Ultrasound-Assisted Extraction And Purification Of Flavonoids From Mulberry Leaves And Inhibition Of Xanthine Oxide Enzyme

Mulberry has a long history of use in China and is the first drug and food variety approved by the National Health and Health Committee.The active substances in mulberry leaves have obvious effects on oxidation,blood sugar lowering and blood fat reduction.Flavonoids are one of the main active substances in mulberry leaves,and have been used as natural pigments and antioxidants in food and cosmetics.Based on the better development and utilization of mulberry leaves,this study studied the extraction and purification of flavonoids from mulberry leaves,and analyzed the antioxidant properties of mulberry leaves flavonoids and the inhibition of xanthine oxide enzymes.The compounds in the flavonoids of mulberry leaves were qualitatively analyzed using the method of LC-MS/MS.In addition,the binding mode of three flavonoids?Rutin,Isoquercetin,Astragalin?to xanthine oxide enzyme active sites was studied by molecular docking.The results of the study have a certain role in promoting the economic value of mulberry leaves,which proves that mulberry leaves can be developed as a potential drug or functional food for preventing or treating gout.The mulberry leaves were dried and pulverized,and the total flavonoids were extracted from the mulberry leaves by ultrasonic assisted extraction.The optimal extraction process conditions were obtained by single factor experiment and response surface experiment optimization.The results showed that the optimal extraction process was ethanol concentration 80%,solid-liquid ratio 1:30?g/mL?,ultrasonic time75 min,ultrasonic temperature 40°C,extraction rate up to 2.694%.The crude extract of mulberry leaves flavonoids was subjected to 3 times of alcohol precipitation and then purified again with macroporous resin.The purification process conditions were studied by static adsorption combined with dynamic adsorption.The experimental results show that the optimal process conditions are:the concentration of the sample solution is 1 mg/mL,pH=4,the loading flow rate is 2mL/min,the eluent is 70%ethanol solution,the elution flow rate is 2 mL/min,and the purity is 12.3%increased to 33.8%.The flavonoids of mulberry leaves were qualitatively analyzed by LC-MS/MS.The mobile phase was composed of 0.1%aqueous formic acid?solvent A?and acetonitrile?solvent B?.A total of 12 compounds were identified,including 6flavonoids,5 phenolic acids,and 1 coumaric acid.The in vitro antioxidant activity of mulberry leaves flavonoids was determined by three different methods,namely,the removal of superoxide,hydrogen peroxide and hydroxyl radicals,with ascorbic acid as a positive control.The results showed that the IC₅₀0 of flavonoids was 0.33±0.006mg/mL,45±0.8?g/mL and2.54±0.05mg/mL,respectively.The inhibition of flavonoids and flavonoids on the xanthine oxide enzymes showed that mulberry leaves flavonoids,Rutin,Isoquercetin and Astragalin inhibited the xanthine oxide enzyme,and the inhibitory activities of mulberry leaves flavonoids and Rutin were compared.high.The results of molecular docking using Discovery Studio 2.5 software show that Rutin,Isoquercetin and Astragalin can lose some of their activity through hydrogen bonding and?-?stacking with xanthine oxide enzyme.The results of kinetic inhibition of xanthine oxide enzyme enzyme on mulberry leaves flavonoids showed that mulberry leaves flavonoids exhibited reversible mixed competitive inhibition.