| The black chokeberry is rich in anthocyanins resources,and anthocyanins,as a type of natural antioxidants,is bound to prevent and protect Alzheimer's disease which is related to oxidative stress.Therefore,in order to isolate the high-purity and strong anti-oxidantion anthocyanins from Aronia melanocarpa,the extraction,purification,identification and antioxidant activity of anthocyanins in the black chokeberry were studied in this research.Then,the anthocyanins was used to further studied protective effects on oxidative stress and apoptosis induced by?-amyloid peptide(1-42)in Alzheimer's disease cell model(SH-SY5Y).The fruit weight,total soluble solids,extraction rate,total anthocyanin content and total phenolic content from Aronia melanocarpa were measured.The results showed that the fruit weight was 99.44 g/80 fruits(FW),the total soluble solid content was 16.63 Brix%,the extraction rate was 2.5%,and the contents of total anthocyanin and total phenolic were 375.87mg/100g(FW)and 1856.88 mg/100g(FW),respectively.Ethanol was selected as the extractant,and the anthocyanin in Aronia melanocarpa was extracted by ultrasonic wave.Based on the results of single factor test,the response surface analysis was used to optimize the extraction process.After the adjustment of actual tests,the optimum values of each factor were determined as follows:ethanol volume fraction was 75%,extraction temperature was42°C,extraction time was 36 min,and the absorbance value was 0.578 under this condition which was close to the theoretical value.The purification conditions of anthocyanins from Aronia melanocarpa were optimized by macroporous resin,and the purification effect of NKA-9 macroporous resin was found to be the best.The purification conditions of NKA-9 macroporous resin were optimized,with the optimal conditions following:the adsorption equilibrium time was 1 h,the desorption equilibrium time was 1 h,the extract was diluted 4 times,50%ethanol(v/v)was eluted,and the loading flow rate was 4 r/min,the elution flow rate was 4 r/min,the sample loading was450 mL,and the amount of distilled water was 2 BV.The anthocyanins purified by macroporous resin were identified by HPLC-MS,and 7 kinds of anthocyanin were identified such as the cyanidin-3,5-dihexoside,cyanidin-hexoside dimer,cyanidin-3-O-(galactoside,glucoside,arabinoside,xyloside)and delphinidin-3-O-rutinoside.Among them,cyanidin-3,5-dihexoside and cyanidin-hexosaside dimer were identified for the first time in Aronia melanocarpa,and delphinis-3-O-rutinoside was only reported once.The semi-preparative reversed-phase high performance liquid chromatography(HPLC)was used to further purify the Aronia melanocarpa anthocyanins,the anthocyanins was identified based on the standards and quantified based on the calibration curves of cyanidin-3-xyloside using the external standard method using high performance liquid chromatography(HPLC).The results showed that cyanidin-3-O-(galactoside,glucoside,arabinoside,xyloside)were identified by HPLC,and the content of the anthocyanin mixture was determined to be 930.30mg/g(DW),including cyanidin-3-O-galactoside 497.87 mg/g(DW),cyanidin-3-O-glucoside33.32 mg/g(DW),cyanidin-3-O-arabinoside 320.47 mg/g(DW)and cyanidin-3-O-xyloside78.64 mg/g(DW).The total antioxidant capacity of anthocyanins with different purities,including Vc,Aronia melanocarpa juice(Aronia melanocarpa),Aronia melanocarpa anthocyanins extracts purified by absorbent resin(A-Anthocyanins-A),Aronia melanocarpa anthocyanins extracts purified by absorbent resin and semi-preparative RPLC(A-Anthocyanins-R),?Beilei‘L.caerulea juice(L.caerulea),?Beilei‘L.caerulea anthocyanins extracts purified by absorbent resin(L-Anthocyanins-A),were assessed by using T-AOC,ABTS and FRAP kit respectively.The results showed that the antioxidant capacity of anthocyanins could be significantly improved by increasing the purity of anthocyanins,and the anthocyanins purified by RPLC(A-Anthocyanins-R)have the highest antioxidant capacity.Then purified anthocyanins(A-Anthocyanins-R)were tested with lard to investigate its potential applicatiion in food,the result showed that anthocyanin can effectively delay the oxidation of lard and has a good application in food.The protective effects of anthocyanins on the SH-SY5Y Alzheimer's disease cell model were studied from the perspective of oxidative stress.Through MTT assay,we found that A?1-42-42 can induce damage to SH-SY5Y cells,result in the decrease of cell viability,and anthocyanins concentration in the range of 10?g/mL to 100?g/mL can impair the damage induced by A?1-42 in SH-SY5Y cells.Through the oxidative stress related index test,A?1-42can increase the production of intracellular ROS and reduce the content of superoxide dismutase(SOD)in the cells,thereby cause oxidative stress in cells.In this experiment,20?g/mL,40?g/mL and 60?g/mL anthocyanins were selected to study the protection of anthocyanins on cellular oxidative stress.It was found that anthocyanins can reduce intracellular ROS production and increase intracellular SOD content,thereby attenuate the oxidative stress induced by A?1-42 in SH-SY5Y cells,and the protective effect was dose-dependent.In addition,this chapter studied the expression of Nrf2-regulated antioxidant proteins and genes by western blot,realtime PCR.It was found that the protein and mRNA expression levels of Nrf2,HO-1 and NQO1 were decreased after A?1-42 treatment to SH-SY5Y cells.In the drug protection groups,anthocyanins up-regulated the protein and mRNA expression levels of Nrf2,HO-1 and NQO1.Through literatures analysis,we concluded that anthocyanins can activate Nrf2 metabolic pathway and up-regulate the mRNA and protein expression of Nrf2,HO-1,NQO1,and reduce the level of ROS in the cells,thereby effectively prevent Alzheimer's disease from oxidative stress damage induced by A?1-42 in SH-SY5Y cells.This mechanism of protecting Alzheimer's disease cell model from A?1-42-induced apoptosis through Aronia melanocarpa anthocyanins was explored via the Ca2+homeostasis and the mitochondrial pathway.Through the research results,we can concluded that the anthocyanins in Aronia melanocarpa could up-regulate the gene and protein expression of Calmodulin,maintain the intracellular calcium content,thereby down-regulate the gene and protein expression of Cytochrome C,Caspase 9,Cleaved caspase 3,and decrease the rate of apoptosis.In addition,anthocyanins can up-regulate the gene and protein expression of BcL-2,down-regulate the gene and protein expression of Bax,thus regulate the permeability of mitochondrial membrane and calcium release channels,reduce the influx of calcium ions into mitochondria,reduce intracellular ROS levels,ATP levels,maintain mitochondrial membrane potential in a normal level,and ensure the function of mitochondrial work properly,thereby protect cells from apoptosis.The above results indicated that anthocyanins in Aronia melanocarpa can protect SH-SY5Y cells from the apoptosis induced by A?1-42 effectively,and this protective effect is dependent on the dose of anthocyanins.When the anthocyanins content is 60?g/mL,the protection effect of anthocyanins is the best. |
PDF Full Text Request