Studies On Extraction,Purification And Bioactivities Of Flavonoids In Blueberry

Anthocyanins and flavonols are two important flavonoids in blueberry. Because of containing so many kinds of anthocyanins and flavonols, blueberries become functional health food that people pay much attention to.This experiment used blueberries as raw materials to study extraction, purification and separation.The anthocyanins and flavonols samples were studied by the structure identification and analysis of activities of anthocyanins. All this done is in order to provide theoretical basis and some scientific message for developing and utilizing blueberry.Anthocyanins and flavonols were extracted by ultrasonic assistant extraction methods.In the extraction process, response surface design was used to study the method of conditions of ultrasonic assistant extraction methods.The optimal extraction conditions of flavonols and anthocyanins were determined as follows:ultrasonic power512.7W, extraction time29.8min, liquid to solid ratio9.5:1mL/g. Under a modified condition:ultrasonic power510W, extraction time30min, liquid to solid ratio10:1mL/g, the experimental yields of flavonols and anthocyanins were0.806mg/g and2.903mg/g respectively, which were agreed closely with the predicted values (0.804mg/g and2.899mg/g).In the purification process, macroporous adsorption resin and Sephadex LH-20medium voltage column chromatography method were used to study purification.Comparing with the anthocyanins’ adsorption rate and desorption rate of six kinds of macroporous to choose the suitable macroporous resins,the static tests results showed:D101was selected as the best macroporous resins.By D101macroporous adsorption resin medium voltage column chromatography researching dynamic test,the appropriate conditions were as follow:the concentration0.073mg/mL, injection volume10mL,adsorption flow rate5.0mL/min, elution solvent80%ethanol. Under the conditions, the anthocyanins recovery rate was96.61%and the purity was75.58%; Comparing with the flavonols’adsorption rate and desorption rate of seven kinds of macroporous to choose the suitable macroporous resins,the static tests results showed:S-8was selected as the best macroporous resins.By S-8macroporous adsorption resin medium voltage column chromatography researching dynamic test,the appropriate conditions were as follow:the concentration0.362mg/mL, injection volume10mL.adsorption flow rate2.0mL/min, elution solvent95%ethanol. Under the conditions, the flavonols recovery rate was92.83%and the purity was55.58%%.The Sephadex LH-20column was applied to the process of separation. The best separation conditions of anthocyanins were as follow:the concentration1.0mg/mL, injection volume lOmL,20%、35%、50%methanol gradient elution.20%methanol, elution flow1.0mL/min,35%、50%methanol, elution flow3.0mL/min.Anthocyanin samples made from the above methods and conditions were identified.The analysis results showed the sample I was cyanidin-3-O-glucoside, and cyanidin-3-O-glucoside was measured by HPLC method and its purity was90.88%. The best separation conditions of flavonols were as follow:the concentration1.0mg/mL, injection volume10mL,50%、70%、100%methanol gradient elution. Flavonols sample I made from the above methods and conditions were identified.The analysis results showed the sample I was quercetin, quercetin was measured by HPLC method and its purity was89.10%,and sample II may be myricetin.Anthocyanins purified by macroporous resin were investigated on the inhibitory action on the non-enzyme glycosylation from the three stages of the reaction.The results showed that anthocyanins had inhibitory action on the non-enzyme glycosylation, and inhibitory action grew with the grow of the concentration of anthocyanins, along with the extend of time,inhibition rate first grew then decreased.When concentration of anthocyanins0.2mg/mL, inhibition rate of NBT reduction reached11.80%and the the second carbonyl compounds generated inhibition rate was39.44%, the inhibitory rate of the non-enzyme glycosylation was45.44%on the third day. This results indicated that the inhibitory action on the non-enzyme glycosylation mainly occurred at the last stage of the the reaction and the inhibition of NBT reduction is not obvious.