Isolation And Purfication Of Oligomeric Proanthocyanidins From Green Mulberryand Their Study Of Antioxidant And Hypoglucemic Activities

Green mulberry belongs to Morus,a fruit from mulberry tree,It is also known as yellow mulberry,which refers to the mulberry in the green fruit stage As a traditional Chinese medicinal material,Its effects are to clear the liver and brighten the eyes,complement blood and spirit,promote fluid production,and quench thirst.Modern research has shown that green mulberry exhibits higher antioxidant potential than mature mulberry,which is related to the higher content of proanthocyanidins in green mulberry than in mature mulberry.China,as a large country in mulberry breeding and silkworm rearing,has abundant mulberry resources.In order to better utilize the by product resource of green mulberry,this article explored the extraction,separation,and purification methods of oligo-procyanidin from green mulberry,as well as the antioxidant and hypoglycemic activities of its isolated components.The main results are as follows:（1）Using ethanol as the extraction agent to extract proanthocyanidins from green mulberry,the optimal extraction process obtained by single factor experiment and response surface simulation analysis is as follows:Under the conditions of solid-liquid ratio of 1:31,ethanol concentration of 62%,PH value of 5.78,temperature of 40℃and extraction time of 90 min,the yield of anthocyanin in green mulberry was 12.56±0.15mg/g.（2）Using solvent extraction method to remove impurities and enrich the extract of proanthocyanidins from green mulberry,obtaining the crude extract of oligomeric proanthocyanidins from green mulberry.The effects of the solvent system,the mobile phase flow rate,the host velocity,and the amount of charge on the separation effect of high-speed countercurrent chromatography were investigated,and the best preparation method was achieved:Under the conditions of the n-hexane-ethyl acetate water solvent system（1∶20∶20,V/V/V）,the flow rate of the mobile phase was 2 mL/min,the host velocity was 850 r/min,the loading volume was 200 mg,High speed countercurrent chromatography was used to separate the crude extract of oligomeric proanthocyanidins and five components were obtained in 500 minutes.The main components of different fractions were analyzed by high performance liquid chromatography and mass spectrometry.Among them,F1 contained catechin,proanthocyanidins B2,proanthocyanidins A1,quercetin,protocatechuic acid and other substances;F2 contained substances such as epicatechin gallate and chlorogenic acid;F3 contained substances such as proanthocyanidin B2;F4 is the main component of epigallocatechin;F5 is the main component of epicatechin.（3）The antioxidant activities of different components were investigated in vitro,The ability of DPPH free radical,hydroxyl free radical,and ABTS⁺free radical scavenging of the five fractions separated by high-speed countercurrent chromatography was investigated.At the same time,the proanthocyanidins content in each fraction and the total antioxidant capacity of the fractions were measured.Meanwhile,The different zones corresponded to the content of prohormones and the amount of antioxidants.Experimentally,all components were found to have some antioxidant capacity,ABTS⁺free radical and the DPPH free radical of F5 and F4 had a quenching capacity that approached that of the same vitamin C concentration,and the free radical scavenging capacity of the hydroxyl group was stronger than that of the same vitamin C concentration,Thus,the higher the procyanidins content of each component,the greater the total antioxidant capacity of the constituents.（4）The study of active effects onα-glucosidase andα-glucoase of different separated components and the in vitro sugar effect of different components showed that each of the surface components could have constant inhibition of the activity ofα-glucosidase andα-glucoase,and was also compared with the activity ofα-glucoase,Inhibition ofα-glucosidase activity of oligomeric green mulberry proanthocyanidins resulted in improved F3 activity,Enzymatic inhibition of F4 and F5 atαglycosidase was much stronger than with the concentration of acarbose and the other components,the concentration of IC₅₀alue of 0.064,0.058,0.058 mg/mL.Inhibition of F4 and F5byα-amylase activity was shown to be superior to that of acarbose,with IC₅₀values of0.320 and 0.468 mg/mL.Through the analysis ofα-glucosidase inhibition kinetics of F3,F4 and F5,F3 and F4 components belonged to reversible mixed inhibitors ofα-glucosidase,and F5 components belonged to reversible competitive inhibitors ofα-glucosidase,and the three components had a single inhibition site or a single class of inhibition site onα-glucosidase.The inhibitory effect of monomer onα-glucosidase was studied by molecular docking simulation experiment and the relevant conclusions of enzyme kinetic inhibition experiment were verified.