| Anemarrhena asphodeloides Bge.,as a traditional Chinese medicine(TCM),is an important component of various herbal TCM formulations,such as Compound Cassia peony Decoction,Guan Guan Pill and Cassia twig peony zhimu soup,and has been used in the treatment of diabetes for thousands of years,however,the underlying mechanism has not been fully elucidated.Polysaccharides is one of the main active component of Rhizoma Anemarrhenae.Studies have shown that the crude polysaccharides or the water extract of Rhizoma Anemarrhenae has certain hypoglycemic effects,but there are few reports on the structure of homogenous Rhizoma Anemarrhenae polysaccharide and its mechanism in treating diabetes and its effects on intestinal flora.Therefore,this thesis systematically isolated,purified and identified the polysaccharides from Anemarrhena asphodeloides and investigated its potential hypoglycemic mechanisms using insulin resistance Hep G2 cell model and db/db diabetic mouse model and throguh its effects on intestinal flora,which can provide a theoretical basis for the development and utilization of the polysaccharides from Anemarrhena asphodeloides.(1)The polysaccharides were separated and purified by stepped-alcohol precipitation and DEAE-52,and the resulting two homogeneous polysaccharide(AABP-1B and AABP-2B)were collected.The molecular weight of AABP-1B was 105 k Da,which composed of mannose,glucose,galactose,galacturonic acid,arabinose,and rhamnose,with a molar ratio of 6.2:1.1:0.5:1.6:0.3:0.5.The molecular weight of AABP-2B was 5.8 k Da,which composed of mannose and glucose,with a molar ratio of 7.2:2.8.The fine structures of AABP-1B and AABP-2B were further elucidated using methylation and NMR(1D and 2D NMR).AABP-1B consisted of 2,6)-?-D-Manp-(1 backbone,branched at C-2 position by various of sugar residues,including 4,6)-?-D-Galp-(1,T-?-L-Rhap-(1,4,6)-?-D-Galp-(1,T-?-D-Glcp-(1,3,6)-?-D-Manp-(1,4)-?-D-Gal Ap-(1,4)-?-D-Gal Ap-(1 and 3)-?-L-Araf-(1.AABP-2B consists of 4)-?-D-Manp-(1?4,6)-?-D-Glcp-(1?4)-?-D-Glcp-(1?T-?-D-Glcp-(1?T-?-D-Manp-(1?3,6)-?-D-Manp-(1 and 2)-?-D-Manp-(1.AABP-2B is glucomannan and the backbone of AABP-2B was mainly composed of 4)-?-D-Manp-(1,4,6)-?-D-Glcp-(1 and3,6)-?-D-Manp-(1.(2)Inhibiting digestive enzymes and an insulin resistance Hep G2 cell model(IR-Hep G2)were established to investigate the in vitro hypoglycemic activity of anemone polysaccharide.The results showed that both AABP-1B and AABP-2B could inhibit the activities of?-amylase and ?-glucosidase.The activity of acid polysaccharide AABP-1B in inhibiting?-amylase and ?-glucosidase was higher than that of AABP-2B.The cell experiment results showed that AABP-1B and AABP-2B treatment promoted glucose uptake in IR-Hep G2 cells and enhanced pyruvate kinase and hexokinase activities in IR-Hep G2 cells.Furthermore,insulin resistance was regulated by PI3K/Akt signaling pathway.AABP-1B and AABP-2B treatment up-regulated ir S-1 m RNA,PI3 K m RNA,Akt m RNA expression,and down-regulated GSK-3? m RNA expression,activated glycogen synthase(GS),and promoted Hep G2 cells to absorb glucose and synthesize liver glycogen.(3)The changes of AABP-1B and AABP-2B during digestion and their effects on intestinal flora were investigated by simulating in vitro digestion and fermentation experiments.After oral digestion,the molecular weight of AABP-1B and AABP-2B did not change significantly,indicating that the two polysaccharides were not digested by saliva.AABP-1B was partially hydrolyzed during simulated gastrointestinal digestion,resulting in a slight decrease in molecular weight and an increase in reducing sugar content.However,the molecular weight and reducing sugar content of AABP-2B did not change significantly during the simulated digestion.In addition,neither polysaccharide produced free monosaccharides during the whole digestion process,indicating that AABP-1B and AABP-2B could pass through the upper digestive system without being broken down.In vitro fermentation results showed that AABP-1B and AABP-2B could regulate the structure of intestinal flora by promoting the production of SCFAs.Acidic polysaccharide AABP-1B produced higher acetic acid than neutral polysaccharide AABP-2B after fermentation,while glucose-rich AABP-2B produced higher propionic acid than AABP-1B.In addition,The ratio of Firmicutes/Bacteroides was significantly reduced.Among them,the decrease of Firmicutes/Bacteroidetes ratio is positively correlated with the reduction of host energy absorption,which may help suppress obesity.(4)The hypoglycemic effects of AABP-1B and AABP-2B were investigated through in vivo animal experiments using db/db mice.Results showed that AABP-1B and AABP-2B treatment significantly reduced mice body weight,fasting blood glucose and fasting insulin concentration,and improved liver insulin resistance by PI3K/Akt pathway.Besides,intake of anemone polysaccharide reduced the content of TC,LDL-C,TG and FFA and increased the concentration of HDL-C in serum.Moreover,the activity of liver antioxidant enzymes(CAT,GSH-Px and SOD)was increased and the content of MDA was decreased after AABP-1B and AABP-2B manipulation.Furthermore,Anemarrhena asphodeloides polysaccharide intervention significantly affected mice fecal intestinal flora.The ratio of Firmicutes/Bacteroides was significantly reduced.Among them,the decrease of Firmicutes/Bacteroidetes ratio is positively correlated with the reduction of host energy absorption,which may help suppress obesity.Besides,AABP-1BA and ABP-2B could regulate the blood sugar level and improve insulin resistance of db/db mice by increasing the abundance of Bifidobacterium,Lactobacillus,Bifidobacterium and Akkermansia. |
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