| Epimedium is a traditional Chinese medicinal material,which is distributed in mountainous areas of Shaanxi,Sichuan,Guizhou and other provinces.The varieties of Epimedium included in Chinese Pharmacopoeia(2020 edition)were E.brevicornum Maxim.),E.pubescens Maxim.,E.sagittatum Maxim.,E.koreanum Nakai and E.Wushanense T.S.Ying.Epimedium wushanense T.S.Ying was newly added in 2010,and it is characterized by high content of active ingredient epimedin C up to 4?7%,and icariin content often less than 0.5%or even undetectable.Icariin is the signature component of its extracts and downstream preparations.Compared with epimedin C,its structure is only less than one rhamnosyl group at the C-3position.However,the classical activity and the market price of drug substance are much higher than that of epimedin C.In the present study,the C-3 terminal rhamnosyl group was mostly removed by acid hydrolysis,microbial fermentation and enzyme catalysis to convert the epimedin C into icariin,but there existed such problems as environmental pollution,many by-products and non-recycling of enzymes.In this study,a new process for the preparation of icariin based on immobilized enzyme and two-phase continuous conversion system was developed using Epimedium wushanense T.S.Ying,which is mainly produced in Bashan Mountains,as the material,to provide support for the production of icariin.The main experiments and results are as follows:(1)Rapid preparation process of epimedin C in Epimedium wushanense T.S.Ying.The static adsorption and desorption experiments showed that HPD-5000 macroporous adsorption resin had the best adsorption and desorption properties for epimedin C,which were 51.73mg/g and 96.07%,respectively.Dynamic experimental optimization confirmed that the concentration of sample in the upper column liquid was 3.5 mg/m L,the volumetric flow rate was 6.0 BV/h,and the maximum loading volume was 11.3 BV.After impurity removal with5.0 BV 10%ethanol of 9.0 BV/h and elution with 70%ethanol,the purity of the concentrated product in the elution section,epimedin C,was 55.27%,and the yield was 81.78%.The crude product was purified by dynamic axial compression column chromatography(DAC-HB50).The optimal separation conditions were as follows:acetonitrile:water(24:76),detection wavelength of 270 nm,flow rate of 100 m L/min,injection volume of 5 m L,and sample concentration of 100 g/L.After purification,the purity and yield of epimedin C were 98.91%and 80.36%,respectively.The structure of the product was confirmed by NMR and HRMS.(2)Screening of hydrolase and optimization of hydrolysis condition.Using the conversion rate of epimedin C and the yield of icariin as the indicators,four different hydrolases were screened and the enzymatic hydrolysis conditions were optimized.The results showed that?-L-rhamnosidase was the optimal hydrolase,and the optimal conditions for free enzyme hydrolysis were as follows:temperature 50?,phosphate buffer p H 4.5,substrate/enzyme mass ratio 1:10,and hydrolysis time 96 h.The hydrolysis efficiency of epimedin C reached 97.36%.The structure of the main product was identified as icariin by NMR and HRMS,and the yield of icariin was 95.77%.(3)Optimization of immobilization conditions of?-L-rhamnocidase and its application.The covalent bond coupling method was adopted,and the optimal immobilization conditions were optimized as follows:in the first step,mesoporous silica SBA-15 was used as a carrier,and aminated SBA-15(SBA-15-NH2)was obtained by aminating it with silanization reagents(APTES);In the second step,7%glutaraldehyde(GA)was added at 35?as a coupling agent to react with the amino group for 4 h with a Schiff base,and an aldehyde group was introduced to obtain an aldehyde-functionalized SBA-15(SBA-15-CHO).In the third step,50mg of carrier SBA-15-CHO and 16 mg of?-L-rhamnocidase were added into phosphate buffer liquid system with p H of 3.5 at 35?for Schiff base reaction for 4 h to obtain the immobilized?-L-rhamnocidase(SBA-15-ENZ)by covalent bonding.The immobilized?-L-rhamnosidase exhibited an enzyme loading rate,enzyme activity recovery rate and enzyme activity of 80.75%,66.25%and 3.3125 U/g,respectively.When 50 mg of immobilized enzyme was added into phosphate buffer p H 4.5 at 50?and substrate concentration of 200?g/m L for hydrolysis for 32 h,the conversion rate still reached 64.21%after four times of reuse.The characterization data of FI-IR and N2 adsorption and desorption indicated that the SBA-15 carrier had completed the functional modification of the group and the immobilization of the enzyme.XRD and SEM data showed that the original hexagonal mesoporous and rod-like structure of SBA-15 remained unchanged after modification and enzyme immobilization.In summary,the results above indicated that?-L-rhamnocidase immobilized on SBA-15 carrier had good stability.(4)Study on the preparation of icariin by hydrolysis of epimedin C in two-phase system.Determine butyl acetate as that optimal organic solvent;When the volume ratio of buffer solution to butyl acetate was 1:3,the mass ratio of substrate to enzyme was 1:30,the concentration of substrate was 2 mg/m L,and the mixture was stirred for hydrolysis for 12 h,After the enzyme solution was recycled for four times,the conversion rates of epimedin C were 97.12%,95.09%,88.41%and 75.24%,respectively.The organic phase transfer rate of the product icariin reached 90.42%.The optimized process was applied to the hydrolysis of Epimedium wushanense T.S.Ying extract and the volume amplification was conducted for500 times.The conversion rate of epimedin C and the organic phase transfer rate of the product icariin were not significantly decreased.The high-efficiency separation from compounds such as epimedin A and epimedin B was achieved,and the purity of icariin reached more than 80%.The process realizes the integration of the generation of the weak polar icariin and the organic phase transfer,reduces the product inhibition in the enzymolysis reaction process,has good stability,low equipment requirement and wide application prospect in scale-up production. |
PDF Full Text Request