Studies On Extraction And Antioxidant Properties Of Black Raspberry Anthocyanin

Black raspberry(Heritage)is large-scalely cultivated in China and exported to abroad,which serves as a natural food plays important roles in health care and disease prevention because of its rich nutrition,especially riched in anthocyanins.Anthocyanins have excellent free radical scavenging ability,and can be used in the fields of anti-oxidation,anti-inflammatory,anti-tumor and hypoglycemic.Heritage black raspberry shows outstanding nutritional value and commercial value owing to the higher anthocyanin contents than that of others.Therefore,Heritage black raspberry was chosen as the research object to analysis its constituent and color value.The response surface methodology was used to optimize the extraction process,which can provid theoretical model for scientific extraction of anthocyanin from black raspberry.Additionally,the abilities of antioxidant activity in vitro and inhibiting oxidative stress effect on cell level were performed to evaluate the antioxidant properties of black raspberry.These researches provided theoretical foundation for comprehensive exploitation of Heritage black raspberries.Main works are listed as followings:(1)Single factor experiment was designed for anthocyanin extraction from black raspberry with alcohol-water as extraction solvent solution and the independent variable included different ethanol concentration,water bath time and extraction temperature.By optimizing the extraction process,ethanol concentration of 33.58%,extraction time of 66.32 min,and solid-liquid ratio of 6.92 mL/g were obtained.The relative error of extraction products between response surface optimization and actual operation is 2.75%.Black raspberry anthocyanins color value was 34.13 that was improved 15.95 times than that of before purification.Anthocyanin content extracted from black raspberry was 756±11 mg/100 g,which were much higher than that of Bristol black raspberry(325.5 mg/100g),smoke 73 grape(313.9 mg/100g)and black bean(383.6 mg/ 100g).(2)A total of 5 kinds of anthocyanins were determinated using HPLC-MS and analysed by mass spectrum database.,They were Cyanidin-3-glucoside,Cyanidin-3-arabinose,Cyanidin-3-malonylglucoside,Cyanidin-3-ortodioxalyglucoside and Cyanidin-3-(6′′-cafteoyl sophoroside)-5-glucoside)respectively.Among them,the Cyanidin-3-glucoside was the main composition of anthocyanins in black raspberry,accounting for 69.32% of the total content.These components are the most common anthocyanins in plants and their glycosylation groups and acylation structures have strong antioxidant properties.(3)The black raspberry anthocyanins showed excellent reducing ability and scavenging ability in DPPH,ABTS,O2-· and ·OH free radical scavenging ability tests.IC50 of black raspberry anthocyanin obtained in O2-· and ·OH scavenging ability test were 41.79 μg/mL and 137.54 μg/m L,respectively,which are lower than IC50 of Hainan Syzygium(151.051 μg/m L,85.0 μg/m L).Black raspberry anthocyanin displayed excellent DPPH and ABTS free radical scavenging abilities and IC50 of back raspberry anthocyanin were 14.11 μg/m L and 14.95 μg/m L,respectively,which was much lower than IC50 of black raspberry wine of 130 μg/m L and 198 μg/m L.Generally,the pecking order of radicals scavenging abilities of black raspberry anthocyanin were: DPPH>ABTS>O2-·>·OH.(4)According to the method of cell antioxidant(CAA),an oxidative damage model of GA induced EC-109 cells was established.To investigate the antioxidant activities of black raspberry anthocyanin in cell,apoptosis,reduced glutathione decreasing,reactive oxygen release,mitochondrial membrane potential changes and nucleus damage were evaluated.The results suggested that GA(8 mM)could lead to survival rate of EC-109 cells decreased significantly,the consumption of reduced glutathione(GSH)increased,the nucleus pycnosis,broken,even apoptotic bodies formed,the reactive oxygen species(ROS)exploded and mitochondrial membrane potential(MPP)declined.However,black raspberry anthocyanins(10 μg/mL,40 μg/m L,80 μg/m L)pretreatment can significantly reduce cell apoptotic induced by GA,inhibite GSH consumption,prompte the karyomorphism get more satiation,reduce apoptosis body,protect the nuclei from damage,greatly reduce the production of ROS,repaire the mitochondrial membrane and enhance the membrane potential.These results demonstrated that black raspberry anthocyanins showed protection on oxidative stress injury of EC-109 cells induced by GA,which suggested that black raspberry anthocyanin dispayed excellent antioxidant ability in cell.