Study On Extraction,purification And Hyperlipidemic And Prebiotics Activity Of Red Raspberry Anthocyanins

In this study,the parameters of extraction and purification of red raspberries anthocyanins were optimized and their effects on hyperlipidemia and intestinal microorganisms were investigated.First,single-factor tests and a response surface test were conducted to optimize the crude raspberry anthocyanin extraction.Second,the purification conditions were optimized.The components of the crude anthocyanins were explored by liquid chromatography-mass spectrometry.Then,four anthocyanin monomers were successfully separated by preparative liquid chromatography.The effects of the crude extracts on hyperlipidemia were studied in animal experiments,and transcriptomics was used to study the hypolipidemic mechanism.Finally,the effects of the four monomers on intestinal flora were also studied in vitro.The main conclusions were as follows:1. The anthocyanins were enzymatically extracted from red raspberry.Four single-factor experiments were conducted to explore the extraction parameters in terms of the yield of anthocyanins.Based on the single factor experiments,a response surface analysis was employed to explore the optimal extraction conditions.The results were as follows:pectinase concentration 57.03 U/m L,solid-liquid ratio 19.85 g/ml,temperature 38.00 ~oC and extration time 57.35 min.The predicted yield was 402.51mg/g.Five kinds of macroporous resins(DM301,D101,HP-20,X-5 and AB-8)were used to conduct static adsorption and desorption experiments.The results indicated that X-5 showed the highest adsorption and desorption activity for the anthocyanins.The yield and purity of the anthocyanins obtained after purification were 0.28‰and 60.08%,respectively.2. The components of the crude extract of the red raspberry anthocyanins were identified by LC-MS/MS and the main components were analyzed by the mass spectrometry.There were 14 kinds of anthocyanins and their total relative abundance added up to 60.08%.Other polyphenols such as procyanindin B2(11.44%)and L-epicatechin(6.49%)were identified.In addition,there were a small amount of acid substances such as ellagic acid(7.16%),citric acid(4.07%),p-coumaric acid(1.92%)and asiatic acid(0.15%).The crude extract was further separated by the semi-preparative liquid chromatography.Four kinds of the anthocyanin monomers were successfully separated and detected by the analytical liquid chromatography.The results of MS detection indicated that the monomers were cyanidin-3-O-di-hexoside,cyanidin-3-O-glucoside,cyanidin-3-O-rutinose and cyanidin-3-O-glucoside-rutinoside,respectively.3. The effects of the red raspberry extract(RRE)intake on hyperlipidemia mice induced by a high-fat diet were studied.After 8 weeks of intragastric administration,the body weight and adipose tissue mass of mice receiving RRE were significantly decreased compared with those without RRE treatment(p<0.05).In addition,RRE treatment significantly(p<0.05)reduced triglyceride and total cholesterol levels in hyperlipidemia mice.Through transcriptome analysis and fluorescent quantitative PCR verification,it was confirmed that RRE could regulate the expression of Ppar?,Hmgcr,Ldlr,Cyp7a1,Acsl3,Pnpla2 and Pin4 genes to lower blood lipids.According to KEGG pathway analysis,target genes such as Cyp7a1 and Pin4 were further regulated by the activation of Ppar?resulting from RRE supplementation.Meanwhile,liver cholesterol synthesis and conversion were inhibited by the expressions of Hmgcr and Cyp7a1 genes regulated by RRE intake,and Ldlr gene was down regulated to limit the transport of cholesterol.In addition,RRE treatment could accelerate the conversion from triglyceride to fatty acid.In short,RRE intake would be a protection against diet-induced hyperlipidemia.4. The effects of the four monomers on the intestinal flora of patients with hyperlipidemia in vitro were further studied.Through in vitro co-culture of the four monomers and intestinal microorganisms,the four monomers could significantly increase the number of Bifidobacterium and Lactobacillus,and had no significant effect on the population of E.coli.After co-cultivation with anthocyanin monomers,the lipased,glucosidase and amylase in the intestinal flora were significantly inhibited,and the cellulase had no significantly change.In addition,compared with the blank group,the content of ferulic acid,p-coumaric acid,caffeic acid and gallic acid in the anthocyanin treatment group increased significantly.In conclusion,the four anthocyanins could improve the intestinal environment of hyperlipidemia populations and improve hyperlipidemia.