Study On The Aqueous Two-phase Extraction Coupled With Enzymatic Hydrolysis Of Flavonoids

Based on the high efficiency,mild conditionsof enzymatic hydrolysis-assisted extraction and the demixing effect and selectively of aqueous two-phase extraction,a rapid,effective and environmentally friendly biological extraction and separation technology that the enzymatic hydrolysis coupled aqueous two-phase extraction technology was established.And it was applied to the extraction of flavonoids from the seed of Cuscuta chinensis Lam.and epimedium.The main research contents were as follows:The enzymatic hydrolysis coupled aqueous two-phase system(ATPS)was used to extract flavonoids from the seed of Cuscuta chinensis Lam..28%(w/w)ethanol-18%(w/w)ammonium sulfate aqueous phase system was used as the best extraction system.The enzymatic hydrolysis and ATPS were combined,and it was found that the extraction effect with the addition of enzyme was significantly better than that use of single enzyme.By researching the effect of enzyme species on the extraction rate of flavonoids,it was found that the extraction efficiency of the composite enzyme consisting of 5%(w/w)cellulase and 5%(w/w)protease was more significant than that of single enzyme extraction.In addition,the qualities of Semen cuscutae from different habitats were found to be different.Compared with the flavonoids of Semen cuscutae in Tai'an,Shandong Province,although the content of hyperoside in the flavonoids of Semen cuscutae in Inner Mongolia is smaller,the content of astragalin and quercetin is relatively higher.The antioxidant activity of Semen cuscutae flavonoids was evaluated by the scavenging rate of 1,1-diphenyl-2-picrylhydrazyl(DPPH)free radical.The flavonoids exhibited very strong concentration-dependent antioxidant potentials against DPPH radicals.In the experiment,the kinetics of scavenging DPPH free radicals by flavonoids were determined,and the results were consistent with the first order reaction kinetics.Compared with heated reflux solvent extraction(HRSE)and ultrasound-assisted extraction(UAE),the extraction rate of flavonoids in this method is higher,reaching 18.75 mg/g.The method has the characteristics of low cost,easy to operate and mild experimental conditions.It is an environmentally friendly and efficient extraction and separation method.The aqueous two-phase system consisted of 28%(w/w)ethanol and 18%(w/w)ammonium sulfate was used to extract icariin from Epimedium extract,and the content of icariin was 2.95%.Subsequently,enzymatic hydrolysis coupled with aqueous two-phase extraction was applied to biotransformation and product extraction of total flavonoids from epimedium.In the experiment,pectinase was used to hydrolyze the total flavonoids of Epimedium.The factors affecting the enzymatic transformation were investigated,including enzyme dosage,enzymatic hydrolysis time and enzymatic hydrolysis temperature.In addition,the enzymatic properties were investigated,and it was found that glucose had a certain inhibitory effect on the enzymatic hydrolysis ability of the enzyme,the organic reagent ethanol can make the enzyme activity lose,and adding ammonium sulfate to the sample solution in advance can improve the enzyme activity and promote the hydrolysis of total flavonoids in Epimedium.Ultimately determine the best process conditions for the enzyme dosage was 12 mg,the substrate concentration was 1.07 mg/mL,the 0.8 g ammonium sulfate was added in enzyme solution in advance,the time of enzymolysis was 3 h,the condition of enzyme solution temperature is 52.5?,and the conversion rate of epimedium glycoside in the Epimedium total flavonoids was over 99.0%.In addition,other unknown flavonoids in the total flavonoids of Epimedium also undergo hydrolysis transformation,and the specific transformation regularity needs further research.