Study On Transforming Of Epimedium Flavonoids By Immobilized Enzymes

Yinyanghuo(Herba Epimdii)listed in the Chinese Pharmacopoeia officially,is one of the Chinese traditional herbals.The main active chemical constituents are four kinds of flavonoids,namely,epiedin A,epimedin B,epimedin C and icariin.According to study of modern pharmacology,Yinyanghuo is potentially important in treating osteoporosis,rheumatism,arthritis and breast lump,etc.However,these kinds of active flavones are poorly absorbed by oral and need to be transformed into metabolites sagittatoside A,sagittatoside B,2"-O-rahmonosylicarside ?,baohuoside ? and anhydroicaritin.Individual intestinal enzymes are different and sometimes even insufficient,leading to affecting absorption.In our preliminary study,metabolites of these epimedium flavonoids could be obtained by enzyme transformation in vitro.However,activities of free enzymes are easy to lose and price of enzymes is high,which are main obstacle for application of free enzymes.In recent years,immobilized enzymes were studied much,because they are reusable,easy to separate and more stable.Transformation using immobilized enzymes avoids losing activities easily,unreusable and high production costs of free enzymes.Compared to chemistry methods,immobilized enzymes are green for environment,high efficiency and high choosing,so we can obtain trace components of Chinese traditional medicine.In this work,we studied three immobilized ezymes,used for transforming epimedium fl avonoids.Firstly,?-glycosidase and snailase were immobilized by barium alginate.Their enzymology and reaction conditions were compared.Immobilized snailase had much higher conversion efficiency than immobilized ?-glycosidase.Therefore,snailase was used for new immobilized method,including microemulsion-gel mesoporous immobilized snailase and mesoporous silicon immobilized snailase.Finally,the best immobilized enzyme was selected and was used for transforming epimedium flavonoids.The products of epimedium flavonoids were test by MTT in vitro.These results were meaningful for obtaining highly active epimedium flavonoids products and study of new drugs for antitumor.In this paper,we firstly give two reviews,namely application of immobilized enzymes in the field of Chinese traditional medicine transformation and stdudy of antitumor effects of epimedium flavonoids.Then experimental researches were presented as follows:1.Enzyme immobilized on barium alginate balls transform epimedium flavonoids1.1 Preparation of enzymes immobilized on barium alginate ballsThe preparation conditions of barium alginate balls were as follows:1.5%BaCl2,1.0%alginate sodium and drppping height of 4.0 cm.The roundness of barium alginate balls could up to 0.987 and were about 2 mm in diameter.Based on the maximum envelopment rate and combination rate of immobilized enzymes,we chose the final concentration of ?-glycosidase was 0.3 mg·mL-1 and final concentration of snailase was 0.4 mg·mL-1.1.2 Preparation of enzymes immobilized on microcapsule of barium alginate-chitosanThe prepared conditons of chitosan shell were as follows:1.0%,12.0 kD chitosan,1.0%Na-TPP,4.0%hole agents of anhydrous sodium chloride.Immobilized enzyme on barium alginate balls were added into the above chitosan,used for preparing immobilized enzymes on microcapsule.1.3 Evaluation of immobilized enzymesThen,the morphology of immobilized enzymes was characterized and enzyme properties were studied including reuseness,storage stability and thermal stability.The results showed that immobilized enzymes we prepared had good properties of reuseness,storage stability and thermal stability.1.4 Enzyme immobilized on barium alginate balls transform epimedium flavonoids(1)Optimal conditons for reactions of enzyme immobilized on barium alginate ballsPhosphate buffer solution pH was 5.0,reaction temperature of 50? for ?-glycosidase and 60? for snailase.(2)Optimal conditons for transforming epimedium flavonoidsQuality of enzyme and substrate was 1:1,substrate concentration of 0.1 mg·mL-1,phosphate buffer solution pH of 5.0.Immobilized ?-glycosidase on barium alginate balls reacted at 50? and reacted 6.0 hour.Immobilized snailase on barium alginate balls reacted at 60? and reacted 2 h.The results showed that transformation efficiency of immobilized snailase was about three times more than immobilized ?-glycosidase.(3)Optimal conditons for transforming epimedium total flavonoids using enzyme immobilized on barium alginate ballsQuality of enzyme and substrate was 1:1,substrate concentration of 1.0 mg·mL-1,phosphate buffer solution pH of 5.0,reaction temperature of 50? and reaction time of 2 h.Epimedium flavonoids and their transformation products were identified by 1H-NMR and 13C-NMR.2.Microspheres immobilized snailase prepared by method of microemulsion-coacervation for transform icariin2.1 Preparation and evaluation of immobilized snailaseThe optimum conditions were as follows:1.0%of barium alginate,1.0%of BaCl2,0.4 mg·mL-1 of snailase solution,ultrasonic washing 30 min by water and repeated 3?5 times.Microspheres immobilized snailase were characterized by scanning electron microscope(SEM).The results showed that the irregular holes were presented on the surface of microspheres.Partical size of microspheres was(157.33±2.01)?m,n=5.Enzyme activities of microspheres immobilized snailase could keep 80%after stored 7 days at 25? and enzyme activities could keep more than 50%after 21 days storage.Microspheres immobilized snailase were used three times to transforming icariin and the average transformation rate is 75.61%.In addition,microspheres immobilized snailase had good thermal stability and pH stability.2.2 Immobilized snailase transform epimedium flavonoidsTemperature was 50?,pH was 5.0 and concentration of icariin was 0.5 mg·mL-1 and mass ratio of enzyme and icariin was 3:1.After reacting 2 h,icariin could be compeletly transformed.3.Immobilized snailase on mesoporous silicon and used for transforming icariin3.1 Preparation and evaluation of immobilized snailase on mesoporous siliconBased on the maximum combination rate,we chose mass ratio of snailase and mesoporous silicon was 1:1,reacted 10 h to prepare immoblilized snailase on mesoporous silicon.The immobilized snailase on mesoporous silicon could retain more than 50%residual enzyme activities after using six times.The residual activities were still more than 80%after stored at 4? for six weeks.Immobilized snailase on mesoporous had good thermal stabilities.Compared to the aboved immoblilized enzymes,immobilized snailase on mesoporous silicon had better enzyme properties.3.2 Optimum reaction conditions for transforming icariinTwo-phase reaction system was comprised of 40%DMSO(organic phase)and 60%phosphate buffer of pH 5.0(water phase).The mass ratio of immoblilized snailase on mesoporous silicon and icariin was 1:1.Icariin could almost be transformed after reaction 2 h at 50?.4.Analysis of cytotoxicity in vitro4.1 Cytotoxicity of epimedium total flavonoids in vitroThe cytotoxicity of epimedium total flavonoids and products were monitored in A549,HepG2,MCF-7 and Hela cells using MTT assay.The results showed that products appeared higher cytotoxic than epimedium total flavonoids and had the highest cytotoxicity effect on MCF-7 cells.Therefore,MCF-7 cells were used for a further study.4.2 Cytotoxicity of different monomer flavonoids in herba epimdiiThe MTT showed that cytotoxicity of correspounding products was significantly higher than monomer flavonoids substrates at all the concentrations we used.Otherwise,baohuoside I appeared the greatest cytotoxic effect in all the epimedium monomer flavonoids.