Extraction And Biotransformation Of Main Flavonoids From Epimedium

Epimedium is a commonly used Chinese herb medicine whose main active ingredients are flavonoids,including Epimedin C(triosaccharide),icariin(disaccharide),Baohuoside Ⅰ(monosaccharide),and icaritin(sugar-free),etc.The content of polyglycosides in epimedium flavonoids is relatively high,but the permeability in vivo is poor and the absorption is not good.Monoglycoside or non-glucoside flavonoids are well absorbed,but their natural content in Epimedium is usually low,so they cannot be prepared in large quantities by separation and purification.Therefore,this paper takes Epimedium as the research object.While studying the extraction and purification of its main flavonoids,it focuses on the transformation of Epimedium polysaccharide base flavonoids into monosaccharide or glucose-free flavonoids using enzymes,in order to establish an effective method for the preparation of high-value flavonoids.The main research contents are as follows:1.Extraction of main flavonoids from Epimedium.HPLC was established for the determination of Epimedium flavonoids.The effects of different extraction conditions(ethanol concentration,solid-liquid ratio,extraction time and extraction temperature)on the extraction amount of main flavonoids(Icariin,Epimedin C and Baohuoside Ⅰ)in Epimedium were studied.Using single factor and orthogonal experiment optimization,the optimal extraction conditions were determined as follows: solid-liquid ratio 1:25,Ethanol concentration of 60%,extraction temperature 60℃;Extraction time 60 min.Verification experiment proved that the process was stable and reliable,and the total extraction amount of three flavonoids was 25.44 mg/g.2.Isolation and purification of main flavonoids from Epimedium.The macroporous adsorption resin method was used for separation and purification.HPD100 macroporous resin was used for separation and purification of main flavonoids(Icariin,Epimedin C and Baohuoside Ⅰ)of Epimedium through static adsorption and desorption experiments.The optimal purification process was determined as follows: the quality concentration of the upper column solution is 4mg/ml,the upper sample flow velocity and the elution flow rate are all 1 BV/h,3 BV30% ethanol solution removes pigment and other impurities.Use 50% ethanol to elute with 6BV,obtaining the Epimedin C and icariin.The total purity of the two compounds increased from 26.16% to 77.14%,with yield rates of 72.9%.Then use 80% ethanol to elute with 3 BV,obtaining Baohuoside Ⅰ,its purity increased from 1.93% to 47.33%,and the gain rate was 76.1%.3.Biotransformation of Epimedium flavonoids.We studied snail enzymes,α-L-rhamnosidase and β-glucosidase for biotransformation of Epimedin C and Icariin.The results showed that Baohuoside Ⅰ could be prepared by α-rhamnosidase and β-glucosidase hydrolysis of Epimedin C,and the conversion rate was 59.8%.And icaritin was prepared by α-L-rhamnosidase and snail enzyme hydrolysis with a conversion rate of 17.8%.Icariin was hydrolyzed by β-glucosidase to prepare Baohuoside Ⅰ,and the conversion rate was 69%.And icaritin was prepared by snail enzymatic hydrolysis with a conversion rate of 38.6%.4.Determination of bioactivity of Epimedium flavonoids.DPPH method,ABTS method and hydroxyl radical method were used to compare the antioxidant activities of four kinds of Epimedium flavonoids.The results showed that the antioxidant activity of the four samples in a certain concentration range was proportional to the concentration.Icaritin showed relatively strong antioxidant activity,followed by Baohuoside Ⅰ,Icariin and Epimedin C,respectively.Then,the four flavonoids were tested by filter paper method.The results showed that Baohuoside Ⅰ had good inhibitory effect on Escherichia coli and Staphylococcus aureus,while Epimedin C had no inhibitory effect on the two bacteria.