m M/h).The quercetin,isoquercitrin,polydatin,arbutin,astragalin,hyperoside and myricetin could be synthesized the corresponding single ester derivatives,but the conversion rate was very different(65.18-98.71%).However there was no enzymatic acylation of the cyanidin 3-O-glucoside,dihydromyricetin,taxifolin and baicalin,it may be related with the structural differences of substrates.