Biocatalytic Synthesis And Physiological Activities Of Puerarin Esters

Puerarin is a kind of isoflavonoids,which has many biological and pharmacological activities,such as anti-cancer,anti-oxidation and cardiovascular/cerebrovascular regulatory activities.However,due to the low fat-solublility,the use of puerarin is greatly limited.It was found that both the physicochemical and the physiological properties of puerarin can be improved by selective modifications.Chemical methods are mainly used for the modification of puerarin,but suffered from their numerous steps,large pollution and low selectivity.And the reports of enzymatic synthesis were not comprehensive.In this dissertation,two types biocataiytic?lipase and whole cell biocatalyst?were tested about the acylation of puerarin in non aqueous phase.At the same time,the effect of several key reaction factors on the biocatalytic acylation of puerarin was discussed,and the physiological activity of puerarin was explored.Studies have shown that immobilized lipase Novozym 435,Lipozyme ^IMTL and Lipozyme ^IMRM could efficiently catalyze the acylation reaction of puerarin.Novozym 435catalyzed acylation of puerarin as a model,THF as reaction solvent,2 mg/m L Novozym 435,substrate:the ratio of acyl donor at 1:30 and water content 0,reaction 6 h,substrate conversion rate reached 99.5%.The reaction products were identified by separation and purification.The analysis of TOF-MS,FT-IR and ¹³C-NMR confirmed that the acylation reaction occurs mainly at the 6''-OH position of sugar moiety of pueratin,the corresponding products were 6''-O-acetyl puerarin,6''-O-propionyl puerarin,6''-O-butyryl puerarin,6''-O-pivalyl puerarin,6''-O-hexanoyl puerarin,6''-O-octanyl puerarin,6''-O-lauryl puerarin,and6''-O-myrisyl puerarin,respectively.And conversion rate and the initial rate of the esters were99.8%,96.9%,96.5%,98.1%,67.6%,50.5%,45.3%;23.56 mmol/?L·h?,12.05mmol/?L·h?,2.91mmol/?L·h?,5.7mmol/?L·h?,0.97mmol/?L·h?,0.45mmol/?L·h?,0.32mmol/?L·h?,respectivey.Both microbial species and medium components have obvious effects on the catalytic activity of the whole cell catalyst.Among the selected 12 strains,Aspergillus oryzae GIM3.4826 prepared by culture medium containing soybean oil showed high catalytic activity in the synthesis reaction of puerarin,and the conversion rate of the puerarin esters synthesis was82.7%.In this model,the effect of key reaction factors on the whole cell reaction was studied,and the optimum conditions were as follows:using THF as the reaction solvent,the dosage of the whole cell catalyst was 50 mg/m L,the molar ratio of substrate was 30:1,the temperature was 40?,the speed was 200 r/min,and the reaction time was 24 h.Under these conditions,the initial rate and conversion rate of reaction reached 4.21 mmol/?L·h?and 94.2%,regioselectivity>98%respectively.Preparative-scale experiments showed the possibility for industrial production of puerarin esters with Novozym 435 and A.oryzae whole cells.However,the operation stability test showed that activity of A.oryzae had more loss after three batches of duplication,which may be due to the toxic effects of organic solvents on microbiological cells,and further study was needed to improve its operating stability.Studies on the physiological activity of puerarin showed that the liposoluble property of acylated products was greatly improved compared with puerarin by LogP.The acylation products of puerarin had not significantly increased the ability of scavenging free radicals?DPPH,ABTS?in the traditional antioxidant evaluation system,but its inhibitory effect on AAPH induced red blood cell oxidation hemolysis had a better antioxidant effect than puerarin.Due to the enhancement of lipophilicity,puerarin esters could be more easily gathered in the cell surface and get inside the cells,so as to better protect cells avoided the attack of free radicals.This research showed practical importance,providing a new route for the acylation modification of puerarin,which was helpful to solve the complex problems of protecting and deprotecting procedures used in the traditional chemical methods for acylation of puerarin.It provided data support about application of medicine and other fields.