The Dynamic Of Anthocyanin Profile Of Blueberry During Development And The Protective Mechanism On AD Cell Apoptosis By Cyanidin-3-O-Glucoside

The study of the mechanism of action of blueberry anthocyanins on AD is key significance for the development of deep-processing products containing blueberry anthocyanins,and the adjuvant prevention and treatment of AD.Blueberry fruits of different varieties at different development stages were used as experimental materials.First,the analysis of types of anthocyanin monomers,content of monomers and differential metabolites at different stages of blueberries were conducted using metabolomics.Second,the analysis of genes related to the blueberry anthocyanin biosynthesis pathways was conducted using transcriptomes.‘Misty’ blueberries at maturity stage were selected as raw materials,of which the extraction and purification conditions were optimized to obtain high purify cyanidin-3-O-glucoside.Finally,the prevention and therapeutic mechanisms of cyanidin-3-O-glucoside on apoptosis produced by Aβ1-42 induced AD cell models were investigated.The study results are as follows:(1)The types and content changes of anthocyanin monomers and differential expression of F3 H,F3’H,DFR,F3’5’H,ANS,UFGT and FLS of anthocyanin biosynthetic pathways in the four blueberry varieties at different development stages were analyzed.The results showed that 32 anthocyanin monomers,including cyanidin,delphinidin,pelargonindin,malvidin,peonidin and petunidin,were detected in the four blueberry varieties for the first time,as well as six proanthocyanidins and three dihydroflavonols.The study of differential metabolities showed that,as the fruit developed,most of the differential metabolites showed an up-regulated,except for ‘Britewell’ Blue and ‘Gardenblue’ from young fruit to color-turning stage.Differences in the metabolites between varieties at the same stage were significant.In general,the content of most anthocyanin monomers showed an increasing trend in all four blueberry varieties and reached the highest level at maturity stage,while the content of proanthocyanidins and dihydroflavonols dwindled.The q RT-PCR results showed that,although the expression of seven genes related to the anthocyanin biosynthesis pathways differed among the four blueberry varieties at different development stages,it was highly consistent with the variation of blueberry anthocyanins.The results also showed that the‘Misty’ blueberries at maturity stage had the highest content of cyanidin-3-O-glucoside.(2)The ultrasonic-microwave-assisted extraction of anthocyanins from blueberries was optimized by Plackett-Burman analysis and Box-Behken response surface design.According to the results,the best conditions for the extraction of anthocyanins from ‘Misty’ blueberries were determined as follows: microwave power was 185 W,ultrasonic power was 250 W,extraction temperature was 42 ℃,extraction time was 42 min,extraction 1 time and liquid ratio was 25∶1,under which the extraction amount of anthocyanins was 241.87 mg/ 100 g.The preliminary purification of the crude extract of anthocyanins was performed with column chromatography,and the best conditions were: adsorption equilibrium time was 2 h,desorption equilibrium time was 2 h,dilution ratio was 4,volume fraction of eluent(ethanol)was 60 %,sample flow rate was 4 r/min,sample quantity was 9,elution flow rate 4 r/min,distilled water was 2.5 BV,under such conditions,the purity of anthocyanins measured by p H difference method was 75.21%.Through further purification was performed by preparative high performance liquid chromatography to yield cyanidin-3-O-glucoside monomer with a purity of 93.73 %.The antioxidant properties of anthocyanins purified from blueberry juice,crude extract of blueberry anthocyanins and macro-porous resin,and anthocyanins purified by HPLC were measured with hydroxyl radicals.The results showed that the antioxidant properties of anthocyanins were dependent on purity.(3)Aβ1-42 induced damage in human neuroblastoma SH-SY5 Y cells was used to construct a cell model of AD.Cyanidin-3-O-glucoside monomer was used to investigate the mechanism of protection against AD by regulating Ca2+ mediated mitochondrial dysfunction.The results showed that,cyanidin-3-O-glucoside was able to down-regulate the expression of genes and proteins of cytochrome C,caspase 9,caspase 3,cleaved caspase 3,and reduce the rate of apoptosis.In addition,it was able to up-regulate the expression of bcl-2 gene and protein,down-regulate the expression of bax gene and protein,regulate the mitochondrial membrane permeability and calcium release channels,mitigate and reduce the influx of calcium ions into mitochondria,maintain the intracellular calcium ion content,reduce the intracellular ROS level and ATP level,maintain the mitochondrial membrane potential at normal level,and ensure the normal mitochondrial function to protect cells from apoptosis.Therefore,cyanidin-3-O-glucoside can effectively prevent and treat Aβ1-42 induced apoptosis of human neuroblastoma SH-SY5 Y cells,and its preventive and protective properties are dependent on the dose of cyanidin-3-O-glucoside.The best protective effect of cyanidin-3-O-glucoside was60 μg/m L.Comparing to the therapeutic effect of cyanidin-3-O-glucoside on Aβ1-42 induced apoptosis of human neuroblastoma SH-SY5 Y cells,the preventive effect of cyanidin-3-O-glucoside was significantly better.