Study On Extraction,Purification,Structure Characterization And Hypoglycemic Activity Of Auricularia Auricularia Polysaccharide

Auricularia auricularia was an important edible fungus with homology of medicine and food.It was rich in polysaccharides,proteins,essential amino acids and various trace elements.Previous studies have shown that polysaccharides,an important bioactive component in Auricularia auriculata,had a variety of biological activities,such as antioxidant,hypidemic,immune enhancement,tumor inhibition and so on.However,systematic studies on the hypoglycemic activity of Auricularia auricularia polysaccharides were lacking,especially on the hypoglycemic mechanism.In this study,the chemical structure of Auricularia auricularia polysaccharides was studied,and the hypoglycemic mechanism of it was systematically studied from metabolomics,metagenomic and related protein expression levels,in order to prevent diabetes,relieve insulin resistance,and achieve high value-added utilization of Auricularia auricularia polysaccharides.Based on this,in this paper,Auricularia auricularia polysaccharide(AAP)was extracted from Auricularia auricularia by hot water extraction and ethanol precipitation method.Response surface optimization experiment was used to obtain the optimal extraction conditions.After purification,two components AAP-1 and AAP-2 were obtained,and their physical and chemical properties,structure characterization and in vitro antioxidant and hypoglycemic activities were compared.AAP with good hypoglycemic activity was screened out and C57BL/6J male mice were used as experimental objects to construct a type 2 diabetes model to study its hypoglycemic activity in vivo.According to the results of relevant physiological and biochemical indicators,AAP had good hypoglycemic and insulin resistance relieving effects.Combined with metabolomics,metagenomics and PI3K/AKT signaling pathway related genes and protein expression levels,the hypoglycemic mechanism was discussed.The specific research results were as follows:(1)Single factor and response surface methodology were used to optimize the extraction process of AAP.The optimal extraction conditions were obtained as follows:extraction temperature 70℃,extraction time 4 h,solid-liquid ratio 1:90(g/mL).Under these conditions,the average yield of AAP was 8.17%.The total sugar content,protein content and uronic acid content of AAP were 71.4%,2.66%and 5.73%,respectively.AAP-1 and AAP-2 were separated and purified by DEAE-52 and G-100,and their total sugar content was 85.1%and 90.2%,respectively.Protein contents were 1.32%and 1.05%,respectively.The contents of uronic acid were 15.84%and 2.35%,respectively.(2)The structures of AAP-1 and AAP-2 were preliminarily characterized.The monosaccharide composition of AAP-1 mainly consisted of 18.43%glucose,7.3%xylose,41.15%mannose and 26.3%glucuronic acid,which are acidic polysaccharides.AAP-2 was composed of fucose,galactose,glucose,mannose and rhamnose,with the molar percentages of 12.54%,19.15%,50.5%,10%and 3.75%,respectively.The two polysaccharide components had characteristic absorption peaks of polysaccharides and belonged to pyranose sugar.HPGPC results showed that the Mw(Da)of AAP-1 and AAP-2 were 6.02×106 Da and 1.49×106 Da,respectively.Methylation results showed that AAP-1 was mainly composed of 10 different sugar residues,and its main glycosidic bond composition included:→4,6)—Glc(p)UA—(1→(16.819%),→2,6)—Man(p)—(1→(15.772%),→6)—Xyl(p)—(1→(13.407%)and Glc(p)—(1→(10.957%);The glycosidic bond of AAP-2 mainly includes:→3)-Gal(p)-(1→(18.988%),Glc(p)-(1→(14.34%),→6)-Fuc(p)-(1→(12.28%)and→2,3)-Glc(p)-(1→(11.641%).SEM results showed that AAP-1 and AAP-2 showed similar lamellar structure,and the surface of AAP-2 was more smooth and compact.(3)In vitro antioxidant experiments showed that AAP,AAP-1 and AAP-2 all had reducing power and scavenging activities of DPPH,hydroxyl radical and superoxide anion.The antioxidant activities of AAP in vitro were significantly higher than those of the other two polysaccharide components.The hypoglycemic activity of AAP,AAP-1 and AAP-2 on α-glucosidase was higher than that of α-amylase,and the hypoglycemic activity of AAP was significantly higher than that of other polysaccharides.(4)T2DM model was established in C57BL/6J male mice induced by high-fat diet combined with STZ.After intervention of different doses of AAP,the symptoms of "three more and one less" were alleviated.Compared with MD group,FBG,OTGG,FINS and HOMA-IR in AAP group were significantly decreased(p<0.01),and AAP-M group had the best hypoglycemic effect.Compared with the NC group,the contents of TG,TC and LDL-C in MD group were significantly increased,while HDL-C was significantly decreased.Liver antioxidant assay showed that AAP had antioxidant effect in vivo.Compared with MD group,liver glycogen content,T-SOD,CAT and GSH-Px activities in AAP-M group were significantly increased(p<0.01),MDA content significantly decreased(p<0.01),the results showed that AAP reduced the oxidative stress injury in T2DM mice,and AAP-M treatment group had the best effect.Pancreatic and liver histopathological observation of the MD group showed atrophy of islet cells,some islets had degeneration and necrosis,and liver cells showed steatosis.However,AAP protection could repair pancreatic tissue damage to varying degrees and relieve hepatocellular lesions.(5)The changes of serum metabolites in different treatment groups were evaluated by UHPLC-Q exactive HFX-MS metabonomics analysis method.PCA and OPLS-DA analysis showed that metabolic disorders occurred in diabetic mice,and metabolites of diabetic mice were significantly improved after AAP intervention.Based on p<0.05 and VIP>1,45 potential biomarkers were selected.KEGG pathway enrichment analysis suggested that AAP regulates arginine and proline metabolic pathways,biosynthesis of aminoacyl-TRNA(amino acid metabolism),unsaturated fatty acid synthesis,glycerophospholipid metabolism and primary bile acid(BA)biosynthesis to improve metabolic disorders in diabetic mice.(6)The gut microflora of different groups of mice were compared by 16S rRNA high-throughput sequencing technology found AAP could regulate the gut microflora structure of diabetic mice.Intaked of AAP could decreased the relative abundance of Firmicutes,increased the abundance of Bacteroidetes at the phylum level,and increased the relative abundance of beneficial bacteria producing short-chain fatty acids at the genus level,including Lachnospiraceae,Faecalibaculum,Dubosiella,Allpprevotella and so on to promote the production of short-chain fatty acids into the human circulation and improve the disorder of glucose and lipid metabolism.(7)WB and RT-qPCR results indicated that AAP could reduce insulin resistance,reduce oxidative stress injury and improve glucose and lipid metabolism by activating PI3K/AKT signaling pathway,up-regulating PI3K and AKT mRNA expression and protein expression levels.In this study,the effects of AAP on insulin resistance and hypoglycemic activity were studied,and the structures of AAP-1 and AAP-2 were preliminarily characterized.Based on metabonomics,16S rRNA gut microflora and gene and protein expressions of PI3K/AKT signaling pathway,the mechanism of hypoglycemic effect and alleviating insulin resistance of AAP was explored.It also provided new ideas for the prevention,adjuvant treatment of T2DM,alleviation of insulin resistance and the realization of high value-added utilization of Auricularia auricularia.