Biotransformation Of Icariin Based On Immobilized Enzyme On SiO₂

Icariin is the main active flavonoid component of the traditional Chinese medicine Epimedium,which has a significant anti-liver cancer effect.Previous research by the research group found that icariin can be hydrolyzedto baohuoside?by intestinal enzymes?bacteria?after oral administration to the intestine,and the intestinal absorption and pharmacological activity of baohuoside?is better than icariin.In the pathological state,due to the decrease in activity and quantity of the intestinal enzyme?bacteria?icariin cannot be effectively hydrolyzed to baohuoside?,and its efficacy will be badly affected.In order to solve the above problems,this paper proposes the following two solutions:?1?in vitro biotransformation of icariin using immobilized enzymes to efficiently obtain the better-absorbed and more active baohuoside?;?2?Oral immobilized enzymes as the in vivo biotransformer of icariin to promote the real-time enzymatic hydrolysis and absorption of icariin in the intestinal tract and improve the antitumor efficacy.Therefore,in this study,immobilized snailase on cross-linked nano-SiO₂and immobilized?-glucosidase on cross-linked mesoporous silicon SBA-15 were prepared.The preparation technology,physical and chemical characterization,optimal enzymatic hydrolysis conditions and the enzymatic properties were studied,and an efficient and stable in vitro biotransformation process of icariin was established,which laid the experimental foundation for green industrial production of baohuoside?.In addition,the immobilized cellulase on crosslinked mesoporous silicon SBA-15 was also prepared by adsorption-crosslinking method,and the preparation process,physical and chemical characterization,biomimetic enzymatic hydrolysis in vitro,and the in vivo fluorescence tracer distribution behavior of immobilized enzyme after intragastric administered to mice were investigated,which provide a experimental basis for improving theintestinal enzymatic hydrolysis,absorption and anti-liver cancer efficacy of icariin.The main research contents of this article were follows:1.In vitro transformation of icariin by immobilized snailase on cross-linked nano-SiO₂Using 3-aminopropyltriethoxysilane?APTES?as amino modifier and glutaraldehyde as cross-linking agent,the immobilized snailase on cross-linked nano-SiO₂was prepared by covalent coupling method,and its preparation process,physical and chemical characterization,optimum enzymatic conditions and enzymatic properties were investigated.The results showed that the best preparation process for immobilized snail enzyme is:SiO₂/APTES feed ratio was 3?1,glutaraldehyde mass fraction was 0.25%,enzyme loading ratio was 1?3,and immobilization time was 6 h.The enzyme activity of the immobilized enzyme prepared under the above preparation conditions was 151.0?mol/h/g,the enzyme activity recovery rate was 91.0%,the enzyme loading amount was 0.270 g/g,and the fixation rate was 81.8%.The results of TEM,FTIR,elemental analysis and thermogravimetric analysis showed that the snailase was successfully immobilized on the surface of the crosslinked nano-SiO₂.The optimum enzymatic conditions for the immobilized enzyme were p H 5.0,conversion temperature 60°C,the ratio of enzyme to substrate mass 1?2,and the conversion time12 h.The enzymatic kinetic parameters Vmax was 0.43?g/min and Km was 0.78mmol/L.After 5 times of repeated use,the remaining relative enzyme activity remained above 70%,but the thermal stability was not significantly improved compared to that of the free enzyme.The immobilized snailase on the crosslinked nano-SiO₂has good mechanical strength and enzymatic properties,and can still maintain good catalytic performance after repeated use.The total enzymatic hydrolysis efficiency of the immobilized snailase was greatly improved compared to that of the free enzyme,and it can be used for the efficient in vitro conversion of icariin to abtain baohuoside?.2.In vitro transformation of icariin by immobilized?-Glucosidase onmesoporous SiO₂Using?-glucosidase as hydrolase of the icariin,mesoporous SiO₂SBA-15 as immobilized carrier,and chitosan as mesoporous blocking agent,immobilized?-Glucosidase on the cross-linked SBA-15 was prepared by adsorption-crosslinking method.The preparation technology,physical and chemical characterization,optimal enzymatic conditions and enzymatic properties were investigated.The results showed that the optimal preparation p H of immobilized?-glucosidase was 6.0,the immobilization time was 8 h,the enzyme concentration was 7 mg/m L,and the amount of chitosan was 0.1 mg/mg SBA-15.The enzyme activity of the immobilized?-glucosidase was 439.2?mol/h/g,the enzyme activity recovery rate was 93.7%,the enzyme loading amount was 1.120 g/g,and the fixation rate was 92.8%.TEM,SEM,FTIR and TGA results showed that the enzyme was successfully immobilized in the mesoporous channels without affecting the morphology of the carrier.The optimal enzymatic conditions for the immobilized?-glucosidase were p H 6.0,conversion temperature 50°C,substrate concentration 0.5 mg/m L,and conversion time 8 h.Enzymatic kinetic parameters V_maxwas 10.24?g/min and K_mwas 16.15 mmol/L.The thermal stability of the enzyme was significantly improved after immobilization,and the remaining relative enzyme activity remained above 60%after 5 times of reuse.The immobilized?-glucosidase oncross-linked SBA-15 has the advantages of simpler preparation process,higher enzyme loading and better enzymatic performance,and is more suitable for the efficient transformation of icariin into baohuoside?in vitro.3.In vitro enzymatic hydrolysis of icariin by immobilized cellulase on cross-linked mesoporous SiO₂Immobilized cellulase on cross-linked SBA-15 was prepared by using cheap and readily available cellulase as the hydrolase of icariin,mesoporous SiO₂SBA-15 as immobilized carrier,and chitosan as mesoporous blocking agent.The preparation process,physical and chemical characterization,and in vitro enzymolysis of immobilized cellulose were investigated.The results showed that the optimal preparation p H of immobilized cellulase was 4.4,the immobilization time was 6 h,the enzyme concentration was 5 mg/m L,and the amount of chitosan was 0.1 mg/mg SBA-15.The enzymatic activity of the immobilized cellulase was 469.8?mol/h/g,the enzyme activity recovery rate was 84.1%,the enzyme loading amount was 0.620 g/g,and the immobilization rate was 61.7%.TEM,SEM,FTIR,TGA and N₂adsorption-desorption results showed that the enzyme was successfully immobilized in the mesoporous channels without affecting the carrier morphology of the carrier.The results of bionic enzymatic hydrolysis in vitro showed that the immobilized cellulase maintained good enzymatic hydrolysis activity in both artificial gastric juice and artificial intestinal fluid,and the optimal enzymatic hydrolysis ratio of icariin to the icariin was 4?1.The immobilized cellulase on cross-linked SBA-15 has the advantages of simple preparation process,strong catalytic activity,good stability,safety and non-toxicity,etc.It can be used orally as an in vivo bioreactor of prodrug icariin to promote the real-time enzymatic hydrolysis and absorption of icariin in the intestinal tract.4.In vivo fluorescence tracing study of immobilized cellulose on the cross-linked mesoporous SiO₂FITC labeled cellulase?FITC-Cel?was prepared by covalent coupling method using FITC as fluorescence tracer,and then FITC-Cel was fixed on the chitosan crosslinked SBA-15?FITC-Cel-SBA-15?.The in vivo distribution of FITC-Cel-SBA-15in mice was studied by small animal imaging fluorescence tracer.The results showed that the fluorescence signals of FITC-Cel-SBA-15 were mainly concentrated in the gastrointestinal tract,and the fluorescence intensity of FITC-Cel-SBA-15 was significantly stronger than that of FITC-Cel in the gastrointestinal tract after 4 hours.The immobilized cellulase on the cross-linked SBA-15 has strong intestinal retention capacity,which is helpful to promote the hydrolysis of icariin into baohuoside?in the intestinal tract,and then improve the anti-hepatoma effect.