Screening And Preparation Of α-Glucosidase Inhibitors From Epimedii Folium Extracts

α-glucosidase,located in villous epithelial cells of small intestine,can specifically cutα-1,4 glycosidic bonds to release glucose and increase the level of postprandial blood glucose.Therefore,reduction of postprandial hyperglycemia by inhibitingα-glucosidase activity is an effective approach for treatment of type 2 diabetes mellitus.α-Glucosidase inhibitors play a crucial role in lowering blood glucose,reducing glycolated hemoglobin levels and incidence of diabetes complications.Hypoglycemic drugs used in clinical practice,such as acarbose and voglibose,often cause liver damage,abdominal cramps or gastrointestinal problems.Natural medicines have attracted broad attention worldwide Rapid screening and obtaining safe and efficientα-glucosidase inhibitors from them has become a research hotspot in the treatment of type II diabetes and the development of hypoglycemic health food.Traditional Chinese medicine Epimedii Folium is an important source ofα-glucosidase inhibitors.For example,preparations containing Epimedii Folium,Antithirst Formula and Tongmai Jiangtang Capsules have been used to relieve diabetic syndrome.However,the specific components of Epimedium that play an inhibitory role in enzyme activity are still not clear.Therefore,in this study,we constructed an immobilizedα-glucosidase by covalent binding,screenedα-glucosidase inhibitors from Epimedii Folium extract by"ligand fishing",and then identified their chemical structures and studied their hypoglycemic mechanism in vitro.A two-phase enzymatic hydrolysis system and a novel continuous enzymatic hydrolysis technology based on immobilized enzyme were constructed and optimized respectively to obtain baohuoside I,an active component of hypoglycemic in Epimedii Folium.My research includes following four parts:1.The UPLC-UV analysis methods for eight flavonoid glycosides in Epimedii Folium,including epimedin A,epimedin B,epimedin C,icariin,sagittatoside A,sagittatoside B,2"-O-rhamnosyl icariside II and baohuoside I,were established,and the methodology was investigated.Chromatographic conditions were as follows:ACQUITY UPLC BEH-C18 column(150 mm L.×2.1 mm I.D.,1.7μm);ddetection wavelength was 270 nm;flow rate was 0.2 m L/min;injection volume was 1μL;column temperature was 35°C;mbile phase was composed of acetonitrile and 0.1%formic acid solution.Eight flavonoid glycosides were separated from baseline under the chromatographic conditions(R>1.5),and the impurity in Epimedium extract and other substances produced during enzymatic hydrolysis did not interfere with the determination of flavonoid glycosides.In the concentration range from 0.7812μg/m L to 200.0μg/m L,the linear relationship between peak area and concentration was good(r~2≥0.9999).RSD of precision,reproducibility and stability tests were all less than 2%.Average recoveries of eight flavonoid glycosides ranged from 97%to 103%,with RSD less than 2%.These results indicated that the established UPLC-UV method could accurately and reliably determine the content of eight flavonoid glycosides,which provided a reliable method for the identification of hypoglycemic active components in Epimedii Folium extract and the identification and purity determination of enzymatic hydrolysate.2.Magnetic nanoparticles immobilizedα-glucosidase were prepared,and then they were used to"fish"α-glucosidase inhibitors from Epimedii Folium extract.Structure of the active compounds were identified and mechanism of inhibitingα-glucosidase activity was preliminarily discussed.Firstly,the conditions ofα-glucosidase immobilization were optimized by single factor test and response surface Box-Behnken design.The optimal conditions were as follows:buffer p H was 6.8,α-glucosidase concentration was 1.2 mg/m L,and immobilization time was 4.2 hrs.Secondly,a model for screeningα-glucosidase inhibitors based on immobilized enzyme was established.Then eight active compounds were screened from Epimedii Folium extract and they were identified as epimedin A,epimedin B,epimedin C,icariin,sagittatoside A,sagittatoside B,2"-O-rhamnosyl icariside II and baohuoside I via UPLC-ESI-QQQ-MS analysis.In vitro enzyme activity inhibition assay and molecular docking simulation results showed that baohuoside I and sagittatoside B inhibitedα-glucosidase activity more strongly than acarbose,and higher than other six active compounds.All eight active compounds inhibitedα-glucosidase activity via binding to the active central site ofα-glucosidase through hydrophobicity and hydrogen bonding.Among them,baohuoside I had the lowest binding free energy,showing a strong enzyme inhibition activity.3.Two-phase enzymatic hydrolysis technology for efficient and convenient preparation of rare baohuoside I was established and optimized,and engineering research and revenue estimation were carried out.Firstly,on the basis of conventional enzymatic hydrolysis conditions,the biphasic enzymatic hydrolysis system was constructed with the propyl acetate/buffer liquid ratio of 21:and then the conditions were optimized.Results showed that after hydrolyzing 6.0 hrs at 60°C and standing for1.0 hr,icariin conversion rate and baohuoside I transfer rate were 99.8%and 80%,respectively.Moreover,hydrolytic capacity of icariin of this system was increased from0.5 mg/m L to 100 mg/m L.When the amount of icariin was halved and buffer containingβ-glucanase was repeated for 3 times,the conversion rate of icariin was still as high as89.5%.In addition,in 50-fold and 100-fold amplification system,the conversion rate of icariin was 99.9%and 98.5%,the yield of baohuoside I was 69%,and the purity of baohuoside I was above 98.0%.According to experimental process,process flow and workshop design of biphasic enzymatic hydrolysis of icariin with an annual output of1 ton of baohuoside I were carried out,and the cost accounting and profit estimation were carried out according to the actual situation and the design drawings.4.A packed bed reactor based on immobilizedβ-glucanase is planned to be constructed to prepare baohuoside I,continuously and eco-friendly.β-glucanase was immobilized on chitosan gel sphere using glutaraldehyde as crosslinking agent.Results of single factor analysis showed that when glutaraldehyde concentration was 1.0%,cross-linking time was 2.0 hrs,concentration ofβ-glucanase was 1.5 mg/m L and immobilization time was 10.0 hrs,immobilization rate ofβ-glucanase,activity recovery of immobilizedβ-glucanase and relative conversion rate of icariin reached the highest levels.Key factors affecting hydrolysis rate were found during construction of this system.The factors included physicochemical properties of baohuoside I and icariin,purity and properties of enzyme,stability of immobilized enzyme and the way of sample loading,etc.,which provided references and ideas for subsequent studies on continuous preparation of active secondary glycosides via immobilized enzyme packed bed bioreactor.