Preparation Of Secondary Flavonol Glycosides Of Epimedii Folium Based On Enzymatic Hydrolysis

Epimedii Folium?EF?comprises the dried leaves of four Epimedium plants,including E.brevicornu Maxim.,E.sagittatum?Sieb.et Zucc.?Maxim.,E.pubescens Maxim.and E.koreanum Nakai.The herb is one of the most recognized traditional herbal medicines for the treatment of cardiovascular diseases,bone loss and impotence.It has been revealed that its important bioactive constituents are flavonol glycosides,such as icariin,epimedin A,epimedin B and epimedin C.However,it's difficult for these original flavonol glycosides to pass through small intestine and play therapeutic effects due to their great hydrophilicity.Baohuoside I,sagittatoside A,sagittatoside B and 2''-O-rhamnosyl icariside II,the products of specific hydrolysis of glycosidic bond?-OR₂?at C-7 position on original flavonol glycosides mentioned above,are secondary flavonol glycosides existed in EF.They could be readily transported into plasma and play an important bioactive role in vivo.Thus,it is promising to develop these flavonoids to be new molecular entities and the convenient preparation of these bioactive components is a crucial task.In previous investigation,many methods have been used to produce secondary flavonol glycosides,including column chromatography,acid hydrolysis,biotransformation and enzymatic hydrolysis.However,column chromatography is not the preferred method for large scale preparation of secondary flavonol glycosides because of their low contents in raw materials and similar polarity.Acid hydrolysis often produces byproducts such as icaritin and hardly obtains secondary flavonol glycosides.In addition,biotransformation possesses mild reaction conditions,but it is not appropriate for industrial application because of its time-consuming and hardly controlled procedures.Alternatively,enzymatic hydrolysis is specific,clean and has been applied for preparing sagittatoside B.However,conventional enzymatic hydrolysis system could not reach an ideal hydrolysis efficiency.Besides,enzymes'reusability is significantly reduced after extracting products by using organic solvents.In order to improve hydrolysis efficiency and reuse enzyme,we established biphase enzymatic hydrolysis system and applied it for preparing secondary flavonol glycosides.Apart from this,immobilized?-glucanase was prepared and applied for obtaining sagittatoside A.Work includes the following four parts:1.HPLC-UV method for analyzing flavonol glycosides of Epimedii Folium was established.Flavonol glycosides'analytical conditions were as follows:chromatographic column:Agilent ZORBAX SB-C18?150 mm L.×4.6 mm I.D.,5?m?;mobile phases:acetonitrile?A?and water?B?;elution programs:0 min,28%A:72%B?8 min,28%A:72%B?17 min,50%A:50%B?21 min,28%A:72%B?25 min,28%A:72%B;flow rate:1.0 mL/min;injection volume:20?L;column temperature:40°C;UV detection wavelength:270 nm.Under these conditions,flavonol glycosides could be well separated from interference on baseline?R>1.5?.Then linerarity,precision,repeatability,stability and accuracy were investigated.Results indicated that linearity was good within the range of 1.563-200.0?g/mL?r²>0.9995?and the method was precise?RSD<2%,n=6?,repeatable?RSD<2%,n=6?,and accurate because recoveries were between 95%and 105%?RSD<2%?.And the prepared samples were stable within 24 hrs?RSD<2%?.Consequently,the established HPLC method was demonstrated to be accurate and reliable.2.Construction of biphase enzymatic hydrolysis system for preparing secondary flavonol glycosides by hydrolyzing original flavonol glycosides of Epimedii Folium.At first,the enzyme that best hydrolyzed original flavonol glycosides?epimedin A and epimedin C?was selected from five commercial enzymes,followed by optimization of the selected enzyme's hydrolysis conditions.Then,the most appropriate organic reagent was screened to establish biphase enzymatic hydrolysis system,and the hydrolysis conditions such as reaction duration and volume ratio of organic solvent to buffer were optimized.Finally,enzyme solution's reusability was investigated.Then,secondary flavonol glycosides could be obtained after the distillation of organic solvent under reduced pressure.As a result,?-glucanase was selected from five enzymes based on their catalysis performance.The optimal hydrolysis conditions of?-glucanase were that,namely,mass ratio of enzyme/original flavonol glycosides was 1:2,hydrolysis duration was 40 mins,reaction temperature was 60°C and pH of buffer was 4.5.Then,propyl acetate was selected to establish biphase enzymatic system.The relative conversion ratio was still above 70%when?-glucanase was reused by seven times.Accordingly,the biphase enzymatic hydrolysis system was demonstrated to improve efficiency and reduce cost.It can reuse enzyme and organic solvent,suggesting its vast prospect.3.In order to further reduce cost,using Epimedium extract as substrate,secondary flavonol glycosides were prepared by biphase enzymatic hydrolysis.At first,using content of sagittatoside A,sagittatoside B and 2''-O-rhamnosyl icariside II and baohuoside I as indexes,the mass ratio of Epimedium extract/?-glucanase,hydrolysis duration and volume ratio of propyl acetate/buffer were optimized in biphase hydrolysis system.Besides,The obtained hydrolysates were isolated and purified by polyamide column chromatography to prepare monomer secondary flavonol glycosides.Results showed that the optimized mass ratio of Epimedium extract/?-glucanase,enzymatic duration and volume ratio of propyl acetate/buffer in biphase enzymatic hydrolysis system were 1:1,3 hrs and 2:1 respectively.Under these optimal hydrolysis conditions,original flavonol glycosides were converted into secondary flavonol glycosides by biphase enzymatic hydrolysis,and the transfer ratios of sagittatoside A,sagittatoside B,2''-O-rhamnosyl icariside II and baohuoside I were 86.2%,90.0%,89.6%and80.2%respectively.Finally,two monomers F1 and F2 obtained by polyamide column chromatography were identified as baohuoside I and sagittatoside A.In summary,this study offered a more economic method for preparing secondary flavonol glycosides in industry.4.To apply enzyme immobilization in preparing secondary flavonol glycosides of Epimedii Folium,immobilized?-glucanase was produced and used to hydrolyze epimedin A.At first,the immobilized?-glucanase was prepared by two different methods.On the one hand,?-glucanase was immobilized in calcium alginate gel by crosslink-embedding method.On the other hand,The amino-functionalized magnetic nanoparticles were prepared and used as carriers for immobilization of?-glucanase by covalent binding method,which was applied for hydrolyzing epimedin A and preparing sagittatoside A.Then,the immobilization conditions were optimized according to immobilization yield and conversion ratio of epimedin A.As a result,when the concentration of?-glucanase was 5 mg/mL,the immobilization yield by crosslink-embedding method was 53.5%and the prepared immobilized enzyme could hydrolyze 33.8%of epimedin A.While the mass ratio of?-glucanase/amino-functionalized magnetic nanoparticles was 10:1?mg/g?,the immobilization yield by covalent binding method was 86.2%and conversion ratio of epimedin A was 30.2%.Thus,there were no obvious difference between immobilized?-glucanase's catalysis capacity of epimedin A by two immobilization methods,but the immobilization yield by covalent binding method is distinctly higher than that by crosslink-embedding method.This research offers a novel idea for preparing secondary flavonol glycosides based on enzymatic hydrolysis method and has a potential of development.