Preparation Of Baohuoside ? And Diosgenin Based On Biphase Enzymatic Hydrolysis

Baohuoside ? is one of the main active compounds in Epimedii Folium,which has various bioactivities,such as anti-tumor,anti osteoporosis,antioxidation and hypertension-relieving.Many attempts have been made to prepare baohuoside I,in previous investigations;typically,chromatographic purification,acid hydrolysis and enzymatic hydrolysis have been explored.However,chromatographic separation is not preferred at the industrial level because of the very low content of baohuoside I in raw materials and many compounds which possess similar polarity with baohuoside I;moreover,by-products are often generated with baohuoside I using acid hydrolysis,which is unfavorable to prepare baohuoside I with high purity.Diosgenin is an important compound for the synthesis of steroid hormone drugs in pharmaceutical industry.In raw materials,diosgenin is usually connected with glucose and rhamnose in the form of saponins;hence,cutting off glycosidic bond is the only way to obtain diosgenin.Traditionallly,acid hydrolysis is used to produce diosgenin from the tuber of Dioscorea zingiberensis C.H.Wright;however,waste acid water is often generated during production,which is not conducive to protect the environment.The aim of this study was to construct a biphase enzymatic hydrolysis system which was used to prepare baohuoside I and diosgenin from icariin and saponins respectively.Work competed are as follows:1.Screening of commercial enzymes and optimization of enzymatic hydrolysis conditions.Firstly,taking the conversion rate of icariin as an index to compare the effect of?-glucosidase,?-dextranase,cellulase,naringinase and glucoamylase on hydrolyzing icariin and screen out the?-glucosidase.Then,the enzymatic hydrolysis conditions of?-glucosidase were optimized by single factor experiment.Results indicated that the optimum condition was that,namely,mass ratio of enzyme/icariin was 3.2:1,reaction time was 60 mins,buffer pH was 4.5 and temperature was 60?.2.Construction of biphase enzymatic hydrolysis system for preparing baohuoside I by hydrolyzing icariin.At first,the organic reagents and duration were optimized to construct biphasic reaction system;then,the investigations on processing capacity of icariin,volume ratio of organic reagent/buffer and recycling performance were conducted in both conventional system and biphase hydrolysis system;furthermore,the ESI-MS,¹H-NMR and ¹³C-NMR were used to determine the structure of product;finally,amplification experiments were also carried out to provide a reference for industrial production of baohuoside I.Results indicated that compared with conventional enzymatic hydrolysis,as time set in 2 hrs,the processing capacity and conversion rate of icariin could increase by 1.5 times and 2%respectively in novel system;moreover,enzyme solution could also be recycled at least 2 times under the conditions of reducing processing capacity of icariin or extending reaction time;furthermore,the amplification times?5 times and 10 times?of biphasic system had little effect on the conversion rate of icariin.In summary,it is more efficient and convenient to prepare baohuoside I by hydrolyzing icariin in biphase enzymatic hydrolysis system,and this novel method possessed good potential for preparing baohuoside I in industrial level.3.Preparation of enzymes for hydrolyzing saponins and optimization of conditions.Firstly,activity screening between commercial enzymes and enzymes from mould was conducted to screen out the optimum enzyme;then,the single factor experiment was used to optimize the fermentation conditions and enzyme hydrolysis conditions.Results indicated that the optimized fermentation condition was as follows,namely,inoculation amount was 1 mL,fermentation time was 6 d,fermentation temperature was 33?and naringin amount was 0.25 g;the optimized hydrolysis condition was as follows,specifically,enzyme/saponin was 0.05:1;duration was 36 hrs;buffer pH was 5.5 and temperature was 50?,based on that,the yield of diosgenin was 1.51%.4.Study on hydrolyzing saponins by biphase enzymatic hydrolysis.First of all,reaction duration and enzyme amount were optimized to improve the yield of diosgenin;after that,the effect of volume ratio of petroleum ether/buffer and the performance of enzyme recovery on diosgenin yield was investigated and the comparison between conventional enzymatic hydrolysis and novel method was operated;lastly,the ESI-MS and NMR were used to determine the structure of product.Results indicated that as the time,enzyme amount and volume ratio of petroleum ether/buffer were set as 36 hrs,4.8mg and 2:1 individually,the yield of diosgenin was 1.52%;besides,compared with the conventional enzymatic hydrolysis,enzyme solution could also be recycled at least 2times by reducing processing capacity of saponins.Consequencely,biphase enzymatic hydrolysis was a cleaner and more convenient method to produce diosgenin and showed a good application prospect in industry.